Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Sterol-Dependent Induction of Plant Defense Responses by a Microbe-Associated Molecular Pattern from Trichoderma viride

Plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. An ethylene-inducing xylanase (Eix) of Trichoderma viride is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). We demonstrate that tomato cyclopropyl isomerase (SlCPI), an enzyme involved in sterol biosynthesis, interacts with the LeEix2 receptor. Moreover, we examined the role of SlCPI in signaling during the LeEix/Eix defense response. We found that SlCPI is an important factor in the regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis, and the induction of pathogenesis-related protein expression in the case of LeEix/Eix. Our results also suggest that changes in the sterol composition reduce LeEix internalization, thereby attenuating the induction of plant defense responses.Plant innate immunity is activated upon the recognition of pathogen- and microbe-associated molecular patterns by surface-localized immune receptors or the stimulation of cytoplasmic immune receptors by pathogen effector proteins (Jones and Dangl, 2006; Thomma et al., 2011). Leucine-rich repeat (LRR) receptor kinases and leucine-rich repeat receptor proteins (LRR-RLPs) respond to conserved microbe-associated molecular patterns by producing a defense response upon detection (Altenbach and Robatzek, 2007; Bittel and Robatzek, 2007; Robatzek et al., 2007; Geldner and Robatzek, 2008). One such LRR-RLP is the ethylene-inducing xylanase (Eix) receptor LeEix2. The fungal protein Eix (Dean et al., 1989) is a well-known protein elicitor of defense response reactions in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum; Bailey et al., 1990; Avni et al., 1994). Eix induces ethylene biosynthesis, extensive electrolyte leakage, pathogenesis-related (PR) gene expression, reactive oxygen species (ROS), and the hypersensitive response (HR; Bailey et al., 1990; Ron et al., 2000). Eix was shown to specifically bind to the plasma membrane of responsive cultivars of both tomato and tobacco (Hanania and Avni, 1997). The response to Eix in tobacco and tomato cultivars is controlled by an LRR-RLP encoded by a single locus, termed LeEix (Ron and Avni, 2004). Previously, we showed that Eix triggers internalization of the LeEix2 receptor and its localization to endosomes (Bar and Avni, 2009).Endocytic processes and vesicular transport in general require the participation of membrane components that form transport vesicles with a capability to store and process a number of molecules known to participate in cell signaling (Anderson, 1993; Patel et al., 2008; Hansen and Nichols, 2010). Sterols are lipophilic membrane components that have many important functions in an array of eukaryotes. Changes in membrane-bound sterol levels and composition can have effects on the activity of membrane proteins and on signal transduction processes. The interaction between sterols and phospholipids forms microdomains termed lipid rafts (Simons and Ikonen, 1997). In response to cellular stimuli, lipid rafts can change the protein microenvironment, leading to the initiation of signaling cascades (Simons and Toomre, 2000; Mongrand et al., 2010; Simon-Plas et al., 2011). Sterols also provide precursors for the biosynthesis of steroid hormones such as mammalian estrogens and glucocorticoids and plant brassinosteroids (Bishop and Yokota, 2001; Benveniste, 2004; Suzuki and Muranaka, 2007). One of the enzymes involved in sterol biosynthesis is cyclopropyl isomerase (CPI; Lovato et al., 2000; Benveniste, 2004).A variety of endocytic pathways have been described in mammalian and fungal cells that differ mainly in the size, shape, and composition of endocytic vesicles and in the participation of different proteins (Conner and Schmid, 2003; Soldati and Schliwa, 2006). Cholesterol, the main mammalian sterol, has an important role in most internalization steps through both caveolae and clathrin-coated pits (Murata et al., 1995; Subtil et al., 1999). Cholesterol depletion alters endocytic structures and reduces the polar delivery of target proteins (Keller and Simons, 1998; Pichler and Riezman, 2004). Plant sterols are reported to be internalized into endosomes and distributed throughout the endocytic pathway in an actin-dependent manner (Grebe et al., 2003). The sterol endocytic pathway has been shown to interrupt the internalization, trafficking, and polar recycling of PIN2, an auxin efflux facilitator and polarity marker, in developing root epidermal cells of Arabidopsis (Arabidopsis thaliana; Grebe et al., 2003; Men et al., 2008). Sterols were shown to function in the trafficking of an ATP-binding cassette (ABCB19) from the trans-Golgi to the plasma membrane (Yang et al., 2013).Here, we report the isolation of SlCPI, which interacts with the LeEix2 receptor. Modulating the expression or function of SlCPI affects the induction of plant defense responses mediated by Eix.     

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.