Plants cope with environmental challenges by rapidly triggering and synchronizing mechanisms governing stress-specific and general stress response (GSR) networks. The GSR acts rapidly and transiently in response to various stresses, but the underpinning mechanisms have remained elusive. To define GSR regulatory components we have exploited the Rapid Stress Response Element (RSRE), a previously established functional GSR motif, using Arabidopsis plants expressing a 4xRSRE::Luciferase (RSRE::LUC) reporter. Initially, we searched public microarray datasets and found an enrichment of RSRE in promoter sequences of stress genes. Next, we treated RSRE::LUC plants with wounding and a range of rapidly stress-inducible hormones and detected a robust LUC activity solely in response to wounding. Application of two Ca2+ burst inducers, flagellin22 (flg22) and oligogalacturonic acid, activated RSRE strongly and systemically, while the Ca2+ chelator EGTA significantly reduced wound induction of RSRE::LUC. In line with the signaling function of Ca2+ in transduction events leading to activation of RSRE, we examined role of CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATORs (CAMTAs) in RSRE induction. Transient expression assays displayed CAMTA3 induction of RSRE and not that of the mutated element mRSRE. Treatment of selected camta mutant lines integrated into RSRE::LUC parent plant, with wounding, flg22, and freezing, established a differential function of these CAMTAs in potentiating the activity of RSRE. Wound response studies using camta double mutants revealed cooperative function of CAMTAs2 and 4 with CAMTA 3 in the RSRE regulation. These studies provide insight into governing components of transduction events and reveal transcriptional modules that tune expression of a key GSR motif.

The general stress response (GSR) is an evolutionarily conserved rapid and transient transcriptional reprograming of genes central for transducing environmental signals into cellular responses, leading to metabolic and physiological readjustments to cope with prevailing conditions. Defining the regulatory components of the GSR will provide crucial insight into the design principles of early stressresponse modules and their role in orchestrating master regulators of adaptive responses. Overaccumulation of methylerythritol cyclodiphosphate (MEcPP), a bifunctional chemical entity serving as both a precursor of isoprenoids produced by the plastidial methylerythritol phosphate (MEP) pathway and a stress-specific retrograde signal, in ceh1 (constitutively expressing hydroperoxide lyase1)-mutant plants leads to large-scale transcriptional alterations. Bioinformatic analyses of microarray data in ceh1 plants established the overrepresentation of a stress-responsive cis element and key GSR marker, the rapid stress response element (RSRE), in the promoters of robustly induced genes. ceh1 plants carrying an established 4×RSRE:Luciferase reporter for monitoring the GSR support constitutive activation of the response in this mutant background. Genetics and pharmacological approaches confirmed the specificity of MEcPP in RSRE induction via the transcription factor CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3), in a calcium-dependent manner. Moreover, CAMTA3-dependent activation of IRE1a (inositol-requiring protein-1) and bZIP60 (basic leucine zipper 60), two RSRE containing unfolded protein-response genes, bridges MEcPP-mediated GSR induction to the potentiation of protein-folding homeostasis in the endoplasmic reticulum. These findings introduce the notion of transcriptional regulation by a key plastidial retrograde signaling metabolite that induces nuclear GSR, thereby offering a window into the role of interorgannellar communication in shaping cellular adaptive responses.S tress-triggered transcriptional reprogramming plays fundamental roles in transducing stress signals and ultimately enabling adaptive responses through readjustments of the appropriate physiological and metabolic processes. The initial transcriptional reprograming known as the "general stress response" (GSR), at times referred to as the "cellular stress response" or "core stress response," is a recognized evolutionarily conserved stress response present across kingdoms (1-5).The GSR, a rapid and transient transcriptional reprogramming, is induced by a wide variety of stresses imposed upon organisms by environmental forces on macromolecules such as membrane lipids, proteins, and/or DNA (6). Bioinformatic analysis of the promoters of the rapid wound-response genes (5 min after mechanical damage) in plants led to the identification of an overrepresented functional cis-element, the rapid stress response element (RSRE), which is analogous to the yeast stress response element (STRE) (4, 7). A reporter line containing luciferase (LUC) driven by a synthet...

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.

SummaryTargeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.

Synthetic promoters, introduced stably or transiently into plants, are an invaluable tool for the identification of functional regulatory elements and the corresponding transcription factor(s) that regulate the amplitude, spatial distribution, and temporal patterns of gene expression. Here, we present a protocol describing the steps required to identify and characterize putative cis-regulatory elements. These steps include application of computational tools to identify putative elements, construction of a synthetic promoter upstream of luciferase, identification of transcription factors that regulate the element, testing the functionality of the element introduced transiently and/or stably into the species of interest followed by high-throughput luciferase screening assays, and subsequent data processing and statistical analysis.