Characterization of a novel cyclase-like gene family involved in controlling stress tolerance in rice.

A novel cyclase-like gene family (CYL) encodes proteins containing cyclase domain, but their functions are largely unknown. We report the systematic identification and characterization of CYL genes in the rice genome. Five putative CYL protein sequences (OsCYL1 to 4b) were identified. These sequences and other CYL homologs were classified into four subgroups based on phylogenetic analysis. Distinct diversification of these CYL proteins exists between plants and non-plants. The CYL family has conserved exon-intron structures, and the organizations of putative motifs in plants are specifically diverse. All OsCYL genes were expressed in a wide range of tissues or organs and were responsive to at least one of the abiotic stresses and hormone treatments applied. Protein OsCYL4a is targeted to the cell membrane. The overexpression of one stress-responsive gene OsCYL4a in rice resulted in decreased tolerance to salt, drought, cold, and oxidative stress. The expression levels of some abiotic stress-responsive factors, including H2O2-accumulating negative factors DST and OsSKIPa in OsCYL4a-overexpressing plants, were reduced compared with the wild type under normal condition and drought stress. These results suggest that rice CYL family may be functionally conserved polyketide cyclase, resulting in the rapid accumulation of reactive oxygen species to decrease tolerance to abiotic stresses.

Analyses of Ca2+ dynamics using a ubiquitin-10 promoter-driven Yellow Cameleon 3.6 indicator reveal reliable transgene expression and differences in cytoplasmic Ca2+ responses in Arabidopsis and rice (Oryza sativa) roots.

Ca(2+) signatures are central to developmental processes and adaptive responses in plants. However, high-resolution studies of Ca(2+) dynamics using genetically encoded Ca(2+) indicators (GECIs) such as Yellow Cameleon (YC) proteins have so far not been conducted in important model crops such as rice (Oryza sativa). We conducted a comparative study of 35S and ubiquitin-10 (UBQ10) promoter functionality in Arabidopsis thaliana and O. sativa plants expressing the Ca(2+) indicator Yellow Cameleon 3.6 (YC3.6) under control of the UBQ10 or 35S promoter. Ca(2+) signatures in roots of both species were analyzed during exposure to hyperpolarization/depolarization cycles or in response to application of the amino acid glutamate. We found a superior performance of the UBQ10 promoter with regard to expression pattern, levels and expression stabilities in both species. We observed remarkable differences between the two species in the spatiotemporal parameters of the observed Ca(2+) signatures. Rice appeared in general to respond with a lower maximal signal amplitude but greatly increased signal duration when compared with Arabidopsis. Our results identify important advantages to using the UBQ10 promoter in Arabidopsis and rice and in T-DNA mutant backgrounds. Moreover, the observed differences in Ca(2+) signaling in the two species underscore the need for comparative studies to achieve a comprehensive understanding of Ca(2+) signaling in plants.

Mutant resources for the functional analysis of the rice genome.

Rice is one of the most important crops worldwide, both as a staple food and as a model system for genomic research. In order to systematically assign functions to all predicted genes in the rice genome, a large number of rice mutant lines, including those created by T-DNA insertion, Ds/dSpm tagging, Tos17 tagging, and chemical/irradiation mutagenesis, have been generated by groups around the world. In this study, we have reviewed the current status of mutant resources for functional analysis of the rice genome. A total of 246 566 flanking sequence tags from rice mutant libraries with T-DNA, Ds/dSpm, or Tos17 insertion have been collected and analyzed. The results show that, among 211 470 unique hits, inserts located in the genic region account for 68.16%, and 60.49% of nuclear genes contain at least one insertion. Currently, 57% of non-transposable-element-related genes in rice have insertional tags. In addition, chemical/irradiation-induced rice mutant libraries have contributed a lot to both gene identification and new technology for the identification of mutant sites. In this review, we summarize how these tools have been used to generate a large collection of mutants. In addition, we discuss the merits of classic mutation strategies. In order to achieve saturation of mutagenesis in rice, DNA targeting, and new resources like RiceFox for gene functional identification are reviewed from a perspective of the future generation of rice mutant resources.

Increased leaf angle1, a Raf-like MAPKKK that interacts with a nuclear protein family, regulates mechanical tissue formation in the Lamina joint of rice.

Mitogen-activated protein kinase kinase kinases (MAPKKKs), which function at the top level of mitogen-activated protein kinase cascades, are clustered into three groups. However, no Group C Raf-like MAPKKKs have yet been functionally identified. We report here the characterization of a rice (Oryza sativa) mutant, increased leaf angle1 (ila1), resulting from a T-DNA insertion in a Group C MAPKKK gene. The increased leaf angle in ila1 is caused by abnormal vascular bundle formation and cell wall composition in the leaf lamina joint, as distinct from the mechanism observed in brassinosteroid-related mutants. Phosphorylation assays revealed that ILA1 is a functional kinase with Ser/Thr kinase activity. ILA1 is predominantly resident in the nucleus and expressed in the vascular bundles of leaf lamina joints. Yeast two-hybrid screening identified six closely related ILA1 interacting proteins (IIPs) of unknown function. Using representative IIPs, the interaction of ILA1 and IIPs was confirmed in vivo. IIPs were localized in the nucleus and showed transactivation activity. Furthermore, ILA1 could phosphorylate IIP4, indicating that IIPs may be the downstream substrates of ILA1. Microarray analyses of leaf lamina joints provided additional evidence for alterations in mechanical strength in ila1. ILA1 is thus a key factor regulating mechanical tissue formation at the leaf lamina joint.

Identification of a cluster of PR4-like genes involved in stress responses in rice.

PR4 proteins constitute a pathogenesis-related (PR) protein family with a conserved BARWIN domain. In this study, we analyzed PR4-homologous genes in rice (Oryza sativa L.) and identified five putative PR4 genes designated as OsPR4a-e. The five PR4 genes are located in tandem on chromosome 11 and constitute a gene cluster with high sequence similarity to each other. The OsPR4 proteins have high sequence similarity to reported PR4 proteins from monocotyledonous species and are predicted to be class II PR4 proteins. Distinct diversification of plant PR4 proteins exists between monocotyledonous and dicotyledonous plants. Except for OsPR4e, which was not detected with any transcript, the other four OsPR4 genes showed diverse temporal-spatial expression patterns, and their expressions are responsive to Magnaporthe grisea infection. Interestingly, the OsPR4 genes are also responsive to abiotic stresses. Their expression levels were strongly induced by at least one of the stress treatments including drought, salt, cold, wounding, heat shock, and ultraviolet. The transcript levels of OsPR4 genes were also induced by some phytohormones such as abscisic acid and jasmonic acid. Transgenic rice with overexpression of OsPR4a showed enhanced tolerance to drought at both seedling and reproductive stages. We conclude that rice PR4 genes are also involved in abiotic stress responses and tolerance in addition to their responsiveness to pathogen attacks.

Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice.

Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative ß-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.