Physiological characteristics of IRR 400 series rubber clones ( Hevea brasiliensis Muell. Arg.) on drought stress [version 1; peer review: 2 approved with reservations]

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 determine the mechanism of drought-tolerant clones, based on physiological characteristics, to obtain character selection and drought-tolerant clones early. 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


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 that affects plants and can reduce yield and productivity in almost all plants in the world. 2 Hence, it becomes most important compared with other environmental factors because it interferes with plant growth and development and disrupts production and performance. Water is part of the protoplasm and makes up 85-90% of the total weight of the plant tissue. Water is a vital reagent in photosynthesis and hydrolysis reactions. In addition, 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 is found in the Amazon basin, South America. 4 Optimal conditions for the growth of rubber plants are high temperature (28 AE 2 o C), high humidity, and rainfall of 2000-4000 mm/year. 5 Rubber plantations in marginal areas, such as the northeastern states of India, southern China, northern and northeastern Thailand, and eastern Indonesia, experience abiotic stresses such as drought. Indonesia has a wide drought area of about 122.1 million ha, and it is not optimally exploited due to limited water resources.
The response caused by drought is quite complex because it involves changes in morphology, physiology, and metabolism. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, stem elongation, leaf senescence, and stomatal opening. 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, D-onomitol, and mannitol maintain the growth and productivity of a plant experiencing drought stress. [9][10][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 plays an important role in the detoxification of these reactive oxygen species (ROS), and enables 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. In times of drought stress, lack of sufficient water combined with the increased CO 2 in the atmosphere can cause plant death. 13 Based on bioinformatics, there are 20 proteins related to drought stress in rubber plants. 14 This study aims to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to obtain character selection and drought-tolerant clones early.

Data analysis
Observations were carries out six times on physiological characteristics, with time intervals every 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, total, 17 proline, [18][19][20][21] super peroxidase dismutase (SOD), 22 peroxidase dismutase (POD), 23 APX enzyme, 24 and hydrogen peroxide (H 2 O 2 ). 22 A step-by-step description of the procedure to analyze sugar content, proline, chlorophyll a, b, total, SOD, POD, H2O2 and APX has been deposited in prototocol.io and is available at dx.doi.org/10.17504/protocols.io.5jyl8je1dg2w/v1.

Total sugar content (μM)
The total sugar content analysis showed a significant effect in all the observations except the first one ( Table 1).
The total sugar content in the six observations carried out on tested clones were consistent. The RRIC 100 clone had the highest total sugar content four times, and the IRR 425 clone had the lowest three times.
The total sugar content analysis at different water levels generally showed a significant effect, except for the initial observation. This indicates that, in most of the six observations, water content affects the total sugar content of the tested clones ( Table 2).
Analysis of the total sugar content due to the interaction between the type of clone and water content level (30%, 60%, 90%) showed significant differences, except in the first observation (Table 3). What is interesting about these results is that the highest accumulation of total sugar is seen in the application of 30% water content. Meanwhile the effect on the different types of clones was quite diverse. 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 fourth). The two clones, RRIC 100 and IRR 429, also had the lowest total sugar in the fifth and sixth observations, respectively. The complete dataset of total sugar content is displayed in Supplementary Table 1.
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 shows 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.
Proline (mgg -1 ) Table 4 depicts the proline analysis of clone types treated with different water contents and shows that there were significantly different effects in all observations.
The results of proline analysis at different water content percentages showed significantly different effects in all observations, as shown in Table 5.  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 6.
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 of IRR 400 series, RRIC 100, and BPM 24 is shown in Figure 2. The complete dataset of proline can be seen in Supplementary Table 2.
Chlorophyll a (μgmg -1 ) The chlorophyll a analysis on the different clone types showed a significant effect, except for the first observation, as displayed in Table 7. Table 8 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 9). The complete dataset of chlorophyll a is depicted in Supplementary Table 3.
The results of the chlorophyll b analysis with different clone types showed significantly different results, except for the first observation (Table 10).
The results of analysis of chlorophyll b levels at the given water contents showed a significant effect in all six observations ( Table 11).
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 12.
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. The complete dataset of chlorophyll b can be seen in Supplementary Table 4.
Chlorophyll total (μgmg -1 ) The analysis results of chlorophyll total with different clone types showed significantly different results except for one observation (Table 13).
The results of chlorophyll total analysis with the given water content showed a significant effect in all six observations, as depicted in Table 14.
The analysis of chlorophyll total 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 15). The complete dataset of chlorophyll total can be seen in Supplementary Table 5.
Orthogonal polynomial regression shows a linear curve, where the water content at 30% has the highest total chlorophyll value. The linear curve shows that the total chlorophyll content increases with the decreasing water content. The orthogonal polynomial linear curve pattern of chlorophyll total of several clones of IRR 400 series, RRIC 100, and BPM 24 can be seen in Figure 4.    Hydrogen peroxidase/H 2 O 2 (μmolg -1 ) The results of the H 2 O 2 analysis with different types of clones showed a significantly different effect in two of the observations (third and fourth) ( Table 16).
The results of H 2 O 2 analysis at different water content levels did not show significant differences in any of the observation ( Table 17).
The analysis of H 2 O 2 levels (μmolg -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 18). The complete dataset of H 2 O 2 is displayed in Supplementary Table 6.
The effect of water content on the H 2 O 2 characteristic shows a linear regression curve based on orthogonal polynomials. This indicates that the lower the water content, the higher the concentration of H 2 O 2 . The linear regression pattern between H 2 O 2 content and water content can be seen in Figure 5. Ascorbate peroxidase/APX (unitsmg -1 ) The results of APX analysis with different clone types were not significantly different in any of the observations ( Table 19).
The analysis results of APX at different water content levels were not significantly different in any of the observations ( Table 20).
The analysis of APX levels (unitmg -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 21). The complete dataset of APX can be seen in Supplementary Table 7.
Superoxide Dismutase/SOD (unitmg -1 ) The SOD analysis with clone types showed a significant difference in three of the observations (Table 22).
Analysis of SOD (unitmg -1 ) levels at different water content levels showed significant differences in three of the observations, as depicted in Table 23.   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 24). The complete dataset of SOD can be seen in Supplementary Table 8.

Peroxide dismutase/POD (unitsmg -1 )
The POD analysis with different types of clones showed significant differences in two observations, as shown in Table 25.
The analysis of POD levels at different water content levels showed a significant difference in one observation, as depicted in Table 26.
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 27). The complete dataset of POD can be seen in Supplementary Table 9.

Discussion
Physiological characteristics that arise due to plant activities in certain environments are observable and enable growth and development. 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 showed its ability to produce total sugar content when stressed. The RRIC 100 is a dry tolerant clone in the field. The increase in total sugar content was seen in most of observations of 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 produced in drought-stressed soybeans 26 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 four observations. Meanwhile, the IRR 440 had the lowest proline levels in four observations. Assessing by 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 antioxidative defense molecule and causes stress signaling. 12 It is classified as an osmoprotectant, which causes increased hyperosmolarity and increased activity of antioxidant enzymes. 28 Increased proline content in droughtstress plants can provide high energy to increase plant growth in water-deficit conditions. 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 the six observations. The clones had high proline levels when treated with 30% water content. This shows that the clonal factor still has to be tested in other environments against drought stress. The proline content has been shown to increase about 10-fold in mungbean, 31 maize, 32 millet, 12,33,34 nyamplung, 35 and soybean 26 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][36][37] 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 addition, when stressed, the lateral roots will grow more to take advantage of the surface water. Even though the plants are grown in greenhouses, the water supplied into the planting media will not go down because the planting media is designed to not have holes, gaps, or place for water to come out. In addition, the surface of the polybag is also covered by plastic to minimize the occurrence of evapotranspiration from the growing media.
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 an ROS. The presence of these enzymes in almost all cells plays an important role in ROS detoxification for plant survival. 38 H 2 O 2 has several important roles in various biochemical and physiological processes. Long plant life and long growth processes result in H 2 O 2 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. 39 High levels of H 2 O 2 cause oxidative stress, which then causes cell damage and death. 40 However, optimal levels of H 2 O 2 can increase tolerance to abiotic stresses through modulation of various physiological processes, including photosynthesis, opening and closing of stomata, osmotic adjustment, and ROS detoxification. 39,40 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 H 2 O 2 triggers the occurrence of chitinase proteins that can produce calcium homeostasis, ion channels, phosphatases, transcription factors, and abscisic acid (ABA), signaling responses to stress. 41 APX in ascorbate-glutathione (AsA-GSH) cycling enzymes is responsible for the decomposition of H 2 O 2 produced by SOD in different cellular organelles. APX plays a key role in both drought stress response and recovery after drought. 41,42 APX is an integral component of the (ASC-GSH) cycle. APX performs the same function in the cytosol and chloroplasts. APX reduces H 2 O 2 to H 2 O and docosahexaenoic acid (DHA), using AsA as a reducing agent.
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). 43 APX is widely distributed and has a better affinity to H 2 O 2 , especially in terms of more efficient uptake of H 2 O 2 in times of stress. [43][44][45][46] Thought 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. A water content of 30% showed relatively the same SOD activity as 60% and 90% in all observations. This indicates the SOD 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. 47 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 of 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 H 2 O 2 to produce optimal protection. Tolerance of some genotypes to environmental stresses has been associated with higher antioxidant enzyme activity. Drought-tolerant species of pigeon pea (Cajanus cajan), 48 wheat (Triticum aestivum), 49,50 and black bean (Phaseolus mungo) 47 have higher SOD, POD, and CAT activities than droughtsensitive 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 as scions. 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
The tolerance ability of the IRR 400 series rubber clones to drought stress was determined by observing the two characteristics of total sugar and proline levels. Furthermore, chlorophyll a, b, and total, H 2 O 2 , APX SOD, and POD should not be used as markers of drought stress tolerance in rubber trees. The concentrations of total sugar and proline were higher when the plants were treated with a lower water content.

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 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. 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.

Results
Similarly, Table 4,5 and 6 (of Proline) 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 plantsprovides 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'?