Combined small RNA and degradome sequencing reveals complex microRNA regulation of catechin biosynthesis in tea (Camellia sinensis) - PubMed (original) (raw)

Combined small RNA and degradome sequencing reveals complex microRNA regulation of catechin biosynthesis in tea (Camellia sinensis)

Ping Sun et al. PLoS One. 2017.

Abstract

MicroRNAs are endogenous non-coding small RNAs playing crucial regulatory roles in plants. Tea, a globally popular non-alcoholic drink, is rich in health-enhancing catechins. In this study, 69 conserved and 47 novel miRNAs targeting 644 genes were identified by high-throughout sequencing. Predicted target genes of miRNAs were mainly involved in plant growth, signal transduction, morphogenesis and defense. To further identify targets of tea miRNAs, degradome sequencing and RNA ligase-mediated rapid amplification of 5'cDNA ends (RLM-RACE) were applied. Using degradome sequencing, 26 genes mainly involved in transcription factor, resistance protein and signal transduction protein synthesis were identified as potential miRNA targets, with 5 genes subsequently verified. Quantitative real-time PCR (qRT-PCR) revealed that the expression patterns of novel-miR1, novel-miR2, csn-miR160a, csn-miR162a, csn-miR394 and csn-miR396a were negatively correlated with catechin content. The expression of six miRNAs (csn-miRNA167a, csn-miR2593e, csn-miR4380a, csn-miR3444b, csn-miR5251 and csn-miR7777-5p.1) and their target genes involved in catechin biosynthesis were also analyzed by qRT-PCR. Negative and positive correlations were found between these miRNAs and catechin contents, while positive correlations were found between their target genes and catechin content. This result suggests that these miRNAs may negatively regulate catechin biosynthesis by down-regulating their biosynthesis-related target genes. Taken together, our results indicate that miRNAs are crucial regulators in tea, with the results of 5'-RLM-RACE and expression analyses revealing the important role of miRNAs in catechin anabolism. Our findings should facilitate future research to elucidate the function of miRNAs in catechin biosynthesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Size distribution of miRNAs by high-throughput sequencing.

(a) Size distribution of ‘1005’ tea total miRNAs; (b) Size distribution of strain ‘1005’ conserved miRNAs; (c) Size distribution of strain ‘1005’ novel miRNAs.

Fig 2

Fig 2. Gene ontology of the predicted targets.

Categorization of target genes was accomplished according to the cellular component (a), the molecular function (b) and biological process (c).

Fig 3

Fig 3. The mRNA cleavage sites of degradome sequence.

Boxes indicate the cleavage sites; the numbers indicate the fraction of cloned PCR products terminating at different positions.

Fig 4

Fig 4. The contents of catechin and expression levels of miRNAs and target genes in tea leaves of different maturity.

A, first leaves; B, third leaves; C, old leaves. The X axis in Fig 4 indicates the position of leaves; the Y axis in Fig 4a indicates percentages of catechin in dry tea leaves; the Y axis in Fig 4b-e indicates relative expression.

Fig 5

Fig 5. Expression of miRNAs at different levels of maturity.

A, first leaves; B, third leaves; C, old leaves.

Fig 6

Fig 6. The mRNA cleavage sites identified by 5’RLM-RACE.

Boxes indicate the cleavage sites; the numbers indicate the fraction of cloned PCR products terminating at different positions.

Fig 7

Fig 7. The potential patterns of miRNAs that regulated cetechins synthesis in tea cultivar 1005.

The arrows pointing down represent down-regulating on catechins content.

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Grants and funding

This work was funded by the National Natural Science Foundation of China (project no. 31170651) and Major Science and Technology project in Fujian Province (2015NZ 0002-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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