Diversification of JAZ-MYC signaling function in immune metabolism.

Jasmonate (JA) re-programs metabolism to confer resistance to diverse environmental threats. Jasmonate stimulates the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of MYC transcription factors. In Arabidopsis thaliana, MYC and JAZ are encoded by 4 and 13 genes, respectively. The extent to which expansion of the MYC and JAZ families has contributed to functional diversification of JA responses is not well understood. Here, we investigated the role of MYC and JAZ paralogs in controlling the production of defense compounds derived from aromatic amino acids (AAAs). Analysis of loss-of-function and dominant myc mutations identified MYC3 and MYC4 as the major regulators of JA-induced tryptophan metabolism. We developed a JAZ family-based, forward genetics approach to screen randomized jaz polymutants for allelic combinations that enhance tryptophan biosynthetic capacity. We found that mutants defective in all members (JAZ1/2/5/6) of JAZ group I over-accumulate AAA-derived defense compounds, constitutively express marker genes for the JA-ethylene branch of immunity and are more resistant to necrotrophic pathogens but not insect herbivores. In defining JAZ and MYC paralogs that regulate the production of amino-acid-derived defense compounds, our results provide insight into the specificity of JA signaling in immunity.

MYC transcription factors coordinate tryptophan-dependent defence responses and compromise seed yield in Arabidopsis.

Robust plant immunity negatively affects other fitness traits, including growth and seed production. Jasmonate (JA) confers broad-spectrum protection against plant consumers by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which in turn relieves repression on transcription factors (TFs) coincident with reduced growth and fecundity. The molecular mechanisms underlying JA-mediated decreases in fitness remain largely unknown. To assess the contribution of MYC TFs to growth and reproductive fitness at high levels of defence, we mutated three MYC genes in a JAZ-deficient mutant (jazD) of Arabidopsis thaliana that exhibits strong defence and low seed yield. Genetic epistasis analysis showed that de-repression of MYC TFs in jazD not only conferred strong resistance to insect herbivory but also reduced shoot and root growth, fruit size and seed yield. We also provided evidence that the JAZ-MYC module coordinates the supply of tryptophan with the production of indole glucosinolates and the proliferation of endoplasmic reticulum bodies that metabolise glucosinolates through the action of ß-glucosidases. Our results establish MYCs as major regulators of growth- and reproductive-defence trade-offs and further indicate that these factors coordinate tryptophan availability with the production of amino acid-derived defence compounds.

A Phytochrome B-Independent Pathway Restricts Growth at High Levels of Jasmonate Defense.

The plant hormone jasmonate (JA) promotes resistance to biotic stress by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which relieves repression on MYC transcription factors that execute defense programs. JA-triggered depletion of JAZ proteins in Arabidopsis (Arabidopsis thaliana) is also associated with reduced growth and seed production, but the mechanisms underlying these pleiotropic growth effects remain unclear. Here, we investigated this question using an Arabidopsis JAZ-deficient mutant (jazD; jaz1-jaz7, jaz9, jaz10, and jaz 13) that exhibits high levels of defense and strong growth inhibition. Genetic suppressor screens for mutations that uncouple growth-defense tradeoffs in the jazD mutant identified nine independent causal mutations in the red-light receptor phytochrome B (phyB). Unlike the ability of the phyB mutations to completely uncouple the mild growth-defense phenotypes in a jaz mutant (jazQ) defective in JAZ1, JAZ3, JAZ4, JAZ9, and JAZ10, phyB null alleles only weakly alleviated the growth and reproductive defects in the jazD mutant. phyB-independent growth restriction of the jazD mutant was tightly correlated with upregulation of the Trp biosynthetic pathway but not with changes in central carbon metabolism. Interestingly, jazD and jazD phyB plants were insensitive to a chemical inhibitor of Trp biosynthesis, which is a phenotype previously observed in plants expressing hyperactive MYC transcription factors that cannot bind JAZ repressors. These data provide evidence that the mechanisms underlying JA-mediated growth-defense balance depend on the level of defense, and they further establish an association between growth inhibition at high levels of defense and dysregulation of Trp biosynthesis.

JAZ repressors of metabolic defense promote growth and reproductive fitness in Arabidopsis.

Plant immune responses mediated by the hormone jasmonoyl-l-isoleucine (JA-Ile) are metabolically costly and often linked to reduced growth. Although it is known that JA-Ile activates defense responses by triggering the degradation of JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor proteins, expansion of the JAZ gene family in vascular plants has hampered efforts to understand how this hormone impacts growth and other physiological tasks over the course of ontogeny. Here, we combined mutations within the 13-member Arabidopsis JAZ gene family to investigate the effects of chronic JAZ deficiency on growth, defense, and reproductive output. A higher-order mutant (jaz decuple, jazD) defective in 10 JAZ genes (JAZ1-7, -9, -10, and -13) exhibited robust resistance to insect herbivores and fungal pathogens, which was accompanied by slow vegetative growth and poor reproductive performance. Metabolic phenotypes of jazD discerned from global transcript and protein profiling were indicative of elevated carbon partitioning to amino acid-, protein-, and endoplasmic reticulum body-based defenses controlled by the JA-Ile and ethylene branches of immunity. Resource allocation to a strong defense sink in jazD leaves was associated with increased respiration and hallmarks of carbon starvation but no overt changes in photosynthetic rate. Depletion of the remaining JAZ repressors in jazD further exaggerated growth stunting, nearly abolished seed production and, under extreme conditions, caused spreading necrotic lesions and tissue death. Our results demonstrate that JAZ proteins promote growth and reproductive success at least in part by preventing catastrophic metabolic effects of an unrestrained immune response.

Determinants of nucleosome positioning and their influence on plant gene expression.

Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome-depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer-specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression level, a phenomenon also found in rice but not in yeast. Among the 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially preconditioned to enable rapid response without changing chromatin state significantly. Our study provides a global assessment of the joint contribution of nucleosome occupancy and motif sequences that are likely cis-elements to the control of gene expression in plants. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.

Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module.

The plant hormone jasmonate (JA) promotes the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins to relieve repression on diverse transcription factors (TFs) that execute JA responses. However, little is known about how combinatorial complexity among JAZ-TF interactions maintains control over myriad aspects of growth, development, reproduction, and immunity. We used loss-of-function mutations to define epistatic interactions within the core JA signaling pathway and to investigate the contribution of MYC TFs to JA responses in Arabidopsis thaliana. Constitutive JA signaling in a jaz quintuple mutant (jazQ) was largely eliminated by mutations that block JA synthesis or perception. Comparison of jazQ and a jazQ myc2 myc3 myc4 octuple mutant validated known functions of MYC2/3/4 in root growth, chlorophyll degradation, and susceptibility to the pathogen Pseudomonas syringae. We found that MYC TFs also control both the enhanced resistance of jazQ leaves to insect herbivory and restricted leaf growth of jazQ. Epistatic transcriptional profiles mirrored these phenotypes and further showed that triterpenoid biosynthetic and glucosinolate catabolic genes are up-regulated in jazQ independently of MYC TFs. Our study highlights the utility of genetic epistasis to unravel the complexities of JAZ-TF interactions and demonstrates that MYC TFs exert master control over a JAZ-repressible transcriptional hierarchy that governs growth-defense balance.

Temporal Dynamics of Growth and Photosynthesis Suppression in Response to Jasmonate Signaling.

Biotic stress constrains plant productivity in natural and agricultural ecosystems. Repression of photosynthetic genes is a conserved plant response to biotic attack, but how this transcriptional reprogramming is linked to changes in photosynthesis and the transition from growth- to defense-oriented metabolism is poorly understood. Here, we used a combination of noninvasive chlorophyll fluorescence imaging technology and RNA sequencing to determine the effect of the defense hormone jasmonate (JA) on the growth, photosynthetic efficiency, and gene expression of Arabidopsis (Arabidopsis thaliana) rosette leaves. High temporal resolution was achieved through treatment with coronatine (COR), a high-affinity agonist of the JA receptor. We show that leaf growth is rapidly arrested after COR treatment and that this effect is tightly correlated with changes in the expression of genes involved in growth, photosynthesis, and defense. Rapid COR-induced expression of defense genes occurred concomitantly with the repression of photosynthetic genes but was not associated with a reduced quantum efficiency of photosystem II. These findings support the view that photosynthetic capacity is maintained during the period in which stress-induced JA signaling redirects metabolism from growth to defense. Chlorophyll fluorescence images captured in a multiscale time series, however, revealed a transient COR-induced decrease in quantum efficiency of photosystem II at dawn of the day after treatment. Physiological studies suggest that this response results from delayed stomatal opening at the night-day transition. These collective results establish a high-resolution temporal view of how a major stress response pathway modulates plant growth and photosynthesis and highlight the utility of chlorophyll fluorescence imaging for revealing transient stress-induced perturbations in photosynthetic performance.

Influence of Genotype, Environment, and Gypsy Moth Herbivory on Local and Systemic Chemical Defenses in Trembling Aspen (Populus tremuloides).

Numerous studies have explored the impacts of intraspecific genetic variation and environment on the induction of plant chemical defenses by herbivory. Relatively few, however, have considered how those factors affect within-plant distribution of induced defenses. This work examined the impacts of plant genotype and soil nutrients on the local and systemic phytochemical responses of trembling aspen (Populus tremuloides) to defoliation by gypsy moth (Lymantria dispar). We deployed larvae onto foliage on individual tree branches for 15 days and then measured chemistry in leaves from: 1) branches receiving damage, 2) undamaged branches of insect-damaged trees, and 3) branches of undamaged control trees. The relationship between post-herbivory phytochemical variation and insect performance also was examined. Plant genotype, soil nutrients, and damage all influenced phytochemistry, with genotype and soil nutrients being stronger determinants than damage. Generally, insect damage decreased foliar nitrogen, increased levels of salicinoids and condensed tannins, but had little effect on levels of a Kunitz trypsin inhibitor, TI3. The largest damage-mediated tannin increases occurred in leaves on branches receiving damage, whereas the largest salicinoid increases occurred in leaves of adjacent, undamaged branches. Foliar nitrogen and the salicinoid tremulacin had the strongest positive and negative relationships, respectively, with insect growth. Overall, plant genetics and environment concomitantly influenced both local and systemic phytochemical responses to herbivory. These findings suggest that herbivory can contribute to phytochemical heterogeneity in aspen foliage, which may in turn influence future patterns of herbivory and nutrient cycling over larger spatial scales.

Optimal timepoint sampling in high-throughput gene expression experiments.

MOTIVATION: Determining the best sampling rates (which maximize information yield and minimize cost) for time-series high-throughput gene expression experiments is a challenging optimization problem. Although existing approaches provide insight into the design of optimal sampling rates, our ability to utilize existing differential gene expression data to discover optimal timepoints is compelling. RESULTS: We present a new data-integrative model, Optimal Timepoint Selection (OTS), to address the sampling rate problem. Three experiments were run on two different datasets in order to test the performance of OTS, including iterative-online and a top-up sampling approaches. In all of the experiments, OTS outperformed the best existing timepoint selection approaches, suggesting that it can optimize the distribution of a limited number of timepoints, potentially leading to better biological insights about the resulting gene expression patterns. AVAILABILITY: OTS is available at www.msu.edu/∼jinchen/OTS.

Stress-responsive mitogen-activated protein kinases interact with the EAR motif of a poplar zinc finger protein and mediate its degradation through the 26S proteasome.

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

Photosynthetic and respiratory changes in leaves of poplar elicited by rust infection.

Poplars are challenged by a wide range of pathogens during their lifespan, and have an innate immunity system that activates defence responses to restrict pathogen growth. Large-scale expression studies of poplar-rust interactions have shown concerted transcriptional changes during defence responses, as in other plant pathosystems. Detailed analysis of expression profiles of metabolic pathways in these studies indicates that photosynthesis and respiration are also important components of the poplar response to rust infection. This is consistent with our current understanding of plant pathogen interactions as defence responses impose substantive demands for resources and energy that are met by reorganization of primary metabolism. This review applies the results of poplar transcriptome analyses to current research describing how plants divert energy from plant primary metabolism for resistance mechanisms.

Resolution of growth-defense conflict: mechanistic insights from jasmonate signaling.

Induced plant resistance depends on the production of specialized metabolites that repel attack by biotic aggressors and is often associated with reduced growth of vegetative tissues. Despite progress in understanding the signal transduction networks that control growth-defense tradeoffs, much remains to be learned about how growth rate is coordinated with changes in metabolism during growth-to-defense transitions. Here, we highlight recent advances in jasmonate research to suggest how a major branch of plant immunity is dynamically regulated to calibrate growth-defense balance with shifts in carbon availability. We review evidence that diminished growth, as an integral facet of induced resistance, may optimize the temporal and spatial expression of defense compounds without compromising other critical roles of central metabolism. New insights into the evolution of jasmonate signaling further suggest that opposing selective pressures associated with too much or too little defense may have shaped the emergence of a modular jasmonate pathway that integrates primary and specialized metabolism through the control of repressor-transcription factor complexes. A better understanding of the mechanistic basis of growth-defense balance has important implications for boosting plant productivity, including insights into how these tradeoffs may be uncoupled for agricultural improvement.

Modularity in Jasmonate Signaling for Multistress Resilience.

The plant hormone jasmonate coordinates immune and growth responses to increase plant survival in unpredictable environments. The core jasmonate signaling pathway comprises several functional modules, including a repertoire of COI1-JAZ (CORONATINE INSENSITIVE1-JASMONATE-ZIM DOMAIN) coreceptors that couple jasmonoyl-l-isoleucine perception to the degradation of JAZ repressors, JAZ-interacting transcription factors that execute physiological responses, and multiple negative feedback loops to ensure timely termination of these responses. Here, we review the jasmonate signaling pathway with an emphasis on understanding how transcriptional responses are specific, tunable, and evolvable. We explore emerging evidence that JAZ proteins integrate multiple informational cues and mediate crosstalk by propagating changes in protein-protein interaction networks. We also discuss recent insights into the evolution of jasmonate signaling and highlight how plant-associated organisms manipulate the pathway to subvert host immunity. Finally, we consider how this mechanistic foundation can accelerate the rational design of jasmonate signaling for improving crop resilience and harnessing the wellspring of specialized plant metabolites.

Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth-Defense Tradeoffs.

Plant growth is often constrained by the limited availability of resources in the microenvironment. Despite the continuous threat of attack from insect herbivores and pathogens, investment in defense represents a lost opportunity to expand photosynthetic capacity in leaves and absorption of nutrients and water by roots. To mitigate the metabolic expenditure on defense, plants have evolved inducible defense strategies. The plant hormone jasmonate (JA) is a key regulator of many inducible defenses. Synthesis of JA in response to perceived danger leads to the deployment of a variety of defensive structures and compounds, along with a potent inhibition of growth. Genetic studies have established an important role for JA in mediating tradeoffs between growth and defense. However, several gaps remain in understanding of how JA signaling inhibits growth, either through direct transcriptional control of JA-response genes or crosstalk with other signaling pathways. Here, we highlight recent progress in uncovering the role of JA in controlling growth-defense balance and its relationship to resource acquisition and allocation. We also discuss tradeoffs in the context of the ability of JA to promote increased leaf mass per area (LMA), which is a key indicator of leaf construction costs and leaf life span.

Rewiring of jasmonate and phytochrome B signalling uncouples plant growth-defense tradeoffs.

Plants resist infection and herbivory with innate immune responses that are often associated with reduced growth. Despite the importance of growth-defense tradeoffs in shaping plant productivity in natural and agricultural ecosystems, the molecular mechanisms that link growth and immunity are poorly understood. Here, we demonstrate that growth-defense tradeoffs mediated by the hormone jasmonate are uncoupled in an Arabidopsis mutant (jazQ phyB) lacking a quintet of Jasmonate ZIM-domain transcriptional repressors and the photoreceptor phyB. Analysis of epistatic interactions between jazQ and phyB reveal that growth inhibition associated with enhanced anti-insect resistance is likely not caused by diversion of photoassimilates from growth to defense but rather by a conserved transcriptional network that is hardwired to attenuate growth upon activation of jasmonate signalling. The ability to unlock growth-defense tradeoffs through relief of transcription repression provides an approach to assemble functional plant traits in new and potentially useful ways.

Induction of acid phosphatase transcripts, protein and enzymatic activity by simulated herbivory of hybrid poplar.

Herbivory and wounding upregulate a large suite of defense genes in hybrid poplar leaves. A strongly wound- and herbivore-induced gene with high similarity to Arabidopsis vegetative storage proteins (VSPs) and acid phosphatase (AP) was identified among genes strongly expressed during the poplar herbivore defense response. Phylogenetic analysis showed that the putative poplar acid phosphatase (PtdAP1) gene is part of an eight-member AP gene family in poplar, and is most closely related to a functionally characterized soybean nodule AP. Unlike the other poplar APs, PtdAP1 is expressed in variety of tissues, as observed in an analysis of EST data. Following wounding, the gene shows an expression profile similar to other known poplar defense genes such as protease inhibitors, chitinase, and polyphenol oxidase. Significantly, we show for the first time that subsequent to the wound-induction of PtdAP1 transcripts, AP protein and activity increase in extracts of leaves and other tissues. Although its mechanism of action is as yet unknown, these results suggest in hybrid poplar PtdAP1 is likely a component of the defense response against leaf-eating herbivores.

Functional analysis of the Kunitz trypsin inhibitor family in poplar reveals biochemical diversity and multiplicity in defense against herbivores.

We investigated the functional and biochemical variability of Kunitz trypsin inhibitor (KTI) genes of Populus trichocarpa x Populus deltoides. Phylogenetic analysis, expressed sequence tag databases, and western-blot analysis confirmed that these genes belong to a large and diverse gene family with complex expression patterns. Five wound- and herbivore-induced genes representing the diversity of the KTI gene family were selected for functional analysis and shown to produce active KTI proteins in Escherichia coli. These recombinant KTI proteins were all biochemically distinct and showed clear differences in efficacy against trypsin-, chymotrypsin-, and elastase-type proteases, suggesting functional specialization of different members of this gene family. The in vitro stability of the KTIs in the presence of reducing agents and elevated temperature also varied widely, emphasizing the biochemical differences of these proteins. Significantly, the properties of the recombinant KTI proteins were not predictable from primary amino acid sequence data. Proteases in midgut extracts of Malacosoma disstria, a lepidopteran pest of Populus, were strongly inhibited by at least two of the KTI gene products. This study suggests that the large diversity in the poplar (Populus spp.) KTI family is important for biochemical and functional specialization, which may be important in the maintenance of pest resistance in long-lived plants such as poplar.

Insect regurgitant and wounding elicit similar defense responses in poplar leaves: not something to spit at?

How plants perceive insect attacks is an area of active research. Numerous studies have shown that regurgitant from feeding insects elicits a defense response in plants, which is often assumed to be distinct from a wound response. We have characterized the inducible defense response in hybrid poplar and found it to be qualitatively similar between wounding and application of regurgitant from forest tent caterpillar. We suggest that this is likely attributable to our wounding treatment which is much more intense compared to most other studies. These overlapping responses appear to be activated via jasmonic acid signaling, and we speculate that they are both triggered by elicitors of plant origin. Wounding would release such elicitor molecules when leaf cells are disrupted, and regurgitant may contain them in a modified or processed form. This hypothesis could explain why some other necrosis-inducing stresses also induce herbivore defense genes.

Molecular analysis of poplar defense against herbivory: comparison of wound- and insect elicitor-induced gene expression.

In order to characterize defense responses of hybrid poplar (Populus trichocarpax P. deltoides), we profiled leaf transcript patterns elicited by wounding and by regurgitant from forest tent caterpillar (FTC; Malacosoma disstria), a Lepidopteran defoliator of poplars. Macroarrays were used to compare transcript profiles. Both FTC-regurgitant (FTC-R) and mechanical wounding with pliers elicited expression of a variety of genes, and for these genes our analysis indicated that these treatments induced qualitatively similar responses. Similarly, a comparison of responses of directly treated and systemically induced leaves indicated extensive overlap in the sets of induced genes. FTC-R was found to contain the insect-derived elicitor volicitin. The simulated herbivory treatments resulted in the induction of genes involved in poplar defense and secondary metabolism. We also identified wound-responsive genes with roles in primary metabolism, including a putative invertase, lipase, and acyl-activating enzyme; some of these genes may have roles in defense signaling. In addition, we found three unknown genes containing a ZIM motif which may represent novel transcription factors.

Gene expression profiling of systemically wound-induced defenses in hybrid poplar.

As part of an ongoing effort to identify genes involved in poplar defense responses, and to provide a resource for comparative analysis of woody and non-woody plant defense, we generated expressed sequence tags (ESTs) from a library constructed from systemically wounded leaves of hybrid poplar (Populus trichocarpa x P. deltoides). Partial sequences were obtained from the 5' ends of 928 individual cDNAs, which could be grouped into 565 non-overlapping sequences. Of these, 447 sequences were singletons, while the remainder fell into 118 clusters containing up to 17 partially overlapping ESTs. Approximately 81% of the EST sequences showed similarity to previously described sequences in public databases. Of these, the distribution of gene functions within the EST set indicated that approximately 11% of the ESTs encode proteins potentially involved in defense or secondary metabolism, while photosynthesis and primary metabolism accounted for 45% of the expressed genes. Two types of defense proteins, Kunitz trypsin inhibitors and chitinases, were found among the ten most abundant ESTs, indicating the significant impact of wounding on the leaf transcriptome and suggesting that these functions are important for hybrid poplar defense. In the course of this work, three new wound-inducible Kunitz trypsin inhibitor-like genes and two new chitinase-like genes were characterized. A suite of other systemically wound-induced genes were identified using northern and macroarray analysis, indicating diversity and multiplicity in the induced defense response. Overall, we demonstrate that defense-related genes of hybrid poplar have a variety of functions, and show remarkably diverse expression patterns upon wounding.