The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific,Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis

Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.     

Lipoxygenase6-Dependent Oxylipin Synthesis in Roots Is Required for Abiotic and Biotic Stress Resistance of Arabidopsis

Jasmonates are oxylipin signals that play important roles in the development of fertile flowers and in defense against pathogens and herbivores in leaves. The aim of this work was to understand the synthesis and function of jasmonates in roots. Grafting experiments with a jasmonate-deficient mutant demonstrated that roots produce jasmonates independently of leaves, despite low expression of biosynthetic enzymes. Levels of 12-oxo-phytodienoic acid, jasmonic acid, and its isoleucine derivative increased in roots upon osmotic and drought stress. Wounding resulted in a decrease of preformed 12-oxo-phytodienoic acid concomitant with an increase of jasmonic acid and jasmonoyl-isoleucine. 13-Lipoxygenases catalyze the first step of lipid oxidation leading to jasmonate production. Analysis of 13-lipoxygenase-deficient mutant lines showed that only one of the four 13-lipoxygenases, LOX6, is responsible and essential for stress-induced jasmonate accumulation in roots. In addition, LOX6 was required for production of basal 12-oxo-phytodienoic acid in leaves and roots. Loss-of-function mutants of LOX6 were more attractive to a detritivorous crustacean and more sensitive to drought, indicating that LOX6-derived oxylipins are important for the responses to abiotic and biotic factors.Oxylipins are ubiquitous signaling molecules that are derived from polyunsaturated fatty acids by enzymatic and nonenzymatic processes. In plants, the biosynthesis and function of oxylipins of the jasmonate family in aboveground tissues has been investigated in detail. Jasmonates comprise 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives of JA. In leaves, jasmonates accumulate in response to abiotic factors such as wounding, drought, osmotic stress, darkness, and ozone and during interactions with organisms such as herbivores, pathogens, and mutualistic organisms (Wasternack, 2007). The relevance of jasmonates in wound response, ozone tolerance, and the defense against herbivores and necrotrophic pathogens in leaves has been well investigated using mutants in JA biosynthesis and signaling (Browse, 2009a). In addition, jasmonates play an important role in flower development, and Arabidopsis (Arabidopsis thaliana) mutants in the JA pathway are male sterile (Browse, 2009b). The first step in jasmonate biosynthesis is catalyzed by 13-lipoxygenases (LOXs). The resulting 13(S)-hydroperoxyoctadecatrienoic acid (13-HPOTE) is converted by allene oxide synthase (AOS) and allene oxide cyclase to OPDA (Wasternack, 2007). These enzymatic steps are located in plastids. OPDA is transported to peroxisomes and converted to JA. JA can be further metabolized to different derivatives that take place mainly in the cytosol. The conjugation of JA with Ile is an important step because jasmonoyl-Ile (JA-Ile) has been identified as a biologically active jasmonate (Staswick and Tiryaki, 2004). OPDA is also biologically active without conversion to JA derivatives. In contrast to all other jasmonates, the OPDA structure contains an electrophilic α,β-unsaturated carbonyl group that renders OPDA more reactive than JA. Therefore, OPDA is classified as a reactive electrophile species with unique signaling properties different from other jasmonates (Farmer and Davoine, 2007).Of the six lipoxygenase genes present in Arabidopsis, four genes encode 13-LOX. For the respective enzymes LOX2, LOX3, LOX4, and LOX6, it was shown that linolenic acid is the preferred substrate and that 13-HPOTE is formed in vitro (Bannenberg et al., 2009). All four enzymes are proposed to be located in plastids. LOX2 is highly expressed in leaves; expression is up-regulated by jasmonates and stress treatments such as wounding and osmotic stress (Bell and Mullet, 1993; Seltmann et al., 2010a). LOX2 was shown to contribute the majority of jasmonate synthesis upon wounding and osmotic stress and during senescence in leaves (Bell et al., 1995; Glauser et al., 2009). LOX2 is also responsible for the accumulation of arabidopsides (Glauser et al., 2009), which are galactolipids containing esterified OPDA in plastids by direct oxidation of galactolipids (Zoeller et al., 2012). LOX3 and LOX4 are required for the development of fertile flowers (Caldelari et al., 2011). LOX6 shows overall low expression (Bannenberg et al., 2009). Recently, it was reported that LOX6 contributes to the fast accumulation of JA and JA-Ile in wounded leaves and is required for the fast increase of JA and JA-Ile in distal leaves after wounding (Chauvin et al., 2013).In contrast to leaves and flowers, little is known on jasmonate biosynthesis and function in roots. Expression of the plastid-localized enzymes of jasmonate synthesis LOX2, AOS, and allene oxide cyclase2 is very low in roots (Zimmermann et al., 2004). By contrast, enzymes such as 9-LOX and α-dioxygenase1 are strongly expressed in roots. These enzymes are involved in the biosynthesis of oxylipins different from jasmonates, and 9-LOX products have been shown to regulate lateral root development because mutants in LOX1 and LOX5 produce more lateral roots (Vellosillo et al., 2007). However, jasmonate function in roots is still obscure. Here, we analyzed jasmonate accumulation in roots upon different stress treatments and show that mutants defective in LOX6 are impaired in stress-induced jasmonate synthesis and are more susceptible to drought and detritivore feeding.     

The Coronatine Toxin of Pseudomonas syringae Is a Multifunctional Suppressor of Arabidopsis Defense

The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box–containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.     

Maintenance of Chloroplast Structure and Function by Overexpression of the Rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE Gene Leads to Enhanced Salt Tolerance in Tobacco

In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant’s response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response.Salt stress is a major environmental factor that poses a serious threat to crop yield and future food production (Møller and Tester, 2007). When plants are exposed to salinity, they suffer two primary obstacles: low external water potentials and high concentrations of toxic ions (Hirayama and Mihara, 1987). These obstacles generally lead to the disruption of various enzymatic processes, changes in membrane lipid composition, alteration in chloroplast structure and function, impairment of photosynthetic capacity, and inhibition of plant growth (Brown and Dupont, 1989; Elkahoui et al., 2004; Munns and Tester, 2008; Sui et al., 2010; Shu et al., 2012).Membranes are the primary matrix for numerous physiological and biochemical activities, and plants easily change their membrane lipid compositions in response to environmental stresses (Harwood, 1996). A number of studies have proved that salt stress can induce changes in plant membrane lipids (Huflejt et al., 1990; Elkahoui et al., 2004; Sui et al., 2010). Sui et al. (2010) found that, in Suaeda salsa, salt stress increased the proportion of phosphatidylglycerol and reduced the proportion of galactolipids. Similar results were observed in Catharanthus roseus cultured cell suspensions, which showed an increase in phospholipid content and a decrease in galactolipid content that were more obvious under 100 mm NaCl than under 50 mm (Elkahoui et al., 2004). Meanwhile, it was shown that salt tolerance in plants is strongly linked with their membrane lipid composition and especially with their galactolipid content, which is positively related to salt tolerance (Hirayama and Mihara, 1987).In plants, galactolipids are major constituents of the photosynthetic membrane, which is the most abundant membrane in nature (Lee, 2000). Two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the predominant photosynthetic membrane lipid building blocks, accounting for about 52 mol % and 26 mol % of thylakoid membrane lipids, respectively (Block et al., 1983). These galactolipids are also the major lipids in cyanobacteria, suggesting that MGDG and DGDG are important for all oxygenic photosynthetic organisms (Hölzl and Dörmann, 2007). They are components of PSII and are the indispensable matrix for photosynthesis (Mizusawa and Wada, 2012). Their unique characteristics also give them distinctive packing properties that are important for the organization of thylakoid membranes (Lee, 2000). MGDG has a conical shape due to its smaller head group and a high content of unsaturated fatty acids, giving it nonbilayer-forming characteristics (Webb and Green, 1991; Aronsson et al., 2008); this feature is assumed to play an important structural role in the dense packing of proteins in the membrane (Williams, 1998; Garab et al., 2000). In contrast to MGDG, DGDG has a cylindrical shape that is typical for most plastid lipids and is considered a bilayer-prone lipid (Aronsson et al., 2008); this feature is involved in lipid-mediated contacts between adjacent trimers of light-harvesting complex II (LHCII) when they are packed into two-dimensional crystalline arrays (Lee, 2000). In addition to forming important membrane structures in the thylakoids, MGDG and DGDG are also present in extraplastidic membranes, including the plasma membrane, tonoplasts, endoplasmic reticulum, and Golgi membranes, indicating the crucial role of these galactoglycerolipids in higher plant membrane systems (Yoshida and Uemura, 1986; Brown and Dupont, 1989; Härtel et al., 2000).Two enzymes are involved in the biosynthesis of these galactoglycerolipids: MGDG synthase (MGD), which transfers a galactosyl residue from UDP-Gal to diacylglycerol, and DGDG synthase, which catalyzes the further galactosylation of MGDG to form DGDG (Shimojima et al., 1997; Dörmann et al., 1999; Shimojima and Ohta, 2011). Thus, MGD is the key enzyme in the biosynthesis of both galactolipids and, consequently, also in the formation of photosynthetic membranes (Nakamura et al., 2010). A number of studies have revealed that MGD is vital for plant growth and development. The loss of MGD function in plants leads to a pale-green phenotype, defects in the chloroplast ultrastructure, disruption in the photosynthetic membranes, and complete impairment of photosynthetic ability and photoautotrophic growth, suggesting a unique role for MGD in chlorophyll formation, the structural organization of the plastidic membranes, and photosynthetic growth (Jarvis et al., 2000; Kobayashi et al., 2007; Botté et al., 2011; Myers et al., 2011). The crucial role of MGD under environmental stresses, including phosphorus deficiency and wounding, is also well studied (Kobayashi et al., 2004, 2009a, 2009b; Moellering and Benning, 2011). However, although a number of studies have proved that salt stress can induce changes in plant membrane lipids (Huflejt et al., 1990; Elkahoui et al., 2004; Sui et al., 2010), little is known about the role of MGD and the involvement of galactolipids in response to salt stress.To investigate the function of MGD in plant salt tolerance, we cloned the relevant gene, which is called OsMGD (Qi et al., 2004), from rice (Oryza sativa ‘FR13A’). This gene has a high similarity to Arabidopsis (Arabidopsis thaliana) MGD2 and MGD3, and its expression is induced by several environmental stresses, including salt, drought, and submergence (Qi et al., 2004; Benning and Ohta, 2005). We then generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD and investigated the salt tolerance ability in the transgenic lines and wild-type plants. The results of this study demonstrate that an increase in galactolipid content in leaves is beneficial for maintaining chloroplast structure and function and leads to enhanced salt tolerance in tobacco.     

The Arabidopsis DELLA RGA-LIKE3 Is a Direct Target of MYC2 and Modulates Jasmonate Signaling Responses

Gibberellins (GAs) are plant hormones involved in the regulation of plant growth in response to endogenous and environmental signals. GA promotes growth by stimulating the degradation of nuclear growth–repressing DELLA proteins. In Arabidopsis thaliana, DELLAs consist of a small family of five proteins that display distinct but also overlapping functions in repressing GA responses. This study reveals that DELLA RGA-LIKE3 (RGL3) protein is essential to fully enhance the jasmonate (JA)-mediated responses. We show that JA rapidly induces RGL3 expression in a CORONATINE INSENSITIVE1 (COI1)– and JASMONATE INSENSITIVE1 (JIN1/MYC2)–dependent manner. In addition, we demonstrate that MYC2 binds directly to RGL3 promoter. Furthermore, we show that RGL3 (like the other DELLAs) interacts with JA ZIM-domain (JAZ) proteins, key repressors of JA signaling. These findings suggest that JA/MYC2-dependent accumulation of RGL3 represses JAZ activity, which in turn enhances the expression of JA-responsive genes. Accordingly, we show that induction of primary JA-responsive genes is reduced in the rgl3-5 mutant and enhanced in transgenic lines overexpressing RGL3. Hence, RGL3 positively regulates JA-mediated resistance to the necrotroph Botrytis cinerea and susceptibility to the hemibiotroph Pseudomonas syringae. We propose that JA-mediated induction of RGL3 expression is of adaptive significance and might represent a recent functional diversification of the DELLAs.     

Functional Convergence of Oxylipin and Abscisic Acid Pathways Controls Stomatal Closure 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.To colonize a diverse range of environments successfully, plants have developed converging functional pathways to synthesize an array of secondary metabolites for their protection against hostile conditions. For example, in response to environmental challenges, the oxylipin pathway induces the de novo synthesis of biologically active compounds called “oxylipins,” derivatives of oxygenated polyunsaturated fatty acids (Feussner and Wasternack, 2002; Howe and Schilmiller, 2002). Among the oxylipin pathways, the enzymes allene oxide synthase (AOS) and hydroperoxide lyase (HPL) are considered to partition two major branches that compete for the same substrates and are critical plant stress response pathways (Chehab et al., 2008).Production of the AOS pathway metabolites 12-oxo-phytodienoic acid (12-OPDA) and jasmonic acid (JA) originates from α-linolenic acid of chloroplast membranes (Feussner and Wasternack, 2002). Oxygenation of α-linolenic acid by a 13-lipoxygenase followed by the action of AOS forms an unstable allene oxide that is subsequently cyclized by an allene oxide cyclase to form 12-OPDA (Stenzel et al., 2012). 12-OPDA is the end product of the plastid-localized part of the pathway (Stintzi and Browse, 2000; Schaller and Stintzi, 2009). 12-OPDA is then translocated to the peroxisome where it is reduced by 12-OPDA reductase3 (OPR3) and subsequently activated by CoA ester prior to undergoing three rounds of β-oxidation to form JA (Schaller et al., 2000; Koo et al., 2006; Kienow et al., 2008). 12-OPDA is also a signaling molecule with both overlapping and distinct functions from JA. The Arabidopsis (Arabidopsis thaliana) opr3 mutant is deficient in JA synthesis but accumulates 12-OPDA and displays wild-type resistance to the dipteran Bradysia impatiens and to the fungal pathogen Alternaria brassicicola, generally considered JA-dependent responses (Stintzi et al., 2001). In addition, expression studies have identified genes induced by 12-OPDA but not by JA or methyl jasmonate (MeJA; Kramell et al., 2000; Stintzi et al., 2001; Taki et al., 2005; Ribot et al., 2008). These studies collectively show that 12-OPDA mediates gene expression with or without the canonical JA signaling framework (Stintzi et al., 2001; Taki et al., 2005; Ribot et al., 2008).The HPL branch of the oxylipin pathway produces aldehydes and corresponding alcohols. The first enzyme in the pathway is encoded by one or more HPL genes, differing in their subcellular localization, including microsomes (Pérez et al., 1999), lipid bodies (Mita et al., 2005), and the outer envelope of chloroplasts (Froehlich et al., 2001), and in some cases, with no specific localization in a particular organelle (Noordermeer et al., 2000). This variation in the number of genes and subcellular localization of their encoded enzymes is suggestive of the differential regulation of this pathway and, ultimately, the diversity of their responses, potentially tailored to the nature of stimuli.We have previously identified three rice (Oryza sativa) HPLs (HPL1 through HPL3) differing in their enzyme kinetics and substrate preference. Expression of these enzymes in Arabidopsis accession Columbia (Col-0), a natural hpl loss-of-function mutant, reestablished the production of the pathway metabolites (Chehab et al., 2006) and revealed the key role of HPL-derived metabolites in plant stress signaling (Chehab et al., 2008).The HPL and AOS branches of the oxylipin pathway do not function independently; the signaling crosstalk between them is key to fine tuning plant adaptive responses to a diverse range of perturbations (Halitschke et al., 2004; Liu et al., 2012; Scala et al., 2013).To gain deeper insight into the role of AOS- and HPL-derived metabolites in fine-tuning plant stress responses, we have (1) characterized the corresponding oxylipin signatures in response to wounding and drought in three Arabidopsis ecotypes, (2) generated a range of transgenic lines that produce varying blends of oxylipins tailored to the nature of the stress, (3) elucidated a JA-independent role for 12-OPDA in enhanced drought tolerance in part via regulation of stomatal aperture, and (4) reexamined the 12-OPDA-mediated regulation of stomatal aperture, alone or in combination with abscisic acid (ABA) in the model system Arabidopsis as well as in two crop species, namely tomato (Solanum lycopersicum) and Brassica napus. Unexpectedly, these analyses have identified drought as a stress signal that uncouples the conversion of 12-OPDA to JA and have revealed that 12-OPDA is a previously unrecognized regulator of stomatal closure in response to drought. This function of 12-OPDA, however, is most effective when combined with ABA, a phytohormone known to be essential for plant-adaptive responses to drought stress (Seki et al., 2007).     

Regulation of Jasmonate-Induced Leaf Senescence by Antagonism between bHLH Subgroup IIIe and IIId Factors in Arabidopsis

Plants initiate leaf senescence to relocate nutrients and energy from aging leaves to developing tissues or storage organs for growth, reproduction, and defense. Leaf senescence, the final stage of leaf development, is regulated by various environmental stresses, developmental cues, and endogenous hormone signals. Jasmonate (JA), a lipid-derived phytohormone essential for plant defense and plant development, serves as an important endogenous signal to activate senescence-associated gene expression and induce leaf senescence. This study revealed one of the mechanisms underlying JA-induced leaf senescence: antagonistic interactions of the bHLH subgroup IIIe factors MYC2, MYC3, and MYC4 with the bHLH subgroup IIId factors bHLH03, bHLH13, bHLH14, and bHLH17. We showed that MYC2, MYC3, and MYC4 function redundantly to activate JA-induced leaf senescence. MYC2 binds to and activates the promoter of its target gene SAG29 (SENESCENCE-ASSOCIATED GENE29) to activate JA-induced leaf senescence. Interestingly, plants have evolved an elaborate feedback regulation mechanism to modulate JA-induced leaf senescence: The bHLH subgroup IIId factors (bHLH03, bHLH13, bHLH14, and bHLH17) bind to the promoter of SAG29 and repress its expression to attenuate MYC2/MYC3/MYC4-activated JA-induced leaf senescence. The antagonistic regulation by activators and repressors would mediate JA-induced leaf senescence at proper level suitable for plant survival in fluctuating environmental conditions.     

Axial and Radial Oxylipin Transport

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.Both animals and plants produce potently active lipid-derived mediators in response to wounding. These oxylipins (oxygenated lipid derivatives) include leukotrienes and prostaglandins in animals (Funk, 2001) and jasmonates in plants (Wasternack and Hause, 2013). Although these regulators frequently show structural similarities (many are cyclopentenone and cyclopentanone lipids), they operate through different signaling pathways often involving large protein complexes. For example, prostaglandins signal in part through G protein-coupled receptor complexes (Furuyashiki and Narumiya, 2011; Kalinski, 2012), and plant jasmonate signaling operates through the Skp/Cullin/F-box CORONATINE INSENSITIVE1 complex (Browse, 2009). Many oxylipins produced in response to tissue damage in metazoans act as paracrine signals to elicit defense responses in distal undamaged cells (Funk, 2001). Similarly, it is possible that jasmonates, including the biologically active derivative jasmonoyl-Ile (JA-Ile; Fonseca et al., 2009), might be transported from cell to cell in plants. However, to date, the majority of studies on oxylipin transport in plants have used exogenous jasmonates, and it remains unclear to what extent these compounds are transported between cells and tissues when produced endogenously.Based on the fact that jasmonic acid (JA) or methyl jasmonate treatments can affect defense gene expression at a distance to the sites of their application, JA was proposed to operate as a paracrine signal capable of being transported from cell to cell in tomato (Solanum lycopersicum) leaves (Farmer et al., 1992). Similar conclusions were drawn for JA in wild tobacco (Nicotiana sylvestris; Zhang and Baldwin, 1997). Isotope-labeling experiments using exogenous jasmonates have indicated JA/JA-Ile transport away from the site of application to distal tissues and even distal organs (Zhang and Baldwin, 1997; Thorpe et al., 2007; Sato et al., 2011). Additionally, grafting experiments in tomato were consistent with long-distance transport of JA/JA precursors (Li et al., 2002; Schilmiller and Howe, 2005), although other studies did not find evidence for JA transport from wounded leaves to distal unwounded leaves (Strassner et al., 2002). Concerning Arabidopsis (Arabidopsis thaliana), Koo et al. (2009) concluded that JA-Ile accumulation detected in leaves distal to the wound site resulted mainly from de novo synthesis in undamaged leaves rather than from the transport of JA/JA-Ile from the wound site. Recently, a transporter (GLUCOSINOLATE TRANSPORTER1) capable of importing JA-Ile (but not JA) into Xenopus laevis oocytes has been described (Saito et al., 2015), further supporting the possibility that jasmonates move between cells.In addition to the transport of jasmonates, there is much evidence consistent with other wound signaling mechanisms that lead to JA synthesis and JA-mediated defense responses at various distances from wounds. That is, wound-activated signaling pathways can be classified as those working near the damage site (i.e. local responses) and those operating distal to it (Rhodes et al., 2006; Wu et al., 2007). Both these types of wound responses can be difficult to study, because several types of events (including the transport of jasmonates) may contribute to JA signaling. However, there has been some progress in understanding long-distance signaling leading to distal wound responses. These mechanisms include electrical and potentially, hydraulic signaling (for review, see Koo and Howe, 2009; Farmer et al., 2014). Membrane hyperpolarizations have been recorded in wounded plants (Zimmermann et al., 2009); however, their relationship with JA synthesis or JA responses has not yet been reported. In Arabidopsis, wounding of adult-phase rosettes stimulates the leaf-to-leaf propagation of signals that (1) can be detected with surface electrodes as cell membrane depolarizations; (2) are propagated from leaf to leaf in a mechanism that requires several clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes, including GLR3.3 and GLR3.6; and (3) can induce JA and JA-Ile accumulation in distal unwounded sites (Mousavi et al., 2013). However, even when electrical signals were compromised in both the wounded and distal leaves of a glr3.3 glr3.6 double mutant, JA responses were affected only in the distal leaf; local responses in the damaged leaf itself were similar to the wild type (Mousavi et al., 2013). Therefore, certain clade 3 GLRs operate in rosette-stage plants to extend the range of the wound response, and even if these genes are mutated, wounded rosette leaves still produce jasmonates. In summary, both jasmonates made near wounds and jasmonates made far from wounds in response to distal signals might be subject to transport within the plant.This study focused on the mobility of endogenous jasmonates produced in response to wounding. Here, we ask: how mobile are endogenous jasmonates generated in aboveground tissues in response to wounding? Our analysis was conducted throughout the vegetative phase and included different tissues that ranged from embryonic leaves (cotyledons) and roots to expanded rosette leaves. We investigated whether a glr3.3 glr3.6 double mutant that reduces leaf-to-leaf signal propagation in the adult phase (Mousavi et al., 2013) could also reduce cotyledon-to-root wound signaling in seedlings. Results from these electrophysiology experiments then led us to investigate whether JA (or JA precursors) can translocate from wounded cotyledons to roots. To do this, we used two approaches. One was based on mutants in 13-LIPOXYGENASEs (13-LOXs) that are necessary for an early step in the synthesis of the JA precursor oxophytodienoic acid. All four 13-LOXs in Arabidopsis (LOX2, LOX3, LOX4, and LOX6) are known to contribute to JA synthesis in vivo (Chauvin et al., 2013). First, LOX2 is responsible for the synthesis of a large pool of JA in wounded leaves (Bell et al., 1995), and it also produces precursors for the synthesis of arabidopside defense-related metabolites (Glauser et al., 2009). Second, LOX3 and LOX4 act together to produce the JA required for full male fertility (Caldelari et al., 2011). Third, LOX6 produces jasmonates in roots that are first separated from aerial tissues and then wounded (Grebner et al., 2013). We tested the impact of mutations in the different 13-LOXs on root JA signaling after cotyledon wounding. This was followed by grafting experiments between the wild type and the JA-deficient mutant allene oxide synthase (aos; Park et al., 2002) to test whether JA (or JA precursors) could translocate axially from wounded shoots into undamaged roots.In addition to its role in wounded roots (Grebner et al., 2013), LOX6 has been implicated in long-distance wound signaling in the adult-phase rosette, where it is necessary for most of the rapid distal expression of the JA-responsive gene JASMONATE ZIM-DOMAIN10 (JAZ10) when another leaf is wounded (Chauvin et al., 2013). This and the fact that the LOX6 promoter is active principally in xylem contact cells (Chauvin et al., 2013) provided us with the opportunity to investigate oxylipin transport within leaves. We confirmed the cellular localization of the LOX6 polypeptide with a LOX6-GUS fusion protein. We then used a lox234 triple mutant expressing a JAZ10 reporter to test whether jasmonates could be exported from xylem contact cells. These experiments led to unique insights into the transport of jasmonates across different leaf cell layers.