Extracellular DNA Is Required for Root Tip Resistance to Fungal Infection

Plant defense involves a complex array of biochemical interactions, many of which occur in the extracellular environment. The apical 1- to 2-mm root tip housing apical and root cap meristems is resistant to infection by most pathogens, so growth and gravity sensing often proceed normally even when other sites on the root are invaded. The mechanism of this resistance is unknown but appears to involve a mucilaginous matrix or “slime” composed of proteins, polysaccharides, and detached living cells called “border cells.” Here, we report that extracellular DNA (exDNA) is a component of root cap slime and that exDNA degradation during inoculation by a fungal pathogen results in loss of root tip resistance to infection. Most root tips (>95%) escape infection even when immersed in inoculum from the root-rotting pathogen Nectria haematococca. By contrast, 100% of inoculated root tips treated with DNase I developed necrosis. Treatment with BAL31, an exonuclease that digests DNA more slowly than DNase I, also resulted in increased root tip infection, but the onset of infection was delayed. Control root tips or fungal spores treated with nuclease alone exhibited normal morphology and growth. Pea (Pisum sativum) root tips incubated with [³²P]dCTP during a 1-h period when no cell death occurs yielded root cap slime containing ³²P-labeled exDNA. Our results suggest that exDNA is a previously unrecognized component of plant defense, an observation that is in accordance with the recent discovery that exDNA from white blood cells plays a key role in the vertebrate immune response against microbial pathogens.Root diseases caused by soil-borne plant pathogens are a perennial source of crop loss worldwide (Bruehl, 1986; Curl and Truelove, 1986). These diseases are of increasing concern, as pesticides like methyl bromide are removed from the market due to environmental concerns (Gilreath et al., 2005). One possible alternative means of crop protection is to exploit natural mechanisms of root disease resistance (Nelson, 1990; Goswami and Punja, 2008; Shittu et al., 2009). Direct observation of root systems under diverse conditions has revealed that root tips, in general, are resistant to infection even when lesions are initiated elsewhere on the same plant root (Foster et al., 1983; Bruehl, 1986; Curl and Truelove, 1986; Smith et al., 1992; Gunawardena et al., 2005; Wen et al., 2007). This form of disease resistance is important for crop production because root growth and its directional movement in response to gravity, water, and other signals can proceed normally as long as the root tip is not invaded. The 1- to 2-mm apical region of roots houses the root meristems required for root growth and cap development, and when infection does occur, root development ceases irreversibly within a few hours even in the absence of severe necrosis (Gunawardena and Hawes, 2002). Mechanisms underlying root tip resistance to infection are unclear, but the phenomenon appears to involve root cap “slime,” a mucilaginous matrix produced by the root cap (Morré et al., 1967; Rougier et al., 1979; Foster, 1982; Chaboud, 1983; Guinel and McCully, 1986; Moody et al., 1988; Knee et al., 2001; Barlow, 2003; Iijima et al., 2008). Within the root cap slime of cereals, legumes, and most other crop species are specialized populations of living cells called root “border cells” (Supplemental Fig. S1; Hawes et al., 2000). Border cell numbers increase in response to pathogens and toxins such as aluminum, and the cell populations maintain a high rate of metabolic activity even after detachment from the root cap periphery (Brigham et al., 1995; Miyasaka and Hawes, 2000).As border cells detach from roots of cereals and legumes, a complex of more than 100 proteins, termed the root cap secretome, is synthesized and exported from living cells into the matrix ensheathing the root tip (Brigham et al., 1995). The profile of secreted proteins changes in response to challenge with soil-borne bacteria (De-la-Peña et al., 2008). In pea (Pisum sativum), root tip resistance to infection is abolished in response to proteolytic degradation of the root cap secretome (Wen et al., 2007). In addition to an array of antimicrobial enzymes and other proteins known to be components of the extracellular matrix and apoplast of higher plants, the DNA-binding protein histone H4 unexpectedly was found to be present among the secreted proteins (Wen et al., 2007). One explanation for the presence of histone is global leakage of material from disrupted nuclei in dead cells, but no cell death occurs during delivery of the secretome (Brigham et al., 1995; Wen et al., 2007). An alternative explanation for the presence of a secreted DNA-binding protein is that extracellular DNA (exDNA) also is present in root cap slime.exDNA has long been known to be a component of slimy biological matrices ranging from purulent localized human infections to bacterial capsules, biofilms, and snail exudate (Sherry and Goeller, 1950; Leuchtenberger and Schrader, 1952; Braun and Whallon, 1954; Smithies and Gibbons, 1955; Catlin, 1956; Fahy et al., 1993; Allesen-Holm et al., 2006; Spoering and Gilmore, 2006; Qin et al., 2007; Izano et al., 2008). Specialized white blood cells in humans and other species including fish recently have been shown to deploy a complex neutrophil extracellular “trap” (NET), composed of DNA and a collection of enzymes, in response to infection (Brinkmann et al., 2004; Brinkmann and Zychlinsky, 2007; Palić et al., 2007; Wartha et al., 2007; Yousefi et al., 2008). NETs appear to kill bacterial, fungal, and protozoan pathogens by localizing them within a matrix of antimicrobial peptides and proteins (Urban et al., 2006; Wartha et al., 2007; Guimaraes-Costa et al., 2009). Several extracellular peptides and proteins implicated in neutrophil function, including histone, also are present within the pea root cap secretome (Wen et al., 2007). exDNA linked with extracellular histone is a structural component of NETs, and treatment with DNase destroys NET integrity and function (Wartha et al., 2007). Moreover, human pathogens including group A Streptococcus and Streptococcus pneumoniae release extracellular DNase (Sherry and Goeller, 1950). When these activities are eliminated by mutagenesis of the encoding genes, bacteria lose their normal ability to escape the NET and multiply at the site of infection (Sumby et al., 2005; Buchanan et al., 2006). Here, we report that, in addition to histone and other secretome proteins, exDNA also is a component of root cap slime. When this exDNA is digested enzymatically, root tip resistance to infection is abolished.     

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.     

Focus on Ethylene: Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.Aerenchyma formation is a morphological adaptation of plants to complete submergence and waterlogging of the soil, and facilitates internal gas diffusion (Armstrong, 1979; Jackson and Armstrong, 1999; Colmer, 2003; Voesenek et al., 2006; Bailey-Serres and Voesenek, 2008; Licausi and Perata, 2009; Sauter, 2013; Voesenek and Bailey-Serres, 2015). To adapt to waterlogging in soil, rice (Oryza sativa) develops lysigenous aerenchyma in shoots (Matsukura et al., 2000; Colmer and Pedersen, 2008; Steffens et al., 2011) and roots (Jackson et al., 1985b; Justin and Armstrong, 1991; Kawai et al., 1998), which is formed by programmed cell death and subsequent lysis of some cortical cells (Jackson and Armstrong, 1999; Evans, 2004; Yamauchi et al., 2013). In rice roots, lysigenous aerenchyma is constitutively formed under aerobic conditions (Jackson et al., 1985b), and its formation is further induced under oxygen-deficient conditions (Colmer et al., 2006; Shiono et al., 2011). The former and latter are designated constitutive and inducible lysigenous aerenchyma formation, respectively (Colmer and Voesenek, 2009). The gaseous plant hormone ethylene regulates adaptive growth responses of plants to submergence (Voesenek and Blom, 1989; Voesenek et al., 1993; Visser et al., 1996a,b; Lorbiecke and Sauter, 1999; Hattori et al., 2009; Steffens and Sauter, 2009; van Veen et al., 2013). Ethylene also induces lysigenous aerenchyma formation in roots of some gramineous plants (Drew et al., 2000; Shiono et al., 2008). The treatment of roots with ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid [ACC]) stimulates aerenchyma formation in rice (Justin and Armstrong, 1991; Colmer et al., 2006; Yukiyoshi and Karahara, 2014), maize (Zea mays; Drew et al., 1981; Jackson et al., 1985a; Takahashi et al., 2015), and wheat (Triticum aestivum; Yamauchi et al., 2014a,b). Moreover, treatment of roots with inhibitors of ethylene action or ethylene biosynthesis effectively blocks aerenchyma formation under hypoxic conditions in maize (Drew et al., 1981; Konings, 1982; Jackson et al., 1985a; Rajhi et al., 2011).Ethylene biosynthesis is accomplished by two main successive enzymatic reactions: conversion of S-adenosyl-Met to ACC by 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and conversion of ACC to ethylene by 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; Yang and Hoffman, 1984). The activities of both enzymes are enhanced during aerenchyma formation under hypoxic conditions in maize root (He et al., 1996). Since the ACC content in roots of maize is increased by oxygen deficiency and is strongly correlated with ethylene production (Atwell et al., 1988), ACC biosynthesis is essential for ethylene production during aerenchyma formation in roots. In fact, exogenously supplied ACC induced ethylene production in roots of maize (Drew et al., 1979; Konings, 1982; Atwell et al., 1988) and wheat (Yamauchi et al., 2014b), even under aerobic conditions. Ethylene production in plants is inversely related to oxygen concentration (Yang and Hoffman, 1984). Under anoxic conditions, the oxidation of ACC to ethylene by ACO, which requires oxygen, is almost completely repressed (Yip et al., 1988; Tonutti and Ramina, 1991). Indeed, anoxic conditions stimulate neither ethylene production nor aerenchyma formation in maize adventitious roots (Drew et al., 1979). Therefore, it is unlikely that the root tissues forming inducible aerenchyma are anoxic, and that the ACO-mediated step is repressed. Moreover, aerenchyma is constitutively formed in rice roots even under aerobic conditions (Jackson et al., 1985b), and thus, after the onset of waterlogging, oxygen can be immediately supplied to the apical regions of roots through the constitutively formed aerenchyma.Very-long-chain fatty acids (VLCFAs; ≥20 carbons) are major constituents of sphingolipids, cuticular waxes, and suberin in plants (Franke and Schreiber, 2007; Kunst and Samuels, 2009). In addition to their structural functions, VLCFAs directly or indirectly participate in several physiological processes (Zheng et al., 2005; Reina-Pinto et al., 2009; Roudier et al., 2010; Ito et al., 2011; Nobusawa et al., 2013; Tsuda et al., 2013), including the regulation of ethylene biosynthesis (Qin et al., 2007). During fiber cell elongation in cotton ovules, ethylene biosynthesis is enhanced by treatment with saturated VLCFAs, especially 24-carbon fatty acids, and is suppressed by an inhibitor of VLCFA biosynthesis (Qin et al., 2007). The first rate-limiting step in VLCFA biosynthesis is condensation of acyl-CoA with malonyl-CoA by β-ketoacyl-CoA synthase (KCS; Joubès et al., 2008). KCS enzymes are thought to determine the substrate and tissue specificities of fatty acid elongation (Joubès et al., 2008). The Arabidopsis (Arabidopsis thaliana) genome has 21 KCS genes (Joubès et al., 2008). In the Arabidopsis cut1 mutant, which has a defect in the gene encoding CUT1 that is required for cuticular wax production (i.e. one of the KCS genes), the expression of AtACO genes and growth of root cells were reduced when compared with the wild type (Qin et al., 2007). Furthermore, expression of the AtACO genes was rescued by exogenously supplied saturated VLCFAs (Qin et al., 2007). These observations imply that VLCFAs or their derivatives work as regulatory factors for gene expression during some physiological processes in plants.reduced culm number1 (rcn1) was first identified as a rice mutant with a low tillering rate in a paddy field (Takamure and Kinoshita, 1985; Yasuno et al., 2007). The rcn1 (rcn1-2) mutant has a single nucleotide substitution in the gene encoding a member of the ATP-binding cassette (ABC) transporter subfamily G, RCN1/OsABCG5, causing an Ala-684Pro substitution (Yasuno et al., 2009). The mutation results in several mutant phenotypes, although the substrates of RCN1/OsABCG5 have not been determined (Ureshi et al., 2012; Funabiki et al., 2013; Matsuda et al., 2014). We previously found that the rcn1 mutant has abnormal root morphology, such as shorter root length and brownish appearance of roots, under stagnant (deoxygenated) conditions (which mimics oxygen-deficient conditions in waterlogged soils). We also found that the rcn1 mutant accumulates less of the major suberin monomers originating from VLCFAs in the outer part of adventitious roots, and this results in a reduction of a functional apoplastic barrier in the root hypodermis (Shiono et al., 2014a).The objective of this study was to elucidate the molecular basis of inducible aerenchyma formation. To this end, we examined lysigenous aerenchyma formation and ACC, ethylene, and VLCFA accumulation and their biosyntheses in rcn1 roots. Based on the results of these studies, we propose that VLCFAs are involved in inducible aerenchyma formation through the enhancement of ethylene biosynthesis in rice roots.     

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.