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.     

Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.The directed flow of auxin from cell to cell, through tissues and organs, from sites of synthesis to sites of action underlies the coordination of many processes during plant growth and development. Arabidopsis (Arabidopsis thaliana) PIN-FORMED (PIN) genes were the first found to be necessary for the phenomenon known as polar auxin transport (Okada et al., 1991; Chen et al., 1998; Gälweiler et al., 1998). Asymmetric localization of PIN proteins to the downstream ends of each cell in auxin-transporting tissues was correctly suggested to be a molecular component of the efflux mechanisms (Gälweiler et al., 1998) originally hypothesized as necessary for a directionally biased, or polar movement of auxin through tissues (Rubery and Sheldrake, 1974; Raven, 1975; Goldsmith, 1977; Goldsmith et al., 1981). Other members of the eight-gene PIN family in Arabidopsis were subsequently shown to affect auxin distribution in various tissues and stages of development (Křeček et al., 2009).Shortly after the breakthrough work on PIN1, members of the B subfamily of ATP-binding cassette (ABCB) transporters were discovered to be equally necessary for the phenomenon of polar auxin transport. They were originally called P-GLYCOPROTEIN1 (Dudler and Hertig, 1992; Sidler et al., 1998) and MULTIDRUG RESISTANCE1 (Noh et al., 2001) and ultimately renamed AtABCB1 and AtABCB19, respectively (Verrier et al., 2008). The connection between ABCB transporters and auxin transport was first made through the analysis of Arabidopsis knockout mutants. Polar auxin flow through abcb19 mutant stems is impaired by approximately 80% compared with the wild type and further reduced in abcb1 abcb19 double mutants (Noh et al., 2001). Resultant effects on development include abnormal hypocotyl tropisms (Noh et al., 2003) and the photomorphogenic control of hypocotyl elongation (Wu et al., 2010). Import of indole-3-acetic acid (IAA) to cotyledons through the petiole is reduced by 50% in abcb19 mutants, and this is correlated with an equivalent reduction in cotyledon blade expansion (Lewis et al., 2009). In roots, loss of ABCB19 greatly impairs auxin flow toward the tip without any detectable effect on shootward flow (Lewis et al., 2007). Surprisingly, the only defect detected in abcb19 primary roots associated with this major disruption of auxin transport is greater meandering of the tip during elongation down a vertical agar surface; gravitropism is unaffected (Lewis et al., 2007). Outgrowth of lateral roots, although not their initiation, depends significantly on ABCB19-mediated tipward auxin transport (Wu et al., 2007). The emergence of adventitious roots at the base of hypocotyls from which roots have been excised from Arabidopsis seedlings depends strongly on ABCB19-mediated auxin accumulation at the sites of primordium initiation (Sukumar et al., 2013).The ABCB19 protein is present predominantly in the central cylinder and cortex of the root, consistent with its role in rootward auxin transport (Lewis et al., 2007; Mravec et al., 2008), whereas the closely related ABCB4 is restricted to the lateral root cap and epidermis (Cho et al., 2007), where it functions in shootward auxin transport (Lewis et al., 2007). Loss of ABCB4 function alters the timing and spatial pattern of gravitropic curvature development, apparently because the gravity-induced auxin gradient across the root is less rapidly dissipated by normal shootward (basipetal) transport of the hormone through the elongation zone (Lewis et al., 2007). Root hairs are significantly longer in abcb4 mutants, a phenotype attributed to auxin accumulation due to impaired efflux (Cho et al., 2007). ABCB4 is reported to conduct auxin influx or efflux, depending on the prevailing external auxin concentration (Kubeš et al., 2012).Noh et al. (2001) originally isolated ABCB19 in a molecular screen for genes encoding an ion channel activity in Arabidopsis cells shown by patch-clamp electrophysiology to be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). The rationale for the screen was that a plant challenged with a channel blocker would overexpress the gene encoding the blocked activity. A hypothesis emerging from the Noh et al. (2001) study is that ABCB19 encodes such an ion channel, which is required for polar auxin transport. If true, NPPB would be established as a blocker of polar auxin transport.Pharmacological inhibitors, used for decades in auxin transport research, have some advantages over mutations. Mutations can create complicating pleiotropic effects by inhibiting the process throughout development, while inhibitors can be used to impose an effect at a specific time. 1-Naphthylphthalamic acid (NPA) is the most commonly used inhibitor of polar auxin transport (Katekar and Geissler, 1980), but others are being discovered (Rojas-Pierce et al., 2007; Kim et al., 2010; Tsuda et al., 2011). Inhibitors are especially useful when their targets are well defined, which would be the case if NPPB blocked ABCB19 and induced its expression as hypothesized. The experiments reported here were designed to test this hypothesis with electrophysiological measurements of ABCB19 transport activity, radiotracer measurements of polar auxin transport in roots, levels of fluorescently tagged ABCB19 proteins, auxin reporter expression patterns, and machine-vision measurements of a root growth response that depends on auxin redistribution.     

The TRANSPORT INHIBITOR RESPONSE2 Gene Is Required for Auxin Synthesis and Diverse Aspects of Plant Development

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.Auxin is known to play an important role in plant development (Davies, 1995). However, many aspects of auxin biology remain poorly understood. Auxin is synthesized primarily in young tissues, such as cotyledons, leaves, and roots (Ljung et al., 2001, 2005), and transported to other tissues where it is perceived by members of the TRANSPORT INHIBITOR RESPONSE1 (TIR1) auxin receptor family. Recent studies have dramatically increased our knowledge of auxin transport and signaling (Quint and Gray, 2006; Vieten et al., 2007). However, the pathways of auxin synthesis and their regulation are still relatively unclear.Several indole-3-acetic acid (IAA) biosynthetic pathways have been proposed in plants based on research in plant-associated bacteria (Patten and Glick, 1996; Woodward and Bartel, 2005; Spaepen et al., 2007). There are two major types of pathways: the Trp-dependent and Trp-independent pathways. It has been hypothesized that plants have four Trp-dependent pathways that are generally named after an intermediate. In bacteria, the indole-3-pyruvic acid (IPA) pathway, one of the Trp-dependent pathways, has been described in detail (Koga, 1995; Spaepen et al., 2007). The current model for the IPA pathway involves a Trp aminotransferase oxidatively transaminating Trp to IPA. Subsequently, an IPA decarboxylase converts IPA to indole-3-acetaldehyde, and indole-3-acetaldehyde is oxidized to IAA. The IPA pathway is considered a major IAA biosynthetic pathway in plants, since potential intermediates have been isolated from different species (Sheldrake, 1973; Cooney and Nonhebel, 1991; Koga, 1995; Tam and Normanly, 1998). In addition, Trp transamination activity has been found in many plants (Gamborg, 1965; Forest and Wightman, 1972; Truelsen, 1973). Recently, two groups reported the identification of a gene called TAA1. This gene encodes an aminotransferase that converts Trp to IPA and functions in IAA biosynthesis (Stepanova et al., 2008; Tao et al., 2008).To identify genes that are required for auxin synthesis, transport, and signaling, we previously screened for Arabidopsis (Arabidopsis thaliana) mutants that are resistant to auxin transport inhibitors, such as N-1-napthylpthalamic (NPA; Ruegger et al., 1997). The treatment of seedlings with NPA results in auxin accumulation in the root tip (Ljung et al., 2005). Thus, mutants that are resistant to NPA may have defects in synthesis, transport, or response because roots of these mutants are expected to have lower levels of IAA or reduced sensitivity to IAA. This screen succeeded in isolating mutations in seven genes with weak NPA-resistant phenotypes, including genes related to auxin signaling (TIR1), auxin transport (TIR3), and auxin synthesis (TIR7; Ruegger et al., 1997, 1998; Ljung et al., 2005).Here, we describe the characterization of TIR2, a gene whose function is required for auxin synthesis. Genetic and physiological analyses of the tir2 mutant suggest that TIR2 is required for the Trp-dependent auxin synthesis pathway and functions as a Trp aminotransferase. Molecular cloning of TIR2 reveals that the gene is identical to TAA1 (Stepanova et al., 2008; Tao et al., 2008). We show that auxin regulates expression of TIR2 in a tissue-specific manner. Furthermore, we show that TIR2 is required for temperature-dependent hypocotyl elongation and that high temperature positively regulates expression of the TIR2 gene, suggesting that temperature regulates hypocotyl elongation directly by stimulating auxin synthesis. Finally, we provide evidence that TIR2 functions in a positive regulatory loop required for root gravitropism.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.