Focus Issue on Calcium Signaling: Identification of Cyclic GMP-Activated Nonselective Ca2+-Permeable Cation Channels and Associated CNGC5 and CNGC6 Genes in Arabidopsis Guard Cells

Cytosolic Ca²⁺ in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca²⁺ increases result from Ca²⁺ influx through Ca²⁺-permeable channels in the plasma membrane and Ca²⁺ release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca²⁺-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3′,5′-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg²⁺ as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba²⁺, Ca²⁺, and Na⁺ ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca²⁺-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO₂ concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca²⁺-permeable cation channels in the plasma membrane of Arabidopsis guard cells.Plants lose water via transpiration and take in CO₂ for photosynthesis through stomatal pores. Each stomatal pore is surrounded by two guard cells, and stomatal movements are driven by the change of turgor pressure in guard cells. The intracellular second messenger Ca²⁺ functions in guard cell signal transduction (Schroeder and Hagiwara, 1989; McAinsh et al., 1990; Webb et al., 1996; Grabov and Blatt, 1998; Allen et al., 1999; MacRobbie, 2000; Mori et al., 2006; Young et al., 2006; Siegel et al., 2009; Chen et al., 2010; Hubbard et al., 2012). Plasma membrane ion channel activity and gene expression in guard cells are finely regulated by the intracellular free calcium concentration ([Ca2+]_cyt; Schroeder and Hagiwara, 1989; Webb et al., 2001; Allen et al., 2002; Siegel et al., 2009; Kim et al., 2010; Stange et al., 2010). Ca²⁺-dependent protein kinases (CPKs) function as targets of the cytosolic Ca²⁺ signal, and several members of the CPK family have been shown to function in stimulus-induced stomatal closing, including the Arabidopsis (Arabidopsis thaliana) CPK3, CPK4, CPK6, CPK10, and CPK11 proteins (Mori et al., 2006; Zhu et al., 2007; Zou et al., 2010; Brandt et al., 2012; Hubbard et al., 2012). Further research found that several CPKs could activate the S-type anion channel SLAC1 in Xenopus laevis oocytes, including CPK21, CPK23, and CPK6 (Geiger et al., 2010; Brandt et al., 2012). At the same time, the Ca²⁺-independent protein kinase Open Stomata1 mediates stomatal closing and activates the S-type anion channel SLAC1 (Mustilli et al., 2002; Yoshida et al., 2002; Geiger et al., 2009; Lee et al., 2009; Xue et al., 2011), indicating that both Ca²⁺-dependent and Ca²⁺-independent pathways function in guard cells.Multiple essential factors of guard cell abscisic acid (ABA) signal transduction function in the regulation of Ca²⁺-permeable channels and [Ca2+]_cyt elevations, including Abscisic Acid Insensitive1 (ABI1), ABI2, Enhanced Response to Abscisic Acid1 (ERA1), the NADPH oxidases AtrbohD and AtrbohF, the Guard Cell Hydrogen Peroxide-Resistant1 (GHR1) receptor kinase, as well as the Ca²⁺-activated CPK6 protein kinase (Pei et al., 1998; Allen et al., 1999, 2002; Kwak et al., 2003; Miao et al., 2006; Mori et al., 2006; Hua et al., 2012). [Ca2+]_cyt increases result from both Ca²⁺ release from intracellular Ca²⁺ stores (McAinsh et al., 1992) and Ca²⁺ influx across the plasma membrane (Hamilton et al., 2000; Pei et al., 2000; Murata et al., 2001; Kwak et al., 2003; Hua et al., 2012). Electrophysiological analyses have characterized nonselective Ca²⁺-permeable channel activity in the plasma membrane of guard cells (Schroeder and Hagiwara, 1990; Hamilton et al., 2000; Pei et al., 2000; Murata et al., 2001; Köhler and Blatt, 2002; Miao et al., 2006; Mori et al., 2006; Suh et al., 2007; Vahisalu et al., 2008; Hua et al., 2012). However, the genetic identities of Ca²⁺-permeable channels in the plasma membrane of guard cells have remained unknown despite over two decades of research on these channel activities.The Arabidopsis genome includes 20 genes encoding cyclic nucleotide-gated channel (CNGC) homologs and 20 genes encoding homologs to animal Glu receptor channels (Lacombe et al., 2001; Kaplan et al., 2007; Ward et al., 2009), which have been proposed to function in plant cells as cation channels (Schuurink et al., 1998; Arazi et al., 1999; Köhler et al., 1999). Recent research has demonstrated functions of specific Glu receptor channels in mediating Ca²⁺ channel activity (Michard et al., 2011; Vincill et al., 2012). Previous studies have shown cAMP activation of nonselective cation currents in guard cells (Lemtiri-Chlieh and Berkowitz, 2004; Ali et al., 2007). However, only a few studies have shown the disappearance of a defined plasma membrane Ca²⁺ channel activity in plants upon mutation of candidate Ca²⁺ channel genes (Ali et al., 2007; Michard et al., 2011; Laohavisit et al., 2012; Vincill et al., 2012). Some CNGCs have been found to be involved in cation nutrient intake, including monovalent cation intake (Guo et al., 2010; Caballero et al., 2012), salt tolerance (Guo et al., 2008; Kugler et al., 2009), programmed cell death and pathogen responses (Clough et al., 2000; Balagué et al., 2003; Urquhart et al., 2007; Abdel-Hamid et al., 2013), thermal sensing (Finka et al., 2012; Gao et al., 2012), and pollen tube growth (Chang et al., 2007; Frietsch et al., 2007; Tunc-Ozdemir et al., 2013a, 2013b). Direct in vivo disappearance of Ca²⁺ channel activity in cngc disruption mutants has been demonstrated in only a few cases thus far (Ali et al., 2007; Gao et al., 2012). In this research, we show that CNGC5 and CNGC6 are required for a cyclic GMP (cGMP)-activated nonselective Ca²⁺-permeable cation channel activity in the plasma membrane of Arabidopsis guard cells.     

Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases

Abscisic acid (ABA) induces stomatal closure and inhibits light-induced stomatal opening. The mechanisms in these two processes are not necessarily the same. It has been postulated that the ABA receptors involved in opening inhibition are different from those involved in closure induction. Here, we provide evidence that four recently identified ABA receptors (PYRABACTIN RESISTANCE1 [PYR1], PYRABACTIN RESISTANCE-LIKE1 [PYL1], PYL2, and PYL4) are not sufficient for opening inhibition in Arabidopsis (Arabidopsis thaliana). ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant. ABA inhibition of the opening of the mutant’s stomata remained intact. ABA did not induce either the production of reactive oxygen species and nitric oxide or the alkalization of the cytosol in the quadruple mutant, in accordance with the closure phenotype. Whole cell patch-clamp analysis of inward-rectifying K⁺ current in guard cells showed a partial inhibition by ABA, indicating that the ABA sensitivity of the mutant was not fully impaired. ABA substantially inhibited blue light-induced phosphorylation of H⁺-ATPase in guard cells in both the mutant and the wild type. On the other hand, in a knockout mutant of the SNF1-related protein kinase, srk2e, stomatal opening and closure, reactive oxygen species and nitric oxide production, cytosolic alkalization, inward-rectifying K⁺ current inactivation, and H⁺-ATPase phosphorylation were not sensitive to ABA.The phytohormone abscisic acid (ABA), which is synthesized in response to abiotic stresses, plays a key role in the drought hardiness of plants. Reducing transpirational water loss through stomatal pores is a major ABA response (Schroeder et al., 2001). ABA promotes the closure of open stomata and inhibits the opening of closed stomata. These effects are not simply the reverse of one another (Allen et al., 1999; Wang et al., 2001; Mishra et al., 2006).A class of receptors of ABA was identified (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009; Nishimura et al., 2010). The sensitivity of stomata to ABA was strongly decreased in quadruple and sextuple mutants of the ABA receptor genes PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE-LIKE/REGULATORY COMPONENT OF ABSCISIC ACID RECEPTOR (PYR/PYL/RCAR; Nishimura et al., 2010; Gonzalez-Guzman et al., 2012). The PYR/PYL/RCAR receptors are involved in the early ABA signaling events, in which a sequence of interactions of the receptors with PROTEIN PHOSPHATASE 2Cs (PP2Cs) and subfamily 2 SNF1-RELATED PROTEIN KINASES (SnRK2s) leads to the activation of downstream ABA signaling targets in guard cells (Cutler et al., 2010; Kim et al., 2010; Weiner et al., 2010). Studies of Commelina communis and Vicia faba suggested that the ABA receptors involved in stomatal opening are not the same as the ABA receptors involved in stomatal closure (Allan et al., 1994; Anderson et al., 1994; Assmann, 1994; Schwartz et al., 1994). The roles of PYR/PYL/RCAR in either stomatal opening or closure remained to be elucidated.Blue light induces stomatal opening through the activation of plasma membrane H⁺-ATPase in guard cells that generates an inside-negative electrochemical gradient across the plasma membrane and drives K⁺ uptake through voltage-dependent inward-rectifying K⁺ channels (Assmann et al., 1985; Shimazaki et al., 1986; Blatt, 1987; Schroeder et al., 1987; Thiel et al., 1992). Phosphorylation of the penultimate Thr of the plasma membrane H⁺-ATPase is a prerequisite for blue light-induced activation of the H⁺-ATPase (Kinoshita and Shimazaki, 1999, 2002). ABA inhibits H⁺-ATPase activity through dephosphorylation of the penultimate Thr in the C terminus of the H⁺-ATPase in guard cells, resulting in prevention of the opening (Goh et al., 1996; Zhang et al., 2004; Hayashi et al., 2011). Inward-rectifying K⁺ currents (I_Kin) of guard cells are negatively regulated by ABA in addition to through the decline of the H⁺ pump-driven membrane potential difference (Schroeder and Hagiwara, 1989; Blatt, 1990; McAinsh et al., 1990; Schwartz et al., 1994; Grabov and Blatt, 1999; Saito et al., 2008). This down-regulation of ion transporters by ABA is essential for the inhibition of stomatal opening.A series of second messengers has been shown to mediate ABA-induced stomatal closure. Reactive oxygen species (ROS) produced by NADPH oxidases play a crucial role in ABA signaling in guard cells (Pei et al., 2000; Zhang et al., 2001; Kwak et al., 2003; Sirichandra et al., 2009; Jannat et al., 2011). Nitric oxide (NO) is an essential signaling component in ABA-induced stomatal closure (Desikan et al., 2002; Guo et al., 2003; Garcia-Mata and Lamattina, 2007; Neill et al., 2008). Alkalization of cytosolic pH in guard cells is postulated to mediate ABA-induced stomatal closure in Arabidopsis (Arabidopsis thaliana) and Pisum sativum and Paphiopedilum species (Irving et al., 1992; Gehring et al., 1997; Grabov and Blatt, 1997; Suhita et al., 2004; Gonugunta et al., 2008). These second messengers transduce environmental signals to ion channels and ion transporters that create the driving force for stomatal movements (Ward et al., 1995; MacRobbie, 1998; Garcia-Mata et al., 2003).In this study, we examined the mobilization of second messengers, the inactivation of I_Kin, and the suppression of H⁺-ATPase phosphorylation evoked by ABA in Arabidopsis mutants to clarify the downstream signaling events of ABA signaling in guard cells. The mutants included a quadruple mutant of PYR/PYL/RCARs, pyr1/pyl1/pyl2/pyl4, and a mutant of a SnRK2 kinase, srk2e.     

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.     

Involvement of the Sieve Element Cytoskeleton in Electrical Responses to Cold Shocks

This study dealt with the visualization of the sieve element (SE) cytoskeleton and its involvement in electrical responses to local cold shocks, exemplifying the role of the cytoskeleton in Ca²⁺-triggered signal cascades in SEs. High-affinity fluorescent phalloidin as well as immunocytochemistry using anti-actin antibodies demonstrated a fully developed parietal actin meshwork in SEs. The involvement of the cytoskeleton in electrical responses and forisome conformation changes as indicators of Ca²⁺ influx was investigated by the application of cold shocks in the presence of diverse actin disruptors (latrunculin A and cytochalasin D). Under control conditions, cold shocks elicited a graded initial voltage transient, ΔV₁, reduced by external La³⁺ in keeping with the involvement of Ca²⁺ channels, and a second voltage transient, ΔV₂. Cytochalasin D had no effect on ΔV₁, while ΔV₁ was significantly reduced with 500 nm latrunculin A. Forisome dispersion was triggered by cold shocks of 4°C or greater, which was indicative of an all-or-none behavior. Forisome dispersion was suppressed by incubation with latrunculin A. In conclusion, the cytoskeleton controls cold shock-induced Ca²⁺ influx into SEs, leading to forisome dispersion and sieve plate occlusion in fava bean (Vicia faba).It has been argued for a long time that sieve elements (SEs) are devoid of a cytoskeleton (Parthasarathy and Pesacreta, 1980; Thorsch and Esau, 1981; Evert, 1990), but more recent biochemical and cytological studies favor the opposite view. Actin as well as profilin were detected in phloem exudates of various monocot and dicot species (Schobert et al., 1998, 2000), while immunocytochemical tests showed the presence of actin and tubulin in phloem exudates of pumpkin (Cucurbita maxima; Kulikova and Puryaseva, 2002). Proteome analyses gave further credence to the occurrence of microfilaments in SEs in castor bean (Ricinus communis; profilin; Barnes et al., 2004), pumpkin (actin; Walz et al., 2004), canola (Brassica napus; actin, profilin1 and profilin2, actin-depolymerizing factor4; Giavalisco et al., 2006), and rice (Oryza sativa; actin1, actin-depolymerizing factor2, actin depolymerizing-factor3, and actin-depolymerizing factor6; Aki et al., 2008). Moreover, cytological evidence suggests residues of a cytoskeleton in SEs; fluorescent immunolabeling identified an actin/myosin system at the sieve plates (Chaffey and Barlow, 2002).Theoretical considerations also call for the presence of a cytoskeleton in SEs. Turnover and addressing of macromolecules (Fisher et al., 1992; Leineweber et al., 2000) requires a local distribution network in SEs. This function was attributed to an endoplasmic reticulum (ER) continuous to the ER strands running through pore plasmodesma units (Blackman et al., 1998) into the companion cells. Although such a mechanism is essentially conceivable, an interaction between the ER and cytoskeleton would provide a more conventional mode of intracellular distribution (Hepler et al., 1990; Boevink et al., 1998; Ueda et al., 2010; Yokota et al., 2011; Chen et al., 2012). Moreover, macromolecular trafficking through pore plasmodesma units (Lucas et al., 2001) was proposed to be executed by actin and myosin (Oparka, 2004), implying the presence of a cytoskeleton in SEs. Despite the massive circumstantial evidence, however, a complete cytoskeleton network and its spatial distribution in SEs have not been visually documented thus far.The existence of an SE cytoskeleton would raise questions regarding its task(s) in this highly specialized cell type. In other plant cells, the cytoskeleton was proposed to be engaged, among others, in ion channel operation and intracellular signaling (Trewavas and Malho, 1997; Mazars et al., 1997, and refs. therein; Thuleau et al., 1998; Örvar et al., 2000; Sangwan et al., 2001; Drøbak et al., 2004; Davies and Stankovic, 2006), as in animal cells (Janmey, 1998; Lange and Gartzke, 2006). For instance, K⁺ fluxes are regulated by actin dynamics (Hwang et al., 1997; Liu and Luan, 1998; Chérel, 2004), while Ca²⁺ influx into the cytoplasm appears to be mediated by voltage-dependent Ca²⁺-permeable channels associated with microtubules (Mazars et al., 1997; Thion et al., 1998) or by mechanosensitive channels possibly associated with microfilaments (Wang et al., 2004; Zhang et al., 2007).Both types of Ca²⁺-permeable channels probably reside in the SE plasma membrane (Knoblauch et al., 2001; Hafke et al., 2007, 2009; Furch et al., 2009), where they are likely involved in Ca²⁺-dependent systemic signaling (Furch et al., 2009; Hafke et al., 2009; van Bel et al., 2011; Hafke and van Bel, 2013). These channels are also putative initiators of Ca²⁺-induced signal transduction in SEs, leading to sieve-plate occlusion in response to local cold shocks (Thorpe et al., 2010). In fava bean (Vicia faba), Ca²⁺-dependent sieve tube occlusion by dispersion of special phloem-specific proteins (P-proteins) known as forisomes has been studied intensely (Knoblauch et al., 2001; Furch et al., 2007, 2009; Thorpe et al., 2010). Thus, apart from its distributive tasks, a cytoskeleton may be of major importance for intracellular signaling cascades in the highly specialized, sparsely equipped SEs.Our objective was to investigate the existence and spatial distribution of an SE cytoskeleton and its engagement in local signaling through Ca²⁺ influx brought about by cold shocks. This study dealt with the visualization of cytoskeletal components in intact sieve tubes using microinjection of fluorescent phalloidin and immunocytochemistry. Confocal laser-scanning micrography (CLSM) and transmission electron microscopy unequivocally showed a parietally located cylindrical actin meshwork. We demonstrated the engagement of the network in local cold shock-induced electrical responses and its association with Ca²⁺ influx, since we found effects of the Ca²⁺ channel blocker La³⁺ and of the cytoskeleton disruptor latrunculin A (LatA) on electrical signatures triggered by cold shocks and, by consequence, on forisome conformation changes.     

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.     

Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress

BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death.BAX INHIBITOR-1 (BI-1) is an endoplasmic reticulum (ER)-based cell death suppressor widely conserved in plants and animals (Xu and Reed, 1998; Kawai et al., 1999). In plants, BI-1 is considered a stress-associated factor, since its expression is stimulated by various stresses (Sanchez et al., 2000; Kawai-Yamada et al., 2001; Matsumura et al., 2003; Watanabe and Lam, 2006; Isbat et al., 2009). Although plants lack the homolog of animal BAX as an inducer of programmed cell death, loss of BI-1 expression results in a severe cell death phenotype under stress conditions, such as fumonisin B1-induced ER stress and disturbance of ion homeostasis (Watanabe and Lam, 2006; Ihara-Ohori et al., 2007). Conversely, plants overexpressing BI-1 exhibit tolerance to cell death induced by various stresses (Kawai-Yamada et al., 2001, 2004; Matsumura et al., 2003; Ihara-Ohori et al., 2007; Watanabe and Lam, 2008; Ishikawa et al., 2010). Moreover, BI-1 overexpression confers not only tolerance to oxidative stress-mediated cell death but also enhanced metabolic acclimation involved in energy and redox balance (Ishikawa et al., 2010). The results of these studies indicate that plant BI-1 is potentially useful for engineering stress-tolerant plants. However, little is known about the mode of action of BI-1 in the cell death regulatory pathway (Ishikawa et al., 2011). While overexpression systems sometimes include artificial or off-site effects, the observation that BI-1 overexpression improves stress tolerance suggests the importance of dissecting plants overexpressing it to further address the molecular basis of BI-1 function and cell death and stress tolerance management.As another approach to understand the molecular function of BI-1, screening of candidates interacting biochemically or functionally with BI-1 has been performed. First, Arabidopsis (Arabidopsis thaliana) BI-1 was confirmed to bind to calmodulin, like barley (Hordeum vulgare) MLO protein, a membrane-bound cell death regulator (Kim et al., 2002; Ihara-Ohori et al., 2007). Since the calmodulin-binding ability of BI-1 and MLO is necessary for their cell death-suppressing activity, Ca²⁺ signaling is critically involved in BI-1- and MLO-mediated cell death regulation (Kim et al., 2002; Kawai-Yamada et al., 2009). More recently, it was also demonstrated that the cell death suppression by BI-1 is mediated, at least in part, through fatty acid hydroxylase (FAH) in a Saccharomyces cerevisiae ectopic expression system (Nagano et al., 2009). In addition, Arabidopsis FAHs (AtFAH1 and AtFAH2) interact with BI-1 via cytochrome b₅ at the ER, resulting in the accumulation of 2-hydroxy fatty acids (2-HFAs) in Arabidopsis plants overexpressing BI-1. 2-HFAs are typical components of the ceramide backbone of sphingolipids (Imai et al., 1995; Pata et al., 2010). Although many functions of plant sphingolipids remain to be elucidated, accumulating evidence clearly indicates that sphingolipids and their metabolism are closely involved in cell death regulation and various stress responses in plants (Ng et al., 2001; Liang et al., 2003; Townley et al., 2005; Chen et al., 2008, 2012; Wang et al., 2008; Saucedo-García et al., 2011; Dutilleul et al., 2012; Kӧnig et al., 2012; Nagano et al., 2012; Mortimer et al., 2013), implying that BI-1 plays a role in cell death regulation through sphingolipid metabolism. Sphingolipids are major components of membrane lipids and are at particularly high concentrations in membrane microdomains, known as lipid rafts in animal cells, which are essential for membrane-mediated signaling and act as a sorting platform for targeted protein traffic (Simons and Toomre, 2000; Staubach and Hanisch, 2011). In mammalian cells, sphingomyelin metabolism in lipid rafts plays a vital role in the initiation of apoptotic cell death (Milhas et al., 2010). Recent studies have demonstrated the presence of raft-like membrane microdomains in plant cells and a role for them in defense responses and targeted protein sorting (Peskan et al., 2000; Fujiwara et al., 2009; Minami et al., 2009; Melser et al., 2010; Markham et al., 2011).This study focused on membrane microdomains in relation to BI-1-mediated sphingolipid metabolism. Our findings indicated that BI-1 alters sphingolipid composition in membrane microdomains, and this is accompanied by dynamic changes in a number of detergent-resistant membrane (DRM) proteins involved in cell death regulation.     

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.     

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

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