Table_1_The Salt Tolerance Related Protein (STRP) Mediates Cold Stress Responses and Abscisic Acid Signalling in Arabidopsis thaliana.xlsx

Low temperature stress is one of the major causes of crop yield reduction in agriculture. The alteration of gene expression pattern and the accumulation of stress-related proteins are the main strategies activated by plants under this unfavourable condition. Here we characterize the Arabidopsis thaliana Salt Tolerance Related Protein (STRP). The protein rapidly accumulates under cold treatment, and this effect is not dependent on transcriptional activation of the STRP gene, but on the inhibition of proteasome-mediated degradation. Subcellular localization of STRP was determined by the transient expression of STRP-YFP in A. thaliana protoplasts. STRP is localized into the cytosol, nucleus, and associated to the plasma membrane. Under cold stress, the membrane-associated fraction decreases, while in the cytosol and in the nucleus STRP levels strongly increase. STRP has high similarity with WCI16, a wheat Late Embryogenesis Abundant (LEA)-like protein. Despite no canonical LEA motifs in the STRP sequence are present, physicochemical characterization demonstrated that STRP shares common features with LEA proteins, being a high hydrophilic unstructured protein, highly soluble after boiling and with cryoprotectant activity. To clarify the physiological function of STRP, we characterized the phenotype and the response to low temperature stress of the strp knockout mutant. The mutation causes an equal impairment of plant growth and development both in physiological and cold stress conditions. The strp mutant is more susceptible to oxidative damage respect to the wild type, showing increased lipid peroxidation and altered membrane integrity. Furthermore, the analysis of Abscisic acid (ABA) effects on protein levels demonstrated that the hormone induces the increase of STRP levels, an effect in part ascribable to its ability to activate STRP expression. ABA treatments showed that the strp mutant displays an ABA hyposensitive phenotype in terms of seed germination, root development, stomata closure and in the expression of ABA-responsive genes. In conclusion, our results demonstrate that STRP acts as a multifunctional protein in the response mechanisms to low temperature, suggesting a crucial role for this protein in stress perception and in the translation of extracellular stimuli in an intracellular response.

低温胁迫是导致农业作物减产的主要诱因之一。在此不良环境条件下，植物主要通过调控基因表达模式、积累胁迫相关蛋白来启动应对策略。本研究针对拟南芥（Arabidopsis thaliana）的盐胁迫耐受相关蛋白（Salt Tolerance Related Protein，STRP）开展了表征分析。该蛋白在低温处理下会快速积累，且这一过程并不依赖于STRP基因的转录激活，而是通过抑制蛋白酶体介导的蛋白质降解实现的。研究通过在拟南芥原生质体中瞬时表达STRP-黄色荧光蛋白（Yellow Fluorescent Protein，YFP）融合蛋白，确定了STRP的亚细胞定位：STRP可定位于细胞质、细胞核，并与质膜相结合。在低温胁迫条件下，与质膜结合的STRP组分占比下降，而细胞质与细胞核中的STRP水平则显著升高。STRP与小麦晚期胚胎富集蛋白（Late Embryogenesis Abundant，LEA）类蛋白WCI16具有较高的序列相似性。尽管STRP的氨基酸序列中不存在典型的LEA基序，但理化性质表征结果显示，STRP与LEA蛋白具有共同特性：属于高度亲水的无序蛋白，煮沸后仍保持高可溶性，且具备低温保护活性。为阐明STRP的生理功能，本研究对strp基因敲除突变体的表型及其对低温胁迫的响应进行了分析。该突变会在正常生理条件与低温胁迫条件下，对植物的生长发育造成同等程度的损伤。与野生型植株相比，strp突变体对氧化损伤更为敏感，表现出脂质过氧化水平升高以及膜完整性异常。此外，通过分析脱落酸（Abscisic acid，ABA）对蛋白水平的影响，研究发现该激素可诱导STRP水平升高，这一效应部分源于其能够激活STRP基因的表达。ABA处理实验结果表明，在种子萌发、根系发育、气孔关闭以及ABA响应基因的表达这几个方面，strp突变体均表现出ABA不敏感表型。综上，本研究结果表明，STRP在低温胁迫响应机制中作为多功能蛋白发挥作用，提示该蛋白在胁迫感知以及将胞外刺激转化为胞内响应的过程中扮演关键角色。