Mutations in SUPPRESSOR OF VARIEGATION1, a factor required for normal chloroplast translation, suppress var2-mediated leaf variegation in Arabidopsis

The Arabidopsis thaliana yellow variegated2 (var2) mutant is variegated due to lack of a chloroplast FtsH-like metalloprotease (FtsH2/VAR2). We have generated suppressors of var2 variegation to gain insight into factors and pathways that interact with VAR2 during chloroplast biogenesis. Here, we describe two such suppressors. Suppression of variegation in the first line, TAG-FN, was caused by disruption of the nuclear gene (SUPPRESSOR OF VARIEGATION1 [SVR1]) for a chloroplast-localized homolog of pseudouridine (Psi) synthase, which isomerizes uridine to Psi in noncoding RNAs. svr1 single mutants were epistatic to var2, and they displayed a phenotypic syndrome that included defects in chloroplast rRNA processing, reduced chloroplast translation, reduced chloroplast protein accumulation, and elevated chloroplast mRNA levels. In the second line (TAG-IE), suppression of variegation was caused by a lesion in SVR2, the gene for the ClpR1 subunit of the chloroplast ClpP/R protease. Like svr1, svr2 was epistatic to var2, and clpR1 mutants had a phenotype that resembled svr1. We propose that an impairment of chloroplast translation in TAG-FN and TAG-IE decreased the demand for VAR2 activity during chloroplast biogenesis and that this resulted in the suppression of var2 variegation. Consistent with this hypothesis, var2 variegation was repressed by chemical inhibitors of chloroplast translation. In planta mutagenesis revealed that SVR1 not only played a role in uridine isomerization but that its physical presence was necessary for proper chloroplast rRNA processing. Our data indicate that defects in chloroplast rRNA processing are a common, but not universal, molecular phenotype associated with suppression of var2 variegation.     

Genetic Interactions Reveal that Specific Defects of Chloroplast Translation are Associated with the Suppression of var2-Mediated Leaf Variegation

Arabidopsis thaliana L. yellow variegated (var2) mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique...     

A <Emphasis Type="Italic">var2</Emphasis> leaf variegation suppressor locus, <Emphasis Type="Italic">SUPPRESSOR OF VARIEGATION3</Emphasis>, encodes a putative chloroplast translation elongation factor that is important for chloroplast development in the cold

Background  

The Arabidopsis var2 mutant displays a unique green and white/yellow leaf variegation phenotype and lacks VAR2, a chloroplast FtsH metalloprotease. We are characterizing second-site var2 genetic suppressors as means to better understand VAR2 function and to study the regulation of chloroplast biogenesis.     

Arabidopsis Chloroplast FtsH,vat2 and Suppressors of var2 Leaf Variegation: a Review

Variegation mutants are ideal model systems to study chloroplast biogenesis.We are interested in variegations whose green and whitesectored leaves arise as a consequence of the action of nuclear recessive genes.In this review,we focus on the Arabidopsis var2 variegation mutant,and discuss recent progress toward understanding the function of VAR2 and the mechanism of var2-mediated variegation.VAR2 is a subunit of the chloroplast FtsH complex,which is involved in turnover of the Photosystem Ⅱ reaction center D1 protein,as well as in other processes required for the development and maintenance of the photosynthetic apparatus.The cells in green sectors of var2have normal-appearing chloroplasts whereas cells in the white sectors have abnormal plastids that lack pigments and organized lameliae.To explain the mechanism of var2 variegation,we have proposed a threshold model in which the formation of chloroplasts is due to the presence of activities/processes that are able to compensate for a lack of VAR2.To gain insight into these activities,second-site suppressor screens have been carried out to obtain mutants with nonvariegation phenotypes.Cloning and characterization of several var2 suppressor lines have uncovered several mechanisms of variegation suppression,including an unexpected link between var2 variegation and chloroplast translation.     

Arabidopsis Chloroplast FtsH,var2 and Suppressors of var2 Leaf Variegation:a Review

Variegation mutants are ideal model systems to study chloroplast biogenesis. We are interested in variegations whose green and whitesectored leaves arise as a consequence of the action of nuclear recessive genes. In this review, we focus on the Arabidopsis var2 variegation mutant, and discuss recent progress toward understanding the function of VAR2 and the mechanism of var2-mediated variegation. VAR2 is a subunit of the chloroplast FtsH complex, which is involved in turnover of the Photosystem II reaction center D1 protein, as well as in other processes required for the development and maintenance of the photosynthetic apparatus. The cells in green sectors of var2 have normal-appearing chloroplasts whereas cells in the white sectors have abnormal plastids that lack pigments and organized lamellae. To explain the mechanism of var2 variegation, we have proposed a threshold model in which the formation of chloroplasts is due to the presence of activities/processes that are able to compensate for a lack of VAR2. To gain insight into these activities, second-site suppressor screens have been carried out to obtain mutants with nonvariegation phenotypes. Cloning and characterization of several var2 suppressor lines have uncovered several mechanisms of variegation suppression, including an unexpected link between var2 variegation and chloroplast translation.     

The balance between protein synthesis and degradation in chloroplasts determines leaf variegation in Arabidopsis yellow variegated mutants

An Arabidopsis thaliana leaf-variegated mutant yellow variegated2 (var2) results from loss of FtsH2, a major component of the chloroplast FtsH complex. FtsH is an ATP-dependent metalloprotease in thylakoid membranes and degrades several chloroplastic proteins. To understand the role of proteolysis by FtsH and mechanisms leading to leaf variegation, we characterized the second-site recessive mutation fu-gaeri1 (fug1) that suppressed leaf variegation of var2. Map-based cloning and subsequent characterization of the FUG1 locus demonstrated that it encodes a protein homologous to prokaryotic translation initiation factor 2 (cpIF2) located in chloroplasts. We show evidence that cpIF2 indeed functions in chloroplast protein synthesis in vivo. Suppression of leaf variegation by fug1 is observed not only in var2 but also in var1 (lacking FtsH5) and var1 var2. Thus, suppression of leaf variegation caused by loss of FtsHs is most likely attributed to reduced protein synthesis in chloroplasts. This hypothesis was further supported by the observation that another viable mutation in chloroplast translation elongation factor G also suppresses leaf variegation in var2. We propose that the balance between protein synthesis and degradation is one of the determining factors leading to the variegated phenotype in Arabidopsis leaves.     

Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.     

Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease

Variegated plants have green- and white-sectored leaves. Cells in the green sectors contain morphologically normal chloroplasts, whereas cells in the white sectors contain non-pigmented plastids that lack organized lamellar structures. Many variegations are caused by mutations in nuclear genes that affect plastid function, yet in only a few cases have the responsible genes been cloned. We show that mutations in the nuclear VAR2 locus of Arabidopsis cause variegation due to loss of a chloroplast thylakoid membrane protein that bears similarity to the FtsH family of AAA proteins (ATPases associated with diverse cellular activities). Escherichia coli FtsH is a chaperone metalloprotease that functions in a number of diverse membrane-associated events. Although FtsH homologs have been identified in multicellular organisms, their functions and activities are largely unknown; we provide genetic in vivo evidence that VAR2 functions in thylakoid membrane biogenesis. We have isolated four var2 alleles and they have allowed us to define domains of the protein that are required for activity. These include two putative ATP-binding sites. VAR2 protein amounts generally correlate with the severity of the var2 mutant phenotype. One allele lacks detectable VAR2 protein, suggesting that the mechanism of var2 variegation involves the action of a redundant activity in the green sectors. We conclude that redundant activities may be a general mechanism to explain nuclear gene-induced plant variegations.     

Roles of the nuclear-encoded chloroplast SMR domain-containing PPR protein SVR7 in photosynthesis and oxidative stress tolerance in Arabidopsis

The mutations of the plastid SMR domaincontaining PPR protein SVR7 were previously reported to cause a specific reduction in the chloroplast ATP synthase levels. Here, we isolated a new mutant allele of SVR7, named svr7-4, in which T-DNA is inserted into the initiation codon of SVR7. The rosette leaves of svr7-4, especially in the juvenile stage, showed a pale green phenotype as a result of a reduction in the chlorophyll levels. The values of P700 and Fv/Fm indicated that the photosynthetic capacities of both PSI and PSII were damaged in svr7-4. Furthermore, we found that the svr7-4 accumulated more reactive oxygen species (ROS) and showed lower photo-oxidative stress tolerance by histochemical staining and hydrogen peroxide bleaching experiments, respectively. The leaves of svr7-4 also had increased anthocyanins accumulation compared to that of wild-type (WT) when floated on water under light. Finally, we found that the expression levels of four abiotic stress-responsive genes including ZAT10, AtAPX1, CAT1 and AtGPX2 were up-regulated in svr7-4. SVR7 was expressed ubiquitously during plant development. These results indicate that SVR7 is important for normal photosynthesis and photo-oxidative stress responses in chloroplasts.     

The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis

Variegated leaves are often caused by a nuclear recessive mutation in higher plants. Characterization of the gene responsible for variegation has shown to provide several pathways involved in plastid differentiation. Here we describe an Arabidopsis variegated mutant isolated by T-DNA tagging. The mutant displayed green and yellow sectors in all green tissues except for cotyledons. Cells in the yellow sector of the mutant contained both normal-appearing and mutant chloroplasts. The isolated mutant was shown to be an allele of the previously reported mutant, yellow variegated (var2). Cloning and molecular characterization of the VAR2 locus revealed that it potentially encodes a chloroplastic homologue of FtsH, an ATP-dependent metalloprotease that belongs to a large protein family involved in various cellular functions. ftsH-like genes appear to comprise a small gene family in Arabidopsis genome, since at least six homologues were found in addition to VAR2. Dispensability of VAR2 was therefore explained by the redundancy of genes coding for FstHs. In the yellow regions of the mutant leaves, accumulation of photosynthetic protein components in the thylakoid membrane appeared to be impaired. Based on the role of FtsH in a protein degradation pathway in plastids, we propose a possibility that VAR2 is required for plastid differentiation by avoiding partial photooxidation of developing chloroplasts.     

SOT1, a pentatricopeptide repeat protein with a small MutS‐related domain,is required for correct processing of plastid 23S–4.5S rRNA precursors in Arabidopsis thaliana

Ribosomal RNA processing is essential for plastid ribosome biogenesis, but is still poorly understood in higher plants. Here, we show that SUPPRESSOR OF THYLAKOID FORMATION1 (SOT1), a plastid‐localized pentatricopeptide repeat (PPR) protein with a small MutS‐related domain, is required for maturation of the 23S–4.5S rRNA dicistron. Loss of SOT1 function leads to slower chloroplast development, suppression of leaf variegation, and abnormal 23S and 4.5S processing. Predictions based on the PPR motif sequences identified the 5′ end of the 23S–4.5S rRNA dicistronic precursor as a putative SOT1 binding site. This was confirmed by electrophoretic mobility shift assay, and by loss of the abundant small RNA ‘footprint’ associated with this site in sot1 mutants. We found that more than half of the 23S–4.5S rRNA dicistrons in sot1 mutants contain eroded and/or unprocessed 5′ and 3′ ends, and that the endonucleolytic cleavage product normally released from the 5′ end of the precursor is absent in a sot1 null mutant. We postulate that SOT1 binding protects the 5′ extremity of the 23S–4.5S rRNA dicistron from exonucleolytic attack, and favours formation of the RNA structure that allows endonucleolytic processing of its 5′ and 3′ ends.     

核基因突变引起的拟南芥菜叶片花斑及其机制

细胞核基因突变引起的植物叶片花斑,是研究细胞器（特别是叶绿体）和细胞核之间信息交流的重要材料,也在园艺科学上有重要的应用价值。综述了拟南芥菜IM．VAR1、VAR2、CHM．CUE1、PAC．ATD2和VAR3等8个细胞核基因突变后引起的叶片花斑,主要包括这些基因所编码的蛋白质以及它们突变以后引起花斑的机制。     

A nucleus-encoded factor, CRR2, is essential for the expression of chloroplast ndhB in Arabidopsis

The chloroplast NDH complex, NAD(P)H dehydrogenase, reduces the plastoquinone pool non-photochemically and is involved in cyclic electron flow around photosystem I (PSI). A transient increase in chlorophyll fluorescence after turning off actinic light is a result of NDH activity. We focused on this subtle change in chlorophyll fluorescence to isolate nuclear mutants affected in chloroplast NDH activity in Arabidopsis by using chlorophyll fluorescence imaging. crr2-1 and crr2-2 (chlororespiratory reduction) are recessive mutant alleles in which accumulation of the NDH complex is impaired. Except for the defect in NDH activity, photosynthetic electron transport was unaffected. CRR2 encodes a member of the plant combinatorial and modular protein (PCMP) family consisting of more than 200 genes in Arabidopsis. CRR2 functions in the intergenic processing of chloroplast RNA between rps7 and ndhB, which is possibly essential for ndhB translation. We have determined the function of a PCMP family member, indicating that the family is closely related to pentatrico-peptide PPR proteins involved in the maturation steps of organellar RNA.     

Photosynthetic Redox Imbalance Governs Leaf Sectoring in the Arabidopsis thaliana Variegation Mutants immutans,spotty, var1, and var2

We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R² = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype.     

The FtsH protease heterocomplex in Arabidopsis: dispensability of type-B protease activity for proper chloroplast development

FtsH is an ATP-dependent metalloprotease present as a hexameric heterocomplex in thylakoid membranes. Encoded in the Arabidopsis thaliana YELLOW VARIEGATED2 (VAR2) locus, FtsH2 is one isoform among major Type A (FtsH1/5) and Type B (FtsH2/8) isomers. Mutants lacking FtsH2 (var2) and FtsH5 (var1) are characterized by a typical leaf-variegated phenotype. The functional importance of the catalytic center (comprised by the zinc binding domain) in FtsH2 was assessed in this study by generating transgenic plants that ectopically expressed FtsH2(488), a proteolytically inactive version of FtsH2. The resulting amino acid substitution inhibited FtsH protease activity in vivo when introduced into Escherichia coli FtsH. By contrast, expression of FtsH2(488) rescued not only leaf variegation in var2 but also seedling lethality in var2 ftsh8, suggesting that the protease activity of Type B isomers is completely dispensable, which implies that the chloroplastic FtsH complex has protease sites in excess and that they act redundantly rather than coordinately. However, expression of FtsH2(488) did not fully rescue leaf variegation in var1 var2 because the overall FtsH levels were reduced under this background. Applying an inducible promoter to our complementation analysis revealed that rescue of leaf variegation indeed depends on the overall amount of FtsH. Our results elucidate protein activity and its amount as important factors for the function of FtsH heterocomplexes that are composed of multiple isoforms in the thylakoid membrane.     

Arabidopsis variegation mutants: new insights into chloroplast biogenesis

Plant variegations are characterized by the presence of white sectors in normally green tissues and organs. Whereas the white sectors contain defective plastids that lack coloured pigments, the green sectors contain morphologically normal chloroplasts. Variegation mutants are defective in chloroplast developmental processes and arise due to mutations in nuclear or organellar genes. Despite their widespread occurrence in nature, only a few variegations have been studied at the molecular level. In this review, recent progress toward understanding two Arabidopsis variegations, immutans (im) and var2 is summarized. Both im and var2 are caused by nuclear recessive mutations and the responsible genes have been cloned and characterized. IMMUTANS functions as a chloroplast terminal oxidase that transfers electrons from the plastoquinol pool to oxygen. It appears to be a versatile electron sink, especially early in chloroplast development, when its function is crucial for carotenoid biosynthesis, and in excess light, when it serves as a 'safety valve'. IM also probably functions in chlororespiration. VAR2 encodes a chloroplast FtsH metalloprotease (termed AtFtsH2). Along with other AtFtsH proteins (AtFtsH1, 5 and 8), it forms complexes in the thylakoid membrane that are probably involved in the process of PSII repair during photoinhibition. A model has been proposed to explain the mechanism of var2 variegation, which suggests that threshold levels of FtsH complexes are required for green sector formation. It is concluded that studies on im and var2 have provided novel insights into nuclear-chloroplast interactions and, especially, into mechanisms of photoprotection.