Abstract
To survive environmental challenges, plants have evolved tightly regulated response networks, including a rapid and transient general stress response (GSR), followed by well-studied stress-specific responses. The mechanisms underpinning the GSR have remained elusive, but a functional cis-element, the rapid stress response element (RSRE), is known to confer transcription of GSR genes rapidly (5 min) and transiently (peaking 90-120 min after stress) in vivo. To investigate signal transduction events in the GSR, we used a 4xRSRE:LUCIFERASE reporter in Arabidopsis (Arabidopsis thaliana), employing complementary approaches of forward and chemical genetic screens, and identified components regulating peak time versus amplitude of RSRE activity. Specifically, we identified a mutant in CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) with reduced RSRE activation, verifying this transcription factor's role in activation of the RSRE-mediated GSR. Furthermore, we isolated a mutant in MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) KINASE KINASE1 (mekk1-5), which displays increased basal and an approximately 60-min earlier peak of wound-induced RSRE activation. The double mekk1/camta3 mutant positioned CAMTA3 downstream of MEKK1 and verified their distinct roles in GSR regulation. mekk1-5 displays programmed cell death and overaccumulates reactive oxygen species and salicylic acid, hallmarks of the hypersensitive response, suggesting that the hypersensitive response may play a role in the RSRE phenotype in this mutant. In addition, chemical inhibition studies suggest that the MAPK network is required for the rapid peak of the RSRE response, distinguishing the impact of chronic (mekk1-5) from transient (chemical inhibition) loss of MAPK signaling. Collectively, these results reveal underlying regulatory components of the plant GSR and further define their distinct roles in the regulation of this key biological process.
Highlights
general stress response (GSR), we used a 4xRSRE:LUCIFERASE reporter in Arabidopsis (Arabidopsis thaliana), employing complementary approaches of forward and chemical genetic screens, and identified components regulating peak time versus amplitude of rapid stress response element (RSRE) activity
Recent genetic approaches and transient expression-based assays have shown that CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR (CAMTA) transcription factors are required for robust 4xRSRE:LUC activity, with CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 (CAMTA3) playing the predominant role of the examined family members (CAMTA1–CAMTA4; Benn et al, 2014)
To identify components of stress signaling leading to RSRE-driven gene transcription, we conducted a forward genetic screen using a population of ethyl methanesulfonate (EMS)-mutagenized 4xRSRE:LUC Arabidopsis
Summary
GSR, we used a 4xRSRE:LUCIFERASE reporter in Arabidopsis (Arabidopsis thaliana), employing complementary approaches of forward and chemical genetic screens, and identified components regulating peak time versus amplitude of RSRE activity. Chemical inhibition studies suggest that the MAPK network is required for the rapid peak of the RSRE response, distinguishing the impact of chronic (mekk1-5) from transient (chemical inhibition) loss of MAPK signaling These results reveal underlying regulatory components of the plant GSR and further define their distinct roles in the regulation of this key biological process. The specific nature of RSRE transcriptional regulation by CAMTAs has remained an enigma; these transcription factors are constitutively transcribed and present in the nucleus, but strong RSRE-driven gene transcription is observed only in response to stress One explanation for this inconsistency could be that rapid calcium ion fluxes, typical of many stresses, lead to CAMTA activation through calmodulin, despite that fact that CAMTA transcription factors do not appear to require calmodulin for either entry into the nucleus or DNA binding (Yang and Poovaiah, 2002). MEKK1 is involved in, but not necessary for, MAPK3 and MAPK6 activation in response to elicitors/pathogens, through MEK4 and MEK5 (Asai et al, 2002; Suarez-Rodriguez et al, 2007)
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