Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II [version 2; peer review: approved] Previously titled: Identification and molecular characterization of a Chlamydomonas reinhardtii mutant that shows a light intensity dependent progressive chlorophyll deficiency

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel). MgChel catalyzes insertion of Mg 2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14 , by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m -2 that in the wild type strain. gun4-I mutant light shift support the findings in cyanobacterial and Arabidopsis mutants This manuscript presents data regarding phenotype of isolated mutants ( 6F14 ), identification of the mutation locus in 6F14 and its complementation.The results revealed that 6F14 is defective in the GUN4 (genome uncoupled 4) gene which regulate MgChel activity. Therefore, the transformation of 6F14 with a GUN4 gene restored the wild type phenotype with over-expressing the GUN4 protein. The authors concluded that 6F14 is the second gun4 mutant identified in


Introduction
Chlamydomonas reinhardtii is a green micro-alga that can grow either heterotrophically using exogenous acetate as a carbon source or photo-autotrophically, using atmospheric CO 2 . It possesses a photosynthetic apparatus very similar to higher plants, has a short and simple haplontic life cycle, can synthesize Chl both light dependently and light independently (unlike most angiosperms) and its genome has been sequenced 1 . In addition, well developed molecular tools exist for genetic manipulations of its genome. All these traits make this alga an elegant model system for dissecting oxygenic photosynthesis 2,3 .
Chl, heme, siroheme, cobalamin, heme d1 and factor F430 are major tetrapyrroles that are involved in wide variety of essential life processes in all living organisms. Chl and heme are synthesized via a common branched pathway 4,5 (outlined in Figure 1). Photosynthetic eukaryotes synthesize 5-aminolevulinic acid (ALA) from glutamine (Glu) bound to tRNA Glu through the C5 pathway consisting of two steps catalyzed by glutamyl-tRNA reductase and glutamate-1semialdehyde aminotransferase 4,5 . ALA is subsequently converted in six steps to PPIX, the last common precursor for both Chl and heme biosynthesis 4,5 . Insertion of Fe 2+ into PPIX by ferrochelatase (FeChel) leads to heme. Insertion of Mg 2+ in PPIX by the heterotrimeric MgChel (comprised of three subunits: CHLD, CHLH and CHLI 6 ) leads to MgPPIX, the first biosynthetic intermediate in the Chl branch 6 . MgPPIX is converted to Pchlide via three enzymatic steps. The reduction of Pchlide to form chlorophyllide (Chlide) can occur by two different mechanisms. One mechanism is catalyzed by the strictly light dependent enzyme NADPH:Pchlide oxidoreductase (LPOR) and occurs in all photosynthetic organisms; it is the only mechanism of Chl formation in angiosperms 7-10 . The second mechanism is catalyzed by the light independent NADPH:Pchlide oxidoreductase (LiPOR) and is present in anoxygenic bacteria, alga, ferns and gymnosperms [11][12][13][14][15][16][17][18][19][20] . The Chlide a undergoes a phytylation reaction, catalyzed by Chl synthase (CS), resulting in the formation of Chl a. In vascular plants and green algae a portion of the Chlide a is converted to Chlide b by Chlide a oxygenase (CAO) prior to phytylation [21][22][23][24] . Chl a is converted to Chl b by CAO via formation of 7-hydroxymethyl chlorophyll a (HCA) and Chl b can be converted back to Chl a via HCA by chlorophyll b reductase (CBR) and 7-hydroxymethyl chlorophyll a reductase (HCAR) 25 . This inter-conversion of Chl a and Chl b, referred to as the "chlorophyll cycle", plays an important role in greening, acclimation to light and senescence 25 .
Stringent control of tetrapyrrole biosynthesis is especially essential for oxygenic photosynthetic organisms that are often prone to oxidative stress. Free Chl, heme and their immediate precursors are highly photo-toxic molecules and generate reactive oxygen species (ROS) under aerobic conditions 26 . Hence most of the cellular Chls are usually bound to the light harvesting complex (LHC) and other photosystem (PS) proteins. Chl is made in the plastid. Most of these Chl binding proteins and enzymes of the tetrapyrrole biosynthetic pathways are encoded by the nuclear genes 5 . Hence a tight coordination of biosynthesis of Chl with its apoprotein is necessary 27 . Chl and heme biosynthesis in plants is under transcriptional, translational and post-translational control at multi level and is accomplished by a complex regulatory network among the chloroplasts, mitochondria and nucleus, that is not well understood [28][29][30] .

Changes from Version 1
We have incorporated some of the suggestions of the reviewers in our revised manuscript. We have changed the title of the paper and have revised the name of our gun4 mutant. It is now named as gun4-II. We have cited the earlier identified Chlamydomonas gun4 mutant in our manuscript text as gun4-I. We have revised the first few sentences in the abstract. We are unable to obtain the gun4-I mutant and hence the comparative physiological experiment suggested by the reviewer, Dr. Jin, cannot be performed and is beyond the scope of our research. We have added two future biochemical experiments that can be performed on the gun4-II mutant. We have accommodated the suggestion of Dr. Yokthongwattana of categorizing some figures as supplementary figures. We have renamed Figure 6- Figure 10 as Figure S1- Figure S5, respectively. The numbering of Figure 11- Figure 17 has changed. We would like to keep the info on HYP1 and HYP2 as it is, in the manuscript. As we currently don't know the exact insertion point of the pUC ori end of the pBC1 vector in the gun4-II genome, we have used HYP1 and HYP2 as marker genes to clarify the extent of deletion/genetic rearrangement surrounding the GUN4 mutation locus. The gene and protein sequences of HYP1 and HYP2 are available on the Phytozome database. The functions of these genes are unknown. Figure 1. A simplified tetrapyrrole biosynthetic pathway. Light regulated steps are in red. Dashed arrows denote multiple enzymatic steps and green arrows point to steps that are positively regulated by the GUN4 protein, respectively. Tetrapyrrole intermediates and enzymes are shown in black and bold black type, respectively. Readers are advised to look in the text for full names of tetrapyrrole intermediates and enzymes, which are abbreviated in this figure.

See referee reports
One of the major research interests of our laboratory is to identify components that play a role in the regulation of Chl biosynthesis under different irradiance conditions. We have generated a random DNA insertional Chlamydomonas mutant library and have screened it to isolate twenty one mutants that are either defective in Chl biosynthesis and/or are incapable of photo-autotrophic growth under different irradiance conditions. One of the isolated mutants (6F14) is a light sensitive mutant which shows a light intensity dependent progressive photo-bleaching and is incapable of photosynthesis under low light intensities (90-100 µmol m -2 s -1 ). Molecular analyses revealed that 6F14 is defective in the GUN4 (genome uncoupled 4) gene which codes for a protein that stimulates MgChel activity. 6F14 is the second gun4 mutant (gun4-II) to be identified in Chlamydomonas 31 . Transformation of 6F14 with a functional copy of the GUN4 gene restored the wild type phenotype. Western analyses show that the two isolated gun4-II complements are over-expressing the GUN4 protein.
Chl analyses show that these gun4-II complements have 50-60% more Chl than that of the wild type strain. In this study, we present our molecular data on the identification of the mutation locus in 6F14 and its complementation.

Algal media and cultures
Chlamydomonas strains 4A+ (a gift from Dr. Krishna Niyogi (UC, Berkeley), gun4-II and gun4-II complements (both generated by our laboratory) were grown either in Tris-Acetate Phosphate (TAP) heterotrophic media or in Sueoka's High Salt (HS) photo-autotrophic media. TAP and HS liquid media and agar plates were prepared in the lab using reagents from Fisher Scientific (Pittsburgh, PA) according to the protocol given in Gorman and Levine (1965) 32 and Sueoka (1960) 33 , respectively. The 4A+ strain and gun4-II complements were maintained on TAP agar plates and TAP + zeocin (Sigma, St. Louis, MO) plates, respectively under dim light intensities (10-15 µmol photons m -2 s -1 ) at 25°C. The final zeocin concentration was 15 µg/ml. The gun4-II mutant (6F14) was maintained in the dim light or in the dark on TAP 1.5% agar plates containing 10 µg/ml of paromomycin (Sigma, St. Louis, MO). Liquid algal cultures used for RNA and genomic DNA extractions and protein analyses were grown in 100 ml flasks on the New Brunswick Scientific Excella E5 platform shaker (Enfield, CT) in TAP media at 150 rpm in the dim light.
Generation of the 6F14 mutant The purified pBC1 plasmid from the DH5α Escherichia coli-pBC1 clone (obtained from Dr. Krishna Niyogi's laboratory at UC, Berkeley) was used for random DNA insertional mutagenesis. This plasmid contains two antibiotic resistance genes: APHVIII and Amp R (Figure 2). APHVIII confers resistance against the antibiotic paromomycin and was used as a selection marker for screening of Chlamydomonas transformants. Amp R was used as a selection marker for screening of E. coli clones harboring the pBC1 plasmid. E. coli was grown in 1 l of Luria Bertani (LB) broth containing 1% tryptone, 0.5% of yeast extract, 1% NaCl and ampicillin (final concentration of ampicillin:100 µg/ml). LB media was prepared in the laboratory using reagents purchased from Fisher (Pittsburgh, PA). Ampicillin was purchased from Fisher (Pittsburgh, PA). The culture was incubated at 37°C overnight. Plasmid purification from E. coli cells was facilitated by a Qiagen plasmid mega kit according to the protocol given in the technical manual (Qiagen, Valencia, CA). Once purified from E. coli, the circular pBC1 vector was linearized with the restriction enzyme KpnI (NEB, Beverly, MA) according to the protocol given in the technical manual. The linearized DNA was purified using a QIAEX II gel extraction kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. All agarose DNA gel electrophoresis was visualized by BioRad Molecular Imager Gel Doc XR+ (BioRad, Hercules, CA). Transformation of parental strain 4A+ by the linearized pBC1 vector was performed utilizing the glass bead transformation technique described by Kindle et al. (1989) 34 and Dent et al. (2005) 2 . Transformants were plated onto fresh TAP agar plates containing 10 µg/ml paromomycin (TAP+P) in the dark. Single colonies of mutants were picked and transferred onto fresh TAP+P plates using a numbered grid layout. Screening of photosynthetic and pigment deficient mutants was done by visual inspection and monitoring of growth under different light intensities in heterotrophic, mixotrophic and photo-autotrophic conditions 2 .
Genomic DNA and RNA extraction 4A+, gun4-II complements and gun4-II were grown in TAP liquid media in the dim light to a cell density of about 5 × 10 6 cells/ml of the culture. Genomic DNA was purified using a phenol-chloroform extraction method 35 . RNA extraction was facilitated by TRIzol reagent from Invitrogen (Carlsbad, CA) following the protocol in the technical manual. DNA and RNA concentrations were measured using a Nanodrop 1000 spectrophotometer from Thermo Fisher Scientific (Wilmington, DE). DNase treatment was performed using Ambion's TURBO DNA-free kit from Invitrogen (Carlsbad, CA) following the protocol in the technical manual to remove genomic DNA from the RNA preparation. Generation of cDNA was performed using Life Technologies Superscript III First-Strand Synthesis System from Invitrogen (Carlsbad, CA) following the protocol in the technical manual.  depending on the T m of the primers. Extension time was varied according to the size of the PCR product amplified. Final extension was set at 72°C for ten minutes. All genomic and reverse transcription PCR products were amplified for a total of thirty-five cycles.
A 50-150 ng sample of genomic DNA or cDNA were used for PCR reactions. For semi-quantitative RT-PCR reactions, 3 µg of total RNA was converted into cDNA and then 150 ng of cDNA templates were used for RT-PCR. Sequences of primers used for genomic and RT-PCR are shown in Table 2- Table 4.
Cloning of the GUN4 gene in the pDBle vector The pDBle vector (obtained from Dr. Saul Purton, University College London, UK) was double-digested with restriction enzymes EcoRI and NdeI (NEB, Beverly, MA) according to the protocol given in the technical manual. The GUN4 gene was amplified using primers given in Table 5. Ligation of the double digested (NdeI and EcoRI digested) GUN4 gene and the NdeI/EcoRI doubledigested pDBle vector was done using the T4 ligase and 1 mM ATP (NEB, Beverly, MA). Chemically competent (CaCl 2 treated) E. coli cells were used for transformation. After transformation, Table 2. List of GUN4 specific primers. These primers were used for GUN4 (Cre05.g246800) genomic DNA PCR on 6F14 and 4A+ and also for DNA sequencing to generate the data in Figure S3 and Figure S4.

Primer name Sequence of primer Location
5´-ATCACATTACGCAACAGTCCGGCT-3´Exon2 Table 1. List of primers used for TAIL (Thermal Asymmetric InterLaced) PCR, verification of TAIL PCR product and DNA sequencing. These primers were used to generate the data in Figure S2 and Figure S3. Single colonies were picked the next day and plasmids were isolated from these clones. Isolated plasmids were double-digested with EcoRI and NdeI to verify the cloning of the GUN4 gene. The GUN4-pDBle construct from the selected clone was sequenced by the UC, Berkeley DNA Sequencing Facility (Berkeley, CA). Chromas Lite (http://technelysium.com.au/) and BLAST were used to analyze DNA sequences.

Primer name Sequence of primer Location
Generation and screening of gun4 complements Complementation of the gun4-II was performed utilizing the glass bead transformation technique described by Kindle et al. 1989 34 . 2 µg of the linearized GUN4-pDBle was used to complement 6F14. Transformed cells were plated onto fresh TAP plates containing 15 µg/ml zeocin (Z) and placed in the dark at 25°C. Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potential gun4 complements. Screening of gun4-II complements was done by Table 3. List of primers used for checking the genomic region upstream of GUN4 (Cre05.g246800) and HYP2 [g5195] gene. These primers were used to generate the data in Figure S5 and Figure 6. HYP2   Table 4. List of primers used for transcript analysis of GUN4 and GUN4 neighboring genes in 6F14. These primers were used to generate the data in Figure 7. The gene loci numbers in Phytozome for the three neighboring genes of GUN4 on chromosome 5 and the control actin gene on chromosome 13 are:

Primer name Sequence of primer Location
and SOXE [Cre05.g246900] and Actin (Cre13.g603700), respectively. Table 5. List of primers used for cloning and complement testing. These primers were used in the experiments that generated the data in Figure 8 and Figure 11 and were also used for GUN4 gene amplification for cloning.

Western analysis
Electrophoretic transfer of the SDS-PAGE resolved proteins onto an Immobilon P-PVDF membrane (Millipore, Billerica, MA) was carried out for 2 hours at a constant current of 400 mA in the transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). The GUN4 polyclonal antibody was raised in rabbit against the full length Chlamydomonas GUN4 mature protein that lacks the first 45 amino acids corresponding to the predicted chloroplast transit peptide 31 . This antibody was generated by Dr. Roberto Bassi's laboratory (University of Verona, Italy) and was provided to us by Dr. Krishna Niyogi (UC, Berkeley). GUN4 primary antibodies were diluted to a ratio of 1:1000 before being used as a primary probe. The secondary antibodies used for Western blotting were conjugated to horseradish peroxidase (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL) and diluted to a ratio of 1:20,000 with the antibody buffer. Western blots were developed by using the Supersignal West Pico chemiluminescent substrate kit (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL).

Cell counts and chlorophyll extraction
Cell density (number of cells per ml of the culture) was calculated by counting the cells using a Neubauer ultraplane hemacytometer (Hausser Scientific, Horsham, PA). Pigments from intact cells were extracted in 80% acetone and cell debris was removed by centrifugation at 10,000 g for 5 minutes. The absorbance of the supernatant was measured with a Beckman Coulter DU 730 Life Science UV/Vis spectrophotometer (Brea, CA). Chl a and b concentrations were determined by Arnon (1949) 38 equations, with corrections as described by Melis et al. (1987) 39 .

Results
Generation and identification of the mutant 6F14 Mutant 6F14 was generated by random insertional mutagenesis of the C. reinhardtii wild type strain 4A+ (137c genetic background).
6F14 was identified as a slightly Chl deficient paromomycin resistant mutant on TAP+P plate in the dark (Figure 3).

Growth analyses of 6F14
Growth analyses in heterotrophic and photo-autotrophic liquid media revealed that 6F14 is light sensitive and shows progressive photo-bleaching with increase in light intensities (Figure 4 and      2.68*10 6 , 3.32*10 -6 2.60*10 6 , 5.56*10 -7 together, the results shown in Figure 4 and Figure S1 show that dark adapted 6F14 is more sensitive to the magnitude of light intensity changes in the environment than the dim light adapted 6F14 ( Figure S1).

Molecular characterization of the mutation in 6F14
The linearized pBC1 plasmid was used to generate 6F14 (Figure 2). To find the insertion of the APHVIII end of the plasmid in 6F14, TAIL PCR method was employed. Figure S2A shows the position of the vector specific TAIL PCR primers and also shows the arbitrary position of the random degenerate primer. A 2.9 kb DNA product from TAIL2 PCR was purified from the agarose gel ( Figure S2B, Table 1). This purified DNA product was used for further PCR using internal primers specific to the 3′ UnTranslated Region (UTR) of the APHVIII gene. The PCR results confirmed that the 2.9 kb DNA product contains the 3′ UTR of the APHVIII gene ( Figure S2C). Sequencing of the 2.9 kb TAIL2 PCR product revealed that the APHVIII end of the plasmid has been inserted 344 bp away from the GUN4 gene (Cre05.g246800) on chromosome 5. The GUN4 locus was cleaved at least at two places ( Figure S3). The first cleavage was about 781 bp away from the 5′ end of the GUN4 gene and the second cleavage was 1131 bp away from the 3′ end of the GUN4 gene. These cleavages were followed by the inversion of the cleaved genomic DNA which then ligated to the 3′ UTR of the GUN4 gene ( Figure S3). Plasmid insertion also led to an addition of 29 bp at the APHVIII end of the plasmid. An addition of 45 bp was found at the breakage point in the 3′ UTR of the GUN4 gene ( Figure S3).
Further genomic DNA PCR analyses with GUN4 specific primers confirmed that the 3′ part of the GUN4 first exon and the 5′ part of the GUN4 second exon were deleted or displaced ( Figure S4). We also used primers specific to the genomic region upstream of the GUN4 gene and primers specific to the 3′ UTR of a hypothetical gene, HYP2, (g5195) located downstream of GUN4 to see the extent of deletion on either side of the GUN4 gene. Our PCR analyses show that a 1.354 kb genomic DNA region, located upstream of GUN4 was deleted/displaced. Additionally, there is a deletion of approximately 526 bp in the 3′ UTR of the downstream HYP2 gene ( Figure S5 and Figure 6). Taken together the data show that plasmid insertion in the 6F14 genome has rearranged the GUN4 locus and has affected a part of the 3′ UTR of the HYP2 gene. We do not yet know the exact location of the pUC ori end of the plasmid in the 6F14 genome ( Figure 2).
Checking for the absence/presence of the transcript of the GUN4 and three neighboring genes of GUN4 Transcript levels of GUN4 and the neighboring genes (HYP1 [Cre05. g246750]; HYP2 [g5195] and SOXE [Cre05.g246900]) were checked using semi-quantitative RT-PCR using GUN4, HYP1, HYP2 and SOXE specific primers, respectively (Figure 7). Reduced levels of HYP1 and HYP2 transcripts were observed in 6F14 compared to TAA that in the wild type (Figure 7). GUN4 transcript is missing in 6F14 as expected (Figure 7). The transcript level of SOXE, the second gene downstream of GUN4, was not affected. Cre05.g246750 and g5195 are genes in the Chlamydomonas database coding for hypothetical proteins. We have named these genes as HYP1 and HYP2 arbitrarily for our study. The SOXE gene codes for sulfocyanin, a blue copper protein. Readers are requested to identify GUN4 and its neighboring genes by the gene locus number (Cre or the g number) in the Phytozome database.

Complementation of gun4-II
We will be referring to 6F14 as gun4-II from here onward. As gun4 specifically lacks a functional GUN4 gene, we cloned the GUN4 gene in the pDBle vector to transform 6F14 (Figure 8, Table 5). The trans GUN4 expression is driven by the constitutive PsaD promoter in the GUN4-pDBle construct. pDBle has two Ble genes that confer resistance to the antibiotic zeocin. Figure 9 shows growth phenotypes of two gun4-II complements (gun4-19 and gun4-27), 6F14 and 4A+. gun4-II complements are not light sensitive and are able to grow and photosynthesize under medium light intensities (300 µmol photons m -2 s -1 ) without photo-bleaching ( Figure 9). As gun4-II complements harbor the Ble gene (from the pDBle vector) and APHVIII gene (derived from the parental strain gun4-II), they can grow both on zeocin and paromomycin media plates unlike gun4-II and 4A+ (Figure 9).
Chl analyses show that under heterotrophic conditions both gun4-II complements have 65-68% more Chl than that of the wild type cells ( Figure 10). Under photo-autotrophic conditions gun4-II complement cells possess 50-60% more Chl than that of the wild type cells (Figure 10). Figure 11A shows a schematic figure of the trans GUN4 gene used for complementation. PCR analyses      Table 5.

A B
* 184 bp of the second exon of the GUN4 gene is deleted. In gun4-II the plasmid insertion outside the GUN4 gene has caused a genetic rearrangement of the GUN4 gene that prevented gene expression ( Figure 6). Transcripts of GUN4 and the neighboring genes of GUN4 in gun4-II were checked by performing semi-quantitative reverse transcription PCR. In gun4-II, the transcript level of the first downstream hypothetical (HYP2) gene was lower than that in the wild type (Figure 7). The plasmid insertion in gun4-II has led to a deletion of part of the 3′ UTR region of the HYP2 gene (270 bp away from the stop codon of the coding region of HYP2; Figure 6). The 3′ UTR is usually responsible for the stability of the transcript. Hence the nature of the deletion in HYP2 explains the decrease in transcript levels of HYP2. GUN4 and the upstream gene, HYP1, are separated from each other by 2.784 kb ( Figure S5). There is a possible deletion/genetic rearrangement in the 5′ genomic region upstream of the GUN4 which does not extend into the HYP1 gene ( Figure S5 and Figure 6). Although the transcription of HYP1 was not hampered, the HYP1 transcript level is lower in our gun4 compared to that in the wild type (Figure 7). Based on the RT-PCR analyses, it is speculated there might be some uncharacterized downstream regulatory sequences present in the 2.784 kb region that might regulate HYP1 transcription. In future, quantitative real time-PCR experiments can be used to accurately quantify transcript levels of HYP1 and HYP2 in gun4-II.
The photosensitive phenotype of our gun4-II mutant resembles that of the earlier identified Chlamydomonas gun4 mutant which we will refer from here onward, as gun4-I. Over-accumulation of photoexcitable PPIX leads to photo-oxidative damage to the cells in presence of light and oxygen 4,26,53 . The light sensitivity of gun4-II is most probably due to an over-accumulation of the PPIX which occurs due to the inactivity of MgChel enzyme as has been shown by Formighieri et al. (2012) 31 in the gun4-I mutant. Future HPLC (High Performance Liquid Chromatography) analyses of steady state tetrapyrrole intermediates in gun4-II will confirm this hypothesis. Formighieri et al. (2012) 31 explored four light conditions (dark, 6-, 50-, and 500 µmol photons m -2 s -1 ) and showed that the gun4-I Chlamydomonas mutant dies under high light (500 µmol photons m -2 s -1 ). These researchers did not explore or clarify the maximum light irradiance condition that can be tolerated by the Chlamydomonas gun4-I mutant in heterotrophic and photosynthetic growth conditions. In this study, we found that gun4-II photo-bleached at 75-80 µmol photons m -2 s -1 and could not tolerate light intensity above 100 µmol photons m -2 s -1 (Figure 4 and Figure 5). The earlier identified C. reinhardtii gun4-I mutant is able to grow in continuous light slightly better than in photoperiodic shifts 31 . In Arabidopsis, the gun4 mutant is seen to exhibit significant improved growth in continuous light compared to periodic shifts in light 30 . In this study, gun4-II and the wild type were adapted to dark or dim light and then shifted to two different light irradiances (40-50 µmol photons m -2 s -1 and 75-80 µmol photons m -2 s -1 ). Cultures exposed to light shifts showed a significant reduction in the Chl content than those grown under a constant light intensity (Figure 4 and Figure S1). Additionally, dark adapted gun4-II showed a significant reduction in the Chl content compared to the dim light adapted gun4-II, when cells were shifted to similar light intensities ( Figure 6). These results show that gun4-II is very sensitive to the magnitude of light intensity fluctuations in the environment unlike the earlier reported using the genomic DNA show that the gun4-II complements possess the functional trans GUN4 gene ( Figure 11B, 11C and 11D). Figure 12A shows a stained protein gel that was loaded on equal Chl basis. Western analyses of the two gun4-II complements with a Chlamydomonas GUN4 specific antibody show that the GUN4 protein is absent in the gun4-II mutant but present in the gun4 complements ( Figure 12B). Western analyses also show that the two gun4-II complements have higher levels of the GUN4 protein compared to that of the wild type ( Figure 12B).  30,46,47 . The porphyrin binding property of GUN4 has been implicated in ROS attenuation but conclusive experimental support is lacking 47 . In higher plants, GUN4 has been implicated as an essential component in a post-translational feedback regulation mechanism that modulates ALA biosynthesis in response to enzymatic activities of the Mg branch of tetrapyrrole biosynthesis as well as to the accumulating Mg porphyrin levels 30 (Figure 1).

Spectrophotometric chlorophyll analyses in
6F14 is the second gun4 mutant (gun4-II) to be identified in C. reinhardtii. The first C. reinhardtii gun4 mutant was identified and characterized in 2012 by Formighieri et al. 31 . In this gun4 mutant, Chlamydomonas gun4-I mutant 31 . Our light shift experimental results support the findings in cyanobacterial and Arabidopsis gun4 mutants 30,[48][49][50] .
By spectrophotometric analysis we have shown that in the dark gun4-II possesses almost similar Chl content like that in the wild type ( Figure 4). This phenotype is very different from that of gun4-I, which possesses 50% of the wild type level of Chl/cell 31 in the dark. Variation in Chl/cell in the dark between the two C. reinhardtii gun4 mutants could possibly be due to a variation of the parental strain's ability to synthesize Chl in the dark. The parental strain used by Formighieri et al. (2012) 31 was cw15mt-. However, the 50% decrease in Chl seen in the gun4-I mutant was determined through HPLC analyses. Hence the discrepancy in Chl content in the two gun4 mutants could be due to the sensitivity of the HPLC method compared to that of the spectrophotometric method used for Chl assays.  Figure 12B). These two gun4-II complements open up new avenues to test if GUN4 has a distinct photoprotective role that is independent from the PPIX-induced GUN4 photo-protective role proposed by several researchers 46,47 . Comparative growth studies, quantitative measurements of GUN4 transcripts by Real Time PCR, GUN4 protein levels by Western analyses and PPIX content by HPLC analyses of the high light-(500 µmol photons m -2 s -1 ) and dim light-(15-20 µmol photons m -2 s -1 ) adapted gun4-II complements and the wild type strain will help to confirm if GUN4 has a distinct photo-protective role that is independent of tetrapyrrole metabolism. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Figure S1. Effect of light shift on the growth of 6F14 and wild type. 6F14 was adapted to dim light (10-15 µmol photons m -2 s -1 ) or dark for one week in TAP media. Dark and dim light adapted cultures were then shifted to 40-50 or 75-80 µmol photons m -2 s -1 . The mean cell density (cells/ml) and the Chlorophyll (Chl) content (nmol Chl per cell) are shown below the culture flasks in red and black numbers, respectively. For each light condition, experiments were performed on three biological replicates of 6F14. Statistical error (±SD) was ≤ 10%. The average Chl content in the dim light and dark adapted 6F14 was 1.7 × 10 -6 and 2.18 × 10 -6 nmol/cell. Primers used for PCR and DNA sequencing are shown by numbered black arrows. Thermal Asymmetric InterLaced1 (TAIL1) PCR was performed using primers 4R and RD1 (a random degenerate primer). (B) TAIL2 PCR was performed using primers 3R and RD1. In lane 1, 10-fold diluted TAIL1 PCR product was used for TAIL2 PCR; Lane 2 is a zero DNA control lane. The 2.9 kb TAIL2 PCR product used for DNA sequencing is highlighted in the red box. Initial DNA sequencing was performed using vector specific primers 2R and 3R (Table 1). (C) Gel purified DNA product (2.9 kb) from TAIL2 PCR was used to verify if the product is specific to the APHVIII gene. PCR primer names are labeled on the top of the gel. PCR product size is labeled. F and R stand for forward and reverse primers, respectively. All primer sequences are shown in Table 1  The genomic DNA sequence obtained by sequencing the 2.9 kb Thermal Asymmetric InterLaced2 (TAIL2) PCR product is highlighted in red. The bold black small arrow indicates insertion point of the pBC1 plasmid. DNA sequencing was performed using GUN4 specific primers 2R, 7F and 7R, 12R and 14R. F and R stand for forward and reverse primers, respectively. Primer sequences are shown in Table 2. DNA gels showing DNA products obtained from genomic DNA PCR using GUN4 specific primers. Lanes 1, 2 and 3 are the zero DNA controls, 6F14 and 4A+, respectively. PCR primer names are labeled on the top of the gel. 14F/3R gives a 517 bp product while 3F/8R gives a 942 bp product. 8F/7R gives a 449 bp product. 7F/12R gives a 313 bp product. 12F/10R gives a 606 bp product and 12F/11R gives a 623 bp product. F and R stand for forward and reverse primers, respectively. Primer sequences are shown in Table 2.  Table 3.

Supplementary materials
they would confuse the reader.

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