The ABI4-Induced Arabidopsis ANAC060 Transcription Factor Attenuates ABA Signaling and Renders Seedlings Sugar Insensitive when Present in the Nucleus

Seedling establishment is inhibited on media containing high levels (∼6%) of glucose or fructose. Genetic loci that overcome the inhibition of seedling growth on high sugar have been identified using natural variation analysis and mutant selection, providing insight into sugar signaling pathways. In this study, a quantitative trait locus (QTL) analysis was performed for seedling sensitivity to high sugar in a Col/C24 F2 population of Arabidopsis thaliana. A glucose and fructose-sensing QTL, GSQ11, was mapped through selective genotyping and confirmed in near-isogenic lines in both Col and C24 backgrounds. Allelism tests and transgenic complementation showed that GSQ11 lies within the ANAC060 gene. The Col ANAC060 allele confers sugar insensitivity and was dominant over the sugar-sensitive C24 allele. Genomic and mRNA analyses showed that a single-nucleotide polymorphism (SNP) in Col ANAC060 affects the splicing patterns of ANAC060 such that 20 additional nucleotides are present in the mRNA. The insertion created a stop codon, resulting in a truncated ANAC60 protein lacking the transmembrane domain (TMD) that is present in the C24 ANAC060 protein. The absence of the TMD results in the nuclear localization of ANAC060. The short version of the ANAC060 protein is found in ∼12% of natural Arabidopsis accessions. Glucose induces GSQ11/ANAC060 expression in a process that requires abscisic acid (ABA) signaling. Chromatin immunoprecipitation-qPCR and transient expression analysis showed that ABI4 directly binds to the GSQ11/ANAC060 promoter to activate transcription. Interestingly, Col ANAC060 reduced ABA sensitivity and Glc-induced ABA accumulation, and ABI4 expression was also reduced in Col ANAC060 lines. Thus, the sugar-ABA signaling cascade induces ANAC060 expression, but the truncated Col ANAC060 protein attenuates ABA induction and ABA signaling. This negative feedback from nuclear ANAC060 on ABA signaling results in sugar insensitivity.

在葡萄糖或果糖浓度约6%的培养基上，拟南芥幼苗的生长会受到显著抑制。研究人员通过自然变异分析与突变体筛选，已鉴定出能够克服高糖环境下幼苗生长抑制的遗传位点，为解析植物糖信号通路提供了关键见解。本研究以拟南芥(Arabidopsis thaliana)Col/C24杂交F₂群体为材料，针对高糖胁迫下的幼苗敏感性开展数量性状位点(quantitative trait locus, QTL)定位分析。通过选择性基因分型，我们定位到一个同时响应葡萄糖与果糖感知的QTL——GSQ11，并在Col和C24遗传背景的近等基因系中验证了该位点的功能。等位性测验与转基因互补实验证实，GSQ11位于ANAC060基因内部。Col背景的ANAC060等位基因赋予植株糖不敏感性，且对糖敏感的C24等位基因呈显性。基因组与mRNA分析显示，Col背景ANAC060基因存在一处单核苷酸多态性(single-nucleotide polymorphism, SNP)，该变异改变了ANAC060的剪接模式，使其mRNA序列多出20个核苷酸。这段额外序列引入了一个终止密码子，最终产生截短的ANAC060蛋白——该蛋白缺失了C24背景ANAC060蛋白所具有的跨膜结构域(transmembrane domain, TMD)。跨膜结构域的缺失使得ANAC060蛋白定位于细胞核中。这种截短形式的ANAC060蛋白在约12%的自然拟南芥种质中存在。葡萄糖可通过依赖脱落酸(abscisic acid, ABA)信号通路的方式诱导GSQ11/ANAC060的表达。染色质免疫沉淀-qPCR(chromatin immunoprecipitation-qPCR)与瞬时表达实验证实，ABI4可直接结合GSQ11/ANAC060的启动子区域以激活其转录。有趣的是，Col背景的ANAC060可降低植株对ABA的敏感性，并抑制葡萄糖诱导的ABA积累；同时，Col背景ANAC060过表达株系中ABI4的表达量也显著降低。由此可见，糖-ABA信号级联反应可诱导ANAC060的表达，而截短的Col背景ANAC060蛋白则会削弱ABA的诱导作用及ABA信号通路。这种来自核定位ANAC060对ABA信号的负反馈调控，最终赋予植株糖不敏感性。