Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants

Accepted Manuscript Title: Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants Authors: Kasia Dinkeloo, Shelton Boyd, Guillaume Pilot PII: DOI: Reference:

S1084-9521(16)30392-5 http://dx.doi.org/doi:10.1016/j.semcdb.2017.07.010 YSCDB 2273

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Seminars in Cell & Developmental Biology

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2-5-2017 30-6-2017 7-7-2017

Please cite this article as: Dinkeloo Kasia, Boyd Shelton, Pilot Guillaume.Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants Authors: Kasia Dinkelooa1, Shelton Boyda1 and Guillaume Pilota* a

Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA 24060, USA * Corresponding author at: Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA 24060, USA E-mail address: [email protected] (G. Pilot) 1

These authors contributed equally.

Abstract: Amino acids are essential components of plant metabolism, not only as constituents of proteins, but also as precursors of important secondary metabolites and carriers of organic nitrogen between the organs of the plant. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. The past few years have seen the identification of a new family of amino acid transporters in Arabidopsis, the characterization of intracellular amino acid transporters, and the discovery of new roles for already known proteins, for instance in hormone transport. While amino acid metabolism needs to be tightly coordinated with amino acid transport activity and carbohydrate metabolism, no gene involved in amino acid sensing in plants has been unequivocally identified to date. This review aims at summarizing the recent data accumulated on the identity and function of amino acid transporters in plants, and discussing the possible identity of amino acid sensors based on data from other organisms. Keywords: Plant Amino acid Transporter Transceptor Sensor Metabolism 1. Introduction Plants absorb both inorganic nitrogen (ammonium and nitrate) and organic nitrogen (amino acids and peptides) from the soil [1]. The first organic nitrogenous molecule produced from inorganic nitrogen is Gln, whose terminal amino group is successively transferred to Glu and other amino acids to synthesize all the nitrogencontaining compounds of the cell. Within the cell, amino acids are synthesized [2], used, and degraded [3] in various compartments (namely chloroplast, cytosol, peroxisomes,

mitochondrion and vacuole), requiring several transport steps across membranes to link the different pathways. Intracellular and intercellular transport of amino acids is mediated by dedicated membrane proteins, amino acid transporters. The first amino acid transporter identified in plants was the Arabidopsis Amino Acid Permease1 (also called Neutral Amino acid Transport system 2, NAT2) gene, published in 1993 by two groups [4,5]. Since then, thanks to advances in yeast functional complementation and genome sequencing, many amino acid transporters from Arabidopsis and other species have been isolated and characterized. This topic is regularly reviewed and the reader is invited to read excellent past articles that summarize our previous knowledge about the roles and functional properties of amino acid transporters [2,6-10]. Briefly, studies of the past decade identified many transporters active in roots, a few transporters connected with seed filling function, and some genes with a proposed role in source to sink translocation of amino acids (Fig. 1): AAP1 and AAP5, together with Lysine and Histidine Transporter 1 (LHT1), LHT6 and Proline Transporter 2 (ProT2) have been shown to have a role in root uptake; AAP2 and AAP6 are involved in xylem-phloem transfer, while AAP2, AAP3, AAP5, ProT1, Cationic Amino Acid Transporter 1 (CAT1), CAT6 and CAT9 contribute to phloem loading and are expressed in vascular tissues; AAP1 and AAP8 have also been shown to be involved in seed loading with amino acids [8-10]. We will focus in the present review on recent advances in the study of amino acid transport. Research completed in the past few years in Arabidopsis and other plant species has increased our knowledge of amino acid translocation from source to sink, for fruit development and seed filling, and transport within the cell. While all the previously known plant amino acid transporters belong to two families, the Amino Acid/Auxin Permease family (AAAP; [11]) and the Amino acid-Polyamine-organoCation family (APC; [12]), a new family of transporters has recently been identified. This family is the Usually Multiple Amino Acids Move In and Out Transporter (UMAMIT, see [13,14]), which is part of the Drug/Metabolite Transporter (DMT) superfamily [15]. As interest in the field of amino acid transport grows, the number of organisms in which it is being studied is expanding. Genome-wide surveys of different species are leading to more direct identification of previously unknown amino acid transporters in rice [16], Selaginalla [17], poplar [18], soybean [19], potato [20] and Ricinus [21]. 2. Amino acid uptake, transport, and partitioning 2.1. Transporters for seed filling, fruit development and source to sink translocation The discovery and study of the UMAMIT transporters has opened a door to understanding amino acid export and import from source leaves to sinks such as new leaves and developing seeds. Beginning with the characterization of Siliques Are Red1 (SiAR1/UMAMIT18) and Walls-are-thin 1 (WAT1/ UMAMIT5), the UMAMIT family has since been shown to contain 47 members, three of which are likely pseudogenes [22]. UMAMIT18 was characterized as a plasma-membrane localized transporter present in vascular tissue as well as developing seeds, and was shown to transport amino acids bidirectionally for accumulation in developing siliques [23]. In addition to UMAMIT18, UMAMIT14, 28 and 29 have been shown to be involved in export from the phloem to the developing embryo. These UMAMITs are expressed in tissues in which amino acid export is required for seed filling [24]. Free amino acids accumulate in the silique of the

corresponding umamit mutants, and mutant seeds are much smaller than wild type seeds [24]. Prior to characterization of the UMAMIT transporters, AAP8 was found to be expressed exclusively in seeds and thought to be implicated in seed filling [25]. Using in situ hybridization, a recent study demonstrated that AAP8 is also expressed in source leaf phloem, suggesting a new role for the protein. More detailed physiological analyses revealed that the effect on seed development is more probably due to reduced loading of the leaf phloem with amino acids than a reduction of import into the seeds, which translates into a weaker sink development [26]. Later studies have shown that UMAMIT18 is also implicated in unloading amino acids from the root phloem [27]. UMAMIT14 was characterized at the same time as an exporter localized in roots with a role in phloem unloading. This gene is expressed in the root pericycle and phloem cells and mediates export of a broad range of amino acids [27]. 2.2. Intracellular amino acid transport While conceptually essential for metabolic activity, little research had been dedicated to understanding how amino acids are transported across membranes at the subcellular level until recently [2,28]. Studies focusing on vacuolar transport in several plant species have become prominent, identifying transporters in tomato, petunia, and Arabidopsis. A proteomics analysis of tomato tonoplast isolated from ripe tomato fruit (which accumulates large amounts of Glu and Asp) led to the isolation of SlCAT9. SlCAT9 is an exchanger that transports Glu and Asp towards the vacuole lumen in exchange for γ-amino butyric acid (GABA). SiCAT9 plays a role in amino acid accumulation during fruit development, which in turn greatly affects the flavor profile of the tomato fruit [29]. In addition to SlCAT9 another CAT subfamily member from tomato, SlCAT2, has been localized to the tonoplast of stamen cells. Based on expression analyses, the authors suggested a role in flower and fruit development [30]. In Arabidopsis, AtCAT2 and AtCAT4 have been shown to localize at the tonoplast when tagged with GFP, and AtCAT2 is implicated in regulation of amino acid levels in leaves [31]. Outside of the CAT family, other amino acid transporters from the AAAP family have recently been shown to localize to the tonoplast [32,33]. These transporters are similar to the yeast Amino acid Vacuolar Transport 3 (AVT3) protein, involved in neutral amino acid transport from the vacuole to the cytosol [34]. One of the Arabidopsis homologs, AtAVT3, has been functionally validated to transport alanine and proline from vacuoles to the cytosol when expressed in yeast [32]. In Arabidopsis, AtAVT3-GFP localized to the vacuolar membrane. Despite these promising results, AtAVT3 does not seem to respond to nitrogen starvation and excess in planta as does the yeast homolog, and its role in amino acid homeostasis remains to be determined [32]. While looking for proteins similar to the Phe transporter from E. coli, pheP, researchers identified a Petunia hybrida plastidial cationic amino acid transporter (PhpCAT), which influences flux distribution through the phenylalanine biosynthetic network by mediating Phe transport from the chloroplast to the cytosol. PhpCAT has been shown to transport all three aromatic amino acids and plays a direct role in controlling levels of phenylalanine and phenylalanine-derived volatiles in petunia [35]. This is the second identified transporter involved in transport of amino acids from the chloroplast, after the Malate/Glu exchanger AtDit2.1 shown to work in conjunction with the 2-oxoglutarate/malate exchanger AtDit1 [36]. Consistent with the CAT family members characterized in tomato and petunia, the Arabidopsis transporter AtCAT9 has

been implicated in intracellular amino acid transport as well: it has been shown to localize to vesicles and may play an unclear role in amino acid homeostasis [37]. 3. Other functional roles of amino acid transporters Some members of amino acid transporter families have been found to transport molecules other than amino acids. A forward genetic screening aiming at finding 1aminocyclopropane-1-carboxylic acid (ACC)-resistant plants isolated the amino acid transporter LHT1 [38]. LHT1 had been previously characterized for its role in amino acid uptake from the soil and leaf mesophyll apoplasm [39,40], and the the lht1 knockout mutant shows an early senescence phenotype, induced defense responses, and disturbance in amino acid homeostasis and redox status [41]. This newly discovered role of LHT1 in ACC transport suggests that some components of the pleiotropic Lht1 phenotype could result from ethylene signaling defects. Interestingly, the authors reported that at least one other member of the LHT family is able to transport ACC [38], suggesting that this property is physiologically relevant. AAC transport by an amino acid transporter is not surprising since ACC is an α-amino acid, whose α-carbon is part of a three carbonring. Substrate specificity of plant amino acid transporters has indeed been shown to be borne by the α-carbon amino and carboxylic groups [42-44], similar to animal amino acid transporters [45-47]. Some members from the AAAP family, namely the AUX1 and the LAX proteins, transport auxin instead of amino acids [48]. While there is some structural relationship between IAA and Trp, the substrate recognition characteristics seem different for these proteins: the auxins do not have any amino group on the α-carbon, and the molecules that compete with IAA transport contain one or two aromatic rings and one carboxylic group [48]. While sequence-related to amino acid transporters, the AUX-LAX proteins have modified substrate-binding properties, which is at least based on a carboxylic group. Another member of an amino acid transporter family, WAT1/UMAMIT5, has been described as a vacuolar transporter involved in the import of IAA from the vacuole to the cytosol [49,50]. The results from these studies suggest that the transport is dependent on the proton-motive force, but the transport mechanism, the Km for IAA, and the substrate specificity remain to be determined. Meticulous study of AtGAT1, a GABA transporter from the AAAP family related to ProTs, has shown that amino acids are not a good substrate of AtGAT1. In addition, it appeared that the presence of a terminal carboxylic group is not required for transport, but that the terminal amino group is critical for transport, independent of the length of the transported molecule [51]. The binding pocket thus seems different from the transporters whose substrates are amino acids, since the latter require the presence of both amino and carboxylic groups on the α-carbon (see above). Some members of the APC family show another substrate specificity: the L-Type Amino acid Transporters/Putrescine Transporters (LAT/PUT) transport polyamines [52], which are amino compounds not structurally related to amino acids, but contain terminal amine groups. Mutants in some LAT/PUT genes display enhanced resistance to the herbicide paraquat, a structural analog of polyamines that induces oxidative stress and cell death. As a final example of the diversity of possible substrates, some amino acid transporters have been shown to transport synthetic compounds: a recent paper showed that the uptake of a derivative of the insecticide fipronyl by Ricinus plants is competitively inhibited by amino acids, suggesting that its transport across membranes is mediated by amino acid importers.

Furthermore, the authors identified some amino acid transporter genes that are induced by the application of the compound, suggesting that they may be good candidates for mediating its uptake [21]. The diversity of substrate specificities of some amino acid transporters from the APC, AAAP and UMAMIT families raises the question of how many members of these families transport substrates other than amino acids. How many of them have an actual role in amino acid transport in the plant? Not enough data is presently available to answer this question, but it will be important to keep in mind this diversity when studying the role of members of these gene families, not limiting the analysis of amino acids. 4. Amino acid transporters as target for crop improvement and resistance to pathogens? 4.1. Manipulation of protein content in storage organs Based on the role of amino acid transporters in long distance transport of amino acids, for instance between leaves to roots or seeds, these genes could be good target for modification in crops (see also [10]). An early successful attempt to utilize amino acid transporters for crop improvement aimed at understanding amino acid translocation to the potato tuber. Suppressing the mRNA accumulation by RNAi of StAAP1 led to up to 50% reduction in amino acid content of the tuber which translated into a ~10-15% reduction in total nitrogen, and interestingly had no major impact on plant photosynthesis or yield [53]. Increasing amino acid supply to the embryo as a way to increase protein content was first tested by over-expressing an AAP-type transporter (VfAAP1) in the cotyledon parenchyma of pea and Vicia plants [54]. The seeds of transformed plants took up more amino acids than the wild type, and contained more free amino acids and proteins, with similar results in the greenhouse and in the field, showing that manipulating amino acid transport is a valid strategy [54,55]. To stimulate amino acid translocation in the plant, another group expressed a similar AAP-type transporter (PsAAP1) in pea, placed under the AtAAP1 promoter, which is active in both leaf phloem and the embryo [56]. The transgenic plants showed increased amino acid and protein accumulation in seeds, in addition to higher yield, suggesting that stimulation of amino acid transport both in the source and sink tissues is required for maximum nitrogen translocation to the seeds [56]. Other recent studies have successfully shown that expression of transporters to increase translocation of nitrogenous compounds (ureides [57] or S-methylmethionine [58]) between the organs of the plant is a good strategy for crop improvement. Use of amino acid transporters for manipulating grain quality does not necessarily requires a transgenic approach. A classical mapping approach of the qPC1 locus in rice revealed identified OsAAP6, which was found to be expressed in roots, leaves and developing seeds. The numerous polymorphisms detected between high- and low-protein varieties of rice correlated with expression of OsAAP6. Furthermore, over-expressing OsAAP6 or suppressing by RNAi its expression definitively proved that this gene was a positive regulator of grain protein content [59]. 4.2. Involvement in plant-pathogen interactions Some amino acid transporters have been found to be important for pathogen feeding on live plant tissues, especially for nematodes. The soybean Rhg1 locus (Resistance to Heterodera glycine), which confers resistance to the soybean cyst

nematode, encodes three genes: an unknown protein, an α-SNAP protein predicted to be involved in membrane trafficking, and an AAAP amino acid transporter (Glyma18g02580, similar to Arabidopsis genes from the AVT6 subfamily of vacuolar transporters). Increasing the copy number of these three genes is critical to confer resistance, but the resistance mechanism is still elusive [60]. It is possible that Glyma18g02580 does not transport amino acids, but a molecule important for triggering nematode resistance. Arabidopsis amino acid transporters were shown to be important for root-knot nematode and cyst nematode development. AtCAT6, induced at the site of infection by root-knot nematode Meloidogyne incognita, was suggested to be involved in supplying amino acid to the feeding structures, but this hypothesis has not been tested experimentally [61]. More recently, mutants of the AtAAP3 and AtAAP6 genes were shown to sustain less growth of the root-knot nematode compared to the wild type, in good agreement with a role in supplying amino acids to the nematodes [62]. The importance of the AtAAP genes was also demonstrated for another nematode, the cyst nematode Heterodera schachtii. After finding and localizing the expression of genes induced during infection, the authors showed that mutants of AtAAP1, AtAAP2 and AtAAP8 carry less females than the Col wild type and the atlht1mutant. Interestingly, probably reflecting the difference between ecotypes, these results were not observed when using the Ws ecotype [63]. The importance of amino acid transporters in sustaining pathogen growth is not surprising since they carry a readily utilizable form of nitrogen, and it is conceivable that alteration in amino acid distribution/transport in mutant plants affects the flow of nitrogen to the pathogen. This tight relationship between amino acid transport and pathogen feeding may be the ground for the following puzzling observations: over-expression of AtCAT1 [64,65] and AtGDU1 [41], or disruption of AtLHT1 [41] lead to constitutive induction of defense responses, evidenced by the expression of pathogenesis-related protein 1 (PR1). While amino acid transport disturbances have been observed for mutants of the AAP family (AAP1 [66], AAP2 [67], AAP6 [68], AAP8 [26]), such a defense response has never been reported in these cases. In another report, a forward genetic screening for Arabidopsis mutants resistant to Phytophtora parasitica led to the identification of the Resistant to Phytophtora parasitica 1 (AtRTP1/AtUMAMIT36) gene, whose expression correlated with susceptibility to the pathogen [69]. Defense responses are activated sooner in the rtp1-1 knockout mutant than in the wild type. Yet, the functional properties of the protein are not known, and the molecular mechanism for its role as a susceptibility factor remains to be determined. It is possible that loss of transport of the corresponding substrate activates defense priming [70], which would explain the resistance. Why altering the expression of some amino acid transporters leads to constitutive defense responses and altering the expression other genes does not is not understood, but it could be related to where these genes are expressed or to their physiological role. Altering amino acid concentration only in specific compartments might trigger a stress response. Application of exogenous amino acids to rice has been shown to induce the expression of defense and stress related genes [71,72]. Similarly, application of amino acids to rice leaves induced systemic resistance to rice blast by induction of defense genes [73]. It is not clear at present why exogenous amino acids induce defense responses, but this may be part of the plant arsenal to defend against pathogens: since many pathogens need to extract amino acids for their own benefit, a surveillance system of such a process would alter the plant cell of the presence of pathogen, much like the

pathogen-associated molecular pattern-triggered immunity, which is the plant’s first response to pathogen-associated molecular patterns [74]. Studies of Brassica napus AAPs have added to information about how amino acid transport and salicylic (SA) / jasmonic acid (JA) levels are linked. Pod-removal decrease the expression of BnAAP1, 2, 4, and 6 in mature leaves, leading to reduced loading of amino acids into the phloem and a higher amino acid content in leaves. The decrease in amino acid transport and phloem loading was correlated with a decrease in SA or SA/JA ratio [75]. These studies add up to the growing body of evidence on the interaction of amino acid metabolism (and hence transport) with defense responses (reviewed in [76]), but the molecular mechanisms remain elusive and will need closer examination. 5. Amino acid sensors in plants Amino acid metabolism is regulated by feedback inhibition and both transcriptional and post-translational enzyme activity (for a review see [2]). Yet, how amino acids are sensed and the corresponding signaling pathways are not understood in plants. Gent and Forde [77] have recently reviewed the status of our knowledge on amino acid sensing in plants. They identified five potential sensing processes involving different types of proteins: (1) the PII protein, which is thought to control Arg biosynthesis and fatty acid metabolism in the plant chloroplast; (2) the TOR signaling pathway, which receives input from unidentified amino acid sensors and carbon status to promote protein synthesis, and is a negative regulator of protein turnover; (3) the GCN2 protein kinase pathway that senses uncharged tRNAs and inhibits protein synthesis (but only a few steps of the signaling cascade are presently identified); (4) genes similar to glutamine synthetase I that could also be involved in amino acid sensing, but here again little is known about their function; (5) glutamate receptors that have been extensively studied in Arabidopsis, and even if their ability to mediate Ca2+ transport to the cytosol upon binding extracellular amino acids is proven, the downstream events and their role as putative amino acid sensor are currently unclear (see [77] and references therein). It thus appears that, while the list of candidate genes is long, no amino acid or nitrogen sensor has been unequivocally identified in plants. We earlier raised the question of plant cells being able to sense alteration of amino acid flow to the apoplasm during pathogen infection, which leads to the question of how cells sense external amino acids. Glutamate receptors are certain candidates, but looking to the other sensors identified in yeast and mammals points to other kinds of proteins that warrant further study. 5.1. Receptors Sensing extracellular nutrients is achieved by receptors that activate signals by directly binding to their substrate. Upon binding, the receptor is subject to a conformational shift, which triggers an interaction with a cytosolic protein that leads to downstream signaling and subsequent cellular response. For instance, the Sulfonylurea Sensitive on YPD 1 (Ssy1) and Peptide TRansport 3 (Ptr3) genes are members of the SPS (Ssy1-Ptr3-Ssy5) amino acid sensing cascade in Saccharomyces cerevisiae. Upon binding external amino acids, the conformation change of Ssy1 signals for the phosphorylation of Ptr3 and Ssy5, and downstream phosphorylation events [78]. Repressing this cascade has been shown to increase NAD homeostasis and extend replication in yeast [79].

Many sensors/receptors share high sequence similarity to transporters, but lack transport properties. For instance, the human Sodium-glucose cotransporter 3 (HsSGLT3) sensor shares similarity to HsSGLT1, a sodium and glucose cotransporter from the same subfamily. Transport assays in Xenopus oocytes revealed that glucose binding to SGLT3 depolarizes the plasma membrane, but glucose is not transported [80]. Mutagenesis of Glu457 in HsSGLT3 to Gln457 (a residue critical for glucose transport [81]) renders HsSGLT3 able to transport glucose similar to HsSGLT1 in Xenopus oocytes, and to alter glucose sensing [82]. Numerous examples of sensors that are similar to transporters suggest that most sensors evolved from transporters by losing transport properties and gaining an ability to interact with intra-cellular components to transduce a signal about nutrient availability [83,84]. 5.2. Transceptors It is conceivable that some proteins sit between these two extreme cases, and have maintained their transport function while having gained sensing properties. These proteins are called transceptors. Transceptor signaling is believed to occur upon nutrient interaction with the protein, which confers a conformational shift leading to substrate transport, and initiates a signal in the cell. Most of our understanding of transceptors has been established from studies in yeast, with a few transceptors having also been identified in plants and mammals. In mammals three transporters have been identified as transceptors, namely glucose transporter Glucose Transporter 2 (GLUT2) [85], and amino acid transporters Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2) [86] and Proton-assisted Amino Acid Transporter 1 (PAT1) [87]. Transceptor function for the human Concentrative nucleoside transporter 1 (CNT1) has also been suggested [88]. In plants, the nitrate transporter NRT1.1 has been extensively studied and is role as a transceptor is well accepted [89]. Establishing the signal output from a transporter has been instrumental for both identifying and characterizing transceptors. The Saccharomyces cerevisiae amino acid transporter General Amino acid Permease 1 (Gap1) was identified for its activation of the Protein Kinase A (PKA) pathway [90], important for cell growth and regulation of metabolically active genes (Fig. 2). Activation of the PKA pathway is measured by the activity of the trehalase. Trehalase activity assays have been used extensively for identifying the signaling capacities of transceptors for sulfate (Sul1, Sul2) [91], ammonium (Mep2) [92], phosphate (Pho84) [93], zinc (Zrt1) and iron (Ftr1) [94]. Correlation of transport activity with signaling does not establish a transporter as a transceptor. It is necessary to prove that the identified transporter is the origin of the signal. Null mutations for Gap1 complemented with compensatory accumulation of amino acids in the yeast cytosol revealed no activation of the PKA pathway [90]. The role as a transceptor of Mep2, a Saccharomyces cerevisiae ammonium transporter, was confirmed using the non-metabolized ammonium analog, methylamine. Transport of methylamine was capable of eliciting activation of the PKA pathway upon Mep2 transport and without further metabolism [95]. Sufficient intracellular accumulation of nutrient coupled with no signaling event in the absence of the transceptors gene confirms the transporters function for signaling. Site directed mutagenesis has also been employed for understanding the mechanism of transceptor sensing. Introducing point mutations affecting distinct steps of transceptor activity has helped with understanding how critical the transporter’s

stereochemistry and conformational state are for initiating a signaling event. AtNRT1.1 Pro492 is required for nitrate transport but not for signaling by this plant nitrate transceptor [89,96]. Importantly, complete transport of substrate across the membrane is not (always) required for sensing and signaling. Asp358 in Pho84, a yeast phosphate transceptor, is critical for transport [93]. Mutagenizing Asp358 to Glu completely abolished Pho84 transport activity, but Pho84 retained the ability to activate of the PKA pathway, effectively dissociating transport and signaling [93]. A similar effect was reported for the Gap1 amino acid transceptor by the finding that certain amino acid analogs were found capable of uncoupling transport and signaling [97,98]. Interaction of Gap1 with these molecules was sufficient for initiating a conformational change of the protein, while complete transport was not required [98]. Expression of most transceptors is sensitive to nutrient availability. Transceptor expression is generally high when the respective nutrient concentration is low and repressed when nutrients are available. Replenishing nutrients after starvation initiates rapid endocytosis and vacuolar degradation by oligo-ubiquitination, as demonstrated for Gap1 [99], Zrt1 [94], Ftr1 [94], and Mep2 [92], among others. Like signaling, endocytosis and oligo-ubiquitination can be uncoupled by various substrate analogs. Overlap of various analogs in the Gap1 binding region revealed distinct conformational changes leading to differential effects on endocytosis and degradation in addition to transport and sensing [97]. The regulatory mechanism behind transceptor trafficking and expression has been extensively studied for Gap1 and has been reviewed in detail [100]. In addition, it becomes clear that transceptor sensing activity is complex and requires a very specific mechanism that is sensitive to protein confirmation, phosphorylation, and protonation following nutrient binding [101,102]. 5.3. Role of transceptors to the mTOR pathway The Target of Rapamycin (TOR) signaling pathway functions as an energy status reporter to regulate cell metabolism in mammals and in yeast. The TOR pathway is composed of two complexes in mammals, TORC1 and TORC2 [103]. Homologs of the TORC1 complex have been identified in higher plants and algae but not for mTORC2 [103]. The TOR pathway is highly complex, and its regulation is highly sensitive to many nutrients such as amino acids [104]. The accumulation and availability of amino acids are proposed to be sensed by transceptors leading to TOR activation [105]. In mammals, the amino acid transceptors SNAT2 and PAT1 have been found to affect the activity of the mTOR pathway. Downstream signaling interactions identified by TAP-tagged SNAT2 protein complexes in HEK293 cell lines showed decreased cell proliferation [106]. Deregulating SNAT2 transport activity by substrate competition using a non-metabolized System A amino acid analogue (methylaminoisobutyric acid) also led to reduced cell proliferation, due to affected SNAT transceptor activity towards mTOR signaling [106]. Accumulation of amino acids within the lumen of lysosome is essential for amino acidinduced activation of mTOR [105]. Once accumulated, amino acids signal for the activity of the V-ATPase, which controls the formation of the RAG complex responsible for recruiting mTOR to the lysosome. Recent studies showed the translocation of PAT1 from the cell surface to the lysosome upon PI3K/Akt/Rheb cascade activation (Fig. 2). The PAT1 transceptor senses amino acid intraluminal concentrations and complexes with Rag-GTPases, which leads to activation of mTOR and regulation of cell proliferation [107].

5.4. Is there evidence of any amino acid transceptor in plants? Unfortunately, there is currently no evidence for the existence of amino acid transceptors in plants. A nitrate transceptor has been identified and characterized, AtNRT1;1 [108], while the physiology of the Arabidopsis mutant for the SULTR1;2 gene is consistent with a function of the protein as a transceptor [109]. It is possible that within all the genes of the three main families of amino acid transporters, AAAP, APC and UMAMIT (~110 genes), some of them evolved as transceptors, or as pure sensors, without any transport function. This would explain why transport properties of several putative amino acid transporters could not be identified in heterologous systems. Alternatively, a presently unidentified gene family could represent the long sought-after sensors/transceptors. The importance of TOR in the regulation of amino acid metabolism in plants is demonstrated [103,110,111], and it is possible that, as in mammals, plant amino acid transporters play a role in the regulation of TOR activity. Proving that a transporter is endowed with sensing properties is not a simple task, requiring several different layers of proof. First, one needs to prove that the substrate is not sensed by other proteins within the cell upon transport. Most transceptors have been identified by finding point mutations that uncouple transport and sensing, followed by confirming inability to restore signaling by complementation from another transporter with similar specificity. Additionally, the identification of selective inhibitors, which affect transport but not signaling, provides substantial evidence for a role as a transceptor [112]. Currently, too little is known about the effects of supplying external amino acids to plants, which could provide clues about the existence and identity of a signaling cascade. Based on what is known in yeast and mammals, it is very likely that amino acid transceptors exist in plants, but their identification will require extensive work. Acknowledgements This work was supported by a grant from the National Science Foundation (IOS1353366), the Hatch Program of the National Institute of Food and Agriculture (VA135908) and the Virginia Agricultural Experiment Station, to G.P.

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Figure legend Fig. 1. Summary of the role of the characterized amino acid transporters in plants. The role in Uptake by roots, Phloem export in root, Phloem loading, Xylem-Phloem transfer, Seed Development and Intracellular Transport is depicted for amino acid transporters recently identified and mentioned in the main text (in rounded boxes) and for amino acid transporters previously reviewed. Black arrows refer to direction of transport when known. References about the roles of AAP1, AAP2, AAP5, AAP6, AAP8, ProT2, LHT1, LHT6, CAT1, CAT6 and DiT2.1 in the plant can be found in the reviews by Tegeder and Rentsch (2010) and Tegeder (2012, 2014) [8-10]. Seed cross section drawing is based on published micrographs [24].

Fig. 2. Transceptors and the corresponding signaling events in mammals and yeast. Amino acid transceptors (dark blue) transport amino acids from the extracellular space (in yeast – left side) or lysosome lumen (in mammals – right side) to the cytosol, and interact with cellular components to signal availability of amino acids in these compartments. The protein kinase A (PKA) pathway in yeast, and the V-ATPAse and then the mTOR pathway in mammals are components of the signaling cascades activated by the amino acid transceptor. The TOR complex is anchored to the membrane by interaction with the lipid-modified Ragulator complex and the small GTPAse Rheb. RagA to RagD are small GTPAse proteins which recruit TORC to the membrane.

Outputs:

Outputs:

Cell growth Protein synthesis Amino acid transport activity Trehalose concentration

Cell growth Protein synthesis Amino acid transport activity

H+

TPK2

PKA

TPK1

Bcy1

mTORC1

TPK3

AA

Ragulator

RagA RagB

GTP

RagC RagD GDP

Cytosol

Extracellular Space

Fig. 2

Cytosol

V-ATPase

GTP

AA

YEAST

RheB

Lysosome lumen

AA

MAMMALS