Biotic and abiotic stresses limit agricultural yields, and plants are often simultaneously exposed to multiple stresses. Combinations of stresses such as heat and drought or cold and high light intensity have profound effects on crop performance and yields. Thus, delineation of the regulatory networks and metabolic pathways responding to single and multiple concurrent stresses is required for breeding and engineering crop stress tolerance. Many studies have described transcriptome changes in response to single stresses. However, exposure of plants to a combination of stress factors may require agonistic or antagonistic responses or responses potentially unrelated to responses to the corresponding single stresses. To analyze such responses, we initially compared transcriptome changes in 10 Arabidopsis (Arabidopsis thaliana) ecotypes using cold, heat, high-light, salt, and flagellin treatments as single stress factors as well as their double combinations. This revealed that some 61% of the transcriptome changes in response to double stresses were not predictable from the responses to single stress treatments. It also showed that plants prioritized between potentially antagonistic responses for only 5% to 10% of the responding transcripts. This indicates that plants have evolved to cope with combinations of stresses and, therefore, may be bred to endure them. In addition, using a subset of this data from the Columbia and Landsberg erecta ecotypes, we have delineated coexpression network modules responding to single and combined stresses.

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1's full kinase activity.