Stress-Induced Cytokinin Synthesis Increases Drought Tolerance through the Coordinated Regulation of Carbon and Nitrogen Assimilation in Rice

The effects of water deficit on carbon and nitrogen metabolism were investigated in flag leaves of wild-type and transgenic rice (Oryza sativa japonica ‘Kitaake’) plants expressing ISOPENTENYLTRANSFERASE (IPT; encoding the enzyme that mediates the rate-limiting step in cytokinin synthesis) under the control of P_SARK, a maturation- and stress-induced promoter. While the wild-type plants displayed inhibition of photosynthesis and nitrogen assimilation during water stress, neither carbon nor nitrogen assimilation was affected by stress in the transgenic P_SARK::IPT plants. In the transgenic plants, photosynthesis was maintained at control levels during stress and the flag leaf showed increased sucrose (Suc) phosphate synthase activity and reduced Suc synthase and invertase activities, leading to increased Suc contents. The sustained carbon assimilation in the transgenic P_SARK::IPT plants was well correlated with enhanced nitrate content, higher nitrate reductase activity, and sustained ammonium contents, indicating that the stress-induced cytokinin synthesis in the transgenic plants played a role in maintaining nitrate acquisition. Protein contents decreased and free amino acids increased in wild-type plants during stress, while protein content was preserved in the transgenic plants. Our results indicate that the stress-induced cytokinin synthesis in the transgenic plants promoted sink strengthening through a cytokinin-dependent coordinated regulation of carbon and nitrogen metabolism that facilitates an enhanced tolerance of the transgenic plants to water deficit.Plant hormones control many aspects of plant growth and development and the responses of plants to abiotic and biotic stresses. Cytokinins (CKs) have been shown to regulate plant cell differentiation, leaf senescence, and other key developmental processes (Sakakibara, 2006). It has also been shown that CKs regulate assimilate partitioning (Ronzhina and Mokronosov, 1994), sink strength (Kuiper, 1993), and source/sink relationships (Roitsch, 1999). The localized expression in tobacco (Nicotiana tabacum) of a promoterless ISOPENTENYLTRANSFERASE (IPT), a gene encoding the enzyme that catalyzes the rate-limiting step in CK synthesis, enhanced the local sink strength and quickly mobilized nutrients to the tissues with elevated CK (Guivarc’h et al., 2002). Changes in sink/source relationships were also observed in CK-deficient tobacco shoots and roots (Werner et al., 2008). Elevated CK levels enhanced the survival of plants under water-stress conditions (Rivero et al., 2007). The overexpression of IPT under the control of SENESCENCE-ASSOCIATED RECEPTOR KINASE (SARK; a maturation- and stress-induced promoter) improved the drought tolerance of both eudicots (Rivero et al., 2007; Qin et al., 2011) and monocots (Peleg et al., 2011). After a water-stress episode during the reproductive stages (pre and post anthesis), transgenic P_SARK::IPT rice (Oryza sativa) plants displayed higher grain yield than the wild type (Peleg et al., 2011). The transgenic P_SARK::IPT rice exhibited a differential expression of genes encoding enzymes associated with hormone synthesis and hormone-regulated pathways. These results suggested that changes in hormone homeostasis induced the modification of source/sink relationships in the transgenic plants, resulting in higher grain yields under stress conditions (Peleg et al., 2011).The maintenance of carbon (C) and nitrogen (N) assimilation is of paramount importance to ensure sink strength and improve stress tolerance without yield penalties. The interactions between C and N metabolism are vital for plant growth and development, and complex mechanisms operate in the plant to coordinate C assimilation with N metabolism (Nunes-Nesi et al., 2010). Thus, plants respond to changes in C and N metabolites through the regulation of translation and posttranslational modification mechanisms. C and N metabolites activate signaling pathways that regulate enzyme and transporter activities that control C and N fluxes, optimizing the plant response to developmental and environmental cues changing source/sink relationships (Coruzzi and Zhou, 2001).Plant hormones affect, either directly or indirectly, these pathways and can act antagonistically or synergistically when responding to environmental stress (Wilkinson et al., 2012). The exposure of plants to water-limiting conditions results in abscisic acid (ABA) synthesis that induces ABA-dependent gene expression (Yamaguchi-Shinozaki and Shinozaki, 2006), triggering the closure of stomata and reducing water loss during drought (Wilkinson and Davies, 2010). Other hormones, in particular CK, salicylic acid, ethylene, and jasmonic acid, also play direct or indirect roles in the plant responses to abiotic stress (Peleg and Blumwald, 2011). Under drought stress, plant CK content decreases, and the reduction in CK increases the plant responses to increasing ABA (Davies and Zhang, 1991), inducing stomata closure and inhibiting photosynthesis (Rivero et al., 2010). Our previous results suggested that the stress-induced CK synthesis, driven by a stress-induced promoter, protected against the deleterious effects of water deficit on the photosynthetic apparatus, allowing higher photosynthetic rates and higher yields after water deficit in tobacco (Rivero et al., 2009) and cotton (Gossypium hirsutum; Kuppu et al., 2013) plants grown in the greenhouse and peanut (Arachis hypogaea) plants grown under field conditions (Qin et al., 2011).Here, we analyzed gene expression profiles, metabolites, and enzymatic and photosynthetic activities of flag leaves of wild-type and transgenic rice expressing P_SARK::IPT exposed to water deficit during the reproductive stage and identified metabolic processes associated with the enhanced tolerance of the transgenic plants to water deficit. Our results indicate that the stress-induced CK synthesis in the transgenic plants promoted sink strengthening through the maintenance and coordination of N and C assimilation during water stress.     

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N⁶-(∆²-isopentenyl)adenine and dimethylated N⁶-(∆²-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N⁶-(∆²-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N⁶-(∆²-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.The balance of phytohormones, such as cytokinins (CKs) and auxins, is finely controlled to maintain proper plant growth and development but is often disturbed following pathogen infection (Robert-Seilaniantz et al., 2007; Pieterse et al., 2012). As a virulence strategy, many phytopathogens synthesize phytohormones that cause aberrant organogenesis and modulate primary carbon metabolism that ultimately aids disease establishment (Jameson, 2000; Robert-Seilaniantz et al., 2007). For several pathogens, CK production is essential for virulence, and they carry genes for CK biosynthesis in a harbored plasmid (Jameson, 2000). Fungal pathogens employ CKs to form green islands with delayed senescence, whereas bacterial pathogens develop gall structures (Sakakibara et al., 2005; Walters et al., 2008; Giron et al., 2013). Rhodococcus fascians is a gram-positive actinomycete that causes symptoms ranging from leaf deformation to differentiated shooty outgrowths known as leafy galls in more than 150 different plant species (Goethals et al., 2001; Stes et al., 2011). In ornamental plants, such infections reduce their value and contribute to economic losses worldwide (Putnam and Miller, 2007). Leafy gall symptoms are reminiscent of CK overproduction and can be partially induced by exogenous application of CKs (Thimann and Sachs, 1966; Eason et al., 1996). Although several CKs have been isolated from R. fascians culture filtrates, a clear correlation with pathogenesis is lacking partially owing to the low concentration of bacterial CKs (Eason et al., 1996). A synergistic action by a mixture of bacterially produced CKs has been proposed, leading to persistent accumulation of CKs locally (Pertry et al., 2009). Nevertheless, to date, no virulence-associated CK analogs have been identified that could contribute to the infection symptoms.Naturally occurring CKs are adenine derivatives with different side chains at the N6 position. Major plant CKs are N⁶-prenylated adenine derivatives such as N⁶-(Δ²-isopentenyl)adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin, collectively known as isoprenoid CKs (Sakakibara, 2006). Among them, iP and tZ are the major CKs in Arabidopsis (Arabidopsis thaliana). iP is synthesized by adenosine phosphate-isopentenyl transferase (IPT) using dimethylallyl diphosphate (DMAPP) and adenosine phosphate as substrates (Sakakibara, 2006). tZ is formed by hydroxylation of the trans-end of the prenyl side chain of the iP nucleotide. CK homeostasis is governed by both biosynthesis and catabolism and has an important regulatory role in plant growth (Sakakibara, 2006; Werner et al., 2006). CK oxidase/dehydrogenase (CKX) is responsible for an irreversible reaction cleaving the unsaturated isoprenoid side chain that results in the formation of adenine and the corresponding aldehyde (Werner et al., 2006). In Arabidopsis, CKs are perceived by a subset of sensory His kinases, ARABIDOPSIS HIS KINASE2 (AHK2) to AHK4, which undergo a His-Asp phosphorelay leading to induction of direct target genes including type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes (Kieber and Schaller, 2010). This two-component signaling system has been implicated in mediating basal and pathogen-induced plant immunity (Choi et al., 2010; Argueso et al., 2012). For instance, infection of Arabidopsis plants by R. fascians reportedly activates type-A ARR5 expression with increased expression of AHK3 and AHK4, resulting in mitotic cell divisions that arrest the infected leaves in a meristematic state to establish a nutrient-rich niche (Depuydt et al., 2008, 2009; Pertry et al., 2010; Stes et al., 2011). As the infection progresses, IPT genes are switched off, whereas the expression of all CKX genes are strongly induced in symptomatic tissues (Depuydt et al., 2008).The virulence determinant of R. fascians is located within the fasciation (fas) locus, an operon encoding several genes involved in CK metabolism, indicating that CKs are essential for this plant-pathogen interaction (Stes et al., 2011). fas4 encodes IPT that catalyzes the rate-limiting step of CK biosynthesis and is vital for virulence (Stes et al., 2013). Interestingly, two methyltransferase-like genes are present upstream of the fas gene, whose functions have been unknown. Despite the presence of the fas genes in R. fascians, fewer known CKs have been detected compared with other gall-causing pathogens such as Pantoea agglomerans, Agrobacterium tumefaciens, and Pseudomonas savastanoi (Goethals et al., 2001). Further, the leafy gall phenotype is unique, not invoked by any of the above-mentioned pathogens, implying that the virulence of R. fascians might not be due to typical CKs alone (Goethals et al., 2001). R. fascians has long been hypothesized to produce CK analogs using similar or modified substrates (Goethals et al., 2001; Galis et al., 2005; Stes et al., 2011), but no such molecules have been discovered so far. Here, we report the identification and mode of biosynthesis for methylated cytokinins (MeCKs) as hormone mimics from R. fascians. These compounds are synthesized by two methyltransferases and FAS4. Their CK-like activity and higher in planta stability suggest a role for the methylated analogs as CK mimics that foster efficient pathogenesis.     

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.     

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.     

Focus Issue on the Plant Physiology of Global Change: A Nitrogen-Regulated Glutamine Amidotransferase (GAT1_2.1) Represses Shoot Branching in Arabidopsis

Shoot branching in plants is regulated by many environmental cues and by specific hormones such as strigolactone (SL). We show that the GAT1_2.1 gene (At1g15040) is repressed over 50-fold by nitrogen stress, and is also involved in branching control. At1g15040 is predicted to encode a class I glutamine amidotransferase (GAT1), a superfamily for which Arabidopsis (Arabidopsis thaliana) has 30 potential members. Most members can be categorized into known biosynthetic pathways, for the amidation of known acceptor molecules (e.g. CTP synthesis). Some members, like GAT1_2.1, are of unknown function, likely involved in amidation of unknown acceptors. A gat1_2.1 mutant exhibits a significant increase in shoot branching, similar to mutants in SL biosynthesis. The results suggest that GAT1_2.1 is not involved in SL biosynthesis since exogenously applied GR24 (a synthetic SL) does not correct the mutant phenotype. The subfamily of GATs (GATase1_2), with At1g15040 as the founding member, appears to be present in all plants (including mosses), but not other organisms. This suggests a plant-specific function such as branching control. We discuss the possibility that the GAT1_2.1 enzyme may activate SLs (e.g. GR24) by amidation, or more likely could embody a new pathway for repression of branching.Shoot branching plays an important role in establishing plant body plans during development and growth, also conferring the flexibility for plants to respond to environmental stresses. The control of bud growth/branching has been studied for many decades with much interest stemming from its value in agriculture. Indeed, many of our domesticated crops have been bred for modified branching to optimize yields. In early studies, auxin synthesized in the shoot apex was proposed to act indirectly to inhibit bud outgrowth, while cytokinin (CK) synthesized in the roots promoted bud outgrowth (Domagalska and Leyser, 2011). Studies on auxin inhibition suggested there should be another signal mediating bud growth control (Hayward et al., 2009; Stirnberg et al., 2010; Domagalska and Leyser, 2011). In the past decade, studies in Arabidopsis (Arabidopsis thaliana) and other plants have addressed this signal. Identification and characterization of mutants with increased branching in garden pea (Pisum sativum), Arabidopsis, rice (Oryza sativa), and Petunia hybrida demonstrated the existence of a long-distance signaling pathway that regulates shoot branching (Beveridge et al., 1996, 1997; Napoli, 1996; Stirnberg et al., 2002, 2007; Sorefan et al., 2003; Booker et al., 2004; Arite et al., 2007; Gomez-Roldan et al., 2008; Umehara et al., 2008, 2010; Lin et al., 2009; Liu et al., 2009, 2011; Zhang et al., 2010). Later, studies on pea (Gomez-Roldan et al., 2008) and rice (Umehara et al., 2008) demonstrated unequivocally that this hormone (or its precursor) is strigolactone (SL). Currently, it is proposed that SL acts downstream of auxin to regulate bud outgrowth (Brewer et al., 2009). It is also likely that SL and auxin have the capacity to modulate each other’s levels and distribution in a dynamic feedback loop required for the branching control (Ferguson and Beveridge, 2009; Hayward et al., 2009; Stirnberg et al., 2010). The interaction between SL and CK during bud outgrowth is less understood, although recent studies in pea indicate that SL and CK act antagonistically on bud growth (Dun et al., 2012).Branching is also modulated in response to environmental conditions, including nutrient supply. Generally, nutrient deficiency in soil causes a reduction in shoot to root ratio, resulting in decreased shoot branching (Lafever, 1981). Under nitrogen or phosphate limitation, elevated levels of SL repress shoot branching in rice, tomato (Solanum lycopersicum), and Arabidopsis (Yoneyama et al., 2007; López-Ráez et al., 2008; Umehara et al., 2008, 2010; Kohlen et al., 2011), and possibly increase lateral root formation (Ruyter-Spira et al., 2011). This makes sense physiologically, diverting resources to roots from shoots to scavenge more nutrients. The basis for modulation of SL levels or nutrient-dependent branching control is not understood.Here, we report a novel gene, GAT1_2.1 (At1g15040), predicted to encode a class I Gln amidotransferase (GAT1) in Arabidopsis, is highly repressed by long-term nitrogen stress (down 57-fold), and that mutation of this gene leads to an enhanced branching phenotype. Thus, this gene may present a link between the nitrogen stress response and branching control.