ERF-VII members exhibit synergistic and separate roles in Arabidopsis

ABSTRACT

The ethylene response factor VII (ERF-VII) transcription factor has been reported to be involved in multiple different stress responses. In a previous study, we showed that ERF74 and ERF75 play a redundant role in the upregulation of RESPIRATORY BURST OXIDASE HOMOLOG D (RbohD) transcription and enhance the oxygen species (ROS) burst during early stages of the stress response. Induction of stress marker genes and ROS-scavenging enzymes under various stress conditions are dependent on this ROS burst. Here, we propose an assumption that ERF71-ERF75 have different functions and act synergistically in response to stresses in Arabidopsis. ERF74 and ERF75 are involved in controlling an RbohD-dependent mechanism in response to different stresses, subsequently maintaining H₂O₂ homeostasis in Arabidopsis as we previously reported. ERF71 and ERF73 may have a role in supervising plant intracellular ROS homeostasis, whereas ERF72 may only act as an activator of ERF74 and ERF75 in the stress response.

The ERF-VII family of transcription factors (TFs) has 5 members in Arabidopsis, namely ERF71 (HRE2), ERF72 (RAP2.3), ERF73 (HRE1), ERF74 (RAP2.12) and ERF75 (RAP2.2).¹ These ERF-VII TFs have been reported in oxidative and osmotic stress,^2,3 Botrytis cinerea infection⁴ and hypoxia.^5-11 They share a similar N-terminal degron motif MCGGAI/V that is regulated by the N-end rule pathway of protein degradation.^12,13 In a previous study, we found for the first time that although ERF74 and ERF75 bind to Hypoxia-Responsive Promoter Element to activate the expression of hypoxia-responsive gene (HRG) expression in hypoxia,¹⁴ this induction is dependent on the ERF74-Rbohd-ROS signaling pathway.¹⁵

ERF71-ERF75 have been reported to play a role in hypoxia, but a recent report showed that ERF71 and ERF73 only play a minor role in the activation of hypoxia-responsive genes in hypoxia.¹⁴ We found that ERF71 and ERF73 may have a role in ROS scavenging in response to hypoxia and other stresses. While we examined the change of intracellular ROS in Arabidopsis protoplasts or plants in flooding and other stresses, we found that erf74;erf75 mutant plants lack a ROS burst in the early stages of different stresses, while erf71;erf73 mutant plants have a normal ROS burst that cannot diminish as with WT in later stage of stress (Fig. 1A). These results indicate that erf71;erf73 mutants seem to minimally perceive H₂O₂ signals and RbohD expression cannot decrease in later stages of high light (HL) and drought stress, which supports this assumption.

Figure 1.

ERF-VII TFs have different functions in the response to stress. (A) Kinetics of changes in H₂O₂ levels using H₂DCFDA by flow cytometry analysis in WT, erf74;erf75 erf71;erf73 protoplasts under different treatments. (B) Kinetics of changes in the viability of protoplasts measured by flow cytometry analysis in WT, erf74;erf75 erf71;erf73 protoplasts under flooding treatment. (C) RT-qPCR analysis of RbohD transcription in WT and erf71;erf73 plants under normal, drought and HL conditions. The expression level of RbohD without treatment in WT was set to 1. Data are means ± SD (n = 4) of 3 independent experiments.

Another interesting result is observed when we test the cell viability of WT, erf74;erf75 and erf71;erf73 plants after flooding treatment. We found that the cell viability of WT and erf71;erf73 mutants was similar after flooding treatment of 3h, while continuous flooding treatment resulted in a more rapid reduction in cell viability of erf71;erf73 (Fig. 1B), possibly due to the disruption of intracellular uncontrolled ROS (Fig. 1C). Taken together, we assume that ERF71 and ERF73 may have a direct role in supervising ROS regulation. The microarray results¹⁶ of Yang et al., (2011) showed that the induction of ROS perception associated genes¹⁷ encoding heat shock factors (Hsfs, At1g74310 and At4g25200) are suppressed in ERF73-RNAi transgenic plants upon hypoxia conditions, partly supporting our assumption. On the other hand, ERF71 and ERF73 have been reported to negatively regulate ROS producing associated peroxidase and cytochrome P450 genes.^16,18

It have been reported that ERF72 was also linked to the hypoxia response. The ERF72 overexpression line had increased expression of HRG in hypoxia, but ERF72 only showed a weak capacity to activate HRPE. When we tested whether ERF71-ERF75 have a role in transcriptional activation of ERF74 and ERF75, we found that ERF72 shows a much stronger activation than the other ERFs. These results indicate that ERF72 may activate the expression of HRG by ERF74 and ERF75.

Another question is why ERF74 and ERF75 regulate HRG expression dependent on the ROS burst. Transcript levels of ERF73 were significantly decreased following DPI treatment and increased following H₂O₂ treatment^18-20 suggesting that the induction of ERF74 and ERF75 in various stresses may be dependent on the ROS burst. Taken together with the results from Fig. 1, we propose that ERF74 and ERF75 play an important role in directly regulating stress response genes, while their expression level is induced by the ROS burst and by ERF-VII family members. This pathway may be related to a signal amplification reaction in plants responding to different stresses.

Conclusion

It has been reported that each ERF-VII overexpressing transgenic plant showed increased tolerance to hypoxia and other stress, whereas its each single or double T-DNA insertion mutant showed decreased tolerance.^2-6,11,17 This suggests that hypoxia and other stress responses act synergistically through ERF71-ERF75. ERF71-ERF75 have different functions in stress responses in Arabidopsis. ERF74 and ERF75 have a role in activating the RbohD-ROS signal pathway to relay the stress signal to downstream effectors and regulate stress response genes, whereas ERF71 and ERF73 may have a direct role in ROS perception by supervising the plant intracellular ROS homeostasis. On the other hand, ERF72 may just participate in the induction of ERF74 and ERF75 in the stress response.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Financial support for this work was obtained from the National Natural Science Foundation of China (Grant nos. 31670670).