Arabidopsis yellow variegated1 (VAR1) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes (ftsh1, ftsh6, and ftsh8) closely related to VAR1 and VAR2. This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1- and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of approximately 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1- and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.

Plastids undergo drastic morphological and physiological changes under different developmental stages and in response to environmental conditions. A key to accomplishing these transitions and maintaining homeostasis is the quality and quantity control of many plastid proteins by proteases and chaperones. Although a limited number of plastid proteases have been identified by biochemical approaches, recent progress in genome information revealed various plant proteases that are of prokaryotic origin and that are localized in chloroplasts. Of these, ATP-dependent proteases such as Clp, FtsH, and Lon are considered the major enzymes involved in processive degradation (gradual degradation to oligopeptides and amino acids). The basic architecture of plant ATP-dependent proteases is very similar to the architechture of bacterial enzymes, such as those in Escherichia coli, but plastid enzymes apparently have extraordinary numbers of isomers. Recent molecular genetic characterization in Arabidopsis has identified differential roles of these isomers. This review covers what is currently known about the types and function of plastid proteases together with our new observations.

In the photosynthetic apparatus, a major target of photodamage is the D1 reaction center protein of photosystem II (PSII). Photosynthetic organisms have developed a PSII repair cycle in which photodamaged D1 is selectively degraded. A thylakoid membrane-bound metalloprotease, FtsH, was shown to play a critical role in this process. Here, the effect of FtsHs in D1 degradation was investigated in Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) or FtsH5 (var1). Because these mutants are characterized by variegated leaves that sometimes complicate biochemical studies, we employed another mutation, fu-gaeri1 (fug1), that suppresses leaf variegation in var1 and var2 to examine D1 degradation. Two-dimensional blue native PAGE showed that var2 has less PSII supercomplex and more PSII intermediate lacking CP43, termed RC47, than the wild type under normal growth light. Moreover, our histochemical and quantitative analyses revealed that chloroplasts in var2 accumulate significant levels of reactive oxygen species, such as superoxide radical and hydrogen peroxide. These results indicate that the lack of FtsH2 leads to impaired D1 degradation at the step of RC47 formation in PSII repair and to photooxidative stress even under nonphotoinhibitory conditions. Our in vivo D1 degradation assays, carried out by nonvariegated var2 fug1 and var1 fug1 leaves, demonstrated that D1 degradation was impaired in different light conditions. Taken together, our results suggest the important role of chloroplastic FtsHs, which was not precisely examined in vivo. Attenuated D1 degradation in the nonvariegated mutants also suggests that leaf variegation seems to be independent of the PSII repair.

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.

Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species. Eukaryotic genomes contain sequences that have originated from DNA viruses and reverse-transcribing viruses, i.e., retroviruses, pararetroviruses (DNA viruses), and transposons. However, the sequences of non-retroviral RNA viruses, which are unable to convert their genomes to DNA, were until recently considered not to be integrated into eukaryotic nuclear genomes. We present evidence for multiple independent events of horizontal gene transfer from a wide range of RNA viruses, including plus-sense, minus-sense, and double-stranded RNA viruses, into the genomes of distantly related plant lineages. Some non-retroviral integrated RNA viral sequences are conserved across genera within a plant family, whereas others are retained only in a limited number of species in a genus. Integration profiles of non-retroviral integrated RNA viral sequences demonstrate the potential of these sequences to serve as powerful molecular tools for deciphering phylogenetic relationships among related plants. Moreover, this study highlights plants co-opting non-retroviral RNA virus sequences, and provides insights into plant genome evolution and interplay between non-reverse-transcribing RNA viruses and their hosts.

Background: A leaf‐variegated mutation var1 of Arabidopsis results in the development of abnormal plastids and the formation of a green/white sector. Genetic analysis of the var1 mutant indicated that it acts synergistically with another mutation var2, suggesting that the two genes are relevant. The VAR2 locus has been shown to encode a chloroplastic FtsH, an ATP‐dependent protease which is possibly involved in the degradation of thylakoid proteins and plastid development.Results: In this study we show that the VAR1 locus encodes a chloroplastic FtsH protein homologous to VAR2. VAR1 contains a conserved motif for ATPase and a metalloprotease characteristic to FtsH proteins, and is targeted into chloroplasts. A VAR1‐fusion protein synthesized in vitro exhibited ATPase activity and partial metalloprotease activity. The maximum yield of photochemistry, measured by chlorophyll fluorescence, showed that the var1 mutants were sensitive to photoinhibitory light exposure at 800 µmol/m²/s.Conclusion: VAR1 and VAR2 comprise an FtsH small gene family together with other FtsH genes in Arabidopsis. VAR1 as well as VAR2 may play an important role in degrading photodamaged subunits in photosystem II. Loss of VAR1 and VAR2 perhaps impairs the photoprotection mechanism and thylakoid development, causing leaf variegation as a consequence.