Table_5_Genome-Wide Identification and Function of Aquaporin Genes During Dormancy and Sprouting Periods of Kernel-Using Apricot (Prunus armeniaca L.).doc

Aquaporins (AQPs) are essential channel proteins that play a major role in plant growth and development, regulate plant water homeostasis, and transport uncharged solutes across biological membranes. In this study, 33 AQP genes were systematically identified from the kernel-using apricot (Prunus armeniaca L.) genome and divided into five subfamilies based on phylogenetic analyses. A total of 14 collinear blocks containing AQP genes between P. armeniaca and Arabidopsis thaliana were identified by synteny analysis, and 30 collinear blocks were identified between P. armeniaca and P. persica. Gene structure and conserved functional motif analyses indicated that the PaAQPs exhibit a conserved exon-intron pattern and that conserved motifs are present within members of each subfamily. Physiological mechanism prediction based on the aromatic/arginine selectivity filter, Froger’s positions, and three-dimensional (3D) protein model construction revealed marked differences in substrate specificity between the members of the five subfamilies of PaAQPs. Promoter analysis of the PaAQP genes for conserved regulatory elements suggested a greater abundance of cis-elements involved in light, hormone, and stress responses, which may reflect the differences in expression patterns of PaAQPs and their various functions associated with plant development and abiotic stress responses. Gene expression patterns of PaAQPs showed that PaPIP1-3, PaPIP2-1, and PaTIP1-1 were highly expressed in flower buds during the dormancy and sprouting stages of P. armeniaca. A PaAQP coexpression network showed that PaAQPs were coexpressed with 14 cold resistance genes and with 16 cold stress-associated genes. The expression pattern of 70% of the PaAQPs coexpressed with cold stress resistance genes was consistent with the four periods [Physiological dormancy (PD), ecological dormancy (ED), sprouting period (SP), and germination stage (GS)] of flower buds of P. armeniaca. Detection of the transient expression of GFP-tagged PaPIP1-1, PaPIP2-3, PaSIP1-3, PaXIP1-2, PaNIP6-1, and PaTIP1-1 revealed that the fusion proteins localized to the plasma membrane. Predictions of an A. thaliana ortholog-based protein–protein interaction network indicated that PaAQP proteins had complex relationships with the cold tolerance pathway, PaNIP6-1 could interact with WRKY6, PaTIP1-1 could interact with TSPO, and PaPIP2-1 could interact with ATHATPLC1G. Interestingly, overexpression of PaPIP1-3 and PaTIP1-1 increased the cold tolerance of and protein accumulation in yeast. Compared with wild-type plants, PaPIP1-3- and PaTIP1-1-overexpressing (OE) Arabidopsis plants exhibited greater tolerance to cold stress, as evidenced by better growth and greater antioxidative enzyme activities. Overall, our study provides insights into the interaction networks, expression patterns, and functional analysis of PaAQP genes in P. armeniaca L. and contributes to the further functional characterization of PaAQPs.

水通道蛋白（Aquaporins, AQPs）是一类关键的通道蛋白，在植物生长发育进程中发挥核心调控作用，既可维持植物水分稳态，亦可介导不带电溶质跨生物膜转运。本研究从仁用杏（Prunus armeniaca L.）基因组中系统鉴定出33个AQP编码基因，并通过系统发育分析将其划分为5个亚家族。通过共线性分析，共鉴定得到仁用杏与拟南芥（Arabidopsis thaliana）间包含AQP基因的共线性模块14个，仁用杏与桃（Prunus persica）间的共线性模块则为30个。基因结构与保守功能基序分析结果显示，PaAQPs（仁用杏AQP基因）具有保守的外显子-内含子排布模式，且各亚家族成员均含有保守基序。基于芳香族精氨酸选择性滤片（aromatic/arginine selectivity filter）、弗罗格位点（Froger’s positions）及三维（3D）蛋白质结构建模开展的生理机制预测表明，PaAQPs的5个亚家族成员在底物特异性上存在显著差异。对PaAQP基因启动子的保守调控元件分析发现，其中富集了大量参与光响应、激素响应与胁迫响应的顺式作用元件，这或可反映PaAQPs的表达模式差异，以及其在植物发育与非生物胁迫响应中所承担的多样功能。PaAQPs的基因表达模式分析显示，PaPIP1-3、PaPIP2-1与PaTIP1-1在仁用杏休眠与萌发阶段的花芽中高表达。PaAQPs共表达网络分析结果表明，PaAQPs可与14个耐寒基因及16个冷胁迫相关基因发生共表达。其中，与冷胁迫耐受基因共表达的PaAQPs中，有70%的表达模式与仁用杏花芽的四个发育时期——生理休眠期（PD）、生态休眠期（ED）、萌芽期（SP）及萌发期（GS）——高度契合。对绿色荧光蛋白（Green Fluorescent Protein, GFP）标记的PaPIP1-1、PaPIP2-3、PaSIP1-3、PaXIP1-2、PaNIP6-1及PaTIP1-1的瞬时表达检测结果显示，上述融合蛋白均定位于质膜。基于拟南芥同源基因的蛋白质相互作用网络预测表明，PaAQP蛋白与耐寒通路存在复杂关联：PaNIP6-1可与WRKY6相互作用，PaTIP1-1可与TSPO相互作用，PaPIP2-1可与ATHATPLC1G相互作用。值得注意的是，过表达PaPIP1-3与PaTIP1-1可提升酵母的耐寒性并增加相关蛋白的积累量。与野生型植株相比，过表达PaPIP1-3与PaTIP1-1的拟南芥过表达（OE）植株对冷胁迫的耐受性更强，具体表现为生长状态更佳、抗氧化酶活性更高。综上，本研究系统解析了仁用杏PaAQP基因的相互作用网络、表达模式与功能特征，为PaAQPs的后续功能研究提供了重要理论参考与研究基础。