Alternative processing of its precursor is related to miR319 decreasing in melon plants exposed to cold

Abstract

miRNAs are fundamental endogenous regulators of gene expression in higher organisms. miRNAs modulate multiple biological processes in plants. Consequently, miRNA accumulation is strictly controlled through miRNA precursor accumulation and processing. Members of the miRNA319 family are ancient ribo-regulators that are essential for plant development and stress responses and exhibit an unusual biogenesis that is characterized by multiple processing of their precursors. The significance of the high conservation of these non-canonical biogenesis pathways remains unknown. Here, we analyze data obtained by massive sRNA sequencing and 5' - RACE to explore the accumulation and infer the processing of members of the miR319 family in melon plants exposed to adverse environmental conditions. Sequence data showed that miR319c was down regulated in response to low temperature. However, the level of its precursor was increased by cold, indicating that miR319c accumulation is not related to the stem loop levels. Furthermore, we found that a decrease in miR319c was inversely correlated with the stable accumulation of an alternative miRNA (#miR319c) derived from multiple processing of the miR319c precursor. Interestingly, the alternative accumulation of miR319c and #miR319c was associated with an additional and non-canonical partial cleavage of the miR319c precursor during its loop-to-base-processing. Analysis of the transcriptional activity showed that miR319c negatively regulated the accumulation of HY5 via TCP2 in melon plants exposed to cold, supporting its involvement in the low temperature signaling pathway associated with anthocyanin biosynthesis. Our results provide new insights regarding the versatility of plant miRNA processing and the mechanisms regulating them as well as the hypothetical mechanism for the response to cold-induced stress in melon, which is based on the alternative regulation of miRNA biogenesis.

Figure 1

miR319 accumulates differentially in cold-exposed melon plants. (A) Graphic representation of the levels of the miR319 family members in melon plants exposed to cold treatments. The accumulation of miRNA in the analyzed samples is shown as the means of the total normalized reads (expressed in reads per million). Error bars show the confidence interval of the difference between means. (B) Relative expression levels (respect to the control) of the miR319 family members expressed as the Log fold change estimated by the statistical testing method edge-R. The boxes represent the range of expression values for the different sequences associated to each member precursor. More details are provided in Table S1.

Figure 2

miR319c levels are not related to pri-miR319c accumulation. Relative accumulation of miR319 precursors in melon plants with respect to untreated controls exposed to a low temperature as estimated by qRT-PCR. The values on the Y-axis represent the mean of the expression (in Log fold change) for each one of the pri-miRNAs. Relative RNA expression was determined by the comparative ΔΔCT method and normalized to the geometric mean the expression of Profilin and ADP-ribosylation factor-like expression used reference controls. Error bars show the confidence interval of the difference between replicates. Control and cold-exposed plants were time-synchronized. The P value was estimated by paired t-Test analysis.

Figure 3

Alternative pri-miR319c processing is observed in cold-exposed melon plants. (A) Graphic representation of the predicted plot derived from conventional processing of the miR319 precursor. According the proposed loop-to-base processing, only plots representing sequences derived from the stable miR319/miR319* duplex are expected. (B) The sRNAs (ranging 20 nt to 25 nt) recovered from the cold-exposed (red line) and control (blue line) melon plants were plotted (allowing only exact matching) onto the four pri-miR319 sequences described in melon. The values on the Y-axis represent the mean of the total reads in each library (normalized in reads per million). The nucleotide positions of the diverse pri-miR319 sequences analyzed are represented on the X-axis.

Figure 4

Accumulation of cold responsive #miR319c and miR319c is negatively correlated. (A) Graphic representation of the levels of the #miR319c sequences in melon plants exposed to cold treatment. The accumulation of miRNAs in the analyzed samples is shown as the mean of the total sequenced sRNAs (normalized in reads per million) (left) and the relative Log fold change accumulation (right) estimated by edge-R analysis. Error bars show the confidence interval of the difference between means. The double bar indicates the values of both miRNA-related sequences (more details in Table S2). (B) Histogram representing the sum of the means for the miR319c- (20 and 21nt length) and #miR319c (20 and 21nt length)-related sequences recovered from cold-exposed (red bars) and control (blue bars) plants. The P values were estimated by paired t-Test analysis.

Figure 5

Processing of the miR319c precursor in melon, as estimated by 5′RACE. (A) Scheme illustrating the method used to identify processed precursor intermediates and the expected fragments. An (*) indicates the partial cut in the 5′-arm. (1 to 4) Represent the four cleavage reactions expected for the canonical loop-to-base-processing of the miR319c precursor. The red bars represent the 5′-RNA-adaptor. Arrows indicate the position of the oligos used in the last PCR amplification. (B) Schemes showing the predicted secondary structure (estimated from sequence > METC022194) and abundance (in percentage) of the cleavage sites detected among processed miR319c precursor recovered from control (upper panel) and cold-exposed (lower panel) plants. Red arrows (filled and doted) show partial processing points in the 5′-arm. Blue and magenta arrows indicate processing points 1 and 2. The processing points related to cuts 3 and 4 are marked with orange arrows. Black arrows indicate the position of the unexpected dicing event involved in the release of unstable miR319c. Gray arrows show the less abundant and unspecific cleavage site.

Figure 6

Cold-induced decrease in miR319c correlates with increased accumulation of well-established members of the low temperature signaling pathway. (A) Histogram showing the means of the relative accumulation of TCP2 mRNA in melon plants exposed to cold as estimated by qRT-PCR-. Error bars show the confidence interval of the difference between means. (B) Representation of the cleaved TCP2 transcript detected by the 5′-RLM-RACE assay. The X-axis indicates the nucleotide position in a selected region (dark magenta box in graphics) of the TCP2 transcripts. The Y-axis shows the relative frequency of clones sequenced showing cleavage in this position. The red asterisk indicates the expected position for TCP2 cleavage mediated by miR319. (C) Relative accumulation with respect to the untreated control for the ELONGATED HIPOCOTYL 5 (HY5), CHALCONE SYNTHASE (CHS) and CHALCONE ISOMERASE (CHI) homologous transcripts in melon plants exposed to low temperature for 11 days as estimated by qRT-PCR. Error bars show the confidence interval of the difference between means. The P value was estimated by paired t-Test analysis.

Figure 7

Proposed model for miR319c function in response to cold in melon. Low temperature induced a decrease of miR319c that promoted a TCP2-mediated increase in HY5 that transcriptionally modulated the accumulation of key components in the anthocyanin biosynthesis pathway (CHS and CHI), which is involved in cold response.

Figure 8

Proposed model for the processing of miR319c in melon. (A) The miR319c precursor is first partially cleaved at the 5′-arm. DCL1 then continues to cut the precursor four more times in a loop-to-base direction until mature miR319c is finally released. The alternative #miR319c/miRNA* duplex is unstable under these processing conditions. (B) In cold-exposed plants, the first partial cleavage is impaired, promoting lower accuracy in the next four DCL1-mediated cuts (mainly cut 4). As a consequence of this altered precursor processing, stable miR319c/miRNA* is inefficiently released and/or accumulated. By contrast, accumulation of alternative #miR319c is increased.