Leaf Variegation of Thylakoid Formation1 Is Suppressed · to elucidate the mechanism underlying...

Leaf Variegation of Thylakoid Formation1 Is Suppressedby Mutations of Specific s-Factors in Arabidopsis1[OPEN]

Fenhong Hu, Ying Zhu, Wenjuan Wu, Ye Xie, and Jirong Huang*

National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, ShanghaiInstitutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

ORCID IDs: 0000-0002-1361-6336 (F.H.); 0000-0001-6509-5723 (Y.X.); 0000-0002-3553-0031 (J.H.).

Thylakoid Formation1 (THF1) has been shown to play roles in chloroplast development, resistance to excessive light, andchlorophyll degradation in Arabidopsis (Arabidopsis thaliana). To elucidate mechanisms underlying THF1-regulated chloroplastdevelopment, we mutagenized thf1 seeds with ethyl methanesulfonate and screened second-site recessive mutations that suppressits leaf variegation phenotype. Here, we characterized a unique suppressor line, 42-6, which displays a leaf virescent phenotype.Map-based cloning and genetic complementation results showed that thf1 variegation was suppressed by a mutation ins-FACTOR6 (SIG6), which is a plastid transcription factor specifically controlling gene expression through the plastid-encodedRNA polymerase. Northern-blot analysis revealed that plastid gene expression was down-regulated in not only 42-6 and sig6 butalso, thf1 at the early stage of chloroplast development. Interestingly, mutations in SIG2 but not in other s-factors also suppressedthf1 leaf variegation. Furthermore, we found that leaf variegation of thf1 and var2 could be suppressed by several virescentmutations, including yellow seedling1, brz-insensitive-pale green2, and nitric oxide-associated protein1, indicating that virescentmutations suppress leaf variegation. Taken together, our results provide unique insights into thf1-mediated leaf variegation,which might be triggered by defects in plastid gene transcription.

Chloroplast biogenesis is a key process for thetransition of plant growth from heterotrophy to auto-trophy. Chloroplasts can arise from proplastids orother various types of plastids, such as etioplasts andchromoplasts, under the light condition. It is wellknown that chloroplast development is controlled bythe coordinated expression of the plastid and nucleargenes. Although significant progress has been madetoward our understanding of molecular mechanismson chloroplast development, it still remains largelyunclear how plastid and nuclear gene expressions arecoordinately regulated. Leaf-variegated mutants,which display green sectors and nongreen sectors inthe same leaf, are recognized as an ideal system forinvestigating the mechanisms of chloroplast forma-tion (Liu et al., 2010c; Putarjunan et al., 2013). The

prominent characteristic of variegation mutants is thatthe severity of leaf variegation depends on environ-mental and developmental cues. To date, a number ofvariegation mutants have been reported in Arabi-dopsis (Arabidopsis thaliana; for review, see Yu et al.,2007; Putarjunan et al., 2013). Among these mutants,variegated1 (var1), var2, and immutans (im) are wellcharacterized. VAR1 and VAR2 encode Filamentationtemperature-sensitive H5 (FtsH5) and FtsH2 subunits,respectively, of the FtsH metalloprotease complex,whereas IM encodes plastid terminal oxidase, whichtransfers electrons from the plastoquinol to molecularoxygen (Carol et al., 1999; Wu et al., 1999; Chen et al.,2000; Sakamoto et al., 2002). The FtsH protease com-posed of types A (FtsH1 and FtsH5/VAR1) and B(FtsH2/VAR2 and FtsH8) subunits plays an importantrole in degradation of the photodamaged D1 protein inthe PSII repair cycle and probably acts as a molecularchaperone in chloroplasts as well (Bailey et al., 2002;Sakamoto et al., 2003). IM has been shown to be aversatile quinol oxidase that is involved in regulationof the plastoquinone pool and carotenoid biosynthesis(Wetzel et al., 1994; Wu et al., 1999; Aluru andRodermel, 2004). Both ftsh2/var2 and im mutants dis-play more severe leaf variegation in high light than inlow light (Rosso et al., 2009). It seems that the leafvariegation phenotype is associated with the defect inbiochemical functions of FtsH2 and IM in photoreac-tion processes, particularly with the activity of PSII.Wang et al. (2004) reported that loss of function ofThylakoid Formation1 (THF1), a homolog of cyano-bacterial photosystem II29 (psb29), led to leaf variegationin Arabidopsis. Our previous data revealed that the

1 This work was supported by the 973 Program (grant nos.2015CB150104 and 2013CB127000) and the National Special Grantfor Transgenic Crops (grant no. 2011ZX08009–003–005).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Jirong Huang ([email protected]).

F.H. designed the experiments, performed most of the experi-ments, analyzed the data, and wrote the article; Y.Z. performed theexperiments and provided technical assistance to F.H.; W.W. con-ceived the original screening and provided technical assistance toF.H.; Y.X. provided technical assistance to F.H.; J.H. conceived theproject and wrote the article.

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thf1 variegation phenotype resulted from the reducedactivity of FtsH protease (Zhang et al., 2009; Wu et al.,2013). How THF1 mutations down-regulate the levelof FtsH protease remains unknown.Suppressor screening in the var2 and thf1 genetic

backgrounds has been shown to be a powerful strategyto elucidate the mechanism underlying variegated leafformation, and a number of suppressor genes has beenidentified to date. These suppressors include recessivegenes coding for Caseinolytic protease C1 (ClpC1),ClpC2, ClpR1, and ClpR4 subunits of chloroplast ClpP/R protease (Park and Rodermel, 2004; Yu et al., 2008; Wuet al., 2013), pseudouridine synthase (Suppressor of Va-riegation1 [SVR1]; Yu et al., 2008), plastid ribosomalproteins L11 (Wu et al., 2013) and L24 (SVR8; Liu et al.,2013), a chloroplast translation initiation factor2 (Fu-gaeri1 [FUG1]; Miura et al., 2007), a chloroplast transla-tion elongation factor G (Snowy cotyledon1 [SCO1];Miura et al., 2007), a type A-like translation elongationfactor (SVR3; Liu et al., 2010a), a pentatricopeptide repeatprotein (SVR7; Liu et al., 2010b), a functionally unknownprotein localized in chloroplasts (SVR4; Yu et al., 2011),and chloroplast peptide deformylase (Peptide deformy-lase 1B [PDF1B]; Adam et al., 2011). All of these varie-gation suppressor proteins are localized in chloroplasts,and most of them function directly in the process ofchloroplast gene expression, such as ribosomal RNA(rRNA) processing and protein translation. However,overexpressing FtsH8 and G protein a-subunit1 rescuesthe leaf variegation phenotype of var2 and thf1 mutants(Yu et al., 2004; Zhang et al., 2009). Based on thesefindings, a threshold level of FtsH protease is suggestedto be required for normal chloroplast development (Chenet al., 2000; Yu et al., 2004). In the suppressor lines, it isplausible to assume that the FtsH threshold level neededfor chloroplast development is lowered, because muta-tions impair chloroplast gene expression and subse-quently, delay chloroplast development (Yu et al., 2008).However, not all mutants that are defective in chloroplastdevelopment are able to suppress leaf variegation, indi-cating that the suppression of leaf variegation is regu-lated in a complicated and specific manner (Liu et al.,2010c). This is consistent with the previously reportedproteomic data showing that the proteome of the sup-pressor line is very similar to that of the single suppressormutant, providing a systemic view on the mechanism ofleaf variegation suppression (Wu et al., 2013).In this study, we isolated another unique suppressor

gene of thf1, namely s-factor6 (sig6). s-factors arebacterial transcription factors that bind to the coreRNA polymerase and initiate RNA synthesis (Helmannand Chamberlin, 1988). Most promoters of housekeep-ing genes are recognized by the primary s-factor s70 inEscherichia coli. Consistently, plant factors have beenreported to form a complex with plastid-encoded RNApolymerase (PEP) in vitro and in vivo (Troxler et al., 1994;Liu and Troxler, 1996; Homann and Link, 2003; Suzukiet al., 2004). The Arabidopsis genome encodes six plastid-localized s-factors (SIGs; SIG1–SIG6) that are clusteredinto the same superfamily of s70 and implement the same

function as those in bacteria (Gruber and Gross, 2003;Lysenko, 2007). Physiological and genetic evidencerevealed that different s-factors function redundantlyand specifically to regulate plastid gene expression inArabidopsis (Kanamaru et al., 2001; Privat et al., 2003;Yao et al., 2003; Nagashima et al., 2004; Tsunoyamaet al., 2004; Favory et al., 2005; Ishizaki et al., 2005;Loschelder et al., 2006; Zghidi et al., 2007). For example,loss of function of SIG2 or SIG6 resulted in slow chlo-roplast development because of inhibition of plastidgene expression, and the sig2 sig6 double mutantshowed a very severe defect in chloroplast developmentand was unable to grow in nature (Shirano et al., 2000;Hanaoka et al., 2003; Ishizaki et al., 2005; Woodsonet al., 2012). SIG5 was reported to control circadianrhythms of transcription of several chloroplast genes(Noordally et al., 2013). In addition, s-factors have beenshown to play an important role in anterograde andretrograde signaling between the nucleus and plastids(Woodson et al., 2012; Oh and Montgomery, 2013).Thus, it is clear that s-factors play multiple roles inplastid gene expression and signal transduction inhigher plants. Here, we showed that the thf1 variegationphenotype was suppressed by mutations in SIG2 orSIG6 but not in other SIGs, indicating the specificity ofSIG genes in controlling chloroplast development.

RESULTS

SIG6 Mutations Suppress Leaf Variegation of thf1

To dissect the genetic network regulating the thf1-mediated chloroplast development, we mutagenized 2mL of thf1 seeds with ethyl methanesulfonate andscreened for suppressor lines that do not display theleaf variegation phenotype. In total, eight suppressorlines were isolated from 8,000 independent M2 lines.Here, we characterized the suppressor line 42-6.Compared with the wild type, thf1 produced varie-gated leaves, whereas the suppressor line 42-6 dis-played a virescent phenotype in newly emergingleaves (Fig. 1A). Chlorophyll content analysis showedthat thf1 and 42-6 mutants contained about 90% and93%, respectively, of the wild type in mature leaves(Fig. 1B). The slow growth rate of thf1 was not alteredin 42-6 (Fig. 1C). As previously reported, transmissionelectron microscopy analysis showed that stacked andstromal thylakoid membranes were observed in thegreen sector but not in the yellow/white sector of thf1leaves (Fig. 1D). In addition, there was no difference inthe ultrastructure of chloroplasts from 42-6 and wild-type mature leaves (Fig. 1D). These results reveal thatthe second mutation in 42-6 rescues chloroplast de-velopment in the yellow sectors of thf1.

To clone the suppressor gene, 42-6 was first back-crossed with thf1 for three generations. Genetic anal-ysis showed that thf1 leaf variegation was suppressedby a single recessive gene in 42-6. Then, 42-6 wascrossed with Landsberg erecta (Ler) to generate the F2mapping population. Map-based cloning results

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revealed that the suppressor gene was located withinthe 94-kb region, which contains 19 annotated genes,between two simple sequence length polymorphism(SSLP) markers (T1J8 and T2N18) on chromosome II(Fig. 2A). We sequenced all of these genes and iden-tified a single nucleotide substitution from G to A inthe fifth exon of the gene locus AT2G36990, whichencodes SIG6. The G-to-A mutation resulted in a ge-netic code switch from the codon of Trp to the stopcodon (Fig. 2A). These results suggest that the functionof SIG6 is disrupted in 42-6.

The single sig6 mutant was isolated from 42-6 andnamed sig6-3. The transcript of SIG6 was not detectedin sig6-3 by northern-blot analysis with the gene-specific probe (Fig. 2B), indicating that sig6-3 is a nullmutant. As previously reported (Ishizaki et al., 2005;Woodson et al., 2012), sig6-3 cotyledons were yellow/white at the early growth stage but gradually turned togreen 8 d after germination (Supplemental Fig. S1). Inaddition, the newly emerging leaf of sig6-3 showed aslightly virescent phenotype, which was observed in42-6. These data suggest that sig6 is epistatic to thf1with regard to chloroplast development.

To confirm the map-based cloning result, the genomicDNA of SIG6 driven by the cauliflower mosaic virus35S promoter was transformed into 42-6. As shown inFigure 2C, overexpressing SIG6 in 42-6 resulted in leafvariegation, indicating that our map-based cloning re-sult is correct. Taken together, our data reveal that sig6is a suppressor for thf1 leaf variegation.

Hypersensitivity of thf1 to Excessive Light Is NotSuppressed by sig6

The thf1 mutant was reported to be hypersensitive toexcessive light (Wang et al., 2004; Keren et al., 2005). Totest whether sig6 was able to suppress the sensitivity ofthf1 to high light, we examined the value of variablePSII fluorescence (Fv)/maximum PSII fluorescence (Fm)in the dark-adapted state, which indicates the maxi-mum quantum yield of PSII photochemistry (PSII ac-tivity), using detached leaves of 30-d-old plants. TheFv/Fm value was reduced more dramatically in thf1 and42-6 than in the wild type and sig6 in excessive light(Fig. 3). When the leaves were treated by high light for 4h, PSII activity was almost undetectable in thf1 and 42-6,whereas it remained at one-half of the original value inthe wild type and sig6. These data indicate that SIG6mutations have no effect on plant sensitivity to exces-sive light. During the dark recovery period, the value ofFv/Fm was recovered much slower in thf1 and 42-6 thanin the wild type and sig6, whereas there was no statis-tically significant difference in the recovery of Fv/Fmbetween 42-6 and thf1. Thus, these results imply thatsig6 cannot suppress thf1 hypersensitivity to high light.

SIG6 Is Predominantly Expressed in Green Tissues

To profile SIG6 expression, the GUS gene driven bythe SIG6 promoter was introduced into wild-typeplants. GUS activity was analyzed using 4-d-old

Figure 1. Characterization of 42-6, oneof the suppressor lines for thf1 leaf va-riegation. A, Phenotypes of 30-d-oldwild-type (WT), thf1, and 42-6 plantsgrown under 8-h-light/16-h-dark cycleconditions with a light intensity of 100mmol photons m22 s21 at 22˚C. The twomagnified images show variegation ofthe thf1 leaf marked by the rectangleand virescence of 42-6 new leavesmarked by the square, respectively.Bars = 1 cm. B, Chlorophyll (Chl) con-tent of leaves from 30-d-old wild-type,thf1, and 42-6 plants shown in A. Errorbars indicate6 SD (n = 3). C, Leaf numberof 60-d-old wild-type, thf1, and 42-6plants grown under 8-h-light/16-h-darkcycle conditions. Error bars indicate 6 SD(n = 10). ***, Significant differences inleaf number between wild-type and mu-tant plants (P, 0.001; Student’s t test). D,Ultrastructure of chloroplasts from matureleaves of 30-d-old wild-type plants, thf1white sectors, thf1 green sectors, and 42-6plants. Bars = 1 mm.

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seedlings grown in the dark or light and 8-week-oldmature plants. Our results showed that the activity ofthe SIG6 promoter was high in all green tissues, suchas young seedlings, leaves, flowers, and siliques, butnot in nonphotosynthetic roots (Fig. 4, A–C). By con-trast, GUS activity was detected only in the cotyledonfrom etiolated seedlings (Fig. 4D). We also examinedthe expression of SIG6 in various tissues or underdifferent stresses using northern-blot analysis withspecific probes against SIG6. Consistent with the re-sults of GUS analysis, SIG6 mRNA accumulatedabundantly in all tissues, except for the root (Fig. 4E).SIG6 expression was induced moderately by NaCl andcold stresses but significantly suppressed by abscisicacid (ABA; Fig. 4F). Thus, our data suggest that SIG6 ispredominantly expressed in photosynthetically activetissues and can be up- or down-regulated by differentstimuli.

Plastid Gene Expression Is Regulated Differentially byTHF1 and SIG6

It is well known that s-factors initiate gene tran-scription by recognizing a specific set of promoters forthe PEP in higher plants (Isono et al., 1997; Fujiwaraet al., 2000). To elucidate the mechanism by whichchloroplast development in thf1 was rescued by sig6,we analyzed plastid gene expression in wild-type, thf1,sig6, and 42-6 seedlings at the early stage of chloroplastdevelopment. Our results showed that, in 4-d-oldseedlings, transcript levels of the examined PEP-de-pendent genes were significantly lower in thf1 and sig6than in the wild type (Fig. 5, A and B), whereas those ofnuclear-encoded RNA polymerase (NEP)-dependentgenes increased in thf1 and sig6 compared with thewild type (Fig. 5C). This result is consistent with thegeneral observation that the inhibition of PEP-depen-dent gene expression promotes the expression of NEP-dependent genes (Kanamaru et al., 2001; Zhou et al.,2009; Yagi et al., 2012). Interestingly, transcript levels ofthe PEP-dependent genes accumulated quite similarlybetween thf1 and sig6 and apparently lower in 42-6 thanin the thf1 and sig6 single mutants (Fig. 5; SupplementalFig. S2), indicating that THF1 and SIG6 regulate PEP-dependent gene expression in a redundant manner. In8-d-old seedlings, the difference in expression of themost plastid genes examined was small between thewild type and mutants, whereas expression of somegenes including cytochrome b6 and glutamyl tRNA wasobviously lower in 42-6 and sig6 than in the wild typeand thf1. These results indicate that SIG6 has a moreprofound influence on plastid gene expression thanTHF1. In addition, we also examined expression ofsome nuclear genes with products that are targeted tochloroplasts. The mRNA levels of genes encoding lightharvesting complex PSII subunit6, light harvestingcomplex PSI subunit1, and ribose bisphosphate car-boxylase small chains were not apparently affected by

Figure 2. Identification of the suppressor gene sig6. A, Map-basedcloning of the sig6 gene. The suppressor gene was mapped to the 94-kb region between two SSLP markers (T1J8 and T2N18) on chromo-some II (Chr II). A substitution of G to A (red arrow) was identified inthe gene At2g36990, which encodes SIG6. B, Northern-blot analysis ofthe transcript levels of SIG6 in 30-d-old wild-type (WT), thf1, 42-6, andsig6-3 plants. The level of rRNA was used as a loading control. Themolecular weight of the SIG6 transcript is about 2 kb. C, Comple-mentation of 42-6 by the SIG6 genomic DNA. Transgenic plants dis-play the same leaf variegation as thf1.

Figure 3. Hypersensitivity of thf1 to high light is not rescued by sig6.The values of Fv/Fm were measured in detached leaves from 30-d-oldwild-type (WT), thf1, 42-6, and sig6 plants grown in soil under anapproximately 100 mmol photons m22 s21 photoperiod (16-h-light/8-h-dark cycle) at 22˚C. Error bars indicate 6 SD (n = 10).



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mutations of THF1 and SIG6 in 4- and 8-d-old seedlingscompared with the wild type (Fig. 5D). Thus, our re-sults suggest that plastid gene expression is regulatedby THF1 and SIG6 in a redundant manner at the earlystage of chloroplast development.

SIG2 Mutations Suppress thf1 Leaf Variegation

The Arabidopsis genome has six s-factor genes: SIG1to SIG6. To investigate whether other s-factors are in-volved in suppressing thf1 leaf variegation, we identi-fied other sig single mutants from the ArabidopsisBiological Resource Center seed stocks and made theirdouble mutants with thf1 through a genetic cross. Inagreement with previous reports (Shirano et al., 2000;Privat et al., 2003; Favory et al., 2005; Zghidi et al.,2007; Lai et al., 2011), sig2 displayed a pale-greenphenotype, whereas sig1, sig3, sig4, and sig5 had novisible phenotypes (Fig. 6A). Except for sig2 thf1,other double mutants sig1 thf1, sig3 thf1, sig4 thf1, andsig5 thf1 still exhibited the leaf variegation phenotype(Fig. 6B). These results indicate that, other than sig6, aloss-of-function mutation sig2 can also suppress thf1leaf variegation.

Virescent Mutants Suppress thf1 Variegation

Because both sig2 and sig6 mutants display the leafvirescent phenotype, we tested whether other vires-cent mutations suppress thf1 leaf variegation. To dothis, we selected three virescent mutants (no-associatedprotein1 [noa1; Guo et al., 2003], yellow seedling1 [ys1;Zhou et al., 2009], and brz-insensitive-pale green2 [bpg2;Komatsu et al., 2010]) and constructed their double

mutants with thf1. Similar to that previously reported,chloroplast development was slower in cotyledonsand young leaves of 8-d-old ys1, noa1, and bpg2 seed-lings compared with the wild type (Fig. 7A). In 30-d-old plants, the virescent phenotype was observedmainly in newly emerging noa1 and bpg2 leaves butnot in ys1 leaves (Fig. 7A). The thf1 noa1, thf1 ys1, andthf1 bpg2 double mutants did not display variegatedleaves (Fig. 7B), suggesting that leaf variegation can besuppressed by virescent mutations.

Leaf Variegation of var2 Is Suppressed by sig6 and ys1

To test whether suppressor genes for thf1 are func-tional in var2, we made var2 sig6 and var2 ys1 doublemutants. As shown in Figure 8, both sig6 and ys1 wereable to suppress var2 variegation, supporting ourprevious results that suppressor genes for thf1 vari-egation are functional in var2 and vice versa. Thesedata also suggest that THF1 and VAR2 function in thesame pathway to regulate chloroplast development.

DISCUSSION

In higher plants, plastid genes are transcribed bytwo different types of RNA polymerase, namely NEPand PEP. PEP is responsible for transcribing photo-synthetic genes, such as psbA and psbD. Miura et al.(2010) reported that numerous genes related to pho-tosynthesis and chloroplast functions were repressedin the white sectors of var2. A dramatic decrease inplastid gene expression was also observed in the eti-olated seedlings of thf1 (Wu et al., 2011). It remainsunclear whether leaf variegation is associated with the

Figure 4. Expression patterns of AtSIG6. A to D,Analysis of GUS activity driven by the nativeSIG6 gene promoter in 4-d-old seedlings (A),leaves (B), flowers (C), and 4-d-old dark-grownseedling (D). E, Northern-blot analysis of SIG6transcripts (2.0 kb) in various tissues from 8-d-oldseedlings and 8-week-old plants. The level of25S rRNA is shown as a loading control. F,Northern-blot analysis of SIG6 transcripts (2.0kb). Eight-day-old seedlings were treated withdeficiency of phosphate (2P), 50 mM ABA, 100mM NaCl, 10% (w/v) polyethylene glycol 6000(PEG), 100 mM hydrogen peroxide (H2O2), and6˚C (cold) for 12 h. The level of 25S rRNA isshown as a loading control. CK, Without treat-ment.


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defect in plastid gene expression. The genetic screenfor suppressor genes of var2 and thf1 variegationrevealed that chloroplast development is highly linkedto several key biological processes in chloroplasts, suchas protein quality control monitored by Clp protease,ribosomal assembly, and protein translation. The ulti-mate influence of these suppressors may be convergeddirectly or indirectly on plastid gene expression. Al-though suppressor genes of leaf variegation have beenshown to function in almost every step of plastid geneexpression, not all genes involved in this process areable to suppress leaf variegation. Thus, it is possiblethat leaf variegation results from a specific defect inplastid gene expression.In this study, we identified a unique suppressor

gene sig6 for thf1-mediated leaf variegation. Consistentwith previous reports (Ishizaki et al., 2005; Woodsonet al., 2012), the identified sig6 mutant displays a clearvirescent phenotype, particularly in cotyledons andnewly emerging leaves. In Arabidopsis, s-factors havebeen suggested to be localized in plastids (Isono et al.,1997; Fujiwara et al., 2000; Hara et al., 2001; Yao et al.,2003). Bioinformatic analysis showed that s-factors areconserved only in the C-terminal region that is

responsible for the promoter recognition, whereas theN-terminal region is highly variable both in length andsequence, indicating that individual plastid s-factorshave a distinctive role in temporally and spatiallyregulating plastid gene expression. Indeed, geneticanalysis showed that SIG2 and SIG6, which function ina partially redundant manner, are essential for plastidgene expression during chloroplast development,whereas other s-factors are involved in specific geneexpression for maintaining photosynthetic efficiencyunder various conditions (Nagashima et al., 2004;Shimizu et al., 2010; Noordally et al., 2013). Consis-tently, our data showed that sig6 and sig2, which ex-hibit a virescent phenotype, but not sig1, sig3, sig4, andsig5, which have no obvious phenotype in chloroplastdevelopment, suppressed thf1 leaf variegation. Re-cently, our proteomic analysis revealed that the pro-teome of chloroplasts was more severely affected byvirescent mutations than variegation mutations (Wuet al., 2013). Thus, it seems that plastid gene expressionspecifically regulated by SIG2 and SIG6 is related toleaf variegation.

Figure 6. Mutations in SIG2 but not in other SIGs suppress thf1 leafvariegation. A, Phenotypes of 30-d-old wild-type (WT), thf1, sig1, sig2,sig3, sig4, and sig5 plants. B, Phenotypes of 30-d-old thf1 and itsdouble mutants with sig1 to sig5. All plants were grown in soil undershort-day (8-h-light/16-h-dark cycle) conditions with a light intensity of100 mmol photons m22 s21.

Figure 5. Northern-blot analysis of gene expression in 4- and 8-d-oldwild-type (WT), thf1, 42-6, and sig6 seedlings grown on one-half-strength MS supplemented with 1% (w/v) Suc under an approximately100 mmol photons m22 s21 photoperiod (16-h-light/8-h-dark cycle) at22˚C. A, mRNA levels of the PEP-dependent, photosynthetic-relatedplastid genes. B, mRNA levels of the PEP-dependent plastid transferRNA (tRNA) genes. C, mRNA levels of the NEP-dependent genes. D,mRNA levels of the nuclear-encoded genes. The level of 25S rRNA isshown as a loading control. The molecular weights of gene productsare shown on the right. accD, Carboxyltransferase beta subunit; atpB,ATP synthase beta subunit; Lhca1, light harvesting complex PSI sub-unit1; Lhcb6, light harvesting complex PSII subunit6; petB, cyto-chrome b6; rbcL, ribulose 1,5-bisphosphate carboxylase/oxygenase;RbcS, ribose 1,5-bisphosphate carboxylase small chains; rpoB, RNApolymerase beta subunit; trnE, glutamic tRNA; trnQ, glutamine tRNA;trnW, tryptophan tRNA.



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To date, a number of recessive genes has beenreported to suppress var2 leaf variegation (Park andRodermel, 2004; Mirua et al., 2007; Yu et al., 2008, 2011;Liu et al., 2010a, 2010b; Adam et al., 2011; Wu et al.,2013). It is evident that the majority of them are of avirescent leaf phenotype. These mutants include clpC1,clpR1, fug, sco1, pdf1b, svr3, svr4, and svr7 (Miura et al.,2007; Yu et al., 2008, 2011; Liu et al., 2010a, 2010b;Adam et al., 2011). This raises an interesting questionof whether all virescent mutants are able to suppressleaf variegation. In this study, we tested three reportedvirescent mutants, namely noa1, bpg2, and ys1. Geneticanalysis showed that noa1, bpg2, and ys1 were epistaticto thf1. Additional investigation is required to makesuch a conclusion.

How virescent mutants suppress leaf variegation isa fascinating question. According to the thresholdmodel of FtsH protease, which has gained molecularand genetic evidence to explain the heterogeneity ofchloroplast development (Yu et al., 2004), either anincrease in the FtsH level or a decrease in thethreshold of the FtsH level is able to suppress leafvariegation. It was proposed that the threshold ofFtsH protease is reduced, because the period ofchloroplast development in virescent mutants is ex-tended (Yu et al., 2007; Liu et al., 2010c). However,proteomic and western-blot assays revealed that thelevel of FtsH protease in the suppressor line (thf1clpR4) was actually higher than that in the variegationmutant thf1 (Wu et al., 2013). Up-regulation of FtsHlevels by suppressor genes can take place at a tran-scriptional and/or posttranscriptional level, such asby increasing FtsH gene expression, preprotein im-port into plastids, and subsequent protein folding andcomplex assembly (Wu et al., 2013). In this study, we

found that transcriptional levels of PEP-dependentgenes were much lower in the suppressor line 42-6than in the single thf1 and sig6 mutants at the earlystage of chloroplast development, providing molec-ular evidence to support that plastid development isdelayed in the suppressor line of leaf variegation.Again, western-blot analysis showed that the level ofthe VAR1 subunit of FtsH protease was higher in thesuppressor lines, including 42-6, thf1ys1, and thf1noa1,than in thf1 (Supplemental Fig. S3). Thus, FtsH pro-tease may no longer be a limiting factor for chloro-plast development in suppressor lines. Interestingly,both SIG2 and SIG6 have been reported to be in-volved in retrograde signaling from chloroplasts tothe nucleus (Woodson et al., 2012). It is possible thatretrograde signaling is involved in suppression of leafvariegation.

The nature for FtsH protease to regulate chloroplastbiogenesis still remains unknown. The best character-ized function of FtsH protease is in degradation of thephotodamaged D1 protein during PSII repair (Lindahlet al., 2000; Bailey et al., 2002; Kato and Sakamoto,2009). The extent of variegation was positively corre-lated with an increase in excitation pressure (Rossoet al., 2009), suggesting that loss of function of VAR2leads to chloroplast photooxidation and subsequently,impairs chloroplast biogenesis. The controversy is thata defect in D1 degradation does not necessarily lead toleaf variegation (Miura et al., 2007), and prolamellarbody formation of etioplasts is severely inhibited whenvar2 is grown in the dark (Wu et al., 2013). This evi-dence indicates that the role of VAR2 in D1 degrada-tion is not directly associated with early chloroplastdevelopment. In addition, it is suggested that the roleof FtsH protease in vesicle fusion and/or maintainingthylakoid membranes might be involved in chloroplastbiogenesis (Hugueney et al., 1995; Kato et al., 2012).Considering that leaf variegation of thf1 is attributed toa significant decrease in FtsH protease activity (Zhanget al., 2009) and that the identified genes suppressingleaf variegation of thf1 and var2 can be mutuallyreplaced (Wu et al., 2013), molecular mechanisms forleaf variegation formation will be focused on howVAR2 is involved in thylakoid membrane formation inthe future.

Figure 8. Leaf variegation of var2 is suppressed by sig6 and ys1.Phenotypes of 30-d-old var2, var2 sig6, and var2 ys1 plants grown insoil under short-day (8-h-light/16-h-dark cycle) conditions with a lightintensity of 100 mmol photons m22 s21 at 22˚C. Bar = 1 cm.

Figure 7. Leaf variegation of thf1 is suppressed by virescent mutants.A, The virescent phenotype of 8-d-old (upper) and 30-d-old (lower)ys1, noa1, and bpg2 plants compared with the wild type (WT). B,Phenotypes of 30-d-old thf1, thf1 noa1, thf1 ys1, and thf1 bpg2 plantsgrown in soil under short-day (8-h-light/16-h-dark cycle) conditionswith a light intensity of 100 mmol photons m22 s21 at 22˚C. Red arrowsindicate the yellow sectors of thf1 leaves. Bar = 1 cm.


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http://www.plantphysiol.org/cgi/content/full/pp.15.00549/DC1

MATERIALS AND METHODS

Plant Material and Growth Conditions

All experiments were performed with Arabidopsis (Arabidopsis thaliana)ecotypes Columbia-0 and Ler. Seeds were germinated and grown on 0.7%(w/v) agar plates containing one-half-strength Murashige and Skoog medium(MS) with 1% (w/v) Suc. For additional analysis, plants were transferred tosoil and maintained under the short-day (8 h of light/16 h of dark) or long-day(16 h of light/8 h of dark) conditions, with a light intensity of 100 mmolphotons m22 s21 at 21°C. All of the primers used in this study are listed inSupplemental Table S1. The transfer DNA insertion mutant of sig4(Salk_146777) was identified from the Arabidopsis Biological ResourceCenter seed stocks. Other single mutants used in this study were previouslydescribed: thf1 (Wang et al., 2004), ys1 (Zhou et al., 2009), bpg2 (Komatsuet al., 2010), noa1/nos1 (Guo et al., 2003), sig1 (Lai et al., 2011), sig2 (Ohand Montgomery, 2013), sig3 (Zghidi et al., 2007), and sig5 (Tsunoyamaet al., 2004).

Map-Based Cloning

Suppressors were identified by screening M2 seeds that were mutagenizedby 0.1% (w/v) ethyl methanesulfonate in the thf1 genetic background. Thesuppressor line 42-6 was crossed to Ler, and the F2 seedlings were used asmapping populations. The mutation gene was mapped using SSLP markers,and information is available at The Arabidopsis Information Resource (http://www.arabidopsis.org).

Plasmid Construction and Transformation

Plasmids were constructed using the Gateway Cloning System (Invitrogen).PCR products from AtSIG6 genomic and promoter regions were cloned intothe pENTR SD/D-TOPO entry vector with gene-specific primers. The genomicfragment of SIG6 was then recombined into the pGWB2 destination vector,whereas the promoter fragment was recombined into the pGWB3 destinationvector (Research Institute of Molecular Genetics, Shimane University). Theconstructs were then transformed into the Agrobacterium tumefaciens GV3101strain and introduced into Arabidopsis plants by the floral dip method(Clough and Bent, 1998). Transformants were selected on one-half-strengthMS plates containing 50 mg L21 kanamycin. Transgenic lines were con-firmed by reverse transcription PCR or PCR using gene-specific primers.

Transmission Electron Microscopy

Samples were obtained from the leaves of 30-d-old plants grown in short-day (8 h of light/16 h of dark) conditions. The samples were fixed in a solutionof 4% (w/v) glutaraldehyde, stained, and examined as described byHarris et al.(1994).

Gene Expression Analysis

Total RNA was isolated using the RNAgents Total RNA Isolation System(Promega) in accordance with the manufacturer’s instructions; 0.5 mg of totalRNA was added to 10 mL of reverse transcription reaction, and 1 mL ofcomplementary DNA was used as template for reverse transcription PCR.Northern-blot analysis was performed using gene-specific probes as describedby Huang et al. (2000). Five to ten micrograms of total RNA was separated bydenaturing agarose gel electrophoresis.

Chlorophyll Analysis and ChlorophyllFluorescence Analysis

Chlorophyll was extracted from samples that were kept in 75% (v/v) ac-etone at 4°C for 24 h. Total chlorophyll content and chlorophyll a/b ratios werecalculated according to Lichtenthaler, 1987. Chlorophyll fluorescence wasmeasured with an Imaging PAM 101 (Watzs), and Fv/Fm values were calcu-lated according to the manufacturer’s instructions. Before the measurement,plants were maintained in the dark for 10 min, and the initial Fv/Fm valueswere recorded. Plants were then exposed to high light (800 photons m22 s21)for 4 h using a halogen light source.

Data from this article can be found in the Arabidopsis Genome Initiativeunder accession numbers THF1, At2g20890; YS1, At3g22690; NOA1/NOS1,At3g47450; BPG2, At3g57180; SIG1, At1g64860; SIG2, At1g08540; SIG3,At3g53920; SIG4, At5g13730; SIG5, At5g24120; and SIG6, At2g36990.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Phenotypes of wild-type, thf1, 42-6, and sig6-3cotyledons.

Supplemental Figure S2. Quantification of the northern-blot results in Fig-ure 5.

Supplemental Figure S3. Western-blot analysis of VAR1 content in wild-type, thf1, 42-6, sig6, thf1ys1, ys1, thf1noa1, and noa1 leaves.

Supplemental Table S1. List of the primers used in this study.

ACKNOWLEDGMENTS

We thank Fangqing Guo (Institute of Plant Physiology and Ecology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) forthe gift of the noa1 mutant, Wataru Sakamoto (Research Institute for Biore-sources, Okayama University, Japan) for the antibody against VAR1, and theCore Facility at Shanghai Institute of Plant Physiology and Ecology for ultra-structural analysis of chloroplasts.

Received April 13, 2015; accepted May 19, 2015; published May 21, 2015.

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