PhERF2, an ethylene-responsive element binding factor, plays an essential role in waterlogging tolerance of petunia

Authors: YIN, DONGMEI - Shanghai Institute Of Technology; SUN, DAOYANG - Northwest A&f University; Norris, Ayla; HAN, ZHUQING - Shanghai Institute Of Technology; NI, DIAN - Shanghai Institute Of Technology; Jiang, Cai-Zhong

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2019
Publication Date: 7/1/2019
Citation: Yin, D., Sun, D., Norris, A.M., Han, Z., Ni, D., Jiang, C. 2019. PhERF2, an ethylene-responsive element binding factor, plays an essential role in waterlogging tolerance of petunia. Horticulture Research. 6:83. https://doi.org/10.1038/s41438-019-0165-z.
DOI: https://doi.org/10.1038/s41438-019-0165-z

Interpretive Summary: Global climate change brings about a frequent occurrence of extreme rainfall events, and it increases the demand for improvement of plant tolerance to waterlogging. Plants under waterlogging conditions undergo hypoxic stress with difficulty in oxygen diffusion, resulting in a drop in photosynthesis, respiration, and chlorophyll accumulation. Plants produce metabolic energy through fermentative glycolysis and not oxidative respiration in response to waterlogging stress. In the case of oxygen deficiency, respiration varies from the aerobic to the anaerobic mode, which is implicated in glycolysis and fermentation. The ethylene-responsive element binding factor (ERF) proteins are crucial transcriptional regulators in response to diverse biotic and abiotic stresses in plants. Members of the ERF family regulate stress responses mostly through the direct binding to specific promoter sequences (cis-acting GCC box) of defense-related genes. ERF genes have been identified in a number of model organisms, including Arabidopsis, rice, soybean, and wheat. Constitutive expression of soybean GmERF3 in transgenic tobacco plants reduces susceptibility to high salinity, dehydration, fungal and viral diseases. Ectopic overexpression of JERF1 and JERF3 increases resistance of tobacco to drought and osmotic stress. Transgenic Arabidopsis plants with overexpressed AtERF98 exhibit enhanced tolerance to salt via regulation of ascorbic acid synthesis. Moreover, the biological role of ERFs in waterlogging stress has been reported previously. Overexpression of an ERF gene Sub1A leads to an enhanced tolerance to submergence in rice. Licausi et al. found that double mutants of hre1 and hre2, two hypoxia-inducible ERFs in Arabidopsis, display increased sensitivity to anoxia. HRE1 overexpression improves anoxia tolerance of transgenic Arabidopsis plants. Transgenic Arabidopsis plants constitutively expressing BnERF2.4 from Brassica napus exhibit enhanced submergence tolerance and alleviated oxidative damage. However, the molecular mechanism of how ERFs regulate waterlogging tolerance remains largely unknown. Petunia, a horticultural crop that is highly sensitive to submergence, is an excellent model system for studies of waterlogging responses. In previous work, we identified a cluster of transcription factors during petunia flower development via transcriptomic analysis, including some ERFs. We have recently reported a critical role of PhERF2, a member of ERF family, in antiviral RNA silencing. Here, we report an additional function of PhERF2 in waterlogging tolerance of petunia. We generated transgenic petunia plants with RNAi silencing and overexpression of PhERF2 to study the regulatory role of PhERF2 in waterlogging responses. Compared with WT plants, PhERF2 silencing compromised the tolerance of petunia seedlings to waterlogging, while overexpression of PhERF2 improved the survival of seedlings subjected to waterlogging. PhERF2-RNAi lines exhibited earlier and severer leaf chlorosis and necrosis than WT, whereas plants overexpressing PhERF2 showed promoted growth vigor under waterlogging. Chlorophyll content was dramatically lower in PhERF2-silenced plants than WT or overexpression plants. Typical characteristics of programmed cell death (PCD), DNA condensation, and moon-shaped nuclei were only observed in PhERF2-overexpressing lines but not in PhERF2-RNAi or control lines. Furthermore, transcript abundances of the alcoholic fermentation-related genes ADH1-1, ADH1-2, ADH1-3, PDC1, and PDC2 were higher in PhERF2-overexpressing plants but lower in PhERF2-silenced plants. In contrast, expression of the lactate fermentation-related gene LDH, which participates in the release of lactic acid, was up-regulated in PhERF2-silenced plants. Moreover, electrophoretic mobility shift assay (EMSA) and dual luciferase assay revealed that PhERF2 directly binds to the

Technical Abstract: Ethylene-responsive element binding factors (ERFs) are involved in regulation of various stress responses in plants, but their biological functions in waterlogging stress are largely unclear. In this study, we identified a petunia (Petunia × hybrida) ERF gene, PhERF2 that was significantly induced by waterlogging in wild-type (WT). To study the regulatory role of PhERF2 in waterlogging responses, transgenic petunia plants with RNAi silencing and overexpression of PhERF2 were generated. Compared with WT plants, PhERF2 silencing compromised the tolerance of petunia seedlings to waterlogging, while overexpression of PhERF2 improved the survival of seedlings subjected to waterlogging. PhERF2-RNAi lines exhibited earlier and severer leaf chlorosis and necrosis than WT, whereas plants overexpressing PhERF2 showed promoted growth vigor under waterlogging. Chlorophyll content was dramatically lower in PhERF2-silenced plants than WT or overexpression plants. Typical characteristics of programmed cell death (PCD), DNA condensation, and moon-shaped nuclei were only observed in PhERF2-overexpressing lines but not in PhERF2-RNAi or control lines. Furthermore, transcript abundances of the alcoholic fermentation-related genes ADH1-1, ADH1-2, ADH1-3, PDC1, and PDC2 were higher in PhERF2-overexpressing plants but lower in PhERF2-silenced plants. In contrast, expression of the lactate fermentation-related gene LDH, which participates in the release of lactic acid, was up-regulated in PhERF2-silenced plants. Moreover, electrophoretic mobility shift assay (EMSA) and dual luciferase assay revealed that PhERF2 directly binds to the ADH1-2 promoter. Our results demonstrate that PhERF2 contributes to petunia tolerance to waterlogging through modulation of PCD and alcoholic fermentation system.