GRAS, termed after GAI (gibberellic acid insensitive), RGA (repressor of GA1), and SCR (scarecrow), is a plant-specific transcription factor crucial for plant development and stress response. However, understanding of the functions played by the GRAS members and their target genes in citrus is limited. In this study, we identified a cold stress-responsive GRAS gene from Poncirus trifoliate, designated as PtrPAT1, by yeast one-hybrid library screening using the promoter of PtrBADH-l, a betaine aldehyde dehydrogenase (BADH)-like gene. PtrPAT1, belonging to the PAT1 subfamily, was localized in the nucleus and plasma membrane, exhibited transactivation activity and showed a remarkable upregulation under cold stress. Overexpression of PtrPAT1 elevated BADH activity, increased glycine betaine (GB) accumulation and conferred enhanced cold tolerance in transgenic tobacco plants compared with wild type, while downregulation in trifoliate orange by virus-induced gene silencing (VIGS) resulted in opposite trends. Furthermore, the activities of two antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were significantly increased in the overexpression plants, but remarkably decreased in the VIGS line, consistent with accumulation patterns of the reactive oxygen species (ROS). PtrPAT1 was demonstrated to interact with and activate the PtrBADH-l promoter through the putative PAT1-binding motif with the core sequence of TTTCATGT, indicating that PtrBADH-l is a target gene of PtrPAT1. Taken together, these results demonstrate that PtrPAT1 positively affects cold tolerance through the regulation of GB biosynthesis by modulating PtrBADH-l expression.

中文翻译：

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Poncirus trifoliata 的 GRAS 转录因子 PtrPAT1 在耐寒性中发挥作用，并通过调节 BADH 样基因来调节甘氨酸甜菜碱含量


GRAS 被称为 GAI （赤霉酸不敏感）、RGA （GA1 阻遏物） 和 SCR （稻草人），是一种对植物发育和胁迫反应至关重要的植物特异性转录因子。然而，对 GRAS 成员及其靶基因在柑橘中所发挥的功能的理解是有限的。在这项研究中，我们使用甜菜碱醛脱氢酶 （BADH） 样基因 PtrBADH-l 的启动子，通过酵母单杂交文库筛选，从三叶桥中鉴定出一个冷应激响应性 GRAS 基因，命名为 PtrPAT1。PtrPAT1 属于 PAT1 亚家族，位于细胞核和质膜中，表现出反式激活活性，在低温胁迫下表现出显著的上调。与野生型相比，PtrPAT1 的过表达提高了转基因烟草植株的 BADH 活性，增加了甘氨酸甜菜碱 （GB） 的积累，并赋予了更高的耐寒性，而病毒诱导的基因沉默 （VIGS） 在三叶橙中下调导致了相反的趋势。此外，过氧化物酶 （POD） 和超氧化物歧化酶 （SOD） 这两种抗氧化酶的活性在过表达植物中显著增加，但在 VIGS 系中显著降低，这与活性氧 （ROS） 的积累模式一致。PtrPAT1 经证实通过与 TTTCATGT 核心序列的推定 PAT1 结合基序与 PtrBADH-l 启动子相互作用并激活 PtrBADH-l，表明 PtrBADH-l 是 PtrPAT1 的靶基因。综上所述，这些结果表明，PtrPAT1 通过调节 PtrBADH-l 表达调节 GB 生物合成，对耐寒性产生积极影响。