Katsuhiro Nakayama

Arabidopsis Cor15am Is a Chloroplast Stromal Protein That Has Cryoprotective Activity and Forms Oligomers

Many plants acquire increased freezing tolerance when they are exposed to nonfreezing temperatures of a certain duration. This process is known as cold acclimation and allows plants to protect themselves from freezing injury. A wide variety of polypeptides are induced during cold acclimation, among which is one encoded by COR15A in Arabidopsis (Arabidopsis thaliana). Previous studies showed that the COR15A gene encodes a small, plastid-targeted polypeptide that is processed to a mature form called Cor15am. In this study, we examined the biochemical properties and activities of Cor15am in more detail. We provide evidence that Cor15am localizes almost exclusively to the chloroplast stroma. In addition, the cold-regulated accumulation of Cor15am is affected by chloroplast functionality. Both gel-filtration chromatography and protein cross-linking reveal that Cor15am forms oligomers in the stroma of chloroplasts. Although Cor15am accumulates in response to low temperature, cold acclimation is not a prerequisite for oligomerization of Cor15am. Structural analysis suggests that Cor15am is composed of both ordered and random structures, and can stay soluble with small structural change after boiling and freeze-thaw treatments. Recombinant Cor15am exhibits in vitro cryoprotection of a freeze-labile enzyme, l-lactate dehydrogenase. Furthermore, Cor15am is capable of associating with l-lactate dehydrogenase in vitro and with potential stromal substrates in vivo. On the basis of these results, we propose that Arabidopsis Cor15am is a cryoprotective protein that forms oligomers in the chloroplast stroma, and that direct association of Cor15am with its substrates is part of its cryoprotective mechanism.

Coordination of Plastid Protein Import and Nuclear Gene Expression by Plastid-to-Nucleus Retrograde Signaling

Expression of nuclear-encoded plastid proteins and import of those proteins into plastids are indispensable for plastid biogenesis. One possible cellular mechanism that coordinates these two essential processes is retrograde signaling from plastids to the nucleus. However, the molecular details of how this signaling occurs remain elusive. Using the plastid protein import2 mutant of Arabidopsis (Arabidopsis thaliana), which lacks the atToc159 protein import receptor, we demonstrate that the expression of photosynthesis-related nuclear genes is tightly coordinated with their import into plastids. Down-regulation of photosynthesis-related nuclear genes is also observed in mutants lacking other components of the plastid protein import apparatus. Genetic studies indicate that the coordination of plastid protein import and nuclear gene expression is independent of proposed plastid signaling pathways such as the accumulation of Mg-protoporphyrin IX and the activity of ABA INSENSITIVE4 (ABI4). Instead, it may involve GUN1 and the transcription factor AtGLK. The expression level of AtGLK1 is tightly correlated with the expression of photosynthesis-related nuclear genes in mutants defective in plastid protein import. Furthermore, the activity of GUN1 appears to down-regulate the expression of AtGLK1 when plastids are dysfunctional. Based on these data, we suggest that defects in plastid protein import generate a signal that represses photosynthesis-related nuclear genes through repression of AtGLK1 expression but not through activation of ABI4.

cDNA Microarray Analysis of Rice Anther Genes under Chilling Stress at the Microsporogenesis Stage Revealed Two Genes with DNA Transposon Castaway in the 5′-Flanking Region

Rice is most chilling sensitive at the onset of microspore release. Chilling treatment at this stage causes male sterility. The gene expression profile during the microspore development process under chilling stress was revealed using a microarray that included 8,987 rice cDNAs. As many as 160 cDNAs were up- or down-regulated by chilling during the microspore release stage. RT-PCR analysis of 5 genes confirmed the microarray results. We identified 3 novel genes whose expression levels were remarkably changed by chilling in rice anther. A new cis element that includes a DNA transposon Castaway sequence was found in the 5′ upstream region of two genes which were conspicuously down-regulated by chilling temperatures in rice anther.

Evaluation of the Protective Activities of a Late Embryogenesis Abundant (LEA) Related Protein, Cor15am, during Various Stresses in Vitro

Arabidopsis Cor15am is a late embryogenesis abundant (LEA) related protein that has been shown to exhibit cryoprotective activity in vitro. In this study, we further investigated the mechanisms by which Cor15am protects substrates from inactivation. Although Cor15am did not exhibit refolding activity, it showed protective activity against various stresses in vitro. This might be attributable to the activity of Cor15am in attenuating the aggregation of the substrates. Our data indicate that Cor15am functions as a protectant against various stresses by preventing protein aggregation.

Identification and characterization of Cor413im proteins as novel components of the chloroplast inner envelope

Plastids are surrounded by two membrane layers, the outer and inner envelope membranes, which have various transport and metabolic activities. A number of envelope membrane proteins have been identified by biochemical approaches and have been assigned to specific functions. Despite those efforts, the chloroplast envelope membrane is expected to contain a number of as yet unidentified proteins that may affect specific aspects of plant growth and development. In this report, we identify and characterize a novel class of inner envelope membrane proteins, designated as Cor413 chloroplast inner envelope membrane group (Cor413im). Both in vivo and in vitro studies indicate that Cor413im proteins are targeted to the chloroplast envelope. Biochemical analyses of Cor413im1 demonstrate that it is an integral membrane protein in the inner envelope of chloroplasts. Quantitative real-time PCR analysis reveals that COR413IM1 is more abundant than COR413IM2 in cold-acclimated Arabidopsis leaves. The analyses of T-DNA insertion mutants indicate that a single copy of COR413IM genes is sufficient to provide normal freezing tolerance to Arabidopsis. Based on these data, we propose that Cor413im proteins are novel components that are targeted to the chloroplast inner envelope in response to low temperature.

Molecular Cloning and Characterization of a Novel β-1,3-Glucanase Gene from Rice

Beta-1,3-glucanases are referred to as pathogenesis-related proteins and they are also involved in several developmental processes. We isolated a cDNA for β-1,3-glucanase from rice anther and named it Oryza sativa glucanase 1 (Osg1). Phylogenetic analysis showed that Osg1 belonged to monocotyledonous endo-β-1,3-glucanase subgroup A. RT-PCR analysis revealed that Osg1 transcripts were present in leaves, roots, and anthers.

Retrograde Signaling Pathway from Plastid to Nucleus

Plastids are a diverse group of organelles found in plants and some parasites. Because genes encoding plastid proteins are divided between the nuclear and plastid genomes, coordinated expression of genes in two separate genomes is indispensable for plastid function. To coordinate nuclear gene expression with the functional or metabolic state of plastids, plant cells have acquired a retrograde signaling pathway from plastid to nucleus, also known as the plastid signaling pathway. To date, several metabolic processes within plastids have been shown to affect the expression of nuclear genes. Recent progress in this field has also revealed that the plastid signaling pathway interacts and shares common components with other intracellular signaling pathways. This review summarizes our current knowledge on retrograde signaling from plastid to nucleus in plant cells and its role in plant growth and development.

Expression analysis of genes for callose synthases and Rho-type small GTP-binding proteins that are related to callose synthesis in rice anther.

The most chilling-sensitive stage of rice has been found to be at the onset of microspore release. The microsporocytes produce a wall of callose between the primary cell wall and the plasma membrane, and it has been shown that precise regulation of callose synthesis and degradation in anther is essential for fertile pollen formation. In this study, genes for 10 callose synthases in the rice genome were fully annotated and phylogenetically analyzed. Expression analysis of these genes showed that OsGSL5, an ortholog of microsporogenesis-related AtGSL2, was specifically expressed in anthers, and was notably downregulated by cooling treatment. Gene expression profiles of Rho-type small GTP-binding proteins in rice anther were also analyzed. The mechanisms of callose synthesis in rice pollen formation and its relationships with cool tolerance are discussed.

Susceptibility to Coolness at the Young Microspore Stage under High Nitrogen Supply in Rice (Oryza Sativa L.). Proteome Analysis of Mature Anthers

Abstract In vitro pollen germination experiment using agar plates showed that the growth under high nitrogen conditions enhanced the damage to pollen germination ability caused by the cooling at the young microspore stage. To clarify the physiological factors related to this damage to pollen germination, we performed the comparative proteome analysis of mature anthers and identified proteins that were changed by high nitrogen conditions or high nitrogen plus cooling conditions. Proteins were extracted from mature anther samples and separated by two-dimensional polyacrylamide gel electrophoresis. By comparing anther protein maps of the samples collected from the plants grown under standard nitrogen conditions, high nitrogen conditions and high nitrogen plus cooling conditions, we found 11 protein spots, which varied with the treatment. These protein spots were identified based on the rice proteome database and/or peptide mass fingerprinting (PMF) analysis after digestion with trypsin. Digested samples were analyzed by matrix-assisted laser desorption/ionization-time flight mass spectrometry to produce PMF data. Database searches using these PMF data revealed the identities of 9 proteins. Seven of these proteins were polypeptides involved in cell elongation, stress responses and sugar metabolism. The relation between the fluctuations of these proteins and the decrease in pollen germination are discussed.

Effects of High Nitrogen Supply on the Susceptibility to Coolness at the Young Microspore Stage in Rice (Oryza sativa L.): Gene Expression Analysis in Mature Anthers

abstract Changes in gene expression patterns by high nitrogen (High-N) and High-N plus cooling at the young microspore stage (High-N-cooling) in rice mature anthers were analyzed by semiquantitative RT-PCR with gene specific primers. Gene expression of alpha-expansin 18 (EXPA18) was repressed under High-N-cooling. Beta-expansin 1 (EXPB1), putative aldehyde dehydrogenase (ALDH) and Fructokinase II (FKII) were upregulated under High-N. EXPB1 and FKII were highly expressed under High-N-cooling. Comprehensive examination of gene expression patterns of 26 alpha-expansins (EXPAs) and 16 beta-expansins (EXPB s) showed that all expansins (EXP s)except EXPA12 were expressed in the anthers. Gene expression of EXP s did not change under High-N except EXPA1, EXPB1 and EXPB5 which were upregulated. In total, 18 EXPAs and 6 EXPB s were repressed under High-N-cooling, and among these, EXPA18, EXPA19 and EXPA20 had high similarities in the amino acid sequences, suggesting that these three genes may constitute a distinct functional gene subfamily related to the decrease in the pollen germination ability.