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.

Humic substances that can be obtained from coal resources such as leonardite in a bulk scale have been employed as crop stimulators and soil conditioners. The polymeric organics containing a variety of aromatic and aliphatic structures are known to activate plants in a multifunctional way, thus resulting in enhanced germination rate and abiotic stress resistance concomitant with induction of numerous genes and proteins. Although detailed structural-functional relationship of humic substances for plant stimulations has not been deciphered yet, cutting-edge analytical tools have unraveled critical features of humic architectures that could be linked to the action mechanisms of their plant stimulations. In this review article, we introduce key findings of humic structures and related biological functions that boost plant growth and abiotic stress resistance. Oxygen-based functional groups and plant hormone-like structures combined with labile and recalcitrant carbon backbones are believed to be critical moieties to induce plant stimulations. Some proteins such as HIGH-AFFINITY K+ TRANSPORTER 1, phospholipase A2 and H+-ATPase have been also recognized as key players that could be critically involved in humic substance-driven changes in plant physiology.

Humic acid (HA) is known to consist of various kinds of polymeric organics, their detailed structures can vary depend on sample sources such as organic manure, composts, peat, and lignite brown coal, and largely exists in grassland soils. HA possesses diverse positive effects that not only increase plant growth but also improve soil fertility. Recently, we have manufactured a co-polymeric product of catechol and vanillic acid (CAVA) synthesized artificially, and found that CAVA as a HA mimic increases seed germination and salt tolerance in Arabidopsis. In this study, we examined whether HA or CAVA affects to seedling growth in alfalfa. Foliar application of HA or CAVA increased alfalfa seedling growth including aerial and in root parts. HA or CAVA dramatically enhanced size of leaf and root, whereas HA significantly displayed higher bioactivity than CAVA. Taken together, CAVA acts like as a HA mimic in alfalfa that could apply as an alternation supplement to enhance plant growth and productivity.

Humic acid (HA) is a complex organic matter found in the environments, especially in grassland soils with a high density. The bioactivity of HA to promote plant growth depends largely on its extraction sources. The quality-control of HA and the quality improvements via an artificial synthesis are thus challenging. We recently reported that a polymeric product from fungal laccase-mediated oxidation of catechol and vanillic acid (CAVA) displays a HA-like activity to enhance seed germination and salt stress tolerance in a model plant, Arabidopsis. Here, we examined whether HA or CAVA enhances the growth of Italian ryegrass seedling. Height and fresh weight of the plant with foliar application of HA or CAVA were bigger than those with only water. Interestingly, enhanced root developments were also observed in spite of the foliar treatments of HA or CAVA. Finally, we proved that HA or CAVA promotes the regrowth of Italian ryegrass after cutting. Collectively, CAVA acts as a HA mimic in Italian ryegrass cultivation, and both as a biostimulant enhanced the early growth and regrowth after cutting of Italian ryegrass, which could improve the productivity of forage crops.