Tatyana Savchenko

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.

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.

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.

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.

Intronic T-DNA Insertion Renders Arabidopsis opr3 a Conditional Jasmonic Acid-Producing Mutant

Jasmonic acid and its derived metabolites (JAs) orchestrate plant defense against insects and fungi. 12-Oxo-phytodienoic acid (OPDA), a JA precursor, has also been implicated in plant defense. We sought to define JAs and OPDA functions through comparative defense susceptibility characteristics of three Arabidopsis (Arabidopsis thaliana) genotypes: aos, lacking JAs and OPDA; opda reductase3 (opr3), deficient in JA production but can accumulate OPDA; and transgenics that overexpress OPR3. opr3, like aos, is susceptible to cabbage loopers (Trichoplusia ni) but, relative to aos, opr3 has enhanced resistance to a necrotrophic fungus. Gas chromatography-mass spectrometry reveals that opr3 produces OPDA but no detectable JAs following wounding and looper infestation; unexpectedly, substantial levels of JAs accumulate in opr3 upon fungal infection. Full-length OPR3 transcripts accumulate in fungal-infected opr3, potentially through splicing of the T-DNA containing intron. Fungal resistance correlates with levels of JAs not OPDA; therefore, opr3 resistance to some pests is likely due to JA accumulation, and signaling activities ascribed to OPDA should be reassessed because opr3 can produce JAs. Together these data (1) reinforce the primary role JAs play in plant defense against insects and necrotrophic fungi, (2) argue for a reassessment of signaling activities ascribed to OPDA, and (3) provide evidence that mutants with intron insertions can retain gene function.

Genome-Wide Expression Profiling Arabidopsis at the Stage of Golovinomyces cichoracearum Haustorium Formation

Compatibility between plants and obligate biotrophic fungi requires fungal mechanisms for efficiently obtaining nutrients and counteracting plant defenses under conditions that are expected to induce changes in the host transcriptome. A key step in the proliferation of biotrophic fungi is haustorium differentiation. Here we analyzed global gene expression patterns in Arabidopsis thaliana leaves during the formation of haustoria by Golovinomyces cichoracearum. At this time, the endogenous levels of salicylic acid (SA) and jasmonic acid (JA) were found to be enhanced. The responses of wild-type, npr1-1, and jar1-1 plants were used to categorize the sensitivity of gene expression changes to NPR1 and JAR1, which are components of the SA and JA signaling pathways, respectively. We found that the infection process was the major source of variation, with 70 genes identified as having similarly altered expression patterns regardless of plant genotype. In addition, principal component analysis (PCA) identified genes responding both to infection and to lack of functional JAR1 (17 genes) or NPR1 (18 genes), indicating that the JA and SA signaling pathways function as secondary sources of variation. Participation of these genes in the SA or JA pathways had not been described previously. We found that some of these genes may be sensitive to the balance between the SA and JA pathways, representing novel markers for the elucidation of cross-talk points between these signaling cascades. Conserved putative regulatory motifs were found in the promoter regions of each subset of genes. Collectively, our results indicate that gene expression changes in response to infection by obligate biotrophic fungi may support fungal nutrition by promoting alterations in host metabolism. In addition, these studies provide novel markers for the characterization of defense pathways and susceptibility features under this infection condition.

Genome-wide comparative analysis of the IQD gene families in Arabidopsis thaliana and Oryza sativa

BackgroundCalcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.ResultsWe identified and analyzed 33 and 29 IQD1-like genes in Arabidopsis thaliana and Oryza sativa, respectively. The encoded IQD proteins contain a plant-specific domain of 67 conserved amino acid residues, referred to as the IQ67 domain, which is characterized by a unique and repetitive arrangement of three different calmodulin recruitment motifs, known as the IQ, 1-5-10, and 1-8-14 motifs. We demonstrated calmodulin binding for IQD20, the smallest IQD protein in Arabidopsis, which consists of a C-terminal IQ67 domain and a short N-terminal extension. A striking feature of IQD proteins is the high isoelectric point (~10.3) and frequency of serine residues (~11%). We compared the Arabidopsis and rice IQD gene families in terms of gene structure, chromosome location, predicted protein properties and motifs, phylogenetic relationships, and evolutionary history. The existence of an IQD-like gene in bryophytes suggests that IQD proteins are an ancient family of calmodulin-binding proteins and arose during the early evolution of land plants.ConclusionComparative phylogenetic analyses indicate that the major IQD gene lineages originated before the monocot-eudicot divergence. The extant IQD loci in Arabidopsis primarily resulted from segmental duplication and reflect preferential retention of paralogous genes, which is characteristic for proteins with regulatory functions. Interaction of IQD1 and IQD20 with calmodulin and the presence of predicted calmodulin binding sites in all IQD family members suggest that IQD proteins are a new class of calmodulin targets. The basic isoelectric point of IQD proteins and their frequently predicted nuclear localization suggest that IQD proteins link calcium signaling pathways to the regulation of gene expression. Our comparative genomics analysis of IQD genes and encoded proteins in two model plant species provides the first step towards the functional dissection of this emerging family of putative calmodulin targets.

Insect herbivores selectively suppress the HPL branch of the oxylipin pathway in host plants

Insect herbivores have developed a myriad of strategies to manipulate the defense responses of their host plants. Here we provide evidence that chewing insects differentially alter the oxylipin profiles produced by the two main and competing branches of the plant defensive response pathway, the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) branches, which are responsible for wound-inducible production of jasmonates (JAs), and green leafy volatiles (GLVs) respectively. Specifically, we used three Arabidopsis genotypes that were damaged by mechanical wounding or by insects of various feeding guilds (piercing aphids, generalist chewing caterpillars and specialist chewing caterpillars). We established that emission of GLVs is stimulated by wounding incurred mechanically or by aphids, but release of these volatiles is constitutively impaired by both generalist and specialist chewing insects. Simultaneously, however, these chewing herbivores stimulated JA production, demonstrating targeted insect suppression of the HPL branch of the oxylipin pathway. Use of lines engineered to express HPL constitutively, in conjunction with quantitative RT-PCR-based expression analyses, established a combination of transcriptional and post-transcriptional reprogramming of the HPL pathway genes as the mechanistic basis of insect-mediated suppression of the corresponding metabolites. Feeding studies suggested a potential evolutionary advantage of suppressing GLV production, as caterpillars preferably consumed leaf tissue from plants that had not been primed by these volatile cues.

Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Background: Plant-specific IQD genes encode putative CaM targets of unknown functions. Results: IQD1 interacts with KLCR1, binds to Arabidopsis CaM/CMLs, and localizes to microtubules. Conclusion: IQD1 may act as a scaffold protein recruiting cargo to kinesin motors for directional transport along microtubules. Significance: This work provides novel insight into IQD function and a framework to study plant kinesin regulation. Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.

Drought stress modulates oxylipin signature by eliciting 12-OPDA as a potent regulator of stomatal aperture

Through evolution, plants have developed a myriad of strategies to adapt to environmental perturbations. Using 3 Arabidopsis ecotypes in conjunction with various transgenic and mutant lines, we provide evidence that wounding and drought differentially alter the metabolic signatures derived from the 2 main competing oxylipin-pathway branches, namely the JA and its precursor 12-OPDA produced by Allene oxide synthase (AOS) branch, and aldehydes and corresponding alcohols generated by Hydroperoxide lyase (HPL) branch. Specifically, we show that wounding induces production of both HPL and AOS-derived metabolites whereas, drought stress only elicits production of hexenal but suppresses hexenol, and further uncouples the conversion of 12-OPDA to JA. This finding led to uncovering of 12-OPDA as a functional convergence point of oxylipin and ABA pathways to control stomatal aperture in plant adaptive responses to drought. In addition, using transgenic lines overexpressing plastidial and extraplastidial HPL enzyme establish the strong interdependence of AOS- and HPL-branch pathways, and the importance of this linkage in tailoring plant adaptive responses to the nature of perturbations.