SummaryThe plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here, we present a protein-DNA network between Arabidopsis transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.

Membranes are primary sites of perception of environmental stimuli. Polyunsaturated fatty acids are major structural constituents of membranes that also function as modulators of a multitude of signal transduction pathways evoked by environmental stimuli. Different stresses induce production of a distinct blend of oxygenated polyunsaturated fatty acids, "oxylipins." We employed three Arabidopsis (Arabidopsis thaliana) ecotypes to examine the oxylipin signature in response to specific stresses and determined that wounding and drought differentially alter oxylipin profiles, particularly the allene oxide synthase branch of the oxylipin pathway, responsible for production of jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (12-OPDA). Specifically, wounding induced both 12-OPDA and JA levels, whereas drought induced only the precursor 12-OPDA. Levels of the classical stress phytohormone abscisic acid (ABA) were also mainly enhanced by drought and little by wounding. To explore the role of 12-OPDA in plant drought responses, we generated a range of transgenic lines and exploited the existing mutant plants that differ in their levels of stress-inducible 12-OPDA but display similar ABA levels. The plants producing higher 12-OPDA levels exhibited enhanced drought tolerance and reduced stomatal aperture. Furthermore, exogenously applied ABA and 12-OPDA, individually or combined, promote stomatal closure of ABA and allene oxide synthase biosynthetic mutants, albeit most effectively when combined. Using tomato (Solanum lycopersicum) and Brassica napus verified the potency of this combination in inducing stomatal closure in plants other than Arabidopsis. These data have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed 12-OPDA as a drought-responsive regulator of stomatal closure functioning most effectively together with ABA.

The timely transition of vegetative to reproductive development is coordinated through quantitative regulation of floral pathway genes in response to physiological and environmental cues. Here, we show that the circadian-controlled expression of the Arabidopsis thaliana floral transition regulators FLOWERING LOCUS T (FT) and CONSTANS (CO) is antiphasic to that of BBX19, a transcription factor with two B-Box motifs. Diminished expression of BBX19 by RNA interference accelerates flowering, and constitutive expression of BBX19 delays flowering under inductive photoperiods. This delay is not accompanied by the alteration of CO expression levels but rather by a reduction of transcript levels of FT and the FT-regulated genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1, LEAFY, and FRUITFUL. Similar to CO, BBX19 is expressed in vasculature. BBX19 and CO colocalize in the nucleus and interact physically in vivo. In transient assays, coinfiltration of 10-fold more CO overcomes the BBX19-mediated repression of FT activation. Substitution of the conserved Cys-25 to Ser in the BBX19 Box1 motif abolishes the BBX19-CO interaction and eliminates the negative regulation of flowering time, while the analogous C76S substitution in the Box2 motif is ineffective. Together, these results implicate BBX19 as a circadian clock output that depletes the active CO pool to accurately monitor daylength and precisely time FT expression.

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.

Hypocotyl elongation is a highly coordinated physiological response regulated by myriad internal and external cues. Here, we show that BBX19, a transcriptional regulator with two B-box motifs, is a positive regulator of growth; diminished BBX19 expression by RNA interference reduces hypocotyl length, and its constitutive expression promotes growth. This function of BBX19 is dependent on the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), EARLY FLOWERING3 (ELF3), and PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5. BBX19 is nucleus-colocalized and interacts physically with COP1 and ELF3, a component of the evening complex that represses the expression of PIF4 and PIF5. Moreover, ELF3 protein abundance inversely correlates with BBX19 expression levels in a COP1-dependent manner. By contrast, PIF expression, coinciding with the initiation of hypocotyl growth in the early evening, is positively correlated with the BBX19 transcript abundance. These results collectively establish BBX19 as an adaptor that binds to and recruits ELF3 for degradation by COP1 and, as such, dynamically gates the formation of the evening complex, resulting in derepression of PIF4/5. This finding refines our perspective on how plants grow by providing a molecular link between COP1, ELF3, and PIF4/5 as an underlying mechanism of photomorphogenic development in Arabidopsis thaliana.