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.

The purified pBC1plasmid 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 .

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.

TAIL (Thermal Asymmetric InterLaced) PCR was implemented, following the protocol of Dent et al. (2005) ² . HotStar Taq Plus DNA polymerase kit reagents (Qiagen, Valencia, CA) were used for PCR. The PCR reaction mixture consisted of 1 × PCR buffer, 200 µM of each dNTP, 1 × Q-solution, 2.5 units of HotStar Taq Plus DNA polymerase, 60 pmoles of the random degenerate primer RD1 and 5 pmol of the APHVIII specific primer. Primers were ordered from IDT (Skokie, IL; Table 1). Degenerate primer RD1 has an average T _m of 51°C while the three APHVIII specific primers used had T _m ranging from 58°C to 64°C. PCR cycling programs were created using the program given in Dent et al. (2005) ² . TAIL1 PCR product was diluted 10-fold and 2 µl of the diluted TAIL1 PCR product was used for TAIL2 PCR reactions. The TAIL2 PCR product was gel purified using a QIAEX II gel extraction kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. Purified TAIL2 PCR product was sequenced at the UC, Berkeley DNA Sequencing Facility (Berkeley, CA). All primer sequences are shown in Table 1.

Primers were designed based on genomic DNA sequences available in the Chlamydomonas genome database in Phytozome. Amplifications of genomic DNA and cDNA were executed using the MJ Research PTC-200 Peltier Thermal Cycler (Watertown, MA). HotStar Taq Plus DNA polymerase kit (Qiagen, Valencia, CA) was used for PCR following the cycling conditions given in the Qiagen protocol booklet. Annealing temperature was between 55 and 60°C 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.

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 double-digested 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, E. coli cells were plated on LB+ampicillin (final concentration of ampicillin:100 µg/ml) plates and incubated at 37°C overnight. 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.

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 monitoring the Chl content and growth of complement strains either on TAP or HS plates under medium light (300 µmol photons m ^-2 s ^-1) in the presence or absence of antibiotics zeocin and paromomycin.

Chlamydomonas cells from different strains grown in TAP in the dim light were harvested, washed twice with fresh medium and resuspended in TEN buffer (10 mM Tris-HCl, 10 mM EDTA and 150 mM NaCl; pH 8). Gel lanes were loaded with an equal amount of Chl (4 µg Chl). Resuspended cell suspension was mixed in a 1:1 ratio with the sample solubilization buffer SDS-urea buffer (150 mM Tris-HCl, pH 6.8; 7% w/v SDS; 10% w/v glycerol; 2 M urea; bromophenol blue and 10% β-mercaptoethanol) and were incubated at room temperature for about thirty minutes, with intermittent vortexing. The sample solubilization buffer was prepared according to the protocol of Smith et al. (1990) ³⁶ using reagents from Fisher (Pittsburgh, PA). After incubation, the solubilized protein samples were vortexed and spun at a maximum speed of 20,000 g in a 1.5 ml eppendorf tube (USA Scientific, Ocala, FL) for five minutes at 4°C. The soluble fraction was loaded on a “any kD™ Mini-PROTEAN ^® TGX™ Precast Gel” (BioRad, Hercules, CA) and SDS-PAGE analysis was performed according to Laemmli (1970) ³⁷ using a Page Ruler prestained molecular weight protein ladder (Fermentas, Glen Burnie, Maryland) at a constant current of 80 V for 2 hours. Gels were stained with colloidal Coomassie Gel Code blue stain reagent (Thermo Fisher Scientific, Rockford, IL) for protein visualization.

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 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 .

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 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 Figure 5). In mixotrophic conditions under 10–15 µmol photons m ^-2 s ^-1, 6F14 possesses 58% less Chl/cell than 4A+. At 40–50 µmol photons m ^-2 s ^-1, 6F14 has 72% less Chl/cell than the wild type. At 75–80 µmol photons m ^-2 s ^-1, 6F14 possesses 99% less Chl/cell than the wild type. At 75–80 µmol photons m ^-2 s ^-1, 6F14 starts to photo-bleach and turns yellow; it dies at light intensities 100–120 µmol photons m ^-2 s ^-1 in TAP ( Figure 4).

Figure 5 shows photo-autotrophic cultures of 6F14 and 4A+. 6F14 has the ability to grow photo-autotrophically in HS media in dim light (10–15 µmol of photons m ^-2 s ^-1). However, the mutant grows extremely slowly in comparison to the wild type. When grown at 10–15 µmol photons m ^-2 s ^-1 in HS media, 6F14 possesses 60% less Chl/cell than the wild type. At 40–50 µmol photons m ^-2 s ^-1 in HS media, 6F14 has 79% less Chl/cell than 4A+, and at 75–80 µmol photons m ^-2 s ^-1 in HS media, 6F14 possesses 83% less Chl/cell than the wild type. At 100–120 µmol photons m ^-2 s ^-1 in HS media, 6F14 fails to survive ( Figure 5).

Figure S1 demonstrates that when dim light adapted 6F14 was shifted to 40–50 µmol photons m ^-2 s ^-1 there was no significant change in Chl/cell content ( Figure 4). Dark adapted 6F14 showed a 50% reduction in Chl/cell when moved to 40–50 µmol photons m ^-2 s ^-1. When dim light adapted 6F14 was shifted to 75–80 µmol photons m ^-2 s ^-1, it showed a 98% reduction in Chl/cell while the dark adapted 6F14 failed to survive under 75–80 µmol photons m ^-2 s ^-1. Taken 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).

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).

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 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.

We will be referring to 6F14 as gun4-II from here onward. As gun4-II 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 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).