Abstract
BackgroundMicroRNAs are a class of small, non-coding RNAs that regulate gene expression by binding target mRNA, which leads to cleavage or translational inhibition. The NAC proteins, which include NAM, ATAF, and CUC, are a plant-specific transcription factor family with diverse roles in development and stress regulation. It has been reported that miR164 negatively regulates NAC1 expression, which in turn affects lateral root development in Arabidopsis; however, little is known about the involvement of the maize NAC family and miR164 in lateral root development.ResultsWe collected 175 maize transcripts with NAC domains. Of these, 7 ZmNACs were putative targets for regulation by miR164. We isolated one gene, called TC258020 (designated ZmNAC1) from 2 maize inbred lines, 87-1 and Zong3. ZmNAC1 had a high expression level in roots and showed higher abundance (1.8 fold) in Zong3 relative to 87-1, which had less lateral roots than Zong3. There was a significant correlation between the expression level of ZmNAC1 and the lateral root density in the recombinant inbred line (RIL) population. Transgenic Arabidopsis that overexpressed ZmNAC1 had increased lateral roots in comparison to the wild type. These findings suggest that ZmNAC1 played a significant role in lateral root development. An allelic expression assay showed that trans-regulatory elements were the dominant mediators of ZmNAC1 differential expression in 87-1 and Zong3, and further analysis revealed that miR164 was a trans-element that guided the cleavage of endogenous ZmNAC1 mRNA. Both mature miR164 and miR164 precursors had higher expression in 87-1 than Zong3, which was the opposite of the expression pattern of ZmNAC1. Additionally, the allelic assay showed that the cis-regulatory element most likely affected Zm-miR164b's expression pattern. A β-glucuronidase (GUS) assay showed that the Zm-miR164b promoter had higher GUS activity in 87-1 than in Zong3. In addition, we detected miR164b expression in the RIL population, and the results indicated that miR164b had a higher expression level in the RILs containing 87-1 promoter than those containing Zong3 promoter.ConclusionOur results indicate one possible pathway in maize by which differences in miR164b promoter activity resulted in a different expression pattern for mature miR164 which negatively regulates ZmNAC1 expression in 87-1 and Zong3, thereby contributing to a significantly different lateral root phenotype.
Highlights
MicroRNAs are a class of small, non-coding RNAs that regulate gene expression by binding target mRNA, which leads to cleavage or translational inhibition
Our results showed that GUS activity that was driven by the promoter from 87-1was significantly higher than that driven by the promoter from Zong3 and by the background (Figure 7B), which strongly suggested that the different promoter activities of pri-miR164 resulted in the diversity of pri-mir164 transcript levels that was found between the two inbred lines, which may have led to the difference in mature miR164 expression between 87-1 and Zong3
ZmNAC1 played an important role in lateral root development We isolated and characterized ZmNAC1, which is a member of the NAC-domain gene family and the first NAC gene shown to be involved in maize lateral root development
Summary
MicroRNAs are a class of small, non-coding RNAs that regulate gene expression by binding target mRNA, which leads to cleavage or translational inhibition. The NAC proteins, which include NAM, ATAF, and CUC, are a plant-specific transcription factor family with diverse roles in development and stress regulation. NAM from petunia [1] and CUC2 of Arabidopsis [2] (the first reported NAC genes) are involved in shoot apical meristem (SAM) development. The Arabidopsis NAC gene CUC3 has been reported to contribute to the establishment of the cotyledon boundary and the shoot meristem [6] Another Arabidopsis NAC gene called NAP (NAC-LIKE, activated by AP3/PI) has been characterized as the target of two MADS box genes, APETAL3 and PISTILLATA, which control cell division and cell expansion in stamens and petals [7]. Some NAC genes such as BnNAC from Brassica [10], AtNAC072 (RD26), AtNAC019 and AtNAC055 from Arabidopsis [11], and SNAC1 [12] and SNAC2 [13] from rice were found to be involved in the plant's response to various environmental stresses, including drought, salinity, and/or low temperature
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