Physiological characteristics of IRR 400 series rubber clones (<i>Hevea brasiliensis</i> Muell. Arg.) under drought stress

Syarifah Aini Pasaribu; Mohammad Basyuni; Edison Purba; Yaya Hasanah

doi:10.12688/f1000research.129421.4

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Research Article

Revised

Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

[version 4; peer review: 1 approved, 2 approved with reservations]

Previously titled: Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) on drought stress

Syarifah Aini Pasaribu¹, Mohammad Basyuni ^2,3, Edison Purba⁴, Yaya Hasanah⁴

PUBLISHED 04 Dec 2023

Author details Author details

¹ Unit Research Sungei Putih, Indonesian Rubber Research Institute, Galang, Deliserdang, North Sumatra, 20585, Indonesia
² Center of Excellence for Mangrove, Universitas Sumatera Utara, Medan, 20155, Indonesia
³ Department of Forestry, Universitas Sumatera Utara, Medan, North Sumatra, 20155, Indonesia
⁴ Department of Agrotechnology, Universitas Sumatera Utara, Medan, North Sumatra, 20155, Indonesia

Syarifah Aini Pasaribu
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Mohammad Basyuni
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Validation, Writing – Review & Editing

Edison Purba
Roles: Data Curation, Formal Analysis, Validation, Writing – Review & Editing

Yaya Hasanah
Roles: Data Curation, Formal Analysis, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

Abstract

Background

Drought stress is one of the main causes of plant death. Strategies for plants survival are morphological adaptations, specific signaling pathways, and tolerance mechanisms. Rubber plantations have many uses, such as foreign exchange sources, job sources, forest revitalization, and a source of alternative wood for building materials and furniture. The rubber plant’s response to drought stress is a complex biological process. A tolerant rubber clone in a dry area is the right approach. The present study aimed to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage.

Methods

The first factor examined for this work was clones (IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24) and the second factor was water content (30%, 60%, and 90%). The study was arranged on a factorial randomized block design and repeated three times. Characteristics observed were total sugar (µM), proline (mg/L), chlorophyll a, b, total (µg/mL), hydrogen peroxidase (µmol/g), ascorbate peroxidase (unit/mg), superoxide dismutase (unit/mg), and peroxide dismutase (unit/mg).

Results

The tolerance ability of the IRR 400 series rubber clones to drought stress was determined by observing the characteristics of sugar total and proline. The concentration of total sugar and proline were higher when the plant was treated with a lower water content. The selected clones tolerant to drought stress are RR 425 and IR 434 with high total sugar content and proline. Other characteristics, namely chlorophyll a, b, and total, as well as hydrogen peroxidase, ascorbate peroxidase, super oxide dismutase, peroxide dismutase, cannot be used as selection characteristics for this study.

Conclusions

This drought study of IRR 400 clones with varying water content percentages illustrated that the total sugar and proline characteristics could be used to distinguish tolerance levels from other observed characteristics.

Keywords

rubber, drought stress, water content, adaptation, abiotic stress

Corresponding author: Mohammad Basyuni

Competing interests: No competing interests were disclosed.

Grant information: The Penelitian Disertasi Doktor project No. 11/AMD/E1/KP.PTNB/2021, from the Directorate General of Research and Community Service, the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia. Center of Excellence for Mangrove, Universitas Sumatera Utara covered the APC of this paper.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Pasaribu SA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Pasaribu SA, Basyuni M, Purba E and Hasanah Y. Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress [version 4; peer review: 1 approved, 2 approved with reservations]. F1000Research 2023, 12:106 (https://doi.org/10.12688/f1000research.129421.4) First published: 27 Jan 2023, 12:106 (https://doi.org/10.12688/f1000research.129421.1) Latest published: 04 Dec 2023, 12:106 (https://doi.org/10.12688/f1000research.129421.4)

Revised Amendments from Version 3

We have revised to incoropreta Reviewer 's 1 comments and suggestions. We updated the aim of study in the Abstract and Introduction (the last paragraph). We also revised the first line of 2nd paragraph and 4th paragraph of Introduction. We revised in the 2nd paragraph of Discussion as well as 4th and 5th paragraph in the Discussion. The last revision is the Conclusion.

See the authors' detailed response to the review by Mohamed sathik Thirruvithamkottil
See the authors' detailed response to the review by Maryam Nazari

Introduction

In rubber plants, drought can cause a delayed maturation phase, short tapping period, slow latex flow, dry latex, increased dry tapping grooves, and even tree death.¹ Drought is one of the main abiotic stresses affecting yield and productivity in almost all crops.² Hence, its significance overshadows that of other environmental factors because it interferes with plant growth and development and disrupts production and performance. Water is an important component of the protoplasm and makes up 85%–90% of the total weight of the plant tissue. Water is also a vital reagent in photosynthesis and hydrolysis reactions. Additionally, it acts as a solvent for salts, gases, and other substances transported between cell tissues to maintain cell growth and leaf shape stability.³

One of the primary sources of natural rubber producing plants was found in the Amazon basin, South America.⁴ Optimal conditions for the growth of rubber plants include high temperature (28 ± 2 °C), high humidity, and rainfall of 2000–4000 mm/year.⁵ Rubber plantations in marginal areas, such as Central India, southern China, northern and northeastern Thailand, and eastern Indonesia, experience occasional drought. Indonesia has a wide drought area of about 122.1 million ha, which is not optimally exploited due to limited water resources.

The response caused by drought with regard to plant is quite complex because it involves changes in morphology, physiology, and metabolism. The initial response to drought stress is the loss of turgor pressure, which results in reduced growth rate and leaf senescence. Drought changes the source–sink relationship and affects the translocation of photosynthate to produce fruit quickly for certain crops.⁶ The fastest response to a water deficit is the stomatal closure to protect plants from water shortages. Water deficit results in abscisic acid (ABA) biosynthesis, which triggers stomatal closure and causes a decrease in intracellular CO ₂ levels and the inhibition of photosynthesis.⁷ Water shortages do not always promote these responses in all plant species. Lack of intracellular CO ₂ due to prolonged stomatal closure leads to the accumulation of reactive oxygen and nitrogen species, which damages the photosynthetic apparatus.⁸ Besides that, the presence of osmoprotectants, such as proline, trehalose sugar, glycine betaine, donomitol, and mannitol maintain the growth and productivity of a plant experiencing drought stress.⁹^–¹¹ The presence of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), in cellular and cytoplasmic organelles play an important role in the detoxification of these reactive oxygen species (ROS) and enable plant cells to activate various stress sensors, which will then activate various signal paths.

Inhibited growth is a typical symptom of drought stress.¹² The consequent physiological, biochemical, and molecular changes affect various cellular processes, thereby reducing the quantity and quality of the plant yield. The combined rise in atmospheric CO₂ concentrations and global air tempeature may significantly alter plant water use.¹³ Pasaribu et al, reported based on bio-informatics, there are 20 proteins related to drought stress in rubber plants.¹⁴ This study aims to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage.

Methods

Study area

Analysis of physiological characteristics was carried out at the Physiology and Protection Laboratory of the Unit Research SungeiPutih, Galang, Deli Serdang, North Sumatra. The study was carried out in a greenhouse during June 2020–May 2021. The materials used were red-yellow podzolic soil (water content = 3.9, pH = 4.5, C-organic = 0.92, N = 0.15, P₂O₅ = 2.13), compound fertilizer, Dithane M-45, and Triko-SP Plus. The tools used included polybags (18 × 35 cm and 50×60 cm), a hoe, a soil sieve, bucket, watering can, a 100 kg UK scale, an analytical balance, object glass, deck glass, binocular microscope, water bath, vortex, UV spectrophotometer, filter paper, test tube, gloves, mask, tissue, distilled water, mortar, beaker, micropipettes 1 ml and 100 μl, stirrer, 15-watt lamp, microcentrifuge, microwave, and others.

Stages of growth and development of the clones

The plants age was 6-12 months, planted in polybags measuring 50 × 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease such as Oidium, Colletotrichum, Corynespora and Pestalotiopsis. The leaves were taken between at 08.00-09.00 AM. The average temperature in the greenhouse during the six months observation was 25.9 °C (morning), 33.9 °C (midday) and 30.4 °C (afternoon). The average temperature outside the greenhouse was 22.9 C (morning), 28.1 °C (midday) and 25.5 °C (afternoon). The average humidity was 92.6% (morning), 62.9% (midday) and 69.2% (afternoon).

Experimental design

The study was arranged based on a factorial randomized block design (RBD). The first factor was the type of clone, consisting of seven types, namely C1: IRR 425, C2: IRR 428, C3: IRR 429, C4: IRR 434, C5: IRR 440, C6: RRIC 100, and C7: BPM 24. The second factor was water content, consisting of three levels, namely: W1: 30%, W2: 60%, and W3: 90%. Each experimental unit was repeated three times, and as many as 63 samples were observed.

Data analysis

Observations were carried out six times on physiological characteristics, with an intervals of three weeks. If the test of variance obtained significantly different treatments, then the Tukey distance test of 0.5% was carried out.¹⁵ The characteristics observed were total sugar content,¹⁶ chlorophyll a, b, and total,¹⁷ proline,¹⁸^–²¹ super peroxidase dismutase (SOD),²² peroxidase dismutase (POD),²³ APX enzyme,²⁴ and hydrogen peroxide (H₂O₂).²²

A step-by-step description of the procedure to analyze sugar content, proline, chlorophyll a, b, and total, SOD, POD, H₂O₂, and APX has been deposited in protocols.io and is available at dx.doi.org/10.17504/protocols.io.5jyl8je1dg2w/v1.

Results

Total sugar content (μM)

The total sugar content analysis showed a significant effect in all the observations except the first one (Table 1).

Table 1.

The total sugar content (μM) in different clones, different water content (%), and interactions between clone type and water content (%).

Treatments	Total sugar content (μM)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	158.99	a	65.53	d	90.74	b	105.17	b	198.38	b	175.77	b
IRR 428	189.62	a	97.27	b	92.06	b	127.02	ab	190.27	bc	136.85	e
IRR 429	190.39	a	122.26	a	98.64	b	146.21	a	221.09	a	140.27	e
IRR 434	178.26	a	96.40	b	92.99	b	132.04	ab	190.12	bc	160.50	c
IRR 440	156.32	a	77.94	cd	96.55	b	127.07	ab	183.62	c	151.19	d
RRIC 100	198.05	a	129.34	a	11.90	a	129.26	ab	193.01	bc	184.73	a
BPM 24	178.27	a	87.36	bc	73.28	c	158.75	a	192.28	bc	166.85	c
Water content (WC)
30%	188.24	a	111.68	a	116.79	a	151.64	a	200.57	a	178.83	a
60%	174.91	a	93.05	b	88.02	b	132.12	b	180.29	b	156.78	b
90%	172.51	a	85.03	c	77.69	c	112.89	c	205.76	c	142.74	c
Interactions C × WC
C1WC1	172.96	a	73.56	ghij	94.76	cdefg	117.88	bc	219.11	a	197.64	abc
C1WC2	166.06	a	63.38	ghij	106.65	bcdef	94.52	c	169.08	bcd	186.03	bc
C1WC3	137.94	a	54.66	j	70.81	g	103.12	c	206.96	ab	143.38	fg
C2WC1	186.45	a	107.60	cdef	111.73	bcde	161.67	abc	181.09	bcd	121.82	h
C2WC2	145.41	a	123.18	bc	92.72	cdefg	113.25	bc	180.51	bcd	143.02	fg
C2WC3	236.99	a	61.03	ij	71.72	g	106.15	bc	209.22	ab	145.70	fg
C3WC1	232.61	a	149.85	a	125.52	ab	206.06	a	227.64	a	164.65	de
C3WC2	168.13	a	111.00	cde	84.10	defg	127.88	abc	213.41	ab	134.68	fgh
C3WC3	170.43	a	105.93	cdef	86.32	efg	104.68	bc	222.21	a	121.48	h
C4WC1	178.63	a	107.71	cdef	122.59	abc	147.23	abc	157.15	d	183.03	cd
C4WC2	178.71	a	87.76	efgh	73.33	g	141.14	abc	208.45	ab	165.46	de
C4WC3	177.42	a	93.72	defg	83.05	efg	107.73	bc	204.76	ab	133.02	gh
C5WC1	164.30	a	83.03	ghij	135.69	ab	147.12	abc	209.03	ab	164.55	e
C5WC2	166.67	a	74.74	fghi	76.94	fg	128.34	abc	171.20	cd	152.69	ef
C5WC3	137.98	a	76.06	fghij	77.01	fg	105.74	bc	170.62	cd	136.32	fgh
C6WC1	210.53	a	148.33	ab	151.18	a	146.51	abc	215.12	a	215.48	a
C6WC2	216.43	a	119.44	cd	116.71	bcd	135.66	abc	152.74	d	186.18	bc
C6WC3	167.18	a	120.25	c	76.82	fg	105.62	bc	211.17	ab	152.52	ef
C7WC1	172.19	a	111.64	cde	76.05	fg	135.00	abc	194.85	abc	204.64	ab
C7WC2	182.99	a	66.88	hij	65.70	g	184.08	ab	166.64	cd	129.11	gh
C7WC3	179.63	a	83.56	fghi	78.09	g	157.17	abc	215.35	a	166.80	de

The total sugar content in the six observations carried out on tested clones was consistent. The RRIC 100 clone had the highest total sugar content four times, and the IRR 425 clone had the lowest four times.

The total sugar content analysis at different water levels generally showed a significant effect, except for the initial observation. This indicates that water content affects the total sugar content of the tested clones (Table 1).

Analysis of the total sugar content due to the interaction between the clone type and water content level (30%, 60%, and 90%) showed significant differences, except in the first observation (Table 1).

What is interesting about these results is that the highest accumulation of total sugar was seen in the application of 30% water content. Meanwhile, the effects were quite diverse across the different clone types. The IRR 429 had the highest total sugar in three observations (second, fourth, and fifth). The RRIC 100 had the highest total sugar in two observations (third and sixth). The two clones, RRIC 100 and IRR 429, also had the lowest total sugar in the fifth and sixth observations, respectively.

Two forms of polynomial curves can be the effect of water content and can be shown by the orthogonal polynomial regression obtained from three levels of water content, namely linear and cubic curves. The results of the analysis show that the linear curve shows a real effect. Figure 1 presents the linear curve regression pattern formed in detail. It demonstrates that the lower the water content added to the growing media, the higher the total sugar content derived from the leaf analysis of several rubber clones of IRR 400 series, RRIC 100, and BPM 24.

Figure 1. Pattern of total sugar content linear curve as a result of orthogonal polynomial analysis.

1: 30%; 2: 60%; 3: 90%.

Proline (mg g^-1)

Table 2 depicts the proline analysis of clone types treated with different water content levels and shows significantly different effects in all observations.

Table 2.

Proline levels (mg g^-1) in different clones, different water content (%), and interactions between clone type and water content (%).

Treatments	Proline (mg g^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	11.16	a	9.25	a	11.47	cd	8.11	a	8.95	a	6.86	a
IRR 428	10.38	a	5.74	c	14.93	a	7.61	ab	7.83	b	6.64	ab
IRR 429	10.51	a	9.27	a	11.78	cd	7.14	abc	7.91	b	6.56	ab
IRR 434	7.38	b	7.14	bc	11.92	bc	7.89	a	4.44	d	6.74	a
IRR 440	6.23	b	7.67	ab	8.76	e	6.25	c	5.33	cd	5.69	b
RRIC 100	5.89	b	6.13	bc	9.99	de	6.29	c	5.57	c	5.99	ab
BPM 24	7.02	b	6.96	bc	13.66	ab	6.80	bc	7.30	b	5.98	ab
Water content (WC)
30%	11.02	a	11.27	a	15.17	a	8.30	b	8.95	a	7.97	a
60%	8.88	b	6.92	b	10.78	b	6.99	a	6.77	b	6.36	b
90%	5.21	c	4.20	c	9.32	c	6.18	b	4.56	b	4.72	a
Interactions C × WC
C1WC1	13.82	a	13.65	a	17.04	ab	9.43	abcd	12.32	a	8.29	g
C1WC2	13.57	a	8.21	efgh	8.47	ghi	8.00	a	9.10	efg	7.98	abc
C1WC3	6.09	cde	5.91	defg	8.19	ghi	6.90	bcdefg	5.43	cd	4.30	ab
C2WC1	13.26	a	7.54	defgh	15.69	abc	9.19	bcdefg	10.26	bc	9.13	fg
C2WC2	11.25	ab	4.89	ghi	15.51	abc	6.87	a	6.92	e	6.23	a
C2WC3	6.64	cde	4.79	ghij	13.60	bcd	6.78	bcdefg	6.31	efg	4.55	cdefg
C3WC1	13.55	a	14.87	a	13.53	cdefg	7.76	abcde	11.77	fg	8.69	a
C3WC2	13.28	a	7.63	fghi	12.12	bcde	7.42	defg	7.19	ab	6.10	fg
C3WC3	4.70	def	5.31	defgh	9.70	efgh	6.25	abcdef	4.78	de	4.62	cdefg
C4WC1	8.40	bcd	12.38	hij	17.61	a	8.75	cdefg	5.47	efg	8.42	abcde
C4WC2	6.94	cde	4.85	ab	9.36	fgh	8.41	abc	5.28	hi	7.11	fg
C4WC3	6.79	cde	4.45	hij	8.79	ghi	6.50	ab	2.58	efg	4.69	ab
C5WC1	7.17	cde	11.74	bcde	11.66	defg	7.48	efg	6.88	e	7.14	efg
C5WC2	6.60	cde	8.93	abc	9.27	fgh	5.72	abcdef	6.68	ef	5.22	abcde
C5WC3	4.91	edef	2.33	ij	5.35	i	5.55	fg	2.42	i	4.69	fg
C6WC1	12.10	ab	10.11	defgh	15.94	abc	8.11	g	6.73	ef	6.39	bcdef
C6WC2	4.15	f	6.95	bcd	7.46	hi	5.61	abcd	5.52	efg	5.81	defg
C6WC3	1.42	ef	1.32	j	6.57	hi	5.14	fg	4.46	gh	5.77	defg
C7WC1	8.84	bc	8.62	cdef	14.71	abcd	7.39	abcdef	9.24	c	7.46	abcd
C7WC2	6.35	cde	6.89	fghi	13.25	bcde	6.89	bcdefg	6.71	ef	6.07	cdefg
C7WC3	5.89	cde	5.29	defgh	13.10	cdef	6.11	defg	5.97	efg	4.41	fg

The results of proline analysis at different water content levels showed significantly different effects in all observations (see Table 2).

The proline analysis caused by the interaction between rubber clones IRR 400 series, RRIC 100, and BPM 24 and given water content (30%, 60%, 90%) showed significantly different effects in all observations, as displayed in Table 2.

The assessment of orthogonal polynomial regression showed a linear curve, where the water content at the 30% level had the highest proline value. The orthogonal polynomial linear curve pattern of proline characteristics of several rubbers, namely, IRR 400 series, RRIC 100, and BPM 24, is shown in Figure 2.

Figure 2. Pattern of proline linear curve as a result of orthogonal polynomial analysis.

1: 30%; 2: 60%; 3: 90%.

Chlorophyll a (μg mg^-1)

The chlorophyll-a analysis on the different clone types showed a significant effect, except for the first observation, as displayed in Table 3.

Table 3.

Chlorophyll a levels (μg mg^-1) in different clones, different water content (%), and interactions between clones and water content (%).

Treatments	Chlorophyll-a (μg mg^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	0.25	a	0.60	a	0.46	C	0.60	b	0.62	a	0.72	a
IRR 428	0.23	a	0.48	b	0.42	d	0.48	d	0.50	b	0.48	bc
IRR 429	0.24	a	0.59	a	0.51	b	0.55	c	0.32	d	0.37	d
IRR 434	0.24	a	0.47	b	0.34	f	0.47	d	0.48	bc	0.38	d
IRR 440	0.23	a	0.39	c	0.38	e	0.37	e	0.28	d	0.48	bc
RRIC 100	0.25	a	0.61	a	0.55	a	0.73	a	0.49	b	0.44	c
BPM 24	0.22	a	0.46	b	0.41	de	0.49	d	0.45	c	0.52	b
Water content (WC)
30%	0.28	a	0.64	a	0.54	a	0.67	a	0.57	a	0.59	a
60%	0.23	b	0.51	b	0.44	b	0.48	b	0.42	b	0.47	b
90%	0.20	c	0.40	c	0.33	c	0.43	c	0.36	c	0.39	c
Interactions C × WC
C1WC1	0.32	a	0.74	a	0.66	a	0.74	bc	0.76	a	0.85	a
C1WC2	0.24	ab	0.64	c	0.47	efg	0.64	de	0.56	cde	0.67	b
C1WC3	0.18	b	0.42	efg	0.26	l	0.42	g	0.53	efg	0.63	bc
C2WC1	0.26	a	0.67	bc	0.48	def	0.67	cd	0.65	bc	0.55	cd
C2WC2	0.24	ab	0.42	efg	0.41	fgh	0.42	g	0.44	ghi	0.46	ef
C2WC3	0.19	ab	0.35	gh	0.35	hijk	0.34	h	0.41	hij	0.43	fg
C3WC1	0.29	a	0.75	a	0.63	ab	0.75	b	0.45	ghi	0.47	ef
C3WC2	0.23	ab	0.56	d	0.57	bc	0.46	g	0.28	kl	0.34	h
C3WC3	0.19	ab	0.46	e	0.34	ijk	0.44	g	0.23	l	0.30	h
C4WC1	0.27	ab	0.56	d	0.36	hij	0.56	f	0.62	bcd	0.46	ef
C4WC2	0.22	ab	0.44	ef	0.33	ijkl	0.44	g	0.45	ghi	0.36	gh
C4WC3	0.22	ab	0.43	ef	0.32	jkl	0.43	g	0.37	ij	0.33	h
C5WC1	0.26	ab	0.45	e	0.46	efg	0.45	g	0.34	jk	0.57	cd
C5WC2	0.23	ab	0.37	fgh	0.40	ghi	0.34	h	0.26	kl	0.53	de
C5WC3	0.22	ab	0.35	h	0.28	kl	0.31	h	0.24	l	0.33	h
C6WC1	0.28	ab	0.73	ab	0.65	ab	0.93	a	0.65	b	0.55	cd
C6WC2	0.24	ab	0.64	c	0.54	cd	0.63	def	0.46	fgh	0.43	fg
C6WC3	0.22	ab	0.45	e	0.47	defg	0.62	def	0.37	ij	0.35	h
C7WC1	0.25	ab	0.55	d	0.53	cde	0.57	ef	0.55	def	0.67	b
C7WC2	0.21	ab	0.47	e	0.38	hij	0.45	g	0.46	fgh	0.52	de
C7WC3	0.19	ab	0.36	h	0.33	ijkl	0.44	g	0.34	jk	0.36	gh

Table 3 shows the chlorophyll-a analysis at different water contents, which demonstrated a significant effect in all six observations.

Analysis of chlorophyll-a levels due to the interaction between clones and water content (30%, 60%, 90%) showed significant differences in all six observations (Table 3).

Chlorophyll b (μg mg^-1)

The results of the chlorophyll b analysis with different clone types showed significantly different results, except for the first observation (Table 4).

Table 4.

Chlorophyll b levels (μg mg^-1) in different clones, different water content (%) and interactions between clones and water content (%).

Treatments	Chlorophyll-b (μg mg^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	0.24	a	0.57	a	0.44	b	0.57	a	0.55	ab	0.52	ab
IRR 428	0.22	a	0.50	b	0.42	b	0.51	b	0.52	b	0.42	c
IRR 429	0.21	a	0.53	ab	0.33	c	0.55	ab	0.46	c	0.37	d
IRR 434	0.23	a	0.30	e	0.33	c	0.34	d	0.47	c	0.39	cd
IRR 440	0.23	a	0.45	c	0.44	cb	0.41	c	0.46	c	0.56	a
RRIC 100	0.23	a	0.42	cd	0.45	b	0.55	ab	0.48	c	0.49	b
BPM 24	0.20	a	0.40	d	0.54	a	0.55	ab	0.56	a	0.55	a
Water content (WC)
30%	0.25	a	0.52	a	0.49	a	0.60	a	0.61	a	0.59	a
60%	0.22	b	0.45	b	0.42	b	0.48	b	0.49	b	0.44	b
90%	0.20	c	0.38	c	0.35	c	0.41	c	0.39	c	0.38	c
Interactions C × WC
C1WC1	0.26	abc	0.70	a	0.57	ab	0.70	ab	0.66	ab	0.74	a
C1WC2	0.26	abc	0.55	cd	0.43	cd	0.56	cde	0.63	b	0.46	bcd
C1WC3	0.20	ab	0.46	defg	0.33	e	0.46	efghi	0.36	gh	0.36	efgh
C2WC1	0.25	abc	0.64	ab	0.45	c	0.64	abc	0.64	b	0.54	b
C2WC2	0.23	abc	0.47	defg	0.42	cd	0.47	efgh	0.55	c	0.37	defgh
C2WC3	0.19	c	0.39	ghijk	0.38	cde	0.42	ghij	0.37	gh	0.35	fgh
C3WC1	0.28	a	0.57	bc	0.37	cde	0.61	bcd	0.54	cde	0.46	bcde
C3WC2	0.18	bc	0.53	cde	0.32	e	0.55	cde	0.47	cdef	0.34	gh
C3WC3	0.17	bc	0.48	defg	0.31	e	0.48	efgh	0.36	gh	0.30	h
C4WC1	0.26	abc	0.33	kl	0.34	e	0.37	ijk	0.54	cd	0.45	bcdef
C4WC2	0.21	abc	0.30	kl	0.33	e	0.34	jk	0.46	ef	0.37	defgh
C4WC3	0.21	abc	0.26	l	0.30	e	0.30	k	0.42	fgh	0.35	gh
C5WC1	0.26	ab	0.52	cdef	0.54	b	0.52	defg	0.63	b	0.69	a
C5WC2	0.22	abc	0.46	defg	0.44	c	0.37	ijk	0.41	fgh	0.54	b
C5WC3	0.22	abc	0.36	ijk	0.34	e	0.35	jk	0.35	h	0.45	bcde
C6WC1	0.26	abc	0.45	efgh	0.55	b	0.73	a	0.55	c	0.54	b
C6WC2	0.22	abc	0.43	fghij	0.44	c	0.55	cde	0.46	def	0.48	bc
C6WC3	0.21	abc	0.36	hijk	0.36	de	0.38	hijk	0.43	Fg	0.43	cdefg
C7WC1	0.21	abc	0.45	efghi	0.64	a	0.66	ab	0.73	a	0.70	a
C7WC2	0.20	abc	0.40	ghijk	0.53	b	0.53	def	0.48	cdef	0.54	b
C7WC3	0.19	abc	0.35	jkl	0.44	c	0.45	fghi	0.47	cdef	0.41	cdefg

The results of the analysis of chlorophyll b levels at the given water contents showed a significant effect in all six observations (Table 4).

The analysis of chlorophyll b levels due to the interaction between clones and water content (30%, 60%, 90%) showed significant differences in all six observations, as shown in Table 4.

The assessment of the orthogonal polynomial regression showed a linear curve, where the water content at the 30% level had the highest chlorophyll b value. The orthogonal polynomial linear curve pattern of the chlorophyll b characteristics of several rubber clones of IRR 400 series, RRIC 100, and BPM 24 can be seen in Figure 3.

Figure 3. Pattern of chlorophyll b linear curve as a result of orthogonal polynomial analysis.

1: 30%; 2: 60%; 3: 90%.

Total chlorophyll content (μg mg^-1)

The analysis results of total chlorophyll content with different clone types showed significantly different results except for one observation (Table 5).

Table 5.

Total chlorophyll content levels (μg mg^-1) in different clones, different water content (%), and interactions between clones and water content (%).

Treatments	Total chlorophyll content (μg mg^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	0.48	a	1.17	a	0.90	b	1.17	b	1.17	a	1.24	a
IRR 428	0.46	a	0.98	b	0.83	c	0.99	d	1.02	b	0.90	c
IRR 429	0.45	a	1.12	a	0.84	c	1.10	c	0.78	d	0.74	d
IRR 434	0.46	a	0.77	d	0.66	d	0.81	e	0.95	c	0.77	d
IRR 440	0.47	a	0.84	c	0.82	c	0.78	e	0.74	e	1.04	b
RRIC 100	0.47	a	1.02	b	1.00	a	1.28	a	0.97	bc	0.93	c
BPM 24	0.42	a	0.86	c	0.95	b	1.03	d	1.01	b	1.06	b
Water content (WC)
30%	0.53	a	1.16	a	1.03	a	1.27	a	1.19	a	0.90	a
60%	0.45	b	0.96	b	0.86	b	0.96	b	0.91	b	0.74	b
90%	0.40	c	0.78	c	0.69	c	0.83	c	0.75	c	0.77	c
Interactions C × WC
C1WC1	0.57	a	1.44	a	1.23	a	1.44	b	1.42	a	1.59	a
C1WC2	0.50	abcd	1.19	cd	0.90	bcd	1.19	c	1.19	bc	1.13	cd
C1WC3	0.38	d	0.88	hij	0.59	h	0.88	efg	0.89	efg	0.99	ef
C2WC1	0.51	abcd	1.31	bc	0.93	bcd	1.31	bc	1.29	b	1.09	de
C2WC2	0.48	abcd	0.89	hij	0.84	de	0.89	defg	0.99	d	0.83	gh
C2WC3	0.38	bcd	0.74	klm	0.74	efg	0.76	gh	0.78	h	0.78	ghi
C3WC1	0.57	a	1.33	ab	0.99	bc	1.37	b	0.98	de	0.92	fg
C3WC2	0.41	abcd	1.09	def	0.89	cd	1.01	d	0.75	def	0.69	hij
C3WC3	0.36	cd	0.94	hij	0.64	gh	0.91	def	0.60	gh	0.60	j
C4WC1	0.53	abcd	0.89	hij	0.70	fgh	0.92	de	1.16	c	0.90	fg
C4WC2	0.43	abcd	0.73	klm	0.66	fgh	0.78	fgh	0.91	def	0.73	hij
C4WC3	0.43	abcd	0.70	m	0.62	gh	0.74	h	0.79	gh	0.68	ij
C5WC1	0.52	abc	0.97	ghi	1.00	b	0.97	de	0.97	de	1.26	bc
C5WC2	0.45	abcd	0.83	jk	0.83	de	0.71	h	0.68	i	1.07	de
C5WC3	0.43	abcd	0.71	lm	0.62	gh	0.66	h	0.58	i	0.79	ghi
C6WC1	0.54	ab	1.19	de	1.20	a	1.67	a	1.20	bc	1.09	de
C6WC2	0.45	abcd	1.07	efg	0.98	bc	1.18	c	0.93	de	0.91	fg
C6WC3	0.43	abcd	0.81	jkl	0.82	de	1.01	de	0.79	gh	0.78	ghi
C7WC1	0.46	abcd	1.00	fgh	1.17	a	1.22	c	1.28	b	1.37	b
C7WC2	0.40	abcd	0.87	ij	0.91	bcd	0.99	de	0.94	de	1.05	de
C7WC3	0.39	bcd	0.69	m	0.76	ef	0.89	defg	0.81	fgh	0.76	hi

The results of total chlorophyll content analysis with the given water content showed a significant effect in all six observations, as depicted in Table 5.

The analysis of total chlorophyll content levels due to the interaction between IRR 400 series, RRIC 100, and BPM 24 and water content (30%, 60%, 90%) showed significant differences in all six observations (Table 5). Orthogonal polynomial regression shows a linear curve, where the water content at 30% has the highest total chlorophyll content value. The linear curve shows that the total chlorophyll content increases with the decreasing water content. The orthogonal polynomial linear curve pattern of total chlorophyll content of several clones of IRR 400 series, RRIC 100, and BPM 24 can be seen in Figure 4.

Figure 4. Pattern of total chlorophyll content linear curve as a result of orthogonal polynomial analysis.

1: 30%; 2: 60%; 3: 90%.

Hydrogen peroxidase/H₂O₂ (μmolg^-1)

The results of total the H₂O₂ analysis with different clone types showed a significantly different effect in two of the observations (third and fourth) (Table 6).

Table 6.

H₂O₂ levels (μmolg^-1) in different clones, different water content (%), interactions between clones and water content (%).

Treatments	H₂O₂ (μmol g^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	0.780	a	0.752	a	0.771	ab	0.746	bc	0.738	a	0.736	a
IRR 428	0.801	a	0.790	a	0.759	ab	0.734	c	0.738	a	0.726	a
IRR 429	0.775	a	0.754	a	0.740	b	0.736	c	0.742	a	0.724	a
IRR 434	0.786	a	0.788	a	0.765	ab	0.777	ab	0.750	a	0.719	a
IRR 440	0.790	a	0.806	a	0.768	ab	0.790	a	0.753	a	0.732	a
RRIC 100	0.782	a	0.761	a	0.790	a	0.785	a	0.759	a	0.728	a
BPM 24	0.798	a	0.749	a	0.771	ab	0.768	abc	0.752	a	0.732	a
Water content (WC)
30%	0.794	a	0.780	a	0.764	a	0.756	a	0.740	a	0.729	a
60%	0.788	a	0.770	a	0.767	a	0.757	a	0.752	a	0.725	a
90%	0.780	a	0.764	a	0.767	a	0.774	a	0.751	a	0.730	a
Interactions C × WC
C1WC1	0.783	a	0.730	a	0.771	a	0.737	abc	0.725	a	0.725	a
C1WC2	0.780	a	0.768	a	0.779	a	0.760	bc	0.737	a	0.740	a
C1WC3	0.777	a	0.758	a	0.764	a	0.742	abc	0.752	a	0.743	a
C2WC1	0.827	a	0.842	a	0.767	a	0.730	bc	0.728	a	0.728	a
C2WC2	0.785	a	0.766	a	0.754	a	0.732	bc	0.748	a	0.731	a
C2WC3	0.790	a	0.762	a	0.756	a	0.740	bc	0.736	a	0.718	a
C3WC1	0.773	a	0.773	a	0.734	a	0.725	c	0.745	a	0.725	a
C3WC2	0.767	a	0.730	a	0.732	a	0.732	bc	0.736	a	0.715	a
C3WC3	0.785	a	0.759	a	0.753	a	0.750	abc	0.746	a	0.730	a
C4WC1	0.782	a	0.798	a	0.768	a	0.780	abc	0.735	a	0.727	a
C4WC2	0.796	a	0.799	a	0.773	a	0.799	abc	0.758	a	0.714	a
C4WC3	0.779	a	0.766	a	0.753	a	0.753	abc	0.756	a	0.716	a
C5WC1	0.812	a	0.837	a	0.780	a	0.804	ab	0.744	a	0.740	a
C5WC2	0.779	a	0.776	a	0.762	a	0.748	a	0.751	a	0.716	a
C5WC3	0.780	a	0.806	a	0.762	a	0.817	abc	0.765	a	0.739	a
C6WC1	0.787	a	0.745	a	0.771	a	0.784	abc	0.769	a	0.728	a
C6WC2	0.793	a	0.792	a	0.807	a	0.776	abc	0.745	a	0.729	a
C6WC3	0.767	a	0.746	a	0.793	a	0.795	abc	0.763	a	0.726	a
C7WC1	0.795	a	0.734	a	0.760	a	0.731	a	0.734	a	0.730	a
C7WC2	0.718	a	0.761	a	0.764	a	0.750	abc	0.786	a	0.728	a
C7WC3	0.780	a	0.752	a	0.790	a	0.823	bc	0.737	a	0.739	a

The results of H₂O₂ analysis at different water content levels did not show significant differences in any of the observations (Table 6).

The analysis of H₂O₂ levels (μmol g^-1) due to interactions between IRR 400 series, RRIC 100, and BPM 24 and given water content (30%, 60%, 90%) showed a significantly different effect in just one observation (fourth) (Table 6).

The effect of water content on the H₂O₂ characteristic shows a linear regression curve based on orthogonal polynomials. This indicates that the lower the water content, the higher the concentration of H₂O₂. The linear regression pattern between H₂O₂ content and water content can be seen in Figure 5.

Figure 5. Pattern of H₂O₂ (μmol g^-1) linear curve as a result of orthogonal polynomial analysis.

1: 30%; 2: 60%; 3: 90%.

Ascorbate peroxidase/APX (units mg^-1)

The results of APX analysis with different clone types were not significantly different in any of the observations (Table 7).

Table 7.

APX levels (units mg^-1) in different clones, different water content (%) and interactions between clones and water content (%).

Treatments	APX level (units mg^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	1.48	a	1.13	a	1.01	a	1.21	a	1.29	a	1.14	a
IRR 428	1.37	a	1.20	a	1.06	a	1.32	a	1.41	a	1.03	a
IRR 429	1.25	a	1.32	a	1.01	a	1.23	a	1.41	a	1.11	a
IRR 434	1.30	a	1.29	a	1.05	a	1.44	a	1.40	a	0.96	a
IRR 440	1.27	a	1.29	a	1.15	a	1.36	a	1.34	a	1.12	a
RRIC 100	1.22	a	1.23	a	1.10	a	1.33	a	1.33	a	1.02	a
BPM 24	1.31	a	1.23	a	1.00	a	1.27	a	1.24	a	0.99	a
Water content (WC)
30%	1.38	a	1.15	a	1.05	a	1.27	a	1.34	a	1.05	a
60%	1.32	a	1.34	a	1.07	a	1.35	a	1.38	a	1.03	a
90%	1.24	a	1.24	a	1.05	a	1.31	a	1.33	a	1.09	a
Interactions C × WC
C1WC1	1.58	a	1.18	a	1.03	a	1.03	a	1.23	a	1.17	a
C1WC2	1.42	a	1.24	a	0.95	a	1.25	a	1.27	a	1.08	a
C1WC3	1.44	a	0.98	a	1.05	a	1.36	a	1.36	a	1.17	a
C2WC1	1.47	a	1.08	a	1.04	a	1.45	a	1.55	a	1.08	a
C2WC2	1.53	a	1.20	a	1.10	a	1.31	a	1.27	a	1.04	a
C2WC3	1.10	a	1.30	a	1.06	a	1.20	a	1.42	a	0.97	a
C3WC1	1.38	a	1.23	a	1.01	a	1.22	a	1.43	a	1.08	a
C3WC2	1.25	a	1.35	a	0.91	a	1.40	a	1.45	a	1.11	a
C3WC3	1.12	a	1.37	a	1.11	a	1.08	a	1.36	a	1.15	a
C4WC1	1.45	a	1.16	a	0.98	a	1.59	a	1.34	a	0.96	a
C4WC2	1.25	a	1.46	a	1.13	a	1.32	a	1.56	a	1.00	a
C4WC3	1.21	a	1.24	a	1.04	a	1.42	a	1.32	a	0.93	a
C5WC1	1.38	a	1.22	a	1.09	a	1.31	a	1.12	a	1.06	a
C5WC2	1.17	a	1.40	a	1.23	a	1.36	a	1.41	a	1.05	a
C5WC3	1.26	a	1.26	a	1.15	a	1.42	a	1.48	a	1.25	a
C6WC1	1.15	a	1.06	a	1.11	a	1.18	a	1.52	a	1.01	a
C6WC2	1.26	a	1.46	a	1.23	a	1.45	a	1.37	a	0.91	a
C6WC3	1.25	a	1.17	a	0.97	a	1.35	a	1.10	a	1.15	a
C7WC1	1.24	a	1.11	a	1.06	a	1.14	a	1.16	a	0.97	a
C7WC2	1.38	a	1.23	a	0.95	a	1.37	a	1.30	a	0.98	a
C7WC3	1.30	a	1.35	a	1.00	a	1.32	a	1.26	a	1.01	a

The analysis results of APX at different water content levels were not significantly different in any of the observations (Table 7).

The analysis of APX levels (units mg^-1) due to the interaction between IRR 400 series, RRIC 100, and BPM 24 and water content (30%, 60%, 90%) did not show any significant differences in any of the observations (Table 7).

Superoxide Dismutase/SOD (units mg^-1)

The SOD analysis with clone types showed a significant difference in three of the observations (Table 8).

Table 8.

SOD levels (units mg^-1) in different clones, different water content (%) and interactions between clones and water content (%).

Treatments	SOD (units mg^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	2.43	a	2.56	bc	2.28	a	2.29	a	2.39	a	2.42	bc
IRR 428	2.36	ab	2.57	abc	2.25	a	2.29	a	2.39	a	2.45	bc
IRR 429	2.23	c	2.60	ab	2.21	a	2.33	a	2.44	a	2.44	bc
IRR 434	2.38	a	2.54	bc	2.27	a	2.23	a	2.44	a	2.49	bc
IRR 440	2.34	abc	2.70	a	2.29	a	2.32	a	2.38	a	2.40	c
RRIC 100	2.27	bc	2.44	c	2.28	a	2.29	a	2.34	a	2.62	a
BPM 24	2.32	bc	2.52	bc	2.21	a	2.35	a	2.43	a	2.52	ab
Water content (WC)
30%	2.28	b	2.56	a	2.23	a	2.32	a	2.39	ab	2.49	a
60%	2.42	a	2.53	a	2.26	a	2.28	a	2.45	a	2.43	b
90%	2.30	b	2.59	a	2.27	a	2.30	a	2.36	b	2.52	a
Interaction C × WC
C1W1	2.29	cdefg	2.45	ab	2.24	a	2.43	a	2.36	ab	2.37	fgh
C1W2	2.35	bcdefg	2.60	ab	2.37	a	2.20	a	2.44	ab	2.35	efgh
C1W3	2.63	a	2.62	ab	2.22	a	2.23	a	2.36	ab	2.54	abcdef
C2W1	2.27	cdefg	2.61	ab	2.24	a	2.35	a	2.32	ab	2.37	defgh
C2W2	2.52	abc	2.54	ab	2.24	a	2.29	a	2.59	a	2.38	efgh
C2W3	2.30	defg	2.56	ab	2.26	a	2.23	a	2.28	b	2.61	abcd
C3W1	2.29	cdefg	2.59	ab	2.19	a	2.36	a	2.40	ab	2.44	cdefgh
C3W2	2.24	cdefg	2.54	ab	2.20	a	2.25	a	2.45	ab	2.42	bcdefgh
C3W3	2.17	g	2.67	ab	2.24	a	2.38	a	2.47	ab	2.45	cdefgh
C4W1	2.43	abcd	2.61	ab	2.19	a	2.20	a	2.35	ab	2.66	ab
C4W2	2.49	abcd	2.44	b	2.25	a	2.31	a	2.51	ab	2.36	bcdefgh
C4W3	2.24	efg	2.57	ab	2.36	a	2.20	a	2.46	b	2.45	fgh
C5W1	2.26	defg	2.69	ab	2.39	a	2.29	a	2.42	ab	2.28	gh
C5W2	2.56	ab	2.67	ab	2.25	a	2.31	a	2.44	ab	2.31	h
C5W3	2.19	fg	2.73	b	2.22	a	2.37	a	2.27	b	2.63	abcd
C6W1	2.20	efg	2.46	ab	2.26	a	2.29	a	2.41	ab	2.59	abcde
C6W2	2.40	abcdef	2.44	ab	2.31	a	2.27	a	2.35	b	2.64	abcd
C6W3	2.21	efg	2.43	b	2.27	a	2.31	a	2.27	b	2.61	abcd
C7W1	2.25	efg	2.49	ab	2.12	a	2.33	a	2.47	ab	2.69	a
C7W2	2.35	bcdefg	2.51	ab	2.20	a	2.35	a	2.39	ab	2.53	abcdefg
C7W3	2.36	bcdefg	2.57	ab	2.30	a	2.37	a	2.42	ab	2.33	fgh

Analysis of SOD (units mg^-1) levels at different water content levels showed significant differences in three of the observations, as depicted in Table 8.

The analysis of SOD levels due to interaction between IRR 400 series, RRIC 100, and BPM 24 and water content (30%, 60%, 90%) showed significant differences in two observations (Table 8).

Peroxide dismutase/POD (units mg^-1)

The POD analysis with different types of clones showed significant differences in two observations, as shown in Table 9.

Table 9.

POD levels (units mg^-1) in different clones, different water content (%), interactions between clones and water content (%).

Treatments	POD (units mg^-1)
	Period to
	1		2		3		4		5		6
Clones (C)
IRR 425	0.869	a	0.903	a	0.856	a	0.932	a	0.918	a	0.858	a
IRR 428	0.898	a	0.940	a	0.850	a	0.919	a	0.923	a	0.870	a
IRR 429	0.887	a	0.895	a	0.875	a	0.914	a	0.873	a	0.834	a
IRR 434	0.891	a	0.906	a	0.881	a	0.846	b	0.919	a	0.848	a
IRR 440	0.889	a	0.901	a	0.870	a	0.918	a	0.917	a	0.853	a
RRIC 100	0.881	a	0.904	a	0.898	a	0.907	ab	0.923	a	0.829	a
BPM 24	0.891	a	0.924	a	0.874	a	0.938	a	0.923	a	0.875	a
Water content (WC)
30%	0.874	b	0.912	a	0.874	a	0.912	a	0.919	a	0.864	a
60%	0.895	a	0.915	a	0.875	a	0.906	a	0.913	a	0.862	a
90%	0.891	a	0.904	a	0.867	a	0.914	a	0.909	a	0.831	a
Interaction C × WC
C1W1	0.869	cdef	0.919	a	0.891	a	0.943	a	0.961	a	0.859	a
C1W2	0.813	fgh	0.931	a	0.867	a	0.940	a	0.900	a	0.836	a
C1W3	0.926	fgh	0.860	a	0.810	a	0.915	ab	0.893	a	0.879	a
C2W1	0.901	bcdef	0.939	a	0.803	a	0.918	ab	0.942	a	0.875	a
C2W2	0.919	abc	0.921	a	0.854	a	0.899	ab	0.937	a	0.899	a
C2W3	0.874	bcd	0.961	a	0.893	a	0.940	a	0.889	a	0.836	a
C3W1	0.826	abc	0.897	a	0.855	a	0.915	b	0.903	a	0.835	a
C3W2	0.897	abc	0.882	a	0.912	a	0.930	ab	0.857	a	0.855	a
C3W3	0.938	ab	0.905	a	0.857	a	0.899	ab	0.858	a	0.811	a
C4W1	0.917	abc	0.897	a	0.920	a	0.784	ab	0.854	a	0.900	a
C4W2	0.920	abcd	0.917	a	0.795	a	0.860	ab	0.936	a	0.849	a
C4W3	0.836	ab	0.905	a	0.929	a	0.894	a	0.967	a	0.795	a
C5W1	0.774	h	0.894	a	0.852	a	0.909	ab	0.929	a	0.879	a
C5W2	0.917	abcd	0.904	a	0.878	a	0.915	ab	0.923	a	0.842	a
C5W3	0.974	a	0.904	a	0.879	a	0.929	a	0.900	a	0.837	a
C6W1	0.908	bcd	0.931	a	0.899	a	0.954	a	0.938	a	0.805	a
C6W2	0.944	abcd	0.923	a	0.904	a	0.863	ab	0.900	a	0.872	a
C6W3	0.790	gh	0.858	a	0.890	a	0.902	ab	0.930	a	0.809	a
C7W1	0.922	abc	0.905	a	0.896	a	0.961	a	0.907	a	0.899	a
C7W2	0.854	bcde	0.931	a	0.913	a	0.935	a	0.936	a	0.897	a
C7W3	0.897	defg	0.937	a	0.814	a	0.919	ab	0.925	a	0.848	a

The analysis of POD levels at different water content levels showed a significant difference in one observation, as depicted in Table 9.

The analysis of POD levels due to interaction between IRR 400 series, RRIC 100, and BPM 24 and given water content (30%, 60%, 90%) showed a significant difference in just one observation (Table 9).

Discussion

Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situation.⁹^,¹⁰

The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the strees by producing compounds that render tolerance. The accumulation of osmoprotectants is a key biochemical property in plants tolerant to abiotic stress,¹⁰^, ²¹ and there is clear evidence that osmotic adjustment sustains crop yields under drought stress.⁹ Drought stress causes changes in amino acid metabolism. The accumulated solutes protect cellular proteins, organelles, membranes, and various enzymes against drought stress.

Several physiological characteristics were analyzed to see the effect of water content on IRR 400 series, RRIC 100, and BPM 24 rubber clones. Some of the dissolved substances assessed in this study were total sugar, proline, and chlorophyll (a, b, total). The correlation of total sugar content to each clone showed different effects. Each clone had altered levels of total sugar content under stress conditions. The RRIC 100 is a drought tolerant clone in the field. The increase in total sugar content was seen in most observations of the water content treatment. The interaction between clone type and water content can increase total sugar content, especially when the water content added is 30%. Initial hypotheses suggest that each clone has the ability to adapt to water shortages. The accumulation of soluble sugars in plant cells subjected to drought stress is responsible for the osmotic adjustment.²⁵ Sugar accumulation in drought-stressed plants is controlled by several mechanisms that affect soluble sugar formation and transfer in leaves.²⁶ Similar results of increased total sugar accumulation have been observed in drought-stressed soybeans²⁶ and sugarcane.²⁷

This study showed different proline values among the tested clones. All six observations indicate that clones have the ability to survive drought. The IRR 425 clone had the highest proline levels in three observations. Meanwhile, the IRR 440 had the lowest proline levels in four observations. Assessing the proline characteristic, the initial assumption was that IRR 425 had a stronger adaptation compared with other clones, especially the IRR 440. Regarding different water contents (30%, 60%, 90%), the proline levels at 30% were greater than at 60% and 90%. This indicated that a higher amount of proline accumulated when the water content was lower in the growth medium. Proline is an important amino acid as it is an osmotic-compatible molecule and has the potential to form a defense system to increase drought tolerance. Proline acts as an antioxidative defense molecule and causes stress signaling.¹² It is classified as an osmoprotectant, as it increases hyperosmolarity and antioxidant enzymes’ activity.²⁸ Increased proline content in drought-stress plants can maintain turgor for continued growth.²⁹ Hence, proline accumulation correlates with osmoprotection.³⁰ The interaction between clone types and moisture content indicated that each clone showed a different effect in all six observations. The clones had high proline levels when treated with 30% water content. This shows that the clonal factor still has to be planted in the field which is classified as a drought environment. The proline content has been shown to increase about 10-fold in mungbean,³¹ maize,³² millet,¹²^,³³^,³⁴ nyamplung,³⁵ and soybean²⁶ under drought stress.

Chlorophyll is the main pigment found in chloroplasts.³⁶ The three main functions of chlorophyll in the photosynthesis process are harnessing solar energy, triggering CO ₂ fixation to produce carbohydrates, and providing energy for the ecosystem as a whole. Chlorophyll a and chlorophyll b absorb the most light in the red part (600–700 nm) and absorb the least in the green part (500–600 nm).³⁵^–³⁷ In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. The genotype BC678 and BC404 of corn cultivars showed resistant to drought stress have highest chlorophyll index and higher potential yield.³⁸

This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.³⁹

This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as water treated gradually. Even though the polybags made small holes to drain excess water during its pre-treatment conditions. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Another factor that can be used as a cause of high chlorophyll levels is ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year).

Antioxidants are active substances that naturally detoxify free radicals (ROS). The presence of oxidative stress and an abundance of antioxidants are important activities for metabolic protection when plants are under stress. ROS in the form of free radicals and peroxides are molecules derived from oxygen metabolism. The toxic effects of ROS can be countered by antioxidant enzymatic as well as non-enzymatic systems, such as SOD, CAT, APX, GR, ascorbic acid (AsA), tocopherols, glutathione and phenolic compounds, and others. Typically, each cellular compartment contains more than one enzymatic activity that detoxifies ROS. The presence of these enzymes in almost all cells plays an important role in ROS detoxification for plant survival.⁴⁰

H₂O₂ has several important roles in various biochemical and physiological processes. Long plant life and long growth processes result in H₂O₂ crossing cellular membranes and potentially acting as a signal in the signal transduction pathway of stress. This pathway triggers various responses of the adaptation process in the environment where the plant is cultivated.⁴¹ High levels of H₂O₂ cause oxidative stress, which then causes cell damage and death.⁴² However, optimal levels of H₂O₂ can increase tolerance to abiotic stresses through modulation of various physiological processes, including photosynthesis, opening and closing of stomata, osmotic adjustment, and ROS detoxification.⁴¹^,⁴² ROS detoxification is very important in maintaining the structural and membrane integrity of cellular organelles and keeping them fully functional under stress. The accumulation of optimal amounts of H₂O₂ triggers the occurrence of chitinase proteins that can produce calcium homeostasis, ion channels, phosphatases, transcription factors, and abscisic acid (ABA), signaling responses to stress.⁴³

APX in ascorbate–glutathione (AsA–GSH) cycling enzymes is responsible for the decomposition of H₂O₂ produced by SOD in different cellular organelles. APX plays a key role in both drought stress response and recovery after drought.⁴³^,⁴⁴ APX is an integral component of the (ASC–GSH) cycle. APX performs the same function in the cytosol and chloroplasts. APX reduces H₂O₂ to H₂O and docosahexaenoic acid (DHA), using AsA as a reducing agent.

H_{2} O_{2} + AA \to_{2} H_{2} O + DHA

The APX family consists of five isoforms based on different sites of amino acid formation, such as the cytosol, mitochondria, peroxisomes, and chloroplastids (stroma and thylakoids).⁴⁵ APX is widely distributed and has a better affinity to H₂O₂, especially in terms of more efficient uptake of H₂O₂ in times of stress.⁴⁵^–⁴⁸

Though the SOD levels in each clone showed a significant effect due to water content, it was limited to a few observations because drought affects the metabolic activity of clones. Likewise for the levels of SOD at a given water content. Water content of 30% showed relatively the same SOD activity as 60% and 90% in all observations. This indicates that SOD were formed in low levels in the observations and therefore cannot be used as a marker of tolerance for these tested clones. SOD is one of the key components of cell protection against oxidative stress. The SOD has three different isoenzymes distributed between organelles. Cu/Zn-SOD is predominantly located in the chloroplasts, cytosol, and peroxisomes, whereas FeSOD and MnSOD are mostly found in chloroplasts and mitochondria, respectively.⁴⁹ POD and SOD activities increased sharply in rubber seedlings after being subject to drought stress. This suggests that the photosynthetic activity and lipid integrity of the cell membranes are rapidly attenuated by drought stress. SODs are metalloenzymes that play an important role in ROS reactions, or, in other words, are able to neutralize the negative effects of ROS. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance. Plants that have a higher induced SOD activity show more tolerance to abiotic stresses. Numerous studies have shown that plants are able to better eliminate the negative effects of ROS produced under stressful situations when their SOD activity is higher, provided there are more SOD isoenzymes present.

POD had low values in all six observations of some clones. The low POD indicated that the effect of some water content percentages given during the six observations on several different clones did not have a significant effect. This indicates that the POD characteristics cannot be used as a reference for plant tolerance to drought stress. Plants that produce more POD under conditions of drought stress will be able to survive by eliminating the effects of ROS. In general, the activity of POD and other antioxidant enzymes will automatically have a higher value in tolerant clones/varieties and will have a lower value in susceptible clones/varieties. This indicates that drought-tolerant clones/varieties will be more efficient in removing H₂O₂ to produce optimal protection. The tolerance of some genotypes to environmental stresses has been associated with higher antioxidant enzyme activity. Drought-tolerant species of pigeon pea (Cajanus cajan),⁵⁰ wheat (Triticum aestivum),⁵¹^,⁵² and black bean (Phaseolus mungo)⁴⁹ have higher SOD, POD, and CAT activities than drought-sensitive species. The results of this study indicate that ROS enzymes, which play a crucial role in the drought-tolerance mechanism under the drought treatment, have been identified in clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24. Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale.

Conclusions

Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H₂O₂, APX SOD, and POD did not show any correlation to use as drought tolerance indicators in this experiment.

Data availability

Underlying data

This project contains the following underlying data:

Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (HeveabrasiliensisMuell. Arg.) on Drought Stress. https://doi.org/10.6084/m9.figshare.21708230.v2 ⁵²

• Biochemistry characters.xlsx (datasets for total sugar, chlorophyll (a, b, total), proline, H ₂O ₂, APX, SOD, POD)

Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (HeveabrasiliensisMuell. Arg.) on Drought Stress. https://doi.org/10.6084/m9.figshare.21708275.v2 ⁵³

• Polyortogonal_test.xlsx (datasets for total sugar, chlorophyll (a, b, total), proline, H ₂O ₂, APX, SOD, POD)

Extended data

Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (HeveabrasiliensisMuell. Arg.) on Drought Stress. https://doi.org/10.6084/m9.figshare.21645116.v5 ⁵⁴

This project contains the following extended data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

We thank Choeriyah and Indra Gunawan for their help in sampling campaigns and laboratory analysis.

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53. Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21708230.v2
54. Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21708275.v2
55. Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21645116.v5

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Version 4

VERSION 4 PUBLISHED 27 Jan 2023

Author details Author details

Syarifah Aini Pasaribu
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Mohammad Basyuni
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Validation, Writing – Review & Editing

Edison Purba
Roles: Data Curation, Formal Analysis, Validation, Writing – Review & Editing

Yaya Hasanah
Roles: Data Curation, Formal Analysis, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The Penelitian Disertasi Doktor project No. 11/AMD/E1/KP.PTNB/2021, from the Directorate General of Research and Community Service, the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia. Center of Excellence for Mangrove, Universitas Sumatera Utara covered the APC of this paper.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Pasaribu SA, Basyuni M, Purba E and Hasanah Y. Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress [version 4; peer review: 1 approved, 2 approved with reservations]. F1000Research 2023, 12:106 (https://doi.org/10.12688/f1000research.129421.4)

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Open Peer Review

Current Reviewer Status: ?

Key to Reviewer Statuses VIEW HIDE

ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 4

VERSION 4

PUBLISHED 04 Dec 2023

Revised

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Reviewer Report 02 Jan 2025

Boon Chin Tan, Universiti Malaya, Kuala Lumpur, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.159406.r350669

The manuscript aims to identify drought-tolerant traits to select drought-tolerant rubber clones at the juvenile stage. Overall, the study is conducted in an acceptable manner, with appropriate methodologies for achieving the objectives. The authors have also revised and improved the manuscript accordingly. However, the manuscript lacks sufficient detail in the methodology section, requiring some clarifications and improvements.

Methods
The work was conducted using 6–12-month-old plants. Could this age range introduce variability that may affect comparisons between different clones?

Experimental design
How did the authors implement drought treatment and achieve 30%, 60%, and 90% soil water content levels?
Did the authors measure soil water content? If so, which apparatus was used?
Were any fertilizers applied during the treatment?
Where were the tested clones sourced from?
The term “normal leaves” should be revised to “disease-free leaves.”
“Six months observation” should be changed to “six months' observation”.

Results
The sentence “the highest total sugar content four times, and the IRR 425 clone had the lowest four times” needs revision for clarity, particularly the meaning of "four times."

APX, SOD, and POD (units mg-1): The authors should define the “units” for each assay.

Tables
The unit for “period to” should be specified as “month.”
It is necessary to mention in the methodology that samples were harvested monthly.
The data analysis method should be explained, and the meaning of the letters should be defined in the table footnotes.

Conclusion
“For e.g.” should be changed to “For example”.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Plant biotechnology and physiology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Version 3

VERSION 3

PUBLISHED 12 Oct 2023

Revised

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Reviewer Report 07 Nov 2023

Mohamed sathik Thirruvithamkottil, Rubber Research Institute of India, Rubber Board, Kottayam, Kerala, India

Approved with Reservations

https://doi.org/10.5256/f1000research.157145.r214777

1. Introduction 2nd paragraph,

One of the primary sources of natural rubber is producing plants - here is is not required.

2. signaling pathways and no comma mark (,) is needed before ‘and’

3. In times of drought stress, lack of sufficient water combined with the increased CO ₂ in the atmosphere can cause plant death.¹³

I could not see the death in the reference mentioned above. Please correct it or omit the point as it does not look relevant.

4. The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

Discussion

1. The previous point mentioned below is not addressed

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

2. Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.²⁹

The above statement as per ref no. 29 was verified and in none of the chapters it says that proline can provide high energy. Please clarify

3. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It has a taproot that will grow even deeper to find water farther from the ground.

In my previous comment, I said that it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. Please check or justify.

4. From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

Please justify!

5. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance.

Please justify how the decrease in subtrate binding affinity (to SOD) can impart resistance?

Conclusion

However, the parameters such as chlorophyll a, b, and total, H₂O₂, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment

"Used' should be changed to 'use'

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Plant Physiology, Molecular Biology, Plant Breeding, etc.

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Author Response 04 Dec 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

04 Dec 2023

Author Response

We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by ... Continue reading We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows:

APPROVED WITH RESERVATIONS
info_outline
1. Introduction 2nd paragraph,

One of the primary sources of natural rubber is producing plants - here is is not required.
Is has been deleted
2. signaling pathways and no comma mark (,) is needed before ‘and’
Comma mark (,) has been removed.
3. In times of drought stress, lack of sufficient water combined with the increased CO 2 in the atmosphere can cause plant death.13
The sentence has been corrected to read: The combined rise in atmospheric CO2 concentrations and global air temperature may significantly alter plant water use.
I could not see the death in the reference mentioned above. Please correct it or omit the point as it does not look relevant.

4. The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.
The aim of study has been corrected.
Discussion

1. The previous point mentioned below is not addressed

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.
Thank you for the correction. Drying has been revised to drought.
2. Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.
Thank you for your correction. The sentence has been improved to read: Increased proline content in drought-stress plants can maintain turgor for continued growth.
The above statement as per ref no. was verified and in none of the chapters it says that proline can provide high energy. Please clarify

3. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It has a taproot that will grow even deeper to find water farther from the ground.
In my previous comment, I said that it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. Please check or justify.
The sentence has been revised to meet Reviewer’s suggestions. Done as recommended.
4. From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

Please justify!
We have corrected the sentence to icorporate Reviewer’s comments. Please refer to revised Discussion.
5. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance.

Please justify how the decrease in subtrate binding affinity (to SOD) can impart resistance?
This sentence has been deleted to avoid the obscurity to SOD
Conclusion

However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment

"Used' should be changed to 'use'
“used’ has been revised to ‘use’
We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows:

APPROVED WITH RESERVATIONS
info_outline
1. Introduction 2nd paragraph,

One of the primary sources of natural rubber is producing plants - here is is not required.
Is has been deleted
2. signaling pathways and no comma mark (,) is needed before ‘and’
Comma mark (,) has been removed.
3. In times of drought stress, lack of sufficient water combined with the increased CO 2 in the atmosphere can cause plant death.13
The sentence has been corrected to read: The combined rise in atmospheric CO2 concentrations and global air temperature may significantly alter plant water use.
I could not see the death in the reference mentioned above. Please correct it or omit the point as it does not look relevant.

4. The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.
The aim of study has been corrected.
Discussion

1. The previous point mentioned below is not addressed

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.
Thank you for the correction. Drying has been revised to drought.
2. Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.
Thank you for your correction. The sentence has been improved to read: Increased proline content in drought-stress plants can maintain turgor for continued growth.
The above statement as per ref no. was verified and in none of the chapters it says that proline can provide high energy. Please clarify

3. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It has a taproot that will grow even deeper to find water farther from the ground.
In my previous comment, I said that it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. Please check or justify.
The sentence has been revised to meet Reviewer’s suggestions. Done as recommended.
4. From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

Please justify!
We have corrected the sentence to icorporate Reviewer’s comments. Please refer to revised Discussion.
5. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance.

Please justify how the decrease in subtrate binding affinity (to SOD) can impart resistance?
This sentence has been deleted to avoid the obscurity to SOD
Conclusion

However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment

"Used' should be changed to 'use'
“used’ has been revised to ‘use’
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 04 Dec 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

04 Dec 2023

Author Response

We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by ... Continue reading We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows:

APPROVED WITH RESERVATIONS
info_outline
1. Introduction 2nd paragraph,

One of the primary sources of natural rubber is producing plants - here is is not required.
Is has been deleted
2. signaling pathways and no comma mark (,) is needed before ‘and’
Comma mark (,) has been removed.
3. In times of drought stress, lack of sufficient water combined with the increased CO 2 in the atmosphere can cause plant death.13
The sentence has been corrected to read: The combined rise in atmospheric CO2 concentrations and global air temperature may significantly alter plant water use.
I could not see the death in the reference mentioned above. Please correct it or omit the point as it does not look relevant.

4. The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.
The aim of study has been corrected.
Discussion

1. The previous point mentioned below is not addressed

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.
Thank you for the correction. Drying has been revised to drought.
2. Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.
Thank you for your correction. The sentence has been improved to read: Increased proline content in drought-stress plants can maintain turgor for continued growth.
The above statement as per ref no. was verified and in none of the chapters it says that proline can provide high energy. Please clarify

3. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It has a taproot that will grow even deeper to find water farther from the ground.
In my previous comment, I said that it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. Please check or justify.
The sentence has been revised to meet Reviewer’s suggestions. Done as recommended.
4. From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

Please justify!
We have corrected the sentence to icorporate Reviewer’s comments. Please refer to revised Discussion.
5. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance.

Please justify how the decrease in subtrate binding affinity (to SOD) can impart resistance?
This sentence has been deleted to avoid the obscurity to SOD
Conclusion

However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment

"Used' should be changed to 'use'
“used’ has been revised to ‘use’
We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows:

APPROVED WITH RESERVATIONS
info_outline
1. Introduction 2nd paragraph,

One of the primary sources of natural rubber is producing plants - here is is not required.
Is has been deleted
2. signaling pathways and no comma mark (,) is needed before ‘and’
Comma mark (,) has been removed.
3. In times of drought stress, lack of sufficient water combined with the increased CO 2 in the atmosphere can cause plant death.13
The sentence has been corrected to read: The combined rise in atmospheric CO2 concentrations and global air temperature may significantly alter plant water use.
I could not see the death in the reference mentioned above. Please correct it or omit the point as it does not look relevant.

4. The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.
The aim of study has been corrected.
Discussion

1. The previous point mentioned below is not addressed

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.
Thank you for the correction. Drying has been revised to drought.
2. Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.
Thank you for your correction. The sentence has been improved to read: Increased proline content in drought-stress plants can maintain turgor for continued growth.
The above statement as per ref no. was verified and in none of the chapters it says that proline can provide high energy. Please clarify

3. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It has a taproot that will grow even deeper to find water farther from the ground.
In my previous comment, I said that it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. Please check or justify.
The sentence has been revised to meet Reviewer’s suggestions. Done as recommended.
4. From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

Please justify!
We have corrected the sentence to icorporate Reviewer’s comments. Please refer to revised Discussion.
5. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance.

Please justify how the decrease in subtrate binding affinity (to SOD) can impart resistance?
This sentence has been deleted to avoid the obscurity to SOD
Conclusion

However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment

"Used' should be changed to 'use'
“used’ has been revised to ‘use’
Competing Interests: No competing interests were disclosed. Close
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Version 2

VERSION 2

PUBLISHED 18 Jul 2023

Revised

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Reviewer Report 19 Sep 2023

Mohamed sathik Thirruvithamkottil, Rubber Research Institute of India, Rubber Board, Kottayam, Kerala, India

Approved with Reservations

https://doi.org/10.5256/f1000research.152374.r188303

Introduction second para
1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon..

2. northeastern states of India - does not experience drought, instead say the 'Central India'.

Introduction third para
3. "Based on bio-informatics, there are 20 proteins related to drought stress in rubber..." This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea.

Methods, Stages of growth and development.....
1. second line: insert space between "...Corynespora and"

2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given.

3. Data Analysis: First line - change to 'with an interval of three weeks'.

Results
1. Proline unit mgg^-1 should be changed to mg g^-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript.

2. 'chlorophyll (microgram) ug mg^-1'

3. Instead of "Chlorophyll total change" it to 'Total Chlorophyll content'

4. Ascorbate peroxidase/APX (units mg^-1) - space inserted.

5. SOD. POD - also (units mg^-1)

Discussion - First Paragraph: Please recast it as it does not convey the message properly.

For e.g. You can try like below.
'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.'

The second and third line are mere repetitions of the first line which you can avoid.

Second para: the first line does not convey the message properly.
I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.'

In third para, third line:
"Each clone showed its ability to produce total sugar content when stressed." - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions?

Fourth para, third line from the bottom:
"This shows that the clonal factor still has to be tested in other environments against drought stress." - What do you mean by this sentence? How do you want to test in other environments against drought stress?

6th para...
"This is presumably because the rubber plant is an perennial plant..." - 'an' is not required, instead just use 'a' only.

second line
"It has a taproot that will grow even deeper to find water farther from the ground." - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement?

7th para:
"The condition of this research is a scale research greenhouse..." - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it.

"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably.

SOD
The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable.

In POD para: "clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24." 'as scions' is not needed as when you say clone, that itself denotes it.

"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale." - Instead you can take it to the Conclusion paragraph and explain like below:

'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H₂O₂, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.'

"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line)." - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation.

These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time.

I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck.

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Plant Physiology, Molecular Biology, Plant Breeding, etc.

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Reviewer Report 09 Aug 2023

Maryam Nazari, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Hamadan, Iran

Approved

https://doi.org/10.5256/f1000research.152374.r188304

Thanks to the authors for revising the article accordingly. I have reviewed the new version ... Continue reading

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Version 1

VERSION 1

PUBLISHED 27 Jan 2023

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Reviewer Report 25 Apr 2023

Maryam Nazari, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Hamadan, Iran

Approved with Reservations

https://doi.org/10.5256/f1000research.142106.r164844

There is no explanation about the stages of growth and development of the clones in this manuscript.

Results

According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.
According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

Discussion

The IRR 425 clone had the highest proline levels in three observations not four.
Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Plant breeding

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Author Response 18 Jul 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

18 Jul 2023

Author Response

The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the ... Continue reading The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version:

Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns. In the following pages we address the reviewers’ comments point by point

Methods
There is no explanation about the stages of growth and development of the clones in this manuscript.

R: Thank you for your suggestion. New sentences have been added to incorporate reviewer’s comments.

The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (oidium, colletotrichum, corynespora and pestalotiosis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon).

Results
According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.

R: Thank you for the suggestion (correction made)

According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

R: Correction made

Discussion
The IRR 425 clone had the highest proline levels in three observations, not four.

R: Thank you for the correction. Done as recommended.

Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

R: Correction made

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

R: Thank you for the suggestion (correction made)

Khayatnezhad, et al, 2023 have reported that genotypes corn BC678 and BC404 have the highest chlorophyll index and the amount of yield and are the most resistant genotypes to drought (reference no.38).
The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version:

Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns. In the following pages we address the reviewers’ comments point by point

Methods
There is no explanation about the stages of growth and development of the clones in this manuscript.

R: Thank you for your suggestion. New sentences have been added to incorporate reviewer’s comments.

The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (oidium, colletotrichum, corynespora and pestalotiosis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon).

Results
According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.

R: Thank you for the suggestion (correction made)

According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

R: Correction made

Discussion
The IRR 425 clone had the highest proline levels in three observations, not four.

R: Thank you for the correction. Done as recommended.

Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

R: Correction made

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

R: Thank you for the suggestion (correction made)

Khayatnezhad, et al, 2023 have reported that genotypes corn BC678 and BC404 have the highest chlorophyll index and the amount of yield and are the most resistant genotypes to drought (reference no.38).
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Close
Report a concern
Author Response 12 Oct 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

12 Oct 2023

Author Response

APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, ... Continue reading APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns in the following pages .

Introduction second para
1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon..
R: Correction made

2. northeastern states of India - does not experience drought, instead say the 'Central India'.
R: Correction made

Introduction third para
3. "Based on bio-informatics, there are 20 proteins related to drought stress in rubber..." This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea.
R: Done as recommended

Methods, Stages of growth and development.....
1. second line: insert space between "...Corynespora and"
R: Correction made

2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given.
R: Correction made

3. Data Analysis: First line - change to 'with an interval of three weeks'.
R: Correction made

Results
1. Proline unit mgg-1 should be changed to mg g-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript.
R: Correction made

2. 'chlorophyll (microgram) ug mg-1'
R: Correction made

3. Instead of "Chlorophyll total change" it to 'Total Chlorophyll content'
R: Correction made

4. Ascorbate peroxidase/APX (units mg-1) - space inserted.
R: Correction made

5. SOD. POD - also (units mg-1)
R: Correction made

Discussion - First Paragraph: Please recast it as it does not convey the message properly.
R: Correction made

For e.g. You can try like below.
'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.'
R: Correction made

The second and third line are mere repetitions of the first line which you can avoid.
R: Correction made

(changing the sentence according to the reviewer's suggestions)
Second para: the first line does not convey the message properly.
I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.'
R: Correction made

In third para, third line:
"Each clone showed its ability to produce total sugar content when stressed." - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions?
R: Correction made

Fourth para, third line from the bottom:
"This shows that the clonal factor still has to be tested in other environments against drought stress." - What do you mean by this sentence? How do you want to test in other environments against drought stress?
R: Correction made

6th para...
"This is presumably because the rubber plant is an perennial plant..." - 'an' is not required, instead just use 'a' only.
R: Correction made

(literature 39) /additional literature
second line
"It has a taproot that will grow even deeper to find water farther from the ground." - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement?

7th para:
"The condition of this research is a scale research greenhouse..." - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it.

"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably.
R: Correction made

SOD
The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable.
R: Correction made

In POD para: "clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24." 'as scions' is not needed as when you say clone, that itself denotes it.

"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale." - Instead you can take it to the Conclusion paragraph and explain like below:
R: Correction made

Changing the sentence as suggestion reviewer
'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.'
Thank you for your suggestions.

"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line)." - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation.

These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time.

I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck.
APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns in the following pages .

Introduction second para
1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon..
R: Correction made

2. northeastern states of India - does not experience drought, instead say the 'Central India'.
R: Correction made

Introduction third para
3. "Based on bio-informatics, there are 20 proteins related to drought stress in rubber..." This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea.
R: Done as recommended

Methods, Stages of growth and development.....
1. second line: insert space between "...Corynespora and"
R: Correction made

2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given.
R: Correction made

3. Data Analysis: First line - change to 'with an interval of three weeks'.
R: Correction made

Results
1. Proline unit mgg-1 should be changed to mg g-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript.
R: Correction made

2. 'chlorophyll (microgram) ug mg-1'
R: Correction made

3. Instead of "Chlorophyll total change" it to 'Total Chlorophyll content'
R: Correction made

4. Ascorbate peroxidase/APX (units mg-1) - space inserted.
R: Correction made

5. SOD. POD - also (units mg-1)
R: Correction made

Discussion - First Paragraph: Please recast it as it does not convey the message properly.
R: Correction made

For e.g. You can try like below.
'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.'
R: Correction made

The second and third line are mere repetitions of the first line which you can avoid.
R: Correction made

(changing the sentence according to the reviewer's suggestions)
Second para: the first line does not convey the message properly.
I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.'
R: Correction made

In third para, third line:
"Each clone showed its ability to produce total sugar content when stressed." - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions?
R: Correction made

Fourth para, third line from the bottom:
"This shows that the clonal factor still has to be tested in other environments against drought stress." - What do you mean by this sentence? How do you want to test in other environments against drought stress?
R: Correction made

6th para...
"This is presumably because the rubber plant is an perennial plant..." - 'an' is not required, instead just use 'a' only.
R: Correction made

(literature 39) /additional literature
second line
"It has a taproot that will grow even deeper to find water farther from the ground." - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement?

7th para:
"The condition of this research is a scale research greenhouse..." - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it.

"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably.
R: Correction made

SOD
The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable.
R: Correction made

In POD para: "clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24." 'as scions' is not needed as when you say clone, that itself denotes it.

"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale." - Instead you can take it to the Conclusion paragraph and explain like below:
R: Correction made

Changing the sentence as suggestion reviewer
'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.'
Thank you for your suggestions.

"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line)." - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation.

These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time.

I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck.
Competing Interests: No competing interests were disclosed. Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 18 Jul 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

18 Jul 2023

Author Response

The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the ... Continue reading The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version:

Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns. In the following pages we address the reviewers’ comments point by point

Methods
There is no explanation about the stages of growth and development of the clones in this manuscript.

R: Thank you for your suggestion. New sentences have been added to incorporate reviewer’s comments.

The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (oidium, colletotrichum, corynespora and pestalotiosis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon).

Results
According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.

R: Thank you for the suggestion (correction made)

According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

R: Correction made

Discussion
The IRR 425 clone had the highest proline levels in three observations, not four.

R: Thank you for the correction. Done as recommended.

Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

R: Correction made

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

R: Thank you for the suggestion (correction made)

Khayatnezhad, et al, 2023 have reported that genotypes corn BC678 and BC404 have the highest chlorophyll index and the amount of yield and are the most resistant genotypes to drought (reference no.38).
The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version:

Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns. In the following pages we address the reviewers’ comments point by point

Methods
There is no explanation about the stages of growth and development of the clones in this manuscript.

R: Thank you for your suggestion. New sentences have been added to incorporate reviewer’s comments.

The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (oidium, colletotrichum, corynespora and pestalotiosis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon).

Results
According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.

R: Thank you for the suggestion (correction made)

According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

R: Correction made

Discussion
The IRR 425 clone had the highest proline levels in three observations, not four.

R: Thank you for the correction. Done as recommended.

Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

R: Correction made

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

R: Thank you for the suggestion (correction made)

Khayatnezhad, et al, 2023 have reported that genotypes corn BC678 and BC404 have the highest chlorophyll index and the amount of yield and are the most resistant genotypes to drought (reference no.38).
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Close
Report a concern
Author Response 12 Oct 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

12 Oct 2023

Author Response

APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, ... Continue reading APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns in the following pages .

Introduction second para
1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon..
R: Correction made

2. northeastern states of India - does not experience drought, instead say the 'Central India'.
R: Correction made

Introduction third para
3. "Based on bio-informatics, there are 20 proteins related to drought stress in rubber..." This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea.
R: Done as recommended

Methods, Stages of growth and development.....
1. second line: insert space between "...Corynespora and"
R: Correction made

2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given.
R: Correction made

3. Data Analysis: First line - change to 'with an interval of three weeks'.
R: Correction made

Results
1. Proline unit mgg-1 should be changed to mg g-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript.
R: Correction made

2. 'chlorophyll (microgram) ug mg-1'
R: Correction made

3. Instead of "Chlorophyll total change" it to 'Total Chlorophyll content'
R: Correction made

4. Ascorbate peroxidase/APX (units mg-1) - space inserted.
R: Correction made

5. SOD. POD - also (units mg-1)
R: Correction made

Discussion - First Paragraph: Please recast it as it does not convey the message properly.
R: Correction made

For e.g. You can try like below.
'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.'
R: Correction made

The second and third line are mere repetitions of the first line which you can avoid.
R: Correction made

(changing the sentence according to the reviewer's suggestions)
Second para: the first line does not convey the message properly.
I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.'
R: Correction made

In third para, third line:
"Each clone showed its ability to produce total sugar content when stressed." - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions?
R: Correction made

Fourth para, third line from the bottom:
"This shows that the clonal factor still has to be tested in other environments against drought stress." - What do you mean by this sentence? How do you want to test in other environments against drought stress?
R: Correction made

6th para...
"This is presumably because the rubber plant is an perennial plant..." - 'an' is not required, instead just use 'a' only.
R: Correction made

(literature 39) /additional literature
second line
"It has a taproot that will grow even deeper to find water farther from the ground." - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement?

7th para:
"The condition of this research is a scale research greenhouse..." - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it.

"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably.
R: Correction made

SOD
The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable.
R: Correction made

In POD para: "clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24." 'as scions' is not needed as when you say clone, that itself denotes it.

"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale." - Instead you can take it to the Conclusion paragraph and explain like below:
R: Correction made

Changing the sentence as suggestion reviewer
'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.'
Thank you for your suggestions.

"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line)." - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation.

These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time.

I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck.
APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns in the following pages .

Introduction second para
1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon..
R: Correction made

2. northeastern states of India - does not experience drought, instead say the 'Central India'.
R: Correction made

Introduction third para
3. "Based on bio-informatics, there are 20 proteins related to drought stress in rubber..." This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea.
R: Done as recommended

Methods, Stages of growth and development.....
1. second line: insert space between "...Corynespora and"
R: Correction made

2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given.
R: Correction made

3. Data Analysis: First line - change to 'with an interval of three weeks'.
R: Correction made

Results
1. Proline unit mgg-1 should be changed to mg g-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript.
R: Correction made

2. 'chlorophyll (microgram) ug mg-1'
R: Correction made

3. Instead of "Chlorophyll total change" it to 'Total Chlorophyll content'
R: Correction made

4. Ascorbate peroxidase/APX (units mg-1) - space inserted.
R: Correction made

5. SOD. POD - also (units mg-1)
R: Correction made

Discussion - First Paragraph: Please recast it as it does not convey the message properly.
R: Correction made

For e.g. You can try like below.
'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.'
R: Correction made

The second and third line are mere repetitions of the first line which you can avoid.
R: Correction made

(changing the sentence according to the reviewer's suggestions)
Second para: the first line does not convey the message properly.
I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.'
R: Correction made

In third para, third line:
"Each clone showed its ability to produce total sugar content when stressed." - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions?
R: Correction made

Fourth para, third line from the bottom:
"This shows that the clonal factor still has to be tested in other environments against drought stress." - What do you mean by this sentence? How do you want to test in other environments against drought stress?
R: Correction made

6th para...
"This is presumably because the rubber plant is an perennial plant..." - 'an' is not required, instead just use 'a' only.
R: Correction made

(literature 39) /additional literature
second line
"It has a taproot that will grow even deeper to find water farther from the ground." - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement?

7th para:
"The condition of this research is a scale research greenhouse..." - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it.

"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably.
R: Correction made

SOD
The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable.
R: Correction made

In POD para: "clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24." 'as scions' is not needed as when you say clone, that itself denotes it.

"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale." - Instead you can take it to the Conclusion paragraph and explain like below:
R: Correction made

Changing the sentence as suggestion reviewer
'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.'
Thank you for your suggestions.

"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line)." - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation.

These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time.

I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck.
Competing Interests: No competing interests were disclosed. Close
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Views

Reviewer Report 06 Apr 2023

Mohamed sathik Thirruvithamkottil, Rubber Research Institute of India, Rubber Board, Kottayam, Kerala, India

Approved with Reservations

https://doi.org/10.5256/f1000research.142106.r161733

Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

Abstract:

Background

Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’

7^th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.

The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

Introduction

The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.

Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.

‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.

Methods

Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.

Results

Table 1, 2 and 3

1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?

The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.

Similarly, Table 4,5 and 6 (of Proline) should be combined and all the figures should be given.

Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.

Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.

Table 13, 14 and 15 – should be combined and all the figures should be given.

Table 16, 17 and 18 - should be combined and all the figures should be given.

Similarly for all the tables of APX, SOD, POD.

Discussion

The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.

In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.

Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.

From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

The 6^th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.

At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?

Conclusions

The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.

So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Plant Physiology, Molecular Biology, Plant Breeding, etc.

CITE

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Author Response 18 Jul 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

18 Jul 2023

Author Response

Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the ... Continue reading Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows in bold font:

Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

R: Correction made, please refer to page 1

Abstract:
Background
Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’

R: Thank you for your correction. Done as recommendation, please refer to page 2

7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.

R: Correction made, see page 2

The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

R: Thank you for the correction. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to identify drought tolerant traits in order to select drought tolerant clones at juvenile stage. Please refer to page 2

Introduction
The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.

R: Thank you for your correction. The word stem elongation and stomatal has been removed to make clarity and readability. Please refer to page 2

Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.

R: Thank you for your suggestions. Correction made. Please refer to page 3

‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.

R: Thank you for your suggestions. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, rapid stem growth, and leaf senescence. “Stem elongation” and “stomatal opening” have been deleted.

Methods
Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.

R: Thank you for suggestions. We have added new sentences to incorporate your suggestions: Stages of growth and development of the clones
The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (Oidium, Ccolletotrichum, Ccorynespora and Pestalotiopsis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Please refer to page 4.

Results

Table 1, 2 and 3

1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?

R: Thank you for the correction (correction made as six time period): Observations of physiological characters were observed 6 times with a time distance 21 days between observations. The total samples observed were 63 samples consisting 7 types of clones x 3 types of water concentration and 3 replications. Please refer to Tables 1-9.

The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.

R: Thank you for your suggestions. Done as recommended.

Similarly, Table 4, 5 and 6 (of Proline) should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 13, 14 and 15 – should be combined and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 16, 17 and 18 - should be combined and all the figures should be given.

R: Thank you for the advice. Correction made.

Similarly for all the tables of APX, SOD, POD.

R: Thank you for the advice. Correction made.

Discussion
The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.

R: Thank you for the correction, we have revised accordingly: Plants respond and adapt due to environmental changes that experience continuous drought stress with appropriate physiological and biochemical changes to overcome these stressful conditions. Stress in plants is a physiological situation that is induced when there is a severe or constant change in the environment or when aggressive normal conditions, changing the physiological and adaptive patterns of plants. All these environmental modifications produce physiological reactions in cells of genetic origin.

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

R: Thank you for the suggestion. There are differences in growth between areas that have distinct rainy and dry seasons, such as in Java. The RRIC 100 clone has a faster growth and higher production potential compared to intolerant clones in the area such as the BPM 24 clone.

Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.

R: Thank you for the suggestion, correction made. Similar results of increased total sugar accumulation have been observed.

In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.

R: Thank you for the advice (correction made): similar results of increased total sugar accumulation have been observed.

Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.

R: Thank you for your correction.

We made a mistake in mentioning that the rubber plant is annual plant, it should be a perennial plant. In this study it can be seen that the content of chlorophyll a, b, and total at 30% water content is higher than that at 90% water content. This is presumably because the rubber plant is an perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

The condition of this research is a scale research greenhouse, the water given to the planting medium will not be lost down because the planting medium is designed not to have holes or gaps where the water comes out. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Matter Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old), plant size (large), ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year).

From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

R: Thank you for the correctiom.

It means that the water is added to soil which is placed in polybag covered with more transparent paste which has no gaps. So that the water will remain in the polybag.

The 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.

R: Thank you for the suggestion.

The SOD result should be different between the water content. But in our research, the same results were obtained between different water content.

At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?

R: Thank you for the suggestion. Correction made.

Conclusions
The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.

R: Thank you for the suggestion.

The water content with a concentration of 30% is considered to reflect dry conditions. So it is referred as drought stress. For the observational characters, two characters were obtained, namely total sugar and proline which showed a significant effect from the 6 time observations. As for the other observational characters (Chlorophyll a, b, total, APX, H2O2, SOD and POD) they still showed the same effect when treated with several water concentrations.

So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.

R: Thank you for the suggestions. Revision made.
Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows in bold font:

Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

R: Correction made, please refer to page 1

Abstract:
Background
Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’

R: Thank you for your correction. Done as recommendation, please refer to page 2

7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.

R: Correction made, see page 2

The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

R: Thank you for the correction. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to identify drought tolerant traits in order to select drought tolerant clones at juvenile stage. Please refer to page 2

Introduction
The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.

R: Thank you for your correction. The word stem elongation and stomatal has been removed to make clarity and readability. Please refer to page 2

Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.

R: Thank you for your suggestions. Correction made. Please refer to page 3

‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.

R: Thank you for your suggestions. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, rapid stem growth, and leaf senescence. “Stem elongation” and “stomatal opening” have been deleted.

Methods
Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.

R: Thank you for suggestions. We have added new sentences to incorporate your suggestions: Stages of growth and development of the clones
The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (Oidium, Ccolletotrichum, Ccorynespora and Pestalotiopsis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Please refer to page 4.

Results

Table 1, 2 and 3

1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?

R: Thank you for the correction (correction made as six time period): Observations of physiological characters were observed 6 times with a time distance 21 days between observations. The total samples observed were 63 samples consisting 7 types of clones x 3 types of water concentration and 3 replications. Please refer to Tables 1-9.

The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.

R: Thank you for your suggestions. Done as recommended.

Similarly, Table 4, 5 and 6 (of Proline) should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 13, 14 and 15 – should be combined and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 16, 17 and 18 - should be combined and all the figures should be given.

R: Thank you for the advice. Correction made.

Similarly for all the tables of APX, SOD, POD.

R: Thank you for the advice. Correction made.

Discussion
The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.

R: Thank you for the correction, we have revised accordingly: Plants respond and adapt due to environmental changes that experience continuous drought stress with appropriate physiological and biochemical changes to overcome these stressful conditions. Stress in plants is a physiological situation that is induced when there is a severe or constant change in the environment or when aggressive normal conditions, changing the physiological and adaptive patterns of plants. All these environmental modifications produce physiological reactions in cells of genetic origin.

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

R: Thank you for the suggestion. There are differences in growth between areas that have distinct rainy and dry seasons, such as in Java. The RRIC 100 clone has a faster growth and higher production potential compared to intolerant clones in the area such as the BPM 24 clone.

Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.

R: Thank you for the suggestion, correction made. Similar results of increased total sugar accumulation have been observed.

In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.

R: Thank you for the advice (correction made): similar results of increased total sugar accumulation have been observed.

Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.

R: Thank you for your correction.

We made a mistake in mentioning that the rubber plant is annual plant, it should be a perennial plant. In this study it can be seen that the content of chlorophyll a, b, and total at 30% water content is higher than that at 90% water content. This is presumably because the rubber plant is an perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

The condition of this research is a scale research greenhouse, the water given to the planting medium will not be lost down because the planting medium is designed not to have holes or gaps where the water comes out. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Matter Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old), plant size (large), ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year).

From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

R: Thank you for the correctiom.

It means that the water is added to soil which is placed in polybag covered with more transparent paste which has no gaps. So that the water will remain in the polybag.

The 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.

R: Thank you for the suggestion.

The SOD result should be different between the water content. But in our research, the same results were obtained between different water content.

At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?

R: Thank you for the suggestion. Correction made.

Conclusions
The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.

R: Thank you for the suggestion.

The water content with a concentration of 30% is considered to reflect dry conditions. So it is referred as drought stress. For the observational characters, two characters were obtained, namely total sugar and proline which showed a significant effect from the 6 time observations. As for the other observational characters (Chlorophyll a, b, total, APX, H2O2, SOD and POD) they still showed the same effect when treated with several water concentrations.

So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.

R: Thank you for the suggestions. Revision made.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Close
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COMMENTS ON THIS REPORT

Author Response 18 Jul 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

18 Jul 2023

Author Response

Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the ... Continue reading Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows in bold font:

Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

R: Correction made, please refer to page 1

Abstract:
Background
Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’

R: Thank you for your correction. Done as recommendation, please refer to page 2

7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.

R: Correction made, see page 2

The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

R: Thank you for the correction. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to identify drought tolerant traits in order to select drought tolerant clones at juvenile stage. Please refer to page 2

Introduction
The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.

R: Thank you for your correction. The word stem elongation and stomatal has been removed to make clarity and readability. Please refer to page 2

Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.

R: Thank you for your suggestions. Correction made. Please refer to page 3

‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.

R: Thank you for your suggestions. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, rapid stem growth, and leaf senescence. “Stem elongation” and “stomatal opening” have been deleted.

Methods
Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.

R: Thank you for suggestions. We have added new sentences to incorporate your suggestions: Stages of growth and development of the clones
The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (Oidium, Ccolletotrichum, Ccorynespora and Pestalotiopsis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Please refer to page 4.

Results

Table 1, 2 and 3

1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?

R: Thank you for the correction (correction made as six time period): Observations of physiological characters were observed 6 times with a time distance 21 days between observations. The total samples observed were 63 samples consisting 7 types of clones x 3 types of water concentration and 3 replications. Please refer to Tables 1-9.

The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.

R: Thank you for your suggestions. Done as recommended.

Similarly, Table 4, 5 and 6 (of Proline) should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 13, 14 and 15 – should be combined and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 16, 17 and 18 - should be combined and all the figures should be given.

R: Thank you for the advice. Correction made.

Similarly for all the tables of APX, SOD, POD.

R: Thank you for the advice. Correction made.

Discussion
The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.

R: Thank you for the correction, we have revised accordingly: Plants respond and adapt due to environmental changes that experience continuous drought stress with appropriate physiological and biochemical changes to overcome these stressful conditions. Stress in plants is a physiological situation that is induced when there is a severe or constant change in the environment or when aggressive normal conditions, changing the physiological and adaptive patterns of plants. All these environmental modifications produce physiological reactions in cells of genetic origin.

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

R: Thank you for the suggestion. There are differences in growth between areas that have distinct rainy and dry seasons, such as in Java. The RRIC 100 clone has a faster growth and higher production potential compared to intolerant clones in the area such as the BPM 24 clone.

Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.

R: Thank you for the suggestion, correction made. Similar results of increased total sugar accumulation have been observed.

In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.

R: Thank you for the advice (correction made): similar results of increased total sugar accumulation have been observed.

Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.

R: Thank you for your correction.

We made a mistake in mentioning that the rubber plant is annual plant, it should be a perennial plant. In this study it can be seen that the content of chlorophyll a, b, and total at 30% water content is higher than that at 90% water content. This is presumably because the rubber plant is an perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

The condition of this research is a scale research greenhouse, the water given to the planting medium will not be lost down because the planting medium is designed not to have holes or gaps where the water comes out. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Matter Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old), plant size (large), ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year).

From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

R: Thank you for the correctiom.

It means that the water is added to soil which is placed in polybag covered with more transparent paste which has no gaps. So that the water will remain in the polybag.

The 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.

R: Thank you for the suggestion.

The SOD result should be different between the water content. But in our research, the same results were obtained between different water content.

At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?

R: Thank you for the suggestion. Correction made.

Conclusions
The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.

R: Thank you for the suggestion.

The water content with a concentration of 30% is considered to reflect dry conditions. So it is referred as drought stress. For the observational characters, two characters were obtained, namely total sugar and proline which showed a significant effect from the 6 time observations. As for the other observational characters (Chlorophyll a, b, total, APX, H2O2, SOD and POD) they still showed the same effect when treated with several water concentrations.

So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.

R: Thank you for the suggestions. Revision made.
Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows in bold font:

Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

R: Correction made, please refer to page 1

Abstract:
Background
Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’

R: Thank you for your correction. Done as recommendation, please refer to page 2

7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.

R: Correction made, see page 2

The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

R: Thank you for the correction. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to identify drought tolerant traits in order to select drought tolerant clones at juvenile stage. Please refer to page 2

Introduction
The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.

R: Thank you for your correction. The word stem elongation and stomatal has been removed to make clarity and readability. Please refer to page 2

Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.

R: Thank you for your suggestions. Correction made. Please refer to page 3

‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.

R: Thank you for your suggestions. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, rapid stem growth, and leaf senescence. “Stem elongation” and “stomatal opening” have been deleted.

Methods
Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.

R: Thank you for suggestions. We have added new sentences to incorporate your suggestions: Stages of growth and development of the clones
The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (Oidium, Ccolletotrichum, Ccorynespora and Pestalotiopsis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Please refer to page 4.

Results

Table 1, 2 and 3

1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?

R: Thank you for the correction (correction made as six time period): Observations of physiological characters were observed 6 times with a time distance 21 days between observations. The total samples observed were 63 samples consisting 7 types of clones x 3 types of water concentration and 3 replications. Please refer to Tables 1-9.

The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.

R: Thank you for your suggestions. Done as recommended.

Similarly, Table 4, 5 and 6 (of Proline) should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 13, 14 and 15 – should be combined and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 16, 17 and 18 - should be combined and all the figures should be given.

R: Thank you for the advice. Correction made.

Similarly for all the tables of APX, SOD, POD.

R: Thank you for the advice. Correction made.

Discussion
The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.

R: Thank you for the correction, we have revised accordingly: Plants respond and adapt due to environmental changes that experience continuous drought stress with appropriate physiological and biochemical changes to overcome these stressful conditions. Stress in plants is a physiological situation that is induced when there is a severe or constant change in the environment or when aggressive normal conditions, changing the physiological and adaptive patterns of plants. All these environmental modifications produce physiological reactions in cells of genetic origin.

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

R: Thank you for the suggestion. There are differences in growth between areas that have distinct rainy and dry seasons, such as in Java. The RRIC 100 clone has a faster growth and higher production potential compared to intolerant clones in the area such as the BPM 24 clone.

Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.

R: Thank you for the suggestion, correction made. Similar results of increased total sugar accumulation have been observed.

In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.

R: Thank you for the advice (correction made): similar results of increased total sugar accumulation have been observed.

Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.

R: Thank you for your correction.

We made a mistake in mentioning that the rubber plant is annual plant, it should be a perennial plant. In this study it can be seen that the content of chlorophyll a, b, and total at 30% water content is higher than that at 90% water content. This is presumably because the rubber plant is an perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

The condition of this research is a scale research greenhouse, the water given to the planting medium will not be lost down because the planting medium is designed not to have holes or gaps where the water comes out. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Matter Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old), plant size (large), ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year).

From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

R: Thank you for the correctiom.

It means that the water is added to soil which is placed in polybag covered with more transparent paste which has no gaps. So that the water will remain in the polybag.

The 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.

R: Thank you for the suggestion.

The SOD result should be different between the water content. But in our research, the same results were obtained between different water content.

At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?

R: Thank you for the suggestion. Correction made.

Conclusions
The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.

R: Thank you for the suggestion.

The water content with a concentration of 30% is considered to reflect dry conditions. So it is referred as drought stress. For the observational characters, two characters were obtained, namely total sugar and proline which showed a significant effect from the 6 time observations. As for the other observational characters (Chlorophyll a, b, total, APX, H2O2, SOD and POD) they still showed the same effect when treated with several water concentrations.

So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.

R: Thank you for the suggestions. Revision made.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Close
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Version 4

VERSION 4 PUBLISHED 27 Jan 2023

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Version 4 (revision) 04 Dec 23			read
Version 3 (revision) 12 Oct 23	read
Version 2 (revision) 18 Jul 23	read	read
Version 1 27 Jan 23	read	read

Mohamed sathik Thirruvithamkottil, Rubber Board, Kottayam, India
Maryam Nazari, Bu-Ali Sina University, Hamadan, Iran
Boon Chin Tan, Universiti Malaya, Kuala Lumpur, Malaysia

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02 Jan 2025 | for Version 4

Boon Chin Tan, Universiti Malaya, Kuala Lumpur, Malaysia

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Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Plant biotechnology and physiology

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10 Views

07 Nov 2023 | for Version 3

Mohamed sathik Thirruvithamkottil, Rubber Research Institute of India, Rubber Board, Kottayam, Kerala, India

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Approved With Reservations

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No competing interests were disclosed.

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Plant Physiology, Molecular Biology, Plant Breeding, etc.

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Author Response

04 Dec 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows:

APPROVED WITH RESERVATIONS
info_outline
1. Introduction 2nd paragraph,

One of the primary sources of natural rubber is producing plants - here is is not required.
Is has been deleted
2. signaling pathways and no comma mark (,) is needed before ‘and’
Comma mark (,) has been removed.
3. In times of drought stress, lack of sufficient water combined with the increased CO 2 in the atmosphere can cause plant death.13
The sentence has been corrected to read: The combined rise in atmospheric CO2 concentrations and global air temperature may significantly alter plant water use.
I could not see the death in the reference mentioned above. Please correct it or omit the point as it does not look relevant.

4. The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.
The aim of study has been corrected.
Discussion

1. The previous point mentioned below is not addressed

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.
Thank you for the correction. Drying has been revised to drought.
2. Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.
Thank you for your correction. The sentence has been improved to read: Increased proline content in drought-stress plants can maintain turgor for continued growth.
The above statement as per ref no. was verified and in none of the chapters it says that proline can provide high energy. Please clarify

3. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant. It has a taproot that will grow even deeper to find water farther from the ground.
In my previous comment, I said that it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. Please check or justify.
The sentence has been revised to meet Reviewer’s suggestions. Done as recommended.
4. From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

Please justify!
We have corrected the sentence to icorporate Reviewer’s comments. Please refer to revised Discussion.
5. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance.

Please justify how the decrease in subtrate binding affinity (to SOD) can impart resistance?
This sentence has been deleted to avoid the obscurity to SOD
Conclusion

However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment

"Used' should be changed to 'use'
“used’ has been revised to ‘use’

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19 Sep 2023 | for Version 2

Mohamed sathik Thirruvithamkottil, Rubber Research Institute of India, Rubber Board, Kottayam, Kerala, India

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No competing interests were disclosed.

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09 Aug 2023 | for Version 2

Maryam Nazari, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Hamadan, Iran

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Approved

Thanks to the authors for revising the article accordingly. I have reviewed the new version and I think the author's response and the changes made to improve the article are appropriate.

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No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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19 Views

25 Apr 2023 | for Version 1

Maryam Nazari, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Bu-Ali Sina University, Hamadan, Iran

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Approved With Reservations

There is no explanation about the stages of growth and development of the clones in this manuscript.

Results

According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.
According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

Discussion

The IRR 425 clone had the highest proline levels in three observations not four.
Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Plant breeding

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Author Response

18 Jul 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version:

Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns. In the following pages we address the reviewers’ comments point by point

Methods
There is no explanation about the stages of growth and development of the clones in this manuscript.

R: Thank you for your suggestion. New sentences have been added to incorporate reviewer’s comments.

The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (oidium, colletotrichum, corynespora and pestalotiosis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon).

Results
According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.

R: Thank you for the suggestion (correction made)

According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.

R: Correction made

Discussion
The IRR 425 clone had the highest proline levels in three observations, not four.

R: Thank you for the correction. Done as recommended.

Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;

R: Correction made

"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…"

In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

R: Thank you for the suggestion (correction made)

Khayatnezhad, et al, 2023 have reported that genotypes corn BC678 and BC404 have the highest chlorophyll index and the amount of yield and are the most resistant genotypes to drought (reference no.38).

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Competing Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Response

12 Oct 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

APPROVED WITH RESERVATIONS
Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns in the following pages .

Introduction second para
1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon..
R: Correction made

2. northeastern states of India - does not experience drought, instead say the 'Central India'.
R: Correction made

Introduction third para
3. "Based on bio-informatics, there are 20 proteins related to drought stress in rubber..." This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea.
R: Done as recommended

Methods, Stages of growth and development.....
1. second line: insert space between "...Corynespora and"
R: Correction made

2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given.
R: Correction made

3. Data Analysis: First line - change to 'with an interval of three weeks'.
R: Correction made

Results
1. Proline unit mgg-1 should be changed to mg g-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript.
R: Correction made

2. 'chlorophyll (microgram) ug mg-1'
R: Correction made

3. Instead of "Chlorophyll total change" it to 'Total Chlorophyll content'
R: Correction made

4. Ascorbate peroxidase/APX (units mg-1) - space inserted.
R: Correction made

5. SOD. POD - also (units mg-1)
R: Correction made

Discussion - First Paragraph: Please recast it as it does not convey the message properly.
R: Correction made

For e.g. You can try like below.
'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.'
R: Correction made

The second and third line are mere repetitions of the first line which you can avoid.
R: Correction made

(changing the sentence according to the reviewer's suggestions)
Second para: the first line does not convey the message properly.
I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.'
R: Correction made

In third para, third line:
"Each clone showed its ability to produce total sugar content when stressed." - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions?
R: Correction made

Fourth para, third line from the bottom:
"This shows that the clonal factor still has to be tested in other environments against drought stress." - What do you mean by this sentence? How do you want to test in other environments against drought stress?
R: Correction made

6th para...
"This is presumably because the rubber plant is an perennial plant..." - 'an' is not required, instead just use 'a' only.
R: Correction made

(literature 39) /additional literature
second line
"It has a taproot that will grow even deeper to find water farther from the ground." - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement?

7th para:
"The condition of this research is a scale research greenhouse..." - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it.

"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably.
R: Correction made

SOD
The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable.
R: Correction made

In POD para: "clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24." 'as scions' is not needed as when you say clone, that itself denotes it.

"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale." - Instead you can take it to the Conclusion paragraph and explain like below:
R: Correction made

Changing the sentence as suggestion reviewer
'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.'
Thank you for your suggestions.

"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line)." - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation.

These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time.

I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

33 Views

06 Apr 2023 | for Version 1

Mohamed sathik Thirruvithamkottil, Rubber Research Institute of India, Rubber Board, Kottayam, Kerala, India

33 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Plant Physiology, Molecular Biology, Plant Breeding, etc.

Respond to this report

Responses (1)

Author Response

18 Jul 2023

Mohammad Basyuni, Department of Forestry, Universitas Sumatera Utara, Medan, 20155, Indonesia

Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows in bold font:

Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

R: Correction made, please refer to page 1

Abstract:
Background
Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’

R: Thank you for your correction. Done as recommendation, please refer to page 2

7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.

R: Correction made, see page 2

The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.

R: Thank you for the correction. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to identify drought tolerant traits in order to select drought tolerant clones at juvenile stage. Please refer to page 2

Introduction
The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.

R: Thank you for your correction. The word stem elongation and stomatal has been removed to make clarity and readability. Please refer to page 2

Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.

R: Thank you for your suggestions. Correction made. Please refer to page 3

‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.

R: Thank you for your suggestions. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, rapid stem growth, and leaf senescence. “Stem elongation” and “stomatal opening” have been deleted.

Methods
Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.

R: Thank you for suggestions. We have added new sentences to incorporate your suggestions: Stages of growth and development of the clones
The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (Oidium, Ccolletotrichum, Ccorynespora and Pestalotiopsis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 ^oC (morning), 33.9 ^oC (days) and 30.4 ^oC (afternoon). The average temperature outside the greenhouse was 22.9 ^oC (morning), 28.1 ^oC (days) and 25.5 ^oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Please refer to page 4.

Results

Table 1, 2 and 3

1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?

R: Thank you for the correction (correction made as six time period): Observations of physiological characters were observed 6 times with a time distance 21 days between observations. The total samples observed were 63 samples consisting 7 types of clones x 3 types of water concentration and 3 replications. Please refer to Tables 1-9.

The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.

R: Thank you for your suggestions. Done as recommended.

Similarly, Table 4, 5 and 6 (of Proline) should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.

R: Thank you for the suggestion. Correction made.

Table 13, 14 and 15 – should be combined and all the figures should be given.

R: Thank you for the advice. Done as recommended.

Table 16, 17 and 18 - should be combined and all the figures should be given.

R: Thank you for the advice. Correction made.

Similarly for all the tables of APX, SOD, POD.

R: Thank you for the advice. Correction made.

Discussion
The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.

R: Thank you for the correction, we have revised accordingly: Plants respond and adapt due to environmental changes that experience continuous drought stress with appropriate physiological and biochemical changes to overcome these stressful conditions. Stress in plants is a physiological situation that is induced when there is a severe or constant change in the environment or when aggressive normal conditions, changing the physiological and adaptive patterns of plants. All these environmental modifications produce physiological reactions in cells of genetic origin.

Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.

R: Thank you for the suggestion. There are differences in growth between areas that have distinct rainy and dry seasons, such as in Java. The RRIC 100 clone has a faster growth and higher production potential compared to intolerant clones in the area such as the BPM 24 clone.

Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.

R: Thank you for the suggestion, correction made. Similar results of increased total sugar accumulation have been observed.

In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.

R: Thank you for the advice (correction made): similar results of increased total sugar accumulation have been observed.

Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.

R: Thank you for your correction.

We made a mistake in mentioning that the rubber plant is annual plant, it should be a perennial plant. In this study it can be seen that the content of chlorophyll a, b, and total at 30% water content is higher than that at 90% water content. This is presumably because the rubber plant is an perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface.

The condition of this research is a scale research greenhouse, the water given to the planting medium will not be lost down because the planting medium is designed not to have holes or gaps where the water comes out. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Matter Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old), plant size (large), ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year).

From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.

R: Thank you for the correctiom.

It means that the water is added to soil which is placed in polybag covered with more transparent paste which has no gaps. So that the water will remain in the polybag.

The 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.

R: Thank you for the suggestion.

The SOD result should be different between the water content. But in our research, the same results were obtained between different water content.

At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?

R: Thank you for the suggestion. Correction made.

Conclusions
The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.

R: Thank you for the suggestion.

The water content with a concentration of 30% is considered to reflect dry conditions. So it is referred as drought stress. For the observational characters, two characters were obtained, namely total sugar and proline which showed a significant effect from the 6 time observations. As for the other observational characters (Chlorophyll a, b, total, APX, H2O2, SOD and POD) they still showed the same effect when treated with several water concentrations.

So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.

R: Thank you for the suggestions. Revision made.

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Competing Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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[53] 53. Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21708230.v2

[54] 54. Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21708275.v2

[55] 55. Figshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21645116.v5

Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress

Abstract

Background

Methods

Results

Conclusions

Keywords

Revised Amendments from Version 3

Introduction

Methods

Study area

Stages of growth and development of the clones

Experimental design

Data analysis

Results

Total sugar content (μM)

Table 1.

Figure 1. Pattern of total sugar content linear curve as a result of orthogonal polynomial analysis.

Proline (mg g-1)

Table 2.

Figure 2. Pattern of proline linear curve as a result of orthogonal polynomial analysis.

Chlorophyll a (μg mg-1)

Table 3.

Chlorophyll b (μg mg-1)

Table 4.

Figure 3. Pattern of chlorophyll b linear curve as a result of orthogonal polynomial analysis.

Total chlorophyll content (μg mg-1)

Table 5.

Figure 4. Pattern of total chlorophyll content linear curve as a result of orthogonal polynomial analysis.

Hydrogen peroxidase/H2O2 (μmolg-1)

Table 6.

Figure 5. Pattern of H2O2 (μmol g-1) linear curve as a result of orthogonal polynomial analysis.

Ascorbate peroxidase/APX (units mg-1)

Table 7.

Superoxide Dismutase/SOD (units mg-1)

Table 8.

Peroxide dismutase/POD (units mg-1)

Table 9.

Discussion

Conclusions

Data availability

Underlying data

Extended data

Acknowledgments

References

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Proline (mg g^-1)

Chlorophyll a (μg mg^-1)

Chlorophyll b (μg mg^-1)

Total chlorophyll content (μg mg^-1)

Hydrogen peroxidase/H₂O₂ (μmolg^-1)

Figure 5. Pattern of H₂O₂ (μmol g^-1) linear curve as a result of orthogonal polynomial analysis.

Ascorbate peroxidase/APX (units mg^-1)

Superoxide Dismutase/SOD (units mg^-1)

Peroxide dismutase/POD (units mg^-1)