Nicotiana attenuata SIPK,WIPK, NPR1, and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glucose (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.Jasmonic acid (JA) and some of its precursors and derivatives are signal molecules that function as essential mediators of the plant''s wound, antiherbivore, and antipathogen responses, as well as in growth and development (Farmer, 1994; Creelman and Mullet, 1997; Turner et al., 2002). In unelicited mature leaves, JA is maintained at very low levels, however, upon specific stimulations, its biosynthesis is induced within a few minutes (Glauser et al., 2008). This rapid biosynthetic response must result from the activation of constitutively expressed JA biosynthesis enzymes in unelicited tissue by substrate availability and/or posttranslational modifications. At present, little is known about the molecular mechanisms that activate JA biosynthetic enzymes.According to the canonical mechanism for JA biosynthesis (Vick and Zimmerman, 1983), free α-linolenic acid (18:3^Δ9,12,15, 18:3) forms 13(S)-hydroperoxyoctadecatrienoic acid 13S-(OOH)-18:3] by the action of 13-lipoxygenase (13-LOX) in plastids. 13S-(OOH)-18:3 is converted by allene oxide synthase (AOS) into a highly unstable allene oxide intermediate that is processed by allene oxide cyclase (AOC) to yield (9S,13S)-12-oxo-phytodienoic acid (OPDA). OPDA is transported from the plastid into the peroxisome where it is reduced by the action of OPDA reductase 3 (OPR3) and after three cycles of β-oxidation, (3R,7S)-JA is formed. Due to the large number of enzymes and different cellular compartments involved in JA biosynthesis, it is expected that the pathway is regulated at multiple steps. Resolution of the structures of the tomato (Solanum lycopersicum) OPR3 and Arabidopsis (Arabidopsis thaliana) AOC2 and ACX1 has provided insights into potential regulatory mechanisms for these enzymes (e.g. oligomerization and phosphorylation; Pedersen and Henriksen, 2005; Breithaupt et al., 2006; Hofmann et al., 2006).The identification of two Arabidopsis plastidial glycerolipases, DAD1 and DGL (Ishiguro et al., 2001; Hyun et al., 2008), has provided genetic evidence for the importance of the release of trienoic fatty acids (FAs) from plastidial lipids in the activation of JA biosynthesis. Recently, some oxylipins have been found esterified to galactolipids in Arabidopsis leaves and hence it is possible that in this species preformed precursors could also supply the JA biosynthesis pathway after their release from lipids (Stelmach et al., 2001; Hisamatsu et al., 2003; Buseman et al., 2006). However, lipid-bound oxylipins are not formed in the leaves of all plant families (Böttcher and Weiler, 2007).In Nicotiana attenuata, wound-induced JA production is amplified by the application of lepidopteran larvae (e.g. Manduca sexta) oral secretions (OS) to mechanical wounds. Major elicitors of the OS-mediated response are FA-amino acid conjugates (FACs) that are sufficient to enhance JA production in leaves of this plant species (Halitschke et al., 2001). Recently, several regulatory factors with a potential function upstream of JA biosynthesis have been identified (Ludwig et al., 2005; Takabatake et al., 2006; Schweighofer et al., 2007; Takahashi et al., 2007); however, how these regulators affect JA biosynthesis is at present unknown. For example, wounding and herbivory in Nicotina spp. and tomato activate the mitogen-activated protein kinases salicylate-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK; Seo et al., 1999; Kandoth et al., 2007; Wu et al., 2007). When SIPK and WIPK expression is silenced in tobacco (Nicotiana tabacum), the plants accumulate 60% to 70% less JA than wild type after wounding or OS elicitation (Seo et al., 2007; Wu et al., 2007). Another regulatory component that affects JA production in N. attenuata is Nonexpressor of PR-1 (NPR1), an essential component of the salicylic acid (SA) signal transduction pathway first identified in Arabidopsis (Cao et al., 1994). N. attenuata NPR1-silenced plants accumulate 60% to 70% lower JA levels after elicitation than wild type (Rayapuram and Baldwin, 2007). NPR1 interacts with the JA and ethylene signaling cascades, and a cytosolic role for this factor in the regulation of JA-dependent responses/biosynthesis has been proposed (Spoel et al., 2003).In contrast to the mechanisms acting upstream of JA biosynthesis, the mechanisms mediating downstream JA responses are better characterized (Kazan and Manners, 2008; Browse, 2009). Among the best-characterized regulators of these responses is CORONATIVE INSENSITIVE1 (COI1), a gene that participates in jasmonate perception (Xie et al., 1998) and regulates gene expression through its interaction with the JASMONATE ZIM-DOMAIN repressors (Chini et al., 2007; Thines et al., 2007).To understand the early processes regulating the activation of JA biosynthesis by wounding and FAC elicitation in N. attenuata leaves, we quantified the initial rates of accumulation of plastid-derived JA precursors after these stimuli in wild type and four JA-deficient genotypes previously described: ir-sipk, ir-wipk, ir-npr1, and ir-coi1 (Rayapuram and Baldwin, 2007; Paschold et al., 2008; Meldau et al., 2009). We show that SIPK, WIPK, NPR1, and FACs contribute to the activation of de novo JA biosynthesis by affecting diverse early enzymatic steps in this pathway. The identification of a plastidial glycerolipase A₁ type I family protein (GLA1) essential for JA biosynthesis pointed to this enzyme as one potential target of some of these activating mechanisms.