Se-Hwan Joo

Arabidopsis CYP85A2, a Cytochrome P450, Mediates the Baeyer-Villiger Oxidation of Castasterone to Brassinolide in Brassinosteroid Biosynthesis

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.

OsGSR1 is involved in crosstalk between gibberellins and brassinosteroids in rice

Gibberellins (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.

The Arabidopsis NAC Transcription Factor ANAC096 Cooperates with bZIP-Type Transcription Factors in Dehydration and Osmotic Stress Responses

This work examines the role of the NAC transcription factor ANAC096, finding that ANAC096 interacts with specific bZIP transcription factors to globally affect abscisic acid–responsive transcription during osmotic and drought stresses. 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.

OsLIC, a Novel CCCH-Type Zinc Finger Protein with Transcription Activation, Mediates Rice Architecture via Brassinosteroids Signaling

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.

The Lesion-Mimic Mutant cpr22 Shows Alterations in Abscisic Acid Signaling and Abscisic Acid Insensitivity in a Salicylic Acid-Dependent Manner

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 up-regulated 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.

Interaction of Brassinosteroids with Light Quality and Plant Hormones in Regulating Shoot Growth of Young Sunflower and Arabidopsis Seedlings

Sunflower hypocotyls elongate as light quality changes from the normal red to far-red (R/FR) ratio of sunlight to a lower R/FR ratio. This low R/FR ratio-induced elongation significantly increases endogenous concentrations of indole-3-acetic acid (IAA) and also of three gibberellins (GAs): GA20, GA1, and GA8. Of these, it is likely GA1 that drives low R/FR-induced growth. Brassinosteroids are also involved in shoot growth. Here we tested three R/FR ratios: high, normal, and low. Significant hypocotyl elongation occurred with this stepwise reduction in R/FR ratio, but endogenous castasterone concentrations in the hypocotyls remained unchanged. Brassinolide was also applied to the seedlings and significantly increased hypocotyl growth, though one that was uniform across all three R/FR ratios. Applied brassinolide increased hypocotyl elongation while significantly reducing (usually) levels of IAA, GA20, and GA8, but not that of GA1, which remained constant. Given the above, we conclude that endogenous castasterone does not mediate the hypocotyl growth that is induced by enriching FR light, relative to R light. Similarly, we conclude that the hypocotyl growth that is induced by applied brassinolide does not result from an interaction of brassinolide with changes in light quality. The ability of applied brassinolide to influence IAA, GA20, and GA8 content, yet have no significant effect on GA1, is hard to explain. One speculative hypothesis, though, could involve the brassinolide-induced reductions that occurred for endogenous IAA, given IAA’s known ability to differentially influence the expression levels of GA20ox, GA3ox, and GA2ox, key genes in GA biosynthesis.

Biosynthesis of a cholesterol-derived brassinosteroid, 28-norcastasterone, in Arabidopsis thaliana

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.

Biosynthetic relationship between C28-brassinosteroids and C29-brassinosteroids in rice (Oryza sativa) seedlings

A crude enzyme solution was prepared from young rice seedlings, and the metabolism of C29-brassinosteroids identified from the seedlings was examined. When 28-homoteasterone was added as a substrate, 28-homotyphasterol, teasterone, and 26-nor-28-homoteasterone were characterized as enzyme products by GC-MS/SIM analysis. With 28-homotyphasterol, 28-homoteasterone, typhasterol, 28-homocastasterone, and 26-nor-28-homotyphasterol were formed and identified as products. When 28-homocastasterone was used, castasterone and 26-nor-28-homocastasterone were identified as products. Together with the reduced biological activity of C29-brassinosteroids and their metabolites in the rice lamina inclination assay, these metabolic studies suggest a biosynthetic sequence, 28-homoteasterone↔28-homotyphasterol→28-homocastasterone for C29-brassinosteroid biosynthesis is connected to the biosynthetic sequence teasterone↔typhasterol→castasterone for C28-brassinosteroids by C-28 demethylation, i.e., in order to increase biological activity in the rice plant. Additionally, the C29-brassinosteroids seem to bio-degrade their C-26 demethylated C28-brassinosteroid analogs to reduce brassinosteroid activity in planta. In conclusion, the biosynthesis of C29-brassinosteroids is a likely alternative route to the biologically-active brassinosteroid, castasterone, in rice.

UV absorbent, marmesin, from the bark of Thanakha,Hesperethusa crenulata L.

We used solvent extractions, SiO2 column chromatographies, and HPLC to isolate from the bark of Thanakha (Hesperethusa crenulata L) an active crystalline compound for absorbing UV-A radiation (320 to 380 nm). Analyses of low-and high-resolution FAB-MS revealed a compound, named marmesin, with a formula of C14H14O4 and a molecular mass of 246. To determine its chemical structure, we conducted 300 MMz NMR analyses using various probes,1H,13C, and DEPT13C. Our NMR data showed a structure of 2,3-dihydro-2(1 -hydroxy-1 -methylethyl)-furanocoumarin. This active compound contains UV-absorbing chromophores, an aromatic ring, a double bond at C3-C4, and a carbonyl at C2. Its λmax is 335 nm, indicating that marmesin could be commercially useful as a natural UV-A-filtering product

Regulation ofVrXTH1 expression in mungbean

Xyloglucan endotransglucosylase/hydrolases (XTHs) play roles in plant development by rearranging xyloglucan cross-links. Previously, we isolated a cDNA of XTH,VrXTH1, from mungbean that is thought to be associated with auxin-related growth. Here we report that several factors regulate the expression ofVrXTH1. This gene is predominantly expressed in the elongating regions of seedlings. Environmental stimuli, such as light and temperature, also affect its transcript levels. Because calcium acts as a second messenger in many signal transduction pathways, we examined its involvement in the regulation ofVrXTH1 expression. Interestingly, the application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA; a specific calcium chelator) repressed transcription, regardless of the presence of auxin. Furthermore, treatment with either A23187 (a calcium ionophore) or calcium increased gene expression, suggesting that changes in the amount of intracellular Ca2+ is important to the modification of transcript levels. Taken together, our results demonstrate that the expression ofVrXTH1 is closely related to plant growth and may be modulated by the concentration of cytosolic calcium.