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.     

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.     

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.     

Patterns of Metabolite Changes Identified from Large-Scale Gene Perturbations in Arabidopsis Using a Genome-Scale Metabolic Network

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.Rational and quantitative assessment of metabolic changes in response to genetic modification (GM) is an open question and in need of innovative solutions. Nontargeted metabolite profiling can detect thousands of compounds, but it is not easy to understand the significance of the changed metabolites in the biochemical and biological context of the organism. To better assess the changes in metabolites from nontargeted metabolomics studies, it is important to examine the changed metabolites in the context of the genome-scale metabolic network of the organism.Metabolomics is a technique that aims to quantify all the metabolites in a biological system (Nikolau and Wurtele, 2007; Nicholson and Lindon, 2008; Roessner and Bowne, 2009). It has been used widely in studies ranging from disease diagnosis (Holmes et al., 2008; DeBerardinis and Thompson, 2012) and drug discovery (Cascante et al., 2002; Kell, 2006) to metabolic reconstruction (Feist et al., 2009; Kim et al., 2012) and metabolic engineering (Keasling, 2010; Lee et al., 2011). Metabolomic studies have demonstrated the possibility of identifying gene functions from changes in the relative concentrations of metabolites (metabotypes or metabolic signatures; Ebbels et al., 2004) in various species including yeast (Saccharomyces cerevisiae; Raamsdonk et al., 2001; Allen et al., 2003), Arabidopsis (Arabidopsis thaliana; Brotman et al., 2011), tomato (Solanum lycopersicum; Schauer et al., 2006), and maize (Zea mays; Riedelsheimer et al., 2012). Metabolomics has also been used to better understand how plants interact with their environments (Field and Lake, 2011), including their responses to biotic and abiotic stresses (Dixon et al., 2006; Arbona et al., 2013), and to predict important agronomic traits (Riedelsheimer et al., 2012). Metabolite profiling has been performed on many plant species, including angiosperms such as Arabidopsis, poplar (Populus trichocarpa), and Catharanthus roseus (Sumner et al., 2003; Rischer et al., 2006), basal land plants such as Selaginella moellendorffii and Physcomitrella patens (Erxleben et al., 2012; Yobi et al., 2012), and Chlamydomonas reinhardtii (Fernie et al., 2012; Davis et al., 2013). With the availability of whole genome sequences of various species, metabolomics has the potential to become a useful tool for elucidating the functions of genes using large-scale systematic analyses (Fiehn et al., 2000; Saito and Matsuda, 2010; Hur et al., 2013).Although metabolomics data have the potential for identifying the roles of genes that are associated with metabolic phenotypes, the biochemical mechanisms that link functions of genes with metabolic phenotypes are still poorly characterized. For example, we do not yet know the principles behind how perturbing the expression of a single gene changes the metabolic system as a whole. Large-scale metabolomics data have provided useful resources for linking phenotypes to genotypes (Fiehn et al., 2000; Roessner et al., 2001; Tikunov et al., 2005; Schauer et al., 2006; Lu et al., 2011; Fukushima et al., 2014). For example, Lu et al. (2011) compared morphological and metabolic phenotypes from more than 5,000 Arabidopsis chloroplast mutants using gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Fukushima et al. (2014) generated metabolite profiles from various characterized and uncharacterized mutant plants and clustered the mutants with similar metabolic phenotypes by conducting multidimensional scaling with quantified metabolic phenotypes. Nonetheless, representation and analysis of such a large amount of data remains a challenge for scientific discovery (Lu et al., 2011). In addition, these studies do not examine the topological and functional characteristics of metabolic changes in the context of a genome-scale metabolic network. To understand the relationship between genotype and metabolic phenotype, we need to investigate the metabolic changes caused by perturbing the expression of a gene in a genome-scale metabolic network perspective, because metabolic pathways are not independent biochemical factories but are components of a complex network (Berg et al., 2002; Merico et al., 2009).Much progress has been made in the last 2 decades to represent metabolism at a genome scale (Terzer et al., 2009). The advances in genome sequencing and emerging fields such as biocuration and bioinformatics enabled the representation of genome-scale metabolic network reconstructions for model organisms (Bassel et al., 2012). Genome-scale metabolic models have been built and applied broadly from microbes to plants. The first step toward modeling a genome-scale metabolism in a plant species started with developing a genome-scale metabolic pathway database for Arabidopsis (AraCyc; Mueller et al., 2003) from reference pathway databases (Kanehisa and Goto, 2000; Karp et al., 2002; Zhang et al., 2010). Genome-scale metabolic pathway databases have been built for several plant species (Mueller et al., 2005; Zhang et al., 2005, 2010; Urbanczyk-Wochniak and Sumner, 2007; May et al., 2009; Dharmawardhana et al., 2013; Monaco et al., 2013, 2014; Van Moerkercke et al., 2013; Chae et al., 2014; Jung et al., 2014). Efforts have been made to develop predictive genome-scale metabolic models using enzyme kinetics and stoichiometric flux-balance approaches (Sweetlove et al., 2008). de Oliveira Dal’Molin et al. (2010) developed a genome-scale metabolic model for Arabidopsis and successfully validated the model by predicting the classical photorespiratory cycle as well as known key differences between redox metabolism in photosynthetic and nonphotosynthetic plant cells. Other genome-scale models have been developed for Arabidopsis (Poolman et al., 2009; Radrich et al., 2010; Mintz-Oron et al., 2012), C. reinhardtii (Chang et al., 2011; Dal’Molin et al., 2011), maize (Dal’Molin et al., 2010; Saha et al., 2011), sorghum (Sorghum bicolor; Dal’Molin et al., 2010), and sugarcane (Saccharum officinarum; Dal’Molin et al., 2010). These predictive models have the potential to be applied broadly in fields such as metabolic engineering, drug target discovery, identification of gene function, study of evolutionary processes, risk assessment of genetically modified crops, and interpretations of mutant phenotypes (Feist and Palsson, 2008; Ricroch et al., 2011).Here, we interrogate the metabotypes caused by 136 single gene perturbations of Arabidopsis by analyzing the relative concentration changes of 1,348 chemically identified metabolites using a reconstructed genome-scale metabolic network. We examine the characteristics of the changed metabolites (the metabolites whose relative concentrations were significantly different in mutants relative to the wild type) in the metabolic network to uncover biological and topological consequences of the perturbed genes.     

Wettability,Polarity, and Water Absorption of Holm Oak Leaves: Effect of Leaf Side and Age

Plant trichomes play important protective functions and may have a major influence on leaf surface wettability. With the aim of gaining insight into trichome structure, composition, and function in relation to water-plant surface interactions, we analyzed the adaxial and abaxial leaf surface of holm oak (Quercus ilex) as a model. By measuring the leaf water potential 24 h after the deposition of water drops onto abaxial and adaxial surfaces, evidence for water penetration through the upper leaf side was gained in young and mature leaves. The structure and chemical composition of the abaxial (always present) and adaxial (occurring only in young leaves) trichomes were analyzed by various microscopic and analytical procedures. The adaxial surfaces were wettable and had a high degree of water drop adhesion in contrast to the highly unwettable and water-repellent abaxial holm oak leaf sides. The surface free energy and solubility parameter decreased with leaf age, with higher values determined for the adaxial sides. All holm oak leaf trichomes were covered with a cuticle. The abaxial trichomes were composed of 8% soluble waxes, 49% cutin, and 43% polysaccharides. For the adaxial side, it is concluded that trichomes and the scars after trichome shedding contribute to water uptake, while the abaxial leaf side is highly hydrophobic due to its high degree of pubescence and different trichome structure, composition, and density. Results are interpreted in terms of water-plant surface interactions, plant surface physical chemistry, and plant ecophysiology.Plant surfaces have an important protecting function against multiple biotic and abiotic stress factors (Riederer, 2006). They may, for example, limit the attack of insects (Eigenbrode and Jetter, 2002) or pathogenic fungi (Gniwotta et al., 2005; Łaźniewska et al., 2012), avoid damage caused by high intensities of UV and visible radiation (Reicosky and Hanover, 1978; Karabourniotis and Bormann, 1999), help to regulate leaf temperature (Ehleringer and Björkman, 1978; Ripley et al., 1999), and chiefly prevent plant organs from dehydration (Riederer and Schreiber, 2001).The epidermis of plants has been found to have a major degree of physical and chemical variability and may often contain specialized cells such as trichomes or stomata (Roth-Nebelsick et al., 2009; Javelle et al., 2011). Most aerial organs are covered with an extracellular and generally lipid-rich layer named the cuticle, which is typically composed of waxes embedded in (intracuticular waxes) or deposited on (epicuticular waxes) a biopolymer matrix of cutin, forming a network of cross-esterified hydroxy C₁₆ and/or C₁₈ fatty acids, and/or cutan, with variable amounts of polysaccharides and phenolics (Domínguez et al., 2011; Yeats and Rose, 2013). Different nano- and/or microscale levels of plant surface sculpturing have been observed by scanning electron microscopy (SEM), generally in relation to the topography of epicuticular waxes, cuticular folds, and epidermal cells (Koch and Barthlott, 2009). Such surface features together with their chemical composition (Khayet and Fernández, 2012) may lead to a high degree of roughness and hydrophobicity (Koch and Barthlott, 2009; Konrad et al., 2012). The interactions of plant surfaces with water have been addressed in some investigations (Brewer et al., 1991; Brewer and Smith, 1997; Pandey and Nagar, 2003; Hanba et al., 2004; Dietz et al., 2007; Holder, 2007a, 2007b; Fernández et al., 2011, 2014; Roth-Nebelsick et al., 2012; Wen et al., 2012; Urrego-Pereira et al., 2013) and are a topic of growing interest for plant ecophysiology (Helliker and Griffiths, 2007; Aryal and Neuner, 2010; Limm and Dawson, 2010; Kim and Lee, 2011; Berry and Smith, 2012; Berry et al., 2013; Rosado and Holder, 2013; Helliker, 2014). On the other hand, the mechanisms of foliar uptake of water and solutes by plant surfaces are still not fully understood (Fernández and Eichert, 2009; Burkhardt and Hunsche, 2013), but they may play an important ecophysiological role (Limm et al., 2009; Johnstone and Dawson, 2010; Adamec, 2013; Berry et al., 2014).The importance of trichomes and pubescent layers on water drop-plant surface interactions and on the subsequent potential water uptake into the organs has been analyzed in some investigations (Fahn, 1986; Brewer et al., 1991; Grammatikopoulos and Manetas, 1994; Brewer and Smith, 1997; Pierce et al., 2001; Kenzo et al., 2008; Fernández et al., 2011, 2014; Burrows et al., 2013). Trichomes are unicellular or multicellular and glandular or nonglandular appendages, which originate from epidermal cells only and develop outwards on the surface of plant organs (Werker, 2000). Nonglandular trichomes are categorized according to their morphology and exhibit a major variability in size, morphology, and function. On the other hand, glandular trichomes are classified by the secretory materials they excrete, accumulate, or absorb (Johnson, 1975; Werker, 2000; Wagner et al., 2004). Trichomes can be often found in xeromorphic leaves and in young organs (Fahn, 1986; Karabourniotis et al., 1995). The occurrence of protecting leaf trichomes has been also reported for Mediterranean species such as holm oak (Quercus ilex; Karabourniotis et al., 1995, 1998; Morales et al., 2002; Karioti et al., 2011; Camarero et al., 2012). There is limited information about the nature of the surface of trichomes, but they are also covered with a cuticle similarly to other epidermal cell types (Fernández et al., 2011, 2014).In this study and using holm oak as a model, we assessed, for the first time, the leaf surface-water relations of the abaxial (always pubescent) versus the adaxial (only pubescent in developing leaves and for a few months) surface, including their capacity to absorb surface-deposited water drops. Based on membrane science methodologies (Fernández et al., 2011; Khayet and Fernández, 2012) and following a new integrative approach, the chemical, physical, and anatomical properties of holm oak leaf surfaces and trichomes were analyzed, with the aim of addressing the following questions. Are young and mature adaxial and abaxial leaf surfaces capable of absorbing water deposited as drops on to the surfaces? Are young and mature abaxial and adaxial leaf surfaces similar in relation to their wettability, hydrophobicity, polarity, work of adhesion (W_a) for water, solubility parameter (δ), and surface free energy (γ)? What is the physical and chemical nature of the adaxial versus the abaxial trichomes, chiefly in relation to young leaves?     

Nontransgenic Genome Modification in Plant Cells

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.Methods for genome editing in plant cells have fallen behind the remarkable progress made in whole-genome sequencing projects. The availability of reliable and efficient methods for genome editing would foster gene discovery and functional gene analyses in model plants and the introduction of novel traits in agriculturally important species (Puchta, 2002; Hanin and Paszkowski, 2003; Reiss, 2003; Porteus, 2009). Genome editing in various species is typically achieved by integrating foreign DNA molecules into the target genome by homologous recombination (HR). Genome editing by HR is routine in yeast (Saccharomyces cerevisiae) cells (Scherer and Davis, 1979) and has been adapted for other species, including Drosophila, human cell lines, various fungal species, and mouse embryonic stem cells (Baribault and Kemler, 1989; Venken and Bellen, 2005; Porteus, 2007; Hall et al., 2009; Laible and Alonso-González, 2009; Tenzen et al., 2009). In plants, however, foreign DNA molecules, which are typically delivered by direct gene-transfer methods (e.g. Agrobacterium and microbombardment of plasmid DNA), often integrate into the target cell genome via nonhomologous end joining (NHEJ) and not HR (Ray and Langer, 2002; Britt and May, 2003).Various methods have been developed to indentify and select for rare site-specific foreign DNA integration events or to enhance the rate of HR-mediated DNA integration in plant cells. Novel T-DNA molecules designed to support strong positive- and negative-selection schemes (e.g. Thykjaer et al., 1997; Terada et al., 2002), altering the plant DNA-repair machinery by expressing yeast chromatin remodeling protein (Shaked et al., 2005), and PCR screening of large numbers of transgenic plants (Kempin et al., 1997; Hanin et al., 2001) are just a few of the experimental approaches used to achieve HR-mediated gene targeting in plant species. While successful, these approaches, and others, have resulted in only a limited number of reports describing the successful implementation of HR-mediated gene targeting of native and transgenic sequences in plant cells (for review, see Puchta, 2002; Hanin and Paszkowski, 2003; Reiss, 2003; Porteus, 2009; Weinthal et al., 2010).HR-mediated gene targeting can potentially be enhanced by the induction of genomic double-strand breaks (DSBs). In their pioneering studies, Puchta et al. (1993, 1996) showed that DSB induction by the naturally occurring rare-cutting restriction enzyme I-SceI leads to enhanced HR-mediated DNA repair in plants. Expression of I-SceI and another rare-cutting restriction enzyme (I-CeuI) also led to efficient NHEJ-mediated site-specific mutagenesis and integration of foreign DNA molecules in plants (Salomon and Puchta, 1998; Chilton and Que, 2003; Tzfira et al., 2003). Naturally occurring rare-cutting restriction enzymes thus hold great promise as a tool for genome editing in plant cells (Carroll, 2004; Pâques and Duchateau, 2007). However, their wide application is hindered by the tedious and next to impossible reengineering of such enzymes for novel DNA-target specificities (Pâques and Duchateau, 2007).A viable alternative to the use of rare-cutting restriction enzymes is the zinc finger nucleases (ZFNs), which have been used for genome editing in a wide range of eukaryotic species, including plants (e.g. Bibikova et al., 2001; Porteus and Baltimore, 2003; Lloyd et al., 2005; Urnov et al., 2005; Wright et al., 2005; Beumer et al., 2006; Moehle et al., 2007; Santiago et al., 2008; Shukla et al., 2009; Tovkach et al., 2009; Townsend et al., 2009; Osakabe et al., 2010; Petolino et al., 2010; Zhang et al., 2010). Here too, ZFNs have been used to enhance DNA integration via HR (e.g. Shukla et al., 2009; Townsend et al., 2009) and as an efficient tool for the induction of site-specific mutagenesis (e.g. Lloyd et al., 2005; Zhang et al., 2010) in plant species. The latter is more efficient and simpler to implement in plants as it does not require codelivery of both ZFN-expressing and donor DNA molecules and it relies on NHEJ—the dominant DNA-repair machinery in most plant species (Ray and Langer, 2002; Britt and May, 2003).ZFNs are artificial restriction enzymes composed of a fusion between an artificial Cys₂His₂ zinc-finger protein DNA-binding domain and the cleavage domain of the FokI endonuclease. The DNA-binding domain of ZFNs can be engineered to recognize a variety of DNA sequences (for review, see Durai et al., 2005; Porteus and Carroll, 2005; Carroll et al., 2006). The FokI endonuclease domain functions as a dimer, and digestion of the target DNA requires proper alignment of two ZFN monomers at the target site (Durai et al., 2005; Porteus and Carroll, 2005; Carroll et al., 2006). Efficient and coordinated expression of both monomers is thus required for the production of DSBs in living cells. Transient ZFN expression, by direct gene delivery, is the method of choice for targeted mutagenesis in human and animal cells (e.g. Urnov et al., 2005; Beumer et al., 2006; Meng et al., 2008). Among the different methods used for high and efficient transient ZFN delivery in animal and human cell lines are plasmid injection (Morton et al., 2006; Foley et al., 2009), direct plasmid transfer (Urnov et al., 2005), the use of integrase-defective lentiviral vectors (Lombardo et al., 2007), and mRNA injection (Takasu et al., 2010).In plant species, however, efficient and strong gene expression is often achieved by stable gene transformation. Both transient and stable ZFN expression have been used in gene-targeting experiments in plants (Lloyd et al., 2005; Wright et al., 2005; Maeder et al., 2008; Cai et al., 2009; de Pater et al., 2009; Shukla et al., 2009; Tovkach et al., 2009; Townsend et al., 2009; Osakabe et al., 2010; Petolino et al., 2010; Zhang et al., 2010). In all cases, direct gene-transformation methods, using polyethylene glycol, silicon carbide whiskers, or Agrobacterium, were deployed. Thus, while mutant plants and tissues could be recovered, potentially without any detectable traces of foreign DNA, such plants were generated using a transgenic approach and are therefore still likely to be classified as transgenic. Furthermore, the recovery of mutants in many cases is also dependent on the ability to regenerate plants from protoplasts, a procedure that has only been successfully applied in a limited number of plant species. Therefore, while ZFN technology is a powerful tool for site-specific mutagenesis, its wider implementation for plant improvement may be somewhat limited, both by its restriction to certain plant species and by legislative restrictions imposed on transgenic plants.Here we describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated plants. Our approach is based on the use of a novel Tobacco rattle virus (TRV)-based expression system, which is capable of systemically infecting its host and spreading into a variety of tissues and cells of intact plants, including developing buds and regenerating tissues. We traced the indirect ZFN delivery in infected plants by activation of a mutated reporter gene and we demonstrate that this approach can be used to recover mutated plants.