Hironori Kaminaka

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.

bZIP10-LSD1 antagonism modulates basal defense and cell death in Arabidopsis following infection

Plants use sophisticated strategies to balance responses to oxidative stress. Programmed cell death, including the hypersensitive response (HR) associated with successful pathogen recognition, is one cellular response regulated by reactive oxygen in various cellular contexts. The Arabidopsis basic leucine zipper (bZIP) transcription factor AtbZIP10 shuttles between the nucleus and the cytoplasm and binds consensus G‐ and C‐box DNA sequences. Surprisingly, AtbZIP10 can be retained outside the nucleus by LSD1, a protein that protects Arabidopsis cells from death in the face of oxidative stress signals. We demonstrate that AtbZIP10 is a positive mediator of the uncontrolled cell death observed in lsd1 mutants. AtbZIP10 and LSD1 act antagonistically in both pathogen‐induced HR and basal defense responses. LSD1 likely functions as a cellular hub, where its interaction with AtbZIP10 and additional, as yet unidentified, proteins contributes significantly to plant oxidative stress responses.

Gateway binary vectors with the bialaphos resistance gene, bar, as a selection marker for plant transformation.

We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 and TAP. Selection of Arabidopsis transformants with BASTA® was successfully carried out using both plate-grown and soil-grown seedlings. Transformed rice calli and suspension-cultured tobacco cells were selected on plates containing BASTA® or glufosinate-ammonium. These vectors are compatible with existing pGWB and R4pGWB vectors carrying kanamycin and hygromycin B resistance.

Structure and differential response to abscisic acid of two promoters for the cytosolic copper/zinc- superoxide dismutase genes, SodCcl and SodCc2, in rice protoplasts☆

We determined the 5′‐flanking sequences of two nuclear genes (SodCcl and SodCcl) encoding cytosolic copper/zinc‐superoxide dismutase in rice (Oryza sativa L.). Utilizing transient β‐glucuronidase (GUS) reporter assays, functional promoter—GUS analysis was performed in rice protoplasts exposed to the phytohormone abscisic acid (ABA) or the antioxidant sulfhydryl reagent, dithiothreitol (DTT). Transcriptional activities from both SodCc—GUS fusions were stimulated by DTT, which induces the promoter activity of the tobacco SodCc gene [Proc. Natl. Acad. Sci. USA 90 (1993) 3108–3112]. ABA had no effect on SodCcl—GUS expression but specifically induced the gene expression of the SodCcl—GUS fusion. The simultaneous application of ABA and gibberellin A3, however, abolished the enhancing effect of ABA. These results indicated that two rice SodCc promoters differentially respond to externally supplied ABA and that one of the regulatory factors for plant SodCc expression is ABA in addition to cellular redox‐modulating antioxidants.

Differential Gene Expressions of Rice Superoxide Dismutase Isoforms to Oxidative and Environmental Stresses

Active oxygen species (AOSs) are produced under stress conditions of plant cells. Superoxide dismutase (SOD) catalyzes the first step in the AOS scavenging system. The responses of SOD genes to environmental stresses were analyzed in rice seedlings by the treatments of drought, salinity and chilling. The expressions of abscisic acid (ABA)-inducible genes, Mn-SOD gene (sodA1) and one of the cytosolic Cu/Zn-SOD genes (sodCc2), were strongly induced by the treatment of drought and salinity. While Fe-SOD gene (sodB) and the other cytosolic Cu/Zn-SOD gene (sodCc1) were also induced by ABA. However the mRNA level of sodB was decreased by drought treatment, and sodCc1 gene was not induced by drought and salinity treatments. Plastidic Cu/Zn-SOD gene (sodCp) quickly responded to salinity treatment in the light but not in the dark. In the treatment with hydrogen peroxide, sodCp gene was strongly induced shortly after the treatment. These results suggested that phytohormone and AOSs are associated with the regulation of SOD genes under environmental stresses.

Preparation of high-strength transparent chitosan film reinforced with surface-deacetylated chitin nanofibers.

Surface-deacetylated chitin nanofiber reinforced chitosan films were prepared. The nano-composite films were highly transparent of approximately 84% at 600 nm due to the nanometer-sized fillers and chitosan matrix, which were embedded in the cavities and on the rough surface of the nanofiber networks. Due to the extended crystalline structure, the nanofibers worked effectively as reinforcement filler to improve the Youngs modulus and the tensile strength of the chitosan film. After 10% blending of nanofiber, these properties were increased by 65% and 94%, respectively. Moreover, thermal expansion was also significantly decreased from 35.3 to 26.1 ppm K(-1) after 10% addition of nanofibers. Surface-deacetylated chitin nanofiber and the nano-composite films showed antifungal activity against A. alternata.

Facile preparation of silver nanoparticles immobilized on chitin nanofiber surfaces to endow antifungal activities.

Silver nanoparticles were prepared on chitin nanofiber surfaces by UV light reduction of silver ions. The chitin nanofibers could be efficient substrates to immobilize silver nanoparticles with stable dispersion states. The dispersion and the nanocomposite film with acrylic resin showed characteristic absorption property in the visible light region due to the effect of the silver nanoparticles. Silver nanoparticles endowed strong antifungal activity to chitin nanofibers.

IAA8 involved in lateral root formation interacts with the TIR1 auxin receptor and ARF transcription factors in Arabidopsis.

The expression of auxin-responsive genes is regulated by the TIR1/AFB auxin receptor-dependent degradation of Aux/IAA transcriptional repressors, which interact with auxin-responsive factors (ARFs). Most of the 29 Aux/IAA genes present in Arabidopsis have not been functionally characterized to date. IAA8 appears to have a distinct function from the other Aux/IAA genes, due to its unique transcriptional response to auxin and the stability of its encoded protein. In this study, we characterized the function of Arabidopsis IAA8 in various developmental processes governed by auxin and in the transcriptional regulation of the auxin response. Transgenic plants expressing estrogen-inducible IAA8 (XVE::IAA8) exhibited significantly fewer lateral roots than the wild type, and an IAA8 loss-of-function mutant exhibited significantly more. Ectopic overexpression of IAA8 resulted in abnormal gravitropism. The strong induction of early auxin-responsive marker genes by auxin treatment was delayed by IAA8 overexpression. GFP-fusion analysis revealed that IAA8 localized not only to the nucleus, but, in contrast to other Aux/IAAs, also to the cytosol. Furthermore, we demonstrated that IAA8 interacts with TIR1, in an auxin-dependent fashion, and with ARF proteins, both in yeast and in planta. Taken together, our results show that IAA8 is involved in lateral root formation, and that this process is regulated through the interaction with the TIR1 auxin receptor and ARF transcription factors in the nucleus.

Overexpression of a New Rice Vacuolar Antiporter Regulating Protein OsARP Improves Salt Tolerance in Tobacco

We examined the function of the rice (Oryza sativa L.) antiporter-regulating protein OsARP by overexpressing it in tobacco (Nicotiana tabacum L.). In public databases, this protein was annotated as a putative Os02g0465900 protein of rice. The OsARP gene was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. The transformants were selected for their ability to grow on medium containing kanamycin. Incorporation of the transgene in the genome of tobacco was confirmed by PCR, and its expression was confirmed by Western blot analysis. Transgenic plants had better growth and vigor than non-transgenic plants under salt stress in vitro. Overexpression of OsARP in transgenic tobacco plants resulted in salt tolerance, and the plants had a higher rate of photosynthesis and effective PSII photon yield when compared with the wild type. The OsARP protein was localized in the tonoplast of rice plants. Transgenic plants accumulated more Na+ in their leaf tissue than did wild-type plants. It is conceivable that the toxic effect of Na+ in the cytosol might be reduced by sequestration into vacuoles. The rate of water loss was higher in the wild type than in transgenic plants under salt stress. Increased vacuolar solute accumulation and water retention could confer salt tolerance in transgenic plants. Tonoplast vesicles isolated from OsARP transgenic plants showed Na+/H+ exchange rates 3-fold higher than those of wild-type plants. These results suggest that OsARP on the tonoplasts plays an important role in compartmentation of Na+ into vacuoles. We suggest that OsARP is a new type of protein participating in Na+ uptake in vacuoles.

Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner.

Highlight Allantoin, a stress-related purine metabolite, can activate JA responses via ABA in Arabidopsis, suggesting its possible involvement in the homeostasis of these phytohormones and their interplay in stress signaling.