Genotypic And Functional Analyses Of OsLHT1 In Root Uptake And Allocation Of Amino Acids And Resistance To Magnaporthe Oryzae Infection In Rice

In agricultural soil,nitrogen(N)is present in both inorganic and organic forms that can be interconverted mainly via soil microbe-controlled mineralization and assimilation processes.Ammonium(NH4+)and nitrate(NO3-)are two major inorganic N compounds,while amino acids,peptides and proteins mainly contribute to the organic soil N.Total free amino acid concentrations in soil solution may be up to 150 μM,with aspartate,glutamate,asparagine,glutamine,glycine and alanine being the predominant forms.Rice can absorb both inorganic and organic N.Root uptake of both inorganic and organic N occurs mainly via root hairs conveyed by plasma membrane-localized N transporters on epidermal cells.Root acquired and synthesized amino acids are transported via xylem and phloem to supply sink growth and development.Amino acids are the major transport forms of N for N distribution in rice grown in flooding paddy soil.Movement of amino acids from the location of synthesis(source)to the sites of usage(sink)requires membrane proteins(i.e.transporters)functioning as export or import systems.Two rice subspecies are cultivated worldwide,which are japonica and indica.Genome-wide survey in rice has identified at least 85 genes putatively encoding amino acid transporters(OsAATs),including 19 OsAAPs and 6 OsLHTs genes.However,none of these transporters was ideintified to play a direct role in root amino acid acquisition in rice.In the current study,rice Lysine-Histidine-type Transporter 1 gene(OsLHT1)was identified as a candidate associated with root uptake rate of aspartate among rice germplasms.Biochemical and physiological analyses in yeast and planta revealed that OsLHTl plays critical functions in amino acid uptake and allocation,and resistance to Magnaporthe oryzae as well.The main results are summarized as follows:1.Both japonica and indica subspecies can directly acquire amino acids.When fed for 2 hours with 0.1 mM 15N-aspartate as sole N source in a hydroponic culture system,all tested 68 rice accessions(japonica and indica subspecies)enriched 15N-aspartate in roots,while japonica acquired aspartate with 1.5 fold on the average than indica.2.OsLHT1 is associated with change of root aspartate uptake rate among rice accessions.Gene-based genetic association between 15N-aspartate uptake rate and single-nucleotide polymorphisms(SNPs)of 85 putative OsAAPs genes was analyzed.The resulting Manhattan plot showed one strong nucleotide variation peak among the OsAATs that represent rice Lysine-Histidine-type transporter 1.The japonica cultivars displayed higher 15N-aspartate uptake and higher OsLHT1 expression levels than the indica cultivars,supporting a positive association between OsLHT1 expression and 15N-aspartate uptake.3.OsLHT1 is expressed throughout the root and leaves.OsLHT1 promoter-GUS studies displayed strongest GUS staining in root hairs and lateral roots.Analysis of root cross sections demonstrated the GUS staining throughout the root,indicating OsLHT1 expression in root epidermis,exodermis,sclerenchyma,cortex,endodermis,pericycle of the root stele.GUS staining was also detected in leaf blades,revealing OsLHT1 expression abundantly in major and minor veins,and mesophyll cells surrounding the vasculature.4.OsLHT1 mediates growth of yeast on a broad spectrum of amino acids and transports acidic amino acids and asparagine in a concentration-dependent manner.The Km values for aspartate,glutamate and asparagine were 55 pM,48 μM and 74 μM,respectively,suggesting that OsLHT1 transports these amino acids at high affinity.5.OsLHT1 function affects rice growth on media containing acidic amino acids.Four independent Oslhtl mutant lines using the CRISPR-Cas9 system were generated to investigate the function of OsLHT1.Both Oslht1 mutants and wild-type(WT)plant could grow on the media containing aspartate,glutamate,asparagine and glutamine as a sole N source.When these plants were fed for 30 min with a nutrient solution containing 100 μM 15N-aspartate,15N-asparagine,15N-glutamate or 15N-glutamine as sole N,15N levels in the roots were 40 to 66%lower in Oslht1 mutants compared to WT,supporting a function of OsLHT1 in root import of acidic amino acids and amides.6.OsLHTl plays a role in amino acid allocation from root to shoot.After fed 1 mM 15N-aspartate for 6 hours in a nutrient solution,56%of the total enriched 15N in WT was found in root tissue and 44%in the shoot,while 71%of total acquired 15N remained in the roots and 29%of 15N was detected in the shoots in Oslht1 mutant lines.In addition,no differences of total free amino acid content between WT and Oslhtl mutants were found in roots,while reduction of the amino acids in leaf sheaths and leaf blades of Oslht1 mutants by up to 10%and 9%in comparison to WT.Further,significant decreases of amino acid concentrations in the xylem sap were observed for the mutants.The data indicated that OsLHTl affects root to shoot translocation of amino acids.7.OsLHTl is essential for growth and development of rice plants.Relative to WT,significant decreases were observed for Oslht1 plant height as well as for Oslhtl culm,leaf and panicle biomass during anthesis and at maturity.Oslht1 mutants had low grain number and grain weight,resulting in low grain yield.The mutants accumulated much more N in vegetative tissues at the reproductive growth phase than WT plants,suggesting that OsLHTl affects leaf-to-panicle amino acid translocation.In addition,OsLHT1 mutation resulted in decrease of leaf N accumulation and alteration of grain storage compounds,showing higher protein and amino acids and lower amylose in the grains.The altered storage compounds in Oslhtl grains affected cooked texture and pasting properties of the endosperm starch.Compared with WT,the peak and cool paste viscosity of the Oslhtl starch were all decreased8.OsLHTl is involved in rice resistance to biotic stress.Oslhtl mutants displayed iron-red blades at the tillering stage and thereafter.Leaf metabolomics analysis showed that 92 metabolites were different between WT and Oslhtl mutants,including amino acids and flavonoid compounds that are involved in N metabolism and anthocyanin biosynthesis.Disruption of OsLHT1 activated expression of defense-related genes involved in salicylateand jasmonate-responsive signaling,resulting in higher accumulated reactive oxygen species in the leaves at mutant than that at WT.Furthermore,when performing pathogenicity assays with spraying conidial suspensions of Magnaporthe oryzae onto leaf blades,the Oslht1 mutants showed significantly reduced susceptibility compared with WT.Abundant and typical lesions were found on WT leaves while a few smaller lesions on Oslht1 leaves,demonstrating that loss of OsLHTl activity enhanced the rice resistance against Magnaporthe oryzae infection.Overall,these results together provide the evidence that OsLHTl functions in both root uptake,root-to-shoot and leaf-to-panicle allocation of a broad spectrum of amino acids in rice.It also plays an important role in rice growth,development,grain production and resistance against Magnaporthe oryzae infection.The findings pave a way to apply the useful allelic variations or haplotypes of OsLHT1 gene in improving rice N use efficiency and resistance to biotic stress in future.