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.     

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.     

Processing-Body Movement in Arabidopsis Depends on an Interaction between Myosins and DECAPPING PROTEIN1

Processing (P)-bodies are cytoplasmic RNA protein aggregates responsible for the storage, degradation, and quality control of translationally repressed messenger RNAs in eukaryotic cells. In mammals, P-body-related RNA and protein exchanges are actomyosin dependent, whereas P-body movement requires intact microtubules. In contrast, in plants, P-body motility is actin based. In this study, we show the direct interaction of the P-body core component DECAPPING PROTEIN1 (DCP1) with the tails of different unconventional myosins in Arabidopsis (Arabidopsis thaliana). By performing coexpression studies with AtDCP1, dominant-negative myosin fragments, as well as functional full-length myosin XI-K, the association of P-bodies and myosins was analyzed in detail. Finally, the combination of mutant analyses and characterization of P-body movement patterns showed that myosin XI-K is essential for fast and directed P-body transport. Together, our data indicate that P-body movement in plants is governed by myosin XI members through direct binding to AtDCP1 rather than through an adapter protein, as known for membrane-coated organelles. Interspecies and intraspecies interaction approaches with mammalian and yeast protein homologs suggest that this mechanism is evolutionarily conserved among eukaryotes.Newly synthesized mRNAs are processed and transported from the nucleus into the cytoplasm. Their subsequent fate depends on a regulatory balance between cytoplasmic translation and mRNA degradation. Both processes are modulated by mRNA-binding proteins, which influence the translational efficiency, stability, and localization of mRNA molecules. Over the last decade, it became apparent that messenger ribonucleoproteins that are translationally repressed and associated with the cytoplasmic 5′ to 3′ decapping machinery tend to assemble into a newly discovered class of highly dynamic mRNA granules, termed processing (P)-bodies (Sheth and Parker, 2003; Anderson and Kedersha, 2006; Parker and Sheth, 2007; Franks and Lykke-Andersen, 2008). P-bodies were initially identified as discrete cytoplasmic punctae to which the mouse 5′ to 3′ exonuclease localizes (Bashkirov et al., 1997). Since then, they have been established as major cytoplasmic sites for the storage of translationally repressed mRNAs, 5′ to 3′ mRNA degradation, quality control, and microRNA-dependent gene silencing (Dunckley and Parker, 1999; Sheth and Parker, 2003; Cougot et al., 2004; Sen and Blau, 2005).Although their complete composition is still not known, a set of proteins related to these different functions has been identified and shown to colocalize with each other in these cytoplasmic foci (Ingelfinger et al., 2002; van Dijk et al., 2002; Sheth and Parker, 2003; Eulalio et al., 2007; Parker and Sheth, 2007). P-bodies are commonly described as highly mobile and dynamic structures (Yang et al., 2004; Aizer et al., 2008; Loschi et al., 2009). In Drosophila melanogaster and mammals, they show a microtubule-dependent pattern of motility (Aizer et al., 2008; Loschi et al., 2009), which is inhibited by drugs interfering with the microtubule cytoskeleton (Aizer et al., 2008). Actin, by contrast, is important for the delivery of RNA and P-body components to P-bodies and for their assembly. This is suggested by the colocalization of mammalian myosin Va with the P-body-associated protein eIF4E (for Eukaryotic Translation Initiation Factor4E) and further by the reduction of P-body number and transport of eIF4E to P-bodies when expressing dominant-negative myosin Va fragments (Lindsay and McCaffrey, 2011). An association of myosins with P-bodies was also reported in yeast. In yeast (Saccharomyces cerevisiae), Myo2p was successfully used to immunoprecipitate complex ribonucleoprotein particles, including hundreds of mRNAs and some P-body components, such as DECAPPING PROTEIN1 (Dcp1p), SM-LIKE PROTEIN1, DNA TOPOISOMERASE 2-ASSOCIATED PROTEIN1, DExD/H-BOX HELICASE1, and EXORIBONUCLEASE1 (Chang et al., 2008).In plants, P-body movement appears to be based on the actin cytoskeleton (Hamada et al., 2012), although myosins have not been implicated in P-body movement. In Arabidopsis (Arabidopsis thaliana), the myosin family contains 17 proteins (Reddy and Day, 2001; Peremyslov et al., 2011), which are grouped into two classes, XI and VIII. Members of the class VIII myosins are associated with endosomal structures, the endoplasmic reticulum, and plasmodesmata (Avisar et al., 2008a; Golomb et al., 2008; Sattarzadeh et al., 2008). The 13 class XI myosins show a domain structure that is similar to those of fungi and animal myosin V proteins. They are composed of an N-terminal motor domain, for both actin association as well as ATP hydrolysis (Kinkema et al., 1994; Tominaga et al., 2003), an internal neck domain, comprising a stretch of six calmodulin-binding IQ motifs (Kinkema and Schiefelbein, 1994), and the C-terminal tail region, which contains a coiled-coil domain of variable extent and a bipartite globular cargo-binding domain (Pashkova et al., 2006; Li and Nebenführ, 2007). The Arabidopsis XI myosins have been implicated in the movement of various membranous organelles and vesicles (for review, see Sparkes, 2010, 2011). Colocalization studies and the analysis of movement in myosin mutants revealed that plant XI myosins are involved in the rapid trafficking of Golgi stacks, mitochondria, and peroxisomes (Avisar et al., 2008b, 2009; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008), the long-distance transport of endomembrane vesicles (Peremyslov et al., 2012), plant-specific transport vesicles (Peremyslov et al., 2013), motility of plastids (Natesan et al., 2009; Sattarzadeh et al., 2009), as well as endoplasmic reticulum flow (Peremyslov et al., 2010; Ueda et al., 2010) and remodeling (Sparkes et al., 2009).In the light of recent data available for the mammalian and yeast systems, myosins appear to be good candidates to also mediate the movement of membraneless RNA granules, such as P-bodies. The molecular basis linking P-bodies to the actin cytoskeleton is not known in any system. In this study, we demonstrate that the P-body core component DECAPPING PROTEIN1 (DCP1) physically interacts with myosins in mammals, yeast, and Arabidopsis and also in interspecies experiments. In support of a direct interaction between myosins and P-bodies, Arabidopsis DCP1 (AtDCP1) colocalizes with the full-size myosin XI-K. Overexpression of dominant-negative myosin tail domains inhibited P-body movement efficiently. The analysis of the respective mutants revealed that in two xi-k alleles, P-body movement was almost completely abolished. This defective motility was efficiently restored in triple myosin knockout plants expressing full-size myosin XI-K. Taken together, these data demonstrate that P-body movement is governed by unconventional myosins and that their anchoring to myosins occurs by direct binding to a core component rather than by connecting to specific adapter proteins, as known for eukaryotic organelle transport.     

SNF1-Related Protein Kinases Type 2 Are Involved in Plant Responses to Cadmium Stress

Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd²⁺ showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd²⁺ treatment. Our data show significantly lower Cd²⁺-induced ROS accumulation in the mutants’ roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions.Cadmium is one of the most toxic soil pollutants. Cadmium ions accumulate in plants and affect, via the food chain, animal and human health. In plants, cadmium is taken up by roots and is transported to aerial organs, leading to chromosomal aberrations, growth reduction, and inhibition of photosynthesis, transpiration, nitrogen metabolism, nutrient and water uptake, eventually causing plant death (for review, see DalCorso et al., 2008). Plants are challenged not only by cadmium ions themselves, but also by Cd²⁺-induced harmful effects including oxidative stress (Schützendübel et al., 2001; Olmos et al., 2003; Cho and Seo, 2005; Sharma and Dietz, 2009). The extent of the detrimental effects on plant growth and metabolism depends on the level of cadmium ions present in the surrounding environment and on the plant’s sensitivity to heavy metal stress.Tolerant plants avoid heavy metal uptake and/or induce the expression of genes encoding products involved, directly or indirectly, in heavy metal binding and removal from potentially sensitive sites, by sequestration or efflux (Clemens, 2006). The best-characterized heavy metal binding ligands in plants are thiol-containing compounds metallothioneins and phytochelatins (PCs), whose production is stimulated by Cd²⁺. PCs bind metal ions and transport them to the vacuole, thus reducing the toxicity of the metal in the cytosol (for review, see Cobbett, 2000; Cobbett and Goldsbrough, 2002). PCs are synthesized from reduced glutathione (GSH). Therefore, production of compounds involved in cadmium detoxification and, at the same time, in cadmium tolerance closely depends on sulfur metabolism. So far, our knowledge on the cellular processes induced by cadmium that lead to changes in sulfur metabolism in plants has been rather limited.Protein kinases and phosphatases are considered major signal transduction elements. However, until now only a few of them have been described to be involved in cadmium stress response or sulfur metabolism. For instance, excessive amounts of cadmium or copper activate mitogen-activated protein kinases (MAPKs) in Medicago sativa (Jonak et al., 2004), rice (Oryza sativa; Yeh et al., 2007), and Arabidopsis (Arabidopsis thaliana; Liu et al., 2010). Studies on rice MAPKs involved in heavy metal stress response indicate that the activity of these kinases depends on the oxidative stress induced by Cd²⁺. Moreover, Yeh et al. (2007) suggested that the activation of MAPKs in rice by cadmium or copper required the activity of calcium-dependent protein kinase (CDPK) and PI3 kinase, since the MAPK pathways involved in cadmium and copper stress response could be inhibited by a CDPK antagonist (W7) or a PI3 kinase inhibitor (wortmannin). However, so far the function of the identified kinases in plant adaptation to heavy metal pollution has not been established. There is some information concerning an involvement of CDPK in sulfur metabolism (Liu et al., 2006). Soybean (Glycine max) Ser acetyltransferase (GmSerat2;1), the enzyme that catalyzes the first reaction in the biosynthesis of Cys from Ser, is phosphorylated by CDPK. The phosphorylation has no effect on GmSerat2;1 activity, but it renders the enzyme insensitive to the feedback inhibition by Cys (Liu et al., 2006). There is growing evidence that SnRK2s (for SNF1-related protein kinase2) play a role in the regulation of sulfur metabolism. Most information showing a connection between SnRK2s and sulfur metabolism comes from experiments on the lower plant Chlamydomonas reinhardtii (Davies et al., 1999; Irihimovitch and Stern, 2006; González-Ballester et al., 2008, 2010). SNRK2.1 is considered a general regulator of S-responsive gene expression in C. reinhardtii (González-Ballester et al., 2008).In higher plants the SnRK2 family members are known to be involved in plant response to drought, salinity, and in abscisic acid (ABA)-dependent plant development (Boudsocq and Laurière, 2005; Fujii et al., 2007, 2011; Fujii and Zhu, 2009; Fujita et al., 2009; Nakashima et al., 2009; Kulik et al., 2011). Ten members of the SnRK2 family have been identified in Arabidopsis and in rice (Boudsocq et al., 2004; Kobayashi et al., 2004). All of them, except SnRK2.9 from Arabidopsis, are rapidly activated by treatment with different osmolytes, such as Suc, mannitol, sorbitol, and NaCl, and some of them also by ABA. Results presented by Kimura et al. (2006) suggest that in Arabidopsis, similarly to C. reinhardtii, some SnRK2s are involved in the regulation of S-responsive gene expression and O-acetyl-l-Ser accumulation under limited sulfur supply, indicating that also higher plants’ SnRK2s could be involved in sulfur metabolism.As it was mentioned before, oxidative stress induced by cadmium ions significantly contributes to the metal toxicity. Reactive oxygen species (ROS) can be produced in many different reactions in various compartments of the cell in response to cadmium (Romero-Puertas et al., 2004; Heyno et al., 2008; Tamás et al., 2009). The best-characterized ROS-generating enzymes that take part in the response to cadmium are the plasma-membrane-bound NADPH oxidases (Olmos et al., 2003; Romero-Puertas et al., 2004; Garnier et al., 2006). There are some indications that plant NADPH oxidases are phosphorylated by SnRK2s (Sirichandra et al., 2009), therefore it is highly plausible that SnRK2s play a role in the regulation of ROS accumulation in plants subjected to cadmium stress. Taking into consideration all facts mentioned above we hypothesized that SnRK2s could be involved in the plant response to stress induced by cadmium ions. To verify this conjecture, we analyzed the activity and potential role of selected SnRK2s, in tobacco (Nicotiana tabacum) cells and Arabidopsis plants, in the response to cadmium ions.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

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.     

Expansive Evolution of the TREHALOSE-6-PHOSPHATE PHOSPHATASE Gene Family in Arabidopsis

Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-β-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.The presence of trehalose in a wide variety of organisms and the existence of different biosynthesis pathways suggest a pivotal and ancient role for trehalose metabolism in nature. The most widely distributed metabolic pathway consists of two consecutive enzymatic reactions, with trehalose-6-phosphate (T6P) synthase (TPS) catalyzing the transfer of a glucosyl moiety from UDP-Glc to Glc-6-phosphate to produce T6P and UDP, and T6P phosphatase (TPP) catalyzing dephosphorylation of T6P to trehalose (Cabib and Leloir, 1958; Avonce et al., 2006). Apart from operating as a (reserve) carbon source and structural component in bacteria, fungi, and invertebrates, trehalose also functions as a major stress protectant of proteins and membranes during adverse conditions such as dehydration, high salinity, hypoxia, and nutrient starvation (Elbein et al., 2003). Trehalose accumulation is also observed in a few lower vascular resurrection plants (e.g. Selaginella lepidophylla). Until about a decade ago, higher vascular plants were believed to have lost the ability to produce trehalose, but with the emergence of more sensitive assays, genome sequencing, and the use of yeast (Saccharomyces cerevisiae) mutant complementation, minute amounts of trehalose and T6P, and functional plant enzyme orthologs were found (Goddijn et al., 1997; Vogel et al., 1998; Lunn et al., 2006). In addition, heterologous expression and disruption of trehalose metabolism in plants conferred pleiotropic effects, ranging from altered stress tolerance, leaf morphology, and developmental timing to embryo lethality (Holmström et al., 1996; Goddijn et al., 1997; Romero et al., 1997; Eastmond et al., 2002; Schluepmann et al., 2003; Avonce et al., 2004; Satoh-Nagasawa et al., 2006; Miranda et al., 2007; Chary et al., 2008), pointing to an important regulatory function. The intermediate T6P has been highlighted as a novel signal for carbohydrate status (for review, see Paul, 2008), positively correlating with Suc levels, redox-regulated ADP-Glc pyrophosphorylase activity, and starch biosynthesis (Lunn et al., 2006). Recently, it was reported that T6P inhibits the activity of the SnRK1 protein kinase to activate energy-consuming biosynthetic processes in growing tissue (Zhang et al., 2009) and that it is required for the onset of leaf senescence (Wingler et al., 2012).In most bacterial and eukaryotic species, the TPS and TPP activities are found on separate proteins. Recent phylogenetic and biochemical analyses showed that some archaea and bacteria, such as Cytophaga hutchinsonii, express proteins that have both active TPS and TPP domains resulting from gene fusion, suggesting that such prokaryotic bifunctional proteins are the evolutionary ancestors of the large eukaryotic trehalose biosynthesis enzymes in which one or both domains have subsequently lost their catalytic activity (Avonce et al., 2010). The yeast TPP enzyme Tps2, for example, harbors an inactive N-terminal TPS domain and an active C-terminal TPP domain. In contrast to the single TPS and TPP genes in most microorganisms, the genomes of higher plants encode a remarkably large family of putative trehalose biosynthesis enzyme homologs. These are commonly classified in three distinct subgroups, according to their similarity to the microbial TPS and TPP proteins and/or presence of specific motifs (e.g. conserved phosphatase boxes; Thaller et al., 1998; Leyman et al., 2001; Eastmond et al., 2003). Even primitive plants such as the alga Ostreococcus tauri and the moss Physcomitrella patens already contain members of each of these gene families, pointing to the early establishment and conservation of these proteins in plant evolution (Lunn, 2007; Avonce et al., 2010). In Arabidopsis (Arabidopsis thaliana), the class I TPS proteins (AtTPS1-4) show most similarity to the yeast TPS Tps1, but also have a C-terminal domain with limited similarity to TPPs. However, only one of these, AtTPS1, appears to have heterologous enzymatic TPS activity in yeast (Blázquez et al., 1998; Vandesteene et al., 2010). Strikingly, AtTPS1 is the only class I enzyme with an N-terminal extension that seems to operate as an autoinhibitory domain (Van Dijck et al., 2002). The class II TPS proteins (AtTPS5-11) are similar bipartite proteins with a TPS-like domain but a more conserved TPP domain. They appear to lack both heterologous TPS and TPP activity (Ramon et al., 2009). The high level of conservation of putative substrate-binding residues in class I and class II proteins, however, suggests that substrates might still bind (Avonce et al., 2006; Lunn, 2007; Ramon et al., 2009; Vandesteene et al., 2010). Together with the specific expression patterns of the class I genes (van Dijken et al., 2004; Geelen et al., 2007; Vandesteene et al., 2010) and the extensive expression regulation of all class II members by plant carbon status (Baena-González et al., 2007; Usadel et al., 2008; Ramon et al., 2009), this suggests tissue-specific regulatory functions for these proteins in metabolic regulation of plant growth and development. Finally, Arabidopsis also harbors a family of 10 smaller proteins (AtTPPA-J; 320–385 amino acids) with limited similarity to the class I and class II proteins (795–942 amino acids). Like class II proteins, they contain the phosphatase box consensus sequences, characteristic of the l-2-haloacid dehalogenase (HAD) super family of enzymes, which includes a wide range of phosphatases and hydrolases (Thaller et al., 1998). It has been suggested that the origin of these plant TPP genes is different from the origin of the class I and II genes (Avonce et al., 2010) and that plants recruited the TPP genes after their divergence from fungi, most probably from proteobacteria or actinobacteria. Consistently, homologous TPP proteins are present in proteobacteria such as Rhodopherax ferrireducens (Avonce et al., 2010). To date, only a few of these single-domain plant TPP proteins have been subject to biochemical characterization, e.g. TPPA and TPPB from Arabidopsis (Vogel et al., 1998), OsTPP1 and OsTPP2 from rice (Oryza sativa; Pramanik and Imai, 2005; Shima et al., 2007), and RAMOSA3 (RA3) from maize (Zea mays; Satoh-Nagasawa et al., 2006).The phenotypic alterations observed in plants fed with trehalose or genetically modified in trehalose biosynthesis, suggest a pivotal role for trehalose metabolism in integrating the metabolic status with growth and development. Disruption of the only known active TPS enzyme in Arabidopsis (AtTPS1) results in embryo lethality (Eastmond et al., 2002) and, when rescued to bridge embryogenesis, causes a strong disruption of vegetative and generative development and abscisic acid (ABA) hypersensitivity (van Dijken et al., 2004; Gómez et al., 2010). Overexpressing AtTPS1 on the other hand renders seedlings sugar and ABA insensitive (Avonce et al., 2004, 2005). These observations strongly link trehalose metabolism with ABA signaling. Interestingly, a mutation of a TPP gene in maize, RA3, results in a distinct phenotype, with incorrect axillary meristem identity and determinacy in both male and female inflorescences (Satoh-Nagasawa et al., 2006). Arabidopsis plants with overall increased T6P levels, such as OtsA (Escherichia coli TPS) overexpression plants, similarly show increased inflorescence branching (Schluepmann et al., 2003; van Dijken et al., 2004).To better understand why higher plants harbor such a large number of putative TPP proteins, we have made a comprehensive study of the 10 Arabidopsis TPP genes and gene products, combining phylogenetic approaches and yeast growth complementation assays, together with a detailed analysis of all 10 TPP gene expression profiles in Arabidopsis, and a more detailed single AtTPP mutant phenotypic analysis.