Light Activates Brassinosteroid Biosynthesis to Promote Hook Opening and Petiole Development in Arabidopsis thaliana.

Although brassinosteroids (BRs) have been proposed to be negative regulators of photomorphogenesis, their physiological role therein has remained elusive. We studied light-induced photomorphogenic development in the presence of the BR biosynthesis inhibitor, brassinazole (Brz). Hook opening was inhibited in the presence of Brz; this inhibition was reversed in the presence of brassinolide (BL). Hook opening was accompanied by cell expansion on the inner (concave) side of the hook. This cell expansion was inhibited in the presence of Brz but was restored upon the addition of BL. We then evaluated light-induced organ-specific expression of three BR biosynthesis genes, DWF4, BR6ox1 and BR6ox2, and a BR-responsive gene, SAUR-AC1, during the photomorphogenesis of Arabidopsis. Expression of these genes was induced, particularly in the hook region, in response to illumination. The induction peaked after 3 h of light exposure and preceded hook opening. Phytochrome-deficient mutants, hy1, hy2 and phyAphyB, and a light-signaling mutant, hy5, were defective in light-induced expression of BR6ox1, BR6ox2 and SAUR-AC1. Light induced both expression of BR6ox genes and petiole development. Petiole development was inhibited in the presence of Brz. Our results largely contradict the early view that BRs are negative regulators of photomorphogenesis. Our data collectively suggest that light activates the expression of BR biosynthesis genes in the hook region via a phytochrome-signaling pathway and HY5 and that BR biosynthesis is essential for hook opening and petiole development during photomorphogenesis.

Loose Plant Architecture1 (LPA1) determines lamina joint bending by suppressing auxin signalling that interacts with C-22-hydroxylated and 6-deoxo brassinosteroids in rice.

Lamina inclination is a key agronomical character that determines plant architecture and is sensitive to auxin and brassinosteroids (BRs). Loose Plant Architecture1 (LPA1) in rice (Oryza sativa) and its Arabidopsis homologues (SGR5/AtIDD15) have been reported to control plant architecture and auxin homeostasis. This study explores the role of LPA1 in determining lamina inclination in rice. LPA1 acts as a positive regulator to suppress lamina bending. Genetic and biochemical data indicate that LPA1 suppresses the auxin signalling that interacts with C-22-hydroxylated and 6-deoxo BRs, which regulates lamina inclination independently of OsBRI1. Mutant lpa1 plants are hypersensitive to indole-3-acetic acid (IAA) during the lamina inclination response, which is suppressed by the brassinazole (Brz) inhibitor of C-22 hydroxylase involved in BR synthesis. A strong synergic effect is detected between lpa1 and d2 (the defective mutant for catalysis of C-23-hydroxylated BRs) during IAA-mediated lamina inclination. No significant interaction between LPA1 and OsBRI1 was identified. The lpa1 mutant is sensitive to C-22-hydroxylated and 6-deoxo BRs in the d61-1 (rice BRI1 mutant) background. We present evidence verifying that two independent pathways function via either BRs or BRI1 to determine IAA-mediated lamina inclination in rice. RNA sequencing analysis and qRT-PCR indicate that LPA1 influences the expression of three OsPIN genes (OsPIN1a, OsPIN1c and OsPIN3a), which suggests that auxin flux might be an important factor in LPA1-mediated lamina inclination in rice.

Rice CYP734As function as multisubstrate and multifunctional enzymes in brassinosteroid catabolism.

Catabolism of brassinosteroids regulates the endogenous level of bioactive brassinosteroids. In Arabidopsis thaliana, bioactive brassinosteroids such as castasterone (CS) and brassinolide (BL) are inactivated mainly by two cytochrome P450 monooxygenases, CYP734A1/BAS1 and CYP72C1/SOB7/CHI2/SHK1; CYP734A1/BAS1 inactivates CS and BL by means of C-26 hydroxylation. Here, we characterized CYP734A orthologs from Oryza sativa (rice). Overexpression of rice CYP734As in transgenic rice gave typical brassinosteroid-deficient phenotypes. These transformants were deficient in both the bioactive CS and its precursors downstream of the C-22 hydroxylation step. Consistent with this result, recombinant rice CYP734As utilized a range of C-22 hydroxylated brassinosteroid intermediates as substrates. In addition, rice CYP734As can catalyze hydroxylation and the second and third oxidations to produce aldehyde and carboxylate groups at C-26 in vitro. These results indicate that rice CYP734As are multifunctional, multisubstrate enzymes that control the endogenous bioactive brassinosteroid content both by direct inactivation of CS and by the suppression of CS biosynthesis by decreasing the levels of brassinosteroid precursors.

Auxin stimulates DWARF4 expression and brassinosteroid biosynthesis in Arabidopsis.

Brassinosteroids (BRs) are growth-promoting steroidal hormones. Despite the importance of BRs in plant biology, the signal that initiates BR biosynthesis remains unknown. Among the enzymes involved in BR biosynthesis in Arabidopsis (Arabidopsis thaliana), DWARF4 catalyzes the rate-determining step. Through both the histochemical analysis of DWF4pro:GUS plants and the direct measurement of endogenous BR content, we discovered that BR biosynthesis is stimulated by auxin. When DWF4pro:GUS was subjected to auxin dose-response tests and a time-course analysis, GUS activity started to increase at an auxin concentration of 10 nm, rising noticeably after 1 h of auxin treatment. In addition, the analysis of the DWF4pro:GUS line in BR- and auxin-mutant backgrounds revealed that the induction by auxin requires auxin-signaling pathways but not BRs, which implies that auxin signaling directly controls BR biosynthesis. Furthermore, chromatin immunoprecipitation assays confirmed that auxin inhibits the binding of the transcriptional repressor, BZR1, to the DWF4 promoter. A microarray analysis that was designed to examine the transcriptomes after treatment with auxin alone or auxin plus brassinazole (a BR biosynthetic inhibitor) revealed that genes previously characterized as being auxin responsive are not properly regulated when BR biosynthesis is disrupted by brassinazole. Therefore, our results support the idea that auxin regulates BR biosynthesis, and that auxin thus relies on synthesized BRs for some of its growth-promoting effects in Arabidopsis.

Constitutive activation of brassinosteroid signaling in the Arabidopsis elongated-D/bak1 mutant.

Defects in brassinosteroid (BR) biosynthetic or signaling genes result in dwarfed plants, whereas overexpression of these genes increases overall stature. An Arabidopsis elongated-D (elg-D) mutant shares phenotypic similarities with BR overexpression lines, suggesting its implication in BR pathways. Here, we determine how elg-D affects BR signaling. Since elg-D rescued dwarfism in bri1-5 plants, a BR receptor mutant, but not in BR-insensitive bin2/dwf12-1D plants, elg-D appears to act between bri1-5 and bin2/dwf12-1D in BR signaling. We found that elg-D had an increased response to epi-brassinolide (epi-BL); that the BES1 transcription factor was shifted toward the dephosphorylated form in elg-D; that the expression of a BR responsive gene, SAUR-AC1, was upregulated in elg-D; and that transcription of BR biosynthetic genes, DWF4 and CPD, was downregulated by feedback inhibition. Thus, endogenous levels of CS and BL as well as biosynthetic intermediates were reduced by the elg-D mutation, whereas basal levels of BR signaling were elevated. Map-based cloning and sequencing revealed that elg-D is allelic to the BR co-receptor protein, BAK1, and has an Asp(122) to Asn substitution in the third repeat of the extracellular leucine-rich repeat (LRR) domain. In agreement with the finding that BAK1/ELG is involved in the perception of pathogen-associated molecular patterns (PAMPs), the bak1/elg-D plants exhibited increased Pseudomonas syringae growth. Therefore, bak1/elg-D promotes Arabidopsis growth by stimulating BR signaling at the expense of its readiness to respond to biotic stress factors. The BAK1/ELG BR co-receptor thus plays an important role in BR signaling that is mediated by its LRR domain.

The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access.

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.

Involvement of C-22-hydroxylated brassinosteroids in auxin-induced lamina joint bending in rice.

The rice lamina joint is ideal material for investigating the activity of brassinosteroids (BRs) and auxin because of its high sensitivity to these compounds. Using a series of rice BR biosynthetic and receptor mutants, we conducted lamina joint tests to elucidate the mechanism of cross-talk between BR and auxin signaling in lamina joint bending. In BR biosynthetic mutants d2 and brd1, which are defective in C-23 hydroxylase and C-6 oxidase, respectively, the lamina joint response to auxin was significantly higher than that of wild-type plants. The other BR-biosynthetic mutants, brd2, osdwarf4 and d11, which are defective in C-22-hydroxylated BRs, showed less or no response to auxin. These results suggest that C-22-hydroxylated BRs are involved in auxin-induced lamina joint bending. The results were supported by the observation that inhibition of the hyper-response to auxin in d2 was reduced by treatment with brassinazole, which inhibits the function of DWARF4, the C-22 hydroxylase. In d61, which is defective in OsBRI1, a possible BR receptor in rice, the bending angle of the lamina joint in response to auxin and C-22-hydroxylated 6-deoxoBRs was nearly the same as that in wild-type plants. This implies that C-22-hydroxylated BRs function in auxin signaling independently of OsBRI1. From these observations, we propose that C-22-hydroxylated BRs participate in auxin signaling via a novel OsBRI1-independent signaling pathway.

Erect leaves caused by brassinosteroid deficiency increase biomass production and grain yield in rice.

New cultivars with very erect leaves, which increase light capture for photosynthesis and nitrogen storage for grain filling, may have increased grain yields. Here we show that the erect leaf phenotype of a rice brassinosteroid-deficient mutant, osdwarf4-1, is associated with enhanced grain yields under conditions of dense planting, even without extra fertilizer. Molecular and biochemical studies reveal that two different cytochrome P450s, CYP90B2/OsDWARF4 and CYP724B1/D11, function redundantly in C-22 hydroxylation, the rate-limiting step of brassinosteroid biosynthesis. Therefore, despite the central role of brassinosteroids in plant growth and development, mutation of OsDWARF4 alone causes only limited defects in brassinosteroid biosynthesis and plant morphology. These results suggest that regulated genetic modulation of brassinosteroid biosynthesis can improve crops without the negative environmental effects of fertilizers.

Castasterone is a likely end product of brassinosteroid biosynthetic pathway in rice.

Brassinolide is known to be the most biologically active compound among more than 50 brassinosteroids identified to date. However, brassinolide has not been detected in rice. To determine if this is due to the lack of the brassinolide synthase function in the rice CYP85A enzyme, we performed analyses to study metabolic conversion using a yeast strain harboring the rice CYP85A1 gene. In repeated feeding tests where the substrates were used, the biosynthetic pathway progressed only up to the synthesis of castasterone, not of brassinolide. Phylogenetic analysis of the CYP85 amino acid sequences revealed that duplication of the CYP85 gene has occurred in most dicotyledonous plant genomes; further, 1 of the 2 copies of CYP85 is evolving to develop a brassinolide synthase function. However, only a single copy of this gene is found in the currently available genome sequences of graminaceous plants; this is a likely explanation for the absence of an endogenous pool of brassinolide in rice plants.

Brz220 interacts with DWF4, a cytochrome P450 monooxygenase in brassinosteroid biosynthesis, and exerts biological activity.

Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.

Brachytic 1 of barley (Hordeum vulgare L.) encodes the α subunit of heterotrimeric G protein.

Physiological and molecular biological analysis of the dwarf barley (Hordeum vulgare L.) mutant brachytic 1 (brh1) was conducted. The root responses of brh1 to brassinolide were weaker than those of wild type, but the responses of leaf segments of dark-grown plants were not. Responses of brh1 to gibberellin A3 were similar to or slightly stronger than those of wild type. Endogenous levels of these hormones in young seedlings were not clearly different between brh1 and wild type. Skotomorphogeneses of brh1 were similar to those of wild type. Some of these physiological characteristics of brh1 resemble those of the dwarf rice (Oryza sativa L.) mutant daikoku (dwarf1; d1), whose dwarfism is caused by a mutation in the heterotrimeric G protein α (Gα) subunit. A database search indicated that the barley Gα gene is located near the locus where Brh1 has already been genetically mapped. Sequences of the Gα gene and cDNAs of five brh1 alleles contained substitutions and a deletion that lead to the production of abnormal Gα proteins. These results indicate that the phenotype of brh1, similarly to that of d1, is caused by mutations in an orthologous gene encoding Gα.

A mammalian steroid action inhibitor spironolactone retards plant growth by inhibition of brassinosteroid action and induces light-induced gene expression in the dark.

We screened steroid derivatives and found that spironolactone, an inhibitor of both 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and aldosterone receptor, is an inhibitor of phytohormone brassinosteroid (BR) action in plants. Under both dark and light growing conditions, spironolactone induced morphological changes in Arabidopsis, characteristic of brassinosteroid-deficient mutants. Spironolactone-treated plants were also nearly restored to the wild-type phenotype by treatment with additional BRs. In the spironolactone-treated Arabidopsis, the CPD gene in the BR biosynthesis pathway was up-regulated, probably due to feedback regulation caused by BR-deficiency. Spironolactone-treated tobacco plants grown in the dark showed expression of light-regulated genes as was observed in the deficient mutant. These data suggest that spironolactone inhibits brassinosteroid action probably due to the blockage of biosynthesis and exerts its activity against plants. Thus, spironolactone, in conjunction with brassinosteroid-deficient mutants, can be used to clarify the function of BRs in plants and characterize mutants. The spironolactone action site was also investigated by feeding BR biosynthesis intermediates to Arabidopsis grown in the dark, and the results are discussed.

Biosynthesis of cholestanol in higher plants.

To understand the early steps of C(27) brassinosteroid biosynthesis, metabolic experiments were performed with Arabidopsis thaliana and Nicotiana tabacum seedlings, and with cultured Catharanthus roseus cells. [26, 28-2H(6)]Campestanol, [26-2H(3)]cholesterol, and [26-2H(3)]cholestanol were administered to each plant, and the resulting metabolites were analyzed by gas chromatography-mass spectrometry. In all the species examined, [2H(3)]cholestanol was identified as a metabolite of [2H(6)]campestanol, and [2H(3)]cholest-4-en-3-one and [2H(3)]cholestanol were identified as metabolites of [2H(3)]cholesterol. This study revealed that cholestanol (C(27) sterol) was biosynthesized from both cholesterol (C(27) sterol) and campestanol (C(28) sterol). It was also demonstrated that cholestanol was converted to 6-oxocholestanol, and campestanol was converted to 6-oxocampestanol.

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris.

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.

Overexpression of 3ß-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in Arabidopsis.

Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3ß-hydroxysteroid dehydrogenases/C-4 decarboxylases (3ßHSD/D). To understand the role of 3ßHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3ßHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3ßHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.

Brassinosteroids regulate grain filling in rice.

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO(2) assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.

Roles of brassinosteroids and related mRNAs in pea seed growth and germination.

The levels of endogenous brassinosteroids (BRs) and the expression of the biosynthesis/metabolism/perception genes involved have been investigated during the development and germination of pea (Pisum sativum) seeds. When seeds were rapidly growing, the level of biologically active BRs (brassinolide [BL] and castasterone [CS]) and the transcript levels of two BR C-6 oxidases (CYP85A1 and CYP85A6) reached a maximum, suggesting the significance of BL and CS in seed development. In the early stages of germination, CS, but not BL, appeared and its level increased in the growing tissues in which the transcript level of CYP85A1 and CYP85A6 was high, suggesting the significance of CS in seed germination and early seedling growth of pea. 6-Deoxocathasterone (6-deoxoCT) was the quantitatively major BR in mature seeds. At the early stage of germination, the level of 6-deoxoCT was specifically decreased, whereas the levels of downstream intermediates were increased. It seems that 6-deoxoCT is the major storage BR and is utilized during germination and early growth stages. The level of the mRNAs of BR biosynthesis and perception genes fluctuated during seed development. In mature seeds, most of mRNAs were present, but the level was generally lower compared with immature seeds. However, CYP90A9 mRNA rapidly increased during seed development and reached the maximum in mature seeds. The mRNAs stored in mature pea seeds seem to be utilized when seeds germinate. However, it was found that de novo transcription of mRNAs also starts as early as during seed imbibition.

BEN1, a gene encoding a dihydroflavonol 4-reductase (DFR)-like protein, regulates the levels of brassinosteroids in Arabidopsis thaliana.

The ben1-1D (bri1-5 enhanced 1-1dominant) mutant was identified via an activation-tagging screen for bri1-5 extragenic modifiers. bri1-5 is a weak mutant allele of the brassinosteroid receptor gene, BRI1. Overexpression of BEN1 greatly enhances the defective phenotypes of bri1-5 plants. Removal of BEN1 by gene disruption in a Col-0 wild-type background, on the other hand, promotes the elongation of organs. Because BEN1 encodes a novel protein homologous to dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (BAN), BEN1 is probably involved in a brassinosteroid metabolic pathway. Analyses of brassinosteroid profiles demonstrated that BEN1 is indeed responsible for regulating the levels of several brassinosteroids, including typhasterol, castasterone and brassinolide. In vivo feeding and in vitro biochemical assays suggest that BEN1 is probably involved in a new mechanism to regulate brassinosteroid levels. These results provide additional insight into the regulatory mechanisms of bioactive brassinosteroids.

Co-regulation of brassinosteroid biosynthesis-related genes during xylem cell differentiation.

To understand the regulatory mechanisms of brassinosteroid (BR) biosynthesis in specific plant developmental processes, we first investigated the accumulation profiles of BRs and sterols in xylem differentiation in a Zinnia culture. The amounts of many substances in the late C28 sterol biosynthetic pathway to campesterol (CR), such as episterol and 24-methylenecholesterol, as well as those in the BR-specific biosynthetic pathway from CR to brassinolide (BL), were elevated in close association with tracheary element differentiation. Among them, 6-deoxotyphasterol (6-deoxoTY) accumulated to unusually high levels within cells cultured in tracheary element-inductive medium, while castasterone (CS) was not elevated either within or outside cells. To identify the molecular basis of this co-up-regulation of BRs and C28 sterols, we isolated Zinnia genes for the key enzymes of BR biosynthesis, ZeSTE1, ZeDIM, ZeDWF4, ZeCPD1 and ZeCPD2. RNA gel blot analysis of these genes indicated a coordinated increase in transcripts for ZeSTE1, ZeDIM, ZeDWF4 and ZeCPD1, and a tracheary element differentiation-specific increase in transcripts for ZeDWF4 and ZeCPD1. In situ hybridization experiments of ZeDWF4 and ZeCPD1 mRNAs revealed their preferential accumulation in procambium cells, immature xylem cells and xylem parenchyma cells. These results suggest that BR biosynthesis during tracheary element differentiation may be regulated by the coordinated regulation of broad sterol biosynthesis and specific regulation of BR biosynthesis, which occurs in part by elevated transcript levels of genes encoding BR biosynthetic enzymes, specifically ZeDWF4 and ZeCPD1. These data provide new insights into the regulation of BR biosynthesis and BR signaling during plant development.

The rice SPINDLY gene functions as a negative regulator of gibberellin signaling by controlling the suppressive function of the DELLA protein, SLR1, and modulating brassinosteroid synthesis.

SPINDLY (SPY) encodes an O-linked N-acetylglucosamine transferase that is considered to be a negative regulator of gibberellin (GA) signaling through an unknown mechanism. To understand the function of SPY in GA signaling in rice, we isolated a rice SPINDLY homolog (OsSPY) and produced knockdown transgenic plants in which OsSPY expression was reduced by introducing its antisense or RNAi construct. In knockdown plants, the enhanced elongation of lower internodes was correlated with decreased levels of OsSPY expression, similar to the spindly phenotype of Arabidopsis spy mutants, suggesting that OsSPY also functions as a negative factor in GA signaling in rice. The suppressive function of OsSPY in GA signaling was supported by the findings that the dwarfism was partially rescued and OsGA20ox2 (GA20 oxidase) expression was reduced in GA-deficient and GA-insensitive mutants by the knockdown of OsSPY function. The suppression of OsSPY function in a GA-insensitive mutant, gid2, also caused an increase in the phosphorylation of a rice DELLA protein, SLR1, but did not change the amount of SLR1. This indicates that the function of OsSPY in GA signaling is not via changes in the amount or stability of SLR1, but probably involves control of the suppressive function of SLR1. In addition to the GA-related phenotypes, OsSPY antisense and RNAi plants showed increased lamina joint bending, which is a brassinosteroid-related phenotype, indicating that OsSPY may play roles both in GA signaling and in the brassinosteroid pathway.