Ryo Matsushima

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 Endoplasmic Reticulum-Derived Structure That Is Induced under Stress Conditions in Arabidopsis

The endoplasmic reticulum (ER) body is a characteristic structure derived from ER and is referred to as a proteinase-sorting system that assists the plant cell under various stress conditions. Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing green fluorescent protein fused with an ER retention signal. ER bodies were widely distributed in the epidermal cells of whole seedlings. In contrast, rosette leaves had no ER bodies. We found that wound stress induced the formation of many ER bodies in rosette leaves. ER bodies were also induced by treatment with methyl jasmonate (MeJA), a plant hormone involved in the defense against wounding and chewing by insects. The induction of ER bodies was suppressed by ethylene. An electron microscopic analysis showed that typical ER bodies were induced in the non-transgenic rosette leaves treated with MeJA. An experiment using coi1 and etr1-4 mutant plants showed that the induction of ER bodies was strictly coupled with the signal transduction of MeJA and ethylene. These results suggested that the formation of ER bodies is a novel and unique type of endomembrane system in the response of plant cells to environmental stresses. It is possible that the biological function of ER bodies is related to defense systems in higher plants.

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.

NAI1 Gene Encodes a Basic-Helix-Loop-Helix–Type Putative Transcription Factor That Regulates the Formation of an Endoplasmic Reticulum–Derived Structure, the ER Body

Plant cells develop various types of endoplasmic reticulum (ER)–derived structures with specific functions. ER body, an ER-derived compartment in Arabidopsis thaliana, is a spindle-shaped structure. The NAI1 gene regulates the development of ER bodies because mutation of NAI1 abolishes the formation of ER bodies. To better understand the role of NAI1, we cloned the NAI1 gene using a positional cloning strategy. The nai1-1 mutant had a single nucleotide change at an intron acceptor site of At2g22770 (NAI1 gene). Because of this mutation, aberrant splicing of NAI1 mRNA occurs in the nai1-1 mutant. NAI1 encodes a transcription factor that has a basic-helix-loop-helix (bHLH) domain. Transient expression of NAI1 induced ER bodies in the nai1-1 mutant. Two-dimensional electrophoresis and RT-PCR analyses showed that a putative lectin was depressed at both the mRNA and protein levels in nai1 mutants, as was a β-glucosidase (PYK10). Our results provide direct evidence that a bHLH protein plays a role in the formation of ER bodies.

Diversity and Formation of Endoplasmic Reticulum-Derived Compartments in Plants. Are These Compartments Specific to Plant Cells?

Unlike animals, plants are not able to escape from adverse circumstances. To cope with external stresses, plants modulate the endomembrane systems, especially the endoplasmic reticulum (ER), which is the most flexible and adaptable organelle ([Staehelin, 1997][1]). Alternatively, plants generate a

A Conserved, Mg2+-Dependent Exonuclease Degrades Organelle DNA during Arabidopsis Pollen Development

Extrachromosomal DNAs, present in plastids and mitochondria, are present in multiple copies and appear to be degraded in the mature pollen of most angiosperm species. This study, by deciphering a tissue-specific organelle DNA degradation mechanism, identifies the organellar nuclease that degrades these extrachromosomal DNAs during Arabidopsis pollen development. In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg2+-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.

A Rapid, Direct Observation Method to Isolate Mutants with Defects in Starch Grain Morphology in Rice

Starch forms transparent grains, referred to as starch grains (SGs), in amyloplasts. Despite the simple glucose polymer composition of starch, SGs exhibit different morphologies depending on plant species, especially in the endosperm of the Poaceae family. This study reports a novel method for preparing thin sections of endosperm without chemical fixation or resin embedding that allowed us to visualize subcellular SGs clearly. Using this method, we observed the SG morphologies of >5,000 mutagenized rice seeds and were able to isolate mutants in which SGs were morphologically altered. In five mutants, named ssg (substandard starch grain), increased numbers of small SGs (ssg1-ssg3), enlarged SGs (ssg4) and abnormal interior structures of SGs (ssg5) were observed. Amylopectin chain length distribution analysis and identification of the mutated gene suggested a possible allelic relationship between ssg1, ssg2, ssg3 and the previously isolated amylose-extender (ae) mutants, while ssg4 and ssg5 seemed to be novel mutants. Compared with conventional observation methods, the methods developed here are more effective for obtaining fine images of subcellular SGs and are suitable for the observation of a large number of samples.

Intraspecies Variation in the Kanzawa Spider Mite Differentially Affects Induced Defensive Response in Lima Bean Plants

The Kanzawa spider mite, Tetranychus kanzawai, is a polyphagous herbivore that feeds on various plant families, including the Leguminacae. Scars made by the mite on lima bean leaves (Phaseolus lunatus) were classified into two types: white and red. We obtained two strains of mites—“White” and “Red”—by selecting individual mites based on the color of the scars. Damage made by the Red strain induced the expression of genes for both basic chitinase, which was downstream of the jasmonic acid (JA) signaling pathway, and acidic chitinase, which was downstream of the salicylic acid (SA) signaling pathway. White strain mites also induced the expression of the basic chitinase gene in infested leaves but they only slightly induced the acidic chitinase gene. The Red genotype was dominant over the White for the induction of the acidic chitinase gene. The amount of endogenous salicylates in leaves increased significantly when infested by Red strain mites but did not increase when infested by White strain mites. JA and SA are known to be involved in the production of lima bean leaf volatiles induced by T. urticae. The blend of volatiles emitted from leaves infested by the Red strain were qualitatively different from those infested by the White strain, suggesting that the SA and JA signaling pathways are differently involved in the production of lima bean leaf volatiles induced by T. kanzawai of different strains.

Amyloplast-Localized SUBSTANDARD STARCH GRAIN4 Protein Influences the Size of Starch Grains in Rice Endosperm

A novel amyloplast-localized protein, SSG4, influences the size of starch grains in rice endosperm. Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.

Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes

Highlight Starch biosynthetic enzymes in rice endosperm are physically associated with each other and form enzymatically active multiple protein–protein complexes, several of which were common to cereals while others were unique.