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.     

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.     

Pepper Heat Shock Protein 70a Interacts with the Type III Effector AvrBsT and Triggers Plant Cell Death and Immunity

Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.The heat shock protein HSP70 is a ubiquitous essential protein chaperone and one of the most abundant and diverse heat stress proteins in plants. HSP70s are induced by environmental stresses and are required for plants to cope with heat. HSP70s are involved in protein folding, synthesis, translocation, and macromolecular assemblies such as microtubules (Mayer et al., 2001; Hartl and Hayer-Hartl, 2002). HSP70s protect cells from heat stress by preventing protein aggregation and by facilitating the refolding of denatured proteins. Protein stability can decrease under heat stress conditions and expose hydrophobic patches that cause the aggregation of denatured proteins. HSP70s bind to hydrophobic patches of partially unfolded proteins in an ATP-dependent manner and prevent protein aggregation (Mayer and Bukau, 2005). The modular HSP70 structure consists of a N-terminal ATPase domain and a C-terminal peptide-binding domain that contains a β-sandwich subdomain with a peptide-binding cleft and an α-helical latch-like segment (Zhu et al., 1996; Hartl and Hayer-Hartl, 2002).HSP70s are involved in microbial pathogenesis, cell death responses, and immune responses. Diverse RNA viruses induce HSP70 expression in Arabidopsis (Arabidopsis thaliana; Whitham et al., 2003). Cytoplasmic HSP70s enhance the infection of Nicotiana benthamiana by Tobacco mosaic virus, Potato virus X, Cucumber mosaic virus, and Watermelon mosaic virus (Chen et al., 2008). Recently, the coat protein of Tomato yellow leaf curl virus was suggested to recruit host plant HSP70 during virus infection (Gorovits et al., 2013). HSP70s appear to be involved in regulating viral reproduction, protein folding, and movement, which ultimately promotes viral infection (Boevink and Oparka, 2005; Hafrén et al., 2010). The Pseudomonas syringae effector protein Hopl1 directly binds and manipulates host HSP70, which promotes bacterial virulence (Jelenska et al., 2010). The cytosolic/nuclear heat shock cognate 70 (HSC70) chaperone, which is highly homologous to HSP70 (Tavaria et al., 1996), regulates Arabidopsis immune responses together with SGT1 (for the suppressor of the G2 allele of S-phase kinase-associated protein1 [skp1]; Noël et al., 2007). Cytoplasmic HSP70 is required for the Phytophthora infestans INF1-mediated hypersensitive response (HR) and nonhost resistance to Pseudomonas cichorii in N. benthamiana (Kanzaki et al., 2003). HSP70 is proposed to be involved in both positive and negative regulation of cell death. Selective HSP70 depletion from human cell lines activates a tumor-specific death program that is independent of known caspases and p53 tumor-suppressor protein (Nylandsted et al., 2000), whereas HSP70 promotes tumor necrosis factor-mediated apoptosis by binding IkB kinase γ and impairing nuclear factor-κB signaling in Cos-1 cells (Ran et al., 2004). In N. benthamiana, HSP70 is required for tabtoxinine-β-lactam-induced cell death (Ito et al., 2014). However, HSP70 expression is shown to decrease the cell death triggered by salicylic acid (SA) in Nicotiana tabacum protoplasts (Cronjé et al., 2004). Overexpression of mitochondrial HSP70 suppresses heat- and hydrogen peroxide (H₂O₂)-induced programmed cell death in rice (Oryza sativa; Qi et al., 2011).The genus Xanthomonas YopJ-like AvrBsT protein activates effector-triggered immunity (ETI) in Arabidopsis Pitztal 0 plants (Cunnac et al., 2007). AvrBsT is a member of the YopJ/AvrRxv family identified in Xanthomonas campestris pv vesicatoria (Xcv; Lewis et al., 2011). AvrBsT alters phospholipid signaling and activates defense responses in Arabidopsis (Kirik and Mudgett, 2009). AvrBsT is an acetyltransferase that acetylates Arabidopsis ACETYLATED INTERACTING PROTEIN1 (ACIP1), a microtubule-associated protein required for plant immunity (Cheong et al., 2014). Xcv strain Bv5-4a secretes the AvrBsT type III effector protein that induces hypersensitive cell death and strong defense responses in pepper (Capsicum annuum) and N. benthamiana (Orth et al., 2000; Escolar et al., 2001; Kim et al., 2010). AvrBsT-induced HR-like cell death in pepper is likely part of the typical ETI-mediated defense response cascade (Jones and Dangl, 2006; Eitas et al., 2008; Eitas and Dangl, 2010). AvrBsT overexpression in Arabidopsis triggers plant cell death and defense signaling, leading to both disease and defense responses to diverse microbial pathogens (Hwang et al., 2012). Type III effectors such as Hopl1 and AvrBsT are used to identify unknown components of plant defense cascades (Nomura et al., 2006; Block et al., 2008; Jelenska et al., 2010; Kim et al., 2014) that modulate host innate immunity to achieve disease resistance. The pepper SGT1 was identified recently as a host interactor of AvrBsT (Kim et al., 2014). Pepper SGT1 has features of a cochaperone (Shirasu and Schulze-Lefert, 2003), interacts with AvrBsT, and promotes hypersensitive cell death associated with the pepper receptor-like cytoplasmic protein kinase1 (PIK1) phosphorylation cascade.In this study, we used a yeast (Saccharomyces cerevisiae) two-hybrid screen to identify the pepper HSP70a (CaHSP70a) as an interacting partner of the Xanthomonas spp. type III effector AvrBsT. Coimmunoprecipitation and bimolecular fluorescence complementation (BiFC) analyses verify that CaHSP70a interacts with AvrBsT in planta. Transient CaHSP70a overexpression in pepper leaves enhances heat stress sensitivity and leads to a cell death response. Cytoplasmic localization of the AvrBsT-CaHSP70a complex strongly elevates cell death. CaHSP70a expression is rapidly and strongly induced by avrBsT (for avirulent Xcv Dukso1 [Ds1]) infection in pepper. CaHSP70a silencing enhances susceptibility to Xcv infection, attenuates the reactive oxygen species (ROS) burst and cell death response, reduces SA and jasmonic acid (JA) levels, and disrupts expression of the defense response genes C. annuum pathogenesis-related protein1 (CaPR1; Kim and Hwang, 2000), CaPR10 (Choi et al., 2012), and CaDEF1 (for defensin; Do et al., 2004). Taken together, this study demonstrates that CaHSP70a is a target of the Xanthomonas spp. type III effector AvrBsT and acts as a positive regulator of plant cell death and immunity signaling.     

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.     

Plantation Forestry under Global Warming: Hybrid Poplars with Improved Thermotolerance Provide New Insights on the in Vivo Function of Small Heat Shock Protein Chaperones

Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula × Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones.In spite of the vast importance of trees as a renewable source of biomaterials and energy, forestry is largely dominated by traditional approaches worldwide. These have been unable to keep up with demand over the last half century, leading to significant deforestation and global concern over carbon emissions (Bonan, 2008). According to the Food and Agriculture Organization of the United Nations (FAO), the net loss in forest area is now at some 5.2 million ha per year, an alarmingly high rate (Global Forest Resources Assessment, 2010 [www.fao.org/forestry/fra2010/]). Tree farming is gaining momentum to reverse this state of affairs, but current yields must be significantly improved for plantations to become a realistic and sustainable alternative to forest logging (Boerjan, 2005; Fenning et al., 2008; Strauss et al., 2009; Harfouche et al., 2011, 2012). Global warming makes this a challenging goal, as evidenced by the massive losses in crop production attributable to the increased temperatures of the last decades (Lobell et al., 2011). Climate-driven heat stress has also been recently identified as a major cause of forest die off (Anderegg et al., 2013). While climate trends make the establishment of high-yielding plantations more pressing than ever, improving the thermotolerance of valuable genotypes has proven difficult (Neale and Kremer, 2011; Harfouche et al., 2012). Apart from long juvenile periods and insufficient genomic resources, progress in this area has been hampered by our poor understanding of the biochemical mechanisms underlying thermotolerance. Forest biotechnology has great potential in this respect, as promising candidate genes can be safely assessed under controlled conditions. Tree breeding programs will integrate this knowledge in the foreseeable future to help improve elite genotypes and sidestep undesirable phenotypic variation (Fenning et al., 2008; Strauss et al., 2009; Harfouche et al., 2011, 2012). A successful example is the development of freeze-tolerant hybrid eucalyptus trees (Eucalyptus grandis × Eucalyptus urophylla) through a biotechnological approach (Hinchee et al., 2009).Extensive research has shown that high-temperature stress has a negative impact on nearly every aspect of plant growth, development, reproduction, and yield (for review, see Mittler et al., 2012). To protect cell function, plants have evolved a complex metabolic adjustment process known as the heat shock response (HSR; Kotak et al., 2007; Mittler at al., 2012). Alternatively, programmed cell death is activated in specific cells, resulting in leaf shedding or abortion of reproductive organs (Qu et al., 2009; Blanvillain et al., 2011). Whereas research in forest trees remains scarce, the ubiquitous and conserved HSR has been thoroughly studied in herbaceous plants (Larkindale and Vierling, 2008; Hu et al., 2009; Finka et al., 2011; Mittler et al., 2012). It is now well established that it involves multiple biochemical and regulatory pathways aimed at minimizing damage and preserving cellular homeostasis. Heat stress promotes protein unfolding and aggregation, and a major component of the HSR is the induction of molecular chaperones of the heat shock protein (HSP) family. HSPs share the ability to recognize and bind other proteins in nonnative states, thereby preventing or reversing aggregation as well as promoting efficient refolding pathways. Moreover, HSPs facilitate the degradation of proteins that cannot be properly folded by facilitating their delivery to cellular proteases.The most abundant and heterogeneous HSPs in plants are the widespread small heat shock proteins (sHSPs). At least 10 separate families have been described in both monocots and dicots, which include proteins localized to the cytoplasm (four classes), nucleus, chloroplasts, mitochondria, endoplasmic reticulum, and peroxisomes (for review, see Basha et al. [2012]; Waters [2013]). Along with their remarkable up-regulation under stressful conditions, the above features suggest important protective roles in virtually all cellular compartments. Yet, sHSPs remain one of the least well-understood families of molecular chaperones. With monomeric sizes of approximately 12 to 43 kD, plant sHSPs share a signature α-crystallin domain of approximately 90 amino acids and the ability to form large oligomers with a dynamic quaternary structure. The majority of structural and functional information derives from studies of cytosolic class I (CI) members, like wheat (Triticum aestivum) TaHSP16.9, for which the crystal structure of the native dodecamer has been solved (van Montfort et al., 2001). While researchers have demonstrated that most sHSPs can act as ATP-independent molecular chaperones, understanding their in vivo mode of action is complex (Basha et al., 2012; Waters, 2013). The prevailing view is that they prevent irreversible protein aggregation by interacting with unfolded proteins generated during stress. Recent evidence indicates that sHSP oligomers bind up to an equal weight of substrate proteins through a unique mechanism that involves substrate- and temperature-dependent remodeling of their own quaternary structure (Jaya et al., 2009; Stengel et al., 2010). Subsequent refolding of the substrates requires, at least in some cases, interaction with high-M_r ATP-dependent chaperones (Haslbeck et al., 2005; Basha et al., 2012). Thus, sHSPs would act primarily as holdases that keep nonnative proteins in a competent state until other downstream chaperones facilitate refolding. In vivo sHSP substrates remain to be identified in higher plants, although different lines of evidence suggest that they are structurally diverse (Basha et al., 2004; Haslbeck et al., 2004; Cheng et al., 2008; Jaya et al., 2009). Likewise, the role of sHSPs in mechanisms of thermotolerance has not yet been established. Reverse genetic approaches have led to inconsistent results in Arabidopsis (Arabidopsis thaliana) and other herbaceous species, ranging from variable protection (Malik et al., 1999; Sanmiya et al., 2004; Zhao et al., 2007; Jiang et al., 2009) to no detectable effects at all (Härndahl et al., 1999; Sun et al., 2001).CsHSP17.5, a major CI sHSP in chestnut trees (Castanea sativa), exhibits features that make it a good candidate to improve heat stress adaptation. First, its accumulation in adult trees increases substantially during spring and early summer, reaching a peak at the hottest time of the year (field conditions). Second, the corresponding gene is strongly activated by heat stress (growth chambers). Third, it significantly improves Escherichia coli viability under heat stress conditions. Fourth, it accumulates at unusually high levels in both stems and seeds, where it reaches levels comparable to those of major storage proteins (Collada et al., 1997; Soto et al., 1999; Lopez-Matas et al., 2004). Besides suggesting a relevant role in thermotolerance, these features indicate that CsHSP17.5 can accumulate in vivo for long periods of time. In vitro experiments have shown, additionally, that the native protein can stand repeated cycles of stressful conditions without losing chaperone activity (Lopez-Matas et al., 2004). The fact that CsHSP17.5 has never been identified as an allergen, despite accumulating so abundantly in mature chestnuts (according to FAO, humans consume over half a million metric tons a year), adds to its biotechnological potential. Here, we show that constitutive overexpression of native CsHSP17.5 substantially enhances the basal thermotolerance of hybrid poplar (Populus tremula × Populus alba) without causing negative pleiotropic effects that might affect plantation yields. A significant improvement of plant performance in vitro and ex vitro was also observed, even in the absence of thermal stress. Our results shed new light on the in vivo roles of sHSPs. In addition, they represent, to our knowledge, the first example of biotechnological manipulation of thermotolerance in forest trees.