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.

Figure 1.

Targets of smRNAs identified by degradome sequencing are shown as target plots (t-plots) by identical reads. Signature abundance (absolute number) along the indicated transcript is plotted. Red dots on the x axis indicate predicted cleavage sites. Red lines indicate signatures produced by miRNA-directed cleavage. A to C, The t-plots for category I (A), category II (B), and category III (C). Cleavage frequency as determined by gene-specific 5′ RACE at the indicated position is shown below the t-plots. In ARF transcripts with dual target sites for tasiRNA-ARF (B and C), only the second (3′) cleavage site was validated by 5′ RACE. D, Cleavage abundance on the TAS3b precursor by tae-miR390.

Figure 2.

Differentially expressed smRNAs in response to cold stress in spikes of the wheat TGMS line. Relative fold change (FC) values greater than 1.5 or less than 0.5 are highlighted in red. The asterisks indicate significant differences between the cold stress and control samples (* P < 0.01, ** P < 0.05).

Figure 3.

qPCR analysis of target genes in spikes from the TGMS line in cold and control conditions. Error bars indicate sd.

Figure 4.

Self-organizing map (SOM) clusters of expression profiles. Wheat unigenes differentially expressed in cold and control conditions were subjected to SOM clustering based on their relative expression levels in both conditions. The y axis represents normalized log₂ transcript expression levels. The x axis represents anther developmental stages in cold (L) and control (C) treatments. A, The SOM cluster includes ARF gene EST Contig1892, which was targeted by tasiRNA-ARF. B, The SOM cluster includes ARF genes EST Contig9875 and Contig4296, which were targeted by tae-miR167d and tasiRNA-ARF, respectively.

Figure 5.

Differential accumulation of miRNA/tasiRNA-ARF in anthers of the wheat TGMS line. Cross-sections of anthers in anther tissue microarray were hybridized with 5′ and 3′ double-labeled locked nucleic acid-modified oligonucleotides detecting tae-miR172a (A–F), tae-miR396a (G–L), tae-miR167d (M–R), and TAS3a-5′D6(+) (S–X). Anther developmental stage and treatment conditions (L, cold stress; C, control condition) are shown in parentheses. The locked nucleic acid probe complementary to Caenorhabditis elegans let-7 served as a negative control. Brown staining shows probe localization. ML, Middle layer; Ta, tapetum; V, vascular bundles. Bars = 50 µm.