Katayoon Dehesh

Retrograde Signaling by the Plastidial Metabolite MEcPP Regulates Expression of Nuclear Stress-Response Genes

Plastid-derived signals are known to coordinate expression of nuclear genes encoding plastid-localized proteins in a process termed retrograde signaling. To date, the identity of retrograde-signaling molecules has remained elusive. Here, we show that methylerythritol cyclodiphosphate (MEcPP), a precursor of isoprenoids produced by the plastidial methylerythritol phosphate (MEP) pathway, elicits the expression of selected stress-responsive nuclear-encoded plastidial proteins. Genetic and pharmacological manipulations of the individual MEP pathway metabolite levels demonstrate the high specificity of MEcPP as an inducer of these targeted stress-responsive genes. We further demonstrate that abiotic stresses elevate MEcPP levels, eliciting the expression of the aforementioned genes. We propose that the MEP pathway, in addition to producing isoprenoids, functions as a stress sensor and a coordinator of expression of targeted stress-responsive nuclear genes via modulation of the levels of MEcPP, a specific and critical retrograde-signaling metabolite.

An Arabidopsis gene regulatory network for secondary cell wall synthesis

The 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 thaliana 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.

Mechanical Stress Induces Biotic and Abiotic Stress Responses via a Novel cis-Element

Plants are continuously exposed to a myriad of abiotic and biotic stresses. However, the molecular mechanisms by which these stress signals are perceived and transduced are poorly understood. To begin to identify primary stress signal transduction components, we have focused on genes that respond rapidly (within 5 min) to stress signals. Because it has been hypothesized that detection of physical stress is a mechanism common to mounting a response against a broad range of environmental stresses, we have utilized mechanical wounding as the stress stimulus and performed whole genome microarray analysis of Arabidopsis thaliana leaf tissue. This led to the identification of a number of rapid wound responsive (RWR) genes. Comparison of RWR genes with published abiotic and biotic stress microarray datasets demonstrates a large overlap across a wide range of environmental stresses. Interestingly, RWR genes also exhibit a striking level and pattern of circadian regulation, with induced and repressed genes displaying antiphasic rhythms. Using bioinformatic analysis, we identified a novel motif overrepresented in the promoters of RWR genes, herein designated as the Rapid Stress Response Element (RSRE). We demonstrate in transgenic plants that multimerized RSREs are sufficient to confer a rapid response to both biotic and abiotic stresses in vivo, thereby establishing the functional involvement of this motif in primary transcriptional stress responses. Collectively, our data provide evidence for a novel cis-element that is distributed across the promoters of an array of diverse stress-responsive genes, poised to respond immediately and coordinately to stress signals. This structure suggests that plants may have a transcriptional network resembling the general stress signaling pathway in yeast and that the RSRE element may provide the key to this coordinate regulation.

phyB is evolutionarily conserved and constitutively expressed in rice seedling shoots.

SummarySouthern blot analysis indicates that the rice genome contains single copies of genes encoding type A (phyA) and type B (phyB) phytochromes. We have isolated overlapping cDNA and genomic clones encoding the entire phyB polypeptide. This monocot sequence is more closely related to phyB from the dicot, Arabidopsis (73% amino acid sequence identity), than it is to the phyA gene in the rice genome (50% identity). These data support the proposal that phyA and phyB subfamilies diverged early in plant evolution and that subsequent divergence accompanied the evolution of monocots and dicots. Moreover, since rice and Arabidopsis phyB polypeptides are more closely related to one another (73% identity) than are monocot and dicot phyA sequences (63–65% identity), it appears that phyB has evolved more slowly than phyA. Sequence conservation between phyA and phyB is greatest in a central core region surrounding the chromophore attachment site, and least toward the amino-terminal and carboxy-terminal ends of the polypeptides, although hydropathy analysis suggests that the overall structure of the two phytochromes has been conserved. Gene-specific Northern blot analysis indicates that, whereas phyA is negatively regulated by phytochrome in rice seedling shoots in the manner typical of monocots, phyB is constitutively expressed irrespective of light treatment. In consequence, phyA and phyB transcripts are equally abundant in fully green tissue. Since Arabidopsis phyB mRNA levels are also unaffected by light, the present results suggest that this mode of regulation is evolutionarily conserved among phyB genes, perhaps reflecting differences in the functional roles of the different phytochrome subfamilies.

Distinct Roles of Jasmonates and Aldehydes in Plant-Defense Responses

Background Many inducible plant-defense responses are activated by jasmonates (JAs), C6-aldehydes, and their corresponding derivatives, produced by the two main competing branches of the oxylipin pathway, the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches, respectively. In addition to competition for substrates, these branch-pathway-derived metabolites have substantial overlap in regulation of gene expression. Past experiments to define the role of C6-aldehydes in plant defense responses were biased towards the exogenous application of the synthetic metabolites or the use of genetic manipulation of HPL expression levels in plant genotypes with intact ability to produce the competing AOS-derived metabolites. To uncouple the roles of the C6-aldehydes and jasmonates in mediating direct and indirect plant-defense responses, we generated Arabidopsis genotypes lacking either one or both of these metabolites. These genotypes were subsequently challenged with a phloem-feeding insect (aphids: Myzus persicae), an insect herbivore (leafminers: Liriomyza trifolii), and two different necrotrophic fungal pathogens (Botrytis cinerea and Alternaria brassicicola). We also characterized the volatiles emitted by these plants upon aphid infestation or mechanical wounding and identified hexenyl acetate as the predominant compound in these volatile blends. Subsequently, we examined the signaling role of this compound in attracting the parasitoid wasp (Aphidius colemani), a natural enemy of aphids. Principal Findings This study conclusively establishes that jasmonates and C6-aldehydes play distinct roles in plant defense responses. The jasmonates are indispensable metabolites in mediating the activation of direct plant-defense responses, whereas the C6-aldehyes are not. On the other hand, hexenyl acetate, an acetylated C6-aldehyde, is the predominant wound-inducible volatile signal that mediates indirect defense responses by directing tritrophic (plant-herbivore-natural enemy) interactions. Significance The data suggest that jasmonates and hexenyl acetate play distinct roles in mediating direct and indirect plant-defense responses. The potential advantage of this “division of labor” is to ensure the most effective defense strategy that minimizes incurred damages at a reduced metabolic cost.

The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

Organisms are continuously exposed to a myriad of environmental stresses. Central to an organisms survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.

Arachidonic Acid: An Evolutionarily Conserved Signaling Molecule Modulates Plant Stress Signaling Networks

Oomycete pathogens contain arachidonic acid (AA), an elicitor of defense responses and programmed cell death in plants. Arabidopsis plants engineered to produce AA, or exogenously treated with this fatty acid, displayed altered resistance to biotic challengers resulting from AA’s action on salicylate and jasmonate stress signaling networks. Fatty acid structure affects cellular activities through changes in membrane lipid composition and the generation of a diversity of bioactive derivatives. Eicosapolyenoic acids are released into plants upon infection by oomycete pathogens, suggesting they may elicit plant defenses. We exploited transgenic Arabidopsis thaliana plants (designated EP) producing eicosadienoic, eicosatrienoic, and arachidonic acid (AA), aimed at mimicking pathogen release of these compounds. We also examined their effect on biotic stress resistance by challenging EP plants with fungal, oomycete, and bacterial pathogens and an insect pest. EP plants exhibited enhanced resistance to all biotic challenges, except they were more susceptible to bacteria than the wild type. Levels of jasmonic acid (JA) were elevated and levels of salicylic acid (SA) were reduced in EP plants. Altered expression of JA and SA pathway genes in EP plants shows that eicosapolyenoic acids effectively modulate stress-responsive transcriptional networks. Exogenous application of various fatty acids to wild-type and JA-deficient mutants confirmed AA as the signaling molecule. Moreover, AA treatment elicited heightened expression of general stress-responsive genes. Importantly, tomato (Solanum lycopersicum) leaves treated with AA exhibited reduced susceptibility to Botrytis cinerea infection, confirming AA signaling in other plants. These studies support the role of AA, an ancient metazoan signaling molecule, in eliciting plant stress and defense signaling networks.

Deficiencies in jasmonate-mediated plant defense reveal quantitative variation in Botrytis cinerea pathogenesis.

Despite the described central role of jasmonate signaling in plant defense against necrotrophic pathogens, the existence of intraspecific variation in pathogen capacity to activate or evade plant jasmonate-mediated defenses is rarely considered. Experimental infection of jasmonate-deficient and jasmonate-insensitive Arabidopsis thaliana with diverse isolates of the necrotrophic fungal pathogen Botrytis cinerea revealed pathogen variation for virulence inhibition by jasmonate-mediated plant defenses and induction of plant defense metabolites. Comparison of the transcriptional effects of infection by two distinct B. cinerea isolates showed only minor differences in transcriptional responses of wild-type plants, but notable isolate-specific transcript differences in jasmonate-insensitive plants. These transcriptional differences suggest B. cinerea activation of plant defenses that require plant jasmonate signaling for activity in response to only one of the two B. cinerea isolates tested. Thus, similar infection phenotypes observed in wild-type plants result from different signaling interactions with the plant that are likely integrated by jasmonate signaling.

The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.

Abstract. The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80–90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available.

Functional Convergence of Oxylipin and Abscisic Acid Pathways Controls Stomatal Closure in Response to Drought

Oxylipin affects stomatal closure, functioning most effectively with ABA in response to drought. 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.