Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.     

Suppressor of <Emphasis Type="Italic">bri1-120</Emphasis> mutant allele revealed interrelated and independent actions of brassinosteroid and light signaling

Brassinosteroids (BRs) are plant hormones that affect diverse aspects of plant development. Various BR-biosynthetic or BR-signaling mutants contribute to BR functions and signaling events in many plant species. The BR receptor brassinosteroid-Insensitive 1 (BRI1) plays critical roles in BR signaling. We previously identified a weak bri1 mutant allele, bri1-120, that has a mutation site in the extracellular domain of BRI1. Here, genetic suppressor screening revealed that a PHYB gene mutation led to suppression of ethyl methanesulfonate (EMS)-mutagenized bri1-120. The morphology of bri1-120phyB-1 indicated that compact and rounded phenotypes of bri1-120 were suppressed. However, BR sensitivity of the bri1-120phyB-1 was only recovered in hypocotyl elongation, and overexpression of PHYB in bri1-120 did not enhance bri1-120 phenotypes. To further investigate the relationship between BR and light signalings, we examined the seed germination pattern and hypocotyl growth of bri1-120phyB-1 as compared to that of each single mutant under various light conditions. Seed germination in bri1-120phyB-1 was higher than in both the single mutants. Hypocotyl length in bri1-120phyB-1 was intermediate between that of bri1-120 and phyB-1, whereas sensitivity to red light in bri1-120phyB-1 remained the same as in phyB-1. These results suggest that BR and light signalings affect diverse cellular responses both together and independently, depending on the specific cellular processes.     

High Temperature Stimulates <Emphasis Type="Italic">DWARF4 (DWF4)</Emphasis> Expression to Increase Hypocotyl Elongation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Hypocotyl growth occurs as a result of an interaction between environmental factors and endogenous phytohormones. In Arabidopsis, high temperature promotes auxin synthesis to increase hypocotyl growth. We previously showed that exogenously provided auxin stimulates expression of the brassinosteroid (BR) biosynthetic gene DWARF4. To determine whether temperature-induced hypocotyl elongation depends on BR biosynthesis, we examined the morphological responses to high temperature and the expression pattern of DWF4pro:GUS in different genetic backgrounds, which are as follows: Ws-2 wild-type, iaa19/msg2, bri1-5, and dwf7-1. In contrast to the wild-type, growth of the three genotypes at 29°C did not significantly increase hypocotyl length; whereas, with the exception of iaa19/msg2, the roots were elongated. These results confirm that BR biosynthesis and signaling pathways are required for hypocotyl growth at high temperature. Furthermore, a GUS histochemical assay revealed that a temperature of 29°C greatly increased DWF4pro:GUS expression in the shoot and root tips compared to a temperature of 22°C. Quantitative measurements of GUS activity in DWF4pro:GUS revealed that growth at 29°C is similar to the level of growth after addition of 100 nM IAA to the medium. Our results suggest that temperature-dependent synthesis of free auxin stimulates BR biosynthesis, particularly via the key biosynthetic gene DWF4, and that the BRs thus synthesized are involved in hypocotyl growth at high temperature.     

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.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

Brassinosteroid homeostasis in Arabidopsis is ensured by feedback expressions of multiple genes involved in its metabolism

Homeostasis of brassinosteroids (BRs) is essential for normal growth and development in higher plants. We examined responsiveness of 11 BR metabolic gene expressions to the decrease or increase of endogenous BR contents in Arabidopsis (Arabidopsis thaliana) to expand our knowledge of molecular mechanisms underlying BR homeostasis. Five BR-specific biosynthesis genes (DET2, DWF4, CPD, BR6ox1, and ROT3) and two sterol biosynthesis genes (FK and DWF5) were up-regulated in BR-depleted wild-type plants grown under brassinazole, a BR biosynthesis inhibitor. On the other hand, in BR-excessive wild-type plants that were fed with brassinolide, four BR-specific synthesis genes (DWF4, CPD, BR6ox1, and ROT3) and a sterol synthesis gene (DWF7) were down-regulated and a BR inactivation gene (BAS1) was up-regulated. However, their response to fluctuation of BR levels was highly reduced (DWF4) or nullified (the other eight genes) in a bri1 mutant. Taken together, our results imply that BR homeostasis is maintained through feedback expressions of multiple genes, each of which is involved not only in BR-specific biosynthesis and inactivation, but also in sterol biosynthesis. Our results also indicate that their feedback expressions are under the control of a BRI1-mediated signaling pathway. Moreover, a weak response in the mutant suggests that DWF4 alone is likely to be regulated in other way(s) in addition to BRI1 mediation.     

The Influence of Chemical Genetics on Plant Science: Shedding Light on Functions and Mechanism of Action of Brassinosteroids Using Biosynthesis Inhibitors

When exogenous chemicals allow rapid, conditional, reversible, selective, and dose-dependent control of biological functions, they act like conditional mutations, either inducing or suppressing the formation of a specific phenotype of interest. Exploration of the small molecules that induce the brassinosteroid (BR) deficient-like phenotype in Arabidopsis led us to identify brassinazole as the first candidate for a BR biosynthesis inhibitor. Brassinazole treatment reduced BR content in plant cells. Investigation of target site(s) of brassinazole revealed that the compound directly binds to the DWF4 protein, a cytochrome P450 monooxygenase that catalyzes 22-hydroxylation of the side chain of BRs. These results suggest that brassinazole is a BR biosynthesis inhibitor. There are currently at least two BR biosynthesis inhibitors that act like conditional mutations in BR biosynthesis. They allow the investigation of the functions of BRs in a variety of plant species. Application of BR biosynthesis inhibitors to a standard genetic screen to identify mutants that confer resistance to these inhibitors allowed the identification of new components working in BR signal transduction. This method has advantages over mutant screens using BR-deficient mutants as a background. Development of chemicals that induce phenotypes of interest is now emerging as a useful way to study biological systems in plants and this would be a complement to classical biochemical and genetic methods.     

Antagonistic Regulation of Arabidopsis Growth by Brassinosteroids and Abiotic Stresses

To withstand ever-changing environmental stresses, plants are equipped with phytohormone-mediated stress resistance mechanisms. Salt stress triggers abscisic acid (ABA) signaling, which enhances stress tolerance at the expense of growth. ABA is thought to inhibit the action of growth-promoting hormones, including brassinosteroids (BRs). However, the regulatory mechanisms that coordinate ABA and BR activity remain to be discovered. We noticed that ABA-treated seedlings exhibited small, round leaves and short roots, a phenotype that is characteristic of the BR signaling mutant, brassinosteroid insensitive1-9 (bri1-9). To identify genes that are antagonistically regulated by ABA and BRs, we examined published Arabidopsis microarray data sets. Of the list of genes identified, those upregulated by ABA but downregulated by BRs were enriched with a BRRE motif in their promoter sequences. After validating the microarray data using quantitative RT-PCR, we focused on RD26, which is induced by salt stress. Histochemical analysis of transgenic Arabidopsis plants expressing RD26pro:GUS revealed that the induction of GUS expression after NaCl treatment was suppressed by co-treatment with BRs, but enhanced by co-treatment with propiconazole, a BR biosynthetic inhibitor. Similarly, treatment with bikinin, an inhibitor of BIN2 kinase, not only inhibited RD26 expression, but also reduced the survival rate of the plant following exposure to salt stress. Our results suggest that ABA and BRs act antagonistically on their target genes at or after the BIN2 step in BR signaling pathways, and suggest a mechanism by which plants fine-tune their growth, particularly when stress responses and growth compete for resources.     

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.