Brassinosteroid Mutants of Crops

Plant steroid hormones, brassinosteroids (BRs), were originally isolated from extracts of pollen because of their growth-promoting properties and their potential use for enhancing crop production. Mutants in the biosynthesis, metabolism, and signaling of brassinolide (BL), the most bioactive BR, are important resources in helping to establish BRs essential role in plant growth and development. The dark green and distinctive dwarf phenotype of BR-related mutants identified in pea, tomato, and rice highlights the importance of BRs in crops. These mutants are helping to elucidate both the conserved and the unique features of BR biosynthesis and signaling. Such insights are providing the key knowledge and understanding that will enable the development of strategies towards the production of crops with enhanced qualities.     

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.     

Brassinosteroid Signal Transduction: A Mix of Conservation and Novelty

Brassinosteroids (BRs) are a unique class of plant steroids that are structurally similar to animal steroid hormones and play important roles in plant growth and development. Unlike the animal steroids, which bind to classical intracellular steroid receptors that directly modulate gene activities after translocation into the nucleus, the plant steroids rely on transmembrane receptor kinases to activate a phosphorylation cascade to regulate gene expression. Recent genetic and biochemical studies have identified several critical BR signaling components and revealed a striking mechanistic similarity between the plant steroid signaling pathway and several well-studied animal signaling cascades involving a receptor kinase and glycogen synthase kinase 3 (GSK3). A working model for BR signal transduction proposes that BR initiates its signaling pathway by promoting heterodimerization of two transmembrane receptor-like kinases at the cell surface, leading to inhibition of a GSK3 kinase and subsequent stabilization and nuclear accumulation of two GSK3 substrates that regulate BR-responsive genes. Such a simple model provides a framework for continued investigation of molecular mechanism(s) of plant steroid signaling.     

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.     

BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation

Plant steroid hormones, known as brassinosteroids (BRs), signal through a plasma membrane localized receptor kinase BRI1. We identified bes1, a semidominant suppressor of bri1, which exhibits constitutive BR response phenotypes including long and bending petioles, curly leaves, accelerated senescence, and constitutive expression of BR-response genes. BES1 accumulates in the nucleus in response to BRs. BES1 is phosphorylated and appears to be destabilized by the glycogen synthase kinase-3 (GSK-3) BIN2, a negative regulator of the BR pathway. These results establish a signaling cascade for BRs with similarities to the Wnt pathway, in which signaling through cell surface receptors leads to inactivation of a GSK-3 allowing accumulation of a nuclear protein that regulates target gene expression.     

Brassinosteroid-Mediated Stress Responses

Brassinosteroids (BRs) are a group of naturally occurring plant steroidal compounds with wide-ranging biological activity that offer the unique possibility of increasing crop yields through both changing plant metabolism and protecting plants from environmental stresses. In recent years, genetic and biochemical studies have established an essential role for BRs in plant development, and on this basis BRs have been given the stature of a phytohormone. A remarkable feature of BRs is their potential to increase resistance in plants to a wide spectrum of stresses, such as low and high temperatures, drought, high salt, and pathogen attack. Despite this, only a few studies aimed at understanding the mechanism by which BRs promote stress resistance have been undertaken. Studies of the BR signaling pathway and BR gene-regulating properties indicate that there is cross-talk between BRs and other hormones, including those with established roles in plant defense responses such as abscisic acid, jasmonic acid, and ethylene. Recent studies aimed at understanding how BRs modulate stress responses suggest that complex molecular changes underlie BR-induced stress tolerance in plants. Analyses of these changes should generate exciting results in the future and clarify whether the ability of BRs to increase plant resistance to a range of stresses lies in the complex interactions of BRs with other hormones. Future studies should also elucidate if BRI1, an essential component of the BR receptor, directly participates in stress response signaling through interactions with ligands and proteins involved in plant defense responses.     

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.     

Brassinosteroids antagonize gibberellin- and salicylate-mediated root immunity in rice

Brassinosteroids (BRs) are a unique class of plant steroid hormones that orchestrate myriad growth and developmental processes. Although BRs have long been known to protect plants from a suite of biotic and abiotic stresses, our understanding of the underlying molecular mechanisms is still rudimentary. Aiming to further decipher the molecular logic of BR-modulated immunity, we have examined the dynamics and impact of BRs during infection of rice (Oryza sativa) with the root oomycete Pythium graminicola. Challenging the prevailing view that BRs positively regulate plant innate immunity, we show that P. graminicola exploits BRs as virulence factors and hijacks the rice BR machinery to inflict disease. Moreover, we demonstrate that this immune-suppressive effect of BRs is due, at least in part, to negative cross talk with salicylic acid (SA) and gibberellic acid (GA) pathways. BR-mediated suppression of SA defenses occurred downstream of SA biosynthesis, but upstream of the master defense regulators NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 and OsWRKY45. In contrast, BR alleviated GA-directed immune responses by interfering at multiple levels with GA metabolism, resulting in indirect stabilization of the DELLA protein and central GA repressor SLENDER RICE1 (SLR1). Collectively, these data favor a model whereby P. graminicola coopts the plant BR pathway as a decoy to antagonize effectual SA- and GA-mediated defenses. Our results highlight the importance of BRs in modulating plant immunity and uncover pathogen-mediated manipulation of plant steroid homeostasis as a core virulence strategy.     

Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis

Plant steroid hormones, brassinosteroids (BRs), are perceived by a cell surface receptor kinase, BRI1, but how BR binding leads to regulation of gene expression in the nucleus is unknown. Here we describe the identification of BZR1 as a nuclear component of the BR signal transduction pathway. A dominant mutation bzr1-1D suppresses BR-deficient and BR-insensitive (bri1) phenotypes and enhances feedback inhibition of BR biosynthesis. BZR1 protein accumulates in the nucleus of elongating cells of dark-grown hypocotyls and is stabilized by BR signaling and the bzr1-1D mutation. Our results demonstrate that BZR1 is a positive regulator of the BR signaling pathway that mediates both downstream BR responses and feedback regulation of BR biosynthesis.     

油菜素内酯调控作物农艺性状和非生物胁迫响应的研究进展

植物对不利环境的适应依赖于将外部胁迫信号传递到内部信号通路中,在进化过程中形成一系列的胁迫响应机制。其中,油菜素内酯(brassinosteroids, BRs)是一种类固醇激素,广泛参与植物生长发育和逆境响应过程。BRs被包括受体BRI1和共受体BAK1在内的细胞表面受体感知,继而触发信号级联,导致蛋白激酶BIN2的抑制和转录因子BES1/BZR1的激活,BES1/BZR1可直接调控数千个下游响应基因的表达。在模式植物拟南芥中的研究表明,BR的生物合成和信号转导通路成员,特别是BIN2和其下游的转录因子BES1/BZR1,可以被各种环境因子广泛地调节。本文系统总结了BR相关的最新研究进展,对BR的生物合成和信号转导是如何被复杂的环境因子所调节,以及BR与环境因子如何协同调控作物重要农艺性状、冷胁迫和盐胁迫的响应进行了综述。     

Sterols regulate development and gene expression in Arabidopsis

Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::beta-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways.     

油菜素甾醇调控水稻盐胁迫应答的作用研究

油菜素甾醇(BR)作为植物内源激素, 广泛参与植物的生长发育过程及逆境应答。虽然BR调控生长发育的分子机制目前已相对清楚, 但在水稻(Oryza sativa)中, BR在逆境反应中的功能还鲜有报道。该研究系统分析了BR在高盐胁迫过程中的作用, 表明盐胁迫和逆境激素脱落酸可抑制BR合成基因D2和D11的表达, 典型的BR缺陷突变体(如d2-2和d61-1)则表现出对盐胁迫敏感性增强。此外, 通过对BR核心转录因子OsBZR1的过表达株系进行分析, 发现BR可显著诱导OsBZR1的去磷酸化, 盐胁迫对OsBZR1蛋白的积累水平和磷酸化状态均有调控作用。转录组数据分析表明, BR处理前后差异表达基因中有38.4%同时受到盐胁迫调控, 其中91.5%受到BR和高盐一致调控, 并显著富集在应激反应过程中。研究结果表明, BR正调控水稻的耐盐性, 而盐胁迫通过抑制BR合成来限制水稻的生长。     

Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.     

Brassinosteroid biosynthesis anddwarf mutants

Plants enjoy their entire life exactly where they were initially rooted. Because of this fixed life pattern, plants have to devise a different type of strategy than animals to survive the numerous biotic and abiotic challenges. Many different plant hormones that act alone or in concert underpin these mechanisms. Brassinosteroids (BRs) collectively refer to plant-originated 5μ-cholestane steroids that elicit growth stimulation in nano-or micromolar concentrations. BRs that are biosynthesized using sterols as precursors are structurally similar to the cholesterol derived, mammalian steroid hormones, insect molting hormones and ecdysteroids. BRs have been known for decades to be effective in plant growth promotion. However, definitive evidence for their roles in growth and development remained unclear until the recent characterization of BRdwarf mutants isolated fromArabidopsis and other plants. This review aims to provide a cohesive summary of information obtained from the molecular genetic characterization of mutants that are defective in sterol and BR biosynthetic pathways.