Table_5_Genome-Wide Characterization of the Aquaporin Gene Family in Radish and Functional Analysis of RsPIP2-6 Involved in Salt Stress.DOCX

Aquaporins (AQPs) constitute a highly diverse family of channel proteins that transport water and neutral solutes. AQPs play crucial roles in plant development and stress responses. However, the characterization and biological functions of RsAQPs in radish (Raphanus sativus L.) remain elusive. In this study, 61 non-redundant members of AQP-encoding genes were identified from the radish genome database and located on nine chromosomes. Radish AQPs (RsAQPs) were divided into four subfamilies, including 21 plasma membrane intrinsic proteins (PIPs), 19 tonoplast intrinsic proteins (TIPs), 16 NOD-like intrinsic proteins (NIPs), and 5 small basic intrinsic proteins (SIPs), through phylogenetic analysis. All RsAQPs contained highly conserved motifs (motifs 1 and 4) and transmembrane regions, indicating the potential transmembrane transport function of RsAQPs. Tissue- and stage-specific expression patterns of AQP gene analysis based on RNA-seq data revealed that the expression levels of PIPs were generally higher than TIPs, NIPs, and SIPs in radish. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) revealed that seven selected RsPIPs, according to our previous transcriptome data (e.g., RsPIP1-3, 1-6, 2-1, 2-6, 2-10, 2-13, and 2-14), exhibited significant upregulation in roots of salt-tolerant radish genotype. In particular, the transcriptional levels of RsPIP2-6 dramatically increased after 6 h of 150 mM NaCl treatment during the taproot thickening stage. Additionally, overexpression of RsPIP2-6 could enhance salt tolerance by Agrobacterium rhizogenes-mediated transgenic radish hairy roots, which exhibited the mitigatory effects of plant growth reduction, leaf relative water content (RWC) reduction and alleviation of O2– in cells, as shown by nitro blue tetrazolium (NBT) staining, under salt stress. These findings are helpful for deeply dissecting the biological function of RsAQPs on the salt stress response, facilitating practical application and genetic improvement of abiotic stress resistance in radish.

水通道蛋白（Aquaporins, AQPs）是一类高度多样化的通道蛋白家族，负责转运水分与中性溶质。AQPs在植物生长发育及胁迫应答过程中发挥关键作用。然而，萝卜（Raphanus sativus L.）中RsAQPs的功能鉴定与生物学功能仍有待阐明。本研究从萝卜基因组数据库中鉴定得到61个非冗余的AQP编码基因，这些基因定位在9条染色体上。通过系统发育分析，可将萝卜AQPs（RsAQPs）划分为4个亚家族：包括21个质膜内在蛋白（PIPs）、19个液泡膜内在蛋白（TIPs）、16个类结节内在蛋白（NIPs）以及5个小型碱性内在蛋白（SIPs）。所有RsAQPs均含有高度保守的基序（基序1与基序4）及跨膜结构域，提示RsAQPs具备潜在的跨膜转运功能。基于RNA测序（RNA-seq）数据的AQP基因组织与发育阶段特异性表达分析显示，在萝卜中，质膜内在蛋白的表达水平普遍高于液泡膜内在蛋白、类结节内在蛋白与小型碱性内在蛋白。此外，结合前期转录组数据筛选得到的7个RsPIPs（如RsPIP1-3、1-6、2-1、2-6、2-10、2-13及2-14），经实时荧光定量聚合酶链式反应（qRT-PCR）验证发现，这些基因在耐盐萝卜基因型的根系中呈现显著上调表达。其中，RsPIP2-6的转录水平在肉质根膨大期经150 mM NaCl处理6小时后显著升高。进一步研究表明，通过发根农杆菌（Agrobacterium rhizogenes）介导的转基因萝卜毛状根过表达RsPIP2-6可增强植株耐盐性：氯化硝基四氮唑蓝（NBT）染色实验显示，该过表达可缓解盐胁迫下植株生长受抑、叶片相对含水量（RWC）降低以及细胞内超氧阴离子（O₂·⁻）的积累。本研究结果有助于深入解析RsAQPs在萝卜盐胁迫应答中的生物学功能，为萝卜非生物胁迫抗性的遗传改良与实际应用提供理论依据。