SummaryThe transcriptional factor DREB/CBF (dehydration-responsive element/C-repeat-binding) speci®cally interacts with the dehydration-responsive element (DRE)/C-repeat (CRT) cis-acting element (A/GCCGAC) and controls the expression of many stress-inducible genes in Arabidopsis. Transgenic plants overexpressing DREB1A showed activated expression of many stress-inducible genes and improved tolerance to not only drought, salinity, and freezing but also growth retardation. We searched for downstream genes in transgenic plants overexpressing DREB1A using the full-length cDNA microarray and Affymetrix GeneChip array. We con®rmed candidate genes selected by array analyses using RNA gel blot and identi®ed 38 genes as the DREB1A downstream genes, including 20 unreported new downstream genes. Many of the products of these genes were proteins known to function against stress and were probably responsible for the stress tolerance of the transgenic plants. The downstream genes also included genes for protein factors involved in further regulation of signal transduction and gene expression in response to stress. The identi®ed genes were classi®ed into direct downstream genes of DREB1A and the others based on their expression patterns in response to cold stress. We also searched for conserved sequences in the promoter regions of the direct downstream genes and found A/GCCGACNT in their promoter regions from À51 to À450 as a consensus DRE. The recombinant DREB1A protein bound to A/GCCGACNT more ef®ciently than to A/GCCGACNA/G/C.
Although numerous physiological studies have addressed the interactions between brassinosteroids and auxins, little is known about the underlying molecular mechanisms. Using an Affymetrix GeneChip representing approximately 8,300 Arabidopsis genes, we studied comprehensive transcript profiles over 24 h in response to indole-3-acetic acid (IAA) and brassinolide (BL). We identified 409 genes as BL inducible, 276 genes as IAA inducible, and 637 genes in total. These two hormones regulated only 48 genes in common, suggesting that most of the actions of each hormone are mediated by gene expression that is unique to each. IAA-up-regulated genes were enriched in genes regulated in common. They were induced quickly by IAA and more slowly by BL, suggesting divergent physiological roles. Many were early auxin-inducible genes and their homologs, namely SAUR, GH3, and IAA. The comprehensive comparison also identified IAA-and BL-specific genes, which should help to elucidate the specific actions of each hormone. The identified genes were classified using hierarchical clustering based on the similarity of their responses to the two hormones. Gene classification also allowed us to analyze the frequency of cis-elements. The TGTCTC element, a core element of the previously reported auxin response element, was not enriched in genes specifically regulated by IAA but was enriched in the 59-flanking region of genes up-regulated by both IAA and BL. Such gene classification should be useful for predicting the functions of unknown genes, to understand the roles of these two hormones, and the promoter analysis should provide insight into the interaction of transcriptional regulation by the two hormones.
Summary Molecular genetic and physiological studies on brassinosteroid (BR)‐related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR‐deficient mutants of rice, BR‐deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C‐6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C‐6 oxidase gene, DWARF. Introduction of the wild‐type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.
We analyzed global gene expression in Arabidopsis in response to various hormones and in related experiments as part of the AtGenExpress project. The experimental agents included seven basic phytohormones (auxin, cytokinin, gibberellin, brassinosteroid, abscisic acid, jasmonate and ethylene) and their inhibitors. In addition, gene expression was investigated in hormone-related mutants and during seed germination and sulfate starvation. Hormone-inducible genes were identified from the hormone response data. The effects of each hormone and the relevance of the gene lists were verified by comparing expression profiles for the hormone treatments and related experiments using Pearson's correlation coefficient. This approach was also used to analyze the relationships among expression profiles for hormone responses and those included in the AtGenExpress stress-response data set. The expected correlations were observed, indicating that this approach is useful to monitor the hormonal status in the stress-related samples. Global interactions among hormones-inducible genes were analyzed in a pairwise fashion, and several known and novel hormone interactions were detected. Genome-wide transcriptional gene-to-gene correlations, analyzed by hierarchical cluster analysis (HCA), indicated that our data set is useful for identification of clusters of co-expressed genes, and to predict the functions of unknown genes, even if a gene's function is not directly related to the experiments included in AtGenExpress. Our data are available online from AtGenExpressJapan; the results of genome-wide HCA are available from PRIMe. The data set presented here will be a versatile resource for future hormone studies, and constitutes a reference for genome-wide gene expression in Arabidopsis.
IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.
Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. Although insights into the functions of BRs have been provided by recent studies of biosynthesis and sensitivity mutants, the mode of action of BRs is poorly understood. With the use of DNA microarray analysis, we identified BR-regulated genes in the wild type (WT; Columbia) of Arabidopsis and in the BR-deficient mutant, det2. BR-regulated genes generally responded more potently in the det2 mutant than in the WT, and they showed only limited response in a BR-insensitive mutant, bri1. A small group of genes showed stronger responses in the WT than in the det2. Exposure of plants to brassinolide and brassinazole, which is a specific inhibitor of BR biosynthesis, elicited opposite effects on gene expression of the identified genes. The list of BR-regulated genes is constituted of transcription factor genes including the phytochrome-interacting factor 3, auxin-related genes, P450 genes, and genes implicated in cell elongation and cell wall organization. The results presented here provide comprehensive view of the physiological functions of BRs using BR-regulated genes as molecular markers. The list of BR-regulated genes will be useful in the characterization of new mutants and new growth-regulating compounds that are associated with BR function.
SummaryGAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.
Active brassinosteroids, such as brassinolide (BL) and castasterone, are growth promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase encoded by CYP72B1 has been implicated in brassinosteroid catabolism as well as photomorphogenesis. We expressed CYP72B1 in yeast, coupled with brassinosteroid feeding, and established the biochemical function to be the hydroxylation of BL and castasterone, to give 26-hydroxybrassinolide and 26-hydroxycastasterone, respectively. Brassinosteroid feeding experiments with wild-type Arabidopsis, a CYP72B1 null mutant, and a CYP72B1 overexpression line demonstrated that carbon 26 hydroxylation of active brassinosteroids is an endogenous function of CYP72B1. Seedling growth assays demonstrated that 26-hydroxybrassinolide is an inactive brassinosteroid. Genetic and physiological analysis of the hypocotyl response to exogenous BL and varying intensities of white and monochromatic light suggested that CYP72B1 modulates photomorphogenesis primarily through far-red light and to a lesser extent through blueand red-light pathways. CYP72B1 transcript accumulation in dark-grown seedlings was organ specific and down-regulated after 1 h of illumination in dim white, red, and blue light, but not far-red light. CYP72B1 translational fusions with the -glucuronidase reporter gene demonstrated that protein levels increased in the hypocotyl elongation zone when shifted from the dark to far-red light, but not blue or red light. We propose a model in which Arabidopsis seedling development switches from dark-grown development (skotomorphogenesis) to light-grown development (photomorphogenesis) in part by rapid modulation of brassinosteroid sensitivity and levels. CYP72B1 provides an intersection between the light and brassinosteroid pathways mainly by far-red-light-dependent modulation of brassinosteroid levels.Brassinolide (BL) is the most active brassinosteroid, a class of polyhydroxylated plant-specific steroids. The isolation of BL in 1979 identified the structure to be a cholestane derivative (Grove et al., 1979). Animal steroids are likewise cholestane derivates (Mussig and Altmann, 2001). Analysis of BL biosynthetic mutants (det2 and cpd) in Arabidopsis revealed similarities between animal and plant steroids. Rescue of the det2 and cpd pleiotropic phenotypes by exogenous BL established the commonality of steroids as fundamental hormones in both animal and plant development (Li et al., 1996;Szekeres et al., 1996). Cloning of the CPD gene from Arabidopsis demonstrated that animals and plants each use cytochrome P450 monooxygenases (CYP450s) for steroid biosynthesis (Szekeres et al., 1996). Analysis of the human steroid 5␣-reductase (hS5R) and its Arabidopsis ortholog, DET2, demonstrated a common mechanism of steroid hormone activation between animals and plants (Li et al., 1996). Both human isoenzymes of hS5R reduce testosterone to dihydrotestosterone to amplify a weak hormone signal, whereas DET2 reduces BL precursors . Ecdysone, an insect steroid hormone, is structurally similar to...
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