The conversion of castasterone (CS) to brassinolide (BL), a Baeyer-Villiger oxidation, represents the final and rate-limiting step in the biosynthesis of BL in plants. Heterologously expressed Arabidopsis thaliana CYP85A2 in yeast mediated the conversion of CS to BL as well as the C-6 oxidation of brassinosteroids (BRs). This indicated that CYP85A2 is a bifunctional enzyme that possesses BR C-6 oxidase and BL synthase activity. CYP85A2 is thus a cytochrome P450 that mediates Baeyer-Villiger oxidation in plants. Biochemical, physiological, and molecular genetic analyses of Arabidopsis CYP85A2 loss-of-function and overexpression lines demonstrated that CS has to be a bioactive BR that controls the overall growth and development of Arabidopsis plants. Mutant studies also revealed that BL may not always be necessary for normal growth and development but that Arabidopsis plants acquire great benefit in terms of growth and development in the presence of BL
Multiple transcription factors (TFs) play essential roles in plants under abiotic stress, but how these multiple TFs cooperate in abiotic stress responses remains largely unknown. In this study, we provide evidence that the NAC (for NAM, ATAF1/2, and CUC2) TF ANAC096 cooperates with the bZIP-type TFs ABRE binding factor and ABRE binding protein (ABF/AREB) to help plants survive under dehydration and osmotic stress conditions. ANAC096 directly interacts with ABF2 and ABF4, but not with ABF3, both in vitro and in vivo. ANAC096 and ABF2 synergistically activate RD29A transcription. Our genome-wide gene expression analysis revealed that a major proportion of abscisic acid (ABA)-responsive genes are under the transcriptional regulation of ANAC096. We found that the Arabidopsis thaliana anac096 mutant is hyposensitive to exogenous ABA and shows impaired ABA-induced stomatal closure and increased water loss under dehydration stress conditions. Furthermore, we found the anac096 abf2 abf4 triple mutant is much more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double mutant. Based on these results, we propose that ANAC096 is involved in a synergistic relationship with a subset of ABFs for the transcriptional activation of ABA-inducible genes in response to dehydration and osmotic stresses.
SummaryGibberellins (GAs) and brassinosteroids (BRs), two growth-promoting phytohormones, regulate many common physiological processes. Their interactions at the molecular level remain unclear. Here, we demonstrate that OsGSR1, a member of the GAST (GA-stimulated transcript) gene family, is induced by GA and repressed by BR. RNA interference (RNAi) transgenic rice plants with reduced OsGSR1 expression show phenotypes similar to plants deficient in BR, including short primary roots, erect leaves and reduced fertility. The OsGSR1 RNAi transgenic rice shows a reduced level of endogenous BR, and the dwarf phenotype could be rescued by the application of brassinolide. The yeast two-hybrid assay revealed that OsGSR1 interacts with DIM/DWF1, an enzyme that catalyzes the conversion from 24-methylenecholesterol to campesterol in BR biosynthesis. These results suggest that OsGSR1 activates BR synthesis by directly regulating a BR biosynthetic enzyme at the post-translational level. Furthermore, OsGSR1 RNAi plants show a reduced sensitivity to GA treatment, an increased expression of the GA biosynthetic gene OsGA20ox2, which is feedback inhibited by GA signaling, and an elevated level of endogenous GA: together, these suggest that OsGSR1 is a positive regulator of GA signaling. These results demonstrate that OsGSR1 plays important roles in both BR and GA pathways, and also mediates an interaction between the two signaling pathways.
Rice architecture is an important agronomic trait and a major limiting factor for its high productivity. Here we describe a novel CCCH-type zinc finger gene, OsLIC (Oraza sativa
leaf and tiller angle increased controller), which is involved in the regulation of rice plant architecture. OsLIC encoded an ancestral and unique CCCH type zinc finge protein. It has many orthologous in other organisms, ranging from yeast to humane. Suppression of endogenous OsLIC expression resulted in drastically increased leaf and tiller angles, shortened shoot height, and consequently reduced grain production in rice. OsLIC is predominantly expressed in rice collar and tiller bud. Genetic analysis suggested that OsLIC is epistatic to d2-1, whereas d61-1 is epistatic to OsLIC. Interestingly, sterols were significantly higher in level in transgenic shoots than in the wild type. Genome-wide expression analysis indicated that brassinosteroids (BRs) signal transduction was activated in transgenic lines. Moreover, transcription of OsLIC was induced by 24-epibrassinolide. OsLIC, with a single CCCH motif, displayed binding activity to double-stranded DNA and single-stranded polyrA, polyrU and polyrG but not polyrC. It contains a novel conserved EELR domain among eukaryotes and displays transcriptional activation activity in yeast. OsLIC may be a transcription activator to control rice plant architecture.
A number of Arabidopsis (Arabidopsis thaliana) lesion-mimic mutants exhibit alterations in both abiotic stress responses and pathogen resistance. One of these mutants, constitutive expresser of PR genes22 (cpr22), which has a mutation in two cyclic nucleotide-gated ion channels, is a typical lesion-mimic mutant exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense-related genes, and enhanced resistance to various pathogens; the majority of its phenotypes are SA dependent. These defense responses in cpr22 are suppressed under high-humidity conditions and enhanced by low humidity. After shifting plants from high to low humidity, the cpr22 mutant, but not the wild type, showed a rapid increase in SA levels followed by an increase in abscisic acid (ABA) levels. Concomitantly, genes for ABA metabolism were upregulated in the mutant. The expression of a subset of ABA-inducible genes, such as RD29A and KIN1/2, was down-regulated, but that of other genes, like ABI1 and HAB1, was up-regulated in cpr22 after the humidity shift. cpr22 showed reduced responsiveness to ABA not only in abiotic stress responses but also in germination and stomatal closure. Double mutant analysis with nahG plants that degrade SA indicated that these alterations in ABA signaling were attributable to elevated SA levels. Furthermore, cpr22 displayed suppressed drought responses by long-term drought stress. Taken together, these results suggest an effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses in cpr22.
DWARF1 functions as a brassinosteroid C-24 reductase that converts 24-methylene brassinosteroids to 24-methyl brassinosteroids to regulate the endogenous level of an active brassinosteroid, castasterone, to control growth and development in Arabidopsis.
A metabolic study revealed that 28-norcastasterone in Arabidopsis is synthesized from cholesterol via the late C-6 oxidation pathway. On the other hand, the early C-6 oxidation pathway was found to be interrupted because cholestanol is converted to 6-oxocholestanol, but further metabolism to 28-norcathasterone was not observed. The 6-oxoBRs were found to have been produced from the respective 6-deoxoBRs administered to the enzyme solution, thus indicating that these 6-oxoBRs are supplied from the late C-6 oxidation pathway. Heterologously expressed CYP85A1 and CYP85A2 in yeast catalysed this C-6 oxidation, with CYP85A2 being much more efficient than CYP85A1. Abnormal growth of det2 and dwf4 was restored via the application of 28-norcastasterone and closer precursors. Furthermore, det2 and dwf4 could not convert cholesterol to cholestanol and cholestanol to 6-deoxo-28-norcathasterone, respectively. It is, therefore, most likely that the same enzyme system is operant in the synthesis of both 28-norcastasterone and castasterone. In the presence of S-adenosyl-L-methionine, the cell-free enzyme extract catalysed the C-24 methylation of 28-norcastasterone to castasterone, although the conversion rates of 28-norteasterone to teasterone and 28-nortyphasterol to typhasterol were much lower; this suggests that 28-norcastasterone is the primary precursor for the generation of C28-BRs from C27-BRs.
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