The Function And Mechanism Of ZmmiR169q In Regulating Salt Stress Response In Maize

Maize is an important food,feed and industry crop,salt stress can significantly affect maize growth and productivity.miRNAs are key regulators of plant growth and development as well as in stress responses.However,little is known about their regulatory functions affecting the maize salt response.In previous studies,we identified a salt stress-sensitive miRNA,ZmmiR169q.ZmmiR169q is reduced by salt stress and is involved in regulating to salt stress response in maize.On the basis of previous studies,the molecular mechanism of ZmmiR169q in response to salt tolerance in maize was studied.The main results are as follows:1.GUS-staining assay of pmiR169q:GUS showed that the expression of ZmmiR169q was sharply reduced by 1 h high salinity?200 mM NaCl?treatment followed by a gradual increased after 4h,suggested the possibility that ZmmiR169q might act as a salt-responsive signal.2.ZmmiR169q knockdown transgenic maize plants?MIM169q?and overexpression lines?ZmmiR169q OE?and their wild-type plants were treated with 200 mM sodium chloride.The results showed that MIM169q increases seedling survivals,whereas ZmmiR169q OE significantly decreases salt tolerance.These results indicated ZmmiR169q functions as a negative regulator of salt tolerance.3.The transcriptome sequencing of ZmmiR169q OE and the root of WT showed that the antioxidant activity gene mainly composed of peroxidase gene was significantly enriched in ZmmiR169q OE,indicating that ZmmiR169q may participate in the oxidative stress induced by salt stress.We used hydrogen peroxide?H₂O₂?treatments to examine how the expression of ZmmiR169q is affected by excess ROS stress in maize WT plants.qPCR showed that ZmmiR169q expression decreased sharply at 1 h post exposure to 1mM H₂O₂,bottomed at 2 h,and the then increased after 4 h.Further experiments with pmiR169q:GUS transgenic maize plants indicated that exposure to 1mM H₂O₂ resulted in decreased transcription from the miR169 promoter.these results show that increased oxidative stress rapidly reduces both the transcription and overall accumulation of ZmmiR169q in maize roots.4.Our previous study showed that that ZmmiR169q targets 7 NF-YA genes in maize?ZmNF-YA1,ZmNF-YA6,ZmNF-YA7,ZmNF-YA13,and ZmNF-YA14?.We investigated the tissue/cell specificity of ZmmiR169q and ZmNF-YA14 expression and observed strong GUS activity in pmiR169:GUS transgenic plants and pNF-YA14:GUS transgenic plants in the root cortex and endodermis tissues of the mature zone.Further,qPCR analysis showed that ZmNF-YA14 expression was significantly decreased in roots of ZmmiR169q OE plants roots compared to the WT.These findings confirm our previous characterization of ZmNF-YA14 as a target of ZmmiR169q.ZmNF-YA14 overexpression transgenic lines?ZmNF-YA14 OE?showed a significant increase in survivals compared with the WT plants.These results demonstrate the positive regulation of ZmNF-YA14 on increasing maize salt tolerance.5.We investigated ROS levels in leaf tissues and root tissues of different maize lines under normal and 200 mM NaCl treatment by monitoring the accumulation of H₂O₂?3,3?-diaminobenzidine staining?and O^2·-?Nitroblue tetrazolium staining?.Under both normal and salt stress conditions,and compared to the WT,ZmmiR169q OE transgenic roots had increased H₂O₂ and O^2·-whereas ZmNF-YA14 OE transgenic roots had reduced levels of both ROS species.To determine whether the observed increases in ROS levels correlate with increased cell injury,we evaluated the MDA content in the various maize lines upon salt stress.The ZmmiR169q OE transgenic lines?with higher ROS levels?accumulated more MDA than WT plants,whereas the ZmNF-YA14 OE transgenic plants?lower ROS accumulation?expressed less MDA than WT.6.We measured the activities of antioxidant enzymes including SOD,CAT,and POD after treating maize seedings with salt stress for 96 h.The activities of all three enzymes were significantly higher in the ZmNF-YA14 OE transgenic plants than in the WT.In contrast,the activities of all three enzymes were significantly reduced in the ZmmiR169q OE plants.These results demonstrate that upregulated ZmNF-YA14 levels could promote ROS scavenger enzyme activity.7.RNA-seq-based transcriptomics analysis showed that enrichment for GO terms associated with tetrapyrrole binding and antioxidant activity for the differentially expressed gene?DEGs?in ZmNF-YA14 OE.We also found that Zm00001d018618 was among the significantly down-regulated DEGs in ZmmiR169q OE line but among the up-regulated DEGs in the ZmNF-YA14 OE line.Zm00001d018618 is predicted to encode a peroxidase 1 protein named ZmPER1.We conducted qPCR analysis which confirmed our findings from the RNA-seq analysis:the abundance of ZmPER1 increases upon ZmNF-YA14 mRNA accumulation,supporting that ZmPER1 transcription may be positively regulated by ZmNF-YA14.8.We analyzed the cis-elements in the predicted promoter region of the ZmPER1 locus seeking to identify potential binding sites of ZmNF-YA14,three typical CCAAT-boxes were found in the ZmPER1 promoter.yeast single hybrid and ChIP-qPCR assays indicate that ZmNF-YA14 bind to the promoter of Zm PER1 gene directly and promote their expression.