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.     

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1^cisΔ11) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2^cisΔ9,12; 17.9%–44.4% and 7%–13.2%, respectively) and decreases in 20:1^cisΔ11 (38.7%–60.7% and 13.8%–16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3^cisΔ9,12,15) in both the acyl-CoA pool and seed oil of the former (48.4%–48.9% and 5.3%–10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil.Cytosolic low-molecular mass (approximately 10 kD) acyl-coenzyme A-binding protein (ACBP) consists of a four-α-helix domain capable of binding acyl-CoAs with high affinity in a wide range of eukaryotic organisms (Faergeman et al., 2007). It is believed to serve a housekeeping function of maintaining free acyl-CoA concentrations at low nanomolar levels and, thus, prevents micelle formation and the partitioning of acyl-CoA into membranes (Knudsen et al., 1999). This protein is also considered to contribute to another facet of acyl-CoA pool maintenance via its role in the intracellular transport of acyl-CoAs in the aqueous environment of the cytosol (Rasmussen et al., 1994). Moreover, it has also been shown to exhibit more specialized functions in metabolic processes in which acyl-CoA is actively involved, depending on the tissue and physiological state (Guerrero et al., 2006; Xiao and Chye 2011; Yurchenko and Weselake, 2011).In the developing seeds of oleaginous plants, fatty acids (FAs) are synthesized de novo in plastids and are activated to acyl-CoAs upon their transfer to the cytosol, after which time they can undergo additional modifications (e.g. elongation and desaturation) on the membranes of the endoplasmic reticulum (ER; for review, see Rawsthorne, 2002). While FA elongation is performed on the acyl-CoA substrate, the introduction of the second and third double bonds requires the acyl group to be esterified to phosphatidylcholine (PC; Jaworski, 1987). The composition of the acyl-CoA pool, therefore, is highly dynamic and represents a net result of both de novo synthesis and acyl-editing processes (Bates et al., 2009).The acyl-CoA pool provides substrate for acyltransferases involved in the biosynthesis of triacylglycerol (TAG), which is a major component of seed oil (Weselake et al., 2009). More specifically, TAG synthesis typically occurs via a series of acyl-CoA-dependent acylations of a glycerol backbone derived from sn-glycerol-3-phosphate in a pathway known as the sn-glycerol-3-phosphate or Kennedy pathway (for review, see Snyder et al., 2009; Weselake et al., 2009), although acyl-CoA-independent reactions can also be involved in the production of TAG (Stobart et al., 1997; Banaś et al., 2000; Dahlqvist et al., 2000) and thus contribute to its final composition. Low-molecular mass ACBPs have been demonstrated to modulate the activities of Kennedy pathway acyltransferases in a manner dependent upon the ratio of ACBP to acyl-CoA, stimulating TAG biosynthesis under conditions of acyl-CoA excess and inhibiting acyltransferase activities when relative amounts of acyl-CoA are low compared with ACBP, thus regulating the size of the acyl-CoA pool (for review, see Yurchenko and Weselake, 2011).The acyl-CoA pool in seeds is also influenced through a distinct route involving lysophosphatidylcholine acyltransferase (LPCAT), which catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine at the sn-2 position to form PC (Ichihara et al., 1995). Acyl groups esterified to PC become substrates for FA desaturation and other modifications (Miquel and Browse, 1992; Broun et al., 1998) and can then be returned back to the acyl-CoA pool or channeled into TAG through acyl-CoA-independent mechanisms (Stymne and Stobart, 1984; Weselake, 2005; Lager et al., 2013). The efficiency of this acyl group channeling to and from PC is an important determinant of the overall composition of FAs in the acyl-CoA pool and, subsequently, in seed oil.Previously, we demonstrated that the expression of the Brassica napus low-molecular mass ACBP (hereafter referred to as BnACBP) in the presence of Arabidopsis (Arabidopsis thaliana) LPCAT isoforms in an in vitro system enhanced the incorporation of oleic acid (18:1^cisΔ9; hereafter referred to as 18:1) into PC and the release of linoleic acid (18:2^cisΔ9,12; hereafter referred to as 18:2) from PC into acyl-CoA (Yurchenko et al., 2009). In line with these results, the expression of BnACBP complementary DNA (cDNA) in Arabidopsis developing seeds was also shown to result in elevated levels of the polyunsaturated fatty acids (PUFAs) 18:2 and α-linolenic acid (18:3^cisΔ9,12,15; hereafter referred to as 18:3) in seed oil, mainly at the expense of eicosenoic acid (20:1^cisΔ11; hereafter referred to as 20:1) and saturated fatty acids (SFAs; Yurchenko et al., 2009). Based on these findings, BnACBP was proposed to be involved in acyl exchange between acyl-CoA and PC pools, which may affect the rate of FA modifications and, ultimately, the FA composition of seed oil (Yurchenko et al., 2009).In this study, we endeavored to provide further evidence that low-molecular mass ACBP functions in acyl trafficking by investigating whether changes in the FA composition of TAG in Arabidopsis seeds expressing BnACBP were correlated with modifications in the composition of the acyl-CoA pool. In addition, since FA modifications such as elongation and desaturation as well as TAG synthesis occur on ER membranes, we also examined the effect of changing the subcellular localization of BnACBP (from the cytosol to the ER) on the acyl composition of TAG and the acyl-CoA pool in transgenic Arabidopsis. Consequently, we generated localized pools of acyl-CoAs that could be readily accessed by acyltransferases involved in seed oil biosynthesis. Taken together, our findings provide insight into the role of low-molecular mass ACBP in seed oil metabolism and suggest that ACBP (either in its native cytosolic form or as an ER-targeted fusion protein) may serve as a useful tool in biotechnological modifications of FA composition in oil crops.     

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.     

Specialization of Oleosins in Oil Body Dynamics during Seed Development in Arabidopsis Seeds

Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. OBs are covered with specific proteins embedded in a single layer of phospholipids. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development. Analyses were carried out with different genotypes: the wild type and three mutants affected in the accumulation of various oleosins (OLE1, OLE2, and OLE4), three major OB proteins. Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t). Our results indicate that OB size increases sharply during seed maturation, in part by OB fusion, and then decreases until the end of the maturation process. In single, double, and triple mutant backgrounds, the size and spatial distribution of OBs are modified, affecting in turn the total lipid content, which suggests that the oleosins studied have specific functions in the dynamics of lipid accumulation.The seed is a complex, specific structure that allows a quiescent plant embryo to cope with unfavorable germinating conditions and also permits dissemination of the species. To achieve these functions, seeds accumulate reserve compounds that will ensure the survival of the embryo and fuel the growth of the plantlet upon germination. Accumulation of lipids occurs in many eukaryotic cells and is a rather common means of storing carbon and energy. Lipid droplets (LDs) can be found in all eukaryotes, such as yeast (Saccharomyces cerevisiae; Leber et al., 1994), mammals (Murphy, 2001; Hodges and Wu, 2010), Caenorhabditis elegans (Zhang et al., 2010; Mak, 2012), Drosophila melanogaster (Beller et al., 2006, 2010), and plants (Hsieh and Huang, 2004), but also in prokaryotes (Wältermann et al., 2005). The basic structure of an LD is a core of neutral lipids covered by a phospholipid monolayer. LDs differ between species by the set of proteins covering their surface, the nature of the lipids stored, and their turnover. Nevertheless, they apparently always ensure the same function in the cell (i.e. energy storage; Murphy, 2012). In Brassicacea species such as Arabidopsis (Arabidopsis thaliana), seed reserves are mainly composed of carbohydrates, proteins, and lipids (Baud et al., 2002). The lipids are primarily stored as triacylglycerols (TAGs) in LDs, more commonly called oil bodies (OBs; Hsieh and Huang, 2004; Chapman et al., 2012; Chapman and Ohlrogge, 2012) of diameter 0.5 to 2 µm (Tzen et al., 1993).The protein composition of seed OBs has been determined for several plant species, including Brassica napus (Katavic et al., 2006; Jolivet et al., 2009) and Arabidopsis (Jolivet et al., 2004; D’Andréa et al., 2007; Vermachova et al., 2011). In Arabidopsis, 10 proteins have been identified, and seed-specific oleosins represent up to 79% of the OB proteins (Jolivet et al., 2004; D’Andréa et al., 2007; Vermachova et al., 2011). Oleosins are rather small proteins of 18.5 to 21.2 kD with a specific and highly conserved central hydrophobic domain of 72 amino acid residues flanked by hydrophilic domains of variable size and amino acid composition (Qu and Huang, 1990; Tzen et al., 1990, 1992; Huang, 1996; Hsieh and Huang, 2004). It is generally agreed that oleosins cover the OB surface, with their central hydrophobic domain inserted in the TAG through the phospholipid layer (Tzen and Huang, 1992). Besides their structural function in OBs, oleosins may serve as docking stations for other proteins at its surface (Wilfling et al., 2013) and may participate in the biosynthesis and mobilization of plant oils (Parthibane et al., 2012a, 2012b). Oleosins are probably involved in OB stability (Leprince et al., 1998; Shimada et al., 2008) and in the regulation of OB repulsion (Heneen et al., 2008), preventing the coalescence of OBs into a single organelle (Schmidt and Herman, 2008). Nevertheless, the precise functions of oleosins in OB biogenesis and dynamics have not yet been established.Global analysis of seed lipids can be performed using gas chromatography (Li et al., 2006), which allows the precise determination of both lipid content and fatty acid composition. Recently, direct organelle mass spectrometry has been used to visualize the lipid composition of cotton (Gossypium hirsutum) seed OBs (Horn et al., 2011). Nevertheless, in both cases, the methods are destructive. To observe lipid accumulation at the subcellular level, well-known nondestructive techniques for lipid visualization have been adapted to seeds. Third harmonic generation microscopy (Débarre et al., 2006) and label-free coherent anti-Stokes Raman scattering microscopy (Paar et al., 2012) allow dyeless observation of LDs but require very specific equipment. Magnetic resonance imaging enables topographic analysis of lipid distribution in cereal grains (Neuberger et al., 2008) and in submillimeter-sized seeds like those of tobacco (Nicotiana tabacum; Fuchs et al., 2013). Nevertheless, the use of fluorescent dyes such as Nile Red (Greenspan and Fowler, 1985), BODIPY (Pagano et al., 1991), or LipidTOX (Invitrogen) associated with confocal microscopy is also a powerful way to monitor LDs in living organisms.Despite knowledge accumulated on this topic (Brasaemle and Wolins, 2012; Chapman et al., 2012), little is known about OB dynamics during seed maturation. In this article, we investigate this question by monitoring the evolution of OBs in living Arabidopsis embryos over time. This analysis showed a marked change in OB size at 9 to 10 d after flowering (DAF). We then examined single, double, and triple mutants of the major oleosins found in developing seeds (OLE1 [At4g25140], OLE2 [At5g40420], and OLE4 [At3g01570]; Jolivet et al., 2004). We analyzed the OB dynamics in these mutant backgrounds as if they would contain only these three proteins. We show that the lack of specific oleosins influences the dynamics and distribution of OBs during seed maturation, which in turn affects lipid accumulation. These results pave the way for analyzing specific functions of oleosins in the synthesis, growth, and evolution of OBs.