甘蓝型油菜的BR响应及BnBZL2基因的功能分析

对甘蓝型油菜(Brassica napus L.)与拟南芥(Arabidopsis thaliana L.)中保守的油菜素甾醇(Brassinosteroids,BR)信号相关基因进行对比分析,并以甘蓝型油菜品种‘沪油15’为材料,对BR信号通路相关同源基因进行了组织表达分析。结果显示,BR合成基因与信号组分在花和幼嫩种子中表达量更高;低浓度BR处理可以促进幼苗根的生长,高浓度BR处理则起抑制作用; BR合成抑制剂(Brassinozole,BRZ)处理可抑制黑暗条件下幼苗下胚轴的伸长; BR处理可以降低BR合成基因的表达水平,而BRZ处理则相反,表明甘蓝型油菜中BR信号增加能反馈抑制BR的合成。烟草(Nicotiana tabacum L.)瞬时表达实验结果发现,与拟南芥BZR1基因同源的甘蓝型油菜BnBZL2编码蛋白定位在细胞质和细胞核中,BR处理可增加BnBZL2的核定位。蛋白质免疫印迹检测结果显示,BR处理可增加去磷酸化BnBZL2的比例。本研究进一步模拟了拟南芥bzr1-1D功能获得性突变体对BnBZL2蛋白进行点突变(BnBZL2*),并构建载体转化拟南芥,黑暗条件下转基因植株幼苗对BRZ处理不敏感,提示BnBZL2*可提高转基因植株的BR信号水平。本研究结果表明甘蓝型油菜中存在与拟南芥相似且保守的BR信号通路和调控机制。     

B-box protein BBX32 integrates light and brassinosteroid signals to inhibit cotyledon opening

Cotyledon opening is a key morphological change that occurs in seedlings during de-etiolation. Brassinosteroids (BRs) inhibit the opening of cotyledons in darkness while light promotes cotyledon opening. The molecular regulation of the interplay between light and BR to regulate cotyledon opening is not well understood. Here, we show the B-box protein BBX32 negatively regulates light signaling and promotes BR signaling to inhibit cotyledon opening in Arabidopsis (Arabidopsis thaliana). BBX32 is highly expressed in the cotyledons of seedlings during de-etiolation. bbx32 and 35S:BBX32 seedlings exhibit enhanced and reduced cotyledon opening, respectively, in response to both light and brassinazole treatment in dark, suggesting that BBX32 mediates cotyledon opening through both light and BR signaling pathways. BBX32 expression is induced by exogenous BR and is upregulated in bzr1-1D (BRASSINAZOLE RESISTANT1-1D). Our in vitro and in vivo interaction studies suggest that BBX32 physically interacts with BZR1. Further, we found that PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) interacts with BBX32 and promotes BR-mediated cotyledon closure. BBX32, BZR1, and PIF3 regulate the expression of common target genes that modulate the opening and closing of cotyledons. Our work suggests BBX32 integrates light and BR signals to regulate cotyledon opening during de-etiolation.     

Brassinosteroids regulate pectin methylesterase activity and AtPME41 expression in Arabidopsis under chilling stress

Pectin methylesterases (PMEs) are important cell wall enzymes that may play important roles in plant chilling/freezing tolerance. We investigated the possible roles of brassinosteroids (BRs) in regulation of PMEs under chilling stress. Chilling stress or 24-epibrassinolide (eBL) treatments induced significant increases in PME activity in wild type (Col-0) seedlings of Arabidopsis. The chilling-stress-induced increases in PME activity were also found in bzr1-D mutant, a BZR1 stabilized mutant with a constitutively active BR signaling pathway, but not in bri1-116, a BR insensitive null allele of the BR receptor BRI1. The results suggest that the regulation of PME activity in Arabidopsis under chilling stress depends on the BR signaling pathway. Furthermore, we showed that the effect of chilling stress on PME activity was impaired in pme41, a knockout mutant of AtPME41. Semi-quantitative RT-PCR results showed that expression of AtPME41 was induced by chilling stress in wild type plants but not in the bri1-116 mutant. The expression of AtPME41 increased in bzr1-D and eBL treated wild type seedlings, but decreased in bri1-116 seedlings. Furthermore, ion leakage induced by low temperature were dramatically increased in both bri1-116 and pme41, while lipid peroxidation was increased in bri1-116 only. The results suggest that BRs may modulate total PME activity in Arabidopsis under chilling stress by regulating AtPME41 expression. Regulation of PME activity may serve as one of the mechanisms that BR participates in chilling tolerance of plants.     

Brassinosteroid‐mediated reactive oxygen species are essential for tapetum degradation and pollen fertility in tomato

Phytohormone brassinosteroids (BRs) are essential for plant growth and development, but the mechanisms of BR‐mediated pollen development remain largely unknown. In this study, we show that pollen viability, pollen germination and seed number decreased in the BR‐deficient mutant d^{^im}, which has a lesion in the BR biosynthetic gene DWARF (DWF), and in the bzr1 mutant, which is deficient in BR signaling regulator BRASSINAZOLE RESISTANT 1 (BZR1), compared with those in wild‐type plants, whereas plants overexpressing DWF or BZR1 exhibited the opposite effects. Loss or gain of function in the DWF or BZR1 genes altered the timing of reactive oxygen species (ROS) production and programmed cell death (PCD) in tapetal cells, resulting in delayed or premature tapetal degeneration, respectively. Further analysis revealed that BZR1 could directly bind to the promoter of RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1), and that RBOH1‐mediated ROS promote pollen and seed development by triggering PCD and tapetal cell degradation. In contrast, the suppression of RBOH1 compromised BR signaling‐mediated ROS production and pollen development. These findings provide strong evidence that BZR1‐dependent ROS production plays a critical role in the BR‐mediated regulation of tapetal cell degeneration and pollen development in Solanum lycopersicum (tomato) plants.     

BRing it on: new insights into the mechanism of brassinosteroid action

Several recent breakthroughs have filled in key details of the brassinosteroid (BR) response. Identification of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further defined the downstream events in this pathway, confirming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered.     

Two putative BIN2 substrates are nuclear components of brassinosteroid signaling

GSK3 is a highly conserved kinase that negatively regulates many cellular processes by phosphorylating a variety of protein substrates. BIN2 is a GSK3-like kinase in Arabidopsis that functions as a negative regulator of brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex that contains the leucine (Leu)-rich repeat receptor-like kinase BRI1, inactivate BIN2 to relieve its inhibitory effect on unknown downstream BR-signaling components. Using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we discovered a potential BIN2 substrate that is identical to a recently identified BR-signaling protein, BES1. BES1 and its closest homolog, BZR1, which was also uncovered as a potential BR-signaling protein, display specific interactions with BIN2 in yeast. Both BES1 and BZR1 contain many copies of a conserved GSK3 phosphorylation site and can be phosphorylated by BIN2 in vitro via a novel GSK3 phosphorylation mechanism that is independent of a priming phosphorylation or a scaffold protein. Five independent bes1 alleles containing the same proline-233-Leu mutation were identified as semidominant suppressors of two different bri1 mutations. Over-expression of the wild-type BZR1 gene partially complemented bin2/+ mutants and resulted in a BRI1 overexpression phenotype in a BIN2(+) background, whereas overexpression of a mutated BZR1 gene containing the corresponding proline-234-Leu mutation rescued a weak bri1 mutation and led to a bes1-like phenotype. Confocal microscopic analysis indicated that both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process.     

A proteomics study of brassinosteroid response in Arabidopsis

The plant steroid hormones brassinosteroids (BRs) play an important role in a wide range of developmental and physiological processes. How BR signaling regulates diverse processes remains unclear. To understand the molecular details of BR responses, we performed a proteomics study of BR-regulated proteins in Arabidopsis using two-dimensional DIGE coupled with LC-MS/MS. We identified 42 BR-regulated proteins, which are predicted to play potential roles in BR regulation of specific cellular processes, such as signaling, cytoskeleton rearrangement, vesicle trafficking, and biosynthesis of hormones and vitamins. Analyses of the BR-insensitive mutant bri1-116 and BR-hypersensitive mutant bzr1-1D identified five proteins (PATL1, PATL2, THI1, AtMDAR3, and NADP-ME2) affected both by BR treatment and in the mutants, suggesting their importance in BR action. Selected proteins were further studied using insertion knock-out mutants or immunoblotting. Interestingly about 80% of the BR-responsive proteins were not identified in previous microarray studies, and direct comparison between protein and RNA changes in BR mutants revealed a very weak correlation. RT-PCR analysis of selected genes revealed gene-specific kinetic relationships between RNA and protein responses. Furthermore BR-regulated posttranslational modification of BiP2 protein was detected as spot shifts in two-dimensional DIGE. This study provides novel insights into the molecular networks that link BR signaling to specific cellular and physiological responses.