Ester Cross-Link Profiling of the Cutin Polymer of Wild-Type and Cutin Synthase Tomato Mutants Highlights Different Mechanisms of Polymerization

Cuticle function is closely related to the structure of the cutin polymer. However, the structure and formation of this hydrophobic polyester of glycerol and hydroxy/epoxy fatty acids has not been fully resolved. An apoplastic GDSL-lipase known as CUTIN SYNTHASE1 (CUS1) is required for cutin deposition in tomato (Solanum lycopersicum) fruit exocarp. In vitro, CUS1 catalyzes the self-transesterification of 2-monoacylglycerol of 9(10),16-dihydroxyhexadecanoic acid, the major tomato cutin monomer. This reaction releases glycerol and leads to the formation of oligomers with the secondary hydroxyl group remaining nonesterified. To check this mechanism in planta, a benzyl etherification of nonesterified hydroxyl groups of glycerol and hydroxy fatty acids was performed within cutin. Remarkably, in addition to a significant decrease in cutin deposition, mid-chain hydroxyl esterification of the dihydroxyhexadecanoic acid was affected in tomato RNA interference and ethyl methanesulfonate-cus1 mutants. Furthermore, in these mutants, the esterification of both sn-1,3 and sn-2 positions of glycerol was impacted, and their cutin contained a higher molar glycerol-to-dihydroxyhexadecanoic acid ratio. Therefore, in planta, CUS1 can catalyze the esterification of both primary and secondary alcohol groups of cutin monomers, and another enzymatic or nonenzymatic mechanism of polymerization may coexist with CUS1-catalyzed polymerization. This mechanism is poorly efficient with secondary alcohol groups and produces polyesters with lower molecular size. Confocal Raman imaging of benzyl etherified cutins showed that the polymerization is heterogenous at the fruit surface. Finally, by comparing tomato mutants either affected or not in cutin polymerization, we concluded that the level of cutin cross-linking had no significant impact on water permeance.Cuticles are ubiquitous hydrophobic barriers at the surfaces of aerial plant organs. These complex hydrophobic assemblies consist of a biopolymer, cutin, coated and filled with waxes and can also comprise embedded cell wall polysaccharides. Waxes comprise solvent-soluble aliphatic molecules with long hydrocarbon chains, terpenes, and steroids (Kunst and Samuels, 2003; Nawrath, 2006; Pollard et al., 2008; Samuels et al., 2008; Schreiber, 2010; Lee and Suh, 2015). Cutin is an insoluble polyester of ω- and mid-chain hydroxy C₁₆ and C₁₈ fatty acids. Glycerol has also been described as a ubiquitous cutin monomer (Graça et al., 2002; Pollard et al., 2008). In some cuticles, a hydrophobic polymer that is resistant to alkaline hydrolysis (i.e. cutan) has been observed (Gupta et al., 2006; Li-Beisson et al., 2010).Cutin fulfills multiple functions in plants, such as the control of nonstomatal water loss (Sieber et al., 2000) and the permeation of gases and solutes (Kersteins, 1996; Schreiber, 2010). Cutin also plays an essential role in the regulation of cell adhesion during plant development by preventing organ fusion, as observed in Arabidopsis (Arabidopsis thaliana) mutants with cuticle defects (Sieber et al., 2000; Nawrath, 2006), or by participating in hull adhesion in grains (Taketa et al., 2008). Finally, it is generally accepted that plant cuticle and its polymeric skeleton, cutin, are primary barriers to pathogens and that cutin monomers released by fungal cutinase are signaling molecules for both the pathogen and plants (Gilbert et al., 1996; Schweizer et al., 1996; Iwamoto et al., 2002; Yeats and Rose, 2013).The biological functions of cutin are closely controlled by its structure, which is determined by its monomer composition and by the number and position of its ester bonds. Cutin monomer composition can vary according to plant species, developmental stage (Baker et al., 1982; Peschel et al., 2007; Mintz-Oron et al., 2008), organs, and environmental stress (Espelie et al., 1979; Li-Beisson et al., 2009; Panikashvili et al., 2009; Bessire et al., 2011). Actually, cutin monomer composition determines the total number of hydroxyl (OH) groups that are potentially available for the formation of ester bonds and, therefore, the cross-linking of the polyester (Bonaventure et al., 2004; Franke et al., 2005; Peschel et al., 2007). The nonesterified OH groups enhance the hydrophilic character of the cutin polymer, increasing its elasticity (Bargel and Neinhuis, 2005).Whereas cutin monomer composition has been described extensively for different plant species, organs, and development stages, the macromolecular structure of the cutin polyester has been much less thoroughly investigated. In particular, the connectivity between the monomers is a key point for understanding the three-dimensional expansion of the polyester in relation to the polymerization process. Different approaches have been proposed to delineate the polymeric architecture of cutin. Linear dimers were identified after partial alkaline hydrolysis of tomato (Solanum lycopersicum) cutin (Osman et al., 1995). NMR and mass spectrometry analyses of oligomers released after partial depolymerization revealed primary and secondary ester linkages between cutin monomers (Graça et al., 2002; Stark and Tian, 2006) as well as covalent linkages between some cutin OH fatty acids and oligosaccharides (Tian et al., 2008). However, it has been shown that partial hydrolysis does not necessarily release all of the representative building blocks of the entire polymer (Deshmukh et al., 2003). Spectrometric analyses have also been developed for the polymer. Attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy analyses of the methylene and carbonyl stretching vibrations allowed the estimation of an ester cross-linking index for cutin but could not differentiate the primary from the secondary ester linkages (Girard et al., 2012; Heredia-Guerrero et al., 2014). NMR studies have provided evidence of both ω- and mid-chain esters in tomato (Deshmukh et al., 2003).In this regard, tomato fruit has proved to be an interesting model for structural studies of the cutin polymer. Indeed, its astomatous cuticle can be easily isolated and is devoid of cutan. Moreover, tomato cutin composition is dominated by a monomer with two OH groups, 9(10),16-dihydroxyhexadecanoic acid (Baker et al., 1982; Osman et al., 1999; Deshmukh et al., 2003). Accordingly, the cutin monomers can be linked by either a linear (on the primary OH) or a branched (with a secondary OH) pattern. Previous studies have demonstrated that both linear and branched cross-links occur in tomato cutin. However, the relative proportion of the linear versus branched esters remains a matter of debate. Oxidation experiments have indicated that almost all of the primary cutin OH groups (94%) were involved in ester bonds, whereas only 44% of the secondary OH groups were esterified in the cutin polymer (Deas and Holloway, 1977; Kolattukudy, 1977). Conversely, partial depolymerization coupled with NMR studies of the released oligomers indicated that the branched secondary esters were the major form of tomato cutin (Graça and Lamosa, 2010). In addition, none of these studies could decipher the ester links of the glycerol OH groups.Additionally, the role of a GDSL lipase, involved in cutin polymerization, was recently reported using two different experimental approaches (Girard et al., 2012; Yeats et al., 2012). The corresponding GDSL lipase, named SlGDSL1 (Girard et al., 2012) or SlGDSL2 (Yeats et al., 2012), is now named CUTIN SYNTHASE1 (SlCUS1; Yeats et al., 2014). Different mutants affected in the expression of SlCUS1 have been generated and constitute attractive tools to delineate the structure of the cutin polymer (Girard et al., 2012; Petit et al., 2014).It has been further demonstrated that 2-monoacylglycerol (2-MAG), a putative precursor of the cutin polymer in Arabidopsis (Yang et al., 2010), can be used by a heterologously expressed SlCUS1 (Yeats et al., 2012) to produce in vitro linear oligomers in aqueous solution (Yeats et al., 2014). Nevertheless, the question of the mechanism of cutin polymerization in planta is still open. Indeed, SlCUS1 is specifically localized within the cutin matrix (i.e. a hydrophobic environment; Girard et al., 2012; Yeats et al., 2012), which could impact the acyltransferase activity of the enzyme as observed previously for lipases (Sharma et al., 2001).By coupling O-alkylation of the nonesterified OH groups of glycerol and fatty acids in an isolated cutin matrix and by further analyses of O-alkylated and nonalkylated monomers released after depolymerization, we elucidated the ester cross-link pattern of tomato cutin. We also showed at two stages of fruit development and in two different genetic backgrounds that the modulation of SlCUS1 protein level, either through RNA interference (cherry tomato ‘West Virginia 106’ [WVa106]) or mutagenesis (miniature tomato ‘Micro-Tom’), resulted in a strong alteration of the cutin ester cross-link pattern. These results give new insights into the polyester structure. In addition, while CUS1 esterification involves mostly primary OH groups in vitro (Yeats et al., 2014), our data here indicate that, in planta, deficiencies in CUS1 also affect the secondary OH group of 9(10),16-dihydroxyhexadecanoic acid and both the primary and secondary OH groups of glycerol.     

Abscisic Acid Deficiency Causes Changes in Cuticle Permeability and Pectin Composition That Influence Tomato Resistance to Botrytis cinerea

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.Plant defense against pathogens often involves the induction of mechanisms after pathogen recognition, including defense signaling, cell wall strengthening, and localized cell death, but plants also have preformed chemical and structural defense barriers. Fungal pathogens that penetrate the plant tissue directly through the outer surface, rather than via natural plant openings or wounds, must pass through the plant cuticle and epidermal cell wall. Penetration of the host surface happens either by physical means (i.e. by a highly localized pressure in the appressorium) or by chemical means (i.e. by the release of hydrolyzing enzymes). Necrotrophic plant pathogens like Botrytis cinerea typically use the latter strategy. During penetration, they produce cutinases and pectinolytic enzymes such as pectin methylesterases, endopolygalacturonases, and exopolygalacturonases (van Kan, 2006).The cuticle is a hydrophobic barrier that covers the aerial surfaces of the plant. It is mainly composed of cutin, a polyester matrix, and soluble waxes, a complex mixture of hydrophobic material containing very-long-chain fatty acids and their derivatives, embedded into and deposited onto the cutin matrix. It plays an important role in organ development and protection against water loss (Yephremov et al., 1999; Sieber et al., 2000; Kurata et al., 2003; Jung et al., 2006). The cuticle is generally considered as a mere passive physical barrier against pathogen invasion, but it has also been recognized as a potential source of signaling and elicitor molecules (Jenks et al., 1994; Reina-Pinto and Yephremov, 2009). Plant cutin monomers trigger cutinase secretion in pathogenic fungi (Woloshuk and Kolattukudy, 1986), and cutin and wax components initiate appressorium formation and penetration in appressorium-forming pathogens (Kolattukudy et al., 1995; Francis et al., 1996; Gilbert et al., 1996; Fauth et al., 1998; Dickman et al., 2003). In plants, cutin monomers induce pathogenesis-related gene expression and elicit hydrogen peroxide (H₂O₂) synthesis (Fauth et al., 1998; Kim et al., 2008; Park et al., 2008). Transgenic tomato (Solanum lycopersicum) plants expressing the yeast Δ-9 desaturase gene had high levels of cutin monomers that inhibited powdery mildew (Erysiphe polygoni) spore germination, leading to enhanced resistance (Wang et al., 2000). Arabidopsis (Arabidopsis thaliana) plants expressing a fungal cutinase or mutants with a defective cuticle, such as long-chain acyl-CoA synthetase2 and bodyguard, are generally more susceptible to bacteria and equally susceptible to biotrophic fungi but are surprisingly resistant to B. cinerea (Bessire et al., 2007; Chassot et al., 2007; Tang et al., 2007). It has been postulated that a defective or thin cuticle encourages these plants to constitutively express defense-related mechanisms and to secrete antifungal compounds to the plant surface, thereby inhibiting B. cinerea growth (Bessire et al., 2007; Chassot et al., 2007). In addition, cuticle metabolic pathways might directly modulate plant-pathogen interactions by interacting with hormonally regulated defense pathways (Fiebig et al., 2000; Garbay et al., 2007; Mang et al., 2009) or with complex lipid signaling pathways leading to hypersensitive cell death (Raffaele et al., 2008).Once plant pathogens have penetrated the cuticle, they secrete hydrolases that target the plant cell wall (ten Have et al., 1998; Oeser et al., 2002; Vogel et al., 2002; Jakob et al., 2007) that is mainly composed of cellulose, hemicellulose, and pectin (35% of total dry weight). Pectin consists mainly of the polysaccharides homogalacturonan and rhamnogalacturonan I and II. Homogalacturonans are linear chains of α-(1–4)-linked d-GalA residues that can be methylesterified at C-6. Rhamnogalacturonan I and II are more complex, branched polysaccharides. B. cinerea is typically regarded as a pectinolytic pathogen because it possesses an efficient pectinolytic machinery, including a variety of polygalacturonases and pectin methylesterases (PMEs), some of which are important virulence factors (ten Have et al., 1998, 2001; Valette-Collet et al., 2003; Kars et al., 2005). Pectins are a rich source of oligogalacturonides (OGAs), biologically active signaling molecules that can activate plant defense mechanisms (Hahn et al., 1981; Côté and Hahn, 1994; Messiaen and Van Cutsem, 1994; Ridley et al., 2001). The eliciting capacity of the OGAs was shown to depend on their size, which in turn is influenced by the methylesterification pattern of the homogalacturonan fraction (Mathieu et al., 1991; Messiaen and Van Cutsem, 1994). To counteract the activity of fungal pectinases, many plants express polygalacturonase-inhibiting proteins and PME inhibitors, which are localized in the cell wall. The role of these proteins in plant defense against B. cinerea has been extensively demonstrated (Powell et al., 2000; Ferrari et al., 2003; Sicilia et al., 2005; Joubert et al., 2006, 2007; Lionetti et al., 2007). The interaction with the inhibitors not only limits the destructive potential of polygalacturonases but also leads to the accumulation of elicitor-active OGAs (De Lorenzo and Ferrari, 2002). How OGAs are perceived by the plant is still unclear, but in view of the diversity of biological activities and structure requirements, they are thought to be recognized through different proteins, including receptor-like kinases, wall-associated kinases, arabinogalactan proteins, and Pro-rich proteins (Côté and Hahn, 1994; Showalter, 2001; Humphrey et al., 2007).Over the past years, the role of abscisic acid (ABA) in plant-pathogen interactions has gained increased attention. ABA is mostly negatively correlated with resistance against phytopathogens through down-regulation of defense responses orchestrated by salicylic acid, jasmonic acid, and ethylene (Mohr and Cahill, 2001; Audenaert et al., 2002; Mauch-Mani and Mauch, 2005; Asselbergh et al., 2008). In tomato, the ABA-deficient mutant sitiens has an enhanced resistance to B. cinerea (Audenaert et al., 2002) that depends on a timely, localized oxidative burst leading to rapid epidermal cell wall fortification and a faster and higher induction of defense-related gene expression upon infection compared with the wild type (Asselbergh et al., 2007). Moreover, basal defense gene expression is higher in this mutant than in the wild type. As this early response is of vital importance for the resistant reaction of tomato against B. cinerea, we investigated whether alterations in cuticle and/or cell wall, which form the first barrier to the invading pathogen, affect resistance. We demonstrate that the sitiens cuticle is more permeable and that permeability is positively correlated with resistance to B. cinerea. Furthermore, differences in pectin composition and rate of methylesterification occur. Together, these data hint at an unanticipated role for extracellular matrix components in the resistance of tomato against B. cinerea and thus shed new light on the largely unexplored interrelationship between the extracellular matrix and plant-pathogen interactions.     

Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance

Stomata control the exchange of CO₂ and water vapor in land plants. Thus, whereas a constant supply of CO₂ is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO₂ concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.Stomata are functionally specialized microscopic pores that control the essential exchange of CO₂ and H₂O with the environment in land plants. Stomata are found on the surfaces of the majority of the aerial parts of plants, rendering them as the main control point regulating the flow of gases between plants and their surrounding atmosphere. Accordingly, the majority of water loss from plants occurs through stomatal pores, allowing plant transpiration and CO₂ absorption for the photosynthetic process (Bergmann and Sack, 2007; Kim et al., 2010). The maintenance of an adequate water balance through stomatal control is crucial to plants because cell expansion and growth require tissues to remain turgid (Sablowski and Carnier Dornelas, 2014), and minor reductions in cell water volume and turgor pressure will therefore compromise both processes (Thompson, 2005). As a result, the high sensitivity of plant tissues to turgor has prompted the use of reverse genetic studies in attempt to engineer plants with improved performance (Cowan and Troughton, 1971; Xiong et al., 2009; Borland et al., 2014; Franks et al., 2015).In most land plants, not only redox signals invoked by shifts in light quality (Busch, 2014) but also the transport of inorganic ions (e.g. K⁺, Cl⁻, and NO⁻₃) as well as metabolites such as the phytohormone abscisic acid (ABA), Suc, and malate, are important players controlling stomatal movements (Hetherington, 2001; Roelfsema and Hedrich, 2005; Pandey et al., 2007; Blatt et al., 2014; Kollist et al., 2014). In this context, although organic acids in plants is known to support numerous and diverse functions both within and beyond cellular metabolism, only recently have we obtained genetic evidence to support that modulation of guard cell malate and fumarate concentration can greatly influence stomatal movements (Nunes-Nesi et al., 2007; Araújo et al., 2011b; Penfield et al., 2012; Medeiros et al., 2015). Notably malate, in particular, has been considered as a key metabolite and one of the most important organic metabolites involved in guard cell movements (Hedrich and Marten, 1993; Fernie and Martinoia, 2009; Meyer et al., 2010). During stomatal aperture, the flux of malate into guard cells coupled with hexoses generated on starch breakdown lead to decreases in the water potential, and consequently, water uptake by the guard cells ultimately opens the stomata pore (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lee et al., 2008). On the other hand, during stomatal closure, malate is believed to be converted into starch, which has no osmotic activity (Penfield et al., 2012) or, alternatively, is released from the guard cells to the surrounding apoplastic space (Lee et al., 2008; Negi et al., 2008; Vahisalu et al., 2008; Meyer et al., 2010).The role of organic acids on the stomatal movements has been largely demonstrated by studies related to malate transport (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In the last decade, two protein families were identified and functionally characterized to be directly involved with organic acid transport at the guard cell plasma membrane and to be required for stomatal functioning (Lee et al., 2008; Meyer et al., 2010; Sasaki et al., 2010). In summary, AtABCB14, a member of the ABC (ATP binding cassette) family, which is involved in malate transport from apoplast to guard cells, was described as a negative modulator of stomatal closure induced by high CO₂ concentration; notably, exogenous application of malate minimizes this response (Lee et al., 2008). In addition, members of a small gene family, which encode the anion channels SLAC1 (slow anion channel 1) and four SLAC1-homologs (SLAHs) in Arabidopsis (Arabidopsis thaliana), have been described to be involved in stomatal movements. SLAC1 is a well-documented S-type anion channel that preferentially transports chloride and nitrate as opposed to malate (Vahisalu et al., 2008, 2010; Geiger et al., 2010; Du et al., 2011; Brandt et al., 2012; Kusumi et al., 2012). Lack of SLAC1 in Arabidopsis and rice (Oryza sativa) culminated in a failure in stomatal closure in response to high CO₂ levels, low relative humidity, and dark conditions (Negi et al., 2008; Vahisalu et al., 2008; Kusumi et al., 2012). Although mutations in AtSLAC1 impair S-type anion channel functions as a whole, the R-type anion channel remained functional (Vahisalu et al., 2008). Indeed, a member of the aluminum-activated malate transporter (ALMT) family, AtALMT12, an R-type anion channel, has been demonstrated to be involved in malate transport, particularly at the plasma membrane of guard cells (Meyer et al., 2010; Sasaki et al., 2010). Although AtALMT12 is a member of ALMT family, it is not activated by aluminum, and therefore Meyer et al. (2010) proposed to rename it as AtQUAC1 (quick-activating anion channel 1; Imes et al., 2013; Mumm et al., 2013). Hereafter, we will follow this nomenclature. Deficiency of a functional AtQUAC1 has been documented to lead to changes in stomatal closure in response to high levels of CO₂, dark, and ABA (Meyer et al., 2010). Taken together, these studies have clearly demonstrated that both S- and R-type anion channels are key modulators of stomatal movements in response to several environmental factors.Despite a vast number of studies involving the above-mentioned anion channels, little information concerning the metabolic changes caused by their impairment is currently available. Such information is important to understand stomatal movements, mainly considering that organic acids, especially the levels of malate in apoplastic/mesophyll cells, have been highlighted as of key importance in leaf metabolism (Fernie and Martinoia, 2009; Araújo et al., 2011a, 2011b; Lawson et al., 2014; Medeiros et al., 2015). Here, we demonstrate that a disruption in the expression of AtQUAC1, which leads to impaired stomatal closure (Meyer et al., 2010), was accompanied by increases in mesophyll conductance (g_m), which is defined as the conductance for the transfer of CO₂ from the intercellular airspaces (C_i) to the sites of carboxylation in the chloroplastic stroma (C_c). By further characterization of atquac1 knockout plants, we demonstrated that reduced diffusive limitations resulted in higher photosynthetic rates and altered respiration that, in turn, led to enhanced biomass accumulation. Overall, the results obtained are discussed both in terms of the importance of organic acid transport in plant cell metabolism and with regard to the contribution that it plays in the regulation of both stomatal function and growth.     

The Tomato odorless-2 Mutant Is Defective in Trichome-Based Production of Diverse Specialized Metabolites and Broad-Spectrum Resistance to Insect Herbivores

Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) produce a wide array of volatile and nonvolatile specialized metabolites. Many of these compounds contribute to the characteristic aroma of tomato foliage and constitute a key part of the language by which plants communicate with other organisms in natural environments. Here, we describe a novel recessive mutation called odorless-2 (od-2) that was identified on the basis of an altered leaf-aroma phenotype. od-2 plants exhibit pleiotrophic phenotypes, including alterations in the morphology, density, and chemical composition of glandular trichomes. Type VI glandular trichomes isolated from od-2 leaves accumulate only trace levels of monoterpenes, sesquiterpenes, and flavonoids. Other foliar defensive compounds, including acyl sugars, glycoalkaloids, and jasmonate-regulated proteinase inhibitors, are produced in od-2 leaves. Growth of od-2 plants under natural field conditions showed that the mutant is highly susceptible to attack by an indigenous flea beetle, Epitrix cucumeris, and the Colorado potato beetle, Leptinotarsa decemlineata. The increased susceptibility of od-2 plants to Colorado potato beetle larvae and to the solanaceous specialist Manduca sexta was verified in no-choice bioassays. These findings indicate that Od-2 is essential for the synthesis of diverse trichome-borne compounds and further suggest that these compounds influence host plant selection and herbivore community composition under natural conditions.The plant epidermal surface provides a formidable protective barrier to invasion by pathogens and arthropod herbivores. Hair-like protuberances, called trichomes, are among the most conspicuous defense-related structures on the aerial epidermis of leaves, stems, and floral organs. Trichomes are typically classified morphologically as being either nonglandular or glandular. Nonglandular trichomes physically impede the movement of small arthropod herbivores on the plant surface. Molecular and ecological studies indicate that trichome density is both a highly adaptive and a functionally important trait for resistance to herbivory (Kennedy, 2003; Kivimaki et al., 2007). In-depth knowledge of the molecular mechanisms that control trichome development in Arabidopsis (Arabidopsis thaliana), which produces unicellular nonglandular trichomes, has provided significant insight into the genetic basis of variation in trichome habit (Marks, 1997; Karkkainen and Agren, 2002; Yoshida et al., 2009).In contrast to our understanding of nonglandular trichomes, much less is known about the development and ecological function of glandular trichomes, many of which are multicellular. These epidermal structures synthesize a diverse array of specialized (i.e. secondary) metabolites that exert toxic or repellent effects on myriad phytophagous animals (Kennedy, 2003; Shepherd et al., 2005; Schilmiller et al., 2008). Rupture of the cuticle upon insect contact releases gland contents, which can rapidly oxidize to form a sticky exudate that physically entraps small insects. Among the major classes of compounds involved in trichome-mediated resistance are terpenoids, alkaloids, flavonoids, and defensive proteins (Shepherd and Wagner, 2007; Schilmiller et al., 2008). Large-scale sequencing of ESTs isolated from purified glands has provided unprecedented insight into the biochemical pathways that operate in glandular trichomes (Lange et al., 2000; Aziz et al., 2005; Wang et al., 2008, 2009; Xie et al., 2008; Schilmiller et al., 2009a; Dai et al., 2010). Many key biosynthetic genes in these pathways have been identified and characterized (Iijima et al., 2004; Falara et al., 2008; Slocombe et al., 2008; Ben-Israel et al., 2009; Marks et al., 2009; Schilmiller et al., 2009a).Cultivated tomato (Solanum lycopersicum) and its wild relatives produce several different types of nonglandular and glandular trichomes on aerial tissues (Luckwill, 1943; Kang et al., 2010). The chemical composition of glandular trichomes varies significantly within and between tomato species (Antonious, 2001; Schilmiller et al., 2008; Besser et al., 2009). Acyl sugars secreted by Solanum pennellii type IV trichomes provide effective resistance to a wide range of insects (Goffreda et al., 1990; Rodriguez et al., 1993; Juvik et al., 1994). Methyl ketone and sesquiterpene derivatives produced in type VI glands of Solanum habrochaites also exert powerful toxic and repellent effects on numerous insect pests (Williams et al., 1980; Maluf et al., 2001; Antonious and Snyder, 2006). Recent studies indicate that trichomes are also an important component of induced anti-insect defenses that are regulated by the plant hormone jasmonate (JA). For example, the density of type VI trichomes on tomato leaves is regulated by the JA pathway (Li et al., 2004; Boughton et al., 2005; Peiffer et al., 2009). JA also plays a role in controlling the accumulation of defense-related terpenoids in type VI glands (Li et al., 2004; van Schie et al., 2007). Recent studies provide evidence that type VI trichomes accumulate JA and may function as sensors for detecting insect movement on the leaf surface (Peiffer et al., 2009). These collective observations highlight the importance of glandular trichomes in shaping plant-insect relations.Our current understanding of the role of trichomes in mediating S. lycopersicum interaction with arthropod herbivores comes mainly from insect bioassays performed under controlled laboratory conditions (Kennedy, 2003; Li et al., 2004; Bleeker et al., 2009; Peiffer et al., 2009; Kang et al., 2010). Much less is known about the ecological relevance of trichomes in tomato plants grown under more natural conditions in the field. Here, we report the characterization of a tomato mutant, odorless-2 (od-2), that was identified on the basis of an altered leaf-aroma phenotype. This mutant exhibits defects in the development and density of glandular trichomes. Detailed chemical analysis of isolated type VI glands showed that od-2 disrupts the production of diverse specialized metabolites, including volatile terpenes and flavonoids. Consistent with important ecological roles for these compounds in host plant selection and defense, we show that od-2 plants are highly susceptible to natural populations of insect herbivores. Our results suggest that trichome-based chemical defenses play a major role in the resistance of cultivated tomato to opportunistic herbivores and also influence herbivore community composition under natural conditions.     

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.     

Decreased Mitochondrial Activities of Malate Dehydrogenase and Fumarase in Tomato Lead to Altered Root Growth and Architecture via Diverse Mechanisms

Transgenic tomato (Solanum lycopersicum) plants in which either mitochondrial malate dehydrogenase or fumarase was antisense inhibited have previously been characterized to exhibit altered photosynthetic metabolism. Here, we demonstrate that these manipulations also resulted in differences in root growth, with both transgenics being characterized by a dramatic reduction of root dry matter deposition and respiratory activity but opposite changes with respect to root area. A range of physiological, molecular, and biochemical experiments were carried out in order to determine whether changes in root morphology were due to altered metabolism within the root itself, alterations in the nature of the transformants'' root exudation, consequences of alteration in the efficiency of photoassimilate delivery to the root, or a combination of these factors. Grafting experiments in which the transformants were reciprocally grafted to wild-type controls suggested that root length and area were determined by the aerial part of the plant but that biomass was not. Despite the transgenic roots displaying alteration in the expression of phytohormone-associated genes, evaluation of the levels of the hormones themselves revealed that, with the exception of gibberellins, they were largely unaltered. When taken together, these combined experiments suggest that root biomass and growth are retarded by root-specific alterations in metabolism and gibberellin contents. These data are discussed in the context of current models of root growth and biomass partitioning.The structure of the plant tricarboxylic acid (TCA) cycle has been established for decades (Beevers, 1961), and in vitro studies have established regulatory properties of many of its component enzymes (Budde and Randall, 1990; Millar and Leaver, 2000; Studart-Guimarães et al., 2005). That said, relatively little is known, as yet, regarding how this important pathway is regulated in vivo (Fernie et al., 2004a; Sweetlove et al., 2007). Indeed, even fundamental questions concerning the degree to which this pathway operates in illuminated leaves (Tcherkez et al., 2005; Nunes-Nesi et al., 2007a) and the influence it has on organic acid levels in fruits (Burger et al., 2003) remain contentious. Furthermore, in contrast to many other pathways of primary metabolism, the TCA cycle has been subjected to relatively few molecular physiological studies. To date, the functions of pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, succinyl-CoA ligase, fumarase, and malate dehydrogenase have been studied via this approach (Landschütze et al., 1995; Carrari et al., 2003; Yui et al., 2003; Nunes-Nesi et al., 2005, 2007a; Lemaitre et al., 2007; Studart-Guimarães et al., 2007); however, several of these studies were relatively cursory. Despite this fact, they generally corroborate one another, with at least two studies providing clear evidence for an important role of the TCA cycle in flower development (Landschütze et al., 1995; Yui et al., 2003) or in the coordination of photosynthetic and respiratory metabolisms of the illuminated leaf (Carrari et al., 2003; Nunes-Nesi et al., 2005, 2007a).In our own studies on tomato (Solanum lycopersicum), we have observed that modulation of fumarase and mitochondrial malate dehydrogenase activities leads to contrasting shoot phenotypes, with the former displaying stunted growth while the later exhibited an enhanced photosynthetic performance (Nunes-Nesi et al., 2005, 2007a). We were able to demonstrate that the stunted-growth phenotype observed in aerial parts of the fumarase plants was a consequence of altered stomatal function (Nunes-Nesi et al., 2007a), whereas the increased photosynthetic performance of the mitochondrial malate dehydrogenase seems likely to be mediated by the alterations in ascorbate metabolism exhibited by these plants (Nunes-Nesi et al., 2005; Urbanczyk-Wochniak et al., 2006). In keeping with the altered rates of photosynthesis in these antisense plants, the fruit yield of fumarase and mitochondrial malate dehydrogenase plants was decreased and increased, respectively. However, the root biomass of both transgenics was significantly reduced (Nunes-Nesi et al., 2005, 2007a). These observations were somewhat surprising given that it is estimated that 30% to 60% of net photosynthate is transported to root organs (Merckx et al., 1986; Nguyen et al., 1999; Singer et al., 2003). When taken together, these results suggest that the root phenotype must result from either an impairment of translocation or a root-specific effect. Neither of these explanations is without precedence, with inhibition of the expression of Suc transporters (Riesmeier et al., 1993; Gottwald et al., 2000) resulting in dramatically impaired root growth while organic acid exudation itself has been implicated in a wide range of root organ functions, including nutrient acquisition (de la Fuente et al., 1997; Imas et al., 1997; Neumann and Römheld, 1999; López-Bucio et al., 2000; Anoop et al., 2003; Delhaize et al., 2004), metal sequestration (Gillooly et al., 1983; de la Fuente et al., 1997; Cramer and Titus, 2001), and microbial proliferation in the rhizosphere (Lugtenberg et al., 1999; Weisskopf et al., 2005). In addition to the putative mechanisms listed above, the TCA cycle could be anticipated to play a vital role in meeting the high energy demands of nitrogen fixation and polymer biosynthesis associated with rapidly growing heterotrophic organs (Pradet and Raymond, 1983; Dieuaide-Noubhani et al., 1997; Stasolla et al., 2003; Deuschle et al., 2006). In keeping with this theory, alteration of the energy status of roots and other heterotrophic tissue has been documented to positively correlate with elevated biomass production (Anekonda, 2001; Regierer et al., 2002; Carrari et al., 2003; Lovas et al., 2003; Geigenberger et al., 2005). Here, we performed a detailed physiological, molecular, and biochemical evaluation of whole plant and root metabolism of the mitochondrial malate dehydrogenase and fumarate antisense tomato lines. In this manner, we broadly assessed biochemical changes in the root, including the levels of several major phytohormones, as well as dissected which characteristics were influenced by aerial parts of the plant. The results obtained are discussed both with respect to the regulation of the TCA cycle per se and within the context of the determination of root morphology and growth.