Biomedical subjects

Shozo Fujioka

Publications and source records attributed to Shozo Fujioka.

At least 19 recordsLinked to original sources

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

The regulation of DWARF4 expression is likely a critical mechanism in maintaining the homeostasis of bioactive brassinosteroids in Arabidopsis.

Mutants that are defective in brassinosteroid (BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis (Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4:beta-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4:GUS expression was negatively regulated by BRs. DWF4:GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4:GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4:GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSINOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.

Arabidopsis↗

PubMed2006

The role of OsBRI1 and its homologous genes, OsBRL1 and OsBRL3, in rice.

Since first identifying two alleles of a rice (Oryza sativa) brassinosteroid (BR)-insensitive mutant, d61, that were also defective in an orthologous gene in Arabidopsis (Arabidopsis thaliana) BRASSINOSTEROID INSENSITIVE1 (BRI1), we have isolated eight additional alleles, including null mutations, of the rice BRI1 gene OsBRI1. The most severe mutant, d61-4, exhibited severe dwarfism and twisted leaves, although pattern formation and differentiation were normal. This severe shoot phenotype was caused mainly by a defect in cell elongation and the disturbance of cell division after the determination of cell fate. In contrast to its severe shoot phenotype, the d61-4 mutant had a mild root phenotype. Concomitantly, the accumulation of castasterone, the active BR in rice, was up to 30-fold greater in the shoots, while only 1.5-fold greater in the roots. The homologous genes for OsBRI1, OsBRL1 and OsBRL3, were highly expressed in roots but weakly expressed in shoots, and their expression was higher in d61-4 than in the wild type. Based on these observations, we conclude that OsBRI1 is not essential for pattern formation or organ initiation, but is involved in organ development through controlling cell division and elongation. In addition, OsBRL1 and OsBRL3 are at least partly involved in BR perception in the roots.

Alleles↗

PubMed2006

Arabidopsis Aux/IAA genes are involved in brassinosteroid-mediated growth responses in a manner dependent on organ type.

We examined whether auxin/indole-3-acetic acid (Aux/IAA) proteins, which are key players in auxin-signal transduction, are involved in brassinosteroid (BR) responses. iaa7/axr2-1 and iaa17/axr3-3 mutants showed aberrant BR sensitivity and aberrant BR-induced gene expression in an organ-dependent manner. Two auxin inhibitors were tested in terms of BR responses. Yokonolide B inhibited BR responses, whereas p-chlorophenoxyisobutyric acid did not inhibit BR responses. DNA microarray analysis revealed that 108 genes were up-regulated, while only eight genes were down-regulated in iaa7. Among the genes that were up- or down-regulated in axr2, 22% were brassinolide-inducible genes, 20% were auxin-inducible genes, and the majority were sensitive neither to BR nor to auxin. An inhibitor of BR biosynthesis, brassinazole, inhibited auxin induction of the DR5-GUS gene, which consists of a synthetic auxin-response element, a minimum promoter, and a beta-glucuronidase. These results suggest that Aux/IAA proteins function in auxin- and BR-signaling pathways, and that IAA proteins function as the signaling components modulating BR sensitivity in a manner dependent on organ type.

Arabidopsis↗

PubMed2006

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols.

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.

Arabidopsis↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

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.

Biomass↗

PubMed2005

The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids.

Steroid hormones are essential for development, and the precise control of their homeostasis is a prerequisite for normal growth. UDP-glycosyltransferases (UGTs) are considered to play an important regulatory role in the activity of steroids in mammals and insects. This study provides an indication that a UGT accepting plant steroids as substrates functions in brassinosteroid (BR) homeostasis. The UGT73C5 of Arabidopsis thaliana catalyses 23-O-glucosylation of the BRs brassinolide (BL) and castasterone. Transgenic plants overexpressing UGT73C5 displayed BR-deficient phenotypes and contained reduced amounts of BRs. The phenotype, which was already apparent in seedlings, could be rescued by application of BR. In feeding experiments with BL, wild-type seedlings converted BL to the 23-O-glucoside; in the transgenic lines silenced in UGT73C5 expression, no 23-O-glucoside was detected, implying that this UGT is the only enzyme that catalyzes BL-23-O-glucosylation in seedlings. Plant lines in which UGT73C5 expression was altered also displayed hypocotyl phenotypes previously described for seedlings in which BR inactivation by hydroxylation was changed. These data support the hypothesis that 23-O-glucosylation of BL is a function of UGT73C5 in planta, and that glucosylation regulates BR activity.

Animals↗

PubMed2005

TANMEI/EMB2757 encodes a WD repeat protein required for embryo development in Arabidopsis.

We identified the Arabidopsis (Arabidopsis thaliana) tanmei/emb2757 (tan) mutation that causes defects in both embryo and seedling development. tan mutant embryos share many characteristics with the leafy cotyledon (lec) class of mutants in that they accumulate anthocyanin, are intolerant of desiccation, form trichomes on cotyledons, and have reduced accumulation of storage proteins and lipids. Thus, TAN functions both in the early and late phases of embryo development. Moreover, the TAN and LEC genes interact synergistically, suggesting that they do not act in series in the same genetic pathway but, rather, that they have overlapping roles during embryogenesis. tan mutants die as embryos, but immature mutant seeds can be germinated in culture. However, tan mutant seedlings are defective in shoot and root development, their hypocotyls fail to elongate in the dark, and they die as seedlings. We isolated the TAN gene and showed that the predicted polypeptide has seven WD repeat motifs, suggesting that TAN forms complexes with other proteins. Together, these results suggest that TAN interacts with other proteins to control many aspects of embryo development.

Amino Acid Motifs↗

PubMed2005

The Rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone.

We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective.

Amino Acid Sequence↗

PubMed2005

Ectopic endoreduplication caused by sterol alteration results in serrated petals in Arabidopsis.

The Arabidopsis frill1 (frl1) mutant, that has serrated petals and sepals but no other large changes in plant morphology, was studied. The frl1 had a mutation in STEROL METHYLTRANSFERASE 2 and an altered sterol composition. It was found that the frl1 mutation causes ectopic endoreduplication in petal tips that do not normally endoreduplicate. The rosette leaves of frl1 also showed an enhanced level of endoreduplication, but their morphology was hardly affected. These facts suggest that the suppression of endoreduplication is important for petal morphogenesis and the normal sterol composition is required for this suppression.

Arabidopsis↗

PubMed2005

A novel cytochrome P450 is implicated in brassinosteroid biosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seed length.

We have characterized a rice (Oryza sativa) dwarf mutant, dwarf11 (d11), that bears seeds of reduced length. To understand the mechanism by which seed length is regulated, the D11 gene was isolated by a map-based cloning method. The gene was found to encode a novel cytochrome P450 (CYP724B1), which showed homology to enzymes involved in brassinosteroid (BR) biosynthesis. The dwarf phenotype of d11 mutants was restored by the application of the brassinolide (BL). Compared with wild-type plants, the aberrant D11 mRNA accumulated at higher levels in d11 mutants and was dramatically reduced by treatment with BL, implying that the gene is feedback-regulated by BL. Precise determination of the defective step(s) in BR synthesis in d11 mutants proved intractable because of tissue specificity and the complex control of BR accumulation in plants. However, 6-deoxotyphasterol (6-DeoxoTY) and typhasterol (TY), but not any upstream intermediates before these compounds, effectively restored BR response in d11 mutants in a lamina joint bending assay. Multiple lines of evidence together suggest that the D11/CYP724B1 gene plays a role in BR synthesis and may be involved in the supply of 6-DeoxoTY and TY in the BR biosynthesis network in rice.

Amino Acid Sequence↗

PubMed2005

Identification of a novel factor, vanillyl benzyl ether, which inhibits somatic embryogenesis of Japanese larch (Larix leptolepis Gordon).

In contrast to angiosperms, some gymnosperms form well-developed suspensors in somatic embryogenesis. This characteristic makes it easy to study suspensor biology. In cultures with high cell densities, somatic embryogenesis of Japanese larch, especially the suspensor development, is strongly inhibited due to factor(s) that are released by the cells into the culture medium. In this study, we purified and identified one of the inhibitory factors present in high-cell-density conditioned medium (HCM) of larch cells. The factor with the strongest inhibitory activity was purified by dialysis, extraction by ethyl acetate, octadecylsilyl (ODS) column chromatography and high-performance liquid chromatography (HPLC). The inhibitory factor was identified as vanillyl benzyl ether (VBE) by physicochemical analysis. This compound was first isolated from natural resources. Authentic VBE inhibited somatic embryo formation in Japanese larch, and the inhibitory effect in the suspensor was stronger than in the embryo proper. Furthermore, quantification of VBE by HPLC demonstrated that VBE accumulates at high concentrations in HCM. These results suggest that VBE is a novel negative regulator of somatic embryogenesis.

Benzaldehydes↗

PubMed2005

Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1.

Both animals and plants use steroids as signalling molecules during growth and development. Animal steroids are principally recognized by members of the nuclear receptor superfamily of transcription factors. In plants, BRI1, a leucine-rich repeat (LRR) receptor kinase localized to the plasma membrane, is a critical component of a receptor complex for brassinosteroids. Here, we present the first evidence for direct binding of active brassinosteroids to BRI1 using a biotin-tagged photoaffinity castasterone (BPCS), a biosynthetic precursor of brassinolide (the most active of the brassinosteroids). Binding studies using BPCS, (3)H-labelled brassinolide and recombinant BRI1 fragments show that the minimal binding domain for brassinosteroids consists of a 70-amino acid island domain (ID) located between LRR21 and LRR22 in the extracellular domain of BRI1, together with the carboxy-terminal flanking LRR (ID-LRR22). Our results demonstrate that brassinosteroids bind directly to the 94 amino acids comprising ID-LRR22 in the extracellular domain of BRI1, and define a new binding domain for steroid hormones.

Amino Acid Sequence↗

PubMed2005

Plant brassinosteroid hormones.

In animals, a large number of steroid hormones play important roles in numerous processes including reproduction and differentiation. The biologically active plant steroid brassinolide (BL) was first discovered in the pollen of western rape in 1979 (Grove et al., 1979). This finding suggested that BL is indispensable for plant growth and differentiation. To date, more than 50 BL analogs have been identified, and the group has been termed brassinosteroids (BRs) (Fujioka and Yokota, 2003). Brassinosteroids have several biological activities, such as inducing cell elongation when applied at very low concentrations. For this reason, soon after their discovery, they were suggested to be a sixth type of plant hormone; however, for years BRs were not considered true plant hormones. The turning point in BR research was the discovery of the Arabidopsis dwarf mutants det2 and cpd in 1996 (Li et al., 1996; Szekeres et al., 1996). These BR-deficient mutants were found to revert to the wild-type phenotype following BR treatment. Concurrent with the analysis of these mutants, an outline of the biosynthetic pathway of BRs was being elucidated through chemical analysis. Following the isolation of det2 and cpd, a great number of BR-deficient mutants were identified. The mutant genes were found to encode proteins that catalyze the conversion of plant steroids to BR precursors. Eventually, BRs were widely recognized as important plant hormones indispensable for growth and differentiation (Clouse and Sasse, 1998). In parallel, mutants that are insensitive to BRs were isolated (Clouse et al., 1996; Li et al., 1997) with phenotypes very similar to those of the BR-biosynthesis mutants. Investigations of these mutants revealed several mechanisms of BR perception and signal transduction (Bishop and Koncz, 2002; Clouse, 2002). This review describes findings on the effects of BRs on plant growth, BR biosynthesis and catabolism, and BR signal transduction.

Brassinosteroids↗

PubMed2005

Brevicompanine C, cyclo-(D-Ile-L-Trp), and cyclo-(D-Leu-L-Trp), plant growth regulators from Penicillium brevi-compactum.

New plant growth regulators, named brevicompanine C (1), cyclo-(D-Ile-L-Trp) (2), and cyclo-(D-Leu-L-Trp) (3), have been isolated from Penicillium brevi-compactum Dierckx, and their structures have been established by spectroscopic methods including 2D NMR and chiral TLC analysis. Plant growth activities of 1, 2, and 3 have been examined using lettuce seedling bioassay methods. All compounds accelerated the root growth of the seedlings in proportion to their concentration from 1 to 100 mg/L.

Amino Acid Sequence↗

PubMed2005

CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.

Amino Acid Sequence↗

PubMed2005

BAS1 and SOB7 act redundantly to modulate Arabidopsis photomorphogenesis via unique brassinosteroid inactivation mechanisms.

Active brassinosteroids (BRs), such as brassinolide (BL) and castasterone (CS), are growth-promoting plant hormones. An Arabidopsis cytochrome P450 monooxygenase (CYP734A1, formerly CYP72B1), encoded by the BAS1 gene, inactivates BRs and modulates photomorphogenesis. BAS1 was identified as the overexpressed gene responsible for a dominant, BR-deficient mutant, bas1-D. This mutant was isolated in an activation-tagged screen designed to identify redundant genes that might not be identified in classic loss-of-function screens. Here we report the isolation of a second activation-tagged mutant with a BR-deficient phenotype. The mutant phenotype is caused by the overexpression of SOB7 (CYP72C1), a homolog of BAS1. We generated single and double null-mutants of BAS1 and SOB7 to test the hypothesis that these two genes act redundantly to modulate photomorphogenesis. BAS1 and SOB7 act redundantly with respect to light promotion of cotyledon expansion, repression of hypocotyl elongation and flowering time in addition to other phenotypes not regulated by light. We also provide biochemical evidence to suggest that BAS1 and SOB7 act redundantly to reduce the level of active BRs, but have unique mechanisms. Overexpression of SOB7 results in a dramatic reduction in endogenous CS levels, and although single null-mutants of BAS1 and SOB7 have the same level of CS as the wild type, the double null-mutant has twice the amount. Application of BL to overexpression lines of BAS1 or SOB7 results in enhanced metabolism of BL, though only BAS1 overexpression lines confer enhanced conversion to 26-OHBL, suggesting that SOB7 and BAS1 convert BL and CS into unique products.

Amino Acid Sequence↗