Riichiro Yoshida

Expression of AODEF, a B-functional MADS-box gene, in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A.xa0officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.

Cool-Temperature-Induced Chlorosis in Rice Plants (I. Relationship between the Induction and a Disturbance of Etioplast Development)

We have established an experimental system for mimicking the phenomenon of cool-temperature-induced chlorosis (CTIC) in rice plants (Oryza sativa L.). Rice seedlings were initially grown in darkness under cool-temperature conditions and then exposed to light and warm conditions to follow the expression of CTIC. Induction of CTIC in the sensitive cultivar (cv Surjamukhi) was bimodally dependent on the temperatures experienced during the initial growth in darkness. CTIC was maximally induced between 15 and 17[deg]C. A positive correlation was demonstrated between induction of CTIC and the growth activity of shoots during growth in darkness. Electrophoretic and immunoblot analysis revealed that accumulation of NADPH-protochlorophyllide oxidoreductase in plastids was also bimodally dependent on the temperatures during the growth in darkness with minimum accumulation between 15 and 17[deg]C, suggesting that the reduction of NADPH-protochlorophyllide oxidoreductase accumulation in plastids might be closely linked to a disturbance in transformations of plastids to etioplasts during the dark growth under the critical temperatures and thereby to the CTIC phenomenon. This was corroborated by electron microscopic observations. These results suggest that growth is one of the determining factors for the expression of CTIC phenotype in rice under cool temperature.

MAP kinases phosphorylate rice WRKY45.

WRKY45 transcription factor is a central regulator of disease resistance mediated by the salicylic acid (SA) signaling pathway in rice. SA-activated WRKY45 protein induces the accumulation of its own mRNA. However, the mechanism underlying this regulation is still unknown. Here, we report three lines of evidence showing that a mitogen-activated protein kinase (MAPK) cascade is involved in this regulation. An inhibitor of MAPK kinase (MAPKK) suppressed the increase in WRKY45 transcript level in response to SA. Two MAPKs, OsMPK4 and OsMPK6, phosphorylated WRKY45 protein in vitro. The activity of OsMPK6 was rapidly upregulated by SA treatment in rice cells. These results suggest that WRKY45 is regulated by MAPK-dependent phosphorylation in the SA pathway.

Cool Temperature-Induced Chlorosis in Rice Plants (II. Effects of Cool Temperature on the Expression of Plastid-Encoded Genes during Shoot Growth in Darkness).

It has been proposed that cool temperature-induced chlorosis (CTIC) in Indica cultivars of rice (Oryza sativa L.) is caused by cell growth and plastid development being impeded at cool temperatures. Since it is well known that the overall rate of transcription of plastid-encoded genes changes dramatically during the early phases of plastid development, in this study we focused on the patterns of expression of these genes. Northern blot analysis revealed that the level of 16S rRNA is decreased in a CTIC-sensitive rice cultivar grown at a cool temperature. The expression of the gene for the [beta] subunit of plasmid RNA polymerase (rpoB) was shown to be somewhat disturbed, particularly in terms of its resuppression under cool conditions. The level of transcripts or proteins of plastid-encoded photosynthetic genes was also decreased in a CTIC-sensitive cultivar at a cool temperature. These results suggest that the temperature-dependent inhibition of the onset of gene expression encoding the transcription/translation apparatus may be primarily involved in the mechanism causing CTIC.

Punctual coordination: Switching on and off for flowering during a day

The photoperiodic flowering of Arabidopsis is shown to be explained in part by the Bünning’s external coincidence model in which clock-controlled expression of CO and stabilization of CO protein by light have important roles. The floral activators, GI and CO, together with ZTL, FKF and CDF1 have been shown to be central for the induction of FT expression during evening to promote the photoperiodic flowering of Arabidopsis. Here we discuss a role of diurnal accumulation of a floral repressor SVP protein in the repression of the FT and SOC1 expression during daytime. A punctual coordination of the diurnal regulation of both positive and negative regulators by circadian clock appears to be important for the photoperiodic flowering in Arabidopsis.

WRKY45 phosphorylation at threonine 266 acts negatively on WRKY45-dependent blast resistance in rice

ABSTRACT WRKY45 is a central regulator of disease resistance mediated by salicylic acid signaling in rice and its activation involves phosphorylation by OsMPK6. OsMPK6 phosphorylates WRKY45 at Thr266, Ser294, and Ser299 in vitro. Phosphorylation of Ser294 and/or Ser299 is required for full activation of WRKY45, but the importance of Thr266 phosphorylation has remained unknown. Here, we report on the characterization of Thr266 phosphorylation of WRKY45 in rice. Transient expression of mutant WRKY45 revealed that Thr266 is phosphorylated in vivo, together with Ser294/299. Replacement of Thr266 by Asn did not affect the enhanced Magnaporthe oryzae resistance afforded by WRKY45 overexpression. By contrast, replacement by Asp negated the enhancement of M. oryzae resistance. These results suggest that Thr266 phosphorylation acts negatively on WRKY45-dependent disease resistance.