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.     

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.     

Multiscale Metabolic Modeling: Dynamic Flux Balance Analysis on a Whole-Plant Scale

Plant metabolism is characterized by a unique complexity on the cellular, tissue, and organ levels. On a whole-plant scale, changing source and sink relations accompanying plant development add another level of complexity to metabolism. With the aim of achieving a spatiotemporal resolution of source-sink interactions in crop plant metabolism, a multiscale metabolic modeling (MMM) approach was applied that integrates static organ-specific models with a whole-plant dynamic model. Allowing for a dynamic flux balance analysis on a whole-plant scale, the MMM approach was used to decipher the metabolic behavior of source and sink organs during the generative phase of the barley (Hordeum vulgare) plant. It reveals a sink-to-source shift of the barley stem caused by the senescence-related decrease in leaf source capacity, which is not sufficient to meet the nutrient requirements of sink organs such as the growing seed. The MMM platform represents a novel approach for the in silico analysis of metabolism on a whole-plant level, allowing for a systemic, spatiotemporally resolved understanding of metabolic processes involved in carbon partitioning, thus providing a novel tool for studying yield stability and crop improvement.Plants are of vital significance as a source of food (Grusak and DellaPenna, 1999; Rogalski and Carrer, 2011), feed (Lu et al., 2011), energy (Tilman et al., 2006; Parmar et al., 2011), and feedstocks for the chemical industry (Metzger and Bornscheuer, 2006; Kinghorn et al., 2011). Given the close connection between plant metabolism and the usability of plant products, there is a growing interest in understanding and predicting the behavior and regulation of plant metabolic processes. In order to increase crop quality and yield, there is a need for methods guiding the rational redesign of the plant metabolic network (Schwender, 2009).Mathematical modeling of plant metabolism offers new approaches to understand, predict, and modify complex plant metabolic processes. In plant research, the issue of metabolic modeling is constantly gaining attention, and different modeling approaches applied to plant metabolism exist, ranging from highly detailed quantitative to less complex qualitative approaches (for review, see Giersch, 2000; Morgan and Rhodes, 2002; Poolman et al., 2004; Rios-Estepa and Lange, 2007).A widely used modeling approach is flux balance analysis (FBA), which allows the prediction of metabolic capabilities and steady-state fluxes under different environmental and genetic backgrounds using (non)linear optimization (Orth et al., 2010). Assuming steady-state conditions, FBA has the advantage of not requiring the knowledge of kinetic parameters and, therefore, can be applied to model detailed, large-scale systems. In recent years, the FBA approach has been applied to several different plant species, such as maize (Zea mays; Dal’Molin et al., 2010; Saha et al., 2011), barley (Hordeum vulgare; Grafahrend-Belau et al., 2009b; Melkus et al., 2011; Rolletschek et al., 2011), rice (Oryza sativa; Lakshmanan et al., 2013), Arabidopsis (Arabidopsis thaliana; Poolman et al., 2009; de Oliveira Dal’Molin et al., 2010; Radrich et al., 2010; Williams et al., 2010; Mintz-Oron et al., 2012; Cheung et al., 2013), and rapeseed (Brassica napus; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011), as well as algae (Boyle and Morgan, 2009; Cogne et al., 2011; Dal’Molin et al., 2011) and photoautotrophic bacteria (Knoop et al., 2010; Montagud et al., 2010; Boyle and Morgan, 2011). These models have been used to study different aspects of metabolism, including the prediction of optimal metabolic yields and energy efficiencies (Dal’Molin et al., 2010; Boyle and Morgan, 2011), changes in flux under different environmental and genetic backgrounds (Grafahrend-Belau et al., 2009b; Dal’Molin et al., 2010; Melkus et al., 2011), and nonintuitive metabolic pathways that merit subsequent experimental investigations (Poolman et al., 2009; Knoop et al., 2010; Rolletschek et al., 2011). Although FBA of plant metabolic models was shown to be capable of reproducing experimentally determined flux distributions (Williams et al., 2010; Hay and Schwender, 2011b) and generating new insights into metabolic behavior, capacities, and efficiencies (Sweetlove and Ratcliffe, 2011), challenges remain to advance the utility and predictive power of the models.Given that many plant metabolic functions are based on interactions between different subcellular compartments, cell types, tissues, and organs, the reconstruction of organ-specific models and the integration of these models into interacting multiorgan and/or whole-plant models is a prerequisite to get insight into complex plant metabolic processes organized on a whole-plant scale (e.g. source-sink interactions). Almost all FBA models of plant metabolism are restricted to one cell type (Boyle and Morgan, 2009; Knoop et al., 2010; Montagud et al., 2010; Cogne et al., 2011; Dal’Molin et al., 2011), one tissue or one organ (Grafahrend-Belau et al., 2009b; Hay and Schwender, 2011a, 2011b; Pilalis et al., 2011; Mintz-Oron et al., 2012), and only one model exists taking into account the interaction between two cell types by specifying the interaction between mesophyll and bundle sheath cells in C4 photosynthesis (Dal’Molin et al., 2010). So far, no model representing metabolism at the whole-plant scale exists.Considering whole-plant metabolism raises the problem of taking into account temporal and environmental changes in metabolism during plant development and growth. Although classical static FBA is unable to predict the dynamics of metabolic processes, as the network analysis is based on steady-state solutions, time-dependent processes can be taken into account by extending the classical static FBA to a dynamic flux balance analysis (dFBA), as proposed by Mahadevan et al. (2002). The static (SOA) and dynamic optimization approaches introduced in this work provide a framework for analyzing the transience of metabolism by integrating kinetic expressions to dynamically constrain exchange fluxes. Due to the requirement of knowing or estimating a large number of kinetic parameters, so far dFBA has only been applied to a plant metabolic model once, to study the photosynthetic metabolism in the chloroplasts of C3 plants by a simplified model of five biochemical reactions (Luo et al., 2009). Integrating a dynamic model into a static FBA model is an alternative approach to perform dFBA.In this study, a multiscale metabolic modeling (MMM) approach was applied with the aim of achieving a spatiotemporal resolution of cereal crop plant metabolism. To provide a framework for the in silico analysis of the metabolic dynamics of barley on a whole-plant scale, the MMM approach integrates a static multiorgan FBA model and a dynamic whole-plant multiscale functional plant model (FPM) to perform dFBA. The performance of the novel whole-plant MMM approach was tested by studying source-sink interactions during the seed developmental phase of barley plants.     

Salt Stress Reduces Root Meristem Size by Nitric Oxide-Mediated Modulation of Auxin Accumulation and Signaling in Arabidopsis

The development of the plant root system is highly plastic, which allows the plant to adapt to various environmental stresses. Salt stress inhibits root elongation by reducing the size of the root meristem. However, the mechanism underlying this process remains unclear. In this study, we explored whether and how auxin and nitric oxide (NO) are involved in salt-mediated inhibition of root meristem growth in Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic approaches. We found that salt stress significantly reduced root meristem size by down-regulating the expression of PINFORMED (PIN) genes, thereby reducing auxin levels. In addition, salt stress promoted AUXIN RESISTANT3 (AXR3)/INDOLE-3-ACETIC ACID17 (IAA17) stabilization, which repressed auxin signaling during this process. Furthermore, salt stress stimulated NO accumulation, whereas blocking NO production with the inhibitor N^ω-nitro-l-arginine-methylester compromised the salt-mediated reduction of root meristem size, PIN down-regulation, and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated inhibition of root meristem growth. Taken together, these findings suggest that salt stress inhibits root meristem growth by repressing PIN expression (thereby reducing auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via increasing NO levels.Due to agricultural practices and climate change, soil salinity has become a serious factor limiting the productivity and quality of agricultural crops (Zhu, 2007). Worldwide, high salinity in the soil damages approximately 20% of total irrigated lands and takes 1.5 million ha out of production each year (Munns and Tester, 2008). In general, high salinity affects plant growth and development by reducing plant water potential, altering nutrient uptake, and increasing the accumulation of toxic ions (Hasegawa et al., 2000; Munns, 2002; Zhang and Shi, 2013). Together, these effects severely reduce plant growth and survival.Because the root is the first organ to sense high salinity, salt stress plays a direct, important role in modulating root system architecture (Wang et al., 2009). For instance, salt stress negatively regulates root hair formation and gravitropism (Sun et al., 2008; Wang et al., 2008). The role of salt in lateral root formation depends on the NaCl concentration. While high NaCl levels inhibit lateral root formation, lower NaCl levels stimulate lateral root formation in an auxin-dependent manner (Zolla et al., 2010; Ji et al., 2013). The root meristem plays an essential role in sustaining root growth (Perilli et al., 2012). Salt stress inhibits primary root elongation by suppressing root meristem activity (West et al., 2004). However, how this inhibition occurs remains largely unclear.Plant hormones are important intermediary signaling compounds that function downstream of environmental stimuli. Among plant hormones, indole-3-acetic acid (IAA) is thought to play a fundamental role in root system architecture by regulating cell division, expansion, and differentiation. In Arabidopsis (Arabidopsis thaliana) root tips, a distal auxin maximum is formed and maintained by polar auxin transport (PAT), which determines the orientation and extent of cell division in the root meristem as well as root pattern formation (Sabatini et al., 1999). PINFORMED (PIN) proteins, which are components of the auxin efflux machinery, regulate primary root elongation and root meristem size (Blilou et al., 2005; Dello Ioio et al., 2008; Yuan et al., 2013, 2014). The auxin signal transduction pathway is activated by direct binding of auxin to its receptor protein, TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB), promoting the degradation of Aux/IAA proteins, releasing auxin response factors (ARFs), and activating the expression of auxin-responsive genes (Gray et al., 2001; Dharmasiri et al., 2005a; Kepinski and Leyser, 2005). Aux/IAA proteins are short-lived, nuclear-localized proteins that play key roles in auxin signal activation and root growth modulation (Rouse et al., 1998). Other hormones and stresses often regulate auxin signaling by affecting Aux/IAA protein stability (Lim and Kunkel, 2004; Nemhauser et al., 2004; Wang et al., 2007; Kushwah and Laxmi, 2014).Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants (He et al., 2004; Fernández-Marcos et al., 2011; Shi et al., 2012), including important roles in the regulation of root growth and development. NO functions downstream of auxin during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002). Exogenous auxin-induced NO biosynthesis is associated with nitrate reductase activity during lateral root formation, and NO is necessary for auxin-induced lateral root and root hair development (Pagnussat et al., 2002; Lombardo et al., 2006). Pharmacological and genetic analyses in Arabidopsis indicate that NO suppresses primary root growth and root meristem activity (Fernández-Marcos et al., 2011). Additionally, both exogenous application of the NO donor sodium nitroprusside (SNP) and overaccumulation of NO in the mutant chlorophyll a/b binding protein underexpressed1 (cue1)/nitric oxide overproducer1 (nox1) result in reduced PIN1 expression and auxin accumulation in root tips. The auxin receptors protein TIR1 is S-nitrosylated by NO, suggesting that this protein is a direct target of NO in the regulation of root development (Terrile et al., 2012).Because NO is a free radical, NO levels are dynamically regulated by endogenous and environmental cues. Many phytohormones, including abscisic acid, auxin, cytokinin, salicylic acid, jasmonic acid, and ethylene, induce NO biosynthesis (Zottini et al., 2007; Kolbert et al., 2008; Tun et al., 2008; García et al., 2011). In addition, many abiotic and biotic stresses or stimuli, such as cold, heat, salt, drought, heavy metals, and pathogens/elicitors, also stimulate NO biosynthesis (Zhao et al., 2009; Mandal et al., 2012). Salt stress stimulates NO and ONOO^– accumulation in roots (Corpas et al., 2009), but the contribution of NO to root meristem growth under salinity stress has yet to be examined in detail.In this study, we found that salt stress significantly down-regulated the expression of PIN genes and promoted AUXIN RESISTANT3 (AXR3)/IAA17 stabilization. Furthermore, salt stress stimulated NO accumulation, and pharmacological inhibition of NO biosynthesis compromised the salt-mediated reduction in root meristem size. Our results support a model in which salt stress reduces root meristem size by increasing NO accumulation, which represses PIN expression and stabilizes IAA17, thereby reducing auxin levels and repressing auxin signaling.     

Natural Variation of Arabidopsis Root Architecture Reveals Complementing Adaptive Strategies to Potassium Starvation

Root architecture is a highly plastic and environmentally responsive trait that enables plants to counteract nutrient scarcities with different foraging strategies. In potassium (K) deficiency (low K), seedlings of the Arabidopsis (Arabidopsis thaliana) reference accession Columbia (Col-0) show a strong reduction of lateral root elongation. To date, it is not clear whether this is a direct consequence of the lack of K as an osmoticum or a triggered response to maintain the growth of other organs under limiting conditions. In this study, we made use of natural variation within Arabidopsis to look for novel root architectural responses to low K. A comprehensive set of 14 differentially responding root parameters were quantified in K-starved and K-replete plants. We identified a phenotypic gradient that links two extreme strategies of morphological adaptation to low K arising from a major tradeoff between main root (MR) and lateral root elongation. Accessions adopting strategy I (e.g. Col-0) maintained MR growth but compromised lateral root elongation, whereas strategy II genotypes (e.g. Catania-1) arrested MR elongation in favor of lateral branching. K resupply and histochemical staining resolved the temporal and spatial patterns of these responses. Quantitative trait locus analysis of K-dependent root architectures within a Col-0 × Catania-1 recombinant inbred line population identified several loci each of which determined a particular subset of root architectural parameters. Our results indicate the existence of genomic hubs in the coordinated control of root growth in stress conditions and provide resources to facilitate the identification of the underlying genes.The ability of plants to actively respond to nutrient scarcity with changes in root architecture is a fascinating phenomenon. Advances in root research and breeding efforts that focus on the enhancement of root traits have been recognized as principal goals to ensure those high yields necessary to feed an ever-growing human population (Hammer et al., 2009; Den Herder et al., 2010). Indeed, understanding the adaptations of root systems to environmental factors has been pointed out as a key issue in modern agriculture (Den Herder et al., 2010).Potassium (K) is the quantitatively most important cation for plant growth, as it serves as the major osmoticum for cell expansion (Leigh and Wyn Jones, 1984; Amtmann et al., 2006). Moreover, K is essential for many cellular and tissue processes, such as enzymatic activity, transport of minerals and metabolites, and regulation of stomatal aperture (Amtmann et al., 2006). Even in fertilized fields, rapid K uptake by plants can lead to K shortage in the root environment, especially early in the growth season. Root adaptations to K deficiency (low K) take place at the physiological (Armengaud et al., 2004; Shin and Schachtman, 2004; Alemán et al., 2011), metabolic (Armengaud et al., 2009a), and morphological levels. In a classic study, Drew (1975) showed an increase in overall lateral root (LR) growth of barley seedlings, even when K was supplied only to parts of the root system. Conversely, a typical response of Arabidopsis (Arabidopsis thaliana) Columbia (Col-0) seedlings to low K is the drastic reduction of LR elongation (Armengaud et al., 2004; Shin and Schachtman, 2004). Conflicting data have been published on the effect of low K on main root (MR) growth in the same species, ranging from no effect (Shin and Schachtman, 2004) to impaired MR elongation (Jung et al., 2009; Kim et al., 2010). Some components involved in K starvation responses have been identified, such as jasmonates (Armengaud et al., 2004, 2010), reactive oxygen species (Shin and Schachtman, 2004), and ethylene (Jung et al., 2009). However, the molecular identity of a root K sensor acting at the base of the signaling cascade is so far unknown.Genetic variation within species is a useful resource to dissect the genetic components determining phenotypes (Koornneef et al., 2004; Trontin et al., 2011; Weigel, 2012). Natural variation within Arabidopsis has been the basis for many studies on plant morphology, physiology, and development as well as stress response (Alonso-Blanco et al., 2009; Weigel, 2012). Natural variation of root traits such as primary root length (Mouchel et al., 2004; Loudet et al., 2005; Sergeeva et al., 2006), LR length (Loudet et al., 2005), and total root size (Fitz Gerald et al., 2006) have pinpointed genomic regions underlying the phenotypic variation via mapping of quantitative trait loci (QTLs) as a first step toward the identification of novel regulatory genes (Mouchel et al., 2004). This strategy has also been applied to environmental responses, such as growth responses to phosphate starvation (Reymond et al., 2006; Svistoonoff et al., 2007). However, despite their importance for plant growth and their strong effect on overall root architecture, root responses to K deficiency have not been genetically dissected.Here, we show that Arabidopsis accessions follow different strategies to adapt to K starvation. We present the quantification of a comprehensive set of root architectural parameters of Arabidopsis grown in K-sufficient and K-deficient media and the identification of genetic loci, each of which determines the response of a distinct subset of root architectural parameters to K starvation.     

Transposon-Mediated Alteration of TaMATE1B Expression in Wheat Confers Constitutive Citrate Efflux from Root Apices

The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3′ regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance.Intraspecific variation in aluminum (Al3+) tolerance is evident in many crop species and has been shown to be under either simple or complex genetic control (Ma et al., 2004; Magalhaes et al., 2007; Krill et al., 2010; Famoso et al., 2011). In wheat (Triticum aestivum), Al3+ tolerance is primarily associated with efflux of organic anions from root tips, with at least two independent mechanisms being involved. The first and most widely distributed among genotypes is the Al3+-activated efflux of malate from root apices (Delhaize et al., 1993, Ryan et al., 1995). More recently, a second and less prevalent mechanism has been identified that relies on the constitutive efflux of citrate from root apices (Ryan et al., 2009).Organic anions protect roots by chelating and detoxifying Al3+ in the apoplast and rhizosphere around sensitive root apices (Delhaize et al., 1993, 2012; Ryan et al., 2001). For example, Al3+-tolerant genotypes of wheat release significantly more malate than sensitive genotypes (Ryan et al., 1995). The efflux of malate from root apices is mediated by the TaALMT1 (for Al3+-activated malate transporter) gene, which resides on chromosome 4DL (Raman et al., 2005, 2008; Zhou et al., 2007). TaALMT1 encodes an Al3+-activated anion channel permeable to malate located on the plasma membrane of root cells (Sasaki et al., 2004; Yamaguchi et al., 2005; Piñeros et al., 2008; Zhang et al., 2008). Similarly, ALMT1 genes contribute to Al3+ tolerance in Arabidopsis (Arabidopsis thaliana), oilseed rape (Brassica napus), and rye (Secale cereale; Ryan et al., 2011). While all members of the ALMT family characterized to date encode transport proteins, only a minority are involved in Al3+ tolerance. The others contribute to physiological processes related to ionic relations and osmotic adjustment (Kovermann et al., 2007; Gruber et al., 2010; Meyer et al., 2010, 2011; Sasaki et al., 2010).In many plant species, citrate efflux from root apices also confers Al3+ tolerance. The genes controlling citrate efflux were first identified in sorghum (Sorghum bicolor; SbMATE [for multidrug and toxic compound extrusion]; Magalhaes et al., 2007) and barley (Hordeum vulgare; HvAACT1; Furukawa et al., 2007), with additional genes subsequently identified in Arabidopsis (Liu et al., 2009), maize (Zea mays; Maron et al., 2010), rice (Yokosho et al., 2009), and rice bean (Vigna umbellate; Yang et al., 2011). These genes belong to a large and ubiquitous family of MATE genes that were first identified in prokaryotes (Hvorup et al., 2003). SbMATE and HvAACT1 are part of a subset of plant MATE genes that facilitate citrate efflux (Liu et al., 2009; Magalhaes, 2010) with roles in Al3+ tolerance and iron (Fe) nutrition. For example, FRD3 from Arabidopsis and OsFRDL1 from rice both encode MATE proteins located in vascular tissue, where they release citrate into the xylem to enable Fe transport to shoots (Durrett et al., 2007). Recent evidence suggests that the Al3+ tolerance gene in barley, HvAACT1, was coopted from an original function in Fe nutrition by a random mutation. Al3+-tolerant genotypes of barley have a 1-kb insertion in the 5′ untranslated region (UTR) of the HvAACT1 coding region that alters its expression pattern. The insertion extends HvAACT1 expression to the root apices, which, in the presence of Al3+, causes citrate efflux from the apices and enhanced Al3+ tolerance (Fujii et al., 2012).Evidence that citrate efflux confers Al3+ tolerance in some genotypes of wheat was provided by Ryan et al. (2009). The trait was mapped to the long arm of chromosome 4B and cosegregated with an EST showing 94% sequence identity to HvAACT1. This EST was expressed only in the root apices of wheat lines that showed citrate efflux (e.g. cv Carazinho; Ryan et al., 2009), suggesting that a MATE gene encodes citrate efflux from root apices. Here, we describe the isolation and characterization of a MATE gene from wheat named TaMATE1B that encodes a citrate transporter located on the plasma membrane. A large transposable element-like sequence was found to be inserted near the start of the TaMATE1B coding region specifically in genotypes that release citrate. We demonstrate that the fragment functions as a promoter that extends TaMATE1B expression to root apices and discuss how this mutation recruited a gene to confer a new phenotype associated with Al3+ tolerance.     

Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box protein^{S-Phase Kinase-Associated Protein 2B}-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses.Roots have developed adaptive strategies to reprogram their gene expression and metabolic activity in response to heterogeneous soil environments (Osmont et al., 2007). By this way, local environmental stimuli can be integrated into the developmental program of roots (Forde, 2014; Giehl and von Wirén, 2014). In resource-depleted environments, an important heterogeneously distributed soil factor is nutrient availability, which then directs lateral root growth preferentially into nutrient-rich patches (Zhang and Forde, 1998; Lima et al., 2010; Giehl et al., 2012). Such directed lateral root development depends on regulatory networks that integrate both local and systemic signals to coordinate them with the overall plant nutritional status (Ruffel et al., 2011; Guan et al., 2014). As shown by the impact of the N status-dependent regulatory module CLAVATA3/EMBRYO-SURROUNDING REGION-related peptides-CLAVATA1 leucine-rich repeat receptor-like kinase, economizing the costs for root development is pivotal for a resource-efficient strategy in nutrient acquisition (Araya et al., 2014). In recent years, strategies on yield and efficiency improvement have been developed that are primarily based on the manipulation of root system architecture (Gregory et al., 2013; Lynch, 2014; Meister et al., 2014). A common imperative of these strategies is to develop crops that use water and nutrients more efficiently, allowing the reduction of fertilizer input and potentially hazardous environmental contamination.Maize (Zea mays) plays an eminent role in global food, feed, and fuel production, which is also a consequence of its unique root system (Rogers and Benfey, 2015). The genetic analysis of maize root architecture revealed a complex molecular network coordinating root development during the whole lifecycle (for review, see Hochholdinger et al., 2004a, 2004b). Identification of root type-specific lateral root mutants in maize emphasized the existence of regulatory mechanisms involved in the branching of embryonic roots, which are distinct from those in postembryonic roots (Hochholdinger and Feix, 1998; Woll et al., 2005). Under heterogeneous nutrient supplies, nitrate-rich patches increased only the length of lateral roots in primary and seminal roots, whereas they increased both length and density of lateral roots from shoot-borne roots of adult maize plants (Yu et al., 2014a). Remarkably, modulation of the extensive postembryonic shoot-borne root stock has a great potential to improve grain yield and nutrient use efficiency (Hochholdinger and Tuberosa, 2009).Lateral root branching is critical to secure anchorage and ensure adequate uptake of water and nutrients. In maize, these roots originate from concentric single-file layers of pericycle and endodermis cells (Fahn, 1990; Jansen et al., 2012). Lateral root initiation is the result of auxin-dependent cell cycle progression (Beeckman et al., 2001; Jansen et al., 2013a). Most of the molecular changes during the cell cycle like, for instance, the induction of positive regulators, such as cyclins (CYCs) and cyclin-dependent kinases (CDKs), and the repression of Kip-related proteins (KRPs), thus account for a reactivation of the cell cycle (Beeckman et al., 2001; Himanen et al., 2002, 2004). In eukaryotes, ubiquitin-mediated degradation of cell cycle proteins plays a critical role in the regulation of cell division (Hershko, 2005; Jakoby et al., 2006). Conjugation of ubiquitin to a substrate requires the sequential action of three enzymes: ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase (E3). The E3 enzymes are responsible for the specificity of the pathway, and several classes of E3 enzymes have been implicated in cell cycle regulation, including the S-Phase Kinase-Associated Protein1-cullin-F-box protein (SCF) and Really Interesting New Gene (RING) finger-domain ubiquitin ligases (Del Pozo and Manzano, 2014). The F-box protein S-Phase Kinase-Associated Protein 2B (SKP2B) encodes an F-box ubiquitin ligase, which plays an important role in the cell cycle by regulating the stability of KRP1 and pericycle founder cell division during lateral root initiation (Ren et al., 2008; Manzano et al., 2012).It has been shown that auxin is involved in long-distance signaling to adjust root growth in response to local nutrient availability (Giehl et al., 2012), and it is likely to serve in long-distance signaling for local nutrient responses as well (for review, see Rubio et al., 2009; Krouk et al., 2011; Saini et al., 2013; Forde, 2014). Polar auxin transport is instrumental for the generation of local auxin maxima, which guide these cells to switch their developmental program (Vanneste and Friml, 2009; Lavenus et al., 2013). In Arabidopsis (Arabidopsis thaliana), the PIN-FORMED (PIN) family of auxin efflux carrier proteins controls the directionality of auxin flows to maximum formation at the tip or pericycle cells (Benková et al., 2003; Laskowski et al., 2008; Marhavý et al., 2013). Auxin responses in protoxylem or protophloem cells of the basal meristem coincide with the site of lateral root initiation (De Smet et al., 2007; Jansen et al., 2012). In these defined pericycle cells, the phloem pole pericycle founder cells are primed before auxin accumulation occurs (De Smet et al., 2007; Jansen et al., 2012, 2013a). In contrast to dicots, the larger PIN family in monocots has a more divergent phylogenetic structure (Paponov et al., 2005). It is likely that monocot-specific PIN genes regulate monocot-specific morphogenetic processes, such as the development of a complex root system (Wang et al., 2009; Forestan et al., 2012).The molecular control of lateral root initiation of the root system to heterogeneous nitrate availabilities is not yet understood in maize. In this study, the plasticity of lateral root induction in adult shoot-borne roots of maize in response to local high concentration of nitrate was surveyed in an experimental setup that simulated patchy nitrate distribution. RNA-sequencing (RNA-Seq) experiments and cell type-specific gene expression analyses showed that local nitrate triggers progressive cell cycle control during pericycle cell division. In addition, tissue-specific determination of indole-3-acetic acid (IAA) and its metabolites combined with auxin maxima determination by DR5 supported a role of basipetal auxin transport during lateral root initiation in shoot-borne roots. Thereby, this study provides unique insights in how auxin orchestrates cell cycle control under local nitrate stimulation in the shoot-borne root system of maize.