Kenji Oda

The DDF1 transcriptional activator upregulates expression of a gibberellin‐deactivating gene, GA2ox7, under high‐salinity stress in Arabidopsis

High-salinity stress affects plant growth and development. We have previously reported that overexpression of the salinity-responsive DWARF AND DELAYED FLOWERING 1 (DDF1) gene, encoding an AP2 transcription factor of the DREB1/CBF subfamily, causes dwarfism mainly by levels of reducing bioactive gibberellin (GA) in transgenic Arabidopsis. Here, we found that the GA 2-oxidase 7 gene (GA2ox7), which encodes a C20-GA deactivation enzyme, is strongly upregulated in DDF1-overexpressing transgenic plants. A loss-of-function mutation of GA2ox7 (ga2ox7-2) suppressed the dwarf phenotype of DDF1-overexpressing plants, indicating that their GA deficiency is due to overexpression of GA2ox7. Transient overexpression of DDF1 activated the promoter of GA2ox7 in Arabidopsis leaves. A gel shift assay showed that DDF1 binds DRE-like motifs (GCCGAC and ATCGAC) in the GA2ox7 promoter. In Arabidopsis under high-salinity stress, six GA2ox genes, including GA2ox7, were upregulated. Furthermore, the ga2ox7-2 mutant was less growth retarded than wild-type Col under high-salinity stress. These results demonstrate that, under salinity stress, Arabidopsis plants actively reduce endogenous GA levels via the induction of GA 2-oxidase, with the result that growth is repressed for stress adaptation.

Systematic approaches to using the FOX hunting system to identify useful rice genes.

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.

RiceFOX: A database of Arabidopsis mutant lines overexpressing rice full-length cDNA that contains a wide range of trait information to facilitate analysis of gene function

Identification of gene function is important not only for basic research but also for applied science, especially with regard to improvements in crop production. For rapid and efficient elucidation of useful traits, we developed a system named FOX hunting (Full-length cDNA Over-eXpressor gene hunting) using full-length cDNAs (fl-cDNAs). A heterologous expression approach provides a solution for the high-throughput characterization of gene functions in agricultural plant species. Since fl-cDNAs contain all the information of functional mRNAs and proteins, we introduced rice fl-cDNAs into Arabidopsis plants for systematic gain-of-function mutation. We generated >30,000 independent Arabidopsis transgenic lines expressing rice fl-cDNAs (rice FOX Arabidopsis mutant lines). These rice FOX Arabidopsis lines were screened systematically for various criteria such as morphology, photosynthesis, UV resistance, element composition, plant hormone profile, metabolite profile/fingerprinting, bacterial resistance, and heat and salt tolerance. The information obtained from these screenings was compiled into a database named ‘RiceFOX’. This database contains around 18,000 records of rice FOX Arabidopsis lines and allows users to search against all the observed results, ranging from morphological to invisible traits. The number of searchable items is approximately 100; moreover, the rice FOX Arabidopsis lines can be searched by rice and Arabidopsis gene/protein identifiers, sequence similarity to the introduced rice fl-cDNA and traits. The RiceFOX database is available at http://ricefox.psc.riken.jp/.

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice.

Approximately 20 000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13 000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.

CTD phosphatases in the attenuation of wound-induced transcription of jasmonic acid biosynthetic genes in Arabidopsis

Trienoic fatty acids (TAs), the major constituents in plant membrane lipids, play essential roles in stress signalling as precursors of the phytohormone jasmonic acid (JA). Arabidopsis FAD7 encodes a plastidial omega-3 fatty acid desaturase, which catalyses the production of TAs. In coordination with other JA-biosynthetic genes, expression of FAD7 is induced locally by wounding. This provides a feedforward mechanism for the rapid and sustainable accumulation of JA. To identify molecular components involved in this mechanism, a transgenic Arabidopsis line carrying the FAD7 promoter (pFAD7) fused to the firefly luciferase gene (LUC) was constructed. Reciprocal crossing experiments revealed that the induction of FAD7 expression depends largely on JA biosynthesis and the SCF(COI1)-mediated signalling mechanism, whereas JA alone is insufficient for its maximal induction. Full induction required synergistic interactions between JA-dependent and -independent wound signalling mechanisms. A genetic screen for aberrant pFAD7::LUC expression yielded a recessive mutant showing enhanced wound-induced LUC bioluminescence. The mutation was associated with the cpl1 locus encoding an RNA polymerase II C-terminal domain (CTD) phosphatase, and conferred wound hyper-responsiveness on the promoters of several JA-biosynthetic genes. The picture of signalling mechanisms underlying the wound-regulated FAD7 expression, and potential roles of CPL proteins as attenuators of wound-induced JA biosynthesis, are discussed.

A novel chloroplast protein, CEST induces tolerance to multiple environmental stresses and reduces photooxidative damage in transgenic Arabidopsis

Environmental stresses are major factors in limiting plant growth and crop production. To find genes improving salt tolerance, the screening of a large population of transgenic Arabidopsis thaliana that expressed rice full-length cDNAs under salinity stress is reported here. In this study one of the isolated salt-tolerant lines, R07303 was analysed in detail. An uncharacterized rice gene CHLOROPLAST PROTEIN-ENHANCING STRESS TOLERANCE (OsCEST) was integrated in R07303. Newly constructed transgenic Arabidopsis that overexpressed OsCEST or its Arabidopsis homologue AtCEST showed improved tolerance to salinity stress. OsCEST and AtCEST were mainly transcribed in photosynthetic tissues. Green fluorescent protein-fused OsCEST and AtCEST proteins were localized to the chloroplast in the Arabidopsis leaf protoplasts. CEST-overexpressing Arabidopsis showed enhanced tolerance not only to salt stress but also to drought stress, high-temperature stress, and paraquat, which causes photooxidative stress. Under saline conditions, overexpression of CESTs modulated the stress-induced impairment of photosynthetic activity and the peroxidation of lipids. Reduced expression of AtCEST because of double-stranded RNA interference resulted in the impairment of photosynthetic activity, the reduction of green pigment, defects in chloroplast development, and growth retardation under light. This paper discusses the relationship between the chloroplast protein CEST and photooxidative damage.

Action potential and rapid movement in the main pulvinus ofMimosa pudica

Relationships between action potential and rapid bending movement in the main pulvinus ofMimosa pudica L. were studied. The pulvinar action potential consisted of a fast-rising spike of 40 to 70 mV and a long-lasting plateau. It had a nature of propagating in both basipetal and acropetal directions at the rate of about 4 cm/sec. The rapid bending movement occurred 10 to 20 msec after the pulvinar action potential. The action potential and movement were both found only in the lower half of the main pulvinus, no response being observed in the upper half. The results suggested that the excitable cells which elicit the action potential as well as the rapid contraction exist only in the lower half of the pulvinus. Some possible mechanisms concerning the coupling between the electrical excitation and the contraction are discussed.

Assessment of ribozyme-mediated gene suppression using a luciferase reporter gene containing the sequence of Chrysanthemum Stunt Viroid as a target

Transgenic tobacco plants expressing hammerhead ribozyme (Rz), which specifically cleaves Chrysanthemum Stunt Viroid (CSVd) RNA, were produced via Agrobacterium-mediated transformation. To analyze the effects of Rz in cells, fire-fly luciferase (Luc) reporter genes, driven by the Cauliflower Mosaic Virus 35S promoter, which have the entire sense or anti-sense cDNA of the CSVd genome in between the Luc ORF and nopalin synthase terminator were constructed. Using particle bombardment, the Luc reporter constructs were delivered into the leaves of the transgenic tobacco plants. Levels of Luc mRNA and Luc enzyme activity were lower in the transgenic plants than in control wild-type plants, suggesting that the transcript of the Luc reporter gene was made unstable in the presence of Rz. On the other hand, mutations at the cleavage sites of the CSVd sequence in the reporter constructs suppressed the reduction of Luc enzyme activity in transgenic plants. A possibility of post-transcriptional gene silencing was ruled out. A direct evidence of the cleavage activity in cells by the experiment of rapid amplification of 5′ cDNA end could not obtained, but the present results suggest that the levels of Luc mRNA and Luc enzyme activity was affected by the ribozymes.