Dept. of Cell Biology and Genetics, Institute of Biology, Pedagogical University, Podchorążych 2, 31-054 Kraków, Poland.
Polish Academy of Sciences, The Franciszek Górski Institute of Plant Physiology, Niezapominajek 21, 30-239 Kraków, Poland.
J Proteomics. 2017 Oct 3;169:58-72. doi: 10.1016/j.jprot.2017.08.019. Epub 2017 Aug 25.
The changes in protein abundance induced by cold hardening were analysed by 2 DE in ten doubled haploid (DH) lines of winter barley, highly differentiated with respect to freezing tolerance level. Among 45 differential proteins identified by MALDI-TOF/TOF, the majority was classified as related to photosynthesis, carbohydrate metabolism, oxidation-reduction reactions and stress response. Among the detected proteins, higher abundance of RuBisCO large and small subunits, RuBisCO activase, two Oxygen-evolving enhancer proteins, Ferredoxin-NADP reductase, Cytochrome P450-dependent fatty acid hydroxylase and 14-3-3 protein was associated with higher freezing tolerance level. Lower relative level of hypothetical ATP synthase beta subunit, uncharacterized mitochondrial protein AtMg00810 and ribosomal RNA small subunit methyltransferase G also seems to be important. The results of proteomic studies were complemented by the evaluation of photosynthetic apparatus acclimation, showing distinctive differences between the studied genotypes in the number of active PSII reaction centres (RC/CS). Additionally, the analysis of antioxidative enzyme activities suggests the importance of HO as a signalling molecule possibly involved in the initiation of cold-induced plant acclimation. However, in DH lines with high freezing tolerance, HO generation during cold hardening treatment was accompanied by more stable activity of catalase, HO-decomposing enzyme.
In the study, the changes in protein abundance induced by cold hardening treatment were analysed by two-dimensional gel electrophoresis in ten doubled haploid (DH) lines of winter barley. Harnessing DH technology resulted in distinctive widening of genetic variation with respect to freezing tolerance level. Both the cold-hardening effect on the protein pattern in an individual winter barley DH line as well as the variation among the selected DH lines were investigated, which resulted in the identification of 45 differentiated proteins classified as involved in 14 metabolic pathways and cellular processes. Among them, eight proteins: (1) the precursor of RuBisCO large subunit, (2) RuBisCO small subunit (partial), (3) RuBisCO activase small isoform, (4) the precursor of Oxygen-evolving enhancer protein 1-like (predicted protein), (5) Oxygen-evolving enhancer protein 2, (6) the leaf isozyme of Ferredoxin-NADP reductase, (7) hypothetical protein M569_12509 Cytochrome P450-dependent fatty acid hydroxylase-like and (8) hypothetical protein BRADI_1g11290 (14-3-3 protein A-like) were accumulated to a higher level in leaves of cold-hardened seedlings of freezing tolerant winter barley DH lines in comparison with susceptible ones. Three others: (9) hypothetical protein BRADI_5g05668 F1 ATP synthase beta subunit-like, (10) predicted protein uncharacterized mitochondrial protein AtMg00810-like and (11) BnaA02g08010D Ribosomal RNA small subunit methyltransferase G-like were detected at lower level in freezing tolerant seedlings in comparison with susceptible genotypes. The last two were for the first time linked to cold acclimation. The results of complementary analyses indicate that PSII activity and stability of antioxidative enzymes under low temperature are also very important for freezing tolerance acquisition.
通过双向电泳分析了 10 个冬大麦双倍单倍体（DH）系在冷驯化过程中诱导的蛋白质丰度变化，这些 DH 系在耐冻性水平上高度分化。通过 MALDI-TOF/TOF 鉴定了 45 种差异蛋白，其中大多数与光合作用、碳水化合物代谢、氧化还原反应和应激反应有关。在所检测到的蛋白质中，Rubisco 大亚基和小亚基、Rubisco 激活酶、两种放氧增强蛋白、铁氧还蛋白-NADP 还原酶、细胞色素 P450 依赖性脂肪酸羟化酶和 14-3-3 蛋白的丰度较高与较高的耐冻性水平相关。ATP 合酶β亚基、未鉴定的线粒体蛋白 AtMg00810 和核糖体 RNA 小亚基甲基转移酶 G 的相对水平较低似乎也很重要。蛋白质组学研究的结果得到了光合作用装置适应评估的补充，表明在研究的基因型之间 PSII 反应中心（RC/CS）的活性有明显差异。此外，抗氧化酶活性的分析表明，HO 作为一种信号分子的重要性，可能参与了冷诱导植物适应的启动。然而，在耐冻性较高的 DH 系中，冷驯化处理过程中 HO 的产生伴随着过氧化氢酶活性的更稳定，过氧化氢酶是 HO 的分解酶。
在这项研究中，通过双向凝胶电泳分析了 10 个冬大麦双倍单倍体（DH）系在冷驯化处理下诱导的蛋白质丰度变化。利用 DH 技术，在耐冻性水平上显著扩大了遗传变异。研究了单个冬大麦 DH 系的冷驯化效应以及所选 DH 系之间的变异，从而鉴定出 45 种差异蛋白，分为涉及 14 种代谢途径和细胞过程的分类。其中 8 种蛋白：（1）Rubisco 大亚基前体，（2）Rubisco 小亚基（部分），（3）Rubisco 激活酶小同工型，（4）放氧增强蛋白 1 样物的前体（预测蛋白），（5）放氧增强蛋白 2，（6）叶型铁氧还蛋白-NADP 还原酶，（7）细胞色素 P450 依赖性脂肪酸羟化酶样假设蛋白 M569_12509 和（8）假设蛋白 BRADI_1g11290（14-3-3 蛋白 A 样）在耐寒性冬大麦 DH 系的冷驯化幼苗叶片中积累到较高水平，与易感型相比。另外 3 种：（9）假设蛋白 BRADI_5g05668 F1 ATP 合酶β亚基样，（10）预测蛋白未鉴定的线粒体蛋白 AtMg00810 样和（11）BnaA02g08010D 核糖体 RNA 小亚基甲基转移酶 G 样在与易感基因型相比，在耐寒性幼苗中检测到的水平较低。后两种是首次与冷驯化相关联的。互补分析的结果表明，在低温下 PSII 活性和抗氧化酶的稳定性对获得耐冻性也非常重要。
