An Integrated Analysis of microRNAs and the Transcriptome Reveals the Molecular Mechanisms Underlying the Regulation of Leaf Development in Xinyang Maojian Green Tea (Camellia sinensis)

Abstract

Xinyang Maojian (XYMJ) tea is one of the world's most popular green teas; the development of new sprouts directly affects the yield and quality of tea products, especially for XYMJ, which has hairy tips. Here, we used transcriptome and small RNA sequencing to identify mRNAs and miRNAs, respectively, involved in regulating leaf development in different plant tissues (bud, leaf, and stem). We identified a total of 381 conserved miRNAs. Given that no genomic data for XYMJ green tea are available, we compared the sequencing data for XYMJ green tea with genomic data from a closely related species (Tieguanyin) and the Camellia sinensis var. sinensis database; we identified a total of 506 and 485 novel miRNAs, respectively. We also identified 11 sequence-identical novel miRNAs in the tissues of XYMJ tea plants. Correlation analyses revealed 97 miRNA-mRNA pairs involved in leaf growth and development; the csn-miR319-2/csnTCP2 and miR159-csnMYB modules were found to be involved in leaf development in XYMJ green tea. Quantitative real-time PCR was used to validate the expression levels of the miRNAs and mRNAs. The miRNAs and target genes identified in this study might shed new light on the molecular mechanisms underlying the regulation of leaf development in tea plants.

Keywords: Xinyang Maojian; leaf development; miRNA; regulatory network; tea plant.

Figure 1

Nucleotide bias at all positions in conserved miRNAs in XYMJ tea plants. (A) The nucleotide bias of known miRNAs at the first position in XYMJ tea plants. (B) The frequency of A, U, C, and G of known miRNAs at each position. The X-axis shows miRNA nucleotide positions; the Y-axis shows the percentage of each nucleotide at each nucleotide position. Green, brown, blue, and pink correspond to A, U, C, and G, respectively.

Figure 2

Distributions of miRNA of different sizes in XYMJ tea plants. (A) Distribution of novel miRNAs in four databases. (B) Distribution of known miRNAs of different lengths.

Figure 3

DEMs in different tissues. (A) Image showing the sBud, sL1, sL2, and sS1 samples that were collected. (B) Venn diagram showing the DEMs identified in each comparison group in XYMJ tea plants. (C) Number of upregulated and downregulated DEMs in each comparison group. Heat map of novel miRNAs in XYMJ tea plants showing differential expression among tissues compared with the CSS (D) and Tieguanyin (E) genome databases. Red indicates high miRNA expression, and green indicates low miRNA expression. miRNAs in the same cluster show similar expression patterns.

Figure 4

Relative expression of miRNAs and mRNAs in four tissues of XYMJ tea plants using qRT-PCR. (A–H) The expression levels of eight conserved miRNAs in different plant tissues. (I–Q) The expression levels of nine novel miRNAs in different plant tissues. (R–T) The expression levels of three target genes in different plant tissues. Error bars indicate the standard deviation (SD) of three independent biological replicates. Different letters above the bars indicate significant differences at p < 0.05. Means followed by the same letter over the bars are not significantly different at the 0.5% level.

Figure 5

Results of GO and KEGG analyses of DEGs in different tissues of XYMJ tea plants. GO analysis was performed by assigning GO terms to DEGs in three categories: molecular function (MF), biological process (BP), and cellular component (CC). The 20 most enriched KEGG pathways of the target genes of the DEMs in XYMJ tea plants are shown. (A–F) show the GO terms and KEGG pathways for the sBud vs. sS1, sBud vs. sL1, and sBud vs. sL2 comparison groups. The significance of the matched gene ratio is indicated by the q-value. Redder values indicate higher q-values, and greener values indicate lower q-values; the size of the circle indicates the number of target genes.

Figure 6

Analysis of the miRNA–mRNA regulatory network. Circles indicate potential target mRNAs, diamonds indicate miRNAs, and lines indicate miRNA–mRNA relationships. Red circles are upregulated genes, and green diamonds are downregulated genes.