| A large number of secondary metabolites exist in the plant kingdom, and the total number of plant metabolites is estimated to 0.2-1 million. Plant metabolites play essential roles in growth, cellular replenishment and whole-plant resource allocation as well as in their interactions with the environment. Acylation of oxygen- and nitrogen- containing substrates to produce esters and amides, respectively, is one of the most common types of secondary metabolites modification. BAHD acyltransferases, unique in plant, are involved in the biosynthesis of plant secondary metabolites, such as flavonoids, alkaloids, terpenoids, polyamine, anthocyanin and volatile esters.Metabolome analysis is an efficient method for the profiling of plant secondary metabolites. Using LC-MS(Liquid chromatography-mass spectrometry), we have established metabolome databasebased on stepwise MIM-EPI(stepwise multiple ion monitoring-enhanced product ions)and MRM(multiple reaction monitoring)method. More than 800 metabolites could be quantified within 30 minutes. Hundreds of metabolites were detected and a number of which were identified based on authentic standard. We have also constructed the MS2T(MS2 spectral tag)library for different rice tissues.To explore the genetic control of rice metabolome, more than 900 metabolites were detected from two different tissue(flag leaf at the heading date and seeds at 72 h after germination)of 210 RILs(recombinant inbred lines)derived from a cross between ZS97 and MH63. Based on further m QTL(metabolic quantitative trait loci)analysis, 2800 highly resolved m QTLs were found, among which 1884 m QTL for flag leaf, 937 m QTL for germinating seed, respectively. Besides, distinct and overlapped accumulation was observed and complex genetic regulation of metabolism was revealed in two different tissues. To evaluate the quality of our m QTL, analysis of metabolic profiling of 64 selected metabolites was performed using an IL(introgression line)population. 50 m QTLs were confirmed by ILs in flag leaf at least two independent lines. Finallys, we associated 24 candidate genes to various m QTLs. We selected two BAHD acyltransferase Os02g28270 and Os02g28340 as candidate genes for a malonyl flavonoid QTL. Functional identification in vivo ofthese two genes was performed. This study demonstrated a powerful tool and provided a vast amount of high-quality data for understanding the plasticity of plant metabolome, which may help bridge the gap between the genome and phenome.To further elucidate the genetic and biochemical bases of natural variation of rice metabolome, we detected 840 metabolites from 529 rice varieties in the five-leaf stage, and performed metabolic genome-wide association study(m GWAS)based on ~6.4 million SNPs. Significant heterogeneity was found in natural variation of metabolites and their underlying genetic architectures associated with two indica and japonica subspecies of rice. We choose 36 candidate genes via data mining(metabolites identified, the chip data, expression and induced expression profile, and Gaussian graphical modeling(GGM)network and gene co-expression). We functionally identified five BAHD acyltransferase genes as phenolamides actltransferase in vivo and in vitro.The strong association(P = 4.1 × 10-15)between SNP sf0433733272 on chromosome 4 that lies 10 kb downstream of Os04g56910 and the level of N-feruloyl agmatine( Fer-Agm) suggests that Os04g56910 encodes an agmatine hydroxycinnamoyl acyltransferase. The functional annotations of candidate genes were further supported by showing that over-expression of Os04g56910 resulted in specific increases in the corresponding metabolite Fer-Agm, which comfirmed its function that Os04g56910 encodes a acyltransferase that catalyzes N-coupling reaction reaction of agmatine to ferulic in vivo.The significant association(P = 6.3 × 10-22)between SNP sf0921455575 that lies 8 kb upstream of Os09g37200(encoding a putative transferase)specifically with the levels of different N-feruloyl putrescine(Fer-Put)compounds suggests that Os09g37200 encodes a putrescine hydroxycinnamoyl acyltransferase. When Os09g37200 was expressed with a histidine tag at the N-terminus in Escherichia coli BL-21, we were able to detect Fer-Put: acyltransferase activity in the soluble protein extract. Consistent with its in vitro activity, over-expression of Os09g37200 in ZH11, resulted in a substantial increase in Fer-Put accumulation in the transgenic-positive lines compared to control plants, confirming its function in vivo.Using the same approach, the association between SNP sf1125035584 at Os11g42370(encoding a putative transferase)and the level of the unknown metabolite mr1133, together with the results from transgenic analysis, confirm that Os11g42370 is responsible for the accumulation of mr1133.The significant association(P = 6.9 × 10-98)between coumaroyl spermidine(Cou-Spd)with SNP sf1215974303 that lies 4 kb upstream and 21 kb downstream of Os12g27220(encoding a putative transferase)and Os12g27254(encoding a putative transferase), respectively, suggests that these two genes were probably the candidate genes. Phylogenetic analysis indicated that both Os12g27220 and Os12g27254 clustered with At SDT that catalyzes the acylation of the spermidine. Based on results from transgenic analysis, were revealed that that Os12g27220 and Os12g27254 might be responsible for natural variation of Cou-Spd in rice.Our study of m GWAS provides insights into genetic and biochemical bases of rice metabolome and can be used as a powerful complementary tool to classical phenotypic traits mapping for rice improvement. |
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