Identification of proteins that changed phosphorylation intensities in response to SL treatment^a.

aRice d10 mutant XJC seedlings were germinated and grown on medium in the presence of 1.0 μM GR24 for 6 days in darkness. Seedlings grown without exogenous application of GR24 served as a control. Total proteins were extracted from seedlings and resolved by 2-DE. Antibodies against phosphoserine or phosphothreonine were used to probe the proteomes. Phosphoproteins that exhibited differences in intensity between GR24 treatment and the control were excised for identification by mass spectrometry. bRelative molecular mass (Mr) in Da and isoelectric point (pI) of proteins are theoretical. cNumber of matched peptides (MP). dSequence coverage (SC, %) of the identified peptides. eProteins with a Mowse score of more than 61 were considered to be identified at a statistically significant level. Significance threshold p<0.05. fExpectation scores are the probability-based values obtained from database search by MASCOT. Values less than their threshold (p<0.05) are statistically confident with more than 95% certainty. gProtein phosphorylation intensity changes were distinguished as induced phosphorylation (+), induced de-phosphorylation (-) and reduced phosphorylation (↓) in response to GR24 treatment. hFold changes (FC) with standard errors (±SE) from three biological replicates were calculated as the ratio of normalized spot intensities between the treatment and control. iFold changes (FC) in spot intensity with a p-value of less than 0.05 were considered to be statistically significant.a. 以水稻d10突变体XJC幼苗为材料，将其置于添加1.0 μM GR24的培养基中，于黑暗条件下萌发并生长6天。以未外源施加GR24的幼苗作为对照组。提取幼苗总蛋白，通过双向凝胶电泳（two-dimensional gel electrophoresis, 2-DE）进行分离。采用抗磷酸丝氨酸（phosphoserine）及抗磷酸苏氨酸（phosphothreonine）抗体对蛋白质组进行免疫杂交检测。将GR24处理组与对照组之间信号强度存在差异的磷酸化蛋白点切下，采用质谱（mass spectrometry）进行鉴定。 b. 蛋白质的相对分子质量（Relative molecular mass, Mr，单位为道尔顿（Dalton, Da））与等电点（isoelectric point, pI）均为理论预测值。 c. 匹配肽段数（matched peptides, MP）。 d. 鉴定肽段的序列覆盖率（Sequence coverage, SC，单位为%）。 e. 当蛋白质的Mowse得分高于61时，认为其鉴定结果具有统计学显著性；显著性阈值设为p<0.05。 f. 期望得分是通过MASCOT进行数据库检索得到的基于概率的数值；若该数值低于阈值（p<0.05），则鉴定结果的统计学置信度可达95%以上。 g. 响应GR24处理的蛋白质磷酸化强度变化可划分为三类：诱导磷酸化（+）、诱导去磷酸化（-）以及磷酸化水平降低（↓）。 h. 基于三次生物学重复的标准化蛋白点强度比值计算得到倍变化值（Fold changes, FC）及其标准误（standard errors, ±SE），即处理组与对照组的标准化蛋白点强度之比。 i. 当蛋白点强度的倍变化值对应的p值小于0.05时，认为其变化具有统计学显著性。