Rosales Raquel, Romero Irene, Escribano Ma Isabel, Merodio Carmen, Sanchez-Ballesta Ma Teresa
Phytochemistry. 2014 Dec;108:17-25. doi: 10.1016/j.phytochem.2014.10.006.
Dehydrins (DHNs), group II LEA (Late Embryogenesis Abundant) proteins, are among the most commonly observed proteins which accumulate in plants in response to cold and any other environmental factors, causing the dehydration of cells. In previous studies, we isolated a YSK2-type VvcDHN1a gene from table grapes (Vitis vinifera cv. Cardinal) which presented two spliced variants (the spliced, DHN1a_s and the unspliced, DHN1a_u). Their expression was induced by low temperature storage and CO2, although with different accumulation patterns. DHN1a_u codifies for a truncated YS protein lacking Ф- and K-segments, which might affect its functionality. In this work, we expressed both DHN1a_s and DHN1a_u recombinant proteins in Escherichia coli. We carried out a number of in vitro assays to analyze the implications that Ф- and K-segments have in the protective role of VvcDHN1 against different abiotic stresses and their antifungal activity against the fungal pathogen Botrytis cinerea. Our results showed that unlike DHN1a_u, DHN1a_s has a potent cryoprotective effect on lactate dehydrogenase activity, protects malate dehydrogenase against dehydration and partially inhibits B. cinerea growth. Moreover, the DHN1a promoter presented cis-regulatory elements related to cold and drought, as well as biotic stress-related elements. We also observed that both spliced variants interact weakly with DNA, suggesting that K-segments are not involved in DNA binding. Overall, this work highlights the crucial role of Ф- and K-segments in DHNs function in plant response to abiotic stress showing for the first time, the potential role of the V. vinifera DHN1a_s in the protection against freezing and dehydration as well as inhibiting B. cinerea growth.
脱水素（DHNs）是第II组胚胎后期丰富（LEA）蛋白，是植物中最常见的因应对寒冷和其他环境因素导致细胞脱水而积累的蛋白质之一。在先前的研究中，我们从鲜食葡萄（欧亚种葡萄品种“绯红无核”）中分离出一个YSK2型VvcDHN1a基因，该基因有两个剪接变体（剪接后的DHN1a_s和未剪接的DHN1a_u）。它们的表达受低温贮藏和二氧化碳诱导，尽管积累模式不同。DHN1a_u编码一种缺少Ф段和K段的截短YS蛋白，这可能会影响其功能。在这项工作中，我们在大肠杆菌中表达了DHN1a_s和DHN1a_u重组蛋白。我们进行了一系列体外试验，以分析Ф段和K段对VvcDHN1抵御不同非生物胁迫的保护作用及其对真菌病原体灰葡萄孢的抗真菌活性的影响。我们的结果表明，与DHN1a_u不同，DHN1a_s对乳酸脱氢酶活性有强大的冷冻保护作用，保护苹果酸脱氢酶免受脱水影响，并部分抑制灰葡萄孢的生长。此外，DHN1a启动子具有与寒冷和干旱相关的顺式调控元件，以及与生物胁迫相关的元件。我们还观察到两个剪接变体与DNA的相互作用较弱，这表明K段不参与DNA结合。总体而言，这项工作突出了Ф段和K段在脱水素响应非生物胁迫功能中的关键作用，首次展示了欧亚种葡萄DHN1a_s在抗冻、抗脱水以及抑制灰葡萄孢生长方面的潜在作用。
