Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis

Abstract

Background: MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted.

Result: Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response.

Conclusions: We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in 'Yingshuang', and 46 up-regulated miRNA and 45 down-regulated miRNAs in 'Baiye 1' in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

Figure 1

Length distribution of small RNA sequences obtained in the tea plant libraries.

Figure 2

Number of distinct members present in conserved miRNA fimilies in C. sinensis under cold stress.

Figure 3

Expression of miRNA in two tea lines with or without cold stress treatments. Real-time PCR validation through the column chart displays, and line graph shows the differential expression of the same miRNA in tea leaf. Error bars represent standard deviation (n = 3).

Figure 4

Distribution of confirmed miRNA targets, separated by category in conserved miRNAs (A) and novel miRNAs (B).

Figure 5

Summary of common and specific targets between -C and + C libraries, targets of known miRNAs (A) and targets of new miRNA candidates (B).

Figure 6

Mapping of the mRNA cleavage sites by RNA ligase-mediated 5′RACE. Each top strand (black) depicts a miRNA complementary site, and each bottom strand depicts the miRNA (red). Watson-Crick pairing (vertical dashes) and G:U wobble paring (circles) are indicated. RNA ligase--mediated 5′RACE was used to map the cleavage sites. The partial mRNA sequences from the target genes were aligned with the miRNAs. The numbers indicate the fraction of cloned PCR products terminating at different positions.

Figure 7

Gene ontology of the predicted targets for 57 differentially expressed miRNAs. Categorization of miRNA-target genes was performed according to the cellular component (A), molecular function (B) and biological process (C).

Figure 8

Differential expression of miRNAs at four metamorphic stages (CK, 4 h, 12 h, and 24 h) by hierarchical clustering in ‘Yingshuang’. Red indicates that a gene is highly expressed at that stage, whereas green indicates the opposite. The absolute signal intensity ranges from −2.5 to +2.5, with corresponding color changes from blue to green, yellow and red. The signal of expression was detected by microarray with four probe repeats. YS-CK: control; YS-4: 4°C for 4 h; YS-12: 4°C for 12 h; YS-24: 4°C for 24 h.

Figure 9

Differential expression of miRNAs at four metamorphic stages (CK, 4 h, 12 h, and 24 h) by hierarchical clustering in ‘Baiye 1’. Red indicates that a gene is highly expressed at that stage, whereas green indicates the opposite. The absolute signal intensity ranges from −2.5 to +2.5, with corresponding color changes from blue to green, yellow and red. The signal of expression was detected by microarray with four probe repeats. BY-CK: control; BY-4: 4°C for 4 h; BY-12: 4°C for 12 h; BY-24: 4°C for 24 h.