Shoshi Kikuchi

Genome-wide analysis of NAC transcription factor family in rice.

We investigated 151 non-redundant NAC genes in rice and 117 in Arabidopsis. A complete overview of this gene family in rice is presented, including gene structures, phylogenies, genome localizations, and expression profiles. We also performed a comparative analysis of these genes in rice and Arabidopsis. Conserved amino acid residues and phylogeny construction using the NAC conserved domain sequence suggest that OsNAC gene family was classified broadly into two major groups (A and B) and sixteen subgroups in rice. We presented more specific phylogenetic analysis of OsNAC proteins based on the DNA-binding domain and known gene function, respectively. Loss of introns was observed in the segmental duplication. Homologous, paralogous, and orthologous searches of rice and Arabidopsis revealed that the major functional diversification within the NAC gene family predated the divergence of monocots and dicots. The chromosomal localizations of OsNAC genes indicated nine segmental duplication events involving 18 genes; 32 non-redundant OsNAC genes were involved in tandem duplications. Expression levels of this gene family were checked under various abiotic stresses (cold, drought, submergence, laid-down submergence, osmotic, salinity and hormone) and biotic stresses [infection with rice viruses such as RSV (rice stripe virus) and RTSV (rice tungro spherical virus)]. Biotic stresses are novel work and increase the possibilities for finding the best candidate genes. A preliminary search based on our microarray (22K and 44K) data suggested that more than 45 and 26 non-redundant genes in this family were upregulated in response to abiotic and biotic stresses, respectively. All of the genes were further investigated for their stress responsiveness by RT-PCR analysis. Six genes showed preferential expression under both biotic RSV and RTSV stress. Eleven genes were upregulated by at least three abiotic treatments. Our study provides a very useful reference for cloning and functional analysis of members of this gene family in rice.

Visualization by comprehensive microarray analysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells

Plants have a unique transdifferentiation mechanism by which differentiated cells can initiate a new program of differentiation. We used a comprehensive analysis of gene expression in an in vitro zinnia (Zinnia elegans L.) culture model system to gather fundamental information about the gene regulation underlying the transdifferentiation of plant cells. In this model, photosynthetic mesophyll cells isolated from zinnia leaves transdifferentiate into xylem cells in a morphogenic process characterized by features such as secondary-wall formation and programmed cell death. More than 8,000 zinnia cDNA clones were isolated from an equalized cDNA library prepared from cultured cells transdifferentiating into xylem cells. Microarray analysis using these cDNAs revealed several types of unique gene regulation patterns, including: the transient expression of a set of genes during cell isolation, presumably induced by wounding; a rapid reduction in the expression of photosynthetic genes and the rapid induction of protein synthesis-associated genes during the first stage; the preferential induction of auxin-related genes during the subsequent stage; and the transient induction of genes closely associated with particular morphogenetic events, including cell-wall formation and degradation and programmed cell death during the final stage. This analysis also revealed a number of previously uncharacterized genes encoding proteins that function in signal transduction, such as protein kinases and transcription factors that are expressed in a stage-specific manner. These findings provide new clues to the molecular mechanisms of both plant transdifferentiation and wood formation.

Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants

NAC transcription factors are one of the largest families of transcriptional regulators in plants, and members of the NAC gene family have been suggested to play important roles in the regulation of the transcriptional reprogramming associated with plant stress responses. A phylogenetic analysis of NAC genes, with a focus on rice and Arabidopsis, was performed. Herein, we present an overview of the regulation of the stress responsive NAC SNAC/(IX) group of genes that are implicated in the resistance to different stresses. SNAC factors have important roles for the control of biotic and abiotic stresses tolerance and that their overexpression can improve stress tolerance via biotechnological approaches. We also review the recent progress in elucidating the roles of NAC transcription factors in plant biotic and abiotic stresses. Modification of the expression pattern of transcription factor genes and/or changes in their activity contribute to the elaboration of various signaling pathways and regulatory networks. However, a single NAC gene often responds to several stress factors, and their protein products may participate in the regulation of several seemingly disparate processes as negative or positive regulators. Additionally, the NAC proteins function via auto-regulation or cross-regulation is extensively found among NAC genes. These observations assist in the understanding of the complex mechanisms of signaling and transcriptional reprogramming controlled by NAC proteins.

Gene Structures, Classification, and Expression Models of the AP2/EREBP Transcription Factor Family in Rice

We identified 163 AP2/EREBP (APETALA2/ethylene-responsive element-binding protein) genes in rice. We analyzed gene structures, phylogenies, domain duplication, genome localizations and expression profiles. Conserved amino acid residues and phylogeny construction using the AP2/ERF conserved domain sequence suggest that in rice the OsAP2/EREBP gene family can be classified broadly into four subfamilies [AP2, RAV (related to ABI3/VP1), DREB (dehydration-responsive element-binding protein) and ERF (ethylene-responsive factor)]. The chromosomal localizations of the OsAP2/EREBP genes indicated 20 segmental duplication events involving 40 genes; 58 redundant OsAP2/EREBP genes were involved in tandem duplication events. There were fewer introns after segmental duplication. We investigated expression profiles of this gene family under biotic stresses [infection with rice viruses such as rice stripe virus (RSV), rice tungro spherical virus (RTSV) and rice dwarf virus (RDV, three virus strains S, O and D84)], and various abiotic stresses. Symptoms of virus infection were more severe in RSV infection than in RTSV and RDV infection. Responses to biotic stresses are novel findings and these stresses enhance the ability to identify the best candidate genes for further functional analysis. The genes of subgroup B-5 were not induced under abiotic treatments whereas they were activated by the three RDV strains. None of the genes of subgroups A-3 were differentially expressed by any of the biotic stresses. Our 44K and 22K microarray results suggest that 53 and 52 non-redundant genes in this family were up-regulated in response to biotic and abiotic stresses, respectively. We further examined the stress responsiveness of most genes by reverse transcription-PCR. The study results should be useful in selecting candidate genes from specific subgroups for functional analysis.

A rice calcium‐dependent protein kinase OsCPK12 oppositely modulates salt‐stress tolerance and blast disease resistance

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plants response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

Integrated transcriptomics, proteomics, and metabolomics analyses to survey ozone responses in the leaves of rice seedling.

Ozone (O(3)), a serious air pollutant, is known to significantly reduce photosynthesis, growth, and yield and to cause foliar injury and senescence. Here, integrated transcriptomics, proteomics, and metabolomics approaches were applied to investigate the molecular responses of O(3) in the leaves of 2-week-old rice (cv. Nipponbare) seedlings exposed to 0.2 ppm O(3) for a period of 24 h. On the basis of the morphological alteration of O(3)-exposed rice leaves, transcript profiling of rice genes was performed in leaves exposed for 1, 12, and 24 h using rice DNA microarray chip. A total of 1535 nonredundant genes showed altered expression of more than 5-fold over the control, representing 8 main functional categories. Genes involved in information storage and processing (10%) and cellular processing and signaling categories (24%) were highly represented within 1 h of O(3) treatment; transcriptional factor and signal transduction, respectively, were the main subcategories. Genes categorized into information storage and processing (17, 23%), cellular processing and signaling (20, 16%) and metabolism (18, 19%) were mainly regulated at 12 and 24 h; their main subcategories were ribosomal protein, post-translational modification, and signal transduction and secondary metabolites biosynthesis, respectively. Two-dimensional gel electrophoresis-based proteomics analyses in combination with tandem mass spectrometer identified 23 differentially expressed protein spots (21 nonredundant proteins) in leaves exposed to O(3) for 24 h compared to respective control. Identified proteins were found to be involved in cellular processing and signaling (32%), photosynthesis (19%), and defense (14%). Capillary electrophoresis-mass spectrometry-based metabolomic profiling revealed accumulation of amino acids, gamma-aminobutyric acid, and glutathione in O(3) exposed leaves until 24 h over control. This systematic survey showed that O(3) triggers a chain reaction of altered gene, protein and metabolite expressions involved in multiple cellular processes in rice.

Antisense transcripts with rice full-length cDNAs

BackgroundNatural antisense transcripts control gene expression through post-transcriptional gene silencing by annealing to the complementary sequence of the sense transcript. Because many genome and mRNA sequences have become available recently, genome-wide searches for sense-antisense transcripts have been reported, but few plant sense-antisense transcript pairs have been studied. The Rice Full-Length cDNA Sequencing Project has enabled computational searching of a large number of plant sense-antisense transcript pairs.ResultsWe identified sense-antisense transcript pairs from 32,127 full-length rice cDNA sequences produced by this project and public rice mRNA sequences by aligning the cDNA sequences with rice genome sequences. We discovered 687 bidirectional transcript pairs in rice, including sense-antisense transcript pairs. Both sense and antisense strands of 342 pairs (50%) showed homology to at least one expressed sequence tag other than that of the pair. Microarray analysis showed 82 pairs (32%) out of 258 pairs on the microarray were more highly expressed than the median expression intensity of 21,938 rice transcriptional units. Both sense and antisense strands of 594 pairs (86%) had coding potential.ConclusionsThe large number of plant sense-antisense transcript pairs suggests that gene regulation by antisense transcripts occurs in plants and not only in animals. On the basis of our results, experiments should be carried out to analyze the function of plant antisense transcripts.

BRASSINOSTEROID UPREGULATED1, Encoding a Helix-Loop-Helix Protein, Is a Novel Gene Involved in Brassinosteroid Signaling and Controls Bending of the Lamina Joint in Rice

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

A Novel Factor FLOURY ENDOSPERM2 Is Involved in Regulation of Rice Grain Size and Starch Quality

The authors clone the rice FLOURY ENDOSPERM2 (FLO2) gene; flo2 mutants have aberrant endosperm, and FLO2 overexpressors have enlarged grains. Gene expression and protein interaction studies indicate that FLO2, a novel tetratricopeptide repeat containing protein, regulates storage starch and protein gene expression in rice endosperm development and may also play a role in heat tolerance. Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein–protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

Brassinosteroids Antagonize Gibberellin- and Salicylate-Mediated Root Immunity in Rice

Brassinosteroids (BRs) are a unique class of plant steroid hormones that orchestrate myriad growth and developmental processes. Although BRs have long been known to protect plants from a suite of biotic and abiotic stresses, our understanding of the underlying molecular mechanisms is still rudimentary. Aiming to further decipher the molecular logic of BR-modulated immunity, we have examined the dynamics and impact of BRs during infection of rice (Oryza sativa) with the root oomycete Pythium graminicola. Challenging the prevailing view that BRs positively regulate plant innate immunity, we show that P. graminicola exploits BRs as virulence factors and hijacks the rice BR machinery to inflict disease. Moreover, we demonstrate that this immune-suppressive effect of BRs is due, at least in part, to negative cross talk with salicylic acid (SA) and gibberellic acid (GA) pathways. BR-mediated suppression of SA defenses occurred downstream of SA biosynthesis, but upstream of the master defense regulators NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 and OsWRKY45. In contrast, BR alleviated GA-directed immune responses by interfering at multiple levels with GA metabolism, resulting in indirect stabilization of the DELLA protein and central GA repressor SLENDER RICE1 (SLR1). Collectively, these data favor a model whereby P. graminicola coopts the plant BR pathway as a decoy to antagonize effectual SA- and GA-mediated defenses. Our results highlight the importance of BRs in modulating plant immunity and uncover pathogen-mediated manipulation of plant steroid homeostasis as a core virulence strategy.