Abstract
To identify the regulatory network of known and novel microRNAs (miRNAs) and their targets responding to salt stress, a combined analysis of mRNA libraries, small RNA libraries, and degradome libraries were performed. In this study, we used unique molecular identifiers (UMIs), which are more sensitive, accurate, and reproducible than traditional methods of sequencing, to quantify the number of molecules and correct for amplification bias. We identified a total of 312 cotton miRNAs using seedlings at 0, 1, 3, and 6 h after NaCl treatment, including 80 known ghr-miRNAs and 232 novel miRNAs and found 155 miRNAs that displayed significant differential expression under salt stress. Among them, fifty-nine differentially expressed miRNAs were simultaneously induced in two or three tissues, while 66, 11, and 19 were specifically expressed in the roots, leaves, and stems, respectively. It is indicated there were different populations of miRNAs against salt stress in roots, leaves and stems. 399 candidate targets of salt-induced miRNAs showed significant differential expression before and after salt treatment, and 72 targets of 25 miRNAs were verified by degradome sequencing data. Furthermore, the regulatory relationship of miRNA-target gene was validated experimentally via 5′RLM-RACE, proving our data reliability. Gene ontology and KEGG pathway analysis found that salt-responsive miRNA targets among the differentially expressed genes were significantly enriched, and mainly involved in response to the stimulus process and the plant hormone signal transduction pathway. Furthermore, the expression levels of newly identified miRNA mir1 and known miRNAs miR390 and miR393 gradually decreased when subjected to continuous salt stress, while overexpression of these miRNAs both increased sensitivity to salt stress. Those newly identified miRNAs and mRNA pairs were conducive to genetic engineering and better understanding the mechanisms responding to salt stress in cotton.
Highlights
Cotton (Gossypium hirsutum L.) is one of the primary agricultural and cash crops in the world and a major source of fiber and oil
The electric conductivity in leaves was significantly increased at three time points under the 300 mM NaCl treatment as compared to the control and reached the peak after 3 h, but with decreased electric conductivity at 6 than 3 h (Figure 1B); fresh weight and dry weight of seedling leaves decreased gradually and stabilized along with the increased salt treatment time, while plant water content increased gradually (Figure 1C), the changed trend of electric conductivity, fresh weight and dry weight both consisted with the wilting phenotypes of rising first and falling after 300 mM NaCl treatment
These results indicate that known miR390, miR393, and novel identified miRNA novel-mir1 both negatively regulated the response to salt stress in cotton, supporting that the identified salt-induced miRNAs were available, and will enrich the regulatory network of cotton responding to salt stress
Summary
Cotton (Gossypium hirsutum L.) is one of the primary agricultural and cash crops in the world and a major source of fiber and oil. MiRNAs and genes responding to salt stress have the potential for improving environmental adaptation to salinity in plants. The ABP9 gene encodes a bZIP transcription factor, which overexpression in transgenic Arabidopsis plants improved tolerance to drought, salt, and other abiotic stresses (Zhang et al, 2011). B3 DNAbinding domain proteins and auxin response factor (ARF) are involved in the response to environmental changes (Fedoroff, 2002; Himmelbach et al, 2003) Except for those protein-coding genes, miRNAs, endogenous non-coding RNAs (18–25 nt in length), play an important role in regulating target genes at the post-transcriptional or translation level (Rae Eden et al, 2013; Dong et al, 2015). Other miRNAs, including miR159, miR167, miR168, miR171, and miR319, all displayed altered expression levels when exposed to salt stress in plant (Cui et al, 2018; He et al, 2018)
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