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

Abstract

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 F ₂ 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.

Author Summary

In plants, sugars function as signaling molecules that control important processes such as photosynthesis, growth, carbon distribution over different organs and the production of storage compounds. Sugar signaling requires the phytohormone abscisic acid (ABA) and the ABA-induced regulatory transcription factor ABI4. In this study, a genetic analysis identified the transcription factor ANAC060 as an important component in establishing sugar sensitivity. It was found that, in natural Arabidopsis thaliana populations, the ANAC060 protein may occur as a long or a short version due to differential ANAC060 mRNA splicing caused by a single-nucleotide polymorphism (SNP). The long ANAC060 protein with an intact transmembrane domain (TMD) is excluded from the nucleus, whereas the short version lacking the TMD is always present in the nucleus, where it regulates gene expression. Functional analyses indicated that Col ANAC060 is involved in a novel negative feedback loop in the sugar-ABA signaling pathway. In this feedback loop model, ABI4 activates ANAC060 expression, but the nuclear presence of Col ANAC060 suppresses Glc-induced ABA accumulation and ABI4 expression, thereby reducing responsiveness to sugar signals.