The glabra1 Mutation Affects Cuticle Formation and Plant Responses to Microbes

Systemic acquired resistance (SAR) is a form of defense that provides resistance against a broad spectrum of pathogens in plants. Previous work indicates a role for plastidial glycerolipid biosynthesis in SAR. Specifically, mutations in FATTY ACID DESATURASE7 (FAD7), which lead to reduced trienoic fatty acid levels and compromised plastidial lipid biosynthesis, have been associated with defective SAR. We show that the defective SAR in Arabidopsis (Arabidopsis thaliana) fad7-1 plants is not associated with a mutation in FAD7 but rather with a second-site mutation in GLABRA1 (GL1), a gene well known for its role in trichome formation. The compromised SAR in gl1 plants is associated with impairment in their cuticles. Furthermore, mutations in two other components of trichome development, GL3 and TRANSPARENT TESTA GLABRA1, also impaired cuticle development and SAR. This suggests an overlap in the biochemical pathways leading to cuticle and trichome development. Interestingly, exogenous application of gibberellic acid (GA) not only enhanced SAR in wild-type plants but also restored SAR in gl1 plants. In contrast to GA, the defense phytohoromes salicylic acid and jasmonic acid were unable to restore SAR in gl1 plants. GA application increased levels of cuticular components but not trichome formation on gl1 plants, thus implicating cuticle, but not trichomes, as an important component of SAR. Our findings question the prudence of using mutant backgrounds for genetic screens and underscore a need to reevaluate phenotypes previously studied in the gl1 background.Plants have evolved a large array of defense mechanisms to resist infection by pathogens. Upon recognition, the host plant initiates one or more signal transduction pathways that activate various plant defenses and thereby prevent pathogen colonization. In many cases, resistance is associated with increased expression of defense genes, including the pathogenesis-related (PR) genes and the accumulation of salicylic acid (SA) in the inoculated leaf. Induction of these responses is accompanied by localized cell death at the site of pathogen entry, which can often restrict the spread of pathogen to cells within and immediately surrounding the lesions. This phenomenon, known as the hypersensitive response, is one of the earliest visible manifestations of induced defense responses and resembles programmed cell death in animals (Dangl et al., 1996; Gray, 2002; Glazebrook, 2005; Kachroo and Kachroo, 2006). Concurrent with hypersensitive response development, defense reactions are triggered in sites both local and distal from the primary infection. This phenomenon, known as systemic acquired resistance (SAR), is accompanied by a local and systemic increase in SA and jasmonic acid (JA) and a concomitant up-regulation of a large set of defense genes (Durrant and Dong, 2004; Truman et al., 2007; Vlot et al., 2009).SAR involves the generation of a mobile signal in the primary leaves that, upon translocation to the distal tissues, activates defense responses resulting in broad-spectrum resistance. The production of the mobile signal takes places within 3 to 6 h of avirulent pathogen inoculation in the primary leaves (Smith-Becker et al., 1998), and the inoculated leaf must remain attached for at least 4 h after inoculation for immunity to be induced in the systemic tissues (Rasmussen et al., 1991). Mutations compromising SA synthesis or impairing SA, JA, or auxin signaling abolish SAR (Durrant and Dong, 2004; Truman et al., 2007, 2010). SAR is also dependent on the SALICYLIC ACID-BINDING PROTEIN2 (SABP2)-catalyzed conversion of methyl SA to SA in the distal tissues (Kumar and Klessig, 2003). Recent studies have suggested that methyl SA is the mobile signal required to initiate SAR in distal tissues in tobacco (Nicotiana tabacum; Park et al., 2007) and Arabidopsis (Arabidopsis thaliana; Liu et al., 2010), although another group reported a disparity in their findings related to the role of methyl SA in Arabidopsis (Attaran et al., 2009). Notably, the time point of requirement of SABP2 activity (between 48 and 72 h post inoculation; Park et al., 2009) does not coincide with the early generation and/or translocation of the mobile signal into distal tissues (within 6 h post inoculation).The mutations acyl carrier protein4 (acp4), long-chain acyl-CoA synthetase2 (lacs2), and lacs9, which are impaired in fatty acid (FA)/lipid flux (Schnurr et al., 2004; Xia et al., 2009), also compromise SAR (Xia et al., 2009). Detailed characterization has shown that the SAR defect in acp4, lacs2, and lacs9 mutants correlates with their defective cuticles. Analysis of the SAR response in acp4 plants has shown that these plants can generate the mobile signal required for inducing SAR but are unable to respond to it. It is likely that the defective cuticle in these plants impairs their ability to perceive the SAR signal, because mechanical abrasion of cuticles disrupts SAR in wild-type plants (Xia et al., 2009). This SAR-disruptive effect of cuticle abrasion is highly specific, because it does not alter local defenses and hinders SAR only during the time frame during which the mobile signal is translocated to distal tissues.SAR is also compromised in plants that contain a mutation in glycerol-3-phosphate dehydrogenase (Nandi et al., 2004). The glycerol-3-phosphate dehydrogenase (GLY1) reduces dihydroxyacetone phosphate to generate glycerol-3-phosphate, an obligatory component and precursor for the biosynthesis of all plant glycerolipids. Consequently, a mutation in GLY1 results in reduced carbon flux through the prokaryotic pathway of lipid biosynthesis, which leads to a reduction in the hexadecatrienoic (16:3) FAs (Miquel et al., 1998; Kachroo et al., 2004). Carbon flux and SAR are also impaired in plants containing mutations in FATTY ACID DESATURASE7 (FAD7; Chaturvedi et al., 2008). The FAD7 enzyme desaturates 16:2 and 18:2 FA species present on plastidial lipids to 16:3 and 18:3, respectively. Consequently, the fad7 mutant plants accumulate significantly reduced levels of trienoic FAs (16:3 and 18:3). Compromised SAR in mutants affected in certain plastidial FA/lipid pathways has prompted the suggestion that plastidial FA/lipids participate in SAR (Chaturvedi et al., 2008). Such a tempting conclusion is also favored by the fact that SAR requires the DIR1-encoded nonspecific lipid transfer protein, which is required for the generation and/or translocation of the mobile signal (Maldonado et al., 2002). In addition, azelaic acid, a dicarboxylic acid, was recently shown to prime SA biosynthesis and thereby SAR (Jung et al., 2009). The fact that azelaic acid is derived from oleic acid, a FA well known for its role in defense (Kachroo et al., 2003, 2004, 2005, 2007, 2008; Chandra-Shekara et al., 2007; Jiang et al., 2009; Venugopal et al., 2009; Xia et al., 2009), further suggests that FA/lipids might participate in SAR.This study was undertaken to reexamine the role of the FA/lipid pathways in SAR and to determine the nature of the FA/lipid species mediating SAR in fad7-1 plants. Our results show that impaired FA/lipid flux is not associated with compromised SAR in fad7-1 plants but, rather, with an abnormal cuticle, which is the result of a nonallelic mutation in the GLABRA1 (GL1) gene. Besides GL1, other mutations affecting trichome formation also compromised cuticle and thereby SAR. A compensatory effect of exogenous GA on gl1 plants suggests that GA might participate in resistance to bacterial pathogens by restoring cuticle formation.