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Gokhan Kir Huaxun Ye Hilde Nelissen Anjanasree K. Neelakandan Andree S. Kusnandar Anding Luo Dirk Inzé Anne W. Sylvester Yanhai Yin Philip W. Becraft 《Plant physiology》2015,169(1):826-839
Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.Brassinosteroids (BRs) are ubiquitous plant hormones that promote plant growth by regulating cell elongation and division (Clouse, 1996; Clouse et al., 1996). BRs have other diverse roles, including enhancing tracheary element differentiation, stimulating ATPase activity, controlling microtubule orientation, and controlling flowering time, fertility, and leaf development (Iwasaki and Shibaoka, 1991; Clouse et al., 1996; Li et al., 1996; Schumacher et al., 1999; Catterou et al., 2001; Oh et al., 2011). BRs also function in tolerance to both biotic and abiotic stresses such as extreme temperatures, drought, and pathogens (Krishna, 2003).Deficiencies in BR biosynthesis or signaling produce characteristic dwarf plant phenotypes (Clouse et al., 1996; Szekeres et al., 1996; Fujioka et al., 1997). Plant height is an important agricultural trait, as seen in the Green Revolution, where semidwarf mutants contributed to increased yields in small-grain crops (Salas Fernandez et al., 2009). BR-deficient dwarf rice (Oryza sativa) produced increased grain and biomass yields because the erect leaf habit allowed higher planting densities under field conditions (Sakamoto et al., 2006). In fact, Green Revolution Uzu barley (Hordeum vulgare) is based on a mutation of the UZU1 gene, which encodes a homolog of BRASSINOSTEROID INSENSITIVE1 (BRI1), a BR receptor (Chono et al., 2003).Genes functioning in BR pathways have been identified by the analysis of dwarf mutants in several species, including Arabidopsis (Arabidopsis thaliana) and rice. Arabidopsis bri1 mutants are shortened, have reduced apical dominance, and are male sterile (Clouse et al., 1996). BRI1 encodes a Leu-rich repeat (LRR) receptor-like kinase that is located in the plasma membrane and contains an extracellular domain responsible for BR binding, a transmembrane sequence, and a cytoplasmic protein kinase domain (Li and Chory, 1997; Vert et al., 2005; Belkhadir and Chory, 2006). The island domain and subsequent LRR 22 are critical for BR binding (Kinoshita et al., 2005; Hothorn et al., 2011; She et al., 2011). Phosphorylation of the conserved residues Ser-1044 and Thr-1049 in the kinase activation loop activates the BRI1 kinase (Wang et al., 2005), while dephosphorylation of BRI1 by PROTEIN PHOSPHATASE2A inhibits its function (Wu et al., 2011).BRI1 is partially redundant in BR signaling with related BRASSINOSTEROID INSENSITIVE1-LIKE RECEPTOR KINASE (BRL) paralogs, both in Arabidopsis and rice. In Arabidopsis, even though null alleles of brl1 or brl3 did not show obvious phenotypic defects in shoots, they enhanced the developmental defects of a weak bri1-5 mutant. In contrast to ubiquitously expressed BRI1, BRL1, BRL2, and BRL3 are tissue specific, mostly expressed in vascular tissues, while BRL1 and BRL3 are also expressed in root apices (Caño-Delgado et al., 2004; Zhou et al., 2004; Fàbregas et al., 2013). Both BRL1 and BRL3 can bind brassinolide (BL; Caño-Delgado et al., 2004). In rice, OsBRI1 is similar to the Arabidopsis BRI1 gene, and phenotypes of OsBRI1 rice mutants include dwarf plants with shortened internodes, erect leaves that are twisted and dark green, and photomorphogenesis in the dark (Yamamuro et al., 2000). There are three BR receptors in rice as well, and while OsBRI1 is universally expressed in all organs, OsBRL1 and OsBRL3 are expressed mostly in roots (Nakamura et al., 2006).To date, two mutant genes of the BR biosynthetic pathway have been reported in maize (Zea
mays). A classic dwarf mutant, nana plant1 (na1), has a mutation in a DE-ETIOLATED2 homologous gene, which encodes a 5α-reductase enzyme in the BR biosynthesis pathway (Hartwig et al., 2011), while the brassinosteroid-dependent1 (brd1) gene encodes brassinosteroid C-6 oxidase (Makarevitch et al., 2012). The maize BR-deficient mutants have shortened internodes, twisted, dark green, erect leaves, and feminized male flowers (Hartwig et al., 2011; Makarevitch et al., 2012). However, no genes in BR signaling have yet been reported in maize. Understanding BR signaling in maize might help improve this important crop for the production of biofuels, biomass, and grain yield. Here, we took a transgenic RNA interference (RNAi) approach to generate maize plants partially deficient for BRI1. These knockdown lines demonstrate that BRI1 functions are generally conserved in maize compared with other plant species, but they also exhibit unique phenotypes, suggesting either that maize possesses novel BR-regulated developmental processes or that aspects of maize morphology reveal processes not evident in other plants. 相似文献
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Norman B. Best Thomas Hartwig Josh Budka Shozo Fujioka Gurmukh Johal Burkhard Schulz Brian P. Dilkes 《Plant physiology》2016,171(4):2633-2647
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Identification and Functional Analysis of Tomato BRI1 and BAK1 Receptor Kinase Phosphorylation Sites
Vikramjit S. Bajwa Xiaofeng Wang R. Kevin Blackburn Michael B. Goshe Srijeet K. Mitra Elisabeth L. Williams Gerard J. Bishop Sergei Krasnyanski George Allen Steven C. Huber Steven D. Clouse 《Plant physiology》2013,163(1):30-42
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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. 相似文献
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Ana Domínguez-Ferreras Marta Kiss-Papp Anna Kristina Jehle Georg Felix Delphine Chinchilla 《Plant physiology》2015,168(3):1106-1121
The membrane-bound BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) is a common coreceptor in plants and regulates distinct cellular programs ranging from growth and development to defense against pathogens. BAK1 functions through binding to ligand-stimulated transmembrane receptors and activating their kinase domains via transphosphorylation. In the absence of microbes, BAK1 activity may be suppressed by different mechanisms, like interaction with the regulatory BIR (for BAK1-INTERACTING RECEPTOR-LIKE KINASE) proteins. Here, we demonstrated that BAK1 overexpression in Arabidopsis (Arabidopsis thaliana) could cause detrimental effects on plant development, including growth arrest, leaf necrosis, and reduced seed production. Further analysis using an inducible expression system showed that BAK1 accumulation quickly stimulated immune responses, even under axenic conditions, and led to increased resistance to pathogenic Pseudomonas syringae pv tomato DC3000. Intriguingly, our study also revealed that the plasma membrane-associated BAK1 ectodomain was sufficient to induce autoimmunity, indicating a novel mode of action for BAK1 in immunity control. We postulate that an excess of BAK1 or its ectodomain could trigger immune receptor activation in the absence of microbes through unbalancing regulatory interactions, including those with BIRs. Consistently, mutation of SUPPRESSOR OF BIR1-1, which encodes an emerging positive regulator of transmembrane receptors in plants, suppressed the effects of BAK1 overexpression. In conclusion, our findings unravel a new role for the BAK1 ectodomain in the tight regulation of Arabidopsis immune receptors necessary to avoid inappropriate activation of immunity.Plants rely on their innate immune system to detect microbes and mount an active defense against pathogens. The plant immune system is traditionally considered to be composed of two layers (Jones and Dangl, 2006). The first one is based on the activity of pattern-recognition receptors (PRRs) that can detect microbe-associated molecular patterns (MAMPs) and trigger what is termed pattern-triggered immunity (PTI; Boller and Felix, 2009). Many plant pathogens can suppress this basal defense response using virulence factors termed effectors. In a second layer of defense, plants can make use of resistance (R) proteins to recognize the presence of pathogen effectors resulting in effector-triggered immunity (ETI), which resembles an accelerated and amplified PTI response (Jones and Dangl, 2006).Plants utilize plasma membrane-associated receptor-like proteins (RLPs) or receptor-like kinases (RLKs) as PRRs to sense specific signals through their ectodomains (Böhm et al., 2014). RLPs and RLKs require the function of additional RLKs to form active receptor complexes and transfer the external signal to the inside of the cells (Zhang and Thomma, 2013; Cao et al., 2014; Liebrand et al., 2014). The best-known coreceptor is the leucine-rich repeat (LRR)-RLK BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1), which was originally identified as a positive regulator and partner for the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1 (BRI1; Li et al., 2002; Nam and Li, 2002). BRs refer to phytohormones that promote plant growth and development (Fujioka and Yokota, 2003). Thus, loss-of-function mutations in BAK1 negatively impact Arabidopsis (Arabidopsis thaliana) growth due to improper cell elongation. In short, bak1 mutants display compact rosettes with round-shaped leaves and shorter petioles and phenocopy weak bri1 mutations (Li et al., 2002; Nam and Li, 2002). Conversely, certain mutants affected in the BAK1 ectodomain show increased activity in the BR signaling pathway and share phenotypic similarities with BRI1-overexpressing lines (Wang et al., 2001), including elongated hypocotyls, petioles, and leaf blades and an overall increase in height (Jaillais et al., 2011; Chung et al., 2012).Furthermore, BAK1 is involved in the containment of cell death, independently of its function in BR signaling. Arabidopsis bak1 knockout mutants exhibit extensive cell death spreading after microbial infection (Kemmerling et al., 2007). In addition, spontaneous cell death develops in Arabidopsis double mutant plants lacking both BAK1 (also named SOMATIC EMBRYOGENESIS RECEPTOR KINASE3 [SERK3]) and its closest homolog BAK1-LIKE1 (BKK1)/SERK4, causing seedling lethality even in the absence of microbes (He et al., 2007). Similar phenotypes are observed in Arabidopsis, rice (Oryza sativa), and Nicotiana benthamiana by lowering the expression of BAK1 and its homologs (Heese et al., 2007; Jeong et al., 2010; Park et al., 2011). Interestingly, typical defense responses, like the production of reactive oxygen species and constitutive callose deposition, are also detected in those plants, although the basis for this phenomenon remains poorly understood (He et al., 2007; Kemmerling et al., 2007; Park et al., 2011; Gao et al., 2013).On the other hand, BAK1 is widely studied as a key component of immune signaling pathways due to its known association with different PRRs, including RLKs and RLPs (Kim et al., 2013; Böhm et al., 2014). Upon MAMP perception, PRRs induce signaling and physiological defense responses like mitogen-activated protein kinase (MAPK) activation, reactive oxygen species and ethylene production, and modifications in gene expression, all of which contribute to PTI. Among the best-studied examples of BAK1-regulated PRRs are two LRR-receptor kinases, ELONGATION FACTOR Tu RECEPTOR (EFR), which senses the active epitope elf18 of the bacterial elongation factor Tu, and the flagellin receptor FLAGELLIN SENSING2 (FLS2), which senses the active epitope flg22 of bacterial flagellin (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2006; Zipfel et al., 2006). Immediately after flg22 binding to its LRR ectodomain, FLS2 forms a tight complex with BAK1 (Chinchilla et al., 2007; Sun et al., 2013). This heteromerization step may bring the two kinase domains closer and thereby induce, within seconds, the phosphorylation of BAK1 and FLS2 (Schulze et al., 2010; Schwessinger et al., 2011). These steps are sufficient to initiate the immune signaling pathway, even if the ectodomains and kinase domains are switched between FLS2 and BAK1 (Albert et al., 2013).While PRRs, such as FLS2 and EFR, are extremely sensitive to even subnanomolar concentrations of their ligands, a tight control of these receptors is expected, since constitutive activation of defense responses in plants dramatically impairs fitness and growth (Tian et al., 2003; Korves and Bergelson, 2004). However, the mechanisms that underlie the attenuation of PRR activation or prevent these receptors from signaling constitutively remain largely unknown (Macho and Zipfel, 2014). Several independent observations indicate that BAK1 and FLS2 are present in close spatial proximity in preformed complexes at the plasma membrane (Chinchilla et al., 2007; Schulze et al., 2010; Roux et al., 2011). Negative regulation of immune signaling prior to ligand perception could happen within the PRR complex and depend on conformational changes following the association of FLS2 with flg22 (Meindl et al., 2000; Schulze et al., 2010; Mueller et al., 2012). Additionally, other partners might prevent the constitutive interaction of BAK1 with FLS2. Such could be the case for the LRR-RLK BAK1-INTERACTING RECEPTOR-LIKE KINASEs (BIRs): BIR2 was recently discovered as a substrate and negative regulator for BAK1, while the absence of BIR1 leads to the activation of defense induction and strong dwarfism (Gao et al., 2009; Halter et al., 2014b). Furthermore, MAMP signaling may be constrained by phosphatases, as suggested in earlier studies (Felix et al., 1994; Gómez-Gómez et al., 2001) and recently shown for the protein phosphatase 2A, which controls PRR activation likely by modulating the BAK1 phosphostatus (Segonzac et al., 2014). These examples illustrate the variety of mechanisms that may tightly control BAK1 activity.In this work, we show that regulation of BAK1 accumulation is crucial for Arabidopsis fitness, as its overexpression leads to dwarfism and premature death. The phenotype differs from BR mutants and is very reminiscent of or even identical to the autoimmune phenotype of plants showing constitutive activation of R proteins (Oldroyd and Staskawicz, 1998; Bendahmane et al., 2002; Zhang et al., 2003). BAK1 overexpression is associated with constitutive activation of defense pathway(s) involving the general coregulator of RLPs, SUPPRESSOR OF BIR1-1 (SOBIR1; Liebrand et al., 2013, 2014). To our knowledge, this is the first report and comprehensive characterization of such an autoimmunity phenotype for Arabidopsis plants overexpressing BAK1, and it highlights the importance of the regulation of PTI overactivation. 相似文献
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Tianyuan Yang Song Zhang Yibing Hu Fachi Wu Qingdi Hu Guang Chen Jing Cai Ting Wu Nava Moran Ling Yu Guohua Xu 《Plant physiology》2014,166(2):945-959
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. 相似文献
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Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein, we present a simple, material and cost-saving high-performance method for determining endogenous brassinosteroids (BRs) in model plants. This new method enables simultaneous enrichment of a wide range of bioactive BRs such as brassinolide, castasterone, teasterone, and typhasterol with ion exchange solid-phase extraction and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to one-twentieth of previously reported and the overall process could be completed within 1 day compared with previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotypes and mutants of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the BR distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1 gram fresh weight of model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work.Brassinosteroids (BRs) have been considered as the sixth class of endogenous plant hormones with wide occurrence across the plant kingdom (Bajguz and Tretyn, 2003). BRs play a key role in a variety of physiological processes, such as cell elongation, vascular differentiation, reproductive development, photomorphogenesis, stress tolerance, and so on (Hayat, 2010). Recently, it was found that BR deficiency could increase grain yield in rice (Oryza sativa) by more than 30%, which showed a food security-enhancing potential and guided new green revolution in the future (Sakamoto et al., 2006; Wu et al., 2008). Since BRs were first isolated and identified from rape (Brassica napus) pollen in 1970s (Mitchell et al., 1970; Grove et al., 1979), the natural occurrence of more than 60 BRs in a large quantity of plant species has been reported (Hayat, 2010). To date, research on the occurrence of BRs in different plants, physiological properties, and their action modes has made much progress (Fujioka and Yokota, 2003; Symons et al., 2008; Kim and Wang, 2010; Tang et al., 2010; Tong and Chu, 2012). However, so far, only limited information was obtained to understand the molecular mechanism of the physiological role of BRs. For example, although the biosynthetic pathway of C28
BRs has been well established, the biosynthesis of C27 and C29
BRs remains unclear, and some intermediates on their biosynthetic pathways still need to be elucidated (Noguchi et al., 2000; Fujita et al., 2006). The plant physiology research of BRs is speeded up by employing BR mutants on biosynthesis and signaling pathways (Yamamuro et al., 2000; Hong et al., 2003; Kwon and Choe, 2005; Tanabe et al., 2005); however, a simple, high-sensitivity screening, detection, and quantification method for BR analysis is still a bottleneck technique for in-depth studying of the role of BRs during the life cycle of plants.In the past 20 years, most of the detection and identification processes of BRs could be described briefly as the following steps. The harvested plant materials were lyophilized and then ground to a fine powder, followed by the CH3OH/CHCl3 extraction. The concentrate was then partitioned with the CHCl3/H2O system three times. After that, the CHCl3 fraction was subjected to a silica gel column for BR enrichment, and the collected BRs-containing fraction was purified with Sephadex LH-20 column and Sep-Pak Plus C18 cartridge in sequence. At last, the collected fractions were purified with preparative HPLC and then derivatized for analysis with gas chromatography-mass spectrometry (MS) under selected ion monitoring mode (Hong et al., 2005; Nomura et al., 2005; Kim et al., 2006). So far, this protocol has been proven to be workable in most cases and provided a great quantity of valuable data for plant physiological research (Hwang et al., 2006; Lee et al., 2010). However, at least more than 20 g of plant materials were consumed for quantifying/identifying BRs in one plant sample without replicates (Hong et al., 2005; Bancos et al., 2006; Kim et al., 2006), and it is difficult to collect sufficient plant tissues for BR measurement in some rare model plant mutants. In addition, the method involved multiple tedious and labor-intensive steps, which might result in poor recovery and low sensitivity, especially for some labile BR intermediates. The traditional method took one person about 3 to 4 d to treat one batch of samples. Most of the BR measurement experiments were performed without biological replicates using traditional methods due to the disadvantages mentioned above, which discounted the reliability of the results. Therefore, a simple, rapid, and sensitive analysis method for BRs is in urgent need, along with the development of BR research.Recently, several efforts were made to improve the BR determination (Svatos et al., 2004; Huo et al., 2012). The consumption of plant material was reduced to 2 g after modifying the BRs with a new derivatization reagent for further ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-MS detection. However, the purification process consisting of deproteinization and multiple solid-phase extraction (SPE) steps was still quite tedious and couldn’t guarantee covering the four most important bioactive BRs, including brassinolide (BL), castasterone (CS), teasterone (TE), and typhasterol (TY; Fig. 1). In our previous study (Xin et al., 2013), we reported a simple, convenient, and high-sensitivity method for detection of endogenous BRs from real plant materials based on the dual role of specific boronate affinity. Although it was the first time to measure multiple BRs in subgram plant materials and the time duration of the method decreased to one-third of that previously reported, the synthesis of boronate affinity-functionalized magnetic nanoparticles made the method difficult to follow in biological laboratories for routine analysis.Open in a separate windowFigure 1.Chemical structure of four major bioactive BRs.BRs are neutral steroid compounds with a common four-ring cholestane skeleton and hydroxyl groups on A ring and/or the side chain linked to D ring. Especially, the vicinal diol moieties on C22 and C23 sites of BL, CS, TY, and TE allow these bioactive BRs to be derivatized with ionizable reagents for MS response enhancement. Considering the unique physicochemical properties of BRs, we herein developed a simplified high-sensitivity analytical method based on mixed-mode anion exchange (MAX)-cation exchange (MCX) solid phase extraction (SPE) purification, vicinal diol derivatization combined with UPLC-MRM3-MS detection for quantification of BL, CS, TE, and TY in model plants (Fig. 2). The performance of the method was demonstrated by determination of BRs in different tissues of both wild-type and mutant Arabidopsis (Arabidopsis thaliana) and rice.Open in a separate windowFigure 2.Simplified high-sensitivity protocol for quantitative analysis of BRs. IS, Internal standards. [See online article for color version of this figure.] 相似文献
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Jia-Gang Wang Sha Li Xin-Ying Zhao Liang-Zi Zhou Guo-Qiang Huang Chong Feng Yan Zhang 《Plant physiology》2013,162(4):1897-1910
In plant cells, secretory and endocytic routes intersect at the trans-Golgi network (TGN)/early endosome (EE), where cargos are further sorted correctly and in a timely manner. Cargo sorting is essential for plant survival and therefore necessitates complex molecular machinery. Adaptor proteins (APs) play key roles in this process by recruiting coat proteins and selecting cargos for different vesicle carriers. The µ1 subunit of AP-1 in Arabidopsis (Arabidopsis thaliana) was recently identified at the TGN/EE and shown to be essential for cytokinesis. However, little was known about other cellular activities affected by mutations in AP-1 or the developmental consequences of such mutations. We report here that HAPLESS13 (HAP13), the Arabidopsis µ1 adaptin, is essential for protein sorting at the TGN/EE. Functional loss of HAP13 displayed pleiotropic developmental defects, some of which were suggestive of disrupted auxin signaling. Consistent with this, the asymmetric localization of PIN-FORMED2 (PIN2), an auxin transporter, was compromised in the mutant. In addition, cell morphogenesis was disrupted. We further demonstrate that HAP13 is critical for brefeldin A-sensitive but wortmannin-insensitive post-Golgi trafficking. Our results show that HAP13 is a key link in the sophisticated trafficking network in plant cells.Plant cells contain sophisticated endomembrane compartments, including the endoplasmic reticulum, the Golgi, the trans-Golgi network (TGN)/early endosome (EE), the prevacuolar compartments/multivesicular bodies (PVC/MVB), various types of vesicles, and the plasma membrane (PM; Ebine and Ueda, 2009; Richter et al., 2009). Intracellular protein sorting between the various locations in the endomembrane system occurs in both secretory and endocytic routes (Richter et al., 2009; De Marcos Lousa et al., 2012). Vesicles in the secretory route start at the endoplasmic reticulum, passing through the Golgi before reaching the TGN/EE, while vesicles in the endocytic route start from the PM before reaching the TGN/EE (Dhonukshe et al., 2007; Viotti et al., 2010). The TGN/EE in Arabidopsis (Arabidopsis thaliana) is an independent and highly dynamic organelle transiently associated with the Golgi (Dettmer et al., 2006; Lam et al., 2007; Viotti et al., 2010), distinct from the animal TGN. Once reaching the TGN/EE, proteins delivered by their vesicle carriers are subject to further sorting, being incorporated either into vesicles that pass through the PVC/MVB before reaching the vacuole for degradation or into vesicles that enter the secretory pathway for delivery to the PM (Ebine and Ueda, 2009; Richter et al., 2009). Therefore, the TGN/EE is a critical sorting compartment that lies at the intersection of the secretory and endocytic routes.Fine-tuned control of intracellular protein sorting at the TGN/EE is essential for plant development (Geldner et al., 2003; Dhonukshe et al., 2007, 2008; Richter et al., 2007; Kitakura et al., 2011; Wang et al., 2013). An auxin gradient is crucial for pattern formation in plants, whose dynamic maintenance requires the polar localization of auxin efflux carrier PINs through endocytic recycling (Geldner et al., 2003; Blilou et al., 2005; Paciorek et al., 2005; Abas et al., 2006; Jaillais et al., 2006; Dhonukshe et al., 2007; Kleine-Vehn et al., 2008). Receptor-like kinases (RLKs) have also been recognized as major cargos undergoing endocytic trafficking, which are either recycled back to the PM or sent for vacuolar degradation (Geldner and Robatzek, 2008; Irani and Russinova, 2009). RLKs are involved in most if not all developmental processes of plants (De Smet et al., 2009).Intracellular protein sorting relies on sorting signals within cargo proteins and on the molecular machinery that recognizes sorting signals (Boehm and Bonifacino, 2001; Robinson, 2004; Dhonukshe et al., 2007). Adaptor proteins (AP) play a key role (Boehm and Bonifacino, 2001; Robinson, 2004) in the recognition of sorting signals. APs are heterotetrameric protein complexes composed of two large subunits (β and γ/α/δ/ε), a small subunit (σ), and a medium subunit (µ) that is crucial for cargo selection (Boehm and Bonifacino, 2001). APs associate with the cytoplasmic side of secretory and endocytic vesicles, recruiting coat proteins and recognizing sorting signals within cargo proteins for their incorporation into vesicle carriers (Boehm and Bonifacino, 2001). Five APs have been identified so far, classified by their components, subcellular localization, and function (Boehm and Bonifacino, 2001; Robinson, 2004; Hirst et al., 2011). Of the five APs, AP-1 associates with the TGN or recycling endosomes (RE) in yeast and mammals (Huang et al., 2001; Robinson, 2004), mediating the sorting of cargo proteins to compartments of the endosomal-lysosomal system or to the basolateral PM of polarized epithelial cells (Gonzalez and Rodriguez-Boulan, 2009). Knockouts of AP-1 components in multicellular organisms resulted in embryonic lethality (Boehm and Bonifacino, 2001; Robinson, 2004).We show here that the recently identified Arabidopsis µ1 adaptin AP1M2 (Park et al., 2013; Teh et al., 2013) is a key component in the cellular machinery mediating intracellular protein sorting at the TGN/EE. AP1M2 was previously named HAPLESS13 (HAP13), whose mutant allele hap13 showed male gametophytic lethality (Johnson et al., 2004). In recent quests for AP-1 in plants, HAP13/AP1M2 was confirmed as the Arabidopsis µ1 adaptin based on its interaction with other components of the AP-1 complex as well as its localization at the TGN (Park et al., 2013; Teh et al., 2013). A novel mutant allele of HAP13/AP1M2, ap1m2-1, was found to be defective in the intracellular distribution of KNOLLE, leading to defective cytokinesis (Park et al., 2013; Teh et al., 2013). However, it was not clear whether HAP13/AP1M2 mediated other cellular activities and their developmental consequences. Using the same mutant allele, we found that functional loss of HAP13 (hap13-1/ap1m2-1) resulted in a full spectrum of growth defects, suggestive of compromised auxin signaling and of defective RLK signaling. Cell morphogenesis was also disturbed in hap13-1. Importantly, hap13-1 was insensitive to brefeldin A (BFA) washout, indicative of defects in guanine nucleotide exchange factors for ADP-ribosylation factor (ArfGEF)-mediated post-Golgi trafficking. Furthermore, HAP13/AP1M2 showed evolutionarily conserved function during vacuolar fusion, providing additional support to its identity as a µ1 adaptin. These results demonstrate the importance of the Arabidopsis µ1 adaptin for intracellular protein sorting centered on the TGN/EE. 相似文献
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