Beom-Gi Kim

Expression of StMYB1R-1, a Novel Potato Single MYB-like domain Transcription Factor, Increases Drought Tolerance

Potato (Solanum tuberosum) is relatively vulnerable to abiotic stress conditions such as drought, but the tolerance mechanisms for such stresses in potato are largely unknown. To identify stress-related factors in potato, we previously carried out a genetic screen of potato plants exposed to abiotic environmental stress conditions using reverse northern-blot analysis. A cDNA encoding a putative R1-type MYB-like transcription factor (StMYB1R-1) was identified as a putative stress-response gene. Here, the transcript levels of StMYB1R-1 were enhanced in response to several environmental stresses in addition to drought but were unaffected by biotic stresses. The results of intracellular targeting and quadruple 9-mer protein-binding microarray analysis indicated that StMYB1R-1 localizes to the nucleus and binds to the DNA sequence G/AGATAA. Overexpression of a StMYB1R-1 transgene in potato plants improved plant tolerance to drought stress while having no significant effects on other agricultural traits. Transgenic plants exhibited reduced rates of water loss and more rapid stomatal closing than wild-type plants under drought stress conditions. In addition, overexpression of StMYB1R-1 enhanced the expression of drought-regulated genes such as AtHB-7, RD28, ALDH22a1, and ERD1-like. Thus, the expression of StMYB1R-1 in potato enhanced drought tolerance via regulation of water loss. These results indicated that StMYB1R-1 functions as a transcription factor involved in the activation of drought-related genes.

Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of seed dormancy and adaptation to abiotic stresses. Previous work identified OsPYL/RCARs as functional ABA receptors regulating ABA-dependent gene expression in Oryza sativa. OsPYL/RCARs thus are considered to be good candidate genes for improvement of abiotic stress tolerance in crops. This work demonstrates that the cytosolic ABA receptor OsPYL/RCAR5 in O. sativa functions as a positive regulator of abiotic stress-responsive gene expression. The constitutive expression of OsPYL/RCAR5 in rice driven by the Zea mays ubiquitin promoter induced the expression of many stress-responsive genes even under normal growth conditions and resulted in improved drought and salt stress tolerance in rice. However, it slightly reduced plant height under paddy field conditions and severely reduced total seed yield. This suggests that, although exogenous expression of OsPYL/RCAR5 is able to improve abiotic stress tolerance in rice, fine regulation of its expression will be required to avoid deleterious effects on agricultural traits.

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.

Overexpression of the mitogen-activated protein kinase gene OsMAPK33 enhances sensitivity to salt stress in rice ( Oryza sativa L.)

Mitogen-activated protein kinases (MAPK) signalling cascades are activated by extracellular stimuli such as environmental stresses and pathogens in higher eukaryotic plants. To know more about MAPK signalling in plants, a MAPK cDNA clone, OsMAPK33, was isolated from rice. The gene is mainly induced by drought stress. In phylogenetic analysis, OsMAPK33 (Os02g0148100) showed approximately 47–93% identity at the amino acid level with other plant MAPKs. It was found to exhibit organ-specific expression with relatively higher expression in leaves as compared with roots or stems, and to exist as a single copy in the rice genome. To investigate the biological functions of OsMAPK33 in rice MAPK signalling, transgenic rice plants that either overexpressed or suppressed OsMAPK33 were made. Under dehydration conditions, the suppressed lines showed lower osmotic potential compared with that of wild-type plants, suggesting a role of OsMAPK33 in osmotic homeostasis. Nonetheless, the suppressed lines did not display any significant difference in drought tolerance compared with their wild-type plants. With increased salinity, there was still no difference in salt tolerance between OsMAPK33-suppressed lines and their wild-type plants. However, the overexpressing lines showed greater reduction in biomass accumulation and higher sodium uptake into cells, resulting in a lower K+/Na+ ratio inside the cell than that in the wild-type plants and OsMAPK33-suppressed lines. These results suggest that OsMAPK33 could play a negative role in salt tolerance through unfavourable ion homeostasis. Gene expression profiling of OsMAPK33 transgenic lines through rice DNA chip analysis showed that OsMAPK33 altered expression of genes involved in ion transport. Further characterization of downstream components will elucidate various biological functions of this novel rice MAPK.

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.

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Metabolite Profiling of Diverse Rice Germplasm and Identification of Conserved Metabolic Markers of Rice Roots in Response to Long-Term Mild Salinity Stress

The sensitivity of rice to salt stress greatly depends on growth stages, organ types and cultivars. Especially, the roots of young rice seedlings are highly salt-sensitive organs that limit plant growth, even under mild soil salinity conditions. In an attempt to identify metabolic markers of rice roots responding to salt stress, metabolite profiling was performed by 1H-NMR spectroscopy in 38 rice genotypes that varied in biomass accumulation under long-term mild salinity condition. Multivariate statistical analysis showed separation of the control and salt-treated rice roots and rice genotypes with differential growth potential. By quantitative analyses of 1H-NMR data, five conserved salt-responsive metabolic markers of rice roots were identified. Sucrose, allantoin and glutamate accumulated by salt stress, whereas the levels of glutamine and alanine decreased. A positive correlation of metabolite changes with growth potential and salt tolerance of rice genotypes was observed for allantoin and glutamine. Adjustment of nitrogen metabolism in rice roots is likely to be closely related to maintain the growth potential and increase the stress tolerance of rice.

Expression Analysis of Proline Metabolism‐related Genes From Halophyte Arabis stelleri under Osmotic Stress Conditions

Arabis stelleri var. japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana. Using an A. thaliana microarray chip, we determined changes in the expression of approximately 2 800 genes between A. stelleri plants treated with 0.2 M mannitol versus mock-treated plants. The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system, stimulus response, stress response, chemical stimulus response, and defense response. The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A. stelleri var. japonica seedlings treated with 0.2 M mannitol, 0.2 M sorbitol, and 0.2 M NaCl. The expression of Δ¹ -pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol. The proline dehydrogenase gene, which is known to be repressed by dehydration stress and induced by free L-proline, was induced at an early stage by mannitol treatment, but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl. The level of free L-proline accumulation increased progressively in response to treatments with mannitol, sorbitol, and NaCl. Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol. These findings demonstrate that the osmotic tolerance of the novel halophyte, Arabis stelleri, is associated with the accumulation of L-proline.

Ectopic expression of a hot pepper bZIP-like transcription factor in potato enhances drought tolerance without decreasing tuber yield

Over-expression of group A bZIP transcription factor genes in plants improves abiotic stress tolerance but usually reduces yields. Thus, there have been several efforts to overcome yield penalty in transgenic plants. In this study, we characterized that expression of the hot pepper (Capsicum annuum) gene CaBZ1, which encodes a group S bZIP transcription factor, was induced by salt and osmotic stress as well as abscisic acid (ABA). Transgenic potato (Solanum tuberosum) plants over-expressing CaBZ1 exhibited reduced rates of water loss and faster stomatal closure than non transgenic potato plants under drought and ABA treatment conditions. CaBZ1 over-expression in transgenic potato increased the expression of ABA- and stress-related genes (such as CYP707A1, CBF and NAC-like genes) and improved drought stress tolerance. Interestingly, over-expression of CaBZ1 in potato did not produce undesirable growth phenotypes in major agricultural traits such as plant height, leaf size and tuber formation under normal growth conditions. The transgenic potato plants also had higher tuber yields than non transgenic potato plants under drought stress conditions. Thus, CaBZ1 may be useful for improving drought tolerance in tuber crops. This might be the first report of the production of transgenic potato with improved tuber yields under drought conditions.

Rice cyclophilin OsCYP18‐2 is translocated to the nucleus by an interaction with SKIP and enhances drought tolerance in rice and Arabidopsis

Cyclophilin 18-2 (CYP18-2) genes, homologues of human peptidyl-prolyl isomerase-like 1 (PPiL1), are conserved across multicellular organisms and Schizosaccharomyces pombe. Although PPiL1 is known to interact with ski-interacting protein (SKIP), a transcriptional co-regulator and spliceosomal component, there have been no functional analyses of PPiL1 homologues in plants. Rice cyclophilin 18-2 (OsCYP18-2) bound directly to amino acids 56-95 of OsSKIP and its binding was independent of cyclosporin A, a cyclophilin-binding drug. Moreover, OsCYP18-2 exhibited PPIase activity regardless of its interaction with OsSKIP. Therefore, the binding site for OsCYP18-2s interaction with SKIP was distinct from the PPIase active site. OsCYP18-2s interaction with SKIP full-length protein enabled OsCYP18-2s translocation from the cytoplasm into the nucleus and AtSKIP interacted in planta with both AtCYP18-2 and OsCYP18-2. Drought and salt stress induced similar expression of OsCYP18-2 and OsSKIP. Overexpression of OsCYP18-2 in transgenic rice and Arabidopsis thaliana plants enhanced drought tolerance and altered expression and pre-mRNA splicing patterns of stress-related genes in Arabidopsis under drought conditions. Furthermore, OsCYP18-2 caused transcriptional activation with/without OsSKIP in the GAL4 system of yeast; thus the OsSKIP-OsCYP18-2 interaction has an important role in the transcriptional and post-transcriptional regulation of stress-related genes and increases tolerance to drought stress.