Phun Bum Park

The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice

A number of basic leucine zipper (bZIP) transcription factors are known to function in stress signaling in plants but few have thus far been functionally characterized in rice. In our current study in rice, we have newly isolated and characterized the OsABF1 (Oryza sativa ABA responsive element binding factor 1) gene that encodes a bZIP transcription factor. Its expression in seedling shoots and roots was found to be induced by various abiotic stress treatments such as anoxia, salinity, drought, oxidative stress, cold and abscisic acid (ABA). Subcellular localization analysis in maize protoplasts using GFP fusion vectors indicated that OsABF1 is a nuclear protein. In a yeast experiment, OsABF1 was shown to bind to ABA responsive elements (ABREs) and its N-terminal region was necessary to transactivate the downstream reporter gene. The homozygous T-DNA insertional mutants Osabf1-1 and Osabf1-2 were more sensitive in response to drought and salinity treatments than wild type plants. Furthermore, the upregulated expression of some ABA/stress-regulated genes in response to ABA treatment was suppressed in these Osabf1 mutants. Our current results thus suggest that OsABF1 is involved in abiotic stress responses and ABA signaling in rice.

The ABRE-binding bZIP transcription factor OsABF2 is a positive regulator of abiotic stress and ABA signaling in rice

Abscisic acid (ABA) is an important phytohormone involved in abiotic stress tolerance in plants. The group A bZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis but little is known about their functions in rice. In our current study, we have isolated and characterized a group A bZIP transcription factor in rice, OsABF2 (Oryza sativa ABA-responsive element binding factor 2). It was found to be expressed in various tissues in rice and induced by different types of abiotic stress treatments such as drought, salinity, cold, oxidative stress, and ABA. Subcellular localization analysis in maize protoplasts using a GFP fusion vector indicated that OsABF2 is a nuclear protein. In yeast experiments, OsABF2 was shown to bind to ABA-responsive elements (ABREs) and its N-terminal region found to be necessary to transactivate a downstream reporter gene. A homozygous T-DNA insertional mutant of OsABF2 is more sensitive to salinity, drought, and oxidative stress compared with wild type plants. In addition, this Osabf2 mutant showed a significantly decreased sensitivity to high levels of ABA at germination and post-germination. Collectively, our present results indicate that OsABF2 functions as a transcriptional regulator that modulates the expression of abiotic stress-responsive genes through an ABA-dependent pathway.

Increased salt and drought tolerance by D-ononitol production in transgenic Arabidopsis thaliana.

The methylation of myo-inositol forms O-methyl inositol (D-ononitol) when plants are under abiotic stress in a reaction catalyzed by myo-inositol methyltransferase (IMT). D-Ononitol can serve as an osmoprotectant that prevents water loss in plants. We isolated the IMT cDNA from Glycine max and found by RT-PCR analysis that GmIMT transcripts are induced by drought and salinity stress treatments in the leaves of soybean seedlings. We confirmed the protein product of GmIMT and its substrate using a recombinant system in E. coli. Transgenic Arabidopsis plants over-expressing GmIMT displayed improved tolerance to dehydration stress treatment and to a lesser extent high salinity stress treatment. These results indicate that GmIMT is functional in heterologous Arabidopsis plants.

Ricebending lamina 2 (bla2) mutants are defective in a cytochrome P450 (CYP734A6) gene predicted to mediate brassinosteroid catabofism

Desirable morphological traits in rice plants, such as shorter stalks and erect leaves, are being pursued in breeding programs. Rice brassinosteroid (BR)-deficient mutants display the phenotype of reduced heights and erect lamina. Therefore, biotechnology can be used for controlled deactivation of bioactive BRs in specific tissues. Here, we isolated a gene encoding for a BR-deactivating enzyme. Based on its sequence identity with a known enzyme,Arahidopsis Cytochrome P450 734A1, we identified a rice homolog named QsCYP734A6 (LOC_Os01 g29150). A search of the rice T-DNA mutant population for insertional mutants of this gene revealed two alleles. These T-DNA loss-of-function mutants displayed a strongly bending lamina phenotype similar to the morphology of rice plants treated with BRs. These two mutants were namedbending lamina (bla)2-1 andbla2-2. InArahidopsis, the transcript level of a putative BR biosynthetic gene,OsDWARF4, is feed-back down-regulated in response to exogenous application of BRs. Similarly, a steady-state level ofOsDWARF4 transcripts was significantly down-regulated here in the bla2 mutant background. Based on their sequence similarity, the bending-Iamina phenotype in thebla2 mutant, and down-regulation of the BR biosynthetic geneQsDWARF4, we therefore propose that OsCYP734A6 (BLA2) is likely to be involved in BR deactivation.

Structure and expression ofOsMRE11 in rice

In yeast and human cells, the Mre11 complex, which consists of Mre11, Rad50, and Xrs2/Nbs1 proteins, participates in basic aspects of chromosome metabolism, such as the repair of meiotic DNA breaks and telomere maintenance. In this study, we isolated a full-length cDNA clone, pOsMrell, encoding a rice ortholog of the Mre11 protein. Its predicted protein sequence (Mr = 79.2 kDa and pl value = 5.91) contains a metallo-phosphoesterase domain at its N-terminal region, and a single putative DNA binding domain in the central region of the protein, with significant homology to corresponding motifs in human and yeast Mre11 proteins. TheOsMRE11 gene is constitutively expressed in all tissues examined here, including leaves, roots, tillers, and meristems, as well as in undifferentiated callus cells. When 10-d-old rice seedlings were treated with 0.025% methyl methanesulfonate (MMS) or 30 watts of UV-C light, they were apparently damaged by those genotoxic agents, with plants being more seriously injured by the latter. RNA gel blot analysis showed that the level ofOsMRE11 mRNA remained unchanged during the 1- to 4-d incubation period with MMS. In contrast,OsMRE11 expression appeared to increase after 3 d of irradiation. In addition, treatments with salicylic acid and jasmonic acid, two important defense-related hormones, significantly activated theOsMRE11 gene. Based on these results, we discuss the possible functions of the OsMre11 protein in a mechanism by which the stability of rice chromosomes is maintained.

KiwiPME1 encoding pectin methylesterase is specifically expressed in the pollen of a dioecious plant species, kiwifruit (Actinidia chinensis)

Pectin methylesterases (PMEs) mediate demethylesterification of pectic polysaccharides such as homogalacturonan, a major component of the primary plant cell wall, in the apoplasm. The PMEs are implicated in a number of developmental processes, including pollen development and pollen tube growth, through the fine tuning of the methylesterification status of pectin. In this study, we isolated a full-length cDNA (KiwiPME1) encoding PME from kiwifruit and characterized its molecular features. Analyses of the primary protein structure and gene structure revealed that KiwiPME1 encodes a pre-pro-PME protein that is predicted to localize to the outside of the cell and belongs to group 2 (formerly type 1). The KiwiPME1 expression was highly detected in pollen grains of kiwifruit but not in vegetative tissues investigated. Expression pattern analysis of KiwiPME1 among different floral tissues of male and female plants revealed that KiwiPME1 was expressed specifically in the stamens of flower buds in male and female plants, whereas its expression was detected only in the stamens of male plants when the flowers opened. Expression analysis of KiwiPME1 promoter fused to the GUS reporter gene in Arabidopsis displayed a very similar pattern to that in kiwifruit. Our study suggests that the cell wall-localized KiwiPME1 is likely implicated in the pollen development and pollen tube growth of a dioecious species kiwifruit.

Isolation of a novel protein phosphatase2C in rice and its response to gibberellin

Protein phosphatase 2Cs (PP2Cs) have been referred to act as negative modulators of the protein kinase pathways involved in different environmental stress responses and developmental processes. In Arabidopsis, PP2Cs have been extensively studied and some are known to negatively regulate abscisic acid signaling. In rice, PP2Cs are scarcely characterized functionally. Here, we identified a novel PP2C from rice (OsPP2C34), which is highly inducible by gibberellin (GA) and expressed in various tissues. Subcellular localization analysis in maize protoplasts using a green fluorescence protein fusion vector localized OsPP2C34 to the cytosol. Genetic analysis of T-DNA insertional mutants revealed that plant height and internode length were significantly shorter in mutants than in corresponding wild types under GA treatment. The induction of the GA-inducibleα-amylase genes RAmy3E and OsAmy was delayed in mutant plants. The substrate of OsPP2C34 was identified by immunoblotting using anti serine/threonine antibodies. A 65 kDa protein was phosphorylated in Ospp2c34-1 but dephosphorylated in the wild type during early germination stage. Overall, the present results indicated that OsPP2C34 is involved inα-amylase expression of GA signal transduction pathway.

Increased Abiotic Stress Tolerance by Over-expressing OsABF2 in Transgenic Arabidopsis thaliana

The phytohormone abscisic acid (ABA) plays an important role in the adaptive response of plants to abiotic stresses. ABA also regulates many important processes, including seed dormancy, germination, inhibition of cell division, and stomatal closure. OsABF2 (Oryza sativa ABRE binding factor2) is one of the bZIP type transcription factors, which are involved in abiotic stress response and ABA signaling in rice. Expression of OsABF2 is induced by ABA and various stress treatments. Findings show that survival rates of OsABF2 over-expressing Arabidopsis lines were increased under drought, salt, and heat stress conditions. The germination ratio of OsABF2 over-expressing Arabidopsis lines was decreased in the presence of ABA. Results indicate that OsABF2 over-expressing Arabidopsis lines have enhanced abiotic stress tolerance and have increased ABA sensitivity.

Increase in the Chlorophyll Contents by Over-expression of GmNAP1 Gene in Arabidopsis Plant

In the course of a research concerning the molecular mechanism of hypocotyl elongation that occurs during soybean seedling growth in darkness, we have generated a number of ESTs from a cDNA library prepared from the hypocotyls of dark-grown soybean seedlings. Comparison of the ESTs assigned a cDNA clone as a putative plastidic ATP-binding-cassette (ABC) protein homologue. The soybean GmNAP1 protein contains an N-terminal transit peptide which targets it into the chloroplast. The transcription level of the GmNAP1 gene was investigated under continuous red light, continuous far-red light, and complete darkness. The main function of this NAP1 protein is the transport of protoporphyrin IX which is the precursor of chlorophyll from the cytoplasm to the chloroplast. The GmNAP1 gene was transferred into the Arabidopsis under the CaMV 35S promoter. The chlorophyll level of this transgenic Arabidopsis plant was much higher than the chlorophyll level of the wild type Arabidopsis plant.