| Grain filling dominates the formation of rice yield and quality,as is affected by multiple genes and environments.Nitrogen(N)fertilization,the most important practice improving rice yield,has a substaintial effect on grain filling.The poor grain filling of inferior grains caused by insufficient source limits high yield potential of super rice varieties under high nitrogen input.From perspective of source-sink relationship,a better understanding of N regulating mechanism for grain filling helps to lay the foundation of genetic improvement and optimization cultivation on rice yield and quality.A pot experiment with four treatments of N rate was performed including the control non N(NO),low N level(LN),medium N level(MN),high N level(HN)were applied during the whole panicle development stage,using the japonica rice cultivar Ningjing5 as material.We compared physiochemical properties of leaf,grain,and bracts in response to N,and profiled the metabolome and transcriptome of these organs,with the aims of elucidating the physiological and molecular foundation of nitrogen on grain filling.The major results are as follows.(1)Dynamic changes of AA,mainly the protein amino acids,in leaf,grain,and bracts were monitored,and the redistribution rate of protein-N from leaf and bracts and its contribution rate to grain protein-N were calculated accordingly.Results showed:(i)The amino acid composition of bracts were similar to that of leaf,but quite different from that of grain.For example,the bracts and leaf had the same order of the most abundant and least four amino acids,while the grain did not.(ii)The amino acids in response to N,AAs in leaf and bracts were more sensitive to N fertilizer than those in grain.(iii)High protein-N redistribution rate of leaf and bracts was observed,being 69.88%,69.62%and 67.42%for leaf,bracts of superior spikelets and inferior spikelets,respectively.N fertilization significantly increased the redistribution rate of leaf protein-N.Averaged contribution rate of leaf protein-N to grain was 23.38%.Notably,a substantial contribution rate of protein-N in bracts was detected,with the mean values being 10.19%and 9.77%for bracts of superior spikelets and inferior spikelets,respectively.These findings collectively suggest that bracts of rice spikelets be a major source of N remobilization for grain filling,and their importance be acquired when exploring the mechanism underlying nitrogen use efficiency.(2)The metabolomic analysis was performed in leaf,bractsand grain,resulting in 196,189 and 248 defferentially experessed metabolites,respectively,covering most of the central metabolic pathways and partial secondary pathways.(ⅰ)In metabolically,the number of metabolites in leaf and bracts were obviously less than that of grain.One hundred and forty five common compounds were detected in these threeorgans,14,5,59 unique compounds were found in leaf,bracts and grain,respectively.The majority of metabolites in grain and bracts showed a declining trend,while those in leaf did not.Pyruvate and α-ketoglutarate contentsin leaf,bracts and grain decreased as the grain matured.The contents of citrate and isocitrate increased in leaf,while those of succinate,fumarate and malate decreased.The contents of isocitrate,succinate,fumarate and malate in the late stage of grain filling(15DAA)were significantly increased in bracts.All these organic acids contents in grain were significantly decreased.(ⅱ)The metabolomic response to nitrogen,N fertilizer significantly increased most AAs contents during the whole grain filling stage in leaf,the positive effects on AAs occurred before 15DAA and 10DAA in bracts and grain,respectively.Contents of hexose,sucrose,trehalose and sugar alcohol decreased,resulting in the carbon compounds occurring relatively deficient,indicating the flux of carbohydrates into the shikimate pathway or TCA cycle.Nitrogen inceased the content of hexoses,sugar alcohols and most AAs contents,while sucrose and trehalose were decreased in bracts.N had no significant effect on most carbohydrates in grains.(ⅲ)From the two aspects of metabolome and nitrogen response,the bracts are the organs between the source(leaf)and the sink(grain).(3)Transcriptome profilings were conducted in leaf,superior and inferior grains,with 3077,395,and 309 differentially express genes(DEGs)detected,respectively.(ⅰ)In gene expression,the number of DEGs in leaf was smaller than that in grain,and the number of up-regulated DEGs was smaller than that of down-regulated.The number of DEGs in the superior grains was greater than that in the inferior ones,and the up-regulated genes were less than down-regulated genes.(ⅱ)Gene expression in response to nitrogen,the number of DEGs in leaf was much larger than that in grain,and there were no obvious difference between superior and inferior grain.Nitrogen enhanced the ability of nitrogen assimilation and amino acid synthesis in leaves,especially for aromatic amino acids and sulfur-containing amino acids,flowing to lignin synthesis.The expression of photosynthesis related genes in leaf was increased,resulting in a higher photosynthetic capacity.The glycolysis pathway enhanced,while the TCA cycle and oxidative phosphorylation weakened as amount of PEP flowing to aromatic amino acid metabolism.The increased expression of TPP in leaf may lead to a reduction of trehalose 6-phosphate(T6P),which enhanced the expression of starch synthesis genes SS,resulting in a decrease of sucrose,reduced sucrose supply capacity to grain.N fertilizer enhanced the expression of ABA2,NCED and CPS in grains.The ability of IAA synthesis increased in superior grains,while the ability of ethylene synthesis decreased.Photosynthetic capacity enhanced,and the key genes AGPase and SS regulating the starch synthesis in superior grains were significantly up-regulated at the earlygrian filling stage.The expression of TPP in inferior grains was increased,which may lead to reduction of T6P.The expression of hisC that control the synthesis of Phe and Tyr in inferior grains was enhanced.(ⅲ)In general,N enhanced the photosynthetic capacity of the superior grain,the starch synthesis capacity,the IAA synthesis capacity,but weakened the ethylene synthesis.The decomposed ability of T6P increased,while the synthesis ability of Phe and Tyr enhanced in inferior grains.In summary,(ⅰ)In leaf,Nitrogen fertilizer increased the expression of gene related to photosynthetic pigments,photosynthetic phosphorylation and carbon assimilation pathways.Simultaneous,though the glycolytic pathway increased,but TCA cycle and oxidative phosphorylation decreased,which may caused by a mount of PEP flowing to aromatic amino acid metabolism.The ability of nitrogen assimilation and amino acid synthesis were up-regulated,especially for aromatic amino acids and sulfur-containing amino acids,resulting in the vast majority AAs contents significantly increased.(ⅲ)In bracts,nitrogen inceased the content of hexoses,sugar alcohols and most AAs contents,while sucrose and trehalose were decreased.(ⅲ)The response to nitrogen differed from superior and inferior grains.In the superior grain,nitrogen enhanced both the capacity of photosynthesis and starch synthesis.Plant hormone ABA and IAA synthesis capacity also increased,while ethylene synthesis capacity decreased.In inferior grains,the expression of TPP increased,while no significant changes occurred in TPS,which might lead to the reduction of T6P content,thus regulating the metabolism of carbon and nitrogen.The expression of gene hisC that related Phe and Tyr synthesis was enhanced,resulting in improving the relative abundance of the two AAs in protein.The results provided a better understanding of N regulating mechanism for grain filling,and lay the foundation of genetic improvement and optimization cultivation on rice yield and quality. |
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