Role of miRNAs in regulation of SA-mediated upregulation of genes involved in folate and methionine metabolism in foxtail millet

Abstract

The effect of exogenous salicylic acid (SA) on folate metabolism and the related gene regulatory mechanisms is still unclear. In this study, the panicle of foxtail millet treated with different SA concentrations showed that 6 mM SA doubled the 5-methyltetrahydrofolate content compared to that of the control. An untargeted metabolomic analysis revealed that 275 metabolites were enriched in amino acid metabolic pathways. Significantly, the relative content of methionine (Met) after 6 mM SA treatment was 3.14 times higher than the control. Transcriptome analysis revealed that differentially expressed genes were mainly enriched in the folate and amino acid biosynthesis pathways (including Met, Cys, Pro, Ser et al.). The miRNA-mRNA interactions related to the folate and Met metabolic pathways were analyzed and several likely structural gene targets for miRNAs were identified, miRNA-seq analysis revealed that 33 and 51 miRNAs targeted 11 and 15 genes related to the folate and Met pathways, respectively. Eight key genes in the folate metabolism pathway were likely to be up-regulated by 14 new miRNAs and 20 new miRNAs up-regulated the 9 key genes in the Met metabolism pathway. The 6 miRNA-mRNA interactions related to the folate and Met metabolism pathways were verified by qRT-PCR, and consistent with the prediction. The results showed that DHFR1 gene expression level related to folate synthesis was directly up-regulated by Nov-m0139-3p with 3.8 times, but DHFR2 was down-regulated by Nov-m0731-5p with 0.62 times. The expression level of CYSC1 and APIP related to Met synthesis were up-regulated by Nov-m0461-5p and Nov-m0664-3p with 4.27 and 1.32 times, respectively. Our results suggested that exogenous SA could induce the folate and Met accumulated in the panicle of foxtail millet. The higher expression level of DHFR1, FTHFD, CYSC1 and APIP in the folate and Met metabolism pathway and their regulators, including Nov-m0139-3p, Nov-m0717-5p, Nov-m0461-5p and Nov-m0664-3p, could be responsible for these metabolites accumulation. This study lays the theoretical foundation for elucidating the post-transcription regulatory mechanisms of folate and Met metabolism.

Keywords: folate; foxtail millet; methionine metabolism; miRNA; salicylic acid.

Figure 1

The phenotypic characteristics and folate content analyses in the panicle of foxtail millet treated with different concentrations of SA (A): the phenotype of the panicle of foxtail millet with 0, 3, 6 mM SA treatment. (B): the FA (Folate), THF (Tetrahydrofolate), and 5-M-THF (5-methyltetrahydrofolate) content of the panicle of foxtail millet with above SA treatment. “*” indicates significant difference (p < 0.05), “**” and “***” indicate extremely significant differences (p<0.01 and p<0.001).

Figure 2

Differentially accumulated metabolites and KEGG enrichment analyses in the panicle of foxtail millet following SA treatment using untargeted metabolome (A) volcanic map of differential metabolites displayed by comparing 3 mM and 6 mM SA treatment to the control (CK); (B) the KEGG enrichment of differential metabolites analyzed by comparing 3mM SA treatment to CK; (C) the KEGG enrichment of differential metabolites analyzed by comparing 6mM SA treatment to CK.

Figure 3

DEGs and KEGG enrichment analyses in the panicle of foxtail millet following SA treatment (A) the statistics of new and known DEGs; (B) volcano plot of DEGs between 6mM and 0 mM SA treatment; (C) the KEGG enrichment of DEGs.

Figure 4

The expression pattern of DEGs related to the folate and Met metabolic pathway the squares represent the metabolites in the folate metabolism pathway; Yellow background indicates genes in the riboflavin metabolic pathway, green background indicates folate synthesis pathway genes; blue background shows folate-dependent 1C metabolism pathway; solid and dotted line arrow indicated directly and indirectly products synthesis route by downstream enzyme and substances. (A): PYRP2: 5-amino-6-(5-phospho-D-ribitylamino)uracil phosphatase; RFK: bi-functional riboflavin kinase; RIBA1/RIBA2;bi-functional riboflavin biosynthesis protein; MOCS1A/1B: GTP 3’,8-cyclase; HPPK/DHPS1: folate synthesis bi-functional protein; DHFR1/DHFR2: dihydrofolate reductase; FTHFD: formyltetrahydrofolate deformylase; MTRF: methionyl-tRNA formyltransferase; 5-FCL1/5-FCL2: 5-formyltetrahydrofolate cyclo-ligase. (B):CYSC1/CYSK: cysteine synthase; AK: bi-functional aspartokinase/homoserine dehydrogenase; BHMT2: homocysteine S-methyltransferase; metE: 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; DNMT1: DNA (cytosine-5)-methyltransferase; ACS: 1-aminocyclopropane-1-carboxylate synthase; ACO1/2: 1-aminocyclopropane-1-carboxylate oxidase; AMD1: S-adenosylmethionine decarboxylase proenzyme; SRM: spermine synthase; APIP: bi-functional methylthioribulose-1-phosphate dehydratase/enolase-phosphatase; ADI1: 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase; TAT1/TAT2: tyrosine aminotransferase.

Figure 5

The distribution characteristic of miRNAs in the panicle of foxtail millet following SA treatment (A) the sequence length distribution of pre-miRNA; (B) the sequence length distribution of mature-miRNA;(C) the base bias analysis of miRNA at the first nucleotide position; (D) the bias analysis of each base for 21~24 nt miRNA; (E) the distribution of miRNA family members.

Figure 6

The prediction of miRNAs targeted mRNAs related to folate (A) and Met (B) metabolism pathway.

Figure 7

qRT-PCR verification of the expression pattern of miRNAs and DEGs (A) the expression level of DEGs related to folate and Met metabolism pathway were analyzed by qRT-PCR; (B) the expression level of miRNAs and targeted FTHFD, DHFR1/2, PYRP2, CYSC1 and APIP genes were analyzed by qRT-PCR. “*” indicates significant difference (p < 0.05), “**” and “***” indicate extremely significant differences (p<0.01 and p<0.001).

Figure 8

Working model for salicylic acid-mediated miRNA regulation of folate and Met metabolism Note: The gray nodes represent metabolites, the squares represent mRNA/miRNA, and the color of the squares represents the up-regulated or down-regulated of the transcript level of mRNA/miRNA; The metabolites are connected by black arrows, and the dotted line indicates that multiple metabolic processes are omitted.