Iron Is Involved in the Maintenance of Circadian Period Length in Arabidopsis

The homeostasis of iron (Fe) in plants is strictly regulated to maintain an optimal level for plant growth and development but not cause oxidative stress. About 30% of arable land is considered Fe deficient because of calcareous soil that renders Fe unavailable to plants. Under Fe-deficient conditions, Arabidopsis (Arabidopsis thaliana) shows retarded growth, disordered chloroplast development, and delayed flowering time. In this study, we explored the possible connection between Fe availability and the circadian clock in growth and development. Circadian period length in Arabidopsis was longer under Fe-deficient conditions, but the lengthened period was not regulated by the canonical Fe-deficiency signaling pathway involving nitric oxide. However, plants with impaired chloroplast function showed long circadian periods. Fe deficiency and impaired chloroplast function combined did not show additive effects on the circadian period, which suggests that plastid-to-nucleus retrograde signaling is involved in the lengthening of circadian period under Fe deficiency. Expression pattern analyses of the central oscillator genes in mutants defective in CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL or GIGANTEA demonstrated their requirement for Fe deficiency-induced long circadian period. In conclusion, Fe is involved in maintaining the period length of circadian rhythm, possibly by acting on specific central oscillators through a retrograde signaling pathway.Metals such as iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), molybdenum, and nickel are essential for the various biological processes that govern plant growth and development (Marschner, 1995). For example, Fe is required for DNA synthesis, photosynthesis, nitrogen fixation, hormone synthesis, and electron transport in the respiratory chain (Briat and Lobreaux, 1997). Similarly, Cu is an important component of electron-transfer reactions mediated by proteins such as superoxide dismutase, cytochrome oxidase, and plastocyanin (Clemens, 2001). Zn is a cofactor for many enzymes, and many proteins contain Zn-binding structural domains (Clarke and Berg, 1998). Although only minimal quantities of these micronutrients are required by plants, their limited availability in soils can significantly hinder crop production and affect nutritional quality (Grotz and Guerinot, 2002). In the case of Fe, about 30% of arable land worldwide is considered calcareous, rendering Fe in these soils unavailable to plants (Mori, 1999). Understanding of the fundamental processes involving metal uptake and sequestration has increased in recent years, but how the availability of particular metals interacts with internal signals to govern the growth and development of plants is largely unknown.The daily biological rhythms of many organisms are regulated by a near 24-h circadian clock that is synchronized by environmental changes such as light and temperature (Harmer, 2009; Imaizumi, 2010). The circadian clock regulates diverse aspects of plant growth and development. The operation of the circadian clock in plants can basically be divided into three main parts, input, central oscillator, and output pathways, and each part has its own complex networks. In Arabidopsis (Arabidopsis thaliana), the central oscillator is composed of a network of multiple feedback loops that can be divided into the morning, central, and evening loops (Harmer, 2009). The central feedback loop is composed of the morning-expressed genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the evening-expressed gene TIMING OF CAB EXPRESSION1 (TOC1; Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000; Alabadí et al., 2001). Although TOC1 is genetically required for the activation of morning genes (Schaffer et al., 1998; Wang and Tobin, 1998; Strayer et al., 2000), it acts as a repressor and directly regulates the expression of CCA1 and LHY (Gendron et al., 2012; Huang et al., 2012; Pokhilko et al., 2012). In the morning loop, CCA1/LHY form another negative feedback loop with the morning genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9, with PRR9/PRR7 directly repressing the expression of CCA1 and LHY (Farré et al., 2005; Nakamichi et al., 2010). In the evening loop, TOC1 forms a negative feedback loop with GIGANTEA (GI) by repressing its expression, and GI in turn activates the expression of TOC1 through an unknown component, Y (Huq et al., 2000; Mizoguchi et al., 2005). After receiving input signals in the form of environmental cues, the central oscillator of the Arabidopsis circadian clock generates various rhythmic outputs that control various physiological events (Hotta et al., 2007; de Montaigu et al., 2010).The central oscillator controls a range of important physiological output processes such as flowering, stress and hormone responses, and regulation of nutrient acquisition (Haydon et al., 2011). Although the uptake of nutrition in plants is known to be influenced by light and temperature (Lahti et al., 2005; Baligar et al., 2006), the interaction between nutritional status and the circadian clock is less well studied. The homeostasis of Cu is known to influence the regulation of oscillator genes (Andrés-Colás et al., 2010; Peñarrubia et al., 2010). Arabidopsis under excess Cu or overexpressing Cu transporters COPT1 and COPT3 showed increased Cu accumulation and reduced expression of CCA1, LHY, and circadian clock output genes. Defective developmental phenotypes were also observed in these plants. Spatial and temporal control of Cu homeostasis, therefore, may be important for plant environmental fitness (Andrés-Colás et al., 2010). It has also been reported that disordered circadian rhythm affects Fe homeostasis. Tight regulation of Fe homeostasis to maintain an optimal Fe level in plants has been found to be associated with circadian clock regulators such as TIME FOR COFFEE (TIC) that modulates the expression of the ferritin gene AtFer1 (Duc et al., 2009). The expression of AtFer1 was up-regulated with excess Fe. TIC could repress AtFer1 expression under low-Fe conditions in photoperiodic light and dark cycles (Duc et al., 2009). However, whether Fe status feeds back to regulate the circadian clock is uncertain.Although Fe homeostasis in terms of uptake and translocation has been studied for decades, Fe availability is still an agricultural problem worldwide. Revealing the interplay between Fe homeostasis and internal cues such as modulation of the circadian clock can help increase understanding of their contributions to overall plant development. In this work, we investigated the effect of Fe deficiency on the circadian clock and found that it lengthened the circadian period. Our data suggest that the functional status of chloroplasts under Fe deficiency may play a key role in the lengthened circadian period.     

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.     

Putrescine Alleviates Iron Deficiency via NO-Dependent Reutilization of Root Cell-Wall Fe in Arabidopsis

Plants challenged with abiotic stress show enhanced polyamines levels. Here, we show that the polyamine putrescine (Put) plays an important role to alleviate Fe deficiency. The adc2-1 mutant, which is defective in Put biosynthesis, was hypersensitive to Fe deficiency compared with wild type (Col-1 of Arabidopsis [Arabidopsis thaliana]). Exogenous Put decreased the Fe bound to root cell wall, especially to hemicellulose, and increased root and shoot soluble Fe content, thus alleviating the Fe deficiency-induced chlorosis. Intriguingly, exogenous Put induced the accumulation of nitric oxide (NO) under both Fe-sufficient (+Fe) and Fe-deficient (-Fe) conditions, although the ferric-chelate reductase (FCR) activity and the expression of genes related to Fe uptake were induced only under -Fe treatment. The alleviation of Fe deficiency by Put was diminished in the hemicellulose-level decreased mutant-xth31 and in the noa1 and nia1nia2 mutants, in which the endogenous NO levels are reduced, indicating that both NO and hemicellulose are involved in Put-mediated alleviation of Fe deficiency. However, the FCR activity and the expression of genes related to Fe uptake were still up-regulated under -Fe+Put treatment compared with -Fe treatment in xth31, and Put-induced cell wall Fe remobilization was abolished in noa1 and nia1nia2, indicating that Put-regulated cell wall Fe reutilization is dependent on NO. From our results, we conclude that Put is involved in the remobilization of Fe from root cell wall hemicellulose in a process dependent on NO accumulation under Fe-deficient condition in Arabidopsis.Iron is an essential element for plant growth and development, and iron deficiency is the most common micronutrient deficiency in the world. To cope with iron deficiency, plants have evolved two distinct mechanisms for Fe acquisition from the rhizosphere. Strategy I, found in all dicots and monocots with the exception of graminaceous species, is characterized by (1) release of protons to acidify the rhizosphere, which is mediated in Arabidopsis (Arabidopsis thaliana) by the proton-translocating ATPase AHA2 (ARABIDOPSIS PLASMA MEMBRANE H⁺-ATPASE ISOFORM 2; Curie and Briat, 2003; Santi and Schmidt, 2009); (2) inducing ferric chelate reductase activity mediated by FRO2 (FERRIC REDUCTASE OXIDASE2; Robinson et al., 1999); and (3) uptake of Fe²⁺ by the metal transporter IRT1 (IRON REGULATED TRANSPORTER1; Eide et al., 1996; Vert et al., 2002). Strategy II, utilized by graminaceous monocots (Römheld and Marschner, 1986), is characterized by enhanced release of phytosiderophores that form chelates with Fe(III) (Curie and Briat, 2003). However, in addition to Fe acquisition, the mechanisms underlying the mobilization of Fe(III) also are a major challenge for us to understand.Recently, accumulating evidence has shown that phenolic compounds are important for iron mobilization. Rodríguez-Celma et al. (2013) showed that secretion of phenolics is critical for Arabidopsis Fe acquisition from low bioavailability sources, and then Fourcroy et al. (2014) and Schmidt et al. (2014) demonstrated that coumarins are the active compounds in this process. Schmid et al. (2014) confirmed that secretion of coumarins is an essential aspect of Arabidopsis Fe acquisition and provided extensive information on metabolomic changes elicited by Fe deficiency. However, under certain conditions Fe is not readily available, and Fe is difficult to mobilize; thus, Fe stored in the plant needs to be reutilized. For example, phenolics are secreted to remobilize the root apoplastic Fe and improve Fe nutrition in red clover (Trifolium pratense) and rice (Oryza sativa) (Jin et al., 2007; Bashir et al., 2011). Moreover, Lei et al. (2014) reported that the cell wall can be an important Fe source during periods of limited Fe supply. As the first barrier to encounter the soil environment, the cell wall is a pivotal site for most cationic ions in plants (Lozano-Rodríguez et al., 1997; Carrier et al., 2003). Hemicellulose contributes to the overall Al/Cd accumulation in the cell wall of Arabidopsis (Zhu et al., 2012, 2013) and also acts as a Fe pool (Lei et al., 2014). Over 75% of Fe in the root is retained in the cell wall (Bienfait et al., 1985), especially in the hemicellulose fraction (Lei et al., 2014). Thus, the cell wall is not only a site to immobilize an element and restrict its entrance into the cell, but also can serve as a pool to provide the nutrient when the supply from the growth medium is limited. However, the upstream mechanism of Fe reutilization through the cell wall, especially hemicellulose, is still far from clear.The responses to Fe deficiency in plants involve numerous phytohormones and signaling molecules, including auxin (Römheld and Marschner, 1981; Chen et al., 2010), ethylene (García et al., 2010; Wu et al., 2011), and NO (Graziano and Lamattina, 2007; Chen et al., 2010). Polyamines share common substrates with nitric oxide (NO) (Shi and Chan, 2014), and polyamines like spermidine and spermine rapidly induce a burst of NO in various plant species, indicating that NO is a potential intermediate of polyamine-mediated signaling.Polyamines, including putrescine (Put), spermidine, and spermine, are low M_r natural compounds with nitrogen-containing aliphatic structure and influence basic physiological and developmental events, such as cell division and differentiation, rhizogenesis, leaf senescence, zygotic, somatic embryogenesis, and development of flowers and fruits (Feirer et al., 1984; Galston et al., 1995; Bouchereau et al., 1999; Kakkar et al., 2000; Tun et al., 2001; Shi and Chan, 2014). The metabolism of polyamines in plant tissues is subject to strict regulation, and polyamine levels in plant roots change upon exposure to abiotic stress such as salt, drought, low and high temperature, heavy metals (Cu, Cr, Fe, and Ni), and oxidative stresses (Liu et al., 2005; Cheng et al., 2009; Wimalasekera et al., 2011; Tavladoraki et al., 2012).Ample evidence demonstrates the involvement of Put in responses to various types of abiotic stress, such as mineral deficiency in barley (Hordeum vulgare) leaves (Smith, 1973), high osmotic pressure in barley, corn, wheat, and wild oat leaves (Flores and Galston, 1982a), low pH in peeled oat (Avena sativa L. var Victory) leaf (Young and Galston, 1983), potassium deficiency in oat shoot and Arabidopsis thaliana (L.) Heynh (Young and Galston, 1984; Watson and Malmberg, 1996), and cadmium toxicity in oat and bean leaves (Weinstein et al., 1986). In animals, Put is produced either from Orn by Orn decarboxylase or from Arg by Arg decarboxylase (ADC) (Hanfrey et al., 2001). As there is no detectable Orn decarboxylase activity in Arabidopsis, the ADC route is critical for Put biosynthesis. Although there are two genes responsible for ADC activity, Urano et al. (2004) reported that the expression of ADC2 correlates well with the increment of free Put, indicating ADC2 plays an important role in Put biosynthesis in Arabidopsis. However, the role of Put under Fe deficiency in plants remains unknown.In this study, we found that Fe deficiency results in enhanced Put levels. Further, whereas exogenous Put alleviated Fe deficiency, the adc2-1 mutant, in which endogenous Put is decreased, exhibited a Fe deficiency-sensitive phenotype. We demonstrated that Put acts upstream of NO to decrease the Fe binding capacity of the cell wall, especially that of hemicellulose, thus resulting in greater Fe reutilization.     

Auxin Resistant1 and PIN-FORMED2 Protect Lateral Root Formation in Arabidopsis under Iron Stress

A stunted root system is a significant symptom of iron (Fe) toxicity, yet little is known about the effects of excess Fe on lateral root (LR) development. In this work, we show that excess Fe has different effects on LR development in different portions of the Arabidopsis (Arabidopsis thaliana) root system and that inhibitory effects on the LR initiation are only seen in roots newly formed during excess Fe exposure. We show that root tip contact with Fe is both necessary and sufficient for LR inhibition and that the auxin, but not abscisic acid, pathway is engaged centrally in the initial stages of excess Fe exposure. Furthermore, Fe stress significantly reduced PIN-FORMED2 (PIN2)-green fluorescent protein (GFP) expression in root tips, and pin2-1 mutants exhibited significantly fewer LR initiation events under excess Fe than the wild type. Exogenous application of both Fe and glutathione together increased PIN2-GFP expression and the number of LR initiation events compared with Fe treatment alone. The ethylene inhibitor aminoethoxyvinyl-glycine intensified Fe-dependent inhibition of LR formation in the wild type, and this inhibition was significantly reduced in the ethylene overproduction mutant ethylene overproducer1-1. We show that Auxin Resistant1 (AUX1) is a critical component in the mediation of endogenous ethylene effects on LR formation under excess Fe stress. Our findings demonstrate the relationship between excess Fe-dependent PIN2 expression and LR formation and the potential role of AUX1 in ethylene-mediated LR tolerance and suggest that AUX1 and PIN2 protect LR formation in Arabidopsis during the early stages of Fe stress.Iron (Fe) is an essential trace element for plants (Pilon et al., 2009), and species differ greatly in how much Fe they require for optimal growth (Wheeler and Power, 1995; Batty and Younger, 2003). As Fe is frequently limiting, Fe deficiency is more commonly studied than toxicity arising from excess Fe exposure (Lei et al., 2014; Bashir et al., 2015; Briat et al., 2015). Fe is also a major focus for efforts in biofortification by targeting Fe transporters (Zhai et al., 2014; Pinto and Ferreira, 2015). However, the excessive presence of Fe in soils is equally common, in particular in soils characterized by low pH and hypoxic or anoxic conditions (Connolly and Guerinot, 2002). Toxicity arising from excess Fe exposure is recognized as one of the major plant diseases attributable to abiotic factors that impact the development and yield potential in the world’s leading cereal crops, rice (Oryza sativa) and wheat (Triticum aestivum; Becker and Asch, 2005; Khabaz-Saberi et al., 2012). Understanding the mechanisms underlying excess Fe toxicity is therefore essential.Plastic responses in the plant’s root system architecture are known to constitute a major mechanism by which plants cope with fluctuating environments. Lateral roots (LRs), which typically comprise the majority of the root system, contribute pivotally to nutrient acquisition from soil, and modulating LR development is a very important avoidance strategy for plants when confronted with unfavorable edaphic conditions, such as high salinity or heavy metals (Ivanov et al., 2003). In the case of excess exposure to Fe, stunting of the root system is among the chief symptoms of toxicity (Becker and Asch, 2005). However, while some information has been emerging on the primary root axis (Li et al., 2015), the specific role of the plant’s LR apparatus remains poorly studied. Yamauchi and Peng (1995) reported retardation of root growth and a reduction in LR length and number under excess Fe conditions. Recently, Reyt et al. (2015) showed that excess Fe had no significant effect on LR initiation in the LR branching zone and that ferritins play an important role in LR emergence under excess Fe in this portion of the root, although the authors had not investigated LR development in the root portions near the growing tip of the primary root. Because LR initiation is restricted to specific pericycle cell files adjacent to a xylem pole in the basal region of the meristem (De Smet et al., 2007; Fukaki and Tasaka, 2009), and LR formation in this new growing root portion may be more susceptible to stress stimuli, such as observed with exposure to high NH₄⁺ and salt (Duan et al., 2013; Li et al., 2013), it is reasonable to suggest that modulation of LR formation near the growing tip of the primary root is critical to the response to excess Fe stress.In Arabidopsis (Arabidopsis thaliana), the development of LRs proceeds through the following stages: lateral root primordia (LRP) initiation, establishment, emergence, activation into mature LRs, and final maintenance of LR elongation (Fukaki and Tasaka, 2009; Péret et al., 2009). The hormones abscisic acid (ABA) and auxin are important internal negative and positive regulators during LR development, respectively (Fukaki and Tasaka, 2009). ABA has been implicated in LRP emergence and meristem activation independent of auxin (De Smet et al., 2003). Auxin is an important internal positive regulator during LR development (Fukaki and Tasaka, 2009), and auxin transport is critical (Blilou et al., 2005). Mutants in auxin efflux carriers such as PIN-FORMED (PIN) and P-Glycoprotein show significant defects in LR formation (Fukaki and Tasaka, 2009; Péret et al., 2009). For example, LR initiation frequency was significantly reduced in pin2 and pin3 mutants (Dubrovsky et al., 2009), and PIN2 was also shown to be involved in exogenous and endogenous signal-mediated LR development (by brassinosteroid, jasmonate, and fungal challenge; Li et al., 2005; Felten et al., 2009; Sun et al., 2009). Similarly, Auxin Resistant1 (AUX1), an auxin influx carrier, also regulates LRP positioning and initiation (De Smet et al., 2007). While both AUX1 and PIN2 are required specifically for the basipetal transport of auxin through the outer root cell layers (Fukaki and Tasaka, 2009), PIN1 localized at the basal end of vascular cells is responsible for direct acropetal auxin flow in the root stele (Blilou et al., 2005). Recently, the roles of ethylene on LR development have also been highlighted, and the ethylene-mediated LR formation is dependent on the auxin pathway (Ivanchenko et al., 2008; Lewis et al., 2011). Ethylene treatment could mediate fluorescence of AUX1 and PIN2 fluorescent protein fusions at the root tip (Růzicka et al., 2007; Lewis et al., 2011). Although ABA, auxin, and ethylene signals have been implicated as important for LR development, it is not known whether and how the three hormones are involved in the response of LR formation to Fe stress.The previously described phenotypes and physiological processes related to Fe toxicity do not clarify the effect of excess Fe on LR formation. In this study, we employed the Arabidopsis wild type and ABA-, auxin-, and ethylene-related mutants to explore the LR formation response to Fe toxicity and to elucidate the roles of ABA, auxin, and ethylene. Potential mechanisms involved in the early stress response to Fe stress are discussed.     

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.     

HAPLESS13, the Arabidopsis μ1 Adaptin,Is Essential for Protein Sorting at the trans-Golgi Network/Early Endosome

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.     

A Member of the Heavy Metal P-Type ATPase OsHMA5 Is Involved in Xylem Loading of Copper in Rice

Heavy metal-transporting P-type ATPase (HMA) has been implicated in the transport of heavy metals in plants. Here, we report the function and role of an uncharacterized member of HMA, OsHMA5 in rice (Oryza sativa). Knockout of OsHMA5 resulted in a decreased copper (Cu) concentration in the shoots but an increased Cu concentration in the roots at the vegetative stage. At the reproductive stage, the concentration of Cu in the brown rice was significantly lower in the mutants than in the wild-type rice; however, there was no difference in the concentrations of iron, manganese, and zinc between two independent mutants and the wild type. The Cu concentration of xylem sap was lower in the mutants than in the wild-type rice. OsHMA5 was mainly expressed in the roots at the vegetative stage but also in nodes, peduncle, rachis, and husk at the reproductive stage. The expression was up-regulated by excess Cu but not by the deficiency of Cu and other metals, including zinc, iron, and manganese, at the vegetative stage. Analysis of the transgenic rice carrying the OsHMA5 promoter fused with green fluorescent protein revealed that it was localized at the root pericycle cells and xylem region of diffuse vascular bundles in node I, vascular tissues of peduncle, rachis, and husk. Furthermore, immunostaining with an antibody against OsHMA5 revealed that it was localized to the plasma membrane. Expression of OsHMA5 in a Cu transport-defective mutant yeast (Saccharomyces cerevisiae) strain restored the growth. Taken together, OsHMA5 is involved in loading Cu to the xylem of the roots and other organs.Plants require nutrient elements to maintain normal growth and development. A number of different transporters, such as Cation Diffusion Facilitator, Natural resistance-associated macrophage protein, ATP-Binding Cassette, Zinc- and Iron-regulated-like Protein, and P-type ATPase, have been reported to be involved in the uptake, translocation, distribution, and homeostasis of nutrients (Hall and Williams, 2003; Krämer et al., 2007; Palmer and Guerinot, 2009). Among them, heavy metal-transporting P-type ATPase (HMA), the P_1B subfamily of the P-type ATPase superfamily, has been implicated in heavy metal transport (Williams and Mills, 2005; Grotz and Guerinot, 2006; Argüello et al., 2007; Burkhead et al., 2009). There are eight and nine members of P_1B-ATPase in Arabidopsis thaliana and rice (Oryza sativa), respectively (Williams and Mills, 2005). They are divided into two groups: zinc (Zn)/cadmium (Cd)/cobalt/lead (Pb) and copper (Cu)/silver transporters (Williams and Mills, 2005). AtHMA1 to AtHMA4 in A. thaliana and OsHMA1 to OsHMA3 in rice belong to the former group, while AtHMA5 to AtHMA8 and OsHMA4 to OsHMA9 belong to the latter group, although AtHMA1 has also been shown to transport Zn, Cu, and calcium (Axelsen and Palmgren, 2001; Williams and Mills, 2005; Seigneurin-Berny et al., 2006; Moreno et al., 2008; Kim et al., 2009).All members of HMAs in A. thaliana have been functionally characterized. AtHMA1 is involved in delivering Cu to the stroma, exporting Zn²⁺ from the chloroplast, or as a Ca²⁺/heavy metal transporter to the intracellular organelle (Seigneurin-Berny et al., 2006; Moreno et al., 2008; Kim et al., 2009). AtHMA2 and AtHMA4 localized at the pericycle are partially redundant and responsible for the release of Zn into the xylem (xylem loading) as well as Cd (Hussain et al., 2004; Verret et al., 2004; Wong and Cobbett, 2009; Wong et al., 2009), while AtHMA3 localized at the tonoplast plays a role in the detoxification of Zn/Cd/cobalt/Pb by mediating them into the vacuole (Morel et al., 2009; Chao et al., 2012). On the other hand, AtHMA5 is involved in the Cu translocation from roots to shoots or Cu detoxification of roots (Andrés-Colás et al., 2006; Kobayashi et al., 2008). AtHMA6 (PAA1, for P-type ATPase of Arabidopsis1) localized at the chloroplast periphery has been proposed to transport Cu over the chloroplast envelope, whereas AtHMA8 (PAA2) localized at the thylakoid membranes most likely transports Cu into the thylakoid lumen to supply plastocyanin (Shikanai et al., 2003; Abdel-Ghany et al., 2005). Finally, AtHMA7 (RESPONSIVE-TO-ANTAGONIST1) is responsible for delivering Cu to ethylene receptors and Cu homeostasis in the seedlings (Hirayama et al., 1999; Woeste and Kieber, 2000; Binder et al., 2010).By contrast, only three out of nine P-type ATPase members have been functionally characterized in rice. OsHMA2 was recently reported to be involved in the root-shoot translocation of Zn and Cd (Satoh-Nagasawa et al., 2012; Takahashi et al., 2012; Yamaji et al., 2013). Furthermore, OsHMA2 at the node is required for preferential distribution of Zn to young leaves and panicles (Yamaji et al., 2013). OsHMA3 is localized to the tonoplast of the root cells and responsible for the sequestration of Cd into the vacuoles (Ueno et al., 2010; Miyadate et al., 2011). On the other hand, OsHMA9 was mainly expressed in vascular tissues, including the xylem and phloem (Lee et al., 2007). The knockout lines accumulated more Zn, Cu, Pb, and Cd, suggesting its role in the efflux of these metals from the cells (Lee et al., 2007).Some members of P-type ATPase have also been identified in other plant species, including barley (Hordeum vulgare), wheat (Triticum aestivum), Thlaspi caerulescens (Noccaea caerulescens), and Arabidopsis halleri. HvHMA1 from barley might be involved in mobilizing Zn and Cu during the stage of grain filling (Mikkelsen et al., 2012). HvHMA2 from barley and TaHMA2 from wheat showed similar functions as OsHMA2 in rice (Mills et al., 2012; Tan et al., 2013). AhHMA3 in A. halleri, a Zn hyperaccumulator, is probably involved in high Zn accumulation (Becher et al., 2004; Chiang et al., 2006). Furthermore, AhHMA4 for Zn translocation showed a higher expression level (Chiang et al., 2006; Hanikenne et al., 2008). On the other hand, TcHMA3 from ecotype Ganges of T. caerulescens, a Cd hyperaccumulator, plays an important role in the detoxification of Cd by sequestering Cd into the vacuole of the leaves (Ueno et al., 2011). High expression of TcHMA4 (NcHMA4) was also reported in T. caerulescens (Bernard et al., 2004; Papoyan and Kochian, 2004; Craciun et al., 2012).In this study, we investigated the function and role of an uncharacterized member of P-type ATPase in rice, OsHMA5. We found that OsHMA5 is involved in the xylem loading of Cu at both the vegetative and reproductive growth stages.