Uncovering Small RNA-Mediated Responses to Cold Stress in a Wheat Thermosensitive Genic Male-Sterile Line by Deep Sequencing    

Abstract

The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNAs) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 78 unique miRNA sequences from 30 families and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing data sets and TaqMan quantitative polymerase chain reaction results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF [for Auxin-Responsive Factor]) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, the expression of cold stress-responsive smRNAs integrated in the auxin-signaling pathway and their target genes was largely noncorrelated. We investigated the tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress.

Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars

“…[63] On the other hand, AP2 expression showed similar expression profile under cold and control conditions in wheat. [31] These data indicate that the detailed investigation on the role of AP2 under cold stress in tomato is needed.…”

“…[12] These proteins are auxin receptors that target repressors of ARF. [31] The different expression profiles of ARF6 and ARF8, and down regulation of TIR1 and F-box protein in cold-tolerant tomato cultivars suggested that auxin perception and signalling may change in stress conditions based on tolerance. In addition to miR167, there could be a key regulatory role of miR393 in the auxin-signalling pathway under cold stress in tomato.…”

“…Moreover, we investigated the target genes from which they had been previously identified in the literature. AFR6 targeted by miR167 [49] and TIR targeted by miR393 [31,50] were included in target gene expression analysis.…”

“…They also formed a sketch pathway, implying possible regulatory role of miR169. Tang et al [31] found up-regulation of miR167 and role of trans-acting short-interfering RNAÀauxin response factor (tasiRNAÀARF) in regulating the auxin-signalling pathway and possibly in the developmental response to cold stress in wheat. In rice, Jian et al [32] found several cold-regulated miRNAs by direct cloning and sequencing.…”

“…In this study, we investigated cold-induced miRNAs (miR167, miR169, miR172, miR397 and miR393) [7,8,31] by a sensitive method, stemÀloop qRTÀPCR, in cold-treated tomato plants. In order to understand the expression profiles of miRNA target genes, we also retrieved the microarray data of three tomato cultivars from a public database.…”