Regulation of gene expression through sequence-specific interaction between miRNAs and their target mRNAs offers an accurate and inheritable mechanism for plants to respond to environment stimuli . Due to water limitations, drought is a major stress that limits the geographic distribution and yield of many crops. Therefore, extensive effort has been made for discovering genetic elements and mechanisms of drought tolerance, including the discovery of drought-associated miRNAs. As an important drought-tolerant crop in semi-arid and arid areas, cowpea offers a good system for the study of drought tolerance. Here we used deep sequencing of small RNA libraries from two cowpea genotypes and identified 157 miRNAs. By comparing the expression level of miRNAs in drought-stressed sample to control sample, we also identified 30 miRNAs that were upregulated in drought condition and 14 downregulated. This list of drought-associated miRNAs includes miRNA families that were known to be associated with drought in other plant species, indicating that they are involved in conserved drought response pathways. Some miRNA families, including some cowpea-specific miRNAs, were found to be associated with drought for the first time, suggesting that they may be involved in lineage- or species-specific stress response pathways and functions.
We predicted target genes for 32 out of 44 drought-associated miRNAs. The predicted target mRNAs encode proteins of diverse function, most of them being transcription factors (Additional file 4). For most of the conserved miRNAs, it is expected that their targets are also conserved. For example, our results showed that miR156 was upregulated in response to drought in cowpea. MiR156 has been known to be responsive to abiotic stresses and targets SPB transcription factors in Arabidopsis, maize, rice and wheat [24, 39, 41–43]. This miRNA is also involved in the regulation of development during vegetative phase change , indicating that reprogramming of development is a crucial step in plants to cope with drought stress. Another miRNA, miR169, was downregulated in both cowpea genotypes. In Arabidopsis, miR169 was downregulated and its target, a Nuclear Factor Y transcription factor NFYA5, was induced by drought stress . MiR169 most likely functions in a similar way in cowpea to enhance drought tolerance by inducing the expression of NFYA5 orthologs.
The cowpea genotypes studied in this work have different abilities of drought tolerance. Because the two genotypes are highly similar to each other in their genetic composition, their phenotypic variations such as drought tolerance are most likely caused by changes in regulatory processes, rather than changes in proteins . Due to their different geographical origins, the two genotypes are adapted to the particular environmental conditions in their natural habitats. It is thus expected to find constitutive differences, which could be related to metabolism, use of energetic resources, mobilization of biomass, structure of radical system, wax deposition in leaves, membrane stability or density of stomata, among other characteristics. We found that nine miRNAs were predominantly or exclusively expressed in only one genotype, regardless of the treatments. On the other hand, 11 miRNAs were found to be differentially expressed under drought stress in one genotype, but not the other. Changes in miRNA expression are expected to cause changes in the expression of target genes between the two genotypes.
Among miRNAs that had genotype-specific regulation, miR160a and miR160b were upregulated in response to drought in the tolerant, but not in the sensitive cultivar (Table 2). Their putative targets are members of the family of Auxin Response Factors (ARFs). ARFs are key elements in regulation of physiological and morphological mechanisms mediated by auxins that may contribute to stress adaptation . Moreover, negative regulation of ARF10 by miR160 was demonstrated to be critical during seed germination in Arabidopsis thaliana through the crosstalk between auxin and ABA-dependent pathways . On the other hand, two members of the miR2111 family were upregulated by drought in the sensitive, but not in the tolerant cultivar (Table 3). Their putative targets are Kelch repeat-containing F-box proteins that belong to a large family with members known to be involved in response to biotic and abiotic stresses . Furthermore, F-box proteins containing Kelch repeats have been found to be responsive to drought in chickpea, a close relative of cowpea . This suggests that genotype-specific regulation of miRNAs might be part of the reason why some cowpea genotypes have stronger drought tolerance than others.
Among the new miRNA candidates that were identified in this study, ten were regulated by drought stress and target genes were predicted for five of them. For instance, vun_cand030 was downregulated by drought and putatively targets a zinc finger protein. Zinc finger proteins are known to be involved in a variety of functions in development and stress response . Moreover, vun_cand015 was upregulated by drought in the tolerant cultivar and putatively targets a basic-helix-loop-helix (bHLH) transcription factor. These proteins have roles in response to abiotic stresses, such as iron deficiency , freezing, and salt stress . This suggests these new miRNAs may be indeed an integral component of drought response in cowpea.
For many miRNAs there were more than one target predicted. The possibility of a miRNA to have multiple targets is commonly observed. To confirm these predicted targets, we need to perform detailed analysis of cleavage of mRNA targets at the miRNA recognition site by experimental approaches, such as RACE and degradome analysis [52–54]. Once we validate the targets of drought-associated miRNAs, we will be in a better position to link the expression changes of miRNAs and their targets to differences of drought tolerance in cowpea.
Because we do not have the complete cowpea genome sequence, some miRNA genes were not identified, even though they had significant expression in our small RNA libraries. To find out how many miRNA families have been missed, we mapped unique small RNA reads to plant miRNA precursors in the miRBase, allowing up to 2 mismatches. Although we did not miss a large number of miRNAs, we did find that miR2118, miR2911, and miR529 had significant expression in our libraries (Additional file 5). The latter two were also induced by drought stress. MiR529 was identified as drought-associated miRNA in rice . However, contrary to the pattern that we found in cowpea, it was downregulated under drought stress in rice. It is not clear whether it was caused by different sampling time or tissue, or species-specific stress response mechanisms.
Like protein coding genes, many miRNA families possess more than one miRNA gene and miRNA genes from the same family may have either identical or similar but different mature miRNA sequences. During evolutionary process, homologous miRNA genes may functionally diverge from each other. In the set of miRNAs that we identified in cowpea, members from miR166 and miR167 families showed clear evidence for functional diversification. While one member miRNA gene (miR166a, miR167b) was induced by drought stress, another miRNA from the same family (miR166b, miR167a) was significantly downregulated (Additional file 4).