Sungsoon Park

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green- and white-sectored leaves. Cells in the green sectors contain normal-appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.

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 (Ψ) synthase, which isomerizes uridine to Ψ 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.

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.

SUPPRESSOR OF VARIEGATION4, a new var2 suppressor locus, encodes a pioneer protein that is required for chloroplast biogenesis.

VAR2 is an integral thylakoid membrane protein and a member of the versatile FtsH class of metalloproteases in prokaryotes and eukaryotes. Recessive mutations in the VAR2 locus give rise to variegated plants (var2) that contain white sectors with abnormal plastids and green sectors with normal-appearing chloroplasts. In a continuing effort to isolate second-site suppressors of var2 variegation, we characterize in this report ems2505, a suppressor strain that has a virescent phenotype due to a missense mutation in At4g28590, the gene for a pioneer protein. We designated this gene SVR4 (for SUPPRESSOR OF VARIEGATION4) and the mutant allele in ems2505 as svr4-1. We demonstrate that SVR4 is located in chloroplasts and that svr4-1 single mutants are normal with respect to chloroplast anatomy and thylakoid membrane protein accumulation. However, they are modestly impaired in several aspects of photochemistry and have enhanced non-photochemical quenching (NPQ) capacity. A T-DNA insertion allele of SVR4, svr4-2, is seedling-lethal due to an early blockage of chloroplast development. We conclude that SVR4 is essential for chloroplast biogenesis, and hypothesize that SVR4 mediates some aspect of thylakoid structure or function that controls NPQ. We propose that in the suppressor strain, photoinhibitory pressure caused by a lack of VAR2 is ameliorated early in chloroplast development by enhanced NPQ capacity caused by reduced SVR4 activity. This would result in an increase in the number of chloroplasts that are able to surmount a threshold necessary to avoid photo-damage and thereby develop into functional chloroplasts.

Sequences Required for the Activity of PTOX (IMMUTANS), a Plastid Terminal Oxidase IN VITRO AND IN PLANTA MUTAGENESIS OF IRON-BINDING SITES AND A CONSERVED SEQUENCE THAT CORRESPONDS TO EXON 8

The thylakoid membranes of most photosynthetic organisms contain a terminal oxidase (PTOX, the product of the Arabidopsis IMMUTANS gene) that functions in the oxidation of the plastoquinone pool. PTOX and AOX are diiron carboxylate proteins, and based on crystal structures of other members of this protein class, a structural model of PTOX has been proposed in which the ligation sphere of the diiron center is composed of six conserved histidine and glutamate residues. We tested the functional significance of these residues by site-directed mutagenesis of PTOX in vitro and in planta, taking advantage null immutans alleles for the latter studies. These experiments showed that the six iron-binding sites do not tolerate change, even conservative ones. We also examined the significance of a conserved sequence in (or near) the PTOX active site that corresponds precisely to Exon 8 of the IM gene. In vitro and in planta mutagenesis revealed that conserved amino acids within this domain can be altered but that deletion of all or part of the domain abolishes activity. Because protein accumulates normally in the deletion mutants, the data suggest that the conformation of the Exon 8 sequence is important for PTOX activity. An allele of immutans (designated 3639) was identified that lacks the Exon 8 sequence; it does not accumulate PTOX protein. Chloroplast import assays revealed that mutant enzymes lacking Exon 8 have enhanced turnover. We conclude that the Exon 8 domain is required not only for PTOX activity but also for its stability.

Mechanism of REP27 Protein Action in the D1 Protein Turnover and Photosystem II Repair from Photodamage

The function of the REP27 protein (GenBank accession no. EF127650) in the photosystem II (PSII) repair process was elucidated. REP27 is a nucleus-encoded and chloroplast-targeted protein containing two tetratricopeptide repeat (TPR) motifs, two putative transmembrane domains, and an extended carboxyl (C)-terminal region. Cell fractionation and western-blot analysis localized the REP27 protein in the Chlamydomonas reinhardtii chloroplast thylakoids. A folding model for REP27 suggested chloroplast stroma localization for amino- and C-terminal regions as well as the two TPRs. A REP27 gene knockout strain of Chlamydomonas, termed the rep27 mutant, was employed for complementation studies. The rep27 mutant was aberrant in the PSII-repair process and had substantially lower than wild-type levels of D1 protein. Truncated REP27 cDNA constructs were made for complementation of rep27, whereby TPR1, TPR2, TPR1+TPR2, or the C-terminal domains were deleted. rep27-complemented strains minus the TPR motifs showed elevated levels of D1 in thylakoids, comparable to those in the wild type, but the PSII photochemical efficiency of these strains was not restored, suggesting that the functionality of the PSII reaction center could not be recovered in the absence of the TPR motifs. It is suggested that TPR motifs play a role in the functional activation of the newly integrated D1 protein in the PSII reaction center. rep27-complemented strains missing the C-terminal domain showed low levels of D1 protein in thylakoids as well as low PSII photochemical efficiency, comparable to those in the rep27 mutant. Therefore, the C-terminal domain is needed for a de novo biosynthesis and/or assembly of D1 in the photodamaged PSII template. We conclude that REP27 plays a dual role in the regulation of D1 protein turnover by facilitating cotranslational biosynthesis insertion (C-terminal domain) and activation (TPR motifs) of the nascent D1 during the PSII repair process.