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.     

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...     

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.     

An Arabidopsis pentatricopeptide repeat protein, SUPPRESSOR OF VARIEGATION7, is required for FtsH-mediated chloroplast biogenesis

The Arabidopsis (Arabidopsis thaliana) yellow variegated2 (var2) mutant has green- and white-sectored leaves due to loss of VAR2, a subunit of the chloroplast FtsH protease/chaperone complex. Suppressor screens are a valuable tool to gain insight into VAR2 function and the mechanism of var2 variegation. Here, we report the molecular characterization of 004-003, a line in which var2 variegation is suppressed. We found that the suppression phenotype in this line is caused by lack of a chloroplast pentatricopeptide repeat (PPR) protein that we named SUPPRESSOR OF VARIEGATION7 (SVR7). PPR proteins contain tandemly repeated PPR motifs that bind specific RNAs, and they are thought to be central regulators of chloroplast and mitochondrial nucleic acid metabolism in plants. The svr7 mutant has defects in chloroplast ribosomal RNA (rRNA) processing that are different from those in other svr mutants, and these defects are correlated with reductions in the accumulation of some chloroplast proteins, directly or indirectly. We also found that whereas var2 displays a leaf variegation phenotype at 22°C, it has a pronounced chlorosis phenotype at 8°C that is correlated with defects in chloroplast rRNA processing and a drastic reduction in chloroplast protein accumulation. Surprisingly, the cold-induced phenotype of var2 cannot be suppressed by svr7. Our results strengthen the previously established linkage between var2 variegation and chloroplast rRNA processing/chloroplast translation, and they also point toward the possibility that VAR2 mediates different activities in chloroplast biogenesis at normal and chilling temperatures.     

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.     

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.     

Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts

Several chloroplast proteases have been characterized in recent years. The ATP-dependent chloroplast proteases Clp and FtsH stand out because they form multi-subunit complexes consisting of different gene products. Surprisingly, both green and non-green plastids appear to contain a similar soluble Clp core proteolytic complex, consisting of five ClpP proteases, their non-catalytic ClpR homologs, and two ClpS homologs that have unknown function. Analyses of single and double FtsH1, FtsH2, FtsH5 and FtsH8 mutants, and overexpression of FtsH proteins in these Arabidopsis thaliana mutants show partial redundancies within pairs of closely related FtsH thylakoid proteins. The presence of at least one member of each pair is essential for functional accumulation. Other chloroplast proteases have also been identified recently. Future challenges include the identification of substrate recognition mechanisms and elucidating the role of proteases in chloroplast biogenesis and function.     

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.     

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.     

The significance of Arabidopsis AAA proteases for activity and assembly/stability of mitochondrial OXPHOS complexes

The Arabidopsis genome encodes four mitochondrially localized adenosine 5'-triphosphate-dependent metalloproteases called FtsH or AAA proteases. All of them span the inner mitochondrial membrane but the catalytic site of two of them (AtFtsH4 and AtFtsH11) faces the intermembrane space, while AtFtsH3 and AtFtsH10 face the matrix. We used a combination of blue-native polyacrylamide gel electrophoresis and histochemical staining to reveal the consequences of the loss of one of mitochondrial FtsHs on the efficiency of the oxidative phosphorylation system in Arabidopsis mitochondria. To address this issue, we have selected homozygous lines of respective transferred DNA (T-DNA) insertional mutants. A decrease in the activity of complexes I and V but not complex IV was observed in the ftsh mutants, except for the mutant lacking functional FtsH11. The reduced activity of complexes I and V was well correlated with a decreased protein level of these complexes. Western blots experiments using specific antibodies against complex V subunits showed a significant reduction of these subunits only in the ftsh4 mutant. Taken together, our results reveal a role of FtsH3, FtsH4 and FtsH10 proteases in the biogenesis of a plant oxidative phosphorylation system.     

Activation of the heterotrimeric G protein α-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 ( thf1 ), indicating that inactivation or activation of the endogenous G protein α-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Gα-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1 . Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2 . Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1 . This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.     

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 hetero-hexameric nature of a chloroplast AAA+ FtsH protease contributes to its thermodynamic stability

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4:2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that 'type B' subunits (FtsH2 and FtsH8) were 2-3 fold more abundant than 'type A' (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two 'type A' and four 'type B' subunits.     

Allelic characterization of the leaf-variegated mutation <Emphasis Type="Italic">var2</Emphasis> identifies the conserved amino acid residues of FtsH that are important for ATP hydrolysis and proteolysis

Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to be allelic to var2. Each mutant was shown to express a different degree of variegation, and the formation of white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature. VAR1/FtsH5 and VAR2/FtsH2 levels were mutually affected even in the weak alleles, confirming our previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino acid substitution. Three of the five amino acid substitutions occurred in Walker A and B motifs of the ATP-binding site, and one occurred in the central pore motif. These mutations were considered to profoundly suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained no obvious signature motifs, but a neighboring sequence, Gly–Ala–Asp, was highly conserved among the members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these results, we propose that the conserved Gly–Ala–Asp motif plays an important role in FtsH activity. Thus, characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.