New Alleles of Rice <i>ebisu dwarf</i>(<i>d</i>2) Mutant Show both Brassinosteroid-Deficient and -Insensitive Phenotypes

ebisu dwarf (d2) is a mutant caused by mutation in a rice brassinosteroid biosynthetic enzyme gene, CYP90D2/D2, thereby conferring a brassinosteroid-deficient dwarf phenotype. Three newly isolated d2 alleles derived from a Nippon- bare mutant library (d2-3, d2-4, and d2-6) produced more severe dwarf phenotypes than the previously characterized null allele from a Taichung 65 mutant library, d2-1. Linkage analysis and a complementation test clearly indicated that the mutant phenotypes in d2-6 were caused by defects in CYP90D2/D2, and exogenous treatment with brassinolide, a bioactive brassinosteroid, rescued the dwarf phenotype of three Nipponbare-derived d2 mutants. However, the content of endogenous bioactive brassinosteroid, castasterone, and the expression of brassinosteroid-response genes indicated that partial suppression of the brassinosteroid response in addition to a brassinosteroid deficiency has occurred in the Nipponbare-derived d2 mutants. Based on these results, we discuss the possibility that wild-type Nipponbare has some defects in an unknown factor or factors related to the brassinosteroid response in rice.

Brassinosteroids mediate the effect of high temperature during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile rice lines

Brassinosteroids(BRs)play critical roles in a wide range of plant developmental processes.However,it is unknown whether and how BRs mediate the effect of high temperature(HT)stress during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile(PTSGMS)rice(Oryza sativa L.)lines.This study investigated the question.Three pot-grown PTSGMS rice lines were subjected to HT stress during anthesis.The contents of 24-epibrassinolide(24-EBL)and 28-homobrassinolide(28-HBL),the major forms of BR in rice plants,and levels of reactive oxygen species(ROS)or antioxidants(AOS),hydrogen peroxide(H2O2),1-aminocylopropane-1-carboxylic acid(ACC),ascorbic acid(AsA),and catalase activity in pistils,were determined.HT stress significantly reduced the contents of both 24-EBL and 28-EBL relative to those under normal temperatures,but the reduction varied by PTSGMS line.A line with higher BR contents under HT stress showed lower contents of ACC and H2O2,higher catalase activity and AsA content in pistils,and higher fertilization rate,seed-setting rate,and seed yield when the line was crossed with a restorer line,indicating that higher levels of BRs increase HT stress resistance.Applying 24-EBL,28-HBL or an inhibitor of BR biosynthesis confirmed the roles of BRs in response to HT stress.The results suggest that BRs mediate the effect of HT stress on pistil activity during anthesis and alleviate the harm of HT stress by increasing AOS and suppressing ROS generation.

Brassinosteroid-regulated plant growth and development and gene expression in soybean

Brassinosteroids(BRs) are endogenous phytohormones that play important roles in regulating plant growth and development.In this study, we evaluated the effects of brassinolide(BL, one of the active BRs) on soybean and identified roles of the hormone in regulating multiple aspects of plant growth and development.BL application promoted hypocotyl and epicotyl elongation in the light but blocked epicotyl elongation in the dark.High levels of castasterone and BL accumulated in light-grown plants.BL disrupted shoot negative gravitropism, whereas gibberellin did not.BL delayed leaf senescence.Transcriptome analysis showed that BL induced cell wall-modifying genes and auxin-associated genes but suppressed a class of WRKY genes involved in senescence and stress responses,showing the complex roles of BRs in multiple biological processes.

Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene,GhDWF1,from Cotton (Gossypium hirsuturm L.)

Brassinosteroids （BRs） are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene （GhDWF1） involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton （Gossypium hirsutum L.） cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5＇-untranslated region, an ORF of 1 692 bp, and a 120 bp 3＇-untranslated region. The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea, tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD- dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA （day post anthesis） ovule （with fibers） while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules （with fibers） of wildtype （Xuzhou 142） than in ovules of fuzzless-lintless mutant at the same developmental stages （0 and 4 DPA）. The results suggest that the GhDWF1 gene plays a crucial role in fiber development.

Ameliorative Effects of Brassinosteroid on Excess Manganese-Induced Oxidative Stress in Zea mays L. Leaves

Manganese （Mn） is becoming an important factor limiting crop growth and yields especially on acid soils. The present study was designed to explore the hypothesis that brassinosteroid application can enhance the tolerance of maize （Zea mays L.） to Mn stress and if so, whether or not the mechanism underlying involves regulation of antioxidative metabolism in leaves. The effects of 24-epibrassinosteroid （EBR） on the growth, photosynthesis, water status, lipid peroxidation, accumulation of reactive oxygen species, and activities or contents of antioxidant defense system in maize plants under Mn stress were investigated by a pot experiment. At supplemented Mn concentrations of 150-750 mg kg^-1 soil, the growth of plants was inhibited in a concentration-dependent manner. The semi-lethal concentration was 550 mg Mn kgq soil. Foliage application with 0.1 mg L^-1 EBR significantly reduced the decrease in dry mass, chlorophyll content, photosynthetic rate, leaf water content, and water potential of plants grown in the soil spiked with 550 mg kg^-1 Mn. The oxidative stress caused by excess Mn, as reflected by the increase in malondialdehyde （MDA） content and lipoxygenase （LOX, EC 1.13.11.12） activity, accumulation of superoxide radical and H2O2, was greatly decreased by EBR treatment. Further investigations revealed that EBR application enhanced the activities of superoxide dismutase （SOD, EC 1.15.1.1）, peroxidase （POD, EC 1.11.1.7）, catalase （EC 1.11.1.6）, ascorbate peroxidase （APX, EC 1.11. 1.11）, dehydroascorbate reductase （DHAR, EC 1.8.5.1）, and glutathione reductase （GR, EC 1.6.4.2）, and the contents of reduced ascorbate and glutathione, compared with the plants without EBR treatment. It is concluded that the ameliorative effects of EBR on Mn toxicity are due to the upregulation of antioxidative capacity in maize under Mn stress.

Brassinosteroids modulate nitrogen physiological response and promote nitrogen uptake in maize(Zea mays L.)

Brassinosteroids(BRs)are steroid hormones that function in plant growth and development and response to environmental stresses and nutrient supplies.However,few studies have investigated the effect of BRs in modulating the physiological response to nitrogen(N)supply in maize.In the present study,BR signalingdeficient mutant zmbri1-RNAi lines and exogenous application of 2,4-epibrassinolide(e BL)were used to study the role of BRs in the regulation of physiological response in maize seedlings supplied with N.Exogenous application of e BL increased primary root length and plant biomass,but zmbri1 plants showed shorter primary roots and less plant biomass than wild-type plants under low N(LN)and normal N(NN)conditions.LN induced the expression of the BR signaling-associated genes Zm DWF4,Zm CPD,Zm DET2,and Zm BZR1 and the production of longer primary roots than NN.Knockdown of Zm BRI1 weakened the biological effects of LN-induced primary root elongation.e BL treatment increased N accumulation in shoots and roots of maize seedlings exposed to LN or NN treatment.Correspondingly,zmbri1 plants showed lower N accumulation in shoots and roots than wild-type plants.Along with reduced N accumulation,zmbri1 plants showed lower NO3-fluxes and^(15)NO_(3)^(-)uptake.The expression of nitrate transporter(NRT)genes(Zm NPF6.4,Zm NPF6.6,Zm NRT2.1,Zm NRT2.2)was lower in zmbri1 than in wild-type roots,but e BL treatments up-regulated the transcript expression of NRT genes.Thus,BRs modulated N physiological response and regulated the transcript expression of NRT genes to promote N uptake in maize.

Comparison of Effect of Brassinosteroid and Gibberellin Biosynthesis Inhibitors on Growth of Rice Seedlings

Brassinosteroid（BR） and gibberellin（GA） are two predominant plant hormones that regulate plant cell elongation. Mutants disrupt the biosynthesis of these hormones and display different degrees of dwarf phenotypes in rice. Although the role of each plant hormone in promoting the longitudinal growth of plants has been extensively studied using genetic methods, their relationship is still poorly understood. In this study, we used two specific inhibitors targeting BR and GA biosynthesis to investigate the roles of BR and GA in growth of rice seedlings. Yucaizol, a specific inhibitor of BR biosynthesis, and Trinexapac-ethyl, a commercially available inhibitor of GA biosynthesis, were used. The effect of Yucaizol on rice seedlings indicated that Yucaizol significantly retarded stem elongation. The IC_（50） value was found to be approximately 0.8 μmol/L. Yucaizol also induced small leaf angle phenocopy in rice seedlings, similarly to BR-deficient rice, while Trinexapac-ethyl did not. When Yucaizol combined with Trinexapac-ethyl was applied to the rice plants, the mixture of these two inhibitors retarded stem elongation of rice at lower doses. Our results suggest that the use of a BR biosynthesis inhibitor combined with a GA biosynthesis inhibitor may be useful in the development of new technologies for controlling rice plant height.

Brassinosteroid and gibberellin coordinate rice seed germination and embryo growth by regulating glutelin mobilization

Seed germination is the beginning of a new lifecycle,and involves many complex physiological and biochemical reactions including seed reserve mobilization in the endosperm and nutrient transport and reuse in the embryo.Although glutelin is a dominant storage protein in rice,its contribution to seed germination and its regulatory mechanisms are mostly unknown.Gibberellin (GA) and brassinosteroid (BR),two major growth-promoting phytohormones,also play positive roles in controlling seed germination.However,how GA and BR interact and coordinate seed germination and facilitate glutelin mobilization remains unclear.In the present study,biochemical and physiological analyses of seed germination indicated that both GA and BR promote seed germination and post-germination growth.Exogenous application of GA restored germination defects caused by BR deficiency or insensitivity.Proteomic and q RT-PCR results showed that the expression of several glutelin proteins and their encoding genes was induced by BR and GA in the embryo.Expression assays suggested that the increased accumulation of glutelin protein in the embryo was due to the accelerated degradation of glutelin by a cysteine proteinase (REP-1) in the endosperm.The breakdown of glutelin in the endosperm showed a strict positive correspondence with the length of the shoot.The GluA2 mutation led to reduced degradation rate of glutelin and defects in seed germination,and the promotion effect of GA on seed germination was weakened in the glua2mutant.In vitro culture assay of rice embryos showed that glutelin mobilization functioned downstream of the GA and BR pathways to promote shoot elongation.These findings suggest a mechanism that mediates crosstalk between BR and GA in co-regulating rice seed germination and embryo growth.

Brassinosteroids and Plant Responses to Heavy Metal Stress. An Overview

Soil contamination with heavy metals has become a world-wide problem, leading to the loss in agricultural productivity. Plants have a remarkable ability to take up and accumulate heavy metals from their external environment and it is well known that high levels of heavy metals affect different physiological and metabolic processes. Brassinosteroids are considered as the sixth class of plant hormones and they are essential for plant growth and development. These compounds are able of inducing abiotic stress tolerance in plants. In this paper, information about brassinosteroids and plant responses to heavy metal stress is reviewed.

Genetic Background Influences Brassinosteroid-Related Mutant Phenotypes in Rice

In two cases, mutations in the same brassinosteroid-related genes caused different phenotypes in japonica varieties Nipponbare and Taichung 65. The mutant phenotypes were less severe in the Taichung 65 background than in the Nipponbare background. Three newly isolated brassinosteroid-insensitive mutants (d61-1N, d61-11, and d61-12) derived from a Nipponbare mutant library were found to be alleles of d61, which represent defects in the OsBRI1 gene. Although the Nipponbare-derived mutant d61-1N had the same nucleotide substitution as the previously characterized Taichung 65-derived mutant d61-1T, these two mutants showed different phenotypes for plant stature, internode elongation pattern, and seed shape;in each case, d61-1N (in the Nipponbare genetic background) had the more severe mutant phenotype. Similar trends were seen for phenotypes caused by mutants of d2, a brassinosteroid biosynthesis gene. Consistent with these phenotypes, the expression of brassinosteroid-responsive genes was lower in the Nipponbare-derived mutants. These results can be explained by our findings that feed-forward up-regulation of OsBRI1 did not occur in the Nipponbare-derived mutants and that an mPing transposon is inserted into the promoter region of Nipponbare OsBRI1. Based on these results, we conclude that the expression of OsBRI1, especially its feed-forward up-regulation, is misregulated in wild-type Nipponbare and in brassinosteroid-related mutants in a Nipponbare genetic background. Although Nipponbare is a model rice genotype, it can be categorized as an OsBRI1 mutant that has reduced sensitivity to brassinosteroid.

Roles of jasmonates and brassinosteroids in rice responses to high temperature stress——A review

High temperature (HT) stress has become one of the most detrimental stresses in crop production among constantly changing environmental factors.Exploiting approaches to enhance crop thermotolerance would have great significance in assuaging adverse effects of HT stress on crop growth and development.As jasmonates (JAs) and brassinosteroids (BRs) are novel phytohormones and play important roles in responses to biotic and abiotic stresses and in a wide range of plant developmental processes,this paper reviewed the roles and mechanisms of JAs and BRs in mitigating HT stress,with focus on rice (Oryza sativa L.) subjected to HT stress during anthesis.It is demonstrated that JAs alleviate spikelet-opening impairment and BRs ameliorate pistil fertilization ability under HT stress during anthesis of rice,although there are controversial observations.Activating the defense system,enhancing osmotic regulation,protecting photosynthesis,and interacting with other phytohormones,especially with ethylene and abscisic acid,are main physiological mechanisms by which JAs or BRs attenuate HT stress to plants.Elevating levels of JAs or BRs in plants could be considered as an important approach to enhance crop thermotolerance through breeding new varieties.Using JAs or BRs as chemical regulators and adopting proper water and nitrogen management practices could reduce the harm of HT stress to rice.Further research is needed to elucidate the roles of JAs and BRs in different plant tissues in responses to HT stress under different genetic backgrounds and environments,reveal the molecular mechanism underlying JAs and BRs mediating HT stress,understand the cross-talk between phytohormones in modulating HT stress,and establish integrated crop management to minimize the hazard of HT stress in rice production.

Improving Photosynthetic Responses during Recovery from Heat Treatments with Brassinosteroid and Calcium Chloride in Indian Bread Wheat Cultivars

Climate change is expected to unleash severe and frequent heat waves in future, adversely affecting crop productivity. The aim of this study was to examine the effect of two separate episodes of heat stress, mimicking heat wave conditions on the physiology of four Indian bread wheat cultivars and to study the ameliorating effects of epibrassinolide (BR) and calcium chloride on the recovery of these cultivars. The two thermo-tolerant cultivars C306 and K7903 suffered less inhibition of photosystem II efficiency as compared to the two thermo-susceptible cultivars HD2329 and PBW343. Application of BR and calcium chloride resulted in faster recovery in all the four cultivars. Measurement of the minimum fluorescence (Fo) versus temperature curves revealed a higher inflection temperature of Fo (Ti) for the two tolerant cultivars as compared to the susceptible cultivars, emphasizing greater thermo stability of the photosynthetic apparatus. The two thermo-tolerant cultivars showed higher photochemistry (ΦPSII) relative to the two susceptible cultivars. An increase in the steady state fluorescence was observed in both the susceptible cultivars as compared to the tolerant cultivars. Expression analysis revealed faster recovery of the transcripts involved in photosynthesis in tolerant cultivars as compared to susceptible cultivars. Exogenous application of the ameliorating compounds resulted in faster recovery of transcripts in all the cultivars. The result suggested that under severe stress conditions tolerant cultivars showed faster recovery and a better thermo-stability of its photosynthetic apparatus as compared to susceptible cultivars and application of epibrassinolide and calcium chloride could ameliorate the damaging effect of severe temperature stress to a considerable level in all the four cultivars under study.

Expression Profile Analysis of Genes Involved in Brassinosteroid Biosynthesis Pathway in Cotton Fiber Development

Cotton(Gossypium hirsutum L.) is the leading fiber crop and one of the mainstays of the economy in the world.Cotton fibers,as the main product of cotton plants,are unicellular,linear

Brassinosteroids promote seed development and physiological maturity of oilseed rape (Brassica napus L.)

Long developmental stage and late harvest time of winter rapeseed (Brassica napus L.) have great negative effects on rice planting of rice-rapeseed farming system in China. Early maturity improvement of rapeseed is necessary. ‘Zhongshuang 11’, an elite winter rapeseed cultivar, was used in consecutive field experiments during 2010-2012. At initial flowering stage, plants were consecutively sprayed with 0.1 mg/L 2-4-Epibrassinolide(BR) for 3 d. Two hundred sampling pods from different plants were randomly collected to measure seed related indexes with a 4 d interval from 7 to 47 d after peak anthesis (DAPA).Seed color turned light brown at 31 or 35 DAPA after BR treatment, seed dry weight (DWT)was increased while seed moisture content (SMC) was decreased during seed development.DWT almost reached the maximum value when SMC was 33.20% at 31 DAPA in 2010-2011 and 35.29% at 35 DAPA in 2011-2012 growing season after BR treatment. Similarly,the maximum values of standard germination test (SGT), accelerated aging test (AAT)and cold test (CT) were observed at 31 or 35 DAPA after BR treatment respectively. The high yield and seed oil content appeared at 31 or 35 DAPA accompanied with rapid decrease in total non-structural carbohydrate (TNC) in stems and leaves. Our study indicated that BR application advanced maturity of winter rapeseed by 4 to 8 days.

Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development

RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants,but its role in plant growth and development remains unknown. Using cDNA array,we found that transcription of RAV1 is downregulated by epibrassinolide (epiBL) in Arabidopsis suspension cells. RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein,and does not require the functional BRI1,suggesting that this regulation might be through a new BR signaling pathway.Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development,and the underexpression causes an earlier flowering phenotype,implying that RAV1 may function as a negative regulatory component of growth and development.

The brassinosteroid signal transduction pathway

类固醇作为在动物和植物表明分子工作。当动物类固醇荷尔蒙被抄写因素的原子受体家庭察觉时，在植物的 brassinosteroids (BR ) 被房间表面受体 kinase 察觉， BRI1。最近的研究证明了到 BRI1 的细胞外的域的那 BR 绑定与另一受体 kinase 导致 kinase 激活和 dimerization， BAK1。激活的 BRI1 或 BAK1 然后调整，可能间接地， BIN2 kinase 的活动或 BSU1 磷酸酶，它直接调整 phosphorylation 地位和二个相应抄写因素， BZR1 和 BES1 的原子累积。BZR1 和 BES1 直接绑在 BR 应答的基因的倡导者调整他们的表示。表明小径的 BR 为在一般来说由房间表面受体发信号的植物和类固醇发信号的两受体 kinase 成为了一个范例。

Unveiling a new regulator: Vacuolar V-ATPase mediates brassinosteroid signaling in Arabidopsis

Plant growth and development are intricately regulated by hormones such as auxins,gibberellins,jamonates,cytokinins,and brassinosteroids(BRs).BRs are a group of plant steroid hormones that play a vital role in plant growth and response to the environment.BRs regulate many biological processes including cell elongation,cell division,and male fertility,as well as abiotic and biotic stress responses.

Brassinosteroid signaling and molecular crosstalk with nutrients in plants

As sessile organisms,plants have evolved sophisticated mechanisms to optimize their growth and development in response to fluctuating nutrient levels.Brassinosteroids(BRs)are a group of plant steroid hormones that play critical roles in plant growth and developmental processes as well as plant responses to environmental stimuli.Recently,multiple molecular mechanisms have been proposed to explain the integration of BRs with different nutrient signaling processes to coordinate gene expression,metabolism,growth,and survival.Here,we review recent advances in understanding the molecular regulatory mechanisms of the BR signaling pathway and the multifaceted roles of BR in the intertwined sensing,signaling,and metabolic processes of sugar,nitrogen,phosphorus,and iron.Further understanding and exploring these BR-related processes and mechanisms will facilitate advances in crop breeding for higher resource efficiency.

CKL2 mediates the crosstalk between abscisic acid and brassinosteroid signaling to promote swift growth recovery after stress in Arabidopsis

Plants must adapt to the constantly changing environment. Adverse environmental conditions trigger various defensive responses, including growth inhibition mediated by phytohormone abscisic acid(ABA). When the stress recedes, plants must transit rapidly from stress defense to growth recovery, but the underlying mechanisms by which plants switch promptly and accurately between stress resistance and growth are poorly understood. Here, using quantitative phosphoproteomics strategy, we discovered that early ABA signaling activates upstream components of brassinosteroid(BR) signaling through CASEIN KINASE 1-LIKE PROTEIN 2(CKL2).Further investigations showed that CKL2 interacts with and phosphorylates BRASSINOSTEROID INSENSITIVE1(BRI1), the main BR receptor, to maintain the basal activity of the upstream of BR pathway in plants exposed to continuous stress conditions. When stress recedes, the elevated phosphorylation of BRI1 by CKL2 contributes to the swift reactivation of BR signaling, which results in quick growth recovery. These results suggest that CKL2 plays a critical regulatory role in the rapid switch between growth and stress resistance. Our evidence expands the understanding of how plants modulate stress defense and growth by integrating ABA and BR signaling cascades.

The U-box ubiquitin ligase TUD1 promotes brassinosteroid-induced GSK2 degradation in rice

Brassinosteroids(BRs)are a class of steroid hormones with great potential for use in crop improvement.De-repression is usually one of the key events in hormone signaling.However,how the stability of GSK2,the central negative regulator of BR signaling in rice(Oryza sativa),is regulated by BRs remains elusive.Here,we identify the U-box ubiquitin ligase TUD1 as a GSK2-interacting protein by yeast two-hybrid screening.We show that TUD1 is able to directly interact with GSK2 and ubiquitinate the protein.Phenotypes of the tud1 mutant are highly similar to those of plants with constitutively activated GSK2.Consistent with this finding,GSK2 protein accumulates in the tud1 mutant compared with the wild type.In addition,inhibition of BR synthesis promotes GSK2 accumulation and suppresses TUD1 stability.By contrast,BRs can induce GSK2 degradation but promote TUD1 accumulation.Furthermore,the GSK2 degradation process is largely impaired in tud1 in response to BR.In conclusion,our study demonstrates the role of TUD1 in BR-induced GSK2 degradation,thereby advancing our understanding of a critical step in the BR signaling pathway of rice.