Physiology and molecular mode of action of brassinosteroids

Brassinosteroids (BRs) comprise a group of polyhydroxysteroids, which show close structural similarity to steroid hormones from arthropods and mammals. BRs are now accepted as a new class of phytohormones due to their ubiquitous occurrence in plants, their highly effective elicitation of various responses and the identification of mutants defective in BR-biosynthesis or -response. Important steps of BR-biosynthesis were elucidated with precursor-feeding experiments and by the analysis of BR-biosynthesis-deficient mutants. The altered phenotypes of these mutants, particularly in Arabidopsis, revealed the essential nature of BRs for normal growth and development. A major role of BRs is the positive regulation of cell expansion. Furthermore, BRs modulate plant responses to biotic and abiotic stresses and to other phytohormones, and influence differentiation processes of cells and tissues. BR-insensitive mutants such as bri1 hold the potential for uncovering BR-signalling pathway(s) at the molecular level. The identification of BR-regulated genes demonstrates a genetic basis for BR mode of action with reference to their multiple effects. This review focuses on the relevance of BRs to the control of various physiological processes, BR-signalling and underlying molecular mechanisms by considering known mutants.     

The brassinosteroid growth response in pea is not mediated by changes in gibberellin content

The objective of this study was to increase our understanding of the relationship between brassinosteroids (BRs) and gibberellins (GAs) by examining the effects of BR deficiency on the GA biosynthesis pathway in several tissue types of pea (Pisum sativum L.). It was suggested recently that, in Arabidopsis, BRs act as positive regulators of GA 20-oxidation, a key step in GA biosynthesis [Bouquin et al. (2001) Plant Physiol 127:450–458]. However, this may not be the case in pea as GA₂₀ levels were consistently higher in all shoot tissues of BR-deficient (lk and lkb) and BR-response (lka) mutants. The application of brassinolide (BL) to lkb plants reduced GA₂₀ levels, and metabolism studies revealed a reduced conversion of GA₁₉ to GA₂₀ in epi-BL-treated lkb plants. These results indicate that BRs actually negatively regulate GA₂₀ levels in pea. Although GA₂₀ levels are affected by BR levels, this does not result in consistent changes in the level of the bioactive GA, GA₁. Therefore, even though a clear interaction exists between endogenous BR levels and the level of GA₂₀, this interaction may not be biologically significant. In addition to the effect of BRs on GA levels, the effect of altered GA₁ levels on endogenous BR levels was examined. There was no significant difference in BR levels between the GA mutants and the wild type (wt), indicating that altered GA₁ levels have no effect on BR levels in pea. It appears that the BR growth response is not mediated by changes in bioactive GA levels, thus providing further evidence that BRs are important regulators of stem elongation.     

Brassinosteroids promote root growth in Arabidopsis

Although brassinosteroids (BRs) are known to regulate shoot growth, their role in the regulation of root growth is less clear. We show that low concentrations of BRs such as 24-epicastasterone and 24-epibrassinolide promote root elongation in Arabidopsis wild-type plants up to 50% and in BR-deficient mutants such as dwf1-6 (cbb1) and cbb3 (which is allelic to cpd) up to 150%. The growth-stimulating effect of exogenous BRs is not reduced by the auxin transport inhibitor 2,3,5-triidobenzoic acid. BR-deficient mutants show normal gravitropism, and 2,3,5-triidobenzoic acid or higher concentrations of 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid inhibit root growth in the mutants to the same extent as in wild-type plants. Simultaneous administration of 24-epibrassinolide and 2,4-dichlorophenoxyacetic acid results in largely additive effects. Exogenous gibberellins do not promote root elongation in the BR-deficient mutants, and the sensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid is not altered. Thus, the root growth-stimulating effect of BRs appears to be largely independent of auxin and gibberellin action. Furthermore, we analyzed BR interactions with other phytohormones on the gene expression level. Only a limited set of auxin- and ethylene-related genes showed altered expression levels. Genes related to other phytohormones barely showed changes, providing further evidence for an autonomous stimulatory effect of BR on root growth.     

Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor

Screening for brassinosteroid (BR) biosynthesis inhibitors was performed to find chemicals that induce dwarfism in Arabidopsis, mutants that resembled BR biosynthesis mutants that can be rescued by BR. Through this screening experiment, the compound brassinazole was selected as the most potent chemical. In dark-grown Arabidopsis, brassinazole-induced morphological changes were nearly restored to those of wild type by treatment with brassinolide. The structure of brassinazole is similar to pacrobutrazol, a gibberellin biosynthesis inhibitor. However, in assays with cress (Lepidium sativum) plants, brassinazole-treated plants did not show recovery after the addition of gibberellin but showed good recovery after the addition of brassinolide. These data demonstrate that brassinazole is a specific BR biosynthesis inhibitor. Brassinazole-treated cress also showed dwarfism, with altered leaf morphology, including the downward curling and dark green color typical of Arabidopsis BR-deficient mutants, and this dwarfism was reversed by the application of 10 nM brassinolide. This result suggests that BRs are essential for plant growth, and that brassinazole can be used to clarify the function of BRs in plants as a complement to BR-deficient mutants. The brassinazole action site was also investigated by feeding BR biosynthesis intermediates to cress grown in the light.     

Salt tolerance at germination and vegetative growth involves different mechanisms in barnyard grass (Echinochloa crusgalli L.) mutants

In this study we describe the selection and characterization of barnyard grass (Echinochloa crusgalli L.) mutants (fows B) with vegetative salt tolerance as compared to a previously described mutant with salt tolerant germination (fows A3). Salt tolerance of both types of mutants was characterized in two distinctive stages of plant development, germination and vegetative growth. About 46% of fows A3 seeds germinated in 300 mM NaCl but none of the seeds of the wild type or fows B mutants were able to germinate in this salt concentration. However, fows B mutants showed significantly higher fresh weights compared to the wild type and the fows A3 mutant when grown in the presence of 200 mM NaCl for 25 days. This indicated that fows B mutants are more salt tolerant than fows A3 mutant as well the wild type. The vegetative salt tolerance of the fows B mutants depended mainly on maintaining more efficient photosynthetic machinery, by keeping significantly higher chlorophyll and Rubisco contents and accumulating soluble sugars particularly sucrose. In addition, fows B mutants had significantly lower malondialdehyde (MDA) contents than did fows A3 and the wild type. This was apparently the result of higher activities of catalase (CAT) and peroxidase (POD) in fows B mutants compared to the wild type and fows A3, indicating that more efficient control of reactive oxygen species correlates with salt tolerance. However, proline accumulation and K⁺/Na⁺ ratio did seem to be essential to vegetative salt tolerance. The vegetative salt tolerance mechanisms in fows B mutants were weakly expressed in the wild type and fows A3 mutant. The results provide evidence that salt tolerance during germination and vegetative growth could involve different mechanisms.     

Effects of secondary mutation in det2-1 on root growth and development in Arabidopsis

Brassinosteroids (BRs) are plant steroidal hormones that regulate a wide range of developmental processes. Most BR mutants display impaired growth and responses to developmental and environmental stimuli. Here, we found a BR-deficient mutant det2-1 that displayed exceedingly short roots and agravitropic growth, which were not present in other BR mutants. By back-crossing det2-1 with the wild type, we isolated a secondary mutation named det2-1 phenotype modifier 1 (dpm1) and demonstrated that those aberrant phenotypes in the original det2-1 were independent of the BR deficiency. Phenotypic analysis showed that impaired root growth of dpm1 appeared in BR-deficient condition, but not in a normal condition. In the light condition, the mutant showed enhanced shoot growth which was suppressed in the det2-1 background. Starch granules in the columella cells of the root tip were highly accumulated and expanded in dpm1. Agravitropic roots and the expanded starch granules of dpm1 could not be recovered by BR. Taken together, these results suggest that DPM1 is required for gravitropic growth, and that its functions on root and shoot growth are BR-dependent.     

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure–activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G₂) is a highly selective inhibitor of BR biosynthesis, with an IC₅₀ value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.     

Functional analysis of HvSPY,a negative regulator of GA response,in barley aleurone cells and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA₃-induced α-amylase expression. With the exception of HvSPYΔ1–5, the other deletion proteins were partially active in the barley assay, including HvSPYΔ6–9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Loss-of-function of a rice brassinosteroid biosynthetic enzyme,C-6 oxidase,prevents the organized arrangement and polar elongation of cells in the leaves and stem

Molecular genetic and physiological studies on brassinosteroid (BR)-related mutants of dicot plants have revealed that BRs play important roles in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of a rice mutant partially insensitive to BR signaling. To investigate the function of BR in monocots, we identified and characterized BR-deficient mutants of rice, BR-deficient dwarf1 (brd1). The brd1 mutants showed a range of abnormalities in organ development and growth, the most striking of which were defects in the elongation of the stem and leaves. Light microscopic observations revealed that this abnormality was primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. The accumulation profile of BR compounds in the brd1 mutants suggested that these plants may be deficient in the activity of BR C-6 oxidase. Therefore, we cloned a rice gene, OsDWARF, which has a high sequence similarity to the tomato C-6 oxidase gene, DWARF. Introduction of the wild-type OsDWARF gene into brd1 rescued the abnormal phenotype of the mutants. The OsDWARF gene was expressed at a low level in all of the examined tissues, with preferential expression in the leaf sheath, and the expression was negatively regulated by brassinolide treatment. On the basis of these findings, we discuss the biological function of BRs in rice plants.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

Microtubule Orientation in the Brassinosteroid Mutants <Emphasis Type="Italic">lk,lka</Emphasis> and <Emphasis Type="Italic">lkb</Emphasis> of Pea

Short brassinosteroid (BR) mutants lk, lka and lkb of pea (Pisum sativum L.) were investigated by immunofluorescence microscopy to elucidate the role of brassinosteroids in cell elongation via an effect on the microtubules (MTs). This study adds to our knowledge the fact that brassinolide (BL) can cause MT realignment in azuki bean and rescue the MT organization of BR mutants in Arabidopsis. It provides novel information on both cortical and epidermal cells and presents detailed information about the ratios of all MT orientations present, ranging from transverse (perpendicular to the elongating axis) to longitudinal (parallel to the elongating axis). Experiments were conducted in vivo using intact plants with direct application of a small amount of brassinolide (BL) to the internode. Employing a BR-receptor mutant, lka, and the BR-synthesis mutants, lk and lkb, allowed the identification and isolation of any BR-induced responses in the MT cytoskeleton following BL application. Increases in growth rate were noted in all pea lines including WT following BL application. These increases were strong in the BR-synthesis mutants, but weak in the BR-receptor mutant. Immunofluorescence revealed significant differences in the average MT orientation of cortical cells of mutants versus WTs. Importantly, these mutants possessed abundant MTs, unlike the BR-deficient bul1-1 mutant in Arabidopsis. Following BL application, the epidermal and cortical cells of lk and lkb plants showed a large and significant shift in MT orientation towards more transverse, whereas lka plants showed a small and nonsignificant response in these cells. These results suggest that the BR response pathway is linked to the regulation of MT orientation.     

Gibberellin mutants

Research on gibberellin (GA) mutants is reviewed, focusing on reports, published since 1993. The mutants have usually been identified via a shoot elongation screen. This screen exposes mutations influencing GA synthesis, deactivation and reception, and also those acting further down the elongation pathway. Mutations blocking synthesis lead to a dwarf. GA-responsive phenotype. Numerous such mutations are now known. For some steps homologous mutations are known across 4 to 6 model species. Examples include the early step, geranylgeranyl diphosphate to copalyl diphosphate, and the activation step, GA₂₆to GA₁. Several GA-synthesis mutations have now been characterised at the molecular level and all are in structural genes. It is now clear some steps are controlled by gene families with distinct tissue specificity. Further, some enzymes control more than one step in the biosynthetic pathway. The only mutation known to block deactivation. sin in pea, leads to an elongated phenotype. The GA response mutants are less well understood and are a more diverse group. They include elongated mutants with a constitutive GA response (spy in arabidopsis. la cry-s in pea and sln in barley) or an enhanced GA response (phyB in arabidopsis. lv in pea and Ih in cucumber). Short response mutants include at least three types. One group accumulates GAs and are mostly unresponsive to applied GA (gai in arabidopsis. D8 in maize. Rht3 in wheat). A recently identified group, exemplified by Igr in pea and gas in barley, have a short stature and reduced response but attain full responses with very high doses of exogenous GA. How close these mutations act to GA reception remains to be determined. Lastly, a number of mutants with short stature and reduced GA response differ in overall phenotype from GA-deficient plants and cannot be made to mimic wild type even at high GA application rates. These mutations act beyond GA reception and some have already proved useful in elucidating other pathways that affect shoot elongation. For example, the lk and lkb mutations in pea appear to block brassinolide synthesis and this in turn prevents normal GA-mediated elongation responses.     

Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases,Which Control Stomata Development in Arabidopsis thaliana

Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.