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.     

The Presence of Fucogalactoxyloglucan and Its Synthesis in Rice Indicates Conserved Functional Importance in Plants

The predominant structure of the hemicellulose xyloglucan (XyG) found in the cell walls of dicots is a fucogalactoXyG with an XXXG core motif, whereas in the Poaceae (grasses and cereals), the structure of XyG is less xylosylated (XXGG_n core motif) and lacks fucosyl residues. However, specialized tissues of rice (Oryza sativa) also contain fucogalactoXyG. Orthologous genes of the fucogalactoXyG biosynthetic machinery of Arabidopsis (Arabidopsis thaliana) are present in the rice genome. Expression of these rice genes, including fucosyl-, galactosyl-, and acetyltransferases, in the corresponding Arabidopsis mutants confirmed their activity and substrate specificity, indicating that plants in the Poaceae family have the ability to synthesize fucogalactoXyG in vivo. The data presented here provide support for a functional conservation of XyG structure in higher plants.The plant cell wall protects and structurally supports plant cells. The wall consists of a variety of polymers, including polysaccharides, the polyphenol lignin, and glycoproteins. One of the major polysaccharides present in the primary walls (i.e. walls of growing cells) in dicots is xyloglucan (XyG), which consists of a β-1,4-glucan backbone with xylosyl substituents. XyG binds noncovalently to cellulose microfibrils and thereby, is thought to act as a spacer molecule, hindering cellulose microfibrils to aggregate (Carpita and Gibeaut, 1993; Pauly et al., 1999a; Bootten et al., 2004; Cosgrove, 2005; Hayashi and Kaida, 2011; Park and Cosgrove, 2012).The side-chain substitutions on XyG can be structurally diverse depending on plant species, tissue type, and developmental stage of the tissue (Pauly et al., 2001; Hoffman et al., 2005; Peña et al., 2008; Hsieh and Harris, 2009, 2012; Lampugnani et al., 2013; Schultink et al., 2014). A one-letter code nomenclature has been established to specify the XyG side-chain substitutions (Fry et al., 1993; Tuomivaara et al., 20145). According to this nomenclature, an unsubstituted glucosyl residue is indicated by a G, whereas a glucosyl residue substituted with a xylosyl moiety is shown as an X. In most dicots, the xylosyl residue can be further substituted with a galactosyl residue (L), which in turn, can be further decorated with a fucosyl residue (F) and/or an acetyl group (F/L). In some species, the xylosyl residue can be substituted with an arabinosyl moiety (S), and the backbone glucosyl residue can be O-acetylated (G; Jia et al., 2003; Hoffman et al., 2005).Numerous genes have been identified in Arabidopsis (Arabidopsis thaliana) that are involved in fucogalactoXyG biosynthesis (Fig. 1; Pauly et al., 2013; Schultink et al., 2014). The glucan backbone is thought to be synthesized by cellulose synthase-like C (CSLC) family proteins, such as AtCSLC4, as shown by in vitro activity data (Cocuron et al., 2007). Several xylosyltransferases (XXTs) from glycosyl transferase family 34 (GT34) are thought to be responsible for XyG xylosylation. Five of these XXTs in Arabidopsis seem to have XXT activity on XyG in vitro (Faik et al., 2002; Zabotina et al., 2008; Vuttipongchaikij et al., 2012; Mansoori et al., 2015). MURUS3 (MUR3) represents a galactosyltransferase that transfers galactosyl moieties specifically to xylosyl residues adjacent to an unsubstituted glucosyl residue on an XXXG unit, converting it to XXLG, whereas Xyloglucan L-side chain galactosyl Transferase2 (XLT2) was identified as another galactosyltransferase transferring a galactosyl moiety specifically to the second xylosyl residue, resulting in XLXG (Madson et al., 2003; Jensen et al., 2012). Both MUR3 and XLT2 belong to GT47 (Li et al., 2004). MUR2/FUCOSYLTRANSFERASE1 (FUT1) from GT37 was found to harbor fucosyltransferase activity, transferring Fuc from GDP-Fuc to a galactosyl residue adjacent to the unsubstituted glucosyl residue (i.e. onto XXLG but not onto XLXG; Perrin et al., 1999; Vanzin et al., 2002). O-acetylation of the galactosyl residue is mediated by Altered Xyloglucan4 (AXY4) and AXY4L, both of which belong to the Trichome Birefringence-Like (TBL) protein family (Bischoff et al., 2010; Gille et al., 2011; Gille and Pauly, 2012).Open in a separate windowFigure 1.Schematic structures of two types of XyGs and known biosynthetic proteins in Arabidopsis (Hsieh and Harris, 2009; Pauly et al., 2013). The corresponding one-letter code for XyG is shown below the pictograms (Fry et al., 1993; Tuomivaara et al., 2015).XyG found throughout land plants exhibits structural diversity with respect to side-chain substitution patterns (Schultink et al., 2014). Most dicots, such as Arabidopsis, and the noncommelinoid monocots possess a fucogalactoXyG of the XXXG-type XyG structure as shown in Figure 1. However, plant species in the Solanaceae and Poaceae as well as the moss Physcomitrella patens contain a different XyG structure with a reduced level of xylosylation, resulting in an XXGG_n core motif (York et al., 1996; Kato et al., 2004; Gibeaut et al., 2005; Jia et al., 2005; Peña et al., 2008; Hsieh and Harris, 2009). In addition, the glucan backbone can be O-acetylated in plants of Solanaceae and Poaceae families (Gibeaut et al., 2005; Jia et al., 2005). XyG from Solanaceae with an XXGG core motif can be further arabinosylated and/or galactosylated (Jia et al., 2005). No XyGs with an XXGG_n motif backbone have been reported to be fucosylated.The function of structural diversity of XyG substitutions, such as fucosylation and/or altered xylosylation pattern, remains enigmatic. Removing the terminal fucosyl or acetyl moieties in the corresponding Arabidopsis mutants does not lead to any change in plant growth and development (Vanzin et al., 2002; Gille et al., 2011). However, removing galactosyl residues as well as fucosyl and acetyl moieties in the Arabidopsis xlt2 mur3.1 double mutant results in a dwarfed plant (Jensen et al., 2012; Kong et al., 2015). Replacing the galactosyl moiety with an arabinofuranosyl residue by, for example, expressing a tomato (Solanum lycopersicum) arabinosyltransferase in the Arabidopsis xlt2 mur3.1 mutant rescues the growth phenotype and restores wall biomechanics, indicating that galactosylation and arabinosylation in XyG have an equivalent function (Schultink et al., 2013). Recently, fucosylated XyG structures were found in the pollen tubes of tobacco (Nicotiana alata) and tomato, indicating that fucogalactoXyG is likely also present in other Solanaceae plants, albeit restricted to specific tissues (Lampugnani et al., 2013; Dardelle et al., 2015). Although there is circumstantial evidence that fucogalactoXyG is present in cell suspension cultures of rice (Oryza sativa) and cell suspension cultures of fescue (Festuca arundinaceae; McDougall and Fry, 1994; Peña et al., 2008), fucogalactoXyG has not been found in any physiologically relevant plant tissues of members of the Poaceae (Kato et al., 1982; Watanabe et al., 1984; Gibeaut et al., 2005; Hsieh and Harris, 2009; Brennan and Harris, 2011). Here, we provide chemical and genetic evidence that fucogalactoXyG is, indeed, present in plant tissues of a grass (rice) and prove that the rice genome harbors the genes that could be part of the synthetic machinery necessary to produce fucogalactoXyG.     

Grain setting defect1, Encoding a Remorin Protein,Affects the Grain Setting in Rice through Regulating Plasmodesmatal Conductance

Effective grain filling is one of the key determinants of grain setting in rice (Oryza sativa). Grain setting defect1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a transfer DNA insertion mutant (gsd1-Dominant) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests that the phenotype of the gsd1-Dominant mutant is caused by defects in the grain-filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with rice ACTIN1 in association with the PD callose binding protein1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice.Grain filling, a key determinant of grain yield in rice (Oryza sativa), hinges on the successful translocation of photoassimilates from the leaves to the fertilized reproductive organs through the phloem transport system. Symplastic phloem loading, which is one of the main pathways responsible for the transport of photoassimilates in rice, is mediated by plasmodesmata (PD) that connect phloem companion cells with sieve elements and surrounding parenchyma cells (Kaneko et al., 1980; Chonan et al., 1981; Eom et al., 2012). PD are transverse cell wall channels structured with the cytoplasmic sleeve and the modified endoplasmic reticulum desmotubule between neighboring cells (Maule, 2008). A number of proteins affect the structure and functional performance of the PD, which in turn impacts the cell-to-cell transport of small and large molecules through the PD during plant growth, development, and defense (Cilia and Jackson, 2004; Sagi et al., 2005; Lucas et al., 2009; Simpson et al., 2009; Stonebloom et al., 2009). For example, actin and myosin, which link the desmotubule to the plasma membrane (PM) at the neck region of PD, are believed to play a role in regulating PD permeability by controlling PD aperture (White et al., 1994; Ding et al., 1996; Reichelt et al., 1999). Callose deposition can also impact the size of the PD aperture at the neck region (Radford et al., 1998; Levy et al., 2007) and callose synthase genes such as Glucan Synthase-Like7 (GSL7, also named CalS7), GSL8, and GSL12 have been shown to play a role in regulating symplastic trafficking (Guseman et al., 2010; Barratt et al., 2011; Vatén et al., 2011; Xie et al., 2011). Other proteins that have been shown to impact the structure and function of the PD include glycosylphosphatidylinositol (GPI)-anchored proteins, PD callose binding protein1 (PDCB1), which is also associated with callose deposition (Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN2, which limits the molecular flux through the PD by chitin perception (Faulkner et al., 2013). Changes in PD permeability can have major consequences for the translocation of photoassimilates needed for grain filling in rice. However, the genes and molecular mechanisms underlying the symplastic transport of photoassimilates remain poorly characterized.Remorins are a diverse family of plant-specific proteins with conserved C-terminal sequences and highly variable N-terminal sequences. Remorins can be classified into six distinct phylogenetic groups (Raffaele et al., 2007). The functions of most remorins are unknown, but some members of the family have been shown to be involved in immune response through controlling the cell-to-cell spread of microbes. StREM1.3, a remorin that is located in PM rafts and the PD, was shown to impair the cell-to-cell movement of a plant virus X by binding to Triple Gene Block protein1 (Raffaele et al., 2009). Medicago truncatula symbiotic remorin1 (MtSYMREM1), a remorin located at the PM in Medicago truncatula, was shown to facilitate infection and the release of rhizobial bacteria into the host cytoplasm (Lefebvre et al., 2010). Overexpression of LjSYMREM1, the ortholog of MtSYMREM1 in Lotus japonicus, resulted in increased root nodulation (Lefebvre et al., 2010; Tóth et al., 2012). Although a potential association between remorins and PD permeability has been proposed (Raffaele et al., 2009), the diversity observed across remorins, plus the fact that remorin mutants generated through different approaches fail to show obvious phenotypes (Reymond et al., 1996; Bariola et al., 2004), have made it challenging to characterize the function of remorins in cell-to-cell transport.In this study, we identified a rice transfer DNA (T-DNA) insertion mutant (grain setting defect1-Dominant [gsd1-D]), with a grain setting-deficient phenotype caused by overexpression of GSD1, a remorin gene with unknown function. GSD1 is expressed specifically in phloem companion cells and is localized in the PD and PM. We provide evidence to show that overexpression of GSD1 leads to deficient grain setting in rice, likely as a consequence of reduced sugar transport resulting from decreased PD permeability in phloem companion cells.     

An S-Domain Receptor-Like Kinase,OsSIK2, Confers Abiotic Stress Tolerance and Delays Dark-Induced Leaf Senescence in Rice

Receptor-like kinases play important roles in plant development and defense responses; however, their functions in other processes remain unclear. Here, we report that OsSIK2, an S-domain receptor-like kinase from rice (Oryza sativa), is involved in abiotic stress and the senescence process. OsSIK2 is a plasma membrane-localized protein with kinase activity in the presence of Mn²⁺. OsSIK2 is expressed mainly in rice leaf and sheath and can be induced by NaCl, drought, cold, dark, and abscisic acid treatment. Transgenic plants overexpressing OsSIK2 and mutant sik2 exhibit enhanced and reduced tolerance to salt and drought stress, respectively, compared with the controls. Interestingly, a truncated version of OsSIK2 without most of the extracellular region confers higher salt tolerance than the full-length OsSIK2, likely through the activation of different sets of downstream genes. Moreover, seedlings of OsSIK2-overexpressing transgenic plants exhibit early leaf development and a delayed dark-induced senescence phenotype, while mutant sik2 shows the opposite phenotype. The downstream PR-related genes specifically up-regulated by full-length OsSIK2 or the DREB-like genes solely enhanced by truncated OsSIK2 are all induced by salt, drought, and dark treatments. These results indicate that OsSIK2 may integrate stress signals into a developmental program for better adaptive growth under unfavorable conditions. Manipulation of OsSIK2 should facilitate the improvement of production in rice and other crops.Abiotic stress is one of the main factors that cause reductions in crop production; thus, it is important to study the molecular mechanisms of plant responses to the stress in order to improve stress tolerance in crops. When plants are exposed to abiotic stresses, signals are likely first sensed by receptors generally localized in the membrane, and then signals are transduced to the downstream factors and activate different stress responses. In this process, receptor-like kinase (RLK) may be the first sensor or transducer.RLK comprises one of the largest families, with more than 610 and 1,131 members in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), respectively (Shiu et al., 2004). Besides big size, another feature of RLK is its diversity. RLK typically contains an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular kinase domain. Extracellular domains vary greatly to perceive a wide range of signals or stimuli. Intracellular kinase domains are relatively conserved with Ser/Thr kinase activity to transduce signals. Based on the identity of extracellular domains, RLKs are classified into 44 subfamilies. The biggest subfamily is leucine-rich repeats (LRRs), and the other subfamilies include S-domains, Domain of Unknown Function26, CELL WALL-ASSOCIATED KINASE-like, and others (Shiu and Bleecker, 2001).The first RLK was found in maize (Zea mays; Walker and Zhang, 1990), and subsequently, a number of RLKs have been identified. RLK functions in many developmental processes, such as leaf development control by Crinkly4 (Becraft et al., 1996), meristem maintenance by CLAVATA1 (Clark et al., 1997), floral organ abscission by HAESA (Jinn et al., 2000), brassinosteroid (BR) perception by BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED KINASE1 (BAK1; Li et al., 2002; Nam and Li, 2002), organ shape regulation by ERECTA1 (Shpak et al., 2003; van Zanten et al., 2009), reproduction process by FERONIA (Huck et al., 2003; Escobar-Restrepo et al., 2007), and polarization of cell division by PANGLOSS1 (PAN1) and PAN2 (Cartwright et al., 2009; Zhang et al., 2012). In addition, RLK also works in defense responses. Xanthomonas resistance21 (Xa21) confers resistance to Xanthomonas oryzae pv oryzae (Wang et al., 1996); Lr10 LOCUS RECEPTOR KINASE (LRK10) mediates resistance to wheat (Triticum aestivum) rust pathogens (Feuillet et al., 1997, 2003); FLAGELLIN SENSITIVE2 (FLS2) is involved in the perception of the bacterial elicitor flagellin (Gómez-Gómez and Boller, 2000; Göhre et al., 2008; Danna et al., 2011); AVRPPHB SUSCEPTIBLE1 (PBS1) improves tolerance to Pseudomonas syringae strains (Swiderski and Innes, 2001); Pi-d2 enhances rice resistance to blast (Chen et al., 2006). Moreover, RLK also participates in abiotic stress responses. Salt-Induced Receptor-Like Kinase (Srlk) contributes to salt tolerance in the legume Medicago truncatula (de Lorenzo et al., 2009). STRESS-INDUCED PROTEIN KINASE1 (OsSIK1) mediates tolerance to drought and salt stress in rice (Ouyang et al., 2010). RECEPTOR-LIKE PROTEIN KINASE1 is implicated in hydrogen peroxide (H₂O₂) and abiotic stress processes (Osakabe et al., 2010; Lee et al., 2011). ABSCISIC ACID (ABA)- AND OSMOTIC-STRESS-INDUCIBLE RECEPTOR-LIKE CYTOSOLIC KINASE1 interacts with CYSTEINE-RICH REPEAT RLK36 (CRK36) and negatively regulates the ABA signaling pathway (Tanaka et al., 2012).In RLK superfamilies, S-domains belong to a large subfamily with 40 members in Arabidopsis and 147 members in rice (Morillo and Tax, 2006). The extracellular domain contains three modules, B_lectin, S-LOCUS GLYCOPROTEIN, and PAN_APPLE, which are presumed to mediate protein-protein and protein-carbohydrate interactions. Studies in this family are relatively few, and most reports focused on self-incompatibility, best characterized by S-RECEPTOR KINASE (SRK) in Brassica species (McCubbin and Kao, 2000; Takayama et al., 2000). Another member, CALMODULIN-BINDING RECEPTOR-LIKE PROTEIN KINASE1, was reported to be a negative regulator of pathogen resistance in Arabidopsis (Kim et al., 2009a, 2009b).Rice is an important crop, and previously, we studied the roles of a LRR-type RLK gene, OsSIK1, in the stress tolerance of rice plants (Ouyang et al., 2010). Here, we report the characterization of OsSIK2, an S-domain RLK gene, in abiotic stress tolerance and the senescence process. The gene is induced by various stresses. OsSIK2 has kinase activity and is localized in the plasma membrane. OsSIK2 overexpression promotes leaf emergence, improves plant tolerance to salt and drought stress, and delays leaf senescence. Interestingly, when the extracellular region was removed, the truncated version of OsSIK2 had stronger roles in conferring stress tolerance than the full-length version. The differential roles of the two versions may be exerted through the regulation of a common set and different sets of genes. Manipulation of OsSIK2 may improve the adaptive growth of rice plants.     

A Member of the Heavy Metal P-Type ATPase OsHMA5 Is Involved in Xylem Loading of Copper in Rice

Heavy metal-transporting P-type ATPase (HMA) has been implicated in the transport of heavy metals in plants. Here, we report the function and role of an uncharacterized member of HMA, OsHMA5 in rice (Oryza sativa). Knockout of OsHMA5 resulted in a decreased copper (Cu) concentration in the shoots but an increased Cu concentration in the roots at the vegetative stage. At the reproductive stage, the concentration of Cu in the brown rice was significantly lower in the mutants than in the wild-type rice; however, there was no difference in the concentrations of iron, manganese, and zinc between two independent mutants and the wild type. The Cu concentration of xylem sap was lower in the mutants than in the wild-type rice. OsHMA5 was mainly expressed in the roots at the vegetative stage but also in nodes, peduncle, rachis, and husk at the reproductive stage. The expression was up-regulated by excess Cu but not by the deficiency of Cu and other metals, including zinc, iron, and manganese, at the vegetative stage. Analysis of the transgenic rice carrying the OsHMA5 promoter fused with green fluorescent protein revealed that it was localized at the root pericycle cells and xylem region of diffuse vascular bundles in node I, vascular tissues of peduncle, rachis, and husk. Furthermore, immunostaining with an antibody against OsHMA5 revealed that it was localized to the plasma membrane. Expression of OsHMA5 in a Cu transport-defective mutant yeast (Saccharomyces cerevisiae) strain restored the growth. Taken together, OsHMA5 is involved in loading Cu to the xylem of the roots and other organs.Plants require nutrient elements to maintain normal growth and development. A number of different transporters, such as Cation Diffusion Facilitator, Natural resistance-associated macrophage protein, ATP-Binding Cassette, Zinc- and Iron-regulated-like Protein, and P-type ATPase, have been reported to be involved in the uptake, translocation, distribution, and homeostasis of nutrients (Hall and Williams, 2003; Krämer et al., 2007; Palmer and Guerinot, 2009). Among them, heavy metal-transporting P-type ATPase (HMA), the P_1B subfamily of the P-type ATPase superfamily, has been implicated in heavy metal transport (Williams and Mills, 2005; Grotz and Guerinot, 2006; Argüello et al., 2007; Burkhead et al., 2009). There are eight and nine members of P_1B-ATPase in Arabidopsis thaliana and rice (Oryza sativa), respectively (Williams and Mills, 2005). They are divided into two groups: zinc (Zn)/cadmium (Cd)/cobalt/lead (Pb) and copper (Cu)/silver transporters (Williams and Mills, 2005). AtHMA1 to AtHMA4 in A. thaliana and OsHMA1 to OsHMA3 in rice belong to the former group, while AtHMA5 to AtHMA8 and OsHMA4 to OsHMA9 belong to the latter group, although AtHMA1 has also been shown to transport Zn, Cu, and calcium (Axelsen and Palmgren, 2001; Williams and Mills, 2005; Seigneurin-Berny et al., 2006; Moreno et al., 2008; Kim et al., 2009).All members of HMAs in A. thaliana have been functionally characterized. AtHMA1 is involved in delivering Cu to the stroma, exporting Zn²⁺ from the chloroplast, or as a Ca²⁺/heavy metal transporter to the intracellular organelle (Seigneurin-Berny et al., 2006; Moreno et al., 2008; Kim et al., 2009). AtHMA2 and AtHMA4 localized at the pericycle are partially redundant and responsible for the release of Zn into the xylem (xylem loading) as well as Cd (Hussain et al., 2004; Verret et al., 2004; Wong and Cobbett, 2009; Wong et al., 2009), while AtHMA3 localized at the tonoplast plays a role in the detoxification of Zn/Cd/cobalt/Pb by mediating them into the vacuole (Morel et al., 2009; Chao et al., 2012). On the other hand, AtHMA5 is involved in the Cu translocation from roots to shoots or Cu detoxification of roots (Andrés-Colás et al., 2006; Kobayashi et al., 2008). AtHMA6 (PAA1, for P-type ATPase of Arabidopsis1) localized at the chloroplast periphery has been proposed to transport Cu over the chloroplast envelope, whereas AtHMA8 (PAA2) localized at the thylakoid membranes most likely transports Cu into the thylakoid lumen to supply plastocyanin (Shikanai et al., 2003; Abdel-Ghany et al., 2005). Finally, AtHMA7 (RESPONSIVE-TO-ANTAGONIST1) is responsible for delivering Cu to ethylene receptors and Cu homeostasis in the seedlings (Hirayama et al., 1999; Woeste and Kieber, 2000; Binder et al., 2010).By contrast, only three out of nine P-type ATPase members have been functionally characterized in rice. OsHMA2 was recently reported to be involved in the root-shoot translocation of Zn and Cd (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012; Yamaji et al., 2013). Furthermore, OsHMA2 at the node is required for preferential distribution of Zn to young leaves and panicles (Yamaji et al., 2013). OsHMA3 is localized to the tonoplast of the root cells and responsible for the sequestration of Cd into the vacuoles (Ueno et al., 2010; Miyadate et al., 2011). On the other hand, OsHMA9 was mainly expressed in vascular tissues, including the xylem and phloem (Lee et al., 2007). The knockout lines accumulated more Zn, Cu, Pb, and Cd, suggesting its role in the efflux of these metals from the cells (Lee et al., 2007).Some members of P-type ATPase have also been identified in other plant species, including barley (Hordeum vulgare), wheat (Triticum aestivum), Thlaspi caerulescens (Noccaea caerulescens), and Arabidopsis halleri. HvHMA1 from barley might be involved in mobilizing Zn and Cu during the stage of grain filling (Mikkelsen et al., 2012). HvHMA2 from barley and TaHMA2 from wheat showed similar functions as OsHMA2 in rice (Mills et al., 2012; Tan et al., 2013). AhHMA3 in A. halleri, a Zn hyperaccumulator, is probably involved in high Zn accumulation (Becher et al., 2004; Chiang et al., 2006). Furthermore, AhHMA4 for Zn translocation showed a higher expression level (Chiang et al., 2006; Hanikenne et al., 2008). On the other hand, TcHMA3 from ecotype Ganges of T. caerulescens, a Cd hyperaccumulator, plays an important role in the detoxification of Cd by sequestering Cd into the vacuole of the leaves (Ueno et al., 2011). High expression of TcHMA4 (NcHMA4) was also reported in T. caerulescens (Bernard et al., 2004; Papoyan and Kochian, 2004; Craciun et al., 2012).In this study, we investigated the function and role of an uncharacterized member of P-type ATPase in rice, OsHMA5. We found that OsHMA5 is involved in the xylem loading of Cu at both the vegetative and reproductive growth stages.     

AtGEN1 and AtSEND1, Two Paralogs in Arabidopsis,Possess Holliday Junction Resolvase Activity

Holliday junctions (HJs) are physical links between homologous DNA molecules that arise as central intermediary structures during homologous recombination and repair in meiotic and somatic cells. It is necessary for these structures to be resolved to ensure correct chromosome segregation and other functions. In eukaryotes, including plants, homologs of a gene called XPG-like endonuclease1 (GEN1) have been identified that process HJs in a manner analogous to the HJ resolvases of phages, archaea, and bacteria. Here, we report that Arabidopsis (Arabidopsis thaliana), a eukaryotic organism, has two functional GEN1 homologs instead of one. Like all known eukaryotic resolvases, AtGEN1 and Arabidopsis single-strand DNA endonuclease1 both belong to class IV of the Rad2/XPG family of nucleases. Their resolvase activity shares the characteristics of the Escherichia coli radiation and UV sensitive C paradigm for resolvases, which involves resolving HJs by symmetrically oriented incisions in two opposing strands. This leads to ligatable products without the need for further processing. The observation that the sequence context influences the cleavage by the enzymes can be interpreted as a hint for the existence of sequence specificity. The two Arabidopsis paralogs differ in their preferred sequences. The precise cleavage positions observed for the resolution of mobile nicked HJs suggest that these cleavage positions are determined by both the substrate structure and the sequence context at the junction point.To counter the effects of endogenous and exogenous factors that threaten the genome integrity, efficient mechanisms have evolved to ensure the faithful transmission of genetic information (Tuteja et al., 2001). Double-strand breaks, induced by conditions such as ionizing radiation or replication fork (RF) stalling, are among the most deleterious lesions (Jackson and Bartek, 2009). To protect the genome from consequences of these lesions, the cells have ancient double-strand break repair mechanisms, including the homologous recombination (HR) pathway. The HR mechanism is also of great importance in the intentional genetic recombination during sexual reproduction. A key intermediate in HR is the so-called Holliday junction (HJ), a structure that was first suggested in the context of a gene conversion model in fungi (Holliday, 1964) and later shown to arise in somatic and meiotic cells (Szostak et al., 1983; Schwacha and Kleckner, 1995; Cromie et al., 2006; Bzymek et al., 2010).HJs are structures consisting of four DNA strands of two homologous DNA helices (e.g. homologous chromosomes or sister chromatids). They arise through invasion of one single strand from each of two helices into the other double strand. This results in two continuous strands (one per helix) and two strands that cross from one helix into the other. Schematics often depict the HJs with a parallel orientation of the helices, in which the crossing strands cross each other as was originally postulated (Holliday, 1964). However, HJs based on oligonucleotides have been shown to adopt an antiparallel conformation (for review, see Lilley, 2000). In this configuration, the junction resembles the letter H in a lateral view, and the crossing strands actually perform U turns. The crossing strands represent physical links between the two DNA strands involved. If a RF is restored by HR-mediated repair during mitosis, the resulting HJ usually involves the two sister chromatids of one chromosome (Li and Heyer, 2008). In meiosis, the physical links in the shape of HJs arise because of meiotic crossover between homologous chromosomes. In either case, these links must be resolved to ensure unperturbed cell survival.The importance of resolving the HJs for the survival of cells and organisms is highlighted by the phenotypes described for mutants defective for the known pathways of HJ resolution. One of these pathways is the resolution by canonical HJ resolvases, enzymes that cleave the two opposing strands of a HJ in perfectly symmetric positions relative to the junction point, which results in readily ligatable nicked duplex (nD) products (Svendsen and Harper, 2010). This property distinguishes the canonical HJ resolvases from the noncanonical resolvases (see below).The main resolvase of Escherichia coli is radiation and UV sensitive C (RuvC), which is part of the E. coli resolvasome (RuvABC complex; Otsuji et al., 1974; Sharples et al., 1990, 1999). In this complex, a HJ is sandwiched between two RuvA tetramers (Panyutin and Hsieh, 1994). Two RuvB complexes form ATP-dependent motors of branch migration, with two opposing helical arms of the junction threaded through their central openings. For the resolution of the HJ, one RuvA tetramer is replaced by a RuvC homodimer. This homodimer positions two active sites at the center of the junction that are poised to cleave the junction point if a preferred consensus sequence of the form 5′-(A/T)TT^↓(G/C)-3′ is encountered. The requirement for this correct sequence is quite strict; even a single base change can lead to a drastic reduction of the cleavage efficiency (Shah et al., 1994). Isolated EcRuvC is also active in vitro and binds only HJ structures with high specificity. This binding is independent of the sequence context, but the cleavage depends on the specific sequence (Iwasaki et al., 1991; Benson and West, 1994; Dunderdale et al., 1994). The exact cleavage position has been determined to be either one nucleotide 3′ or 5′ from the junction or at the junction point (Bennett and West, 1996; Shida et al., 1996; Osman et al., 2009). The well-characterized EcRuvC is often referred to as a paradigm of canonical HJ resolution.Eukaryotes have evolved a more complex interplay of different HJ resolution pathways (Schwartz and Heyer, 2011; Zakharyevich et al., 2012). A defined complex, consisting of a recombination deficiency Q (RecQ) helicase (AtRECQ4A in Arabidopsis [Arabidopsis thaliana], Bloom syndrome protein in human, and Slow growth suppression1 (Sgs1) in yeast [Saccharomyces cerevisiae]), a type IA topoisomerase (DNA topoisomerase 3-alpha [TOP3A] in Arabidopsis, HsTOPOIIIα in human, and ScTop3 in yeast), and the structural protein RecQ-mediated genome instability1 (AtRMI1 in Arabidopsis, HsRMI1 in human, and ScRmi1 in yeast; RTR complex), mediates the so-called dissolution pathway. The crossing points of a double HJ are brought together by branch migration catalyzed by the helicase followed by decatenation catalyzed by the topoisomerase (Wu and Hickson, 2003; Hartung et al., 2007a, 2008; Mankouri and Hickson, 2007; Yang et al., 2010). In addition to the catalytic activities, a functional RTR complex also requires structural functions based on protein-protein interactions, for which RMI1 plays an essential role (Mullen et al., 2005; Chen and Brill, 2007; Bonnet et al., 2013; Schröpfer et al., 2014). Dissolution leads to noncross-over products and therefore, is a major mechanism in somatic yeast cells (Gangloff et al., 1994; Ira et al., 2003; Matos et al., 2011). In Arabidopsis, the loss of RTR component function leads to elevated rates of HR as well as sensitivity to UV light and methylmethane sulfonate (MMS; Bagherieh-Najjar et al., 2005; Hartung et al., 2007a; Bonnet et al., 2013). Mutants of AtRMI1 and AtTOP3A exhibit severe and unique meiotic phenotypes (Chelysheva et al., 2008; Hartung et al., 2008). This meiosis I arrest is dependent on HR, but the exact nature of the recombination intermediates that are involved remains unclear (Li et al., 2004; Hartung et al., 2007b; Knoll et al., 2014).Dissolution acts in parallel with a second pathway mediated by the structure-specific endonuclease MMS and UV-sensitive protein81 (MUS81) as shown by the fact that the additional mutation of ScSgs1/AtRECQ4A leads to synthetic lethality (Mullen et al., 2001; Hartung et al., 2006; Mannuss et al., 2010). Single mutants of MUS81 in yeast, human, Drosophila melanogaster, and Arabidopsis are sensitive to DNA-damaging agents that perturb RFs and show reduced HR after induction of double-strand breaks (Boddy et al., 2001; Hanada et al., 2006; Hartung et al., 2006). The MUS81 homologs form heterodimers with the noncatalytic subunit essential meiotic endonuclease1 (EME1; ScMms4 in S. cerevisiae). SpMus81-Eme1 was, to our knowledge, the first nuclear endonuclease reported to be capable of resolving HJs (Boddy et al., 2001). The Arabidopsis complexes can be formed with the two different subunits: AtEME1A or AtEME1B (Geuting et al., 2009). AtMUS81-EME1A/B, like the fission yeast ortholog, preferentially cleaves nicked Holliday junctions (nHJs) and 3′-flaps but also shows weaker activity on intact HJs in vitro (Boddy et al., 2001; Osman et al., 2003; Geuting et al., 2009; Schwartz and Heyer, 2011). MUS81 homologs are key players in meiotic cross-over generation (Osman et al., 2003; Berchowitz et al., 2007; Higgins et al., 2008). Although cross-over formation is solely dependent on SpMus81 in fission yeast, this function was shown to be shared with ScYen1 in budding yeast (Osman et al., 2003; Blanco et al., 2010; Ho et al., 2010; Tay and Wu, 2010). Tightly regulated by cell division cycle5-dependent hyperphosphorylation at the end of prophase I, the main activity of ScMus81-Mms4 is timed to coordinate with the formation of chiasmata and HJs that link the homologous chromosomes. This role in meiosis I is shown by the failure of chromosome segregation at the end of meiosis I in ScMus81 mutants (Matos et al., 2011). Interestingly, the chromosomes could be segregated at the end of meiosis II because of the presence of ScYen1. In contrast to canonical HJ resolvases, the hallmark of the MUS81-EME1 cleavage mechanism is the asymmetry of the second incision relative to either a first incision or a preexisting nick. This difference classifies MUS81-EME1 as a noncanonical resolvase. Its products need additional processing by gap-filling or flap-cleaving enzymes to allow religation (Boddy et al., 2001; Geuting et al., 2009).In very recent studies, HsMUS81-EME1 was found to constitute an essential canonical HJ resolvase with HsSLX1-SLX4 (SLX for synthetic lethal of unknown function), in which a first incision is made by HsSLX1-SLX4 followed by the enhanced action of the HsMUS81-EME1 subunits on the resulting nHJ (Garner et al., 2013; Wyatt et al., 2013). HsSLX1-SLX4 had previously been described as a canonical resolvase, albeit producing only a low level of symmetrically cut ligatable products (Fekairi et al., 2009).In addition to the mechanisms described above, an activity resembling that of EcRuvC had long been known to be present in mammalian cell-free extracts. In 2008, the group of Steven C. West succeeded in identifying, to their knowledge, the first nuclear proteins analogous to the EcRuvC paradigm: ScYen1 and Homo sapiens XPG-like endonuclease1 (HsGEN1; Ip et al., 2008). These proteins are members of the large and well-characterized Rad2/XPG family of nucleases. The Rad2/XPG family consists of the Xeroderma pigmentosum group G-complementing protein (XPG) endonucleases of the nucleotide excision repair (class I), the flap endonuclease1 (FEN1) replication-associated flap endonucleases (class II), the exodeoxyribonuclease1 (EXO1) exonucleases of recombination and repair (class III), and class IV (containing the [putative] eukaryotic HJ resolvases). This last class was introduced after the identification of the rice (Oryza sativa) single-strand DNA endonuclease1 (OsSEND-1) based on sequence homology. The class IV members show a domain composition homologous to FEN1 and EXO1, with no spacer region between their N-terminal XPG (XPG-N) and internal XPG (XPG-I) domains, whereas the primary structure of these domains is more similar to the sequence of the nuclease domain of XPG (Furukawa et al., 2003).Although all Rad2/XPG homologs share a common cleavage mechanism as observed for the typical 5′-flap substrate (Tsutakawa et al., 2011; Tsutakawa and Tainer, 2012), the striking evolutionary difference between classes I, II, and III on the one hand and the HJ resolvases (class IV) on the other hand is the ability of class IV members to form homodimers in vitro at their preferred substrate, the HJs (Rass et al., 2010). The homodimer configuration ensures the presence of two active sites positioned on the opposing strands of the HJ, which is necessary for resolution. The mode of eukaryotic HJ resolution is largely similar to the bacterial paradigm: (1) cleavage occurs one nucleotide in the 3′ direction of a static junction point (equivalent to the main cleavage site on 5′-flaps), (2) the incisions occur with almost perfect point symmetry, (3) the incisions result in readily ligatable nDs, and (4) certain sites within a migratable HJ core are preferred, providing evidence for a (yet to be determined) sequence specificity (Ip et al., 2008; Bailly et al., 2010; Rass et al., 2010; Yang et al., 2012).In the absence of MUS81-EME1/Mms4, the proteins HsGEN1, ScYen1, and CeGEN-1 have been shown to play a role in response to replication-associated perturbations, such as MMS- and UV-induced DNA damage (Bailly et al., 2010; Blanco et al., 2010; Tay and Wu, 2010; Gao et al., 2012; Muñoz-Galván et al., 2012). It is also likely that these proteins provide a backup mechanism in mitosis and meiosis, ensuring proper chromosome segregation after a failure of other mechanisms, including MUS81-EME1/Mms4 (Blanco et al., 2010; Matos et al., 2011).Although canonical HJ resolvases in animals and fungi are a current topic of great interest, very little is known about these proteins in plants. In rice, two members of the Rad2/XPG class IV have been described: OsSEND-1 (the founding member) and OsGEN-like (OsGEN-L). OsSEND-1 was shown to digest single-stranded circular DNA, and its expression is induced on MMS-induced genotoxic stress, whereas OsGEN-L is implicated in late spore development (Furukawa et al., 2003; Moritoh et al., 2005). Both studies (Furukawa et al., 2003; Moritoh et al., 2005) proposed putative homologs in other plants, and the gene locus At1g01880 of Arabidopsis, coding for the protein AtGEN1, is considered the ortholog of HsGEN1 and ScYen1 (Ip et al., 2008). However, currently, only OsGEN-L has been further investigated and described to possess in vitro properties similar to both Rad2/XPG nucleases and EcRuvC. This protein shows a well-defined 5′-flap activity as well as a poorly characterized ability, similar to that of EcRuvC, to resolve mobile HJs (Yang et al., 2012).Thus, of two members of Rad2/XPG class IV of plants, only one member has so far been analyzed with respect to a possible HJ resolvase activity. However, Arabidopsis expression data show that both proteins are expressed in plants and do not reveal marked differences (Laubinger et al., 2008). In this study, the goal was, therefore, to characterize the in vitro activities of not only AtGEN1 but also, AtSEND1, focusing on the idea that Arabidopsis and (seed) plants in general might encode not one but actually two HJ resolvases with functional homology to EcRuvC.