UC Riverside

Environmental stresses involving flooding and the accompanying cellular low oxygen (hypoxia) constitute a major limitation to agricultural production worldwide. These stresses hinder the plant's ability to maintain energy production and growth. Yet, previous studies have shown that certain genes of group VII ethylene responsive factor (ERF) family of transcription factors play a functional role in the survival of submergence and low oxygen stresses in Oryza sativa(rice) and Arabidopsis thaliana. This study utilized a transient gene transfection assay of protoplasts to characterize gene networks regulated by the group VII ERFs by measuring the transcriptional activation of various promoters. Experiments revealed that orthologous genes are regulated by the group VII ERFs of both species. In rice, an ERF-type transcription factor, SUB1A, confers submergence tolerance by regulating carbohydrate metabolism, elongation growth, and hormonal responses. SUB1A is linked to the related genes SUB1B and SUB1C. Here, two alleles of SUB1A (SUB1A-1, SUB1A-2) as well as one allele each of the other two genes (SUB1B-1 and SUB1C-1) were shown to be transcriptional activators, and the nuclear localization of SUB1A-1 and SUB1C-1 was demonstrated. In Arabidopsis, core hypoxia responsive genes are governed by the N-end rule pathway of targeted proteolysis, in which proteins of the group VII ERFs (i.e., RAP2.12 and HRE2) are regulated post-translationally under hypoxic conditions. Transactivation experiments demonstrated that the trihelix protein HRA1 negatively regulates the transcriptional activation ability of RAP2.12 in the core hypoxia response. By contrast, HRE2 was not targeted by HRA1 downregulation, but was instead upregulated by RAP2.12 during the stress. Experiments involving truncations of HRA1 reveal that the coil-coil domain is functionally important for HRA1 interaction with group VII ERF complexes. These results with a rapid protoplast assay provide a foundation for future investigations into the low oxygen response gene network regulated by group VII ERF transcription factors binding to cis-regulatory elements, and modulated by the trihelix protein HRA1.