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Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.Starch is the most important carbohydrate used for food and feed purposes and represents the major resource for our diet (Smith, 2008). The total yield of starch in rice (Oryza sativa), corn (Zea mays), wheat (Triticum aestivum), and potato (Solanum tuberosum) exceeds 109 tons per year (Kossmann and Lloyd, 2000; Slattery et al., 2000). In addition to its use in a nonprocessed form, extracted starch is processed in many different ways, for instance as a high-Fru syrup, as a food additive, or for various technical purposes. As a result of this considerable importance, increasing the starch content of plant tissues has been a major goal for many years, with both classical breeding and biotechnological approaches being taken extensively over the last few decades (Martin and Smith, 1995; Regierer et al., 2002).The pathway by which carbon is converted from Suc to starch in the potato tuber is well established (Kruger, 1997; Fernie et al., 2002; Geigenberger et al., 2004; Geigenberger, 2011). Imported Suc is cleaved in the cytosol by Suc synthase, resulting in the formation of UDP-Glc and Fru; the UDP-Glc is subsequently converted to Glc-1-P by UDP-Glc pyrophosphorylase. The second product of the Suc synthase reaction, Fru, is efficiently phosphorylated to Fru-6-P by fructokinase (Renz et al., 1993; Davies et al., 2005). Fru-6-P is freely converted to Glc-6-P, in which form it normally enters the amyloplast (Kammerer et al., 1998; Tauberger et al., 2000; Zhang et al., 2008), and once in the plastid, it is converted to starch via the concerted action of plastidial phosphoglucomutase, ADP-Glc pyrophosphorylase (AGPase), and the various isoforms of starch synthase (Martin and Smith, 1995; Geigenberger, 2011). Of these reactions, although some of the control of starch synthesis resides in the plastidial phosphoglucomutase reaction (Fernie et al., 2001b), the AGPase reaction harbors the highest proportion of control within the linear pathway (Sweetlove et al., 1999; Geigenberger et al., 1999, 2004). In addition, considerable control resides in both the Glc-6-P phosphate antiporter (Zhang et al., 2008) and the amyloplastidial adenylate transporter (Tjaden et al., 1998; Zhang et al., 2008) as well as in reactions external to the pathways, such as the amyloplastidial adenylate kinase (Regierer et al., 2002), cytosolic UMP synthase (Geigenberger et al., 2005), and mitochondrial NAD-malic enzyme (Jenner et al., 2001).As part of our ongoing study of the constituent enzymes of the tricarboxylic acid (TCA) cycle, we made an initially surprising observation that increasing or decreasing the content of malate via a fruit-specific expression of antisense constructs targeted against the mitochondrial malate dehydrogenase or fumarase, respectively, resulted in opposing changes in the levels of starch (Centeno et al., 2011). We were able to demonstrate that these plants were characterized by an altered cellular redox balance and that this led to changes in the activation state of the AGPase reaction. Given that starch only accumulates transiently in tomato (Solanum lycopersicum; Beckles et al., 2001) as a consequence of this activation, the fruits were characterized by altered sugar content at ripening, a fact that dramatically altered their postharvest characteristics (Centeno et al., 2011). Here, we chose to express the antisense fumarase construct in potato in order to ascertain the effect of the manipulation in an organ that linearly accumulates starch across its development. The results obtained are compared and contrasted with those of the tomato fruit and within the context of current models of subcellular redox regulation.  相似文献   

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Carbohydrate metabolism in plants is tightly linked to photosynthesis and is essential for energy and carbon skeleton supply of the entire organism. Thus, the hexose phosphate pools of the cytosol and the chloroplast represent important metabolic resources that are maintained through action of phosphoglucose isomerase (PGI) and phosphoglucose mutase interconverting glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. Here, we investigated the impact of disrupted cytosolic PGI (cPGI) function on plant viability and metabolism. Overexpressing an artificial microRNA targeted against cPGI (amiR-cpgi) resulted in adult plants with vegetative tissue essentially free of cPGI activity. These plants displayed diminished growth compared with the wild type and accumulated excess starch in chloroplasts but maintained low sucrose content in leaves at the end of the night. Moreover, amiR-cpgi plants exhibited increased nonphotochemical chlorophyll a quenching during photosynthesis. In contrast to amiR-cpgi plants, viable transfer DNA insertion mutants disrupted in cPGI function could only be identified as heterozygous individuals. However, homozygous transfer DNA insertion mutants could be isolated among plants ectopically expressing cPGI. Intriguingly, these plants were only fertile when expression was driven by the ubiquitin10 promoter but sterile when the seed-specific unknown seed protein promoter or the Cauliflower mosaic virus 35S promoter were employed. These data show that metabolism is apparently able to compensate for missing cPGI activity in adult amiR-cpgi plants and indicate an essential function for cPGI in plant reproduction. Moreover, our data suggest a feedback regulation in amiR-cpgi plants that fine-tunes cytosolic sucrose metabolism with plastidic starch turnover.Starch and Suc turnover are major pathways of primary metabolism in all higher plants. As such, they are essential for carbohydrate storage and the energy supply of sink tissues and as building blocks for amino acid, fatty acid, or cell wall biosynthesis (Stitt and Zeeman, 2012).A core reaction in both starch and Suc biosynthesis is the reversible interconversion of the hexose phosphate pool metabolites Fru 6-phosphate (Fru6P) and Glc 6-phosphate (Glc6P), which is mediated by phosphoglucose isomerase (PGI). Arabidopsis (Arabidopsis thaliana) contains two isoforms of PGI, one in the plastids and one in the cytosol (Caspar et al., 1985).During the light period, the plastid isoform of PGI (PGI1) is involved in starch biosynthesis by generating Glc6P from the primary photosynthetic product Fru6P. Glc6P is further converted to Glc 1-phosphate (Glc1P) and ADP-glucose via action of phosphoglucomutase (PGM) and ADP-glucose pyrophosphorylase (AGPase), respectively (Stitt and Zeeman, 2012). Finally, transfer of the glucosyl moiety of ADP-glucose to the growing carbohydrate chain of starch is mediated by starch synthases. Any of the enzymatic reactions of this linear pathway is essential for starch synthesis, as illustrated by the virtual absence of transitory starch in chloroplasts of mutant plant lines with impaired function of PGI1 (Yu et al., 2000; Kunz et al., 2010), PGM (Caspar et al., 1985; Kofler et al., 2000), or AGPase (Lin et al., 1988). Interestingly, in a few specific cell types, e.g. leaf guard cells and root columella cells, loss of PGI1 activity can be bypassed by the presence of the plastid Glc6P/phosphate translocator GPT1 (Niewiadomski et al., 2005; Kunz et al., 2010).The cytosolic isoform of PGI (cPGI) is involved in anabolism and catabolism of Suc, the major transport form of carbohydrates in plants. Glc6P and Fru6P interconversion is necessary for both Suc synthesis during the day and during the night. During the day, Suc synthesis in source leaves is fueled mainly by triose phosphates exported from chloroplasts that are eventually converted to Fru6P in the cytosol. However, Fru6P is only one substrate for the Suc-generating enzyme Suc phosphate synthase. The second substrate, UDP-glucose, is synthesized from Fru6P via Glc6P and Glc1P by the cytosolic isoenzymes of PGI1 and PGM as well as UDP-glucose pyrophosphorylase.Because Suc is the major long-distance carbon transport form, its synthesis has to continue throughout the night to supply energy and carbohydrates to all tissues. The nocturnal synthesis of Suc is dependent on breakdown and mobilization of transitory starch from chloroplasts (Zeeman et al., 2007) via export of maltose and Glc (Weber et al., 2000; Niittylä et al., 2004; Weise et al., 2004; Cho et al., 2011). Exported maltose is temporarily integrated into cytosolic heteroglycans (Fettke et al., 2005) mediated by disproportionating enzyme2 (DPE2; Chia et al., 2004; Lu and Sharkey, 2004) yielding Glc and a heteroglycan molecule elongated by an α1-4-bound glucosyl residue. Cytosolic Glc can directly be phosphorylated to Glc6P by the action of hexokinase, while temporarily stored Glc in heteroglycans is released as Glc1P mediated by cytosolic glucan phosphorylase2 (PHS2; Fettke et al., 2004; Lu et al., 2006). Both Glc6P and Glc1P can then be converted to UDP-glucose as during the day.Generation of Fru6P, the second substrate for Suc synthesis, can proceed only to a limited extent from triose phosphates during the night. This limitation is caused mainly by the nocturnal inactivation of Fru 1,6-bisphosphatase (Cséke et al., 1982; Stitt, 1990), a key enzyme in Suc biosynthesis during the day. Hence, in contrast to the situation in the light, cPGI activity is now crucial for providing Fru6P from Glc6P.On the catabolic side, degradation of Suc into its monosaccharides in sink tissues yields both Glc6P and Fru6P, of which only Fru6P can be utilized in glycolytic degradation. Therefore, cPGI is also required for Glc6P conversion to Fru6P in glycolysis, which, in combination with respiration, is the major path of energy production in heterotrophic tissues.Impairment or loss of function of enzymes contributing to the cytosolic hexose phosphate pool has recently been investigated for the Glc1P-forming enzyme PGM (Egli et al., 2010). The Arabidopsis genome encodes three PGM isoforms, with PGM1 localized to plastids and PGM2 and PGM3 localized to the cytosol (Caspar et al., 1985; Egli et al., 2010). Analyses of transfer DNA (T-DNA) mutants showed that homozygous pgm2/pgm3 double mutants were nonviable because of impaired gametophyte development. However, pgm2 and pgm3 single mutants grew like ecotype Columbia (Col-0) wild-type plants, indicating overlapping functions of PGM2 and PGM3 (Egli et al., 2010).By contrast, cPGI is encoded only by a single locus in Arabidopsis (Kawabe et al., 2000). Higher plant mutants reduced in cPGI activity have so far been characterized only in ethyl methanesulfonate-mutagenized Clarkia xantiana (Jones et al., 1986a; Kruckeberg et al., 1989; Neuhaus et al., 1989). The C. xantiana genome encodes for two isoenzymes of cPGI, and homozygous point mutations in each individual cPGI led to significant decrease in cPGI enzyme activity, which was further reduced to a residual activity of 18% in cpgi2/cpgi3 double mutants, where the cPGI3 locus was heterozygous for the mutation (Jones et al., 1986a; Kruckeberg et al., 1989). Detailed physiological analyses of these mutants indicated a negative impact on Suc biosynthesis and elevated starch levels when cPGI activity was decreased at least 3- to 5-fold (Kruckeberg et al., 1989).The physiological impact of decreased or even absent cPGI activity has not been characterized in the genetic model organism Arabidopsis. Here, we show that homozygous T-DNA insertion mutants in the cPGI locus are nonviable and present data from analyses of mature Arabidopsis plants constitutively expressing artificial microRNAs (amiRNAs) targeted against cPGI. These mutants reveal altered photosynthesis, a strong impact on nocturnal leaf starch degradation, and impaired Suc metabolism.  相似文献   

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Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g−1 fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.The nonreducing Glc disaccharide, trehalose [α-d-glucopyranosyl-(1→1)-α-d-glucopyranoside] is widespread in nature. In resurrection plants, fungi, bacteria, and nonvertebrate animals, it performs a role as a carbon source and stress protection compound (Elbein et al., 2003; Paul et al., 2008). In the majority of plants, however, amounts of trehalose are too low to perform this function. Instead, the pathway has developed into a specialized system that regulates and integrates metabolism with growth and development (Schluepmann et al., 2003; Lunn et al., 2006; Ramon and Rolland, 2007; Gómez et al., 2010). This system is indispensible throughout seed and vegetative development (Eastmond et al., 2002; van Dijken et al., 2004; Gómez et al., 2010), and evidence suggests that the critical function is performed by the precursor of trehalose, trehalose 6-P (T6P). There is one known trehalose biosynthesis pathway in plants from the intermediates Glc 6-P and UDP-Glc catalyzed by trehalose phosphate synthase (TPS), which synthesizes T6P. T6P is then converted to trehalose by trehalose phosphate phosphatase (TPP). The regulation of T6P content in plants by TPSs and TPPs is not well understood. TPS1 is thought to account for most TPS catalytic activity in plants (Vandesteene et al., 2010). All 10 TPPs are now known to be catalytically active (Vandesteene et al., 2012); however, their specific contribution to T6P homeostasis is not known. Evidence suggests that T6P is a sugar signal in plants. T6P responds strongly to Suc supply when Suc is fed to seedlings grown in culture and in response to an increase in Suc in illuminated leaves (Lunn et al., 2006). Biosynthetic pathways for cell wall (Gómez et al., 2006) and starch synthesis (Kolbe et al., 2005) are regulated by T6P, supporting the observation that T6P promotes carbon utilization and growth of seedlings at high sugar levels when its content is increased through expression of otsA, a TPS-encoding gene from Escherichia coli (Schluepmann et al., 2003; Paul et al., 2010). In contrast, expression of otsB, a corresponding TPP-encoding gene from E. coli, decreases T6P content and inhibits growth in the presence of high sugar (Schluepmann et al., 2003; Paul et al., 2010). Given the importance of T6P in the regulation of growth and end-product synthesis, targets for its interaction have been eagerly sought.Recently, it was found that T6P inhibits the protein kinase SnRK1 in growing tissues of plants (Zhang et al., 2009; Debast et al., 2011; Delatte et al., 2011; Martínez-Barajas et al., 2011) through an intermediary factor. SnRK1 (AKIN10/AKIN11) is a member of the SNF1-related AMPK group of protein kinases that perform central functions in the regulation of responses of cells to endogenous energy and carbon status (Hardie, 2007). Baena-González et al. (2007) established that over 1000 genes are regulated by SnRK1 involved in biosynthetic, growth, and stress responses. It was observed that, in addition to cell wall and starch synthesis, T6P could regulate amino acid metabolism, protein, and nucleotide synthesis (Zhang et al., 2009) and is most likely connected to hormone signaling (Zhang et al., 2009; Paul et al., 2010). A model is proposed where SnRK1 inhibits growth processes when sugar and energy supplies are scarce, thus enabling survival under starvation stress conditions. When sugar supply is plentiful, T6P accumulates and inhibits SnRK1 blocking expression of genes involved in the stress survival response and inducing genes involved in the feast response, including growth processes. Interestingly, plants with altered SnRK1 activity display similar phenotypes to plants with altered T6P in both growth and developmental processes such that plants with genetically decreased T6P content resemble those with overexpressed SnRK1 and vice versa (Schluepmann et al., 2003; Baena-González et al., 2007; Wingler et al., 2012).Sugars fluctuate widely in plants in response to changes in photosynthesis and in response to environmental variables. Sugar starvation conditions, such as those induced by deep shade, limit growth through lack of sugar availability; SnRK1 would be active under such conditions. High sugar availability, however, does not necessarily indicate good conditions for growth and high growth rates. For example, under low-temperature and limiting nutrient supply, growth is limited in spite of abundant sugar availability (Paul and Stitt, 1993; Usadel et al., 2008). This is termed sink-limited growth, when growth is limited by capacity of sinks, i.e. growing regions to use assimilate. It departs from the famine model of growth regulation by SnRK1. The interrelationship between T6P, SnRK1, and growth is not known under such conditions. Here, we vary growth conditions by temperature and nutrient supply to induce sink-limited growth and feed Suc and Glc at physiological levels (15 mm). We show a strong specific interrelationship between T6P and Suc and SnRK1-regulated gene expression under all conditions irrespective of growth rate. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth. By priming, we mean being in a prepared state with an advanced capacity to activate growth following relief of a growth limitation, such as low temperature. To test that T6P/SnRK1 enable growth recovery following relief from sink limitation, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that T6P responds to Suc induced by growth restriction. This enables growth recovery following relief of limitations downstream of T6P/SnRK1, such as low temperature. Our findings are included in a model for the regulation of growth by the T6P/SnRK1 signaling pathway.  相似文献   

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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.  相似文献   

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

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Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.Isoprenoids constitute a versatile class of compounds fulfilling major physiological functions. They are formed by two pathways in plants, the mevalonate (MVA) pathway in the cytosol (Gershenzon and Croteau, 1993) and the 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids (Gershenzon and Croteau, 1993; Jomaa et al., 1999; Li and Sharkey, 2013b). The MVA pathway is primarily responsible for the synthesis of sesquiterpenes (C15), triterpenes (C30) including brassinosteroids, and even larger molecules such as dolichols (Bick and Lange, 2003; Li and Sharkey, 2013b; Rajabi Memari et al., 2013; Rosenkranz and Schnitzler, 2013). The DXP/MEP pathway is responsible for the synthesis of the simplest isoprenoids, isoprene and 2-methyl-3-buten-2-ol (C5), monoterpenes (C10), diterpenes (C20) including gibberellins and phytol residue of chlorophylls, and tetraterpenes (C40) including carotenoids (Rodríguez-Concepción and Boronat, 2002; Roberts, 2007).Given that in plants both pathways produce ultimately the same substrates, dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP), the pertinent question is to what extent the two pathways can exchange metabolites (Rodríguez-Concepción and Boronat, 2002). There is evidence of a certain exchange of IDP between cytosolic and plastidic compartments, although the contribution of IDP from one compartment to the pathway flux in the other seems to be relatively minor (Schwender et al., 2001; Rodríguez-Concepción and Boronat, 2002; Bick and Lange, 2003). Some studies have further demonstrated that the exchange of IDP is fully bidirectional (De-Eknamkul and Potduang, 2003; Rodríguez-Concepción, 2006), whereas other studies suggest that IDP export from plastids to cytosol operates with a greater efficiency than the opposite transport (Hemmerlin et al., 2003; Laule et al., 2003). However, although the overall intercompartmental exchange of isoprenoid substrates to pathway flux in the given compartment might seem minor under nonstressed conditions, the importance of cross talk among the pathways might increase under stress conditions that specifically inhibit isoprenoid synthesis in one pathway. In fact, the DXP/MEP pathway is strongly linked to photosynthetic metabolism, and therefore, inhibition of photosynthesis under stressful conditions such as heat stress or drought or photoinhibition could inhibit the synthesis of isoprenoids when they are most needed to fulfill their protective function (Loreto and Schnitzler, 2010; Niinemets, 2010; Possell and Loreto, 2013). There is some evidence demonstrating a certain cooperativity among the two isoprenoid synthesis pathways under conditions leading to a reduction of the activity of one of them (Piel et al., 1998; Jux et al., 2001; Page et al., 2004; Rodríguez-Concepción, 2006), but the capacity for such a replacement of function and regulation is poorly understood.Recent studies using genetically modified plants accumulating end products of the DXP/MEP pathway or using natural variation in product accumulation have demonstrated the existence of a potentially important feedback regulation of the DXP/MEP pathway flux by primary end products of the pathway (Banerjee et al., 2013; Ghirardo et al., 2014; Wright et al., 2014). In particular, binding of DMADP and perhaps IDP to DXP synthase, the first enzyme in the DXP/MEP pathway, leads to downregulation of the pathway flux when the end products cannot be used, such as under stress conditions. However, the strength of such a feedback regulation can be importantly modified by accumulation of phosphorylated intermediates of the pathway, such that DMADP and IDP do not accumulate. Previous studies have demonstrated that there is a certain pool of phosphorylated intermediates in vivo, and that this pool can strongly increase under certain conditions, including experimental and genetic modification of DXP/MEP pathway input and output (Li et al., 2011; Rasulov et al., 2011; Li and Sharkey, 2013a; Ghirardo et al., 2014; Wright et al., 2014).It has further been shown that 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (ME-cDP) is the metabolite accumulating in the plastids, and this accumulation can buffer DMADP and IDP changes in the case of varying DXP/MEP pathway input and consumption (Li and Sharkey, 2013a; Wright et al., 2014). A significant part of ME-cDP might even escape to cytosol, implying the existence of another interesting link between cytosolic and chloroplastic processes in isoprenoid synthesis (Wright et al., 2014). Furthermore, as ME-cDP is an important signaling molecule eliciting a number of gene expression responses (Xiao et al., 2012), accumulation of ME-cDP in plastids and flux to cytosol and further to the nucleus is particularly interesting from the perspective of long-term regulation of isoprenoid synthesis, and suggests a coordination of cellular stress responses by the plastidial isoprenoid synthesis pathway.Isoprene-emitting species constitute an exciting model system where a very large DXP/MEP pathway flux goes to isoprene synthesis under physiological conditions (Li and Sharkey, 2013b; Sharkey et al., 2013). In isoprene-emitting species, there is a concomitant use of the primary substrate DMADP between the plastidic synthesis of isoprene and isoprenoids with a larger molecular size, such as phytol residue of chlorophyll (C20) and carotenoids (C40; Ghirardo et al., 2014; Rasulov et al., 2014), and different from nonemitting species, isoprene emitters seem to support a much larger pool of DMADP without the onset of feedback inhibition (Ghirardo et al., 2014; Wright et al., 2014). However, it is poorly understood how inhibition of one branch of the pathway (isoprene versus larger isoprenoids) affects the other, to what extent it can lead to accumulation of phosphorylated intermediates, how it affects the overall pathway flux through the feedback regulation, and what is the possible role of cytosolic import and export of intermediates. These are all relevant questions to gain insight into the control of the partitioning of pathway flux between isoprene and larger isoprenoids and to understand the biological role of isoprene emission.Studies using metabolic inhibitors to deconvolute the factors involved in pathway regulation and understand the biological role of isoprene have so far used inhibitors that block the early steps of the corresponding pathways. In particular, fosmidomycin, a specific inhibitor of DXP reductoisomerase, the enzyme responsible for the synthesis of MEP from DXP, has been used to inhibit the DXP/MEP pathway (Loreto and Velikova, 2001; Sharkey et al., 2001; Loreto et al., 2004). In addition, lovastatin (mevinolin), the inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase that controls the MVA pathway flux, has been used to study the cooperativity of the two pathways (e.g. Laule et al., 2003; Mansouri and Salari, 2014). However, these inhibitors are not suitable to understand how end product accumulation can alter the pathway flux.Bisphosphonates constitute a promising class of inhibitors that could be particularly apt for studies on the effects of the inhibition of the end points of the pathway. They have been demonstrated to inhibit cytosolic farnesyl diphosphate (FDP) synthase activity (Oberhauser et al., 1998; Cromartie et al., 1999; van Beek et al., 1999; Bergstrom et al., 2000; Burke et al., 2004), аs well as geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) synthase activities (Oberhauser et al., 1998; Cromartie et al., 1999; Kloer et al., 2006; No et al., 2012; Lindert et al., 2013). To our knowledge, bisphosphonates have not been used to study the effects of end product accumulation on the pathway flux in isoprene-emitting species, with the exception of one study that investigated the development of isoprene emission capacity through leaf ontogeny (Rasulov et al., 2014).A limitation with any inhibitor study could be a certain nonspecificity, inhibition of additional nondesired reactions, but so far there are no data on such nonspecificity of bisphosphonates. However, there is evidence that diphosphate and its analogs are inhibitors of any ferredoxin (Fd)-dependent reaction (Forti and Meyer, 1969; Bojko and Więckowski, 1999). This could be potentially relevant given that DXP/MEP pathway-reducing steps, at the level of 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBDP) synthase (HDS) and reductase (HDR), directly accept electrons from Fd in light (Eisenreich et al., 2001; Seemann et al., 2006; Li and Sharkey, 2013a). In addition to the DXP/MEP pathway, inhibition at the level of Fd could also affect photosynthetic reactions and thereby alter energy supply for the DXP/MEP pathway.In this study, we have investigated the effects of inhibition of the initial and final steps of the DXP/MEP pathway by fosmidomycin and bisphosphonate inhibitors alendronate and zoledronate in a strong isoprene emitter hybrid aspen (Populus tremula × Populus tremuloides). Alendronate is a highly specific inhibitor of GDP (Lange et al., 2001; Burke et al., 2004) and FDP synthases (Bergstrom et al., 2000; Burke et al., 2004), and a less specific inhibitor of GGDP synthase (Szabo et al., 2002). Zoledronate operates similarly to alendronate, but is a much stronger inhibitor, being operationally active in concentrations several orders of magnitude less than alendronate (Lange et al., 2001; Henneman et al., 2011; Wasko, 2011). A unique in vivo method was used to study dynamic changes in DMADP and phosphorylated intermediate pool sizes (Rasulov et al., 2009a, 2011; Li et al., 2011), and different inhibitors were applied alone or in sequence to study the regulation of the pathway flux in conditions when the flux out of the pathway or into the pathway is curbed and when both the input and the output are curbed. Dynamic model calculations were used to quantitatively evaluate the significance of the cytosolic intermediate input into chloroplastic isoprenoid synthesis under different conditions of the DXP/MEP pathway entrance and exit, and to evaluate the possible nonspecific inhibition of other steps controlling DXP/MEP pathway flux. The study demonstrates the important regulation of DXP/MEP pathway input and output under conditions of end product accumulation and partial cooperativity among chloroplastic and cytosolic isoprenoid synthesis pathways.  相似文献   

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