Distinct roles for mitogen-activated protein kinase signaling and CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR3 in regulating the peak time and amplitude of the plant general stress response

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.

Figure 1.

Mutation in CAMTA3 reduces RSRE activation. A and B, Compared with the parent line, RAR71 (camta3-4) shows approximately 70% reduced 4xRSRE:LUC activity in response to wounding. The color-coded bar in A displays the intensity of LUC activity. Data are means ± se (n = 30). C, camta3-4 has a premature stop in the CAMTA3 gene.

Figure 2.

A single amino acid substitution in MEKK1 constitutively activates RSRE. A and B, CAR98 (mekk1-5) shows constitutive 4xRSRE:LUC activity (A), with peak wound induction shifted approximately 60 min earlier than that of the parent line (B). The color-coded bar in A displays the intensity of LUC activity. Data are means ± se (n ≤ 48). The asterisk indicates significantly different peak times (P < 0.05). C, CAR98 has a single Pro (P)-to-Leu (L) amino acid substitution in MEKK1.

Figure 3.

mekk1-5 is a partial loss-of-function allele of MEKK1. A to C, mekk1-5 displays phenotypes between parent plants and mekk1-1 in size (A), H₂O₂ measured by DAB (B), and PCD assayed using Trypan Blue (C). D, A western blot recognizing phosphorylated MAPKs shows a similar phosphorylation pattern among all three genotypes treated with water (control) or flg22. Ponceau S staining displays protein loading.

Figure 4.

mekk1 mutants accumulate significantly higher SA levels than the parent control. mekk1-5 has statistically significantly more SA than the parent but significantly less than mekk1-1 (P < 0.05). SA was measured via gas chromatography-mass spectrometry after flg22 elicitation. Each bar represents an average of three replicates of 30 to 120 plants + sd. fw, Fresh weight.

Figure 5.

RSRE:LUC activity of camta3/mekk1 resembles camta3-4 in level but mekk1-5 in peak time. A and B, mekk1-5/camta3-4 resembles mekk1-5 in size but camta3-4 in basal and wound-induced 4xRSRE:LUC signal amplitude 1.5 h after wounding. The color-coded bar in B displays the intensity of LUC activity. C, mekk1-5/camta3-4 retains the early-shifted peak of mekk1-5 relative to the parent and camta3-4. Data are averages ± se (n ≥ 30). Asterisks indicate significantly different peak times (P < 0.05).

Figure 6.

Chemical inhibition of MAPK signaling delays RSRE:LUC peak activity. A, RM1 application induces 4xRSRE:LUC activity, with a peak shifted approximately 1 h later than wounding and the DMSO control. B, The MAPK signaling inhibitor PD98059 (RM2) delays flg22-induced 4xRSRE:LUC activation by approximately 1.5 h relative to the DMSO control. Chemical structures are shown in each graph, and the data shown are means ± se (n ≥ 48). Asterisks indicate significant differences in peak times (P < 0.05). NT, No treatment.

Figure 7.

Schematic model displaying the MAPK regulation of GSR. Stress activation of MEKK1 and other MAPKKKs results in the phosphorylation of downstream signaling partners and, ultimately, the RSRE-mediated induction of GSR. The MAPK inhibitor RM2 induces a delay in peak RSRE activity, whereas mekk1-5 accelerates peak time and elevates basal RSRE activity. We propose that the mekk1-5 phenotype is not the direct result of MAPK action but rather indirectly caused by constitutive activation of the HR, specifically the associated increase in ROS, functioning through two independent channels: one via an as-yet-unknown mediator to alter peak time and the other through CAMTAs to induce the RSRE and, hence, the related GSR.