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.

Identification and Characterization of Stress-Responsive TAS3-Derived TasiRNAs in Melon

“…For genome-wide detection of putative TAS loci in melon, the TASI-PREDICTION tool included in the UEA small RNA Workbench (v.4.4) was employed using as input data the reads recovered from sRNA libraries obtained from melon plants exposed to seven biotic and abiotic stress conditions (Bustamante et al 2018, Sanz-Carbonell et al 2019 and the melon genome sequence (Version 3.5.1) [47]. The software parameters established for a more robust prediction of phased siRNAs in melon are detailed in the Materials and Methods section.…”

“…To analyze target expression, total RNA (1.5 μg) extracted from control or treated plants was subjected to DNase treatment (EN0525, Thermo Scientific™) followed by reverse transcription using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific™) according to the manufacturer´s instructions by using of oligo-dT. Then, real-time PCR was performed (by triplicate) as previously described (Bustamante et al, 2018). The efficiency of PCR amplification was derived from a standard curve generated by four 5-fold serial dilution points of cDNA mixed from the two samples.…”

“…It is extensively cultivated in semi-arid regions frequently exposed to changing environmental conditions that affects its production (Wei et al 2013). The recent development of molecular tools such as EST collections (Clepet et al 2011), TILLING platforms (González et al 2011), and genome sequencing and annotation (Garcia-Mas et al 2012, Ruggieri et al 2018, has favored the emergence of melon as a valuable experimental system to conduct significant agriculturalrelated research focused on development (Wu et al 2018) or response to stress (Bustamante et al 2018, Sanz-Carbonell et al 2019. Although, a melon genome region containing two predicted miR390 targets sites able to generate phased-sRNAs and reminiscent to TAS3 genes was previously identified computationally (Gonzalez-Ibeas et al 2011), the role that siRNAs in general and tasiRNAs in particular play in the regulation of the response to stress in melon plants remains in a conundrum.…”

Dynamic architecture and regulatory implications of the miRNA network underlying the response to stress in melon

“…cDNAs were amplified by conventional end point RT-PCR using specific primers to assess for sequence specificity. Then, realtime PCR was performed as described previously (Bustamante et al, 2018). All analyses were performed in triplicate on an ABI 7500 Fast-Real Time qPCR instrument (Applied Biosystems) using a standard protocol.…”

“…Connected to their role in the regulation of development, miRNAs are modulators of the response to both biotic (e.g., bacterial, fungal, and viral pathogenesis; (Chaloner et al, 2016;Zhang et al, 2016;Liu et al, 2017;Brant and Budak, 2018) and abiotic stress conditions (e.g., drought, salinity, nutrient deprivation, cold, or high temperature (Shriram et al, 2016;Zhu et al, 2016;Baek et al, 2017;Bustamante et al, 2018). In addition, recent reports have provided evidence supporting that miRNA biogenesis (Bustamante et al, 2018;Manavella et al, 2019), miRNA turnover and miRNA-RISC assembly (Song et al, 2019) are also susceptible to be controlled by external stimulus.…”