Table_1_Overexpression of abscisic acid-insensitive gene ABI4 from Medicago truncatula, which could interact with ABA2, improved plant cold tolerance mediated by ABA signaling.XLSX

ABI4 is considered an important transcription factor with multiple regulatory functions involved in many biological events. However, its role in abiotic stresses, especially low-temperature-induced stress, is poorly understood. In this study, the MtABI4 gene was derived from M. truncatula, a widely used forage grass. Analysis of subcellular localization indicated that ABI4 was localized in the nucleus. Identification of expression characteristics showed that ABI4 was involved in the regulatory mechanisms of multiple hormones and could be induced by the low temperature. IP-MS assay revealed that MtABI4 protein could interact with xanthoxin dehydrogenase protein (ABA2). The two-hybrid yeast assay and the biomolecular fluorescence complementarity assay further supported this finding. Expression analysis demonstrated that overexpression of MtABI4 induced an increase in ABA2 gene expression both in M. truncatula and Arabidopsis, which in turn increased the ABA level in transgenic plants. In addition, the transgenic lines with the overexpression of MtABI4 exhibited enhanced tolerance to low temperature, including lower malondialdehyde content, electrical conductivity, and cell membrane permeability, compared with the wide-type lines after being cultivated for 5 days in 4°C. Gene expression and enzyme activities of the antioxidant system assay revealed the increased activities of SOD, CAT, MDHAR, and GR, and higher ASA/DHA ratio and GSH/GSSG ratio in transgenic lines. Additionally, overexpression of ABI4 also induced the expression of members of the Inducer of CBF expression genes (ICEs)-C-repeat binding transcription factor genes(CBFs)-Cold regulated genes (CORs) low-temperature response module. In summary, under low-temperature conditions, overexpression of ABI4 could enhance the content of endogenous ABA in plants through interactions with ABA2, which in turn reduced low-temperature damage in plants. This provides a new perspective for further understanding the molecular regulatory mechanism of plant response to low temperature and the improvement of plant cold tolerance.

ABI4被认为是一种具有多重调控功能的转录因子，其参与多种生物事件。然而，其在非生物胁迫，尤其是低温诱导胁迫中的作用尚不明确。在本研究中，从广泛用作牧草的拟南芥（M. truncatula）中提取了MtABI4基因。亚细胞定位分析表明，ABI4定位于细胞核。表达特征鉴定显示，ABI4参与多种激素的调控机制，并可被低温诱导。免疫共沉淀-质谱法（IP-MS）实验揭示了MtABI4蛋白可与黄氧氧化酶蛋白（ABA2）相互作用。双杂交酵母实验和生物分子荧光互补实验进一步证实了这一发现。表达分析表明，过表达MtABI4在拟南芥和Arabidopsis中均诱导了ABA2基因表达的升高，进而增加了转基因植物中的ABA水平。此外，与野生型相比，过表达MtABI4的转基因植株在4°C条件下培养5天后表现出增强的低温耐受性，包括较低的丙二醛含量、电导率和细胞膜透性。抗氧化系统基因表达和酶活性分析显示，转基因植株中SOD、CAT、MDHAR和GR的活性增加，以及ASA/DHA比率和GSH/GSSG比率升高。此外，ABI4的过表达还诱导了诱导CBF表达基因（ICEs）-C重复结合转录因子基因（CBFs）-低温响应基因（CORs）低温响应模块成员的表达。总之，在低温条件下，通过ABA2的相互作用，ABI4过表达能够提高植物中内源ABA的含量，从而降低植物的低温损伤。这为深入理解植物对低温的分子调控机制以及提高植物抗冷性提供了新的视角。