Table_3_Transcriptome analysis of the 2,4-dichlorophenoxyacetic acid (2,4-D)-tolerant cotton chromosome substitution line CS-B15sh and its susceptible parental lines G. hirsutum L. cv. Texas Marker-1 and G. barbadense L. cv. Pima 379.XLSX

The cotton chromosome substitution line, CS-B15sh, exhibits 41% lower injury from 2,4-D when applied at the field recommended rate of 1.12 kg ae ha−1 (1×) than does Texas Marker-1 (TM-1). CS-B15sh was developed in the genetic background of Gossypium hirsutum L. cv TM-1 and has chromosome introgression on the short arm of chromosome 15 from Gossypium barbadense L. cv. Pima 379. In a previous experiment, we observed reduced translocation of [14C]2,4-D outside the treated leaf tissue in CS-B15sh, which contrasted with an increased translocation of the herbicide in the tissues above and below the treated leaf in TM-1. Our results indicate a potential 2,4-D tolerance mechanism in CS-B15sh involving altered movement of 2,4-D. Here, we used RNA sequencing (RNA-seq) to determine the differential expression of genes between 2,4-D-challenged and control plants of the tolerant (CS-B15sh) and susceptible lines (TM-1 and Pima 379). Several components of the 2,4-D/auxin-response pathway—including ubiquitin E3 ligase, PB1|AUX/IAA, ARF transcription factors, and F-box proteins of the SCFTIR1/AFB complex—were upregulated with at least threefold higher expression in TM-1 compared with CS-B15sh, while both Pima 379 and TM-1 showed the same fold change expression for PB1|AUX/IAA mRNA. Some genes associated with herbicide metabolism, including flavin monooxygenase (Gohir.A01G174100) and FAD-linked oxidase (Gohir.D06G002600), exhibited at least a twofold increase in CS-B15sh than in TM-1 (the gene was not expressed in Pima 379), suggesting a potential relationship between the gene’s expression and 2,4-D tolerance. It is interesting to note that glutathione S-transferase was differentially expressed in both CS-B15sh and Pima 379 but not in TM-1, while cytochrome P450 and other genes involved in the oxidation–reduction process were significantly expressed only in CS-B15sh in response to 2,4-D. Gene set enrichment analysis on the union DEGs of the three cotton genotypes revealed the depletion of transcripts involved in photosynthesis and enrichment of transcripts involved in ABA response and signaling.

棉染色体代换系CS-B15sh在按照田间推荐浓度1.12千克/公顷（1倍）喷施2,4-D的情况下，相较于德克萨斯标记-1（TM-1），其损伤降低了41%。CS-B15sh是在Gossypium hirsutum L. cv TM-1的遗传背景下培育而成，并在第15染色体的短臂上引入了Gossypium barbadense L. cv. Pima 379的染色体片段。在先前的研究中，我们观察到CS-B15sh中[14C]2,4-D在处理叶组织外的转移减少，这与TM-1中处理叶上、下组织内除草剂转移增加形成鲜明对比。我们的研究结果提示CS-B15sh中可能存在一种2,4-D耐受机制，涉及2,4-D的转移改变。在本研究中，我们采用RNA测序技术（RNA-seq）来探究耐受性（CS-B15sh）与易感性品种（TM-1和Pima 379）的2,4-D挑战植物与对照植物之间基因表达的差异。2,4-D/生长素响应途径的多个组分，包括泛素E3连接酶、PB1|AUX/IAA、ARF转录因子以及SCFTIR1/AFB复合物中的F-box蛋白，在TM-1中的表达量至少比CS-B15sh高3倍，而Pima 379和TM-1的PB1|AUX/IAA mRNA表达量变化相同。一些与除草剂代谢相关的基因，如黄素单加氧酶（Gohir.A01G174100）和FAD连接的氧化酶（Gohir.D06G002600），在CS-B15sh中的表达量至少比TM-1高2倍（该基因在Pima 379中未表达），这表明基因表达与2,4-D耐受性之间可能存在潜在关系。值得注意的是，谷胱甘肽转移酶在CS-B15sh和Pima 379中表达存在差异，但在TM-1中则没有，而细胞色素P450及其他参与氧化还原过程的基因仅在CS-B15sh中响应2,4-D显著表达。对三个棉花基因型的联合差异表达基因集进行富集分析，揭示了光合作用相关转录本的减少以及ABA反应和信号传导相关转录本的增加。