The chloroplast metalloproteases VAR2 and EGY1 act synergistically to regulate chloroplast development in Arabidopsis

Chloroplast development and photosynthesis require the proper assembly and turnover of photosynthetic protein complexes. Chloroplasts harbor a repertoire of proteases to facilitate proteostasis and development. We have previously used an Arabidopsis leaf variegation mutant, yellow variegated2 (var2), defective in thylakoid FtsH protease complexes, as a tool to dissect the genetic regulation of chloroplast development. Here, we report a new genetic enhancer mutant of var2, enhancer of variegation3–1 (evr3–1). We confirm that EVR3 encodes a chloroplast metalloprotease, reported previously as ethylene-dependent gravitropism-deficient and yellow-green1 (EGY1)/ammonium overly sensitive1 (AMOS1). We observed that mutations in EVR3/EGY1/AMOS1 cause more severe leaf variegation in var2–5 and synthetic lethality in var2–4. Using a modified blue-native PAGE system, we reveal abnormal accumulations of photosystem I, photosystem II, and light-harvesting antenna complexes in EVR3/EGY1/AMOS1 mutants. Moreover, we discover distinct roles of VAR2 and EVR3/EGY1/AMOS1 in the turnover of photosystem II reaction center under high light stress. In summary, our findings indicate that two chloroplast metalloproteases, VAR2/AtFtsH2 and EVR3/EGY1/AMOS1, function coordinately to regulate chloroplast development and reveal new roles of EVR3/EGY1/AMOS1 in regulating chloroplast proteostasis in Arabidopsis.

Chloroplasts are semi-autonomous organelles that originated from ancient prokaryotic cyanobacteria through endosymbiosis (1). During endosymbiosis, the majority of chloroplast genes were transferred to the nuclear genome, giving rise to contemporary chloroplast genomes with only ϳ100 proteincoding genes (2). The separation of genetic information responsible for the ϳ3000 chloroplast-localized proteins necessitates that proteins encoded by the nuclear genome must be synthe-sized in the cytosol, imported into chloroplasts, and assembled with chloroplast genome-encoded subunits to form functional multi-subunit photosynthetic complexes, such as photosystem II (PSII) 3 and photosystem I (PSI) (3). The coordinated expression of the two genomes is regulated at multiple levels, including transcriptional, translational, and posttranslational levels, to maintain the proper stoichiometry between protein subunits encoded by the two genomes (4,5). The proteome of chloroplasts is also strikingly dynamic in response to diverse developmental signals and environmental cues (6). Genetic dissections of chloroplast development using leaf coloration as the phenotypic readout have been proven to be extremely fruitful as a spectrum of leaf color mutants ranging from albino, yellow, pale green, virescent, and variegation can be readily identified in genetic screens (7). These mutants serve as splendid genetic resources in elucidating the regulation of chloroplast development by nuclear-encoded chloroplast proteins. However, how these factors coordinate genetically to regulate chloroplast development remains largely unexplored.
Mutations in chloroplast proteases often result in arrested or delayed chloroplast development, highlighting the importance of proteostasis in chloroplasts (8,9). One of the most intriguing chloroplast proteolytic systems is the thylakoid FtsH complex because of the unique leaf variegation phenotype of Arabidopsis yellow variegated2 (var2) and var1 mutants, defective in thylakoid FtsH complex components VAR2/AtFtsH2 and VAR1/ AtFtsH5, respectively (10,11). In Arabidopsis, thylakoid FtsH complexes are heterohexamers comprised of both type A (AtFtsH1 and VAR1/AtFtsH5) and type B (VAR2/AtFtsH2 and AtFtsH8) subunits, based on their functional redundancy and interchangeability (12,13). In photosynthetic organisms, thylakoid FtsH complexes take part in the PSII repair cycle, particularly the turnover process of D1, the reaction center subunit of PSII (14 -18). The absence of thylakoid FtsH complexes and Deg protease in Arabidopsis leads to inefficient degradation of D1 protein under photoinhibition conditions induced by high light (19 -21). Moreover, thylakoid FtsH complexes are also essential for chloroplast development as the complete loss of either type A or type B FtsH subunits cause lethality (12,13). In addition, the presence of undifferentiated plastids in white sec-tors in var2 also suggests that thylakoid FtsH complexes are involved in thylakoid biogenesis and chloroplast development (22).
To dissect the genetic mechanisms underlying leaf variegation and the regulation of chloroplast development, several research groups have taken advantage of the var2 leaf variegation phenotype and isolated an increasing number of var2 genetic suppressors, which reverse the white sector and variegation phenotype of var2 mutants via extragenic mutations (23)(24)(25)(26)(27). We have identified the SUPPRESSORS OF VARIEGA-TION (SVRs) loci, which encode many components involved in chloroplast translation and gene expression (24,(28)(29)(30)(31)(32)(33)(34). The disruptions of these SVR genes cause a reduction in plastid gene expression and translation and are sufficient for the suppression of variegation phenotypes, thus establishing strong genetic and functional relationships between thylakoid FtsH complexes and plastid gene expression. The identification of a large number of var2 genetic suppressor loci is consistent with the essential nature of thylakoid FtsH complexes, and indicates that VAR2/AtFtsH2 may represent a highly connected genetic network hub (35).
To further explore the functional interaction network of VAR2/AtFtsH2, we systematically screened for var2 genetic enhancer loci, termed ENHANCERS OF VARIEGATION (EVRs). Recently, we showed that mutations in EVR1/RPS21b, which encodes a cytosolic 40S ribosomal protein RPS21, and reduced activities of cytosolic translation enhance var2 leaf variegation, revealing that the balance between cytosolic and chloroplast translation regulates VAR2-mediated chloroplast development (36). Here we report the identification of a new EVR locus, EVR3. Molecular cloning and complementation confirmed that loss-of-function mutations in EVR3 greatly enhance var2 leaf variegation, and EVR3 encodes a chloroplast metalloprotease which was previously reported as ethylene-dependent gravitropism-deficient and yellow-green1 (EGY1) and ammonium overly sensitive1 (AMOS1) (37,38). In addition, we uncovered previously unknown defects in PSI and PSII supercomplex assembly in evr3/egy1/amos1 mutants. Moreover, we discovered that the PSII stability, particularly D1 stability, under high light is significantly compromised in evr3-1 and further worsened in var2-5 evr3-1 double mutant. Our findings establish that VAR2/AtFtsH2 and EVR3/EGY1/AMOS1 coordinate to regulate PSII stability and chloroplast development.

Isolation of a var2-5 genetic enhancer mutant, evr3-1
To unravel the genetic regulatory network of chloroplast development, we took advantage of the leaf variegation phenotype of the var2 mutant and systematically isolated var2 extra-
Finally, we tested the genetic interaction between evr3-1 and var2-4, a likely null allele of var2 (10,12). Interestingly, we identified albino plants that have the var2-4 evr3-1 genotype in the F2 progeny of a cross between evr3-1 and var2-4, but these seedlings do not survive the cotyledon stage (Fig. 1D). Albino plants segregated from the same F2 progeny grown on sucrose-containing medium could develop a few true leaves but were not autotrophic (Fig. S1B). PCR-based genotyping confirmed that these white seedlings were var2-4 evr3-1 double mutants (Fig. S1C). The synthetic lethality observed in var2-4 evr3-1 double mutant, indicated that VAR2 and EVR3 act synergistically to promote chloroplast development and together VAR2 and EVR3 gene activities are essential for establishing phototrophic growth under our growth conditions.

EVR3 is EGY1/AMOS1
To uncover the molecular lesion in evr3-1, a whole genome resequencing strategy was adopted using pooled genomic DNAs of evr3-1 seedlings from a segregating F2 population of a backcross between evr3-1 and WT. A G to A point mutation that would cause a Gly432Glu missense mutation in the protein coded by At5g35220 was identified in evr3-1 (Fig. 2, A and B). At5g35220 was previously reported as EGY1/AMOS1, encoding a chloroplast thylakoid membrane-localized S2P-like metalloprotease (37,38). EGY1/AMOS1 contains a conserved zincbinding motif HEXXH localized between putative TM2 and TM3, and a NPDG motif localized between putative TM6 and TM7 (Fig. S1, D and E) (39). These two motifs are required for the proteolytic activity of S2P in animals and S2P-like homologues in plants (40). Evolutionarily, EGY1/AMOS1 homologues are present in most photosynthetic organisms (Fig. S1, D and E). The mutated Gly 432 in evr3-1 is conserved in photosynthetic organisms and is localized next to the NPDG motif (Fig.  S1E).
More importantly, expression of WT EGY1/AMOS1 under the control of the Cauliflower Mosaic Virus 35S promoter promoter (p35S:EGY1) in evr3-1 single mutant yielded multiple

EVR3/EGY1/AMOS1 is required for the accumulation of antenna proteins during de-etiolation
As a chloroplast protease, EGY1 is expected to have a role in chloroplast proteostasis (37). We noticed that the abundance of antenna proteins, such as LhcA1, LhcA2, and LhcB2 was reduced in evr3-1 (Fig. 1C). This is in agreement with previous report that the disruption of EGY1 leads to significantly decreased levels of LHCI and LHCII antenna proteins (37,41). In contrast, steady state levels of FtsH or cytochrome b 6 f complex subunits were unaffected in evr3-1 compared with those in the WT (Fig. 1C). These observations suggest a specific role of EVR3/EGY1/AMOS1 on the accumulation of antenna proteins. To test if EGY1/AMOS1 is involved in the accumulation of antenna proteins, we utilized a de-etiolation system, which could provide a clear starting time point for the accumulation of photosynthetic proteins. Etiolated seedlings were transferred to light, and the amount of photosynthetic proteins was monitored and quantified during greening. We found that the rate and extent of increase in most photosynthetic subunits, including nuclear genome-encoded subunits such as PetC, RbcS, and VAR2, and also plastid genome-encoded subunits such as D1 and Cytf were similar in WT and evr3-1 during de-etiolation (Fig. 3, A and B). In contrast, LHCI and LHCII antenna proteins, such as LhcA2 and LhcB2, were readily detectable in WT but remained undetectable in evr3-1 after transfer to light for 3 h. 6 h after transfer to light, high levels of antenna proteins accumulated in WT whereas greatly reduced amounts of LhcA2 and LhcB2 were detected in evr3-1 (Fig. 3, A and B). These data uncover a critical role of EGY1 for the efficient accumulation of antenna proteins in response to light, a process critical for germinating seedlings to establish photosynthesis and autotrophic growth.

Loss of EVR3/EGY1/AMOS1 leads to abnormal accumulation of photosystem I complexes
To explore the role of EVR3/EGY1/AMOS1 in chloroplast proteostasis, we probed the status of thylakoid photosynthetic complexes in WT and evr3-1 using blue-native PAGE (BN-PAGE). First, we utilized the classic n-dodecyl-␤-D-maltoside solubilization method and observed a conspicuous reduction of LHCII antenna trimers in evr3-1 (Fig. S4). Next, we employed a BN-PAGE solubilization method based on nonionic detergent digitonin, which can reveal additional protein complex information (42). Surprisingly, in addition to the reduced level of LHCII antenna trimers in evr3-1, the banding pattern of thylakoid photosynthetic complexes was clearly altered in evr3-1 compared with WT, with the most pronounced differences in the region of the 1-D BN-PAGE gel where three major PSI complexes, B1, B2, and B3, were found ( Fig. 4A) (42). B1, B2, and B3 correspond to PSI-LHCI-LHCII, PSI-LHCI, and PSI core, respectively (42). 1-D BN-PAGE showed that B1 was markedly reduced, B2 remained unchanged, and B3 was overaccumulated in evr3-1 compared with those in the WT (Fig. 4,  A and B). Identities of B1, B2, and B3 complexes were validated by immunoblot analyses of proteins extracted from their native gel bands using antibodies against PSI core subunits and antenna proteins (Fig. 4C). We detected PSI core subunits

EGY1 regulates chloroplast development and proteostasis
(PsaD, PsaC, and PsaF), LhcAs and LhcB2 in B1 complexes, and these proteins were less abundant in evr3-1 (Fig. 4, C and D). LhcB2 is absent in B2 complexes, consistent with its PSI-LHCI identity (Fig. 4, C and D). Overall accumulations of PSI core and LHCI proteins in B2 complexes were similar in WT and evr3-1 (Fig. 4, C and D). In the B3 complex, we observed markedly increased levels of PSI core subunits in evr3-1, in contrast to their low presence in WT (Fig. 4, C and D). Finally, to gain a more comprehensive picture of proteins in B1, B2, and B3 complexes, 2-D BN/SDS-PAGEs were performed. Immunoblots of the 2-D gels confirmed that PSI-LHCI-LHCII, i.e. the B1 complex, was reduced in evr3-1, although PSI core, i.e. the B3 complex, was more abundant in evr3-1 (Fig. 4E).
Importantly, the abnormal PSI complexes observed in evr3-1 were reversed to patterns similar to WT in complementation lines (Fig. 4F). Similar PSI defects were also observed in two other egy1 alleles, egy1-2 and egy1-3 (Fig. S5A). These findings indicate that the abnormal accumulation of PSI complexes in evr3-1 is a direct consequence of the lack of functional EVR3/EGY1/AMOS1. Together, these results establish that EVR3/EGY1/AMOS1 is required for the proper accumulation of PSI complexes.

Loss of EVR3/EGY1/AMOS1 leads to more photosystem II dimer and monomer accumulation
Using the digitonin solubilization BN-PAGE procedure, we next examined the status of various PSII complexes in WT, var2, and evr3-1. We observed no conspicuous differences in major PSII complexes between WT and var2 mutants, including both var2-4 and var2-5 (Fig. S6). This observation was further confirmed by silver staining of the 2-D BN/SDS-PAGE gels (Fig. S7). However, we noticed that migration patterns of CP47-containing PSII complexes were not identical in WT and evr3-1 in silver-stained 2-D BN/SDS-PAGE gels (Fig. S7), suggesting abnormalities in the accumulation of PSII complexes in evr3-1. To explore the abnormalities of PSII complex accumulation in evr3-1, we performed immunoblotting of BN-PAGE with antibodies against PSII core subunits D2 and CP47, and LHCII subunit LhcB2. We found that although the amount of PSII monomer LHC trimer complex was similar in WT and evr3-1, PSII dimer and PSII monomer were dramatically overaccumulated in evr3-1 compared with those in the WT (Fig. 5, A-C). Similar PSII defects were also found in egy1-2 and egy1-3 (Fig. S5B). These data revealed that EVR3/EGY1/ AMOS1 is required for the homeostasis of PSII complexes.

VAR2/AtFtsH2 and EVR3/EGY1/AMOS1 regulate PSII stability under high light
Previously, VAR2/AtFtsH2 has been shown to be involved in the repair cycle of PSII subunits, particularly D1 degradation under high light conditions (14 -16). Given the synergistic interaction between evr3-1 and var2, as well as the abnormal accumulations of PSI and PSII complexes in evr3-1, we reasoned that VAR2/AtFtsH2 and EVR3/EGY1/AMOS1 may both function in the PSII repair cycle to control the stability of PSII subunits under high light stress. To monitor the stability of major photosynthetic proteins under high light stress, WT and mutant leaf discs were infiltrated with lincomycin and cycloheximide to block both chloroplast and cytosol translation during high light treatment. After 0-, 2-, and 4-hour high light treatment, levels of representative subunits of major photosynthetic complexes, including PSII (D1, D2, CP47, and CP43), PSI (PsaD and PsaF), LHCI (LhcA1 and LhcA2), LHCII (LhcB2), and cytochrome b 6 f (Cytf), were analyzed with immunoblotting. Cytf, which has been reported to remain stable under high light stress, was included as a control to normalize the loading of immunoblots (43).
Upon high light treatment, although with varied rates, gradual degradations of PSII core subunits (D1, D2, CP47, and CP43) were observed in WT, var2-5, evr3-1, and var2-5 evr3-1 double mutant, whereas PSI subunits remained stable in all the genotypes during our highlight treatment (Fig. 6, A and  B). In var2-5, the degradation of PSII reaction center proteins D1 and D2 was partially blocked compared with WT (Fig. 6, A  and B). These observations are consistent with previous reports (15,16). Degradations of all four PSII core subunits examined were faster in evr3-1 than in WT (Fig. 6, A and B). In addition, antenna proteins, especially LHCI subunits, were less stable in evr3-1 compared with those in WT (Fig. 6, A and B). Interestingly, in var2-5 evr3-1 double mutants, D1 and D2 become less stable with degradation rates similar to those of evr3-1, indicating the blockage of D1 and D2 degradation caused by the var2-5 mutation was bypassed by the loss of EVR3/EGY1/ AMOS1 (Fig. 6, A and B). Our findings indicate that although VAR2 is necessary for the turnover of D1 and D2, EVR3/EGY1/ AMOS1 is required for stabilizing PSII core proteins. PSII repair cycle probably needs coordinated actions of VAR2/At-FtsH2 and EVR3/EGY1/AMOS1 under high light stress.

Discussion
Single gene-based molecular genetic analysis has served as the cornerstone of modern molecular biology. However, comprehensive genetic interactions and networks have to be established to tackle the genotype to phenotype, and ultimately, the genome to phenome question. The fascinating leaf variegation phenotype of the var2 mutant has long attracted geneticists' interest and enables genetic screens for both genetic suppressor and enhancers (23)(24)(25)(26)(27)36). Enhancer screens are powerful tools to reveal close functional relationships (44). In the case of

EGY1 regulates chloroplast development and proteostasis
VAR2/AtFtsH2, it is known that enhancement of chloroplast development defects can be generated by combining var2 mutations with mutations in FtsH subunit genes VAR1/At-FtsH5 and AtFtsH8 (12,13). In this work, we isolated a new var2 enhancer locus, EVR3. We confirmed that EVR3 is identical to the previously reported locus EGY1/AMOS1 (37,38). Initially identified based on pigmentation-deficient and defective in ethylene-stimulated gravitropic responses, EVR3/EGY1/  var2-3, var2-4, and evr3-1 were solubilized with 2% digitonin, resolved by BN-PAGE, and probed with antibodies against D2, CP47, and LhcB2. Positions of different PSII complexes were marked by arrows. PSII s.c., PSII supercomplex; PSII di, PSII dimer; PSII mono, PSII monomer; PSII mono LHCII tri, monomeric PSII core with LHCII trimer. B, quantifications of immunoblots shown in A. In each blot, signal intensity of the indicated protein band from the WT sample was defined as 100%. Data were presented as mean Ϯ S.D. of three blots obtained from independent biological replicates. ***, p Ͻ 0.001; one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test (WT versus mutant). C, schematic depiction of the PSII complexes accumulation defects in evr3-1 shown in A.
Our findings focus on the role of EGY1 in chloroplast. We show that the collective activities of EVR3/EGY1/AMOS1 and VAR2/ AtFtsH2 are essential for chloroplast development in Arabidopsis thaliana (Fig. 1), thus establish a previously unknown functional connection between these two metalloproteases.
Chloroplast thylakoid membrane protein complexes, including PSII, PSI, cytochrome b 6 f, and ATP synthase, catalyze the critical conversion of light energy to chemical energy (48). More importantly, photosynthetic protein complex assemblies are under dynamic regulation by numerous factors, responding to developmental and environmental inputs. For example, to optimize light energy harvest, LHCII trimers, the major light harvesting complexes of PSII, can disassociate from PSII and instead bind to PSI to balance the energy inputs between PSII and PSI, thus constituting the state transition regulation (49). Native gel electrophoresis analysis has been successfully used to probe the status of membrane protein complexes and a modified native gel system based on the mild detergent digitonin has been shown to reveal additional photosynthetic protein complex information (42). Using this system, our detailed native gel analyses revealed previously unreported PSI and PSII assembly defects in evr3/egy1 mutants (Figs. 4 and 5). First, we discovered that the accumulation of PSI-LHCI-LHCII (B1) complex was dramatically reduced in evr3-1 mutants (Fig. 4). This suggests that EGY1 is required for the association of LHCII with PSI under our growth conditions. Concurrent with this, the level of LHCII trimers were also greatly reduced in evr3-1 mutants, potentially leading to reduced availability of LHCII trimers for PSI. The abnormal accumulation of PSI core (B3) complex is puzzling, suggesting that EGY1 may also be necessary for the binding of LHCI to the PSI core complex to form PSI-LHCI (B2) complex (Fig. 4). In addition, PSII assembly is also perturbed in evr3-1 mutants and we discovered abnormal accumulations of PSII dimer and PSII monomer in evr3-1 mutants (Fig. 5). It is interestingly to note that the over-accumulation of these PSII complexes is accompanied also by the reduction of LHCII trimers (Fig. 5). The reduction of LHCII trimers is consistent with reported abnormal accumulation of light harvesting proteins in egy1 mutants (37). Although we lack direct cause and effect evidence, it is possible that the reduced LHCII trimers may trigger the PSI and PSII defects in evr3-1 mutants.
One of the most intriguing regulations of photosynthetic protein complexes is the turnover process of PSII (18). The highly oxidative reactions of PSII put great stress on PSII components, particularly reaction center D1 protein, which is long known to undergo a rapid turnover process (50). Chloroplast FtsH and Deg proteases are intimately involved in the degradation of damaged D1 protein (19 -21). Consistently, the degradation of D1 protein under high light conditions was slowed in var2 mutants, indicating an involvement of VAR2/AtFtsH2 and thylakoid FtsH complexes in regulating D1 degradation (Fig. 6)  (14 -17). Surprisingly, we uncovered a previously unknown involvement of EVR3/EGY1/AMOS1 in the D1 turnover pro-

EGY1 regulates chloroplast development and proteostasis
cess. Under high light conditions, the degradation of D1 was much faster in evr3-1 mutants than in WT (Fig. 6). This finding is counterintuitive as the absence of a protease, EVR3/EGY1/ AMOS1, leads to accelerated protein degradation, and suggests that D1 is not the direct substrate of EVR3/EGY1/AMOS1. Moreover, the slowed degradation of D1 in var2 mutants was somehow bypassed by the mutation in EVR3/EGY1/AMOS1 as evr3-1 var2-5 has a D1 degradation rate similar to that of evr3-1 (Fig. 6). These findings suggest the existence and/or activation of alternative protein degradation capacities in the absence of both VAR2/AtFtsH2 and EVR3/EGY1/AMOS1 to facilitate D1 degradation.
Despite the PSII and PSI assembly defects, mutants of EVR3/ EGY1/AMOS1 display pale green leaf coloration, rather than a leaf variegation phenotype. The synthetic lethal combination of VAR2/AtFtsH2 and EVR3/EGY1/AMOS1 mutations may stem from the abnormal assembly of PSII and PSI, or the aberrant D1 degradation, and eventually leading to an enhancement of var2 leaf variegation phenotype. We have proposed a threshold hypothesis in which the reduced thylakoid FtsH complexes in var2 mutants may generate a sensitized chloroplast state ideal for the identification of factors that act together with VAR2/AtFtsH2 to regulate chloroplast development (32). In the context of the threshold hypothesis, the defects caused by EVR3/EGY1/AMOS1 mutations generate far more dramatic consequences in the more sensitized var2 mutant backgrounds than the EVR3/EGY1/AMOS1 mutation alone. Our genetic and biochemical evidence indicate that VAR2/ AtFtsH2 and EVR3/EGY1/AMOS1 act synergistically to regulate chloroplast development.

Quantification of chlorophyll content
Leaves of 2-week-old plants grown on soil mix were harvested, weighed, and ground in liquid nitrogen. 95% ethanol (v/v) was then added to extract chlorophyll pigments at 4°C for 24 h in the dark. The content of chlorophylls and carotenoids was calculated as described in Ref. 36. Three biological replicates for each genotype were included.

Whole genome resequencing
evr3-1 was backcrossed with Col-0 for five times. In the F2 generation of the last round backcross, seedlings of 60 individuals with evr3-1 phenotype were pooled together to extract DNA using the DNAquick Plant System kit (TIANGEN Biotech). evr3-1 genomic DNA library preparation, genome resequencing, and sequencing data analysis were performed at Novogene. In brief, DNA sequencing library was prepared using the NEBNext ® Ultra TM DNA Library Prep Kit for Illumina ® (New England Biolabs) following the manual provided by the manufacturer. Library was assessed using the Agilent Bioanalyzer 2100 system before sequenced on an Illumina HiSeq4000 platform and 150 bp paired-end reads were generated. Filtered clean reads were aligned to the TAIR10 reference genome. Single nucleotide polymorphisms between the reference genome sequence and pooled F2 mutant DNA sequence were detected and annotated. We selected homozygous nonsynonymous G to A single nucleotide polymorphisms located in the gene regions as candidate mutations. The G to A mutation found in EGY1/AMOS1/At5g35220 was confirmed by conventional Sanger sequencing and was focused for further analysis because of the phenotypic resemblance of evr3-1 with the reported egy1 mutant alleles.

Transgenic lines
For complementation assay, the coding region of EVR3/ At5g35220 was amplified from WT cDNA with primers AT5G35220 F and AT5G35220 R (Table S1). The PCR product was cloned into a modified binary vector pBI111L-intron to place the At5g35220 ORF under the control of 35S promoter (28). The resulting construct was sequenced and used to transform evr3-1 single mutant and var2-5 heterozygous evr3-1 homozygous (var2-5/ϩ evr3-1) using the floral dip method (52). T1 transgenic lines were screened on 1/2 Murashige and Skoog plates with 50 mg liter Ϫ1 kanamycin. Genotypes of the transgenic lines were confirmed by PCR using sequence-specific primers. Genotyping primers are listed in Table S1.

Total protein extraction and immunoblot analysis
Fresh plant materials were weighed, ground in liquid nitrogen, and extracted with 2ϫ SDS sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% ␤-mecaptoethanol, and 0.02% bromphenol blue) at 65°C for 30 min. The volume of 2ϫ SDS sample buffer was normalized based on sample fresh weight. For immunoblotting, total proteins were separated with 12% SDS-PAGE containing 8 M urea, transferred onto PVDF membranes (0.22 m, Millipore), and probed with indicated antibodies. The source of antibodies used in this study is listed in Table S2. Representative immunoblots from three indepen-EGY1 regulates chloroplast development and proteostasis dent biological replicates were shown. Quantification of immunoblots were carried out with the Image Lab software (Bio-Rad). Graphs were generated with the GraphPad Prism 8 software.

Preparation of thylakoid membranes, BN-PAGE, and silver staining
Preparation of thylakoid membranes and blue-native PAGE was performed as described (42), with some modifications in solubilizing thylakoid membranes. Briefly, for solubilization with ␤-dodecylmaltoside, thylakoids equivalent to equal amounts of total protein were solubilized with 25BTH20G buffer (25 mM BisTris-HCl, pH 7.0, 20% glycerol) containing 1% n-dodecyl-␤-D-maltoside (w/v). For solubilization with digitonin, thylakoids equivalent to equal amounts of total protein were solubilized with 2% digitonin (w/v) in 25BTH20G buffer. For 1-D BN-PAGE, solubilized membranes were resolved on 3-12% gradient native PAGE. For 2-D SDS-PAGE, excised lanes from 1-D gels were denatured in 2ϫ SDS sample buffer and resolved on 12% SDS-PAGE containing 8 M urea. Silver staining of 2-D gels was performed as described (42). Representative gel pictures from three independent biological replicates were shown.

High light treatment
Leaf discs excised from the seventh or eighth true leaves of 4-week-old plants were first infiltrated in a solution containing 0.2% (v/v) Tween 20, 1.0 mg ml Ϫ1 lincomycin, and 0.1 mg ml Ϫ1 cycloheximide for 15 min, and then transferred to high light conditions (ϳ1000 mol m Ϫ2 s Ϫ1 ) for 0, 2, and 4 h. Each sample contains at least five leaf discs, and total proteins were extracted and normalized to tissue fresh weight.