Eiko Miura

The variegated mutants lacking chloroplastic FtsHs are defective in D1 degradation and accumulate reactive oxygen species.

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 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.

White Leaf Sectors in yellow variegated2 Are Formed by Viable Cells with Undifferentiated Plastids

The yellow variegated2 (var2) is one of the best-characterized Arabidopsis (Arabidopsis thaliana) mutants showing leaf variegation. Leaf variegation of var2 results from the loss of an ATP-dependent metalloprotease, FtsH2, which is a major component of the FtsH heterocomplex in thylakoid membranes. While the functional role of FtsH2 in protein quality control has been extensively studied, the physiological state of plastids in white tissues of the var2 is not well characterized. Here we show that the white tissue in var2 is neither the result of photobleaching nor enhanced senescence. Visualization of plastids by plastid-targeted green fluorescent protein revealed that plastids in the white sector are distinct and have undifferentiated characteristics. The plastids are also distinct in that they contain large nucleoids, a complex structure of plastid DNA and proteins, that are typically found in undifferentiated plastids. Comparative analyses of protein profiles from green and white tissues suggested that the difference was observed in the proteins related to photosynthesis but not due to proteins of other organelles. Thus, cells in the white tissue are viable and their defect is limited to plastid function. The plastid accumulates normal levels of chloroplast transcripts, whereas a substantial repression of nuclear-encoded photosynthetic genes was evident in the white sector. Based upon these results, we inferred that the white sectors in var2 are made by viable cells that have plastids arrested in thylakoid formation. A proposed model to form the variegated sector in var2 is provided.

Arrested differentiation of proplastids into chloroplasts in variegated leaves characterized by plastid ultrastructure and nucleoid morphology

Leaf variegation is seen in many ornamental plants and is often caused by a cell-lineage type formation of white sectors lacking functional chloroplasts. A mutant showing such leaf variegation is viable and is therefore suitable for studying chloroplast development. In this study, the formation of white sectors was temporally investigated in the Arabidopsis leaf-variegated mutant var2. Green sectors were found to emerge from white sectors after the formation of the first true leaf. Transmission electron microscopic examination of plastid ultrastructures confirmed that the peripheral zone in the var2 shoot meristem contained proplastids but lacked developing chloroplasts that were normally detected in wild type. These data suggest that chloroplast development proceeds very slowly in var2 variegated leaves. A notable feature in var2 is that the plastids in white sectors contain remarkable globular vacuolated membranes and prolamellar body-like structures. Although defective plastids were hardly observed in shoot meristems, they began to accumulate during early leaf development. Consistent with these observations, large plastid nucleoids detected in white sectors by DNA-specific fluorescent dyes were characteristic of those found in proplastids and were clearly distinguished from those in chloroplasts. These results strongly imply that in white sectors, differentiation of plastids into chloroplasts is arrested at the early stage of thylakoid development. Interestingly, large plastid nucleoids were detected in variegated sectors from species other than Arabidopsis. Thus, plastids in variegated leaves appear to share a common feature and represent a novel plastid type.

Comparative transcriptome analysis of green/white variegated sectors in Arabidopsis yellow variegated2: responses to oxidative and other stresses in white sectors

The yellow variegated2 (var2) mutant in Arabidopsis thaliana has been studied as a typical leaf-variegated mutant whose defect results from the lack of FtsH2 metalloprotease in chloroplasts. The var2 green sectors suffer from photo-oxidative stress and accumulate high levels of reactive oxygen species (ROS) because of compromised Photosystem II repair. This study investigated and compared microarray-based expression profiles of green and white sectors of var2 leaves. Results suggest that ROS that accumulate in chloroplasts of var2 green sectors do not cause much significant change in the transcriptional profile related to ROS signalling and scavenging. By contrast, transcriptome in the white sectors apparently differs from those in the green sectors and wild type. Numerous genes related to photosynthesis and chloroplast functions were repressed in the white sectors. Furthermore, many genes related to oxidative stress were up-regulated. Among them, ROS scavenging genes were specifically examined, such as Cu/Zn superoxide dismutase 2 (CSD2), that were apparently up-regulated in white but not in the green sectors. Up-regulation of CSD2 appears to be partly attributable to the lack of a microRNA (miR398) in the white sectors. It was concluded that the white sectors exhibit a response to oxidative and other stresses, including CSD2 up-regulation, which might be commonly found in tissues with abnormal chloroplast differentiation.

Allelic characterization of the leaf-variegated mutation var2 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.

Ion gradients in xylem exudate and guttation fluid related to tissue ion levels along primary leaves of barley.

The concentration of ions in plant cells and tissues is an essential factor in determining physiological function. In the present study, we established that concentration gradients of mobile ions exist in both xylem exudates and tissues within a barley (Hordeum vulgare) primary leaf. For K(+) and NO3 (-) , ion concentrations generally decreased from the leaf base to the tip in both xylem exudates and tissues. Ion gradients were also found for Pi and Cl(-) in the xylem. The hydathode strongly absorbed Pi and re-translocated it to the rest of the plant, whereas Cl(-) was extruded. The ion concentration gradients developed early during leaf growth, increased as the tissue aged and remained under both high and low transpiration conditions. Measurement of the expression profiles of Pi, K(+) and NO3 (-) transporters along the longitudinal axis of the leaf revealed that some transporters are more expressed at the hydathode, but for most transporters, there was no significant variation along the leaf. The mechanisms by which longitudinal ion gradients develop in leaves and their physiological functions are discussed.

Reactive oxygen species derived from impaired quality control of Photosystem II are irrelevant to plasma-membrane NADPH oxidases.

Protein quality control plays an important role in the photosynthetic apparatus because its components receive excess light energy and are susceptible to photooxidative damage. In chloroplasts, photodamage is targeted to the D1 protein of Photosystem II (PSII). The coordinated PSII repair cycle (PSII disassembly, D1 degradation and synthesis, and PSII reassembly) is necessary to mitigate photoinhibition. A thylakoid protease FtsH, which is formed predominantly as a heteromeric complex with two isoforms of FtsH2 and FtsH5 in Arabidopsis, is the major protease involved in PSII repair. A mutant lacking FtsH2 (termed var2) shows compromised D1 degradation. Furthermore, var2 accumulates high levels of chloroplastic reactive oxygen species (cpROS), reflecting photooxidative stress without functional PSII repair. To examine if the cpROS produced in var2 are connected to a ROS signaling pathway mediated by plasma membrane NADPH oxidase (encoded by AtRbohD or AtRbohF), we generated mutants in which either Rboh gene was inactivated under var2 background. Lack of NADPH oxidases had little or no impact on cpROS accumulation. It seems unlikely that cpROS in var2 activate plasma membrane NADPH oxidases to enhance ROS production and the signaling pathway. Mutants that are defective in PSII repair might be valuable for investigating cpROS and their physiological roles.

A Novel Link between Chloroplast Development and Stress Response Lessoned by Leaf-Variegated Mutant

Recessive mutations are known to give rise to cell lineage-type leaf variegation that forms green and white sectors due to arrested chloroplast development. The yellow variegated 2 (var2) mutant in Arabidopsis thaliana has been studied as a typical leaf-variegated mutant whose defect results from the lack of FtsH2 metalloprotease in chloroplasts. To understand physiological properties of variegated sectors, gene expression profiles were investigated between green and white sectors of var2 leaves. Consistent with impaired thylakoid formation, a substantial number of genes related to photosynthesis and chloroplast functions were repressed in white sectors. In addition, many genes were up-regulated in white sectors. Since var2 leaves suffer from photooxidative stress and accumulate high levels of reactive oxygen species (ROS) due to compromised Photosystem II repair, we focused ROS scavenging genes such as Cu/Zn superoxide dismutase 2 (CSD2). Activation of CSD2 was specific to white sectors and appeared to be under the control of copper availability. A lack of thylakoid membranes in white sectors perhaps leads to excess free copper and iron conditions, thus white sectors mimic a copper sufficient condition. We infer that CSD2 acts not only on ROS detoxification but also on copper buffering. Interestingly, an up-regulation of Cu/Zn SOD was commonly observed in various variegated leaves. Our findings thus highlight the crucial role for the control of oxidative stress and free metals in variegated leaf sectors.

Importance of the Balance Between Protein Synthesis and Degradation in Chloroplasts Revealed by the Studies of Arabidopsis Yellow Variegated Mutants

In Arabidopsis, yellow variegated (var) is one of the best characterized among the mutants showing leaf variegation. VAR1 and VAR2 encode different FtsH proteases (FtsH5 and FtsH2, respectively) involved in the quality control of chloroplast proteins, particularly in the turnover of D1 reaction center protein. To investigate the precise role of FtsH in protein degradation and understand how the variegated sectors are formed, we recently identified a suppressor of var2 named fug1. Characterization of the fug1 locus showed that it encodes a chloroplast translation initiation factor 2 (cpIF2) and indeed acts on chloroplast protein synthesis. Based on this suppressor analysis, we hypothesized that the balance between protein synthesis and degradation determines leaf variegation in var mutants. Here, we present another type of evidence that supports our hypothesis, by supplementing var2 with exogenous translational inhibitors.