Abstract
MicroRNA396 (miR396) is a conserved microRNA family that targets growth-regulating factors (GRFs), which play significant roles in plant growth and stress responses. Available evidence justifies the idea that miR396-targeted GRFs have important functions in many plant species; however, no genome-wide analysis of the pitaya (Hylocereus polyrhizus) miR396 gene has yet been reported. Further, its biological functions remain elusive. To uncover the regulatory roles of miR396 and its targets, the hairpin sequence of pitaya miR396b and the open reading frame (ORF) of its target, HpGRF6, were isolated from pitaya. Phylogenetic analysis showed that the precursor miR396b (MIR396b) gene of plants might be clustered into three major groups, and, generally, a more recent evolutionary relationship in the intra-family has been demonstrated. The sequence analysis indicated that the binding site of hpo-miR396b in HpGRF6 is located at the conserved motif which codes the conserved “RSRKPVE” amino acid in the Trp–Arg–Cys (WRC) region. In addition, degradome sequencing analysis confirmed that four GRFs (GRF1, c56908.graph_c0; GRF4, c52862.graph_c0; GRF6, c39378.graph_c0 and GRF9, c54658.graph_c0) are hpo-miR396b targets that are regulated by specific cleavage at the binding site between the 10th and 11th nucleotides from the 5′ terminus of hpo-miR396b. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that hpo-miR396b is down-regulated when confronted with drought stress (15% polyethylene glycol, PEG), and its expression fluctuates under other abiotic stresses, i.e., low temperature (4 ± 1 °C), high temperature (42 ± 1 °C), NaCl (100 mM), and abscisic acid (ABA; 0.38 mM). Conversely, the expression of HpGRF6 showed the opposite trend to exposure to these abiotic stresses. Taken together, hpo-miR396b plays a regulatory role in the control of HpGRF6, which might influence the abiotic stress response of pitaya. This is the first documentation of this role in pitaya and improves the understanding of the molecular mechanisms underlying the tolerance to drought stress in this fruit.
Highlights
MiRNAs are endogenous, single-strand, non-coding, small-molecular-weight RNAs of about 20 nucleotides in length, whose precursors are characterized by a stem loop structure [1,2]
The expression of the miR169 is inhibited by low nitrogen levels in Arabidopsis thaliana, and its target Nuclear transcription factor Yalpha (NFYA), which is part of a network that regulates nitrogen metabolism in combination with Nitrate transporters1.1 (AtNRT1.1) and AtNRT2.1 in A. thaliana, was up-regulated [14]
The expected band at about 240 bp was observed by 2% agarose gel electrophoresis (Figure 1A), which is based on the stem-loop structure of hpo-miR396b
Summary
MiRNAs are endogenous, single-strand, non-coding, small-molecular-weight RNAs of about 20 nucleotides (nt) in length, whose precursors are characterized by a stem loop structure [1,2] They are known to regulate gene expression at the post-transcriptional level through cleavage and/or the translational repression of mRNAs [3]. Available evidence justifies the idea that miRNAs are highly conserved, temporally and tissue- expressed, and lead to the regulation of many biological processes, including those involved in growth, development, and metabolism, as well as abiotic and biotic stress responses in plants [7,8]. Various studies have proven that the regulation of GRFs by miR396 plays an important role in plant growth and development and involves a variety of stress responses [21]. The results generated may further elucidate the roles of miR396b in plants’ adaptation to abiotic stresses
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