Abstract
Low temperature is one of the most important environmental factors that limits the geographical distribution and productivity of grapevine. However, the molecular mechanisms on how grapevine responds to cold stress remains to be elucidated. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play an essential role during plant development and stress responses. Although miRNAs and their targets have been identified in several Vitis species, their participation during cold accumulation in grapevine remains unknown. In this study, two small RNA libraries were generated from micropropagated ‘Muscat Hamburg’ (V. vinifera) plantlets under normal and low temperatures (4°C). A total of 163 known miRNAs and 67 putative novel miRNAs were detected from two small RNA libraries by Solexa sequencing. Forty-four cold-inducible miRNAs were identified through differentially expressed miRNAs (DEMs) analysis; among which, 13 belonged to upregulated DEMs while 31 belonged downregulated DEMs. The expression patterns of the 13 DEMs were verified by real-time RT-PCR analysis. The prediction of the target genes for DEMs indicated that miRNA may regulate transcription factors, including AP2, SBP, MYB, bHLH, GRAS, and bZIP under cold stress. The 5′-RLM RACE were conducted to verify the cleavage site of predicted targets. Seven predicted target genes for four known and three novel vvi-miRNAs showed specific cleavage sites corresponding to their miRNA complementary sequences. The expression pattern of these seven target genes revealed negative correlation with the expression level of the corresponding vvi-miRNAs. Our results indicated that a diverse set of miRNAs in V. vinifera are cold-inducible and may play an important role in cold stress response.
Highlights
MicroRNAs are a class of endogenous small non-coding RNAs (∼21 nt in length) that regulate gene expression at the post-transcriptional level by targeting messenger RNAs for cleavage or translational repression (Chen, 2004; Jones-Rhoades et al, 2006; Mallory and Vaucheret, 2006)
The NCT small RNA library was constructed using the plantlets grown at 25◦C, whereas the cold treated (CT) small RNA library was constructed with micropropagated plantlets subjected to 4◦C for 4 h to observe the change in miRNAs in the early stage during cold stress
We identified 163 known miRNAs and 67 novel miRNAs; among which, 44 were differentially expressed miRNAs (DEMs) during cold stress in V. vinifera
Summary
MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs (∼21 nt in length) that regulate gene expression at the post-transcriptional level by targeting messenger RNAs (mRNA) for cleavage or translational repression (Chen, 2004; Jones-Rhoades et al, 2006; Mallory and Vaucheret, 2006). MiRNAs have been initially identified in Caenorhabditis elegans as genes required for temporal control of developmental events (Lee et al, 1993). Cold-inducible miRNAs in grapevine of miRNAs have been identified in a broad range of mammals, as well as in plants and viruses The functions of these miRNAs range from maintaining genome stability, developmental patterning, and response to environmental stimuli in defense against viruses and bacteria in eukaryotes (Ramachandran and Chen, 2008). MiR395 could be induced by sulfate starvation and targets the ATP sulfurylase genes (APS1, APS3, and APS4) to regulate sulfate metabolism (Jones-Rhoades and Bartel, 2004). Overexpression of Osa-miR319 could lead to enhanced cold tolerance in rice (Yang et al, 2013)
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