Hye Sun Cho

Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803

Positive phototaxis systems have been well studied in bacteria; however, the photoreceptor(s) and their downstream signaling components that are responsible for negative phototaxis are poorly understood. Negative phototaxis sensory systems are important for cyanobacteria, oxygenic photosynthetic organisms that must contend with reactive oxygen species generated by an abundance of pigment photosensitizers. The unicellular cyanobacterium Synechocystis sp. PCC6803 exhibits type IV pilus-dependent negative phototaxis in response to unidirectional UV-A illumination. Using a reverse genetic approach, together with biochemical, molecular genetic, and RNA expression profiling analyses, we show that the cyanobacteriochrome locus (slr1212/uirS) of Synechocystis and two adjacent response regulator loci (slr1213/uirR and the PatA-type regulator slr1214/lsiR) encode a UV-A–activated signaling system that is required for negative phototaxis. We propose that UirS, which is membrane-associated via its ETR1 domain, functions as a UV-A photosensor directing expression of lsiR via release of bound UirR, which targets the lsiR promoter. Constitutive expression of LsiR induces negative phototaxis under conditions that normally promote positive phototaxis. Also induced by other stresses, LsiR thus integrates light inputs from multiple photosensors to determine the direction of movement.

DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4′,6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack

Reactive oxygen species (ROS) are inevitably generated in aerobic organisms as by-products of normal metabolism or as the result of defense and development. ROS readily oxidize methionine (Met) residues in proteins/peptides to form Met-R-sulfoxide or Met-S-sulfoxide, causing inactivation or malfunction of the proteins. A pepper (Capsicum annuum) methionine sulfoxide reductase B2 gene (CaMsrB2) was isolated, and its roles in plant defense were studied. CaMsrB2 was down-regulated upon inoculation with either incompatible or compatible pathogens. The down-regulation, however, was restored to the original expression levels only in a compatible interaction. Gain-of-function studies using tomato (Solanum lycopersicum) plants transformed with CaMsrB2 resulted in enhanced resistance to Phytophthora capsici and Phytophthora infestans. Inversely, loss-of-function studies of CaMsrB2 using virus-induced gene silencing in pepper plants (cv Early Calwonder-30R) resulted in accelerated cell death from an incompatible bacterial pathogen, Xanthomonas axonopodis pv vesicatoria (Xav) race 1, and enhanced susceptibility to a compatible bacterial pathogen, virulent X. axonopodis pv vesicatoria race 3. Measurement of ROS levels in CaMsrB2-silenced pepper plants revealed that suppression of CaMsrB2 increased the production of ROS, which in turn resulted in the acceleration of cell death via accumulation of ROS. In contrast, the CaMsrB2-transgenic tomato plants showed reduced production of hydrogen peroxide. Taken together, our results suggest that the plant MsrBs have novel functions in active defense against pathogens via the regulation of cell redox status.

Classification of rice (oryza sativa l. japonica nipponbare) immunophilins (fkbps, cyps) and expression patterns under water stress

BackgroundFK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.ResultsFKBP and CYP proteins in rice (Oryza sativa cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with Arabidopsis and Chlamydomonas or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of Arabidopsis IMMs. However, some genes were novel, did not match with those of Arabidopsis and Chlamydomonas, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein.ConclusionLike other green photosynthetic organisms such as Arabidopsis (23 FKBPs and 29 CYPs) and Chlamydomonas (23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, Arabidopsis and Chlamydomonas. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice.

CHRK1, a chitinase-related receptor-like kinase, interacts with NtPUB4, an armadillo repeat protein, in tobacco.

CHRK1 is a receptor-like kinase containing a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. The previous study indicated that CHRK1 plays a role in a signaling pathway regulating plant development and the endogenous cytokinin levels. In this study, we identified NtPUB4 as a CHRK1-interacting protein using yeast two-hybrid screening. NtPUB4 contains the U-box and five arm repeats, and is homologous to Arabidopsis AtPUB4 with unknown function and to Brassica arm repeat containing 1 (ARC1) that interacts with SRK receptor-like kinases during self-incompatibility response. The arm repeats of NtPUB4 are important for the interaction with CHRK1. CHRK1-NtPUB4 interaction was confirmed by in vitro binding assay using the recombinant proteins. NtPUB4 exhibited spatial and temporal expression patterns that are very similar to those of CHRK1. Finally, GFP and RFP fusion experiments demonstrated that both CHRK1 and NtPUB4 are localized at the plasma membrane in vivo. These results strongly indicate that NtPUB4 is an interacting partner of CHRK1 receptor-like kinase, and is likely involved in modulating the plant developmental signaling pathway mediated by CHRK1.

The rice thylakoid lumenal cyclophilin OsCYP20-2 confers enhanced environmental stress tolerance in tobacco and Arabidopsis

The role that the putative thylakoid lumenal cyclophilin (CYP) CYP20-2 locates in the thylakoid, and whether CYP20-2 is an essential gene, have not yet been elucidated. Here, we show that CYP20-2 is well conserved in several photosynthetic plants and that the transcript level of the rice OsCYP20-2 gene is highly regulated under abiotic stress. We found that ectopic expression of rice OsCYP20-2 in both tobacco and Arabidopsis confers enhanced tolerance to osmotic stress and extremely high light. Based on these results, we suggest that although the exact biochemical function of OsCYP20-2 in the thylakoid lumen (TL) remains unclear, it may be involved in photosynthetic acclimation to help plants cope with environmental stress; the OsCYP20-2 gene may be a candidate for enhancing multiple abiotic stress tolerance.

Inactivation of Organellar Glutamyl- and Seryl-tRNA Synthetases Leads to Developmental Arrest of Chloroplasts and Mitochondria in Higher Plants

Aminoacyl-tRNA synthetases (ARSs) are key enzymes involved in protein translation, and both cytosolic and organellar forms are present in the genomes of eukaryotes. In this study, we investigated cellular effects of depletion of organellar forms of ARS using virus-induced gene silencing (VIGS) in Nicotiana benthamiana. VIGS of NbERS and NbSRS, which encode organellar GluRS and SerRS, respectively, resulted in a severe leaf-yellowing phenotype. The NbERS and NbSRS genes were ubiquitously expressed in plant tissues, and induced in response to light. Green fluorescent protein (GFP) fusion proteins of the full-length glutamyl-tRNA synthetase (ERS) and seryl-tRNA synthetase (SRS) of Arabidopsis and GFP fusions to the N-terminal extension of these proteins were all dualtargeted to chloroplasts and mitochondria. At the cell level, depletion of NbERS and NbSRS resulted in dramatically reduced numbers of chloroplasts with reduced sizes and chlorophyll content. The numbers and/or physiology of mitochondria were also severely affected. The abnormal chloroplasts lacked most of the thylakoid membranes and appeared to be degenerating, whereas some of them showed doublet morphology, indicating defective chloroplast division. Pulse-field gel electrophoresis analyses demonstrated that chloroplast DNA in subgenomic sizes is the predominant form in the abnormal chloroplasts. Interestingly, despite severe abnormalities in chloroplasts and mitochondria, expression of many nuclear genes encoding chloroplastor mitochondria-targeted proteins, and chlorophyll biosynthesis genes remained unchanged in the ERS and SRS VIGS lines. This is the first report to analyze the effect of ARS disruption on organelle development in plants.

CHRK1, a chitinase-related receptor-like kinase, plays a role in plant development and cytokinin homeostasis in tobacco

CHRK1 encodes a receptor-like kinase that contains a chitinase-related sequence in the extracellular domain in Nicotiana tabacum. In this study, we showed that CHRK1 is mainly expressed in the shoot apex region including leaf primordia and young leaves, and germinating seedlings and vascular tissues, based on GUS activity of transgenic tobacco plants carrying the CHRK1promoter-GUS fusion gene. Transgenic tobacco plants in which CHRK1 expression was suppressed exhibited pleiotrophic developmental abnormality, including formation of proliferating shooty calli from emerging seedlings and severely altered seedling development. At the cellular level, ectopic cell proliferation, reduced cell specificity, and aberrant chloroplast development were observed. The transgenic lines contained 3-fold higher level of cytokinin than the wild-type plants. Consistently, the transgenic seedlings exhibited a typical cytokinin response in the absence of hormone, such as deetiolation under the dark. Based on these results, we propose that CHRK1 is involved in a developmental signaling pathway regulating cell proliferation/differentiation and the endogenous cytokinin levels in tobacco.

Expression patterns of diverse genes in response to gamma irradiation inNicotiana tabacum

We investigated the expression patterns of diverse genes at various time points after gamma irradiation of young tobacco plants. The first group of genes showed stimulation of transcript levels upon gamma irradiation, although their induction patterns varied. This group included glutathione-S-transferase, peroxidase, superoxide dismutase, and catalase. A second group, with post-irradiation reduction of transcripts, included genes encoding cytosolic ascorbate peroxidase, stromal ascorbate peroxidase, and a TMK1 receptor-like kinase. The third group of genes either showed no change in transcript levels or exhibited irregular patterns. These included genes encoding PR1a (pathogenesis-related protein), tobacco Ca++-dependent protein kinase, the β-subunit of translational initiation factor 2B, and CHRK1, a chitinase-related receptor-like kinase. Thus, various genes displayed differential patterns of gene expression in response to gamma irradiation in tobacco plants, thereby suggesting a complex signaling mechanism is involved in the irradiation-induced defense by plants. In addition, many stress-responsive genes exhibited gene expression patterns upon gamma irradiation that differed from those resulting from other biotic and abiotic stresses. With the knowledge of distinctive expression patterns of diverse genes, irradiation-indicating marker plants could be developed by engineering and monitoring multiple radiation-responsive genes.

Multiple genes encoding serine/threonine protein phosphatases and their differential expression in Nicotiana tabacum.

Five cDNA clones encoding the catalytic subunits of Ser/Thr protein phosphatases (PP) of Nicotiana tabacum were described. Among them, three clones (NPP1, NPP2, and NPP3) encoded type 1 PP (PP1), whereas the rest of the clones (NPP4 and NPP5) encoded type 2A PP (PP2A). These cDNA clones exhibited high sequence identity in the PP core region to the corresponding genes from animals and plants. NPP1 mRNA was predominantly expressed in flowers, whereas NPP5 mRNA was mainly detected in leaves and flowers. In contrast, the transcripts of NPP2, NPP3, and NPP4 genes were present in all tissues examined. Throughout flower development, NPP1, NPP2, NPP5 mRNAs were expressed without any significant variation at the steady-state level. Genomic Southern blot showed that tobacco genome contained multiple genes and/or pseudogenes for both type 1 and type 2A phosphatases.