Brassinosteroid Levels Research Articles

Promotion of transcript accumulation of novel Zinnia immature xylem-specific HD-Zip III homeobox genes by brassinosteroids.

We isolated three novel homeobox genes (ZeHB-10, -11 and -12) from Zinnia elegans to elucidate the molecular mechanism underlying vascular system formation. ZeHB-10, -11 and -12 encode for HD-Zip proteins of the class III to which Arabidopsis Athb-8, -9, -14, -15 and IFL1 belong. In situ hybridization analysis demonstrated that the ZeHB-10, -11 and -12 mRNAs accumulated preferentially in procambium and immature xylem cells in 14-day-old plants. Transcripts for the three genes also accumulated in cultured Zinnia cells in a xylogenesis-specific manner. The accumulation of transcripts for all of ZeHB-10, -11 and -12 in cultured Zinnia cells was suppressed strongly by uniconazole, an inhibitor of brassinosteroid synthesis, and such suppression was reversed by the addition of brassinolide, a biologically active brassinosteroid. Thus the expression of ZeHB-10, -11 and -12 may be regulated by endogenous levels of brassinosteroids. Taken together with the fact that ZeHB-10, -11 and -12 proteins can bind to each other in yeast, the roles of HD-Zip III genes in vascular development are discussed.

Hormone levels and response during de-etiolation in pea.

The objective of this study was to increase our understanding of the hormonal regulation of de-etiolation by investigating endogenous hormone levels and response in etiolated pea ( Pisum sativum L.) seedlings after exposure to continuous white light. Recent reports suggest that de-etiolation may result from the down-regulation of an enzyme in the brassinosteroid (BR) biosynthesis pathway in pea. A subsequent review highlighted the need for direct measurements of BR levels to support this hypothesis. We have shown that endogenous castasterone and 6-deoxocastasterone levels are not greatly reduced after exposure to light; indeed, 6-deoxocastasterone levels were actually increased. Similarly, the elongation response to exogenous brassinolide was greater in plants grown in continuous light, or in dark-grown plants that had been transferred into the light, than in plants that were grown in continuous darkness. These results provide further evidence to suggest that BRs do not negatively regulate de-etiolation in pea. However, changes in the levels of several other hormones have also been implicated in light-regulated development. We have simultaneously quantified indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid levels in whole seedlings, which revealed a complex pattern of changes in the levels of these substances after exposure to light. The first and most dramatic of these changes was a significant reduction in GA(1) levels, which reached a minimum 8 h after exposure to light. Whilst GA(1) levels rapidly decreased, IAA levels remained unchanged in the short term after exposure to light, suggesting that GA(1) levels may be the primary factor regulating the reduction in elongation growth during de-etiolation. In the long term after exposure to light, IAA levels underwent a transitory increase, which peaked at 48 h, and had abated by 96 h. However, abscisic acid levels remained unchanged in the first 1 h after exposure to light before undergoing a steady decline over time. The relative importance of these changes in mediating light-induced changes in plant morphology is discussed.

The identification of CVP1 reveals a role for sterols in vascular patterning.

Vascular cell axialization refers to the uniform alignment of vascular strands. In the Arabidopsis cotyledon vascular pattern1 (cvp1) mutant, vascular cells are not arranged in parallel files and are misshapen, suggesting that CVP1 has a role in promoting vascular cell polarity and alignment. Characterization of an allelic series of cvp1 mutations revealed additional functions of CVP1 in organ expansion and elongation. We identified CVP1 and found that it encodes STEROL METHYLTRANSFERASE2 (SMT2), an enzyme in the sterol biosynthetic pathway. SMT2 and the functionally redundant SMT3 act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarization and axialization.

Uncoupling brassinosteroid levels and de-etiolation in pea.

The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1, the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea.

Brassinosteroid의 대사공학

Sterols play two major roles in plants: a bulk component in biological membranes and precursors of plant steroid hormones. Physiological effects of plant steroids, brassinosteroids (BRs), include cell elongation, cell division, stress tolerance, and senescence acceleration. Arabidopsis mutants that carry genetic defects in BR biosynthesis or its signaling display characteristic phenotypes, such as short robust inflorescences, dark-green round leaves, and sterility. Currently there are more than 100 dwarf mutants representing 7 genetic loci in Arabidopsis. Mutants of 6 loci, dwf1/dim1/cbb1, cpd/dwf3, dwf4, dwf5, det2/dwf6, dwf7 are rescued by exogenous application of BRs, whereas bri1/dwf2 shares phenotypes with the above 6 loci but are resistant to BRs. These suggest that the 6 loci are defective in BR biosynthesis, and the one locus is in BR signaling. Biochemical analyses, such as intermediate feeding tests, examining the levels of endogenous BR, and molecular cloning of the genes revealed that dwf7, dwf5, and dwf1 are defective in the three consecutive steps of sterol biosynthesis, from episterol to campesterol via 5-dehydroepisterol. Similarly, det2/dwf6, dwf4, and cpd /dwf3 were shown to be blocked in D<TEX>$^4$</TEX>reduction, 22a-hydroxylation, and 23 a-hydroxylation, respectively. A signaling mutant bril/dwf2 carries mutations in a Leucine-rich repeat receptor kinase. Interestingly, the bri1 mutant was shown to accumulate significant amount of BRs, suggesting that signaling and biosynthesis are dynamically coupled in Arabidopsis. Thus it is likely that transgenic plants over-expressing the rate-limiting step enzyme DWF4 as well as blocking its use by BRIl could dramatically increase the biosynthetic yield of BRs. When applied industrially, BRs will boost new sector of plant biotechnology because of its potential use as a precursor of human steroid hormones, a novel lead compound for cholesterol-lowering effects, and a various application in plant protection.

Roots and shoots of tomato produce 6-deoxo-28-norcathasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone, possible precursors of 28-norcastasterone

Roots and shoots of tomato ( Lycopersicon esculentum) were investigated for the occurrence of biosynthetic precursors of 28-norcastasterone, a C 27 brassinosteroid that we have shown to be present in shoots of tomato. A series of putative precursors, including 6-deoxo-28-norcathasterone, 6-deoxo-28-norteasterone, 3-dehydro-6-deoxo-28-norteasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone, were synthesized and used as GC-MS standards, resulting in the identification of 6-deoxo-28-norcathasterone, 6-deoxo-28-nortyphasterol and 6-deoxo-28-norcastasterone in both roots and shoots. These findings indicate that the biosynthesis of 28-norcastasterone may parallel that of castasterone. The endogenous levels of brassinosteroids differed between roots and shoots, indicating that the biosynthesis of brassinosteroids is differently regulated between these tissues. Regulation of root growth by brassinosteroids is also discussed.

Selective Interaction of Triazole Derivatives with DWF4, a Cytochrome P450 Monooxygenase of the Brassinosteroid Biosynthetic Pathway, Correlates with Brassinosteroid Deficiency in Planta

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.

Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis.

Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22alpha hydroxylase (CYP90B1) step that is the putative rate-limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light- and dark-grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws-2 and AOD4 revealed that dwf4 accumulated the precursors of the 22alpha-hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws-2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild-type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre, are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.

Effect of light quality on the level of endogenous brassinosteroids in rice

Effect of light quality on the level of endogenous brassinosteroids in rice

A putative role for the tomato genes DUMPY and CURL-3 in brassinosteroid biosynthesis and response.

The dumpy (dpy) mutant of tomato (Lycopersicon esculentum Mill.) exhibits short stature, reduced axillary branching, and altered leaf morphology. Application of brassinolide and castasterone rescued the dpy phenotype, as did C-23-hydroxylated, 6-deoxo intermediates of brassinolide biosynthesis. The brassinolide precursors campesterol, campestanol, and 6-deoxocathasterone failed to rescue, suggesting that dpy may be affected in the conversion of 6-deoxocathasterone to 6-deoxoteasterone, similar to the Arabidopsis constitutive photomorphogenesis and dwarfism (cpd) mutant. Measurements of endogenous brassinosteroid levels by gas chromatography-mass spectrometry were consistent with this hypothesis. To examine brassinosteroid-regulated gene expression in dpy, we performed cDNA subtractive hybridization and isolated a novel xyloglucan endotransglycosylase that is regulated by brassinosteroid treatment. The curl-3 (cu-3) mutant (Lycopersicon pimpinellifolium ¿Jusl. Mill.) shows extreme dwarfism, altered leaf morphology, de-etiolation, and reduced fertility, all strikingly similar to the Arabidopsis mutant brassinosteroid insensitive 1 (bri1). Primary root elongation of wild-type L. pimpinellifolium seedlings was strongly inhibited by brassinosteroid application, while cu-3 mutant roots were able to elongate at the same brassinosteroid concentration. Moreover, cu-3 mutants retained sensitivity to indole-3-acetic acid, cytokinins, gibberellin, and abscisic acid while showing hypersensitivity to 2, 4-dichlorophenoxyacetic acid in the root elongation assay. The cu-3 root response to hormones, coupled with its bri1-like phenotype, suggests that cu-3 may also be brassinosteroid insensitive.

BAS1: A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis.

The Arabidopsis bas1-D mutation suppresses the long hypocotyl phenotype caused by mutations in the photoreceptor phytochrome B (phyB). The adult phenotype of bas1-D phyB-4 double mutants mimics that of brassinosteroid biosynthetic and response mutants. bas1-D phyB-4 has reduced levels of brassinosteroids and accumulates 26-hydroxybrassinolide in feeding experiments. The basis for the mutant phenotype is the enhanced expression of a cytochrome P450 (CYP72B1). bas1-D suppresses a phyB-null allele, but not a phyA-null mutation, and partially suppresses a cryptochrome-null mutation. Seedlings with reduced BAS1 expression are hyperresponsive to brassinosteroids in a light-dependent manner and display reduced sensitivity to light under a variety of conditions. Thus, BAS1 represents one of the control points between multiple photoreceptor systems and brassinosteroid signal transduction.

Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids.

Seven dwarf mutants resembling brassinosteroid (BR)-biosynthetic dwarfs were isolated that did not respond significantly to the application of exogenous BRs. Genetic and molecular analyses revealed that these were novel alleles of BRI1 (Brassinosteroid-Insensitive 1), which encodes a receptor kinase that may act as a receptor for BRs or be involved in downstream signaling. The results of morphological and molecular analyses indicated that these represent a range of alleles from weak to null. The endogenous BRs were examined from 5-week-old plants of a null allele (bri1-4) and two weak alleles (bri1-5 and bri1-6). Previous analysis of endogenous BRs in several BR-biosynthetic dwarf mutants revealed that active BRs are deficient in these mutants. However, bri1-4 plants accumulated very high levels of brassinolide, castasterone, and typhasterol (57-, 128-, and 33-fold higher, respectively, than those of wild-type plants). Weaker alleles (bri1-5 and bri1-6) also accumulated considerable levels of brassinolide, castasterone, and typhasterol, but less than the null allele (bri1-4). The levels of 6-deoxoBRs in bri1 mutants were comparable to that of wild type. The accumulation of biologically active BRs may result from the inability to utilize these active BRs, the inability to regulate BR biosynthesis in bri1 mutants, or both. Therefore, BRI1 is required for the homeostasis of endogenous BR levels.

The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis.

Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.

Effect of S-3307 on the levels of sterols,brassinosteroids and gibberellins in pea seedling.

Effect of S-3307 on the levels of sterols,brassinosteroids and gibberellins in pea seedling.