Wing Kin Yip

A Stilbene Synthase Gene (SbSTS1) Is Involved in Host and Nonhost Defense Responses in Sorghum

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.

Nomenclature for Two-Component Signaling Elements of Rice

Plants make use of two-component systems for signal transduction, and these are involved in vital cellular processes such as the responses to cytokinins, ethylene, red/far-red light, and osmosensing ([Schaller et al., 2002][1]). Two-component systems were originally identified in bacteria, and in

An anther-specific dihydroflavonol 4-reductase-like gene (DRL1) is essential for male fertility in Arabidopsis.

Arabidopsis contains only one functional dihydroflavonol 4-reductase (DFR) gene, but several DFR-like genes encoding proteins with the conserved NAD(P)H binding domain. At4g35420, named DRL1 (Dihydroflavonol 4-reductase-like1), is a closely related homolog of the rice anther-specific gene OsDFR2 reported previously. Two T-DNA mutants (drl1-1 and drl1-2) were found to have impaired pollen formation and seed production. Histological analysis revealed defective microspore development after tetrad release in both mutants. Microspore walls were found to rupture, releasing the protoplasts which eventually degenerated. The DRL1 promoter is anther-specific in closed flower buds. Promoter-GUS analysis in transgenic Arabidopsis revealed expression in tapetum, tetrads, and developing microspores, but not in mature anthers. Enhanced yellow fluorescent protein (EYFP)-localization analysis demonstrated that DRL1 is a soluble cytosolic protein that may also be localized in the nucleus. Restoration of male fertility and seed formation was only achieved by a native promoter-DRL1 construct, but not by a 35S-DRL1 construct, demonstrating the importance of spatial and temporal specificities of DRL1 expression. DRL1 may be involved in a novel metabolic pathway essential for pollen wall development. DRL1 homologs were identified as anther- and floral-specific expressed sequence tags from different species, suggesting that DRL1 may have a conserved functional role in male fertility in flowering plants.

Heterologous expression analyses of rice OsCAS in Arabidopsis and in yeast provide evidence for its roles in cyanide detoxification rather than in cysteine synthesis in vivo

While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of β-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 μmol H2S mg−1 protein min−1 and 0.92 μmol Cys mg−1 protein min−1, respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification.

Expression of a microsporocyte-specific gene encoding dihydroflavonol 4-reductase-like protein is developmentally regulated during early microsporogenesis in rice

In an attempt to understand more about the molecular mechanism by which pollen development is regulated and coordinated in rice, we employed a cDNA subtraction strategy to identify genes that differentially expressed between the wild-type, AN-N and its thermosensitive genic male-sterile mutant (TGMS), AN S-1 during microsporogenesis. Eleven clones exhibiting differential expression patterns were isolated and identified. We report here in detail the molecular cloning and characterization of one of the clones, OS-DFR2 which encodes a dihydroflavonol 4-reductase-like protein. RNA gel blot analysis revealed that OS-DFR2 expressed strongly in anthers but no expression was detected in roots, leaves, stems and pistils, indicating that OS-DFR2 was a tissue-specific gene. Furthermore, the expression of OS-DFR2 was developmentally regulated during early microsporogenesis. It accumulated at high levels in anthers undergoing meiosis and reached a maximal value during tetrad formation stage in the wild-type, AN-N. In AN S-1, the expression of OS-DFR2 resembled that of AN-N except its mRNA level is approximately one-fourth of that found in AN-N at the tetrad formation stage. In situ analysis indicated that OS-DFR2 transcript was specifically expressed in microsporocyte of AN-N; whereas OS-DFR2 was present both in microsporocyte and tapetal cells in AN S-1. The significance and possible role played by OS-DFR2 during male gametophyte development in rice is discussed.

Differentially localized rice ethylene receptors OsERS1 and OsETR2 and their potential role during submergence

ABSTRACT Ethylene is gaseous plant hormone that controls a variety of physiologic activities. OsERS1 and OsETR2 are major ethylene receptors in rice that have been reported to have different regulatory functions. The GFP fused N-terminus of OsERS1 and OsETR2 showed differentially localization patterns when transiently expressed in onion epidermal cells. Base on these results, we suggested that OsERS1 could be localized to plasma membranes, whereas OsETR2 could be localized to the endoplasmic reticulum. Furthermore, instead of the constitutive expression profile of OsERS1, OsETR2 is differentially expressed in seedlings of light/dark-grown conditions, submergence or exogenous ethylene treatments. Our results and others support the notion that OsERS1 and OsETR2 could have different roles during rice plant submergence.