An integrative approach to identify hexaploid wheat miRNAome associated with development and tolerance to abiotic stress

Abstract

Background: Wheat is a major staple crop with broad adaptability to a wide range of environmental conditions. This adaptability involves several stress and developmentally responsive genes, in which microRNAs (miRNAs) have emerged as important regulatory factors. However, the currently used approaches to identify miRNAs in this polyploid complex system focus on conserved and highly expressed miRNAs avoiding regularly those that are often lineage-specific, condition-specific, or appeared recently in evolution. In addition, many environmental and biological factors affecting miRNA expression were not yet considered, resulting still in an incomplete repertoire of wheat miRNAs.

Results: We developed a conservation-independent technique based on an integrative approach that combines machine learning, bioinformatic tools, biological insights of known miRNA expression profiles and universal criteria of plant miRNAs to identify miRNAs with more confidence. The developed pipeline can potentially identify novel wheat miRNAs that share features common to several species or that are species specific or clade specific. It allowed the discovery of 199 miRNA candidates associated with different abiotic stresses and development stages. We also highlight from the raw data 267 miRNAs conserved with 43 miRBase families. The predicted miRNAs are highly associated with abiotic stress responses, tolerance and development. GO enrichment analysis showed that they may play biological and physiological roles associated with cold, salt and aluminum (Al) through auxin signaling pathways, regulation of gene expression, ubiquitination, transport, carbohydrates, gibberellins, lipid, glutathione and secondary metabolism, photosynthesis, as well as floral transition and flowering.

Conclusion: This approach provides a broad repertoire of hexaploid wheat miRNAs associated with abiotic stress responses, tolerance and development. These valuable resources of expressed wheat miRNAs will help in elucidating the regulatory mechanisms involved in freezing and Al responses and tolerance mechanisms as well as for development and flowering. In the long term, it may help in breeding stress tolerant plants.

Figure 1

Overview of the wheat miRNA pipeline. The procedure is divided in three parts: producing and sequencing small RNA libraries, the bioinformatic prediction of miRNAs and functional analysis of the predicted miRNAs. The customized or developed steps are marked by stars. Orange boxes specify the data at hand after each given step. For details see Experimental procedure.

Figure 2

Overview of the predicted miRNAs. a) Diagram of the intersection between miRNAs predicted by the novel approach, conserved miRNAs identified by sequence homology, and miRNAs published in the literature; b) Evidence of conserved plant miRNA families present in miRBase including those predicted by our approach (tae, osa, bdi, hvu, ath and ptc correspond respectively to Triticum aestivum, Oryza sativa, Brachypodium distachyon, Hordeum vulgare L., Arabidopsis thaliana and Populus trichocarpa); c) The abundance bins of all predicted miRNAs in the 10 libraries (L1-L10) produced from plants grown under different investigated conditions. The abundance of the identified miRNAs represents the number of reads sequenced in each library and classified on 4 levels: low, 10–99 reads; medium, 100–999 reads; and high, 1000 and up; d) the length distribution of miRNAs associated with miRNAs differentially expressed in different investigated comparisons; e) the number of miRNA targeted genes (presented by EST id or UniRef id) associated with miRNAs differentially expressed in different investigated comparisons. WcvCl, winter wheat cultivar Clair (cold tolerant); ScvBo, spring wheat cultivar Bounty (cold and Al sensitive); WcvAt, winter wheat cultivar Atlas (Al tolerant); A.P, aerial parts; L./Rep. T., leaves and reproductive tissues; N.C, normal conditions; Al, Aluminum; Vern., vernalization; Rep., reproductive; Str. Resp., stress response; Tol., tolerance; Dev. resp., developmentally response; Fl. Trans, floral transition; Flw., flowering. See Additional file 1: Method S1 and Additional file 2: Table S1 for libraries and conditions and Additional file 2: Table S10 for the different investigated comparisons.

Figure 3

Experimental validation of predicted and conserved wheat miRNAs. a) Pre-miRNA secondary structure of miRNA candidates experimentally validated by northern in the investigated libraries; b) northern blot of predicted miRNAs in common between MiRdup* and MIRcheck (CM*M) as well as specifically predicted with MiRdup* tool (SM*); c) northern blot of miRNA candidates identified by both sequence homology against miRBase (conserved miRNAs) and predicted in common between MIRcheck and MiRdup*. Ethidium bromide staining of the rRNAs is shown as gel loading control. L0 represents the control library for Al treatment (L8) in spring wheat Bounty which was not sequenced. The numbers between the parentheses correspond to the expression rank among the 199 predicted miRNAs. The lower value corresponds to the higher read abundance. For more information about the libraries and conditions see Additional file 1: Method S1 and Additional file 2: Table S1.

Figure 4

Differentially expressed miRNAs in response to cold, salt, aluminum and development. a) The differential expression of miRNAs in response to vernalization (presented on log10 adjusted p-value based on the FDR method of Benjamini and Hochberg [80], associated with the log10 of the fold change (FC)). The lines specify the thresholds used to identify the most relevant differentially expressed miRNAs. The blue and red dots correspond respectively to expressed small RNAs and predicted miRNAs; b) the frequencies of differentially expressed miRNAs in response to vernalization, cold, Al, salt and development stage (floral transition and flowering); and those differentially expressed between tolerant and sensitive genotypes; c) Venn diagram of miRNAs regulated under short/long exposure to cold (cold/vernalization, L2/L1 and L7/L6) in leaves, Al (L10/L9 and L8/L9) in roots and salt (L4/L1) in leaves; d) Venn diagram of miRNAs expressed in control plants during vegetative phase under normal conditions (control library L1), plants acclimated up to 56 days at 4°C (vernalized library L2) during vegetative phase and, plants acclimated up to 56 days at 4°C and then transferred to normal conditions under long day photoperiod to induce flowering during the reproductive phase (reproductive library L3). Up, up-regulated miRNAs; Dw, down-regulated miRNAs; Cold/vrn, cold and vernalization responsive miRNAs in spring (L7/L6) and winter wheat (L7/L6), respectively; salt responsive miRNAs in winter wheat (L4/L1); Al responsive miRNAs in spring (L8/L9) and winter (L10/L9) wheat; For tolerance, only differentially expressed miRNAs between cold (L2/L7) and Al (L10/L8) treated libraries are presented. All other abbreviations’ are described in the legend of Figure 2. See Additional file 1: Method S1 and Additional file 2: Table S1 about libraries and conditions and Additional file 2: Table S9 for more information about regulated miRNAs.

Figure 5

GO Slim enrichment for differentially expressed miRNAs in response to abiotic stress and development. Differentially expressed miRNAs with the same or different expression patterns between plants from tolerant and sensitive genotypes under normal and abiotic stress conditions; and between plants at vegetative and reproductive phases were classified into 24 miRNA groups. MiRNA targets are annotated to the best scoring GO Slim terms in Biological process category. The lines are grouped according to their association to cold and vernalization (L2/L1 and L7/L6), Aluminum (L10/L9 and L8/L9) and development (L3/L1 and L3/L2). See Additional file 2: Table S10 for more information about miRNA groups. The value in each case indicates the number of associations miRNA-target- GO for the corresponding GO Slim. The enrichment is presented in four different colors (“brown square symbol” high enrichment (P-value < 10⁻⁵), “orange square symbol” medium enrichment (P-value < 10⁻³), “light orange square symbol” low enrichment (P-value < 0.05) and “white square symbol” no enrichment (P-value ≥ 0.05).