Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi, 110007, India.
Plant Physiol Biochem. 2021 Apr;161:234-247. doi: 10.1016/j.plaphy.2021.02.012. Epub 2021 Feb 19.
Plant cuticle, the site of perception of stress signals, is an extracellular hydrophobic barrier that covers the epidermis of the above-ground parts. This lipidic layer has been explored for its cutin and wax composition. However, reports on the cuticle proteins are scanty. Therefore, leaf cuticle proteins of Brassica juncea isolated using organic solvents (chloroform-methanol, 2:1(v/v)) were analyzed using gel based and quantitative shotgun proteomics. Out of 615 proteins identified, 27% (169) had signal peptides supporting extracellular localization. Bioinformatics tool, QuickGO predicted the involvement of these proteins in catabolism (21%), peptidase activity (13%), oxidoreductase (12%), defense response (9%), fatty acid binding (9%), nutrient reservoir activity (8%), chitin binding (7%) and lipid transport (2%). Myrosinase-catalyzed glucosinolate hydrolysis releases bioactive compounds, which contribute to plant defense. This system is termed as "mustard oil bomb". Myrosinase and its associating protein, GDSL esterase/lipase ESM1 (involved in cuticle structuring and defense) were detected in the cuticle. GDSL-esterase/lipase ESM1 and β-glucanase (an antifreeze protein) showed in vitro activity. Analysis of cuticle extract by nanoliter osmometer-phase contrast microscopy detected antifreeze activity due to non-protein component. Post-translational modification analysis using PTM viewer predicted N-glycosylation (66%), N-terminal proteolysis (40%), and phosphorylation (32%) to be the dominant modification in the classical secretory proteins. N-glycosylation of myrosinase and GDSL esterase/lipase, ESM1 was confirmed by Con A affinoblotting. This study not only identified leaf cuticle proteins, but also laid the foundation for exploring the extracellular glucosinolate-myrosinase system, PTM crosstalk, and antifreeze activity as stress adaptive strategies in B. juncea.
植物表皮是感受胁迫信号的部位，是覆盖地上部分表皮的细胞外疏水屏障。人们研究了这个脂类层的角质和蜡的组成。然而，关于表皮蛋白的报道却很少。因此，本研究使用有机溶剂（氯仿-甲醇，2:1(v/v))分离芥菜叶片表皮蛋白，并通过凝胶基础和定量鸟枪法蛋白质组学进行分析。在鉴定的 615 种蛋白质中，有 27%（169 种）具有支持细胞外定位的信号肽。生物信息学工具 QuickGO 预测这些蛋白质参与了代谢（21%）、肽酶活性（13%）、氧化还原酶（12%）、防御反应（9%）、脂肪酸结合（9%）、营养库活性（8%）、几丁质结合（7%）和脂质转运（2%）。黑芥子酶催化硫代葡萄糖苷水解会释放生物活性化合物，这些化合物有助于植物防御。这个系统被称为“芥子油炸弹”。黑芥子酶及其相关蛋白 GDSL 酯酶/脂肪酶 ESM1（参与表皮结构和防御）在表皮中被检测到。GDSL 酯酶/脂肪酶 ESM1 和β-葡聚糖酶（一种抗冻蛋白）表现出体外活性。纳米升渗透压计相衬显微镜分析表皮提取物检测到由于非蛋白成分而产生的抗冻活性。PTM 查看器预测的翻译后修饰分析表明，在经典分泌蛋白中，N-糖基化（66%）、N 端蛋白水解（40%）和磷酸化（32%）是主要的修饰方式。黑芥子酶和 GDSL 酯酶/脂肪酶 ESM1 的 N-糖基化通过 ConA 亲和印迹得到了证实。本研究不仅鉴定了叶片表皮蛋白，还为探索细胞外硫代葡萄糖苷-黑芥子酶系统、PTM 串扰和抗冻活性作为芥菜应激适应策略奠定了基础。
