Landau Olivia A, Jamison Brendan V, Riechers Dean E
Department of Crop Sciences, University of Illinois, Urbana, Illinois, United States of America.
PLoS One. 2025 Feb 18;20(2):e0319151. doi: 10.1371/journal.pone.0319151. eCollection 2025.
Identification and characterization of genes encoding herbicide-detoxifying enzymes is lacking in allohexaploid wheat (Triticum aestivum L.). Gene expression is frequently induced by herbicide safeners and implies the encoded enzymes serve a role in herbicide metabolism and detoxification. Cloquintocet-mexyl (CM) is a safener commonly utilized with halauxifen-methyl (HM), a synthetic auxin herbicide whose phytotoxic form is halauxifen acid (HA). Our first objective was to identify candidate HA-detoxifying genes via RNA-Seq by comparing untreated and CM-treated leaf tissue. On average, 81% of RNA-Seq library reads mapped uniquely to the reference genome and 76.4% of reads were mapped to a gene. Among the 103 significant differentially expressed genes (DEGs), functional annotations indicate the majority of DEGs encode proteins associated with herbicide or xenobiotic metabolism. This finding was further corroborated by gene ontology (GO) enrichment analysis, where several genes were assigned GO terms indicating oxidoreductase activity (34 genes) and transferase activity (45 genes). One of the significant DEGs is a member of the CYP81A subfamily of cytochrome P450s (CYPs; denoted as CYP81A-5A), which are of interest due to their ability to catalyze synthetic auxin detoxification. To investigate CYP expression induced by HM and/or CM, our second objective was to measure gene-specific expression of CYP81A-5A and its homoeologs (CYP81A-5B and CYP81A-5D) in untreated leaf tissue and leaf tissue treated with CM and HM over time using RT-qPCR. Relative to the reference gene (β-tubulin), basal CYP expression is high, expression among these CYPs varies over time, and expression for all CYPs is CM-inducible but not HM-inducible. Further analysis of CYP81A-5A, such as gene knock-out, overexpression experiments, or in vitro activity assays with purified enzyme are necessary to test the hypotheses that the encoded CYP detoxifies HA and that CM upregulates this reaction.
在异源六倍体小麦（普通小麦）中，缺乏对编码除草剂解毒酶的基因进行鉴定和表征的研究。基因表达常常由除草剂安全剂诱导，这表明所编码的酶在除草剂代谢和解毒过程中发挥作用。氯喹酮甲酯（CM）是一种常用的安全剂，与合成生长素除草剂甲基二氯喹啉酸（HM）一起使用，其植物毒性形式为二氯喹啉酸（HA）。我们的首要目标是通过比较未处理和CM处理的叶片组织，利用RNA测序来鉴定候选的HA解毒基因。平均而言，81%的RNA测序文库读数唯一地映射到参考基因组，76.4%的读数映射到一个基因。在103个显著差异表达基因（DEG）中，功能注释表明大多数DEG编码与除草剂或异生物质代谢相关的蛋白质。基因本体（GO）富集分析进一步证实了这一发现，其中几个基因被赋予了表明氧化还原酶活性（34个基因）和转移酶活性（45个基因）的GO术语。一个显著的DEG是细胞色素P450（CYPs）的CYP81A亚家族成员（表示为CYP81A - 5A），由于它们催化合成生长素解毒的能力而受到关注。为了研究HM和/或CM诱导的CYP表达，我们的第二个目标是使用逆转录定量PCR（RT - qPCR）来测量CYP81A - 5A及其同源基因（CYP81A - 5B和CYP81A - 5D）在未处理的叶片组织以及随着时间推移用CM和HM处理的叶片组织中的基因特异性表达。相对于参考基因（β - 微管蛋白），基础CYP表达较高，这些CYPs之间的表达随时间变化，并且所有CYPs的表达都可被CM诱导，但不能被HM诱导。有必要对CYP81A - 5A进行进一步分析，例如基因敲除、过表达实验或用纯化酶进行体外活性测定，以检验所编码的CYP使HA解毒以及CM上调该反应的假设。
