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.     

The Role of a Potassium Transporter OsHAK5 in Potassium Acquisition and Transport from Roots to Shoots in Rice at Low Potassium Supply Levels

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.Potassium (K) is one of the three most important macronutrients and the most abundant cation in plants. As a major osmoticum in the vacuole, K drives the generation of turgor pressure, enabling cell expansion. In the vascular tissue, K is an important participant in the generation of root pressure (for review, see Wegner, 2014 [including his new hypothesis]). In the phloem, K is critical for the transport of photoassimilates from source to sink (Marschner, 1996; Deeken et al., 2002; Gajdanowicz et al., 2011). In addition, enhancing K absorption and decreasing sodium (Na) accumulation is a major strategy of glycophytes in salt stress tolerance (Maathuis and Amtmann, 1999; Munns and Tester, 2008; Shabala and Cuin, 2008).Plants acquire K through K-permeable proteins at the root surface. Since available K concentration in the soil may vary by 100-fold, plants have developed multiple K uptake systems for adapting to this variability (Epstein et al., 1963; Grabov, 2007; Maathuis, 2009). In a classic K uptake experiment in barley (Hordeum vulgare), root K absorption has been described as a high-affinity and low-affinity biphasic transport process (Epstein et al., 1963). It is generally assumed that the low-affinity transport system (LATS) in the roots mediates K uptake in the millimolar range and that the activity of this system is insensitive to external K concentration (Maathuis and Sanders, 1997; Chérel et al., 2014). In contrast, the high-affinity transport system (HATS) was rapidly up-regulated when the supply of exogenous K was halted (Glass, 1976; Glass and Dunlop, 1978).The membrane transporters for K flux identified in plants are generally classified into three channels and three transporter families based on phylogenetic analysis (Mäser et al., 2001; Véry and Sentenac, 2003; Lebaudy et al., 2007; Alemán et al., 2011). For K uptake, it was predicted that, under most circumstances, K transporters function as HATS, while K-permeable channels mediate LATS (Maathuis and Sanders, 1997). However, a root-expressed K channel in Arabidopsis (Arabidopsis thaliana), Arabidopsis K Transporter1 (AKT1), mediates K absorption over a wide range of external K concentrations (Sentenac et al., 1992; Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), while evidence is accumulating that many K transporters, including members of the K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) family, are low-affinity K transporters (Quintero and Blatt, 1997; Senn et al., 2001), implying that functions of plant K channels and transporters overlap at different K concentration ranges.Out of the three families of K transporters, cation proton antiporter (CPA), high affinity K/Na transporter (HKT), and KT/HAK/KUP, CPA was characterized as a K⁺(Na⁺)/H⁺ antiporter, HKT may cotransport Na and K or transport Na only (Rubio et al., 1995; Uozumi et al., 2000), while KT/HAK/KUP were predicted to be H⁺-coupled K⁺ symporters (Mäser et al., 2001; Lebaudy et al., 2007). KT/HAK/KUP were named by different researchers who first identified and cloned them (Quintero and Blatt, 1997; Santa-María et al., 1997). In plants, the KT/HAK/KUP family is the largest K transporter family, including 13 members in Arabidopsis and 27 members in the rice (Oryza sativa) genome (Rubio et al., 2000; Mäser et al., 2001; Bañuelos et al., 2002; Gupta et al., 2008). Sequence alignments show that genes of this family share relatively low homology to each other. The KT/HAK/KUP family was divided into four major clusters (Rubio et al., 2000; Gupta et al., 2008), and in cluster I and II, they were further separated into A and B groups. Genes of cluster I or II likely exist in all plants, cluster III is composed of genes from both Arabidopsis and rice, while cluster IV includes only four rice genes (Grabov, 2007; Gupta et al., 2008).The functions of KT/HAK/KUP were studied mostly in heterologous expression systems. Transporters of cluster I, such as AtHAK5, HvHAK1, OsHAK1, and OsHAK5, are localized in the plasma membrane (Kim et al., 1998; Bañuelos et al., 2002; Gierth et al., 2005) and exhibit high-affinity K uptake in the yeast Saccharomyces cerevisiae (Santa-María et al., 1997; Fu and Luan, 1998; Rubio et al., 2000) and in Escherichia coli (Horie et al., 2011). Transporters of cluster II, like AtKUP4 (TINY ROOT HAIRS1, TRH1), HvHAK2, OsHAK2, OsHAK7, and OsHAK10, could not complement the K uptake-deficient yeast (Saccharomyces cerevisiae) but were able to mediate K fluxes in a bacterial mutant; they might be tonoplast transporters (Senn et al., 2001; Bañuelos et al., 2002; Rodríguez-Navarro and Rubio, 2006). The function of transporters in clusters III and IV is even less known (Grabov, 2007).Existing data suggest that some KT/HAK/KUP transporters also may respond to salinity stress (Maathuis, 2009). The cluster I transporters of HvHAK1 mediate Na influx (Santa-María et al., 1997), while AtHAK5 expression is inhibited by Na (Rubio et al., 2000; Nieves-Cordones et al., 2010). Expression of OsHAK5 in tobacco (Nicotiana tabacum) BY2 cells enhanced the salt tolerance of these cells by accumulating more K without affecting their Na content (Horie et al., 2011).There are only scarce reports on the physiological function of KT/HAK/KUP in planta. In Arabidopsis, mutation of AtKUP2 (SHORT HYPOCOTYL3) resulted in a short hypocotyl, small leaves, and a short flowering stem (Elumalai et al., 2002), while a loss-of-function mutation of AtKUP4 (TRH1) resulted in short root hairs and a loss of gravity response in the root (Rigas et al., 2001; Desbrosses et al., 2003; Ahn et al., 2004). AtHAK5 is the only system currently known to mediate K uptake at concentrations below 0.01 mm (Rubio et al., 2010) and provides a cesium uptake pathway (Qi et al., 2008). AtHAK5 and AtAKT1 are the two major physiologically relevant molecular entities mediating K uptake into roots in the range between 0.01 and 0.05 mm (Pyo et al., 2010; Rubio et al., 2010). AtAKT1 may contribute to K uptake within the K concentrations that belong to the high-affinity system described by Epstein et al. (1963).Among all 27 members of the KT/HAK/KUP family in rice, OsHAK1, OsHAK5, OsHAK19, and OsHAK20 were grouped in cluster IB (Gupta et al., 2008). These four rice HAK members share 50.9% to 53.4% amino acid identity with AtHAK5. OsHAK1 was expressed in the whole plant, with maximum expression in roots, and was up-regulated by K deficiency; it mediated high-affinity K uptake in yeast (Bañuelos et al., 2002). In this study, we examined the tissue-specific localization and the physiological functions of OsHAK5 in response to variation in K supply and to salt stress in rice. By comparing K uptake and translocation in OsHAK5 knockout (KO) mutants and in OsHAK5-overexpressing lines with those in their respective wild-type lines supplied with different K concentrations, we found that OsHAK5 not only mediates high-affinity K acquisition but also participates in root-to-shoot K transport as well as in K-regulated salt tolerance.     

A Chimeric Arabinogalactan Protein Promotes Somatic Embryogenesis in Cotton Cell Culture

Arabinogalactan proteins (AGPs) are a family of extracellular plant proteoglycans implicated in many aspects of plant growth and development, including in vitro somatic embryogenesis (SE). We found that specific AGPs were produced by cotton (Gossypium hirsutum) calli undergoing SE and that when these AGPs were isolated and incorporated into tissue culture medium, cotton SE was promoted. When the AGPs were partly or fully deglycosylated, SE-promoting activity was not diminished. Testing of AGPs separated by reverse-phase high-performance liquid chromatography revealed that the SE-promoting activity resided in a hydrophobic fraction. We cloned a full-length complementary DNA (cotton PHYTOCYANIN-LIKE ARABINOGALACTAN-PROTEIN1 [GhPLA1]) that encoded the protein backbone of an AGP in the active fraction. It has a chimeric structure comprising an amino-terminal signal sequence, a phytocyanin-like domain, an AGP-like domain, and a hydrophobic carboxyl-terminal domain. Recombinant production of GhPLA1 in tobacco (Nicotiana tabacum) cells enabled us to purify and analyze a single glycosylated AGP and to demonstrate that this chimeric AGP promotes cotton SE. Furthermore, the nonglycosylated phytocyanin-like domain from GhPLA1, which was bacterially produced, also promoted SE, indicating that the glycosylated AGP domain was unnecessary for in vitro activity.Arabinogalactan proteins (AGPs) comprise a diverse group of plant proteoglycans (for review, see Fincher et al., 1993; Nothnagel, 1997; Seifert and Roberts, 2007; Ellis et al., 2010). They are structurally complex, generally consisting of a Pro-, Ala-, Ser-, and Thr-rich protein backbone that is extensively modified, principally by hydroxylation of Pro residues (to Hyp) and subsequent glycosylation through O-linkages with type II arabinogalactans (Tan et al., 2003; Shimizu et al., 2005). Many AGPs also have a C-terminal hydrophobic domain that is processed and replaced with a glycosylphosphatidylinositol (GPI) anchor, which acts to tether the molecule to the extracellular face of the plasma membrane (Schultz et al., 1998). AGPs are also defined by their ability to be bound and precipitated by the synthetic dye β-glucosyl Yariv reagent (β-GlcY) and related molecules (Yariv et al., 1967). These dyes have been useful in isolating, localizing, and quantifying AGPs.AGPs are grouped into three subclasses (Schultz et al., 2002): AGPs have an N-terminal signal sequence, an arabinogalactosylated domain, and a hydrophobic C-terminal domain; “chimeric AGPs” contain at least one arabinogalactosylated domain and a domain with an unrelated motif; while “hybrid AGPs” contain arabinogalactosylated as well as different Pro/Hyp-rich glycoprotein motifs.AGPs are implicated in many aspects of plant cell growth and development. Historically, it was not possible to assign roles to individual AGPs, as tests were conducted with unfractionated mixtures of AGPs. More recently, individual AGPs, mainly from Arabidopsis (Arabidopsis thaliana), have been studied using techniques such as mutant analysis and gene knockout/silencing, providing evidence for roles of individual AGPs in cell expansion, root and seed regeneration, the coordination of vascular development, both male and female gametogenesis, the development of cotton fibers, and as contributors to plant stem strength (Shi et al., 2003; van Hengel and Roberts, 2003; Acosta-García and Vielle-Calzada, 2004; Motose et al., 2004; Yang et al., 2007; Levitin et al., 2008; Coimbra et al., 2009; Li et al., 2010; MacMillan et al., 2010).Conditioned media from in vitro embryogenic cultures contain factors that can promote somatic embryogenesis (SE), implying the presence of secreted signaling molecules (de Vries et al., 1988). There is evidence that secreted AGPs, which are components of conditioned media, are involved in SE. For example, SE in carrot (Daucus carota) and spruce (Picea abies) cell cultures was promoted when AGPs from conditioned media were added exogenously (Kreuger and van Holst, 1993; Egertsdotter and von Arnold, 1995). Subsequent studies showed the association of particular AGP epitopes with SE-promoting activity and the involvement of AGPs in SE for several other species (Kreuger et al., 1995; McCabe et al., 1997; Toonen et al., 1997; Chapman et al., 2000; Saare-Surminski et al., 2000; Ben Amar et al., 2007). There is also evidence that SE-promoting AGPs may be cleaved by an endochitinase (Egertsdotter and von Arnold, 1988; Domon et al., 2000; van Hengel et al., 2001, 2002), but neither the identity of the individual AGP(s) involved in promoting SE nor the mechanism of action has been established.In this study, we focused on SE in cotton (Gossypium hirsutum ‘Coker 315’), which is a limiting step in cotton transformation, and the potential role of AGPs in this process. We show that cotton calli undergoing somatic embryogenesis secrete an AGP fraction that promotes SE when incorporated back into the growth medium. We report the cloning and sequencing of a complementary DNA (cDNA) encoding a chimeric AGP present in this fraction and show that this molecule promotes SE.     

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

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

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

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

S-Nitrosylation of Ascorbate Peroxidase Is Part of Programmed Cell Death Signaling in Tobacco Bright Yellow-2 Cells

Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H₂O₂) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H₂O₂-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H₂O₂-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H₂O₂-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H₂O₂ levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.Nitric oxide (NO) is a gaseous and diffusible redox molecule that acts as a signaling compound in both animal and plant systems (Pacher et al., 2007; Besson-Bard et al., 2008). In plants, NO has been found to play a key role in several physiological processes, such as germination, lateral root development, flowering, senescence, stomatal closure, and growth of pollen tubes (Beligni and Lamattina, 2000; Neill et al., 2002; Correa-Aragunde et al., 2004; He et al., 2004; Prado et al., 2004; Carimi et al., 2005). In addition, NO has been reported to be involved in plant responses to both biotic and abiotic stresses (Leitner et al., 2009; Siddiqui et al., 2011) and in the signaling pathways leading to programmed cell death (PCD; Delledonne et al., 1998; de Pinto et al., 2006; De Michele et al., 2009; Lin et al., 2012; Serrano et al., 2012).The cellular environment may greatly influence the chemical reactivity of NO, giving rise to different biologically active NO-derived compounds, collectively named reactive nitrogen species, which amplify and differentiate its ability to activate physiological and stress-related processes. Many of the biological properties of NO are due to its high affinity with transition metals of metalloproteins as well as its reactivity with reactive oxygen species (ROS; Hill et al., 2010). However, recent evidence suggests that protein S-nitrosylation, due to the addition of NO to reactive Cys thiols, may act as a key mechanism of NO signaling in plants (Wang et al., 2006; Astier et al., 2011). NO is also able to react with reduced glutathione (GSH), the most abundant cellular thiol, thus producing S-nitrosoglutathione (GSNO), which also acts as an endogenous trans-nitrosylating agent. GSNO is also considered as a NO store and donor and, as it is more stable than NO, acts as a long-distance NO transporter through the floematic flux (Malik et al., 2011). S-Nitrosoglutathione reductase (GSNOR), which is an enzyme conserved from bacteria to humans, has been suggested to play a role in regulating S-nitrosothiols (SNO) and the turnover of S-nitrosylated proteins in plants (Liu et al., 2001; Rusterucci et al., 2007).A number of proteins involved in metabolism, stress responses, and redox homeostasis have been identified as potential targets for S-nitrosylation in Arabidopsis (Arabidopsis thaliana; Lindermayr et al., 2005). During the hypersensitive response (HR), 16 proteins were identified to be S-nitrosylated in the seedlings of the same species (Romero-Puertas et al., 2008); in Citrus species, S-nitrosylation of about 50 proteins occurred in the NO-mediated resistance to high salinity (Tanou et al., 2009).However, while the number of candidate proteins for S-nitrosylation is increasing, the functional significance of protein S-nitrosylation has been explained only in a few cases, such as for nonsymbiotic hemoglobin (Perazzolli et al., 2004), glyceraldehyde 3-phosphate dehydrogenase (Lindermayr et al., 2005; Wawer et al., 2010), Met adenosyltransferase (Lindermayr et al., 2006), and metacaspase9 (Belenghi et al., 2007). Of particular interest are the cases in which S-nitrosylation involves enzymes controlling ROS homeostasis. For instance, it has been reported that S-nitrosylation of peroxiredoxin IIE regulates the antioxidant function of this enzyme and might contribute to the HR (Romero-Puertas et al., 2007). It has also been shown that in the immunity response, S-nitrosylation of NADPH oxidase inactivates the enzyme, thus reducing ROS production and controlling HR development (Yun et al., 2011).Recently, S-nitrosylation has also been shown to be involved in PCD of nitric oxide excess1 (noe1) rice (Oryza sativa) plants, which are mutated in the OsCATC gene coding for catalase (Lin et al., 2012). In these plants, which show PCD-like phenotypes under high-light conditions, glyceraldehyde 3-phosphate dehydrogenase and thioredoxin are S-nitrosylated. This suggests that the NO-dependent regulation of these proteins is involved in plant PCD, similar to what occurs in animal apoptosis (Sumbayev, 2003; Hara et al., 2005; Lin et al., 2012). The increase in hydrogen peroxide (H₂O₂) after exposure to high light in noe1 plants is responsible for the production of NO required for leaf cell death induction (Lin et al., 2012). There is a strict relationship between H₂O₂ and NO in PCD activation (Delledonne et al., 2001; de Pinto et al., 2002); however, the mechanism of this interplay is largely still unknown (for review, see Zaninotto et al., 2006; Zhao, 2007; Yoshioka et al., 2011). NO can induce ROS production and vice versa, and their reciprocal modulation in terms of intensity and timing seems to be crucial in determining PCD activation and in controlling HR development (Delledonne et al., 2001; Zhao, 2007; Yun et al., 2011).In previous papers, we demonstrated that heat shock (HS) at 55°C and treatment with 50 mm H₂O₂ promote PCD in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Vacca et al., 2004; de Pinto et al., 2006; Locato et al., 2008). In both experimental conditions, NO production and decrease in cytosolic ascorbate peroxidase (cAPX) were observed as early events in the PCD pathway, and cAPX decrease has been suggested to contribute to determining the redox environment required for PCD (de Pinto et al., 2006; Locato et al., 2008).In this study, the production of nitrosylating agents (NO and GSNO) in the first hours of PCD induction by HS or H₂O₂ treatment in tobacco BY-2 cells and their role in PCD were studied. The possibility that S-nitrosylation could be a first step in regulating cAPX activity and turnover as part of the signaling pathway leading to PCD was also investigated.     

Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants

State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems.In oxygenic photosynthesis, PSII and PSI function in series to convert light energy into the chemical energy that fuels multiple metabolic processes. Most of this light energy is captured by the chlorophyll (Chl) and carotenoid pigments in the light-harvesting antenna complexes (LHCs) that are peripherally associated with the core complexes of both photosystems (Wobbe et al., 2016). However, since the two photosystems exhibit different absorption spectra (Nelson and Yocum, 2006; Nield and Barber, 2006; Qin et al., 2015), PSI or PSII is preferentially excited under naturally fluctuating light intensities and qualities. To optimize photosynthetic electron transfer, the excitation state of the two photosystems must be rebalanced in response to changes in lighting conditions. To achieve this, higher plants and green algae require rapid and precise acclimatory mechanisms to adjust the relative absorption cross-sections of the two photosystems.To date, the phenomenon of state transitions is one of the well-documented short-term acclimatory mechanisms. It allows a mobile portion of the light-harvesting antenna complex II (LHCII) to be allocated to either photosystem, depending on the spectral composition and intensity of the ambient light (Allen and Forsberg, 2001; Rochaix, 2011; Goldschmidt-Clermont and Bassi, 2015; Gollan et al., 2015). State transitions are driven by the redox state of the plastoquinone (PQ) pool (Vener et al., 1997; Zito et al., 1999). When PSI is preferentially excited (by far-red light), the PQ pool is oxidized and all the LHCII is associated with PSII. This allocation of antenna complexes is defined as state I. When light conditions (blue/red light or low light) favor exciton trapping of PSII, the transition from state I to state II occurs. The over-reduced PQ pool triggers the activation of the membrane-localized Ser-Thr kinase STN7, which phosphorylates an N-terminal Thr on each of two major LHCII proteins, LHCB1 and LHCB2 (Allen, 1992; Bellafiore et al., 2005; Shapiguzov et al., 2016). Phosphorylation of LHCII results in the dissociation of LHCII from PSII and triggers its reversible relocation to PSI (Allen, 1992; Rochaix, 2011). Conversely, when the PQ pool is reoxidized, STN7 is inactivated and the constitutively active, thylakoid-associated phosphatase TAP38/PPH1 dephosphorylates LHCII, which then reassociates with PSII (Pribil et al., 2010; Shapiguzov et al., 2010). The physiological significance of state transitions has been demonstrated by the reduction in growth rate seen in the stn7 knock-out mutant under fluctuating light conditions (Bellafiore et al., 2005; Tikkanen et al., 2010).The canonical state transitions model implies spatial and temporal regulation of the allocation of LHC between the two spatially segregated photosystems (Dekker and Boekema, 2005). PSII-LHCII supercomplexes are organized in a tightly packed form in the stacked grana regions of thylakoid membranes, while PSI-LHCI supercomplexes are mainly localized in the nonstacked stromal lamellae and grana margin regions (Dekker and Boekema, 2005; Haferkamp et al., 2010). It has been proposed that, in the grana margin regions, which harbor LHCII and both photosystems, LHCII can migrate rapidly between them (Albertsson et al., 1990; Albertsson, 2001). This idea is supported by the recent discovery of mega complexes containing both photosystems in the grana margin regions (Yokono et al., 2015). Furthermore, phosphorylation of LHCII was found to increase not only the amount of PSI found in the grana margin region of thylakoid membranes (Tikkanen et al., 2008a), but also to modulate the pattern of PSI-PSII megacomplexes under changing light conditions (Suorsa et al., 2015). Nonetheless, open questions remain in relation to the physiological significance of the detection of phosphorylated LHCII in all thylakoid regions, even under the constant light conditions (Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013), although LHCII phosphorylation has been shown to modify the stacking of thylakoid membranes (Chuartzman et al., 2008; Pietrzykowska et al., 2014).State I-to-state II transition is featured by the formation of LHCII-PSI-LHCI supercomplexes, in which LHCII favors the light-harvesting capacity of PSI. Recently, LHCII-PSI-LHCI supercomplexes have been successfully isolated and purified using various detergents (Galka et al., 2012; Drop et al., 2014; Crepin and Caffarri, 2015) or a styrene-maleic acid copolymer (Bell et al., 2015). These findings yielded further insights into the reorganization of supercomplexes associated with state transitions, and it was suggested that phosphorylation of LHCB2 rather than LHCB1 is the essential trigger for the formation of state transition supercomplexes (Leoni et al., 2013; Pietrzykowska et al., 2014; Crepin and Caffarri, 2015; Longoni et al., 2015). Furthermore, characterization of mutants deficient in individual PSI core subunits indicates that PsaH, L, and I are required for docking of LHCII at PSI (Lunde et al., 2000; Zhang and Scheller, 2004; Kouril et al., 2005; Plöchinger et al., 2016).Recently, the state transition capacity has been characterized in the Arabidopsis (Arabidopsis thaliana) mutants with missing LHCI components. Although the Arabidopsis knock-out mutants lacking one of the four LHCI proteins (LHCA1-4) showed enhanced accumulation of LHCII-PSI complexes, the absorption cross-section of PSI under state II conditions was still compromised in the lhca1-4 mutants, and it is suggested that LHCI mediates the detergent-sensitive interaction between ‘extra LHCII’ and PSI (Benson et al., 2015; Grieco et al., 2015). Furthermore, the Arabidopsis mutant ΔLhca lacking all LHCA1-4 proteins was shown to be compensated for the deficiency of LHCI by binding LHCII under state II conditions (Bressan et al., 2016). In spite of this finding, the significant reduction in the absorption cross-section of PSI was still observed in the ΔLhca mutant, suggesting a substantial role of LHCI in light absorption under canopy conditions (Bressan et al., 2016). However, these findings emphasize the acclimatory function of state transitions in balancing light absorption capacity between the two photosystems by modifying their relative antenna size and imply the dynamic and variable organization of PS-LHC supercomplexes.LHC proteins are encoded by the nuclear Lhc superfamily (Jansson, 1994). The biogenesis of LHCs includes the cytoplasmic synthesis of the LHC precursor proteins, their translocation into chloroplasts via the TOC/TIC complex, and their posttranslational targeting and integration into the thylakoid membranes by means of the chloroplast signal recognition particle (cpSRP) machinery (Jarvis and Lopez-Juez, 2013). The posttranslational cpSRP-dependent pathway for the final translocation of LHC proteins into the thylakoid membrane includes interaction of cpSRP43 with LHC apo-proteins and recruitment of cpSRP54 to form a transit complex. Then binding of this tripartite cpSRP transit complex to the SRP receptor cpFtsY follows, which supports docking of the transit complex to thylakoid membranes and its association with the LHC translocase ALB3. Ultimately, ALB3 inserts LHC apo-proteins into the thylakoid membrane (Richter et al., 2010). Importantly, stoichiometric amounts of newly synthesized Chl a and Chl b as well as carotenoid are inserted into the LHC apo-proteins by unknown mechanisms to form the functional LHCs that associate with the core complexes of both photosystems in the thylakoid membranes (Dall’Osto et al., 2015; Wang and Grimm, 2015).The first committed steps in Chl synthesis occur in the Mg branch of the tetrapyrrole biosynthesis pathway. 5-Aminolevulinic acid synthesis provides the precursor for the formation of protoporphyrin IX, which is directed into the Mg branch (Tanaka and Tanaka, 2007; Brzezowski et al., 2015). Chl synthesis ends with the conversion of Chl a to Chl b catalyzed by Chl a oxygenase (CAO; Tanaka et al., 1998; Tomitani et al., 1999). It has been hypothesized that coordination between Chl synthesis and the posttranslational cpSRP pathway is a prerequisite for the efficient integration of Chls into LHC apo-proteins.In this study, we intend to characterize the assembly of LHCs when the availability of Chl molecules or the integration of LHC apo-proteins into thylakoid membranes is limiting. To this end, we compared the assembly of LHCs and the organization of PS-LHC complexes in two different sets of Arabidopsis mutants. Firstly, we used the chlorina1-2 (ch1-2) mutant, which is defective in the CAO gene. The members of the second set of mutants carry knock-out mutations in genes involved in the chloroplast SRP pathway (Richter et al., 2010).Our studies revealed distinct accumulation of PS-LHC supercomplexes between the two sets of mutant relative to wild-type plants. In spite of the defect in synthesis of Chl b, ch1-2 retains predominantly intact PSI-LHCI supercomplexes but has strongly reduced amounts of LHCII. In contrast, the chaos (cpSRP43) mutant exhibits synchronously reduced contents of both LHCI and LHCII, which results in the accumulation of PS core complexes without accompanying LHCs. Thus, the distribution of LHCs in the thylakoid membranes of the two mutants, ch1-2 and chaos, were explored under varying light conditions with the aim of elucidating the influence of modified LHCI/LHCII antenna size on state transitions. Our results contribute to an expanding view on the variety of photosynthetic complexes, which can be observed in Arabidopsis plants with specified mutations in LHC biogenesis.     

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis,Altering Cell Growth and Morphogenesis

Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.The primary walls of growing plant cells are largely constructed of cellulose and noncellulosic matrix polysaccharides that include hemicelluloses and pectins (Carpita and Gibeaut, 1993; Somerville et al., 2004; Cosgrove, 2005). Xyloglucan (XyG) is the most abundant hemicellulose in the primary walls of eudicots and is composed of a β-1,4-glucan backbone with side chains containing Xyl, Gal, and Fuc (Park and Cosgrove, 2015). XyG is synthesized in the Golgi apparatus before being secreted to the apoplast, and its biosynthesis requires several glycosyltransferases, including β-1,4-glucosyltransferase, α-1,6-xylosyltransferase, β-1,2-galactosyltransferase, and α-1,2-fucosyltransferase activities (Zabotina, 2012). Arabidopsis (Arabidopsis thaliana) XYLOGLUCAN XYLOSYLTRANSFERASE1 (XXT1) and XXT2 display xylosyltransferase activity in vitro (Faik et al., 2002; Cavalier and Keegstra, 2006), and strikingly, no XyG is detectable in the walls of xxt1 xxt2 double mutants (Cavalier et al., 2008; Park and Cosgrove, 2012a), suggesting that the activity of XXT1 and XXT2 are required for XyG synthesis, delivery, and/or stability.Much attention has been paid to the interactions between cellulose and XyG over the past 40 years. Currently, there are several hypotheses concerning the nature of these interactions (Park and Cosgrove, 2015). One possibility is that XyGs bind directly to cellulose microfibrils (CMFs). Recent data indicating that crystalline cellulose cores are surrounded with hemicelluloses support this hypothesis (Dick-Pérez et al., 2011). It is also possible that XyG acts as a spacer-molecule to prevent CMFs from aggregating in cell walls (Anderson et al., 2010) or as an adapter to link cellulose with other cell wall components, such as pectin (Cosgrove, 2005; Cavalier et al., 2008). XyG can be covalently linked to pectin (Thompson and Fry, 2000; Popper and Fry, 2005, 2008), and NMR data demonstrate that pectins and cellulose might interact to a greater extent than XyG and cellulose in native walls (Dick-Pérez et al., 2011). Alternative models exist for how XyG-cellulose interactions influence primary wall architecture and mechanics. One such model posits that XyG chains act as load-bearing tethers that bind to CMFs in primary cell walls to form a cellulose-XyG network (Carpita and Gibeaut, 1993; Pauly et al., 1999; Somerville et al., 2004; Cosgrove, 2005). However, results have been accumulating against this tethered network model, leading to an alternative model in which CMFs make direct contact, in some cases mediated by a monolayer of xyloglucan, at limited cell wall sites dubbed “biomechanical hotspots,” which are envisioned as the key sites of cell wall loosening during cell growth (Park and Cosgrove, 2012a; Wang et al., 2013; Park and Cosgrove, 2015). Further molecular, biochemical, and microscopy experiments are required to help distinguish which aspects of the load-bearing, spacer/plasticizer, and/or hotspot models most accurately describe the functions of XyG in primary walls.Cortical microtubules (MTs) direct CMF deposition by guiding cellulose synthase complexes in the plasma membrane (Baskin et al., 2004; Paredez et al., 2006; Emons et al., 2007; Sánchez-Rodriguez et al., 2012), and the patterned deposition of cellulose in the wall in turn can help determine plant cell anisotropic growth and morphogenesis (Baskin, 2005). Disruption of cortical MTs by oryzalin, a MT-depolymerizing drug, alters the alignment of CMFs, suggesting that MTs contribute to CMF organization (Baskin et al., 2004). CELLULOSE SYNTHASE (CESA) genes, including CESA1, CESA3, and CESA6, are required for normal CMF synthesis in primary cell walls (Kohorn et al., 2006; Desprez et al., 2007), and accessory proteins such as COBRA function in cellulose production (Lally et al., 2001). Live-cell imaging from double-labeled YFP-CESA6; CFP-ALPHA-1 TUBULIN (TUA1) Arabidopsis seedlings provides direct evidence that cortical MTs determine the trajectories of cellulose synthesis complexes (CSCs) and patterns of cellulose deposition (Paredez et al., 2006). Additionally, MT organization affects the rotation of cellulose synthase trajectories in the epidermal cells of Arabidopsis hypocotyls (Chan et al., 2010). Recently, additional evidence for direct guidance of CSCs by MTs has been provided by the identification of CSI1/POM2, which binds to both MTs and CESAs (Bringmann et al., 2012; Li et al., 2012). MICROTUBULE ORGANIZATION1 (MOR1) is essential for cortical MT organization (Whittington et al., 2001), but disruption of cortical MTs in the mor1 mutant does not greatly affect CMF organization (Sugimoto et al., 2003), and oryzalin treatment does not abolish CSC motility (Paredez et al., 2006).Conversely, the organization of cortical MTs can be affected by cellulose synthesis. Treatment with isoxaben, a cellulose synthesis inhibitor, results in disorganized cortical MTs in tobacco cells, suggesting that inhibition of cellulose synthesis affects MT organization (Fisher and Cyr, 1998), and treatment with 2,6-dichlorobenzonitrile, another cellulose synthesis inhibitor, alters MT organization in mor1 plants (Himmelspach et al., 2003). Cortical MT orientation in Arabidopsis roots is also altered in two cellulose synthesis-deficient mutants, CESA6^52-isx and kor1-3, suggesting that CSC activity can affect MT arrays (Paredez et al., 2008). Together, these results point to a bidirectional relationship between cellulose synthesis/patterning and MT organization.MTs influence plant organ morphology, but the detailed mechanisms by which they do so are incompletely understood. The dynamics and stability of cortical MTs are also affected by MT-associated proteins (MAPs). MAP18 is a MT destabilizing protein that depolymerizes MTs (Wang et al., 2007), MAP65-1 functions as a MT crosslinker, and MAP70-1 functions in MT assembly (Korolev et al., 2005; Lucas et al., 2011). MAP70-5 stabilizes existing MTs to maintain their length, and its overexpression induces right-handed helical growth (Korolev et al., 2007); likewise, MAP20 overexpression results in helical cell twisting (Rajangam et al., 2008). CLASP promotes microtubule stability, and its mutant is hypersensitive to microtubule-destabilizing drug oryzalin (Ambrose et al., 2007). KATANIN1 (KTN1) is a MT-severing protein that can sever MTs into short fragments and promote the formation of thick MT bundles that ultimately depolymerize (Stoppin-Mellet et al., 2006), and loss of KTN1 function results in reduced responses to mechanical stress (Uyttewaal et al., 2012). In general, cortical MT orientation responds to mechanical signals and can be altered by applying force directly to the shoot apical meristem (Hamant et al., 2008). The application of external mechanical pressure to Arabidopsis leaves also triggers MT bundling (Jacques et al., 2013). Kinesins, including KINESIN-13A (KIN-13A) and FRAGILE FIBER1 (FRA1), have been implicated in cell wall synthesis (Cheung and Wu, 2011; Fujikura et al., 2014). The identification of cell wall receptors and sensors is beginning to reveal how plant cell walls sense and respond to external signals (Humphrey et al., 2007; Ringli, 2010); some of them, such as FEI1, FEI2, THESEUS1 (THE1), FERONIA (FER), HERCULES RECEPTOR KINASE1 (HERK1), WALL ASSOCIATED KINASE1 (WAK1), WAK2, and WAK4, have been characterized (Lally et al., 2001; Decreux and Messiaen, 2005; Kohorn et al., 2006; Xu et al., 2008; Guo et al., 2009; Cheung and Wu, 2011). However, the relationships between wall integrity, cytoskeletal dynamics, and wall synthesis have not yet been fully elucidated.In this study, we analyzed CMF patterning, MT patterning and dynamics, and cellulose biosynthesis in the Arabidopsis xxt1 xxt2 double mutant that lacks detectable XyG and displays altered growth (Cavalier et al., 2008; Park and Cosgrove, 2012a). To investigate whether and how XyG deficiency affects the organization of CMFs and cortical MTs, we observed CMF patterning in xxt1 xxt2 mutants and Col (wild-type) controls using atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and confocal microscopy (Hodick and Kutschera, 1992; Derbyshire et al., 2007; Anderson et al., 2010; Zhang et al., 2014). We also generated transgenic Col and xxt1 xxt2 lines expressing GFP-MAP4 (Marc et al., 1998) and GFP-CESA3 (Desprez et al., 2007), and analyzed MT arrays and cellulose synthesis using live-cell imaging. Our results show that the organization of CMFs is altered, that MTs in xxt1 xxt2 mutants are aberrantly organized and are more sensitive to external mechanical pressure and the MT-depolymerizing drug oryzalin, and that cellulose synthase motility and cellulose content are decreased in xxt1 xxt2 mutants. Furthermore, real-time quantitative RT-PCR measurements indicate that the enhanced sensitivity of cortical MTs to mechanical stress and oryzalin in xxt1 xxt2 plants might be due to altered expression of MT-stabilizing and wall receptor genes. Together, these data provide insights into the connections between the functions of XyG in wall assembly, the mechanical integrity of the cell wall, cytoskeleton-mediated cellular responses to deficiencies in wall biosynthesis, and cell and tissue morphogenesis.     

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.