This study investigates the role of the NAC transcription factor ANAC032 in regulating abscisic acid (ABA)-dependent stress responses and its involvement in sugar signaling pathways. Arabidopsis seedlings with overexpressed or knock-out ANAC032 were examined for their sensitivity to ABA, glucose, and fluridone to elucidate the functional role of ANAC032 in ABA and high glucose-mediated growth retardation. Our results showed that ANAC032 negatively regulates ABA responses, as ANAC-overexpressing plants exhibited higher ABA sensitivity, while anac032 mutants were less sensitive. Under high glucose conditions, anac032 mutants demonstrated hyposensitivity, with germination rates higher than wild-type and ANAC032-overexpressing plants. Additionally, yeast two-hybrid screening identified three NAC proteins, ANAC020, ANAC064, and ANAC074, interact with ANAC032. These findings highlight ANAC032’s role in stress signaling pathways and its potential interactions with other NAC proteins, contributing to a better understanding of transcriptional regulation in plant stress responses and possibly expanding to forage crop development.

The plant-specific NAC transcription factors control various biological processes, including plant development and stress responses. We have isolated an ANAC032 gene, one of the NAC transcription factor family, which was highly activated by multi-abiotic stresses, including high salt and drought in Arabidopsis. Here, we generated transgenic plants constitutively expressing ANAC032 and its knockout to identify the functional roles of ANAC032 in Arabidopsis under abiotic stress responses. The ANAC032-overexpressing plants showed enhanced tolerance to salinity and drought stresses. The anac032 knockout mutants were observed no significant changes under the high salt and drought conditions. We also monitored the expression of high salt and drought stress-responsive genes in the ANAC032 transgenic plants and anac032 mutant. The ANAC032 overexpression upregulated the expression of stress-responsive genes, RD29A and ERD10, under the stresses. Thus, our data identify that transcription factor ANAC032 plays as an enhancer for salinity and drought tolerance through the upregulation of stress-responsive genes and provides useful genetic traits for generating multi-abiotic stress-tolerant forage crops.

Forage crop management is severely challenged by global warming-induced climate changes representing diverse a/biotic stresses. Thus, screening of valuable genetic resources would be applied to develop stress-tolerant forage crops. We isolated two NAC (NAM, ATAF1, ATAF2, CUC2) transcription factors (ANAC032 and ANAC083) transcriptionally activated by multi-abiotic stresses (salt, drought, and cold stresses) from Arabidopsis by microarray analysis. The NAC family is one of the most prominent transcription factor families in plants and functions in various biological processes. The enhanced expressions of two ANACs by multi-abiotic stresses were validated by quantitative RT-PCR analysis. We also confirmed that both ANACs were localized in the nucleus, suggesting that ANAC032 and ANAC083 act as transcription factors to regulate the expression of downstream target genes. Promoter activities of ANAC032 and ANAC083 through histochemical GUS staining again suggested that various abiotic stresses strongly drive both ANACs expressions. Our data suggest that ANAC032 and ANAC083 would be valuable genetic candidates for breeding multi-abiotic stress-tolerant forage crops via the genetic modification of a single gene.