Nan Guo ,Shunan Zhang ,Mingji Gu ,Guohua Xu ,*

a Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding,College of Horticulture and Plant Protection,Yangzhou University,Yangzhou 225009,Jiangsu,China

b State Key Laboratory of Crop Genetics and Germplasm Enhancement,MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China

Keywords:Amino acids Amino acid transporter Grain quality Nitrogen uptake efficiency Nitrogen utilization efficiency Rice architecture

ABSTRACT Amino acids are essential plant compounds serving as the building blocks of proteins,the predominant forms of nitrogen(N)distribution,and signaling molecules.Plant amino acids derive from root acquisition,nitrate reduction,and ammonium assimilation.Many amino acid transporters(AATs)mediating transfer processes of amino acids have been functionally characterized in Arabidopsis,whereas the function and regulation of the vast majority of AATs in rice(Oryza sativa L.)and other crops remain unknown.In this review,we summarize the current understanding of amino acids in the rhizosphere and in metabolism.We describe their function as signal molecules and in regulating plant architecture,flowering time,and defense against abiotic stress and pathogen attack.AATs not only function in root acquisition and translocation of amino acids from source to sink organs,regulating N uptake and use efficiency,but also as transporters of non-amino acid substrates or as amino acid sensors.Several AAT genes show natural variations in their promoter and coding regions that are associated with altered uptake rate of amino acids,grain N content,and tiller number.Development of an amino acid transfer model in plants will advance the manipulation of AATs for improving rice architecture,grain yield and quality,and N-use efficiency.

1.Introduction

Plant roots use both inorganic nitrogen(N;mainly ammonium,and nitrate,)and organic N including amino acids,peptides,and proteins[1-5].Plants grown under upland conditions take up most of their N in the form of nitrate.Root-acquired nitrate is partially reduced in roots and largely transported to shoots,where it is reduced to nitrite by nitrate reductase in the cytoplasm and then further to ammonium by nitrite reductase in plastids[3].Rice(Oryza sativa L.)is commonly grown in flooded paddy soil,whereis the dominant form of inorganic N.The ammonium either directly acquired from soil or derived from nitrate is further assimilated into amino acids via the glutamine synthetase(GS)/glutamine-2-oxoglutarate aminotransferase(GOGAT)cycle in cytoplasm and plastids[3].

Amino acids are present in relatively high amounts in paddy soil and can be acquired by rice roots[6].Following direct uptake or ammonium assimilation via the GS/GOGAT pathway,various amino acids in roots are translocated to shoots in the xylem[3,7].Amino acids are also the major form of N remobilized from transient storage pools or by protein hydrolysis in long-distance transport via the phloem.Amino acids play many roles in plants,including acting as signal molecules,regulating root and shoot architecture,and regulating flowering time and stress defense.N is transported primarily in the form of amino acids in most plants.For this reason,improving amino acid partitioning from source leaves to sink organs may increase N utilization efficiency(NUtE,the fraction of plant-acquired N converted to total plant biomass or grain yield)and seed yield[8].

The movement of amino acids from rhizosphere to seed is mediated by various membrane-localized amino acid transporters(AATs).In Arabidopsis,AATs comprise 9 subfamilies,of which several representative members have been functionally characterized[7,8].Alteration of cellular or subcellular N levels by modification of AAT activities can improve plant productivity,and increase N uptake efficiency(NUpE,the capacity of plant roots to acquire N from the soil)and NUtE,as well as resistance to stresses in a range of N environments[7].However,current knowledge about AATs of rice and other crops is based only on characterization of several amino acid permeases(AAPs)and the lysine-histidine-like transporter(LHT)subfamily.In this review,we follow the route of amino acids from rhizosphere to seed,summarize what is known about function,transport,and regulation of amino acids in rice crop,and address the promise of manipulating AATs in improving rice grain production and N-use efficiency(NUE,the grain yield per unit of N supply in soil,which integrates NUpE and NUtE).

2.Amino acids in rhizosphere of paddy soil

Agricultural soils commonly comprise 0.05%-0.2%N,and more than 95%of total N is present in organic form(as organic matter and soil macro-and microorganisms)with proteinaceous materials(proteins,peptides,and amino acids)as major components[9].Soil microbe-controlled mineralization and assimilation processes dominate interconversion between organic and inorganic forms of N[10].Organic N in soil potentially available for plant roots has two pools:soluble organic N(SON,N extracted from soil by water,KCl,etc.)and dissolved organic N(DON,N dissolved in soil solution or drainage water)[11].SON is the major organic N pool and the primary N source for mineralization.Arable agricultural soils contain about 20-30 kg ha-1of SON,with amino acids being the largest pool[11,12].In soil solution,total free amino acids may be present in concentrations of up to 150μmol L-1,with aspartate,glutamate,asparagine,glutamine,glycine,and alanine being the predominant forms[13,14].

Agricultural soils generally have higher adsorption capacity for amino acids than non-agricultural soils,owing to their higher organic matter and cation-exchange capacity[15].The amounts of total amino acids including free and extractable amino acids in paddy soil are lower than those in upland soil[16].Although the factors affecting amino acid levels in paddy soil are poorly understood,competition of the microorganism community and leaching with flowing water may account for their lower content of amino acids[17-19].

In the rhizosphere,which is the soil surrounding and affected by plants,part of the amino acids come from root exudates and secretion by soil microbes[20-22].After sugars,amino acids are the second most abundant compounds exuded by roots[23].The amino acids in the rhizosphere are competitively utilized by plants and microorganisms.In natural soil ecosystems,more than 80%of land plants form arbuscular mycorrhizal(AM)symbioses with mycorrhizal fungi for increasing nutrient and water absorption and resistance to stresses[24].Owing to their contribution to extending the root system and the competition of mycorrhizal fungi with other microorganisms,plants with mycorrhizal symbionts take up much more inorganic N and amino acids in soil than those without[12,25-27].For example,mycorrhizal rice received more than 40%of its N via the mycorrhizal pathway and an AM-specific nitrate transporter,OsNPF4.5,accounted for approximately 45%of the mycorrhizal nitrate uptake[27].The fungi can convert a large part of the inorganic N to organic form in extraradical mycelium and then transfer the organic N,in particular amino acids,to the root before it is acquired by N transporters of the host plant[28].However,the plant AAT contribution to absorption of amino acids derived from mycelium remains unknown.

Rice develops aerenchyma-based aeration tissues,interconnected gas-filled channels between shoot and root,to adapt to flooded soil environments[29].Released oxygen(O2)and other exudates secreted from roots result in a rhizosphere environment around rice different from those around other plants[29].Although root-released O2increases nitrification,the biological oxidation of ammonium to nitrate,at the root surface[30],the effect of aeration tissues on turnover and amount of amino acids in the rice rhizosphere are still unknown.

Alternative drying and wetting(ADW)management is widely used during the rice growth season to reduce water consumption and improve N supply status[31-33].Compared with continuous flooding of soil,ADW brings more O2into the soil and increases the activity of aerobic microorganisms,resulting in increased N transformation and competition for amino acid uptake between soil microbes and plant roots[12,34-36].However,despite its function in improving rice N acquisition and use efficiency[37-39],rewetting in the paddy field increases loss of soluble organic N including amino acids.

3.Metabolism and functions of amino acids in rice

3.1.Anabolism of amino acid in rice

Glutamine and glutamate derived from ammonium assimilation via the GS-GOGAT pathway are precursors of other amino acids[40,41].GS includes two isoforms:cytosolic GS1 and plastidic GS2,whereas GOGAT has three forms:NADH-GOGAT,NADPHGOGAT,and Fd-GOGAT,based on the electron donor for the conversion of glutamine and 2-oxoglutarate to glutamate[41].In rice,three isoenzymes of cytosolic GS1(GS1;1,GS1;2,and GS1;3)and two NADH-GOGAT(NADH-GOGAT1 and NADH-GOGAT2)have been functionally characterized.OsGS1;2/NADH-GOGAT1 play the primary role in assimilation of ammonium in roots[42-44],while OsGS1;1/OsNADH-GOGAT1/2 are associated with ammonium assimilation in photosynthetic tissues for the remobilization of N in senescing organs[43,45,46].OsGS2 and OsFd-GOGAT are abundant in chloroplast-containing cells and modulate photorespiration associated chlorophyll synthesis and leaf senescence[42,47].OsGS1;3 is expressed in spikelets and functions in natural senescence and seed germination[46].

In rice,asparagine is the second most abundant amino acid in phloem sap and endosperm.It is synthesized via transfer of the amino group of glutamine to aspartate by asparagine synthetase(AS)[45].OsAS1 and OsAS2 are two AS isoenzymes in rice.OsAS1 is responsible for biosynthesis of asparagine in roots[48],whereas OsAS2 is found mainly in the phloem companion and parenchyma cells of leaf blades with a potential role in translocation of asparagine in leaves[49].

Arginine has the highest N-to-carbon ratio among the 21 proteinogenic amino acids,and both glutamate and ornithine are intermediate products of its biosynthetic pathway[50].In rice,disruption of argininosuccinate lyase(OsASL)which catalyzes the last step of arginine biosynthesis,results in reduced root length[51].Given that the short-root phenotype of the osasl mutant could be rescued by exogenous addition of arginine[51],arginine biosynthesis may be associated with the arginine uptake and transport process[52].

Amino acid anabolism is determined by the interplay of the uptake,biosynthesis,and transport of amino acids.Manipulation of candidate genes involved in amino acid anabolism has been demonstrated[53]to be a promising strategy to improve plant NUE.Amino acid biosynthesis and the relevant regulatory events have been studied in depth only in Arabidopsis and not in agronomic crops such as rice[40,54].

3.2.Function as signal molecules

Amino acids function as signaling molecules in regulating N uptake and utilization[55,56].As a preliminary product of ammonium assimilation,glutamine,acting as an N signal,affects N uptake and expression of genes involved in N response in plants[57,58].Glutamine,rather than ammonium itself,is responsible for feedback regulation of ammonium uptake in Arabidopsis[59].However,the effect of amino acids on ammonium uptake in diverse plants is disputed[60].In rice,the ammonium transporter gene OsAMT1;1 shows expression in both shoots and roots,whereas both OsAMT1;2 and OsAMT1;3 are expressed mainly in roots[61].The protein activities and transcriptional expression of these three ammonium transporters are regulated by ammonium and glutamine[61-63].OsAMT1;1 and OsAMT1;2 show a positive and OsAMT1;3 a negative relationship with the content of glutamine instead of ammonium in rice roots[61],suggesting that the effects of glutamine on ammonium uptake and assimilation depend on N status in rice.

As a signal molecule,glutamine functions in sensing plant N status as well as in N metabolism,but the underlying mechanism is still poorly understood.PII is considered[64]to be a sensor for glutamine signaling in bacteria.Arabidopsis PII protein retains the ability to bind small molecules(such as ATP,ADP,and 2-ketoglutarate),but a deficiency of three amino acids in its C-terminal small loop structure leads to impaired glutamine binding structure[64-66].It has been shown[67]that the interaction between PII and N-acetyl glutamate kinase(NAGK,a key enzyme for synthesis of ornithine,a precursor of proline,polyamines,and citrulline)requires the binding of glutamine to the Q loop of PII.However,it is still unknown whether the complex formation of PII(OsGlnB)and OsNAGK1 is glutamine-dependent orindependent in rice[68].

Glutamate functions in neurotransmission in mammals and insects[69,70].But understanding of the mechanisms underlying glutamate sensing and signaling in plants is only emerging.External glutamate can be sensed by root tips to trigger root growth[55,71].Plants inherit glutamate receptor-like(GLR)genes with similarity to the ionotropic glutamate receptor(iGluR)family in mammals.In Arabidopsis,glutamate activates GLR,which converts the signal into changes in cytosolic Ca2+concentration for longdistance signal transmission[72].In the rice genome,the 24 putative GLR genes are categorized into four subfamilies:OsGLR1,OsGLR2,OsGLR3,and OsGLR4[73,74].OsGLRs have been identified[75]as being membrane-localized and involved in regulating ion uptake.OsGLR1.3 is essential for cell proliferation in the root apical meristem and is required for the survival of meristematic cells.Knockout of OsGLR1.3 results in a short-root phenotype[76].Glutamate can trigger Ca2+influx in rice roots[75].It is likely that OsGLRs serve as receptors or Ca2+channels for transducing glutamate signal to initiate root development and ion uptake.However,more experimental evidence is needed to confirm or challenge this hypothesis in rice.

3.3.Function in altering rice root and shoot architecture

Amino acids are generally considered as building blocks for protein synthesis and substrates of secondary metabolites.As one of the N sources,amino acids at suitable levels promote plant growth and development[77].In comparison to 1 mmol L-1NH4NO3,the supply of 1 mmol L-1glutamine as sole N source to rice plants for two weeks resulted in the same height,biomass and amount of accumulated N in the shoot,whereas supply of 1 mmol L-1asparagine increased shoot growth and N accumulation in comparison to free N(Fig.1).The benefits of organic N in plant development could result from the carbon bonus in organic N,which makes N assimilation more efficient[78].The C gained from root-absorbed organic N may directly participate in biomass incorporation and plant metabolism,saving the energy required for C and N assimilation[78].

Amino acids can elicit changes in root morphology[5,58,79,80].The inhibitory effects of exogenous amino acids on root growth were reported more than 80 years ago[81-84]and depend on the types and concentrations of amino acids as well as the plant species[82,85-87].Some amino acids increase root length as a sole N source within a certain concentration range,whereas amino acids mixed with inorganic N in culture solution inhibit root growth[85,88-92].As shown in Fig.1,although external supply of either glutamine or asparagine at concentrations of 0.1 and 1 mmol L-1increased N concentrations in both root and shoot,they dramatically inhibited root growth,particularly at the 1 mmol L-1level at the seedling stage.The effects of endogenous amino acids on root morphology have been characterized by use of mutants defective in amino acid contents.As examples,low free histidine induced a defect of root meristem maintenance and growth[93]and arginine biosynthesis inhibition reduced root length[94].

Amino acids are also involved in altering shoot architecture[95].In rice,provision of additional glutamine increased shoot growth and accumulation of free glutamine and glutamate suppressed tillering[96,97].Tiller bud growth and elongation was promoted by provision of external amino acids at around 1 mmol L-1but inhibited at higher concentrations,particularly by the basic amino acids lysine and arginine[98].The amino acid modulation of root and shoot architecture may be due to altered synthesis and activity of phytohormones.Amino acids can be conjugated to some hormones, including indole-3-acetic acid (IAA), a tryptophan-derived compound,and jasmonic acid(JA)[99,100].IAA-amino acid conjugates have the same hormonal activity as IAA itself to regulate root elongation in Arabidopsis[101].Supplying L-tryptophan to soil increased plant growth by promoting auxin production by soil microorganisms[102].Serotonin,a tryptophan-derived signal molecule,also functions in altering root morphology[103].Some amino acids can directly trigger or inhibit hormones or NO signals in plants[88].In rice,glutamine mediates root morphology by regulating de novo cytokinin biosynthesis,suggesting that glutamine serves as a signal to regulate phytohormones[104].

3.4.Function in regulating flowering time in rice

As endogenous signal compounds,amino acids are involved in regulating flowering time[105,106].However,such regulatory effects may rely on plant species and N status.For example,targeted expression of a soybean GS1 in tobacco delayed flowering,whereas it promoted flowering in Lotus corniculatus[107,108],indicating the complexity of the role of glutamine synthesis in regulating plant flowering.In rice,mutation of OsGS1;1 severely suppressed rice growth and flowering time [109],whereas overexpression of OsGS1.1 promoted rice flowering[110].Increasing the activity of Fd-GOGAT resulted in delay of senescence and prolonged the growth period in rice[111,112].These results strongly support the very recent finding that glutamine,not ammonium itself,promotes flowering time in rice[112].

3.5.Function in regulating plant defense to stresses

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system[113].For example,the acylation of amino acids can increase plant resistance to pathogens and pests by the formation of protective plant metabolites or modulation of plant hormone activity[113].Proline functions in various plant species in defense against both biotic and abiotic stresses,includ-ing drought,salinity,light and UV irradiation,heavy metals,and pathogens[114-116].In rice,proline,glycine,glutamate,and glutamine function in stress response and disease resistance[117-119].Provision of glutamine to rice roots elevated the expression of several transcription factor genes DREB1A,IRO2,and NAC5,which are involved in regulation of stress response[58].External supply of glutamate to a salicylic acid(SA)-deficent mutant induced the expression of SA-responsive genes and rescued susceptibility to rice blast,suggesting that glutamate might act as a provider of defense components by regulating the SA pathway[97,120].

Fig.1.Effect of forms and concentrations of N on rice growth at seedling stage.Rice(cv.Nipponbare)was grown in basal nutrient solution containing 1 mmol L-1 NH4NO3 for 14 days that was then replaced with glutamine(0.1 mmol L-1:0.1-Gln,and 1 mmol L-1:1-Gln)or asparagine(0.1 mmol L-1:0.1-Asn,and 1 mmol L-1:1-Asn,)as sole N source in addition to a continual supply of 1 mmol L-1 NH4NO3(+N)or removal of N(-N)for 14 days.(A)Phenotypes of plants under the N treatments.Scale bar,6 cm.(B)Total N concentration.(C)Length of shoot and root.(D)Total dry weight.Values are means±SD(n=4).One-way ANOVA was used for the statistical analysis(*,P≤0.05).

4.Amino acid transporters in rice

4.1.Classification and molecular evolution

Plant roots may capture amino acids from soil using H+-ATPase driven proton co-transporters[121].Membrane amino acid t ransporters (AATs)mediate both transport across intracellular membranes and translocation of amino acids within the plant[122].In general,plant AATs include two main families,the amino acid/auxin permease(AAAP)superfamily and the amino acid polyamine and choline transporter(APC)superfamily.The AAAP superfamily contains at least seven subfamilies:amino acid permeases(AAPs),lysine-histidine-like transporters (LHTs),proline transporters(ProTs),γ-aminobutyric acid transporters(GATs),aromatic and neutral amino acid-like transporters(ANTs),auxin transporters(AUXs),amino acid transporter-like(ATLs)and amino acid vacuolar transporters(AVTs).The APC superfamily includes subfamilies of the cationic amino acid transporters(CATs),bidirectional acid transporters(BATs)and L-type amino acid transporters(LATs)[123-125].In the rice genome,at least 85 putative genes encoding AATs have been isolated[124].In comparison with identified ATLs in rice[124],ATL-similar sequences in Arabidopsis encode vacuolar transporters[123,126].

Owing to gene duplication,the phenomenon of expanded members of the transporter family is common in plant evolution.However,AAT sequences are quite conserved among plant species.The identity between homologous genes of AATs can be as high as 98%[124,127].As functionally characterized in both Arabidopsis and rice,some AAPs and LHTs are involved in both vascular and nonvascular amino acid transport.AAP transporters seem to be restricted to land plants with their major function in phloem(vascular tissue)loading of amino acids,while LHT transporters appeared before the early land plants.

4.2.Subcellular localization and substrate selectivity

The characterized Arabidopsis AATs have been shown[122,128]to be localized in the plasma membrane,whereas rice AATs have multiple subcellular locations(Table 1).Expression analyses in rice protoplasts show that OsAAP3,OsAAP5,and OsLHT1 are located on the plasma membrane,whereas OsAAP6 are located on the endo-sperm reticulum(ER)instead of the plasma membrane or trans-Golgi network[98,129-131].OsAAP1 is probably located in both the plasma and nuclear membranes,whereas OsAAP4 is observed in both the plasma membrane and the nucleus[132,133].Given that the subcellular localization of AATs expressed in heterogenous systems could be affected by many factors,such as pH,signal peptides,or promoters[129],it is necessary to use multiple expression systems to identify the precise locations of AATs in plant tissues.But the diverse AAT subcellular localization in rice hints that some AATs play roles beyond transport.

Several rice AATs belonging to the AAP and LHT subfamilies have been functionally characterized.These proteins display diverse substrate selectivity and affinity when expressed in rice protoplast or heterologous systems including yeast cells and Xenopus laevis oocytes(Table 1).OsAAP1,OsAAP7,and OsAAP16 have similar substrate specificities and transport a broad spectrum of amino acids[129].OsLHT1 mediates acidic and neutral amino acid transport with high affinity[5,130].In contrast,OsAAP3 displays a preference for basic amino acids[129],while OsAAP5 can take up both basic(Lys,Arg)and neutral(Val,Ala)amino acids[98]and OsAAP4 transports neutral amino acids in rice protoplasts[133].The differing localizations and substrate preferences of AAPs and possibly other AATs may represent the differing requirements of amino acids in different rice tissues and adaptation to varying N supply status.

5.Transporters for amino acid uptake and translocation from source to sink in rice

5.1.Acquisition and partitioning of amino acids in roots

Since the establishment of plant mineral nutrition theory,the contribution of amino acids in soils as N source for plants has been paid little attention[134].In some soil ecosystems with limited levels of inorganic N,such as forest,grassland and arctic or lowfertility soils,amino acids are the dominant form of N[135]and plant roots tend to take up organic rather than inorganic N[136-138].For example,an arctic sedge,Eriophorum vaginatum,prefers and has better ability to absorb amino acids in the field[139].

In the absence of evidence that rice roots can absorb peptides and proteins,amino acid N is generally used as a synonym for organic N available for rice.Rice roots are capable of acquiring a wide range of amino acids[1,6,97,140].Generally,amino acid uptake occurs via root tips and root hairs and the uptake rate is determined primarily by the activities of AATs on the plasma membrane[122].Rice OsLHT1 supported yeast cell growth on a broad range of amino acids and increased the uptake of acidic amino acids and asparagine in the high-affinity range with Km values of 50-80μmol L-1[5].OsLHT1 is expressed in root hairs and lateral roots of rice plants.Short-term uptake analysis of four individual15N-labeled amino acids(aspartate,asparagine,glutamate,or glutamine)as sole N revealed that OsLHT1 participates directly in the process and contributes 40%-66%of the capacity for root amino acid acquisition in rice[5,141].In comparison with a wild type,Osaap6 mutants showed lower root absorption of a range of amino acids[131],indicating that OsAAP6 is also involved in root uptake of amino acids.OsAAP1,OsAAP4 and OsAAP5 are expressed in root epidermis[98,132,133],but their contribution to root acquisition of amino acid from soil has not been confirmed.In Arabidopsis,several AATs have been implicated in root amino acid uptake,including AAP,LHT,and ProT families[7,122,142](Fig.2).Other rice AATs participating in root amino acid absorption await discovery.

Amino acids are among the main components of root exudates produced in response to changes of N availability in soil and N status in plants,though it remains unresolved how the exudation of amino acids is controlled in roots[143].Considering that amino acid concentrations in rice roots are much higher than those in soil solutions,the concentration gradient might theoretically generate a strong driving force for leakage of amino acids from roots,and the process of amino acid exudation does not need exporters or carriers[143].However,it has been shown[80]that root tips can sense changes in specific amino acid concentration and transduce these signals to modify root growth,leading to challenges[143-145]to the idea of root exudation as a purely unregulated passive process.In Arabidopsis,several members of the Usually Multiple Amino Acids Move In and Out Transporter(UMAMIT)family are bidirectional amino acid facilitators and are involved in export of amino acids from phloem of both roots and seeds[146-148].It remains unknown which efflux transporter(s)could work in root epidermis and what is the molecular mechanism involved in root amino acid exudation in rice.

Amino acids are transported in roots via both the symplastic and apoplastic pathways.As shown in Arabidopsis[122]and rice(Fig.2;Table 1),AATs have diverse root cellular localizations,substrate specificities,and uptake capacities for amino acids,possibly accounting for the large number of AATs expressed in roots.In rice,the Casparian strip located in both exodermis and endodermis blocks apoplastic amino acid flow to the stele[149].With the destruction of the cortex cells between the exodermis and endodermis,rice mature roots have a highly developed aerenchyma[150,151].Along with uptake by root epidermis,amino acids may be transported into exodermis cells by AATs importers,released into the apoplast across the aerenchyma by AAT exporters[122,149],and then transported to the endodermis via the symplastic pathway and further released to the root vasculature(Fig.2).OsLHT1 is localized in root epidermis,exodermis,sclerenchyma,cortex cells,and endodermis,supporting the notion that OsLHT1 contributes to the import of apoplastically moving amino acids into root cells[5].

5.2.Transport of amino acids from root to shoot

Following loading into the root xylem,amino acids are translocated to the shoot by‘‘transpiration pull”[152].Amino acids represent the main N forms for long-distance translocation[122,142].Their concentrations in rice xylem can reach 10 mmol L-1and glutamine,glutamate,asparagine and aspartate are the dominant forms[43,131].Amino acid levels in rice aboveground organs are also much higher than those in roots[5].These two observations together support the idea that most amino acids taken up by or synthesized in roots are transported to the shoot via the xylem.The amino acid importers OsLHT1,OsAAP1,OsAAP5,and OsAAP6 are expressed in vascular tissues of roots,indicating their functions in amino acid movement into steles[5,98,131,132].Loss of function of OsLHT1 leads to a decrease in15N-aspartate allocation from root to shoot,demonstrating that restricted root-to-shoot amino acid translocation in Oslht1 knockout plants results from decreased amino acid levels in the xylem sap and leaves[5].Mutation of OsAAP6 also reduced concentrations of total amino acids in xylem sap,supporting the notion that OsAAP6 contributes to translocation of amino acids in rice[131].Xylem loading requires export systems,but efflux transporters for the process of amino acid distribution have not yet been identified in rice.

During long-distance transport in the xylem to the shoot,amino acid exchange between xylem and phloem occurs(Fig.2).During transfer into the xylem parenchyma,amino acids move to phloem parenchyma cells symplastically and are ultimately released into the phloem[122,151,153,154].Amino acids in xylem sap in mature leaves can be transferred first into mesophyll cells by importers and stored briefly in the leaves before being distributed to sinkorgans[122].In Arabidopsis,AtAAP2 and AtAAP6 function in xylem-phloem transfer,while AtLHT1 as an importer contributes to amino acid distribution from apoplast into mesophyll cells[155-157].However,there is no clear evidence identifying the amino acid transporters involved in long-distance distribution in rice.

Table1Rolesof characterized aminoacid transportersin rice.

Fig.2.A distinct amino acid transport pathway in rice and characterized transporters in rice and Arabidopsis.Amino acids(AAs)are taken up into roots and then distributed to the leaf and seed through vascular tissues.Dotted lines show the movement of AAs following the symplastic and/or apoplastic pathways.Amino acid influx and efflux transporters are required for intercellular and intervascular transfer.Transporters with known tissue localization and functions are shown in orange(rice)and gray(Arabidopsis)boxes.Details of rice amino acid transporters can be found in Table 1 and the main text.Roles of amino acid transporters in Arabidopsis are described in reviews[122,142,171].Ep,epidermis;Ex,exodermis;Sl,sclerenchyma layer;Co,cortex;En,endodermis;Pc,pericycle;Xy,xylem;Ph,phloem;Xy-par,xylem parenchyma;Ph-par,phloem parenchyma;Me,mesophyll cell.

5.3.Distribution of amino acids from source leaf to sink organ and seed

In rice,nodes with well-organized vascular systems are key tissues for allocating nutrients.Among them,node I consists of both enlarged vascular bundles(EVBs)and diffuse vascular bundles(DVBs)that control nutrient distribution to flag leaves and panicles[150,158].Transport of nutrients from source tissues to developing seeds requires intervascular transfer from EVBs to DVBs,which presumably requires various transporters[150,151,159].

Amino acids are the primary forms of N transported within the plant and are distributed to flag leaves and panicles through nodes at emergence of the panicles[160,161].OsLHT1 is abundantly expressed in rachis and node I,especially in the vascular bundles of leaves.Its knockout resulted in gradual increased amino acid accumulation in the flag leaf during the stages from anthesis initiation to maturity[5,162].OsLHT1 has been shown[162]to be responsible for phloem loading and allocation from leaf to panicles.OsAPP6 is expressed in vascular bundles in nodes and contributes to the distribution of amino acids to flag leaves and hulls[131].

Amino acid transfer into the seeds requires symplastic phloem unloading[163].The rice seed consists of three main parts:seed coat,embryo,and endosperm[131,164].Given the isolation of the endosperm from the embryo,amino acids released from the seed coat may be transported directly into the embryo or be imported into the endosperm and then released into the seed apoplasm(Fig.2)[122,153].Amino acids imported into the embryo are used to synthesize storage compounds that support growth or seed development.In rice,OsAAP6 as an importer of amino acids is expressed in the endosperm,ovular vascular trace end,and lateral stylar vascular traces,and functions in changing grain storage protein content and total amount of amino acids[131].Amino acid transfer from source leaves to sink organs including seeds also requires exporters,but these remain unidentified in rice.

5.4.Effects on N-use efficiency,grain yield,and quality

Genetic manipulation of AATs to increase amino acid transport from source to sink increased both plant N uptake and utilization efficiency in both pea(Pisum sativum)[8]and Arabidopsis[165].In rice,the functions of OsAAP1,OsAAP3,OsAAP4,OsAAP5,and OsLHT1 as plasma membrane transporters in sustaining N allocation during plant growth and development have been characterized(Table 1).Knockout of OsLHT1 reduced allocation of amino acids from root to shoot,causing high accumulation in roots of amino acids that in turn,acting as feedback signal compounds,further reduced acquisition of ammonium,resulting in lower total NUpE(Fig.3)[162].OsLHT1 also functions as a switch for allocating amino acids to shoots and grain.OsLHT1 mutation reduced the distribution of amino acids to grain and the alteration of grain storage compounds,ultimately leading to reduction in the pasting properties of endosperm starch and lower grain yield and NUtE[5,162].OsAAP1 and OsAAP4 promoted rice growth and grain yield,whereas OsAAP3 and OsAAP5 functioned as negative regulators of grain yield[98,132,133,166].These four transporters may affect rice NUE indirectly by regulating rice shoot architecture(see the sixth part of this review).OsAAP6 increases grain protein content by increasing glutelins,prolamins,globulins,albumins,and total amino acids,thereby improving grain nutritional quality[131].These reports support the idea that engineering AATs to enhance N allocation from source to sink would be an effective strategy for improving crop productivity as well as N use efficiency in a range of N environments[8].

Fig.3.A rice amino acid transporter affecting rice growth and nitrogen use efficiency.OsLHT1 is a rice amino acid transporter that has been characterized in detail in uptake and distribution of nitrogen(as ammonium and amino acids)at multiple growth and development stages[5,162].The values in the figure show the relative amounts(in%)of root uptake of amino acids and ammonium at vegetative growth stage,and final relative distribution(in%)and use efficiency of total N at the mature stage in the paddy field for a wild type(WT)and an Oslht1 mutant.NUpE,nitrogen uptake efficiency(total accumulated N/total supplied fertilizer N);NUtE,nitrogen utilization efficiency(total grain yield/total accumulated N).NUpE was calculated based on a 180 kg N ha-1 application level in paddy field.Values are from Guo et al.[5,162].

6.Roles of rice amino acid transporters beyond transport

6.1.As transporters of non-amino acid substrates or as amino acid sensors

Besides their basic roles as transporters in the acquisition and distribution of amino acids,some AATs can transport other substrates.For example,Arabidopsis LHT1 can transport 1-aminocyclopropane-1-carboxylic acid(ACC),a biosynthetic precursor of ethylene[167].In rice,external supply of aspartate or glutamate reduces primary root growth,while knockout of OsLHT1 prevents the inhibitory effects of aspartate but not the effects of glutamate[5].In view of the substrate diversity of LHT1,it is still unknown whether the growth phenotype of Oslht1 mutant plants is due to hormone signaling or an amino acid transport defect.The transport properties of amino acid transporters have been characterized in diverse heterologous systems,possibly accounting for some inconsistency(Table 1)[5,130].To date,there is no evidence in rice for the roles of amino acids in transporting substrates other than amino acids.Still,it is important to bear this diversity in mind in investigating the physiological functions of AATs in plants.

Although amino acid transport activity and carbohydrate metabolism must be tightly coordinated for efficient metabolism ofamino acids,the genes involved in amino acid sensing in plants have not been unequivocally identified.Some plant transporters can serve as transceptors that play dual roles in transport and sensing specific ions or compounds.For example,in Arabidopsis,NRT1.1(CHL1),a dual-affinity nitrate transporter,have been named as a nitrate transceptor[168].Likewise,plant cells may harbor receptors,such as a glutamate receptor[56],to sense changes in concentration of both external and internal amino acids.In yeast,a general amino acid permease 1(Gap1)uses the same sites for amino acid binding or transport and signaling[169,170].Certain members belonging to the three main AAT families(AAAP,APC,and UMAMIT)may also evolve as transceptors of amino acids in plants[171].

6.2.Enhancing plant resistance to pathogen attack

Amino acid metabolic pathways can regulate plant immunity to pathogens by influencing crosstalk between biosynthesis and signaling of salicylic acid(SA)and/or jasmonic acid(JA)[172-174].Some plant AATs function in suppressing pathogen growth.For example,disruption of LHT1 in Arabidopsis enhanced disease resistance to a broad spectrum of pathogens in a SA-dependent manner[175].Cationic amino acid transporter 1(CAT1)facilitates basic amino acid uptake;its overexpression increased resistance to the hemi-biotrophic bacterial pathogen P.syringae by activating the SA pathway[176].It is not known which AATs in rice function in enhancing plant defense against pathogens.The evidence from Arabidopsis encourages research to identify the relationships between amino acid metabolism and allocation and resistance to various pathogen types,particularly to resolve the molecular mechanisms linking amino acid transport and defense responses in rice.

6.3.Regulating rice shoot architecture

In rice,some AATs function in regulating shoot architecture,including plant height,tiller and kernel number.Physiological and phenotypical analysis of an OsAAP1 overexpression line and its knockout mutants indicated[132]that OsAAP1 promoted neutral amino acid uptake and reallocation and increased effective tiller number and final grain yield.It is known[177-179]that cytokinin promotes,whereas auxin and strigolactone(SL)inhibit,bud growth and shoot branching.As indicated by transcriptome analysis[132],OsAAP1 induced changes of tiller initiation and elongation associated with the auxin,cytokinin,and strigolactone signaling pathways.Similarly,OsAAP4 functions in increasing tiller number and grain yield,probably by altering the expressions of genes in the auxin and/or cytokinin signaling pathways[133].In contrast,OsAAP3 and OsAAP5 function as negative regulators in rice tiller bud growth and grain yield[98,166].Inactivation of OsAAP5 increased the concentrations of cytokinins(cis-zeatin and dihydrozeatin),whereas its overexpression showed opposite effects,indicating that OsAAP5 regulates tiller number by affecting cytokinin levels[98].Likewise,OsAAP3 inactivation promoted growth and elongation of buds and increased tillering,resulting in increased grain yield[166].

7.Natural variation in amino acid transporters in rice germplasm

Asian cultivated rice has two major subspecies:the O.sativa xian group(indica)and geng group(japonica),and 29 million single-nucleotide polymorphisms(SNPs)have been identified among 3024 fully sequenced rice genomes[180].Genome-wide association studies(GWAS)reveal[181,182]that natural variation in specific genes contributes to the diversity of rice growth and development and to rice adaptation to changed environments.

In a large-scale analysis of SNP profiles[5],many individual AATs showed variation among rice germplasm accessions.The sequences of characterized rice amino acid transporters showed divergence and were associated with altered uptake rates of amino acids,grain N content,and tiller number.For example,both geng and xian rice subspecies took up amino acids,but geng at much higher levels.Analysis of the association between15N-aspartate uptake and SNPs of all rice putative OsAATs in 68 rice accessions from geng and xian backgrounds revealed that OsLHT1 is a major gene determining root amino acid acquisition.OsLHT1 also contributes to allocation of N from source leaves to panicle and affects grain quality[162].Analysis of OsLHT1 SNPs in promoter and coding region sequences revealed a total of 10 haplotypes,with clear separation between xian and geng subspecies.Expression of OsLHT1 in geng is commonly higher than that in xian,with evidence of a positive association between root aspartate uptake and expression of OsLHT1[5].

Based on sequence variation among 197 accessions in a rice mini-core collection,OsAAP6 displayed eight haplotypes that were placed into two groups[131].The cultivars in group 1 tended to show higher OsAAP6 expression levels in endosperm and higher grain protein content(GPC)than those in group 2,and natural variation of rice GPC may be due in part to differences in OsAAP6 expression.Similarly,based on the variation of OsAAP3 or OsAAP4 sequences in their encoding and promoter regions among 524 accessions,the rice population could be divided into 25 OsAAP3 and 5 OsAAP4 haplotypes[133,166].Expression of OsAAP3 showed a negative correlation with tiller number in the population,whereas OsAAP4 showed a positive correlation[133,166].OsAAP5,which negatively regulated rice tiller number and grain yield in geng cultivars,showed high divergence in its promoter region between xian and geng[98].Given that altering the expression of OsAAP3 did not affect OsAAP5 expression and vice versa in their transgenic lines,the effects of OsAAP3 and OsAAP5 on tiller number may be independent.It is thus possible that appropriate combination of the divergent OsAAP3 and OsAAP5 haplotypes would improve the rate of initiation,elongation,and total effective tiller number[98,166].

As indicated by natural variation of OsNPF6.5[4]and OsNR2[183]in the rice genome,xian cultivars show higher nitrate absorption and reduction activity and more tolerance to low N supply[4,183]but generally less favorable grain quality[184]than found in geng cultivars.In view of the contrasting inorganic and organic N uptake of xian and geng cultivars[4,5,183],testing whether geng alleles of OsLHT1 can be used to improve the efficiency of amino acid uptake and distribution,as well as grain quality,in xian subspecies is merited.

8.Concluding remarks and future issues

The striking advances in recent decades in understanding the regulation of N use in plants are the identification of multiple transporters,transcription factors,and several plant-specific sensors for nitrate and ammonium[185,186].However,plant roots can also simultaneously acquire and secrete amino acids in the rhizosphere.Currently,it is not possible to quantify the exact amount of root N uptake in organic forms,mainly as amino acids in soil,particularly in rice paddy fields under ADWmanagement.Efficient use of the N inside the rice crop for both yield and quality relies on proper amino acid-centered N metabolism and allocation of N mainly in the form of amino acids.

Most steps of amino acid transport in plants are tightly controlled by membrane AATs.Genetic manipulation of some AATscan increase plant biomass,seed yield,and/or quality as well as NUE,offering a promising strategy for crop improvement.However,the cell-autonomously and systemically regulatory network of AATs expression and activity,as well as signaling and metabolism of amino acids,remains elusive and awaits investigation.Functional characterization of plant AATs has been performed mainly in Arabidopsis,while research on their counterparts in rice and other major crops is scarce.Among total 85 putative AATs that have been annotated in the rice genome,only a few have been characterized as altering either transport or metabolism of amino acids or architecture(Table 1).It is still difficult to identify systems for amino acid import and export and candidate AATs in N signaling in rice.

Long-distance transport of amino acids occurs in the xylem and phloem.The loading and unloading of amino acids in these tissues require the coordinated activity of AATs mediating cellular import and/or export of amino acids.Most characterized plant AATs are involved in influx of amino acids,whereas AATs functioning in unloading of amino acids,particularly in rice,remain to be identified.The AATs responsible for cellular compartmentation of amino acids,such as for vacuolar transport and efflux,are largely unknown[187].For better understanding of proper amino acid allocation,the coordination between symplastic and apoplastic transport processes of amino acids should be investigated.

Optimized coordination of N and carbon partitioning processes is critical for improving plant yield and NUE.Genetic manipulation of AATs is potentially able to alter plant performance under varied N,along with coordinating N assimilation and metabolism,and ultimately increase yield and NUE.Identifying natural variation of AATs among germplasm accessions combined with their phylogenetic analysis and transporter structures may reveal key genes involved in acquisition and source-to-sink translocation of amino acids.For breeding new rice cultivars with high yield and NUE,it is desirable to find combinations of haplotypes that balance the acquisition and distribution of inorganic and organic N.

CRediT authorship contribution statement

Guohua Xuconceived the idea;Nan Guo,Shunan Zhang,andMingji Guprepared the figures and table.All authors wrote and approved the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(31930101),National Key Research and Development Program of China(2016YFD0100700),Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization and the Innovative Research Team Development Plan of the Ministry of Education of China(IRT17R56 and KYT201802),and the Priority Academic Program Development from Jiangsu Government.