The Small Ethylene Response Factors Are Involved In The Regulation Of ABA And Salt Responses In Arabidopsis

Ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, flowering, senescence and abscission, fruit ripening, and root nodules formation, and of plant responses to environmental stimuli including biotic(i.e. pathogen invading) and abiotic stresses(i.e. drought, salt or alkaline stresses). Ethylene Response Factors(ERFs) are plant-specific transcription factors that belong to a subfamily of the AP2(APETALA2)/ERF transcription factor family. There are 4 small ERFs in ERF family, including ERF95, ERF96, ERF97 and ERF98 are ERFs. The samll ERFs are highly conserved at the amino acid level. They all have a DNA binding domain similar to the C-terminal ERF region in other ERFs, and a CMIX-1 domain with unknown function, but lack of an activation or repression domain at their N-terminals found in other ERFs. ERF95 and ERF98 have been shown to involve in the regulation of plant responses to abiotic stresses, and ERF96 and ERF97 in plant defense response in Arabidopsis. We provide evidence here that small ERFs are positive regulators of abscisic acid(ABA) responses in Arabidopsis.Phylogenetic analysi using the full amino acid sequence of the small ERFs showed that ERF95, ERF96 and ERF97 formed a cluster, but ERF98 was distantly related to other three ERFs. By using quantitative RT-PCR, we found that ERF95, ERF97 and ERF98 were expressed in all organs and tissues examined including root, stem, leaf, flower, and mature seed, but highest expression levels were detected in different organs. ERF95 was highly expressed in mature seed, ERF98 in stem, whereas ERF97 in flower and seed. ERF96 was also mainly expressed in flower and seed, however, its expression in root was unexamined. Subcellular localization analysis indicated that ERF95, ERF96, ERF97 and ERF98 are nuclear proteins. Results from the protoplast transfection assay indicated that all ERFs are transcriptional activators, and the EDLL motif-containing C-terminal domain is responsible for their transcriptional activity. Although loss-of-function mutant of erf95 erf96 erf97, erf96 erf97 erf98, erf95 erf96 erf97 erf98 were morphologically similar to wild type plants, transgenic plants overexpressing ERFs had different phenotype from wild type plants. Transgenic plants overexpressing ERF95, ERF96, and ERF98 had smaller rosette size(p<0.01) and were delayed in flowering(p<0.01). In ABA sensitivity assays, we found that ERFs overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including ABI5, ABF3, ABF4, RD29 A, P5 CS, and COR15 A were observed in the transgenic plants in the presence of ABA. Our experiments also showed that water loss in ERFs overexpression plants were slower than that in Col wild type plants. Stomatal closure assays indicated that ERFs overexpression plants had reduced stomatal aperture in the presence of ABA. ERFs overexpression plants had higher instantaneous leaf water-use efficiency compared to Col wild type plants in the presence of ABA. Taken together, our results suggest that ERFs positively regulates ABA responses in Arabidopsis.In addition, we also found that, similar to that of ERF95 and ERF98, ERF96 and ERF97 are also involved in the regulation of plants responses to NaCl stress in Arabidopsis. Under NaCl treatment condition, the ERFs overexpression plants had a better growing status than Col wild type plants; they have a higher shoot fresh weight, and prodeuced more lateral roots. Relative higher transcript levels of some NaCl-responsive genes including RD29 A, P5 CS, COR15 A, KIN1 and RAB18 were observed in the transgenic plants unpon NaCl treatment. We found that, under NaCl treatment condition, ERF96 and ERF97 transgenic plants contained less Na+ and more K+ when compared with that in Col wild type plants, suggesting that small ERFs may regulate plant response to salt stress by affecting the metabolism of Na+ and K+.