Impaired Auxin Biosynthesis in the defective endosperm18 Mutant Is Due to Mutational Loss of Expression in the ZmYuc1 Gene Encoding Endosperm-Specific YUCCA1 Protein in Maize

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.The phytohormone auxin, as a signaling molecule, controls and coordinates numerous aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most predominant in planta auxin and regulates diverse processes, including cell division, cell elongation, formation and maintenance of meristems, vascular tissue differentiation, phototropism, flowering, and endosperm and embryo growth in developing seeds (Davies, 2010). Despite its critical roles, basic components of IAA biosynthesis are poorly understood, compared with transport and signaling aspects. However, the use of appropriate genetic screens in Arabidopsis (Arabidopsis thaliana) and the use of sensitive analytical tools in the identification of metabolic intermediates have led to significant advancements toward a better understanding of biosynthesis. Currently, there are four proposed Trp-dependent pathways of de novo IAA biosynthesis in plants (Woodward and Bartel, 2005; Pollmann et al., 2009; Normanly, 2010); of these, indole-3-pyruvic acid (IPA) was recently suggested to predominate in Arabidopsis (Mashiguchi et al., 2011; Won et al., 2011; Stepanova et al., 2011) and in pea (Pisum sativum) seeds (Tivendale et al., 2012).The first step of the IPA pathway involves the conversion of Trp to IPA by Trp aminotransferases, first demonstrated in Arabidopsis by Stepanova et al. (2008) and Tao et al. (2008). The mutants of Arabidopsis Trp-aminotransferase (taa1) are defective in shade avoidance syndrome due to reduced levels of IAA. In maize (Zea mays), orthologs of the TAA1 gene include an endosperm-specific gene, ZmTar1 (for TA-Related1; Chourey et al., 2010) and Vanishing tassel2 (Vt2), which encode grass-specific Trp aminotransferases (Phillips et al., 2011). The vt2 mutant is marked by severe developmental abnormality, attributed to approximately 60% reduced IAA levels in the mutant seedlings. These results are significant in showing the functionality of the TAR enzyme and the IPA pathway in IAA biosynthesis in maize. Recently, it was suggested that the IPA pathway also involves the YUCCA (YUC) genes, which encode flavin monooxygenases that are now believed to catalyze the conversion of IPA to IAA (Phillips et al., 2011; Mashiguchi et al., 2011; Stepanova et al., 2011; Won et al., 2011; Kriechbaumer et al., 2012). This is based in part on evidence that the Arabidopsis YUC2 protein, expressed in Escherichia coli, converted IPA to IAA in vitro (Mashiguchi et al., 2011). In Arabidopsis, three Yuc genes, Yuc-1, -4, and -10, are expressed in an overlapping fashion in developing seeds and are considered essential in embryogenesis (Cheng et al., 2007); however, single or double mutant yuc1 yuc4 do not show detectable defects in embryogenesis or seed phenotype.Orthologs of the AtYuc genes are now described in several plant groups, including maize (Gallavotti et al., 2008; LeClere et al., 2010). The first Yuc-like gene in maize was isolated through positional cloning of the sparse inflorescence1 (spi1) locus; spi1 mutants showed auxin-deficient-related characteristics in the male inflorescence (Gallavotti et al., 2008). The second gene, ZmYuc1, is highly endosperm specific and its temporal expression pattern coincided with IAA biosynthesis at various stages of seed development (LeClere et al., 2010). In pea, two highly similar PsYuc-like genes, PsYuc1 and PsYuc2, showed seed- and root-specific expression, respectively (Tivendale et al., 2010). Metabolic studies in pea, however, showed that only the roots but not seeds can metabolize Trp to IAA through the proposed TAM pathway (Quittenden et al., 2009; Tivendale et al., 2010).In contrast with many studies on auxin-related mutants that affect vegetative parts of the plant, very limited data are available on auxin mutants affecting seed development, even though seeds accumulate higher levels of IAA than any other tissue of the plant. In maize, endosperm synthesizes nearly 100- to 500-fold higher levels of IAA relative to vegetative tissues (Jensen and Bandurski, 1994; LeClere et al., 2008; Phillips et al., 2011). The significance of the large abundance of IAA in developing endosperm remains to be understood, except that it may be used during the very early stages of seed germination because >90% of the total IAA is in biologically inactive conjugated storage form (Jensen and Bandurski, 1994; LeClere et al., 2008). Such a role in germination is consistent with the fact that there are very few viable seed mutants reported in maize that are linked to IAA deficiency, although single-locus recessive mutants (defective kernels [dek]) with various abnormalities in either embryo or endosperm development and with low IAA levels (measured by ELISA) were reported by Lur and Setter (1993). It is significant in this regard that a viable defective endosperm-B18 (hereafter, de18) was identified as associated with IAA deficiency (Torti et al., 1986). Although not quantified by mass spectrometry, de18 endosperms contained total IAA levels (including conjugates) in the range of 6% to 0.3% of the wild type B37 (hereafter, De18) values, during 12 to 40 d after pollination (DAP). At the early stages, the mutant seed phenotype is <50% of the wild type in seed weight, and throughout seed development, mutant seeds are reduced in kernel size and accumulate less dry matter. Furthermore, application of the synthetic auxin, naphthalene acetic acid, to developing seeds largely rescued the de18 mutant phenotype, indicating impairment in IAA biosynthesis or metabolism as the cause of the phenotypic changes (Torti et al., 1986). Recent cellular-level studies also indicated the IAA deficiency of the de18 endosperm; high levels of immunosignal for IAA were detected in the basal endosperm transfer layer (BETL), aleurone, embryo surrounding region domains, and maternal chalazal tissue in De18 but not in the mutant (Forestan et al., 2010). Overall, the maize de18 and the pea tar2 (Tivendale et al., 2012) mutants are thus far the only seed-specific viable mutants linked to auxin deficiency. The objective of this study is to further extend our knowledge on IAA deficit in the de18 kernels, to specifically analyze temporal expression of two major IAA biosynthetic genes and to elucidate the possible molecular basis of the mutant. Our collective data, based on the cloning and sequencing of ZmYuc1 and on mapping studies, indicate that ZmYuc1 and De18 are tightly associated and that the aberrant YUC1 protein in de18 is the causal basis of IAA deficiency and the small seed phenotype in that mutant.     

Spore Density Determines Infection Strategy by the Plant Pathogenic Fungus Plectosphaerella cucumerina

Necrotrophic and biotrophic pathogens are resisted by different plant defenses. While necrotrophic pathogens are sensitive to jasmonic acid (JA)-dependent resistance, biotrophic pathogens are resisted by salicylic acid (SA)- and reactive oxygen species (ROS)-dependent resistance. Although many pathogens switch from biotrophy to necrotrophy during infection, little is known about the signals triggering this transition. This study is based on the observation that the early colonization pattern and symptom development by the ascomycete pathogen Plectosphaerella cucumerina (P. cucumerina) vary between inoculation methods. Using the Arabidopsis (Arabidopsis thaliana) defense response as a proxy for infection strategy, we examined whether P. cucumerina alternates between hemibiotrophic and necrotrophic lifestyles, depending on initial spore density and distribution on the leaf surface. Untargeted metabolome analysis revealed profound differences in metabolic defense signatures upon different inoculation methods. Quantification of JA and SA, marker gene expression, and cell death confirmed that infection from high spore densities activates JA-dependent defenses with excessive cell death, while infection from low spore densities induces SA-dependent defenses with lower levels of cell death. Phenotyping of Arabidopsis mutants in JA, SA, and ROS signaling confirmed that P. cucumerina is differentially resisted by JA- and SA/ROS-dependent defenses, depending on initial spore density and distribution on the leaf. Furthermore, in situ staining for early callose deposition at the infection sites revealed that necrotrophy by P. cucumerina is associated with elevated host defense. We conclude that P. cucumerina adapts to early-acting plant defenses by switching from a hemibiotrophic to a necrotrophic infection program, thereby gaining an advantage of immunity-related cell death in the host.Plant pathogens are often classified as necrotrophic or biotrophic, depending on their infection strategy (Glazebrook, 2005; Nishimura and Dangl, 2010). Necrotrophic pathogens kill living host cells and use the decayed plant tissue as a substrate to colonize the plant, whereas biotrophic pathogens parasitize living plant cells by employing effector molecules that suppress the host immune system (Pel and Pieterse, 2013). Despite this binary classification, the majority of pathogenic microbes employ a hemibiotrophic infection strategy, which is characterized by an initial biotrophic phase followed by a necrotrophic infection strategy at later stages of infection (Perfect and Green, 2001). The pathogenic fungi Magnaporthe grisea, Sclerotinia sclerotiorum, and Mycosphaerella graminicola, the oomycete Phytophthora infestans, and the bacterial pathogen Pseudomonas syringae are examples of hemibiotrophic plant pathogens (Perfect and Green, 2001; Koeck et al., 2011; van Kan et al., 2014; Kabbage et al., 2015).Despite considerable progress in our understanding of plant resistance to necrotrophic and biotrophic pathogens (Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013), recent debate highlights the dynamic and complex interplay between plant-pathogenic microbes and their hosts, which is raising concerns about the use of infection strategies as a static tool to classify plant pathogens. For instance, the fungal genus Botrytis is often labeled as an archetypal necrotroph, even though there is evidence that it can behave as an endophytic fungus with a biotrophic lifestyle (van Kan et al., 2014). The rice blast fungus Magnaporthe oryzae, which is often classified as a hemibiotrophic leaf pathogen (Perfect and Green, 2001; Koeck et al., 2011), can adopt a purely biotrophic lifestyle when infecting root tissues (Marcel et al., 2010). It remains unclear which signals are responsible for the switch from biotrophy to necrotrophy and whether these signals rely solely on the physiological state of the pathogen, or whether host-derived signals play a role as well (Kabbage et al., 2015).The plant hormones salicylic acid (SA) and jasmonic acid (JA) play a central role in the activation of plant defenses (Glazebrook, 2005; Pieterse et al., 2009, 2012). The first evidence that biotrophic and necrotrophic pathogens are resisted by different immune responses came from Thomma et al. (1998), who demonstrated that Arabidopsis (Arabidopsis thaliana) genotypes impaired in SA signaling show enhanced susceptibility to the biotrophic pathogen Hyaloperonospora arabidopsidis (formerly known as Peronospora parastitica), while JA-insensitive genotypes were more susceptible to the necrotrophic fungus Alternaria brassicicola. In subsequent years, the differential effectiveness of SA- and JA-dependent defense mechanisms has been confirmed in different plant-pathogen interactions, while additional plant hormones, such as ethylene, abscisic acid (ABA), auxins, and cytokinins, have emerged as regulators of SA- and JA-dependent defenses (Bari and Jones, 2009; Cao et al., 2011; Pieterse et al., 2012). Moreover, SA- and JA-dependent defense pathways have been shown to act antagonistically on each other, which allows plants to prioritize an appropriate defense response to attack by biotrophic pathogens, necrotrophic pathogens, or herbivores (Koornneef and Pieterse, 2008; Pieterse et al., 2009; Verhage et al., 2010).In addition to plant hormones, reactive oxygen species (ROS) play an important regulatory role in plant defenses (Torres et al., 2006; Lehmann et al., 2015). Within minutes after the perception of pathogen-associated molecular patterns, NADPH oxidases and apoplastic peroxidases generate early ROS bursts (Torres et al., 2002; Daudi et al., 2012; O’Brien et al., 2012), which activate downstream defense signaling cascades (Apel and Hirt, 2004; Torres et al., 2006; Miller et al., 2009; Mittler et al., 2011; Lehmann et al., 2015). ROS play an important regulatory role in the deposition of callose (Luna et al., 2011; Pastor et al., 2013) and can also stimulate SA-dependent defenses (Chaouch et al., 2010; Yun and Chen, 2011; Wang et al., 2014; Mammarella et al., 2015). However, the spread of SA-induced apoptosis during hyperstimulation of the plant immune system is contained by the ROS-generating NADPH oxidase RBOHD (Torres et al., 2005), presumably to allow for the sufficient generation of SA-dependent defense signals from living cells that are adjacent to apoptotic cells. Nitric oxide (NO) plays an additional role in the regulation of SA/ROS-dependent defense (Trapet et al., 2015). This gaseous molecule can stimulate ROS production and cell death in the absence of SA while preventing excessive ROS production at high cellular SA levels via S-nitrosylation of RBOHD (Yun et al., 2011). Recently, it was shown that pathogen-induced accumulation of NO and ROS promotes the production of azelaic acid, a lipid derivative that primes distal plants for SA-dependent defenses (Wang et al., 2014). Hence, NO, ROS, and SA are intertwined in a complex regulatory network to mount local and systemic resistance against biotrophic pathogens. Interestingly, pathogens with a necrotrophic lifestyle can benefit from ROS/SA-dependent defenses and associated cell death (Govrin and Levine, 2000). For instance, Kabbage et al. (2013) demonstrated that S. sclerotiorum utilizes oxalic acid to repress oxidative defense signaling during initial biotrophic colonization, but it stimulates apoptosis at later stages to advance necrotrophic colonization. Moreover, SA-induced repression of JA-dependent resistance not only benefits necrotrophic pathogens but also hemibiotrophic pathogens after having switched from biotrophy to necrotrophy (Glazebrook, 2005; Pieterse et al., 2009, 2012).Plectosphaerella cucumerina ((P. cucumerina, anamorph Plectosporum tabacinum) anamorph Plectosporum tabacinum) is a filamentous ascomycete fungus that can survive saprophytically in soil by decomposing plant material (Palm et al., 1995). The fungus can cause sudden death and blight disease in a variety of crops (Chen et al., 1999; Harrington et al., 2000). Because P. cucumerina can infect Arabidopsis leaves, the P. cucumerina-Arabidopsis interaction has emerged as a popular model system in which to study plant defense reactions to necrotrophic fungi (Berrocal-Lobo et al., 2002; Ton and Mauch-Mani, 2004; Carlucci et al., 2012; Ramos et al., 2013). Various studies have shown that Arabidopsis deploys a wide range of inducible defense strategies against P. cucumerina, including JA-, SA-, ABA-, and auxin-dependent defenses, glucosinolates (Tierens et al., 2001; Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014), callose deposition (García-Andrade et al., 2011; Gamir et al., 2012, 2014; Sánchez-Vallet et al., 2012), and ROS (Tierens et al., 2002; Sánchez-Vallet et al., 2010; Barna et al., 2012; Gamir et al., 2012, 2014; Pastor et al., 2014). Recent metabolomics studies have revealed large-scale metabolic changes in P. cucumerina-infected Arabidopsis, presumably to mobilize chemical defenses (Sánchez-Vallet et al., 2010; Gamir et al., 2014; Pastor et al., 2014). Furthermore, various chemical agents have been reported to induce resistance against P. cucumerina. These chemicals include β-amino-butyric acid, which primes callose deposition and SA-dependent defenses, benzothiadiazole (BTH or Bion; Görlach et al., 1996; Ton and Mauch-Mani, 2004), which activates SA-related defenses (Lawton et al., 1996; Ton and Mauch-Mani, 2004; Gamir et al., 2014; Luna et al., 2014), JA (Ton and Mauch-Mani, 2004), and ABA, which primes ROS and callose deposition (Ton and Mauch-Mani, 2004; Pastor et al., 2013). However, among all these studies, there is increasing controversy about the exact signaling pathways and defense responses contributing to plant resistance against P. cucumerina. While it is clear that JA and ethylene contribute to basal resistance against the fungus, the exact roles of SA, ABA, and ROS in P. cucumerina resistance vary between studies (Thomma et al., 1998; Ton and Mauch-Mani, 2004; Sánchez-Vallet et al., 2012; Gamir et al., 2014).This study is based on the observation that the disease phenotype during P. cucumerina infection differs according to the inoculation method used. We provide evidence that the fungus follows a hemibiotrophic infection strategy when infecting from relatively low spore densities on the leaf surface. By contrast, when challenged by localized host defense to relatively high spore densities, the fungus switches to a necrotrophic infection program. Our study has uncovered a novel strategy by which plant-pathogenic fungi can take advantage of the early immune response in the host plant.     

The Role of a Potassium Transporter OsHAK5 in Potassium Acquisition and Transport from Roots to Shoots in Rice at Low Potassium Supply Levels

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.Potassium (K) is one of the three most important macronutrients and the most abundant cation in plants. As a major osmoticum in the vacuole, K drives the generation of turgor pressure, enabling cell expansion. In the vascular tissue, K is an important participant in the generation of root pressure (for review, see Wegner, 2014 [including his new hypothesis]). In the phloem, K is critical for the transport of photoassimilates from source to sink (Marschner, 1996; Deeken et al., 2002; Gajdanowicz et al., 2011). In addition, enhancing K absorption and decreasing sodium (Na) accumulation is a major strategy of glycophytes in salt stress tolerance (Maathuis and Amtmann, 1999; Munns and Tester, 2008; Shabala and Cuin, 2008).Plants acquire K through K-permeable proteins at the root surface. Since available K concentration in the soil may vary by 100-fold, plants have developed multiple K uptake systems for adapting to this variability (Epstein et al., 1963; Grabov, 2007; Maathuis, 2009). In a classic K uptake experiment in barley (Hordeum vulgare), root K absorption has been described as a high-affinity and low-affinity biphasic transport process (Epstein et al., 1963). It is generally assumed that the low-affinity transport system (LATS) in the roots mediates K uptake in the millimolar range and that the activity of this system is insensitive to external K concentration (Maathuis and Sanders, 1997; Chérel et al., 2014). In contrast, the high-affinity transport system (HATS) was rapidly up-regulated when the supply of exogenous K was halted (Glass, 1976; Glass and Dunlop, 1978).The membrane transporters for K flux identified in plants are generally classified into three channels and three transporter families based on phylogenetic analysis (Mäser et al., 2001; Véry and Sentenac, 2003; Lebaudy et al., 2007; Alemán et al., 2011). For K uptake, it was predicted that, under most circumstances, K transporters function as HATS, while K-permeable channels mediate LATS (Maathuis and Sanders, 1997). However, a root-expressed K channel in Arabidopsis (Arabidopsis thaliana), Arabidopsis K Transporter1 (AKT1), mediates K absorption over a wide range of external K concentrations (Sentenac et al., 1992; Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), while evidence is accumulating that many K transporters, including members of the K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) family, are low-affinity K transporters (Quintero and Blatt, 1997; Senn et al., 2001), implying that functions of plant K channels and transporters overlap at different K concentration ranges.Out of the three families of K transporters, cation proton antiporter (CPA), high affinity K/Na transporter (HKT), and KT/HAK/KUP, CPA was characterized as a K⁺(Na⁺)/H⁺ antiporter, HKT may cotransport Na and K or transport Na only (Rubio et al., 1995; Uozumi et al., 2000), while KT/HAK/KUP were predicted to be H⁺-coupled K⁺ symporters (Mäser et al., 2001; Lebaudy et al., 2007). KT/HAK/KUP were named by different researchers who first identified and cloned them (Quintero and Blatt, 1997; Santa-María et al., 1997). In plants, the KT/HAK/KUP family is the largest K transporter family, including 13 members in Arabidopsis and 27 members in the rice (Oryza sativa) genome (Rubio et al., 2000; Mäser et al., 2001; Bañuelos et al., 2002; Gupta et al., 2008). Sequence alignments show that genes of this family share relatively low homology to each other. The KT/HAK/KUP family was divided into four major clusters (Rubio et al., 2000; Gupta et al., 2008), and in cluster I and II, they were further separated into A and B groups. Genes of cluster I or II likely exist in all plants, cluster III is composed of genes from both Arabidopsis and rice, while cluster IV includes only four rice genes (Grabov, 2007; Gupta et al., 2008).The functions of KT/HAK/KUP were studied mostly in heterologous expression systems. Transporters of cluster I, such as AtHAK5, HvHAK1, OsHAK1, and OsHAK5, are localized in the plasma membrane (Kim et al., 1998; Bañuelos et al., 2002; Gierth et al., 2005) and exhibit high-affinity K uptake in the yeast Saccharomyces cerevisiae (Santa-María et al., 1997; Fu and Luan, 1998; Rubio et al., 2000) and in Escherichia coli (Horie et al., 2011). Transporters of cluster II, like AtKUP4 (TINY ROOT HAIRS1, TRH1), HvHAK2, OsHAK2, OsHAK7, and OsHAK10, could not complement the K uptake-deficient yeast (Saccharomyces cerevisiae) but were able to mediate K fluxes in a bacterial mutant; they might be tonoplast transporters (Senn et al., 2001; Bañuelos et al., 2002; Rodríguez-Navarro and Rubio, 2006). The function of transporters in clusters III and IV is even less known (Grabov, 2007).Existing data suggest that some KT/HAK/KUP transporters also may respond to salinity stress (Maathuis, 2009). The cluster I transporters of HvHAK1 mediate Na influx (Santa-María et al., 1997), while AtHAK5 expression is inhibited by Na (Rubio et al., 2000; Nieves-Cordones et al., 2010). Expression of OsHAK5 in tobacco (Nicotiana tabacum) BY2 cells enhanced the salt tolerance of these cells by accumulating more K without affecting their Na content (Horie et al., 2011).There are only scarce reports on the physiological function of KT/HAK/KUP in planta. In Arabidopsis, mutation of AtKUP2 (SHORT HYPOCOTYL3) resulted in a short hypocotyl, small leaves, and a short flowering stem (Elumalai et al., 2002), while a loss-of-function mutation of AtKUP4 (TRH1) resulted in short root hairs and a loss of gravity response in the root (Rigas et al., 2001; Desbrosses et al., 2003; Ahn et al., 2004). AtHAK5 is the only system currently known to mediate K uptake at concentrations below 0.01 mm (Rubio et al., 2010) and provides a cesium uptake pathway (Qi et al., 2008). AtHAK5 and AtAKT1 are the two major physiologically relevant molecular entities mediating K uptake into roots in the range between 0.01 and 0.05 mm (Pyo et al., 2010; Rubio et al., 2010). AtAKT1 may contribute to K uptake within the K concentrations that belong to the high-affinity system described by Epstein et al. (1963).Among all 27 members of the KT/HAK/KUP family in rice, OsHAK1, OsHAK5, OsHAK19, and OsHAK20 were grouped in cluster IB (Gupta et al., 2008). These four rice HAK members share 50.9% to 53.4% amino acid identity with AtHAK5. OsHAK1 was expressed in the whole plant, with maximum expression in roots, and was up-regulated by K deficiency; it mediated high-affinity K uptake in yeast (Bañuelos et al., 2002). In this study, we examined the tissue-specific localization and the physiological functions of OsHAK5 in response to variation in K supply and to salt stress in rice. By comparing K uptake and translocation in OsHAK5 knockout (KO) mutants and in OsHAK5-overexpressing lines with those in their respective wild-type lines supplied with different K concentrations, we found that OsHAK5 not only mediates high-affinity K acquisition but also participates in root-to-shoot K transport as well as in K-regulated salt tolerance.     

Tryptophan-Independent Indole-3-Acetic Acid Synthesis: Critical Evaluation of the Evidence

Evidence for Trp-independent IAA synthesis is critically reevaluated in the light of tryptophan synthase proteome data, local IAA synthesis and Trp, indole-3-pyruvate, and IAA turnover.Trp-independent synthesis of indole-3-acetic acid (IAA) was proposed back in the early 1990s based on observations from Trp auxotrophs in maize (Zea mays; Wright et al., 1991) and Arabidopsis (Arabidopsis thaliana; Normanly et al., 1993). Recently, Wang et al. (2015) published new data suggesting that a cytosolic indole synthase (INS) may catalyze the first step separating the Trp-dependent and Trp-independent pathways in Arabidopsis. If this is the case, it would be a major breakthrough; however, in this article, I critically evaluate both recent and older evidence for the Trp-independent route and suggest that the INS is more likely to participate in Trp-dependent IAA production.The original work supporting Trp-independent IAA production was carried out prior to the availability of genome/proteome data and before the discovery that the final step of Trp-dependent IAA synthesis is carried out by a large number of YUCCA homologs operating in a highly localized manner (Zhao, 2008). I argue that experimental data supporting the Trp-independent route needs to be reconsidered in light of complete proteome data. Further, the evidence from feeding labeled compounds should be critically evaluated in light of recent data on the highly localized nature of IAA synthesis as well as older quantitative data on Trp, indole-3-pyruvic acid (IPA), and IAA turnover from my own laboratory (Cooney and Nonhebel, 1991). I conclude that evidence for the Trp-independent route is at best equivocal, and that it is not a conserved source of IAA in angiosperms.Figure 1 shows the major Trp-dependent route for IAA production whereby Trp, produced by the concerted action of Trp synthase α- and β-subunits, is converted to IAA in a further two steps catalyzed by Trp aminotransferase and the flavin monooxygenases commonly known as YUCCA (Mashiguchi et al., 2011; Won et al., 2011). This is compared with the Trp-independent route in which IAA may be produced from free indole by an unknown route (Ouyang et al., 2000; Wang et al., 2015).Open in a separate windowFigure 1.Outline of the major pathway for Trp-dependent IAA synthesis and the proposed Trp-independent route. The role proposed for Trp synthase beta (TSB) homologs discussed in the present paper is also shown. For clarity, reactions are simplified to show only the major compounds relevant to IAA synthesis.The Trp-independent route was originally based on data from Trp auxotrophs that have mutations in genes encoding either the α- or β-subunits of Trp synthase. The α-subunit catalyzes the removal of the side chain from indole-3-glycerol phosphate, passing the indole product directly to the β-subunit where the Trp side chain is created from a Ser substrate (Pan et al., 1997). In plants, this is a chloroplast-localized enzyme. Elevated levels of IAA have been reported in Trp auxotrophs of both maize and Arabidopsis. However, the trp3-1 and trp2-1 mutants of Arabidopsis, deficient in the α- and β-subunits, respectively, only showed an increase in total IAA measured following conjugate hydrolysis. No difference in free IAA levels was found (Normanly et al., 1993). The orange pericarp (orp) maize mutant was reported to have 50 times more IAA than the wild type (Wright et al., 1991). However, this was also total IAA; no data on free IAA were published. Work by Müller and Weiler (2000) indicated that IAA measured following conjugate hydrolysis could have originated via the degradation of indole-3-glycerol phosphate that accumulates in trp3-1 mutants. Further doubt regarding the accuracy of IAA measurements following conjugate hydrolysis has recently been published. Yu et al. (2015) have shown that conjugate hydrolysis treatment substantially overestimates the actual conjugated IAA due to degradation of glucobrassicin and proteins. In addition, neither report (Wright et al., 1991; Normanly et al., 1993) described a high auxin phenotype for the Trp auxotrophs. This contrasts with the superroot1 (sur1) and sur2 mutants, where the accumulation of indole intermediates resulted in a high level of free IAA as well as a high auxin phenotype (Boerjan et al., 1995; Delarue et al., 1998). It is therefore doubtful that Trp auxotrophs actually accumulate more IAA than the wild-type plants.In addition, proteome data have revealed new homologs of TSB in both Arabidopsis and maize that may contribute to Trp production in TSB mutants; these have not been considered in arguments supporting Trp-independent IAA synthesis. Maize orp has mutations in two TSB genes, resulting in a seedling lethal phenotype with high levels of accumulated indole. However, proteome sequence information now indicates that maize has three TSB genes. Plants and bacteria have divergent forms of TSB, type 1 and type 2 (Xie et al., 2001); the major TSB genes responsible for Trp synthase activity in maize and Arabidopsis are type 1. The third maize TSB gene, maize locus ID GRMZM2G054465, is a member of the TSB type 2 group. Its product is reported not to interact directly with a Trp synthase alpha (TSA) subunit but has experimentally demonstrated catalytic activity converting indole and Ser to Trp (Yin et al., 2010). This type 2 TSB may allow orp plants to make sufficient Trp for IAA production from the accumulated free indole.When the original work on trp2 mutants of Arabidopsis was carried out, two TSB genes were known (Last et al., 1991). As the trp2 plants were deficient only in TSB1, they were able to make sufficient Trp to survive under low-light conditions. Full proteome data now indicate that Arabidopsis has four TSB-like genes; in addition to TSB1 and TSB2, there is a third type 1 TSB gene, Arabidopsis locus ID AT5G28237. The product of this gene has not been experimentally characterized. The fourth gene, AT5G38530, encodes a type 2 TSB with demonstrated catalytic activity similar to ZmTSB type 2 mentioned above (Yin et al., 2010). Thus, the trp2 plants may also make enough Trp for IAA production. It is even possible that one of the minor forms of TSB has a specific role in IAA production. Type 2 TSBs are conserved throughout the plant kingdom, and the biological role for this protein is not known (Xie et al., 2001).A phylogenetic analysis of type 1 TSBs is shown in Figure 2. This indicates that the product of AT5G28237 belongs to a eudicot-conserved TSB type 1-like clade, divergent from that containing major experimentally characterized TSBs. A multiple sequence alignment (not shown) reveals that members of this divergent clade have a shortened N terminus with respect to the major chloroplast-localized TSB proteins. A localization prediction carried out in CELLO (Yu et al., 2006) suggests a cytosolic location for these proteins. Examination of EST databases indicates that the genes encoding these proteins are expressed. It is possible that the product of AT5G28237 could interact with the cytosolic INS studied by Wang et al. (2015), or separately with its indole product, to produce Trp that is further converted to IAA.Open in a separate windowFigure 2.Phylogeny of TSB type 1 homologs from Oryza sativa (LOC_Os), Sorghum bicolor (Sobic), Z. mays (GRMZM), Arabidopsis (AT), Brassica rapa (Brara), Solanum lycopersicum (Solyc), Populus trichocarpa (Potri), and Physcomitrella patens (Phpat). Protein sequences were downloaded from Phytozome v10.2 (Goodstein et al., 2012). The phylogenetic analysis was conducted in MEGA6 (http://megasoftware.net; Tamura et al., 2013) with multiple sequence alignment by MUSCLE (Edgar, 2004) and evolutionary history inferred using the neighbor-joining method (Saitou and Nei, 1987). The optimal tree is shown; the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale; the scale bar indicates the number of amino acid substitutions per site. It is rooted with type 1 TSBs from the moss P. patens.The second major line of evidence for Trp-independent IAA synthesis comes from isotopic labeling experiments. Wright et al. (1991) observed greater incorporation of ²H into IAA than Trp in orp seedlings grown on ²H₂O. Normanly et al. (1993) reported higher enrichment of ¹⁵N in IAA than Trp in trp2-1 mutants of Arabidopsis grown on ¹⁵N anthranilate; very poor incorporation of deuterium from ²H-Trp into IAA was reported in the trp2-1 plants. A number of similar reports relating to other plants have been published showing differences in the incorporation of label from Trp into IAA depending on experimental tissue and environmental conditions (e.g. Michalczuk et al., 1992; Rapparini et al., 2002; Sztein et al., 2002). This evidence has been persuasive; however, it assumes a single pool of Trp to which ¹⁵N anthranilate and ²H-Trp contribute and from which IAA is made. If Trp is made at different rates in different parts of the plant, and/or exogenous ²H-Trp does not equilibrate with newly synthesized Trp, then the ratio of ¹⁵N to ²H in Trp will vary in different plant organs/tissues/cells. Trp turnover and thus incorporation of label from ¹⁵N anthranilate are likely to differ substantially throughout the plant, with the highest rates of labeling occurring in cells with high rates of protein synthesis. This would not be a problem for the experiment if IAA is made at equal rates in different parts of the plant, but we know it is not. The Trp aminotransferase/YUCCA pathway of IAA synthesis elegantly shown to be responsible for the bulk of IAA synthesis (Mashiguchi et al., 2011; Won et al., 2011) appears to be locally controlled in Arabidopsis via 11 different YUCCA-encoding genes that have highly localized expression (Zhao, 2008). Adding to the complication is the need for ¹⁵N anthranilate and ²H-Trp to move into and through the plant to regions of Trp and IAA synthesis, respectively. This is likely to occur at different rates due to differing transporter requirements.Data from my own laboratory (Cooney and Nonhebel, 1991) is particularly relevant to this discussion. We monitored incorporation of ²H from deuterated water into IAA and Trp in tomato (S. lycopersicum) shoots. Unlike the other studies, we also measured the incorporation of label into IPA. Our data showed that IPA became labeled at a rate consistent with this compound acting as the major/sole precursor of IAA. Crucially, the proportion of labeled Trp was lower than ²H-IPA. Our interpretation of these data was that IPA and IAA were produced from newly synthesized Trp, and that Trp was not uniformly labeled throughout the shoot. At the time, we suggested different subcellular pools of Trp; this may be the case, but in light of new knowledge of localized IAA synthesis, it is most likely that substantial differences in Trp and IAA turnover in different cells/tissues may be the reason for these observations.The arguments above cast doubt on the existence of the Trp-independent route; however, a recent publication by Wang et al. (2015) claims to provide new evidence for its importance. They present the interesting finding that Arabidopsis plants with a null mutation in INS, a cytosolic TSA homolog previously shown to have indole-3-glycerol phosphate lyase (IGL) activity (Zhang et al., 2008), had reduced levels of IAA. The mutation particularly affected early embryo development. I suggest that the INS may make a contribution to IAA synthesis, but the only specific evidence that it does so via a Trp-independent route is the observation that the ins-1 mutation has an additive effect with the weakly ethylene insensitive8-1 Trp aminotransferase mutation. This evidence is indicative rather than conclusive. The possibility that INS may act in concert with a minor TSB homolog, as suggested in Figure 1, needs to be considered.In addition, Wang et al. (2015) focus on Arabidopsis alone. If INS has a key role in IAA synthesis, then evolutionary theory predicts a conserved protein with wide taxonomic distribution. On the contrary, an exhaustive BLAST search (Altschul et al., 1997) of diverse taxa in Phytozome v10.2 (Goodstein et al., 2012) and GenBank (Benson et al., 2013) revealed that INS orthologs with cytosolic prediction and shortened N terminus occur only in members of the Brassicaceae (Eutrema salsugineum, Arabis alpina, Camelina sativa, Capsella rubella, Brassica napus, Boechera stricta, Arabidopsis lyrata, B. rapa) and in Tarenaya hassleriana from the Brassicaceae sister family, the Cleomaceae. The phylogenetic tree in Figure 3 shows relationships between INS and TSA homologs from several plant species and indicates the separate clade of cytosolic INS homologs in the Brassicaceae. In this diagram, the sequence most closely related to INS from another group is that from tomato. This protein is the only TSA found in tomato and has an unambiguous chloroplast signal peptide. P. trichocarpa and M. truncatula as well as other eudicots outside the Brassicaeae and Cleomaceae also lack cytosolic TSA homologs. Furthermore, INS and its orthologs are phylogenetically distinct from the other experimentally characterized indole-3-glycerol phosphate lyases benzoxazin1 and IGL (Frey et al., 2000) and their orthologs. The latter are restricted to the grasses where they are involved in the production of cyclic hydroxamic acid defense compounds (Frey et al., 2000). The grasses also have an additional separate clade of cytosolic TSA homologs, although work by Kriechbaumer et al. (2008) did not detect any catalytic activity for the product of GRMZM2G046191_T01. The phylogeny of INS and its orthologs would suggest the major role of these proteins may be the production of lineage-specific metabolites such as the indole-derived defense compounds produced in grasses; any role in IAA synthesis may be incidental and restricted to the Brassicaeae and Cleomaceae.Open in a separate windowFigure 3.Phylogeny of TSA homologs from O. sativa (LOC_Os), S. bicolor (Sobic), Z. mays (GRMZM), Arabidopsis (AT), A. lyrata (Alyrata), B. rapa (Brara), S. lycopersicum (Solyc), Medicago truncatula (Medtr), P. trichocarpa (Potri), and P. patens (Phpat). Protein sequences were downloaded from Phytozome v10.2 (Goodstein et al., 2012). The phylogenetic analysis was conducted in MEGA6 (Tamura et al., 2013) with multiple sequence alignment by MUSCLE (Edgar, 2004) and evolutionary history inferred using the neighbor-joining method (Saitou and Nei, 1987). The rooted optimal tree is shown; the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale; the scale bar indicates the number of amino acid substitutions per site. It is rooted with the TSA ortholog from the moss P. patens.In conclusion, I contend that experimental data relating to IAA synthesis in Arabidopsis, including that suggesting the involvement of a cytosolic INS, can be explained by the Trp-dependent IAA synthesis pathway. I show that INS and its orthologs are not found outside the Brassicaceae and a closely related sister clade; any alternative IAA synthesis pathway in which they may be involved is likely to have similar limited taxonomic occurrence. Furthermore, Arabidopsis and its relatives contain two additional TSB homologs that could convert free indole into Trp. Curiously, both of these proteins have a wider taxonomic distribution. A priority for further experimental work should be testing the involvement of minor TSB homologs in IAA synthesis, including the highly conserved type 2 TSBs as well as a eudicot-specific clade of possibly cytosolic type 1 TSBs. Work would also have to establish whether free indole exists in plants other than the Brassicaceae and the grasses. Finally, I argue that isotope-labeling experiments do not provide strong support for the Trp-independent route, as IAA production is highly localized. Previously published data from my laboratory clearly show that the main Trp-dependent IAA precursor IPA becomes more highly labeled from ²H₂O than Trp, even though the latter is produced from Trp in a single reaction. Thus, it cannot be argued that differences in isotope enrichment between Trp and IAA demonstrate the existence of a Trp-independent route.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.     

Disruption of Fumarylacetoacetate Hydrolase Causes Spontaneous Cell Death under Short-Day Conditions in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Deficiency of FAH in animals results in an inborn lethal disorder. However, the role for the Tyr degradation pathway in plants remains to be elucidated. In this study, we isolated an Arabidopsis (Arabidopsis thaliana) short-day sensitive cell death1 (sscd1) mutant that displays a spontaneous cell death phenotype under short-day conditions. The SSCD1 gene was cloned via a map-based cloning approach and found to encode an Arabidopsis putative FAH. The spontaneous cell death phenotype of the sscd1 mutant was completely eliminated by further knockout of the gene encoding the putative homogentisate dioxygenase, which catalyzes homogentisate into maleylacetoacetate (the antepenultimate step) in the Tyr degradation pathway. Furthermore, treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway, mimicked the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under short-day conditions.Programmed cell death (PCD) has been defined as a sequence of genetically regulated events that lead to the elimination of specific cells, tissues, or whole organs (Lockshin and Zakeri, 2004). In plants, PCD is essential for developmental processes and defense responses (Dangl et al., 1996; Greenberg, 1996; Durrant et al., 2007). One well-characterized example of plant PCD is the hypersensitive response occurring during incompatible plant-pathogen interactions (Lam, 2004), which results in cell death to form visible lesions at the site of infection by an avirulent pathogen and consequently limits the pathogen spread (Morel and Dangl, 1997).To date, a large number of mutants that display spontaneous cell death lesions have been identified in barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana; Marchetti et al., 1983; Wolter et al., 1993; Dietrich et al., 1994; Gray et al., 1997). Because lesions form in the absence of pathogen infection, these mutants have been collectively termed as lesion-mimic mutants. Many genes with regulatory roles in PCD and defense responses, including LESION SIMULATING DISEASE1, ACCELERATED CELL DEATH11, and VASCULAR ASSOCIATED DEATH1, have been cloned and characterized (Dietrich et al., 1997; Brodersen et al., 2002; Lorrain et al., 2004).The appearance of spontaneous cell death lesions in some lesion-mimic mutants is dependent on photoperiod. For example, the Arabidopsis mutant lesion simulating disease1 and myoinositol-1-phosphate synthase1 show lesions under long days (LD; Dietrich et al., 1994; Meng et al., 2009), whereas the lesion simulating disease2, lesion initiation1, enhancing RPW8-mediated HR-like cell death1, and lag one homolog1 display lesions under short days (SD; Dietrich et al., 1994; Ishikawa et al., 2003; Wang et al., 2008; Ternes et al., 2011).Blockage of some metabolic pathways in plants may cause cell death and result in lesion formation. For example, the lesion-mimic phenotypes in the Arabidopsis mutants lesion initiation2 and accelerated cell death2 and the maize mutant lesion mimic22 result from an impairment of porphyrin metabolism (Hu et al., 1998; Ishikawa et al., 2001; Mach et al., 2001). Deficiency in fatty acid, sphingolipid, and myoinositol metabolism also causes cell death in Arabidopsis (Mou et al., 2000; Liang et al., 2003; Wang et al., 2008; Meng et al., 2009; Donahue et al., 2010; Berkey et al., 2012).Tyr degradation is an essential five-step pathway in animals (Lindblad et al., 1977). First, Tyr aminotransferase catalyzes the conversion of Tyr into 4-hydroxyphenylpyruvate, which is further transformed into homogentisate by 4-hydroxyphenylpyruvate dioxygenase. Through the sequential action of homogentisate dioxygenase (HGO), maleylacetoacetate isomerase (MAAI), and fumarylacetoacetate hydrolase (FAH), homogentisate is catalyzed to generate fumarate and acetoacetate (Lindblad et al., 1977). Blockage of this pathway in animals results in metabolic disorder diseases (Lindblad et al., 1977; Ruppert et al., 1992; Grompe et al., 1993). For example, human FAH deficiency causes hereditary tyrosinemia type I (HT1), an inborn lethal disease (St-Louis and Tanguay, 1997). Although the homologous genes putatively encoding these enzymes exist in plants (Dixon et al., 2000; Lopukhina et al., 2001; Dixon and Edwards, 2006), it is unclear whether this pathway is essential for plant growth and development.In this study, we report the isolation and characterization of a recessive short-day sensitive cell death1 (sscd1) mutant in Arabidopsis. Map-based cloning of the corresponding gene revealed that SSCD1 encodes the Arabidopsis putative FAH. Further knockout of the gene encoding the Arabidopsis putative HGO completely eliminated the spontaneous cell death phenotype in the sscd1 mutant. Furthermore, we found that treatment of Arabidopsis wild-type seedlings with succinylacetone, an abnormal metabolite caused by loss of FAH in the Tyr degradation pathway (Lindblad et al., 1977), is able to mimic the sscd1 cell death phenotype. These results demonstrate that disruption of FAH leads to cell death in Arabidopsis and suggest that the Tyr degradation pathway is essential for plant survival under SD.     

The Rubisco Small Subunit Is Involved in Tobamovirus Movement and Tm-22-Mediated Extreme Resistance

The multifunctional movement protein (MP) of Tomato mosaic tobamovirus (ToMV) is involved in viral cell-to-cell movement, symptom development, and resistance gene recognition. However, it remains to be elucidated how ToMV MP plays such diverse roles in plants. Here, we show that ToMV MP interacts with the Rubisco small subunit (RbCS) of Nicotiana benthamiana in vitro and in vivo. In susceptible N. benthamiana plants, silencing of NbRbCS enabled ToMV to induce necrosis in inoculated leaves, thus enhancing virus local infectivity. However, the development of systemic viral symptoms was delayed. In transgenic N. benthamiana plants harboring Tobacco mosaic virus resistance-2² (Tm-2²), which mediates extreme resistance to ToMV, silencing of NbRbCS compromised Tm-2²-dependent resistance. ToMV was able to establish efficient local infection but was not able to move systemically. These findings suggest that NbRbCS plays a vital role in tobamovirus movement and plant antiviral defenses.Plant viruses use at least one movement protein (MP) to facilitate viral spread between plant cells via plasmodesmata (PD; Lucas and Gilbertson, 1994; Ghoshroy et al., 1997). Among viral MPs, the MP of tobamoviruses, such as Tobacco mosaic virus (TMV) and its close relative Tomato mosaic virus (ToMV), is the best characterized. TMV MP specifically accumulates in PD and modifies the plasmodesmatal size exclusion limit in mature source leaves or tissues (Wolf et al., 1989; Deom et al., 1990; Ding et al., 1992). TMV MP and viral genomic RNA form a mobile ribonucleoprotein complex that is essential for cell-to-cell movement of viral infection (Watanabe et al., 1984; Deom et al., 1987; Citovsky et al., 1990, 1992; Kiselyova et al., 2001; Kawakami et al., 2004; Waigmann et al., 2007). TMV MP also enhances intercellular RNA silencing (Vogler et al., 2008) and affects viral symptom development, host range, and host susceptibility to virus (Dardick et al., 2000; Bazzini et al., 2007). Furthermore, ToMV MP is identified as an avirulence factor that is recognized by tomato (Solanum lycopersicum) resistance proteins Tobacco mosaic virus resistance-2 (Tm-2) and Tm-2² (Meshi et al., 1989; Lanfermeijer et al., 2004). Indeed, tomato Tm-2² confers extreme resistance against TMV and ToMV in tomato plants and even in heterologous tobacco (Nicotiana tabacum) plants (Lanfermeijer et al., 2003, 2004).To date, several host factors that interact with TMV MP have been identified. These TMV MP-binding host factors include cell wall-associated proteins such as pectin methylesterase (Chen et al., 2000), calreticulin (Meshi et al., 1989), ANK1 (Ueki et al., 2010), and the cellular DnaJ-like protein MPIP1 (Shimizu et al., 2009). Many cytoskeletal components such as actin filaments (McLean et al., 1995), microtubules (Heinlein et al., 1995), and the microtubule-associated proteins MPB2C (Kragler et al., 2003) and EB1a (Brandner et al., 2008) also interact with TMV MP. Most of these factors are involved in TMV cell-to-cell movement.Rubisco catalyzes the first step of CO₂ assimilation in photosynthesis and photorespiration. The Rubisco holoenzyme is a heteropolymer consisting of eight large subunits (RbCLs) and eight small subunits (RbCSs). RbCL was reported to interact with the coat protein of Potato virus Y (Feki et al., 2005). Both RbCS and RbCL were reported to interact with the P3 proteins encoded by several potyviruses, including Shallot yellow stripe virus, Onion yellow dwarf virus, Soybean mosaic virus, and Turnip mosaic virus (Lin et al., 2011). Proteomic analysis of the plant-virus interactome revealed that RbCS participates in the formation of virus complexes of Rice yellow mottle virus (Brizard et al., 2006). However, the biological function of Rubisco in viral infection remains unknown.In this study, we show that RbCS plays an essential role in virus movement, host susceptibility, and Tm-2²-mediated extreme resistance in the ToMV-host plant interaction.