Study On Molecular Mechanism And Application Potential Of MiR156 In Response To Cadmium Stress In Tobacco

With the rapid development of global industrialization and urbanization,soil resources are increasingly polluted by heavy metals.The heavy metals pollution in soil,mainly Cd,not only has an important impact on the yield and quality of crops,but also poses a great threat to human health through the food chain.Phytoremediation is a low-cost,pollution-free and suitable remediation method for large-scale soil pollution,but its practical application is limited by the long remediation cycle.The enhancement of hyperaccumulators by genetic engineering is an effective method to improve the efficiency of phytoremediation.Presently,miR156has been shown to respond to heavy metal stresses such as Cd in several plant species,but its molecular mechanism is not yet clear.In this study,we explored the molecular mechanism of Nta-miR156 in response to Cd stress and the effect of overexpression of Nta-miR156 on Cd tolerance and accumulation in plants using the hyperaccumulators tobacco（Nicotiana tabacum）as material.1.Identification of tobacco miR156 family members and the analysis of the expression patternTo obtain basic information about the members of the tobacco miR156 family,the sequence characteristics and expression patterns of 20 members of the tobacco miR156 family were characterized by bioinformatics methods.The results of multiplex sequence alignment showed that the precursor sequences were highly different from each other while the mature sequences were highly conserved.Except for Nta-MIR156i,all the members of tobacco miR156 family contain stable stem-loop structures and low minimum folding free energy（-44.2 to-63.2 kcal/mol）.In addition,the promoter regions of all members contain cis-acting elements and transcription factor binding sites of varying types and numbers,implying a complex pattern of transcriptional regulation.The target genes of tobacco miR156 mainly aim at Nt SPL genes to perform biological functions,and the target genes of Nta-miR156 members with different core sequences are slightly different.Nta-miR156p and Nta-miR156q were higher expressed only in flower and pod;while Nta-miR156e/f/h/i/l/r were lower expressed only in stem,leaf and root,and the remaining 12 members were highly expressed in all tissues.In addition,the expression of Nta-miR156 was significantly up-regulated in tobacco during 1-3 d under Cd stress,while it returned to the control level after 7 d.Nta-miR156a was selected for the follow-up study based on its representativeness as a family member in response to Cd stress.2.Effect of overexpression of miR156 on biological traits of tobaccoTo ascertain the effect of miR156 on tobacco growth and development,five tobacco lines which stably overexpressed Nta-miR156a（OE Nta-miR156a）were obtained by the Agrobacterium-mediated genetic transformation method.The overexpression of Nta-miR156a caused dramatic changes in the biological traits of tobacco,mainly in the greater number of leaves and branches,reduced leaf photosynthetic pigment content and larger aspect ratio,thinner and shorter node spacing in the stem,and restricted root development.The content of proline,phenolic compounds,and flavonoids was significantly lower in OE Nta-miR156a tobacco lines compared to wild-type（WT）tobacco.In addition,overexpression of Nta-miR156a caused disturbance of metal endostasis in tobacco.Significant reduction of Cu content in three tissues of tobacco leaves,stems and roots,increased accumulation of Mn in leaves,and Fe showing a shift from roots to stems.3.Extraction of miR156-regulated genes involved in metal stress response based on comparative transcriptomicsTo analyze the causes of the differences in OE Nta-miR156a tobacco traits and changes in expression of genes associated with heavy metal resistance,transcriptome sequencing was used to investigate the differences between the two at the molecular level.The results showed that 1456 genes were significantly up-regulated and 1200genes were significantly down-regulated.Among them,the expression of 8 Nt SPL genes was significantly reduced in OE Nta-miR156a tobacco,confirming the targeting relationship between Nta-miR156 and SPL genes in tobacco.In addition,the14 significantly differentially expressed genes may be regulated by Nt SPL transcription factors,including 8 abiotic stress-related genes and 6 photosynthetic pigment system-related genes.There were also 33 genes which encode metal transporter proteins and 3 genes which encode with metal chelating proteins among the differentially expressed genes.GO enrichment analysis showed that differentially expressed genes enriched to molecular functions were mainly associated with transporter protein activity,antioxidant activity and signal transduction activity.KEGG enrichment results showed that differentially expressed genes were mainly enriched in metabolic pathways such as phytohormone signaling,glutathione metabolism,and ascorbic acid metabolism.In addition,the results of q PCR for 8genes showed a high correlation with the sequencing data（R²=0.9408）,affirming the accuracy of the sequencing results.4.Analysis of the potential of Nta-miR156a for phytoremediation applicationsTo further elucidate the application potential of miR156 in soil remediation,the Cd tolerance and accumulation of OE Nta-miR156a Arabidopsis thaliana under Cd stress were analyzed.OE Nta-miR156a Arabidopsis showed stronger Cd tolerance than WT Arabidopsis under 50μM Cd²⁺stress,as evidenced by higher seed germination rate and significantly greater root length,biomass and photosynthetic pigment content than WT.In addition,the cells of OE Nta-miR156a lines under Cd stress were more metabolically active,containing more proline,phenolic compounds,flavonoids,soluble sugars and starch.Moreover,the Cd content in the tissues of OE Nta-miR156a Arabidopsis was 1.61-fold and 1.49-fold higher than that of WT Arabidopsis,showing stronger Cd accumulation.In summary,this study identified 20 members of the tobacco miR156 family based on genome-wide analysis,which differ in gene structure and expression patterns and and can be regulated by the heavy metal Cd.Overexpression of miR156in tobacco resulted in transgenic plants with significant differences in biological traits,substance metabolism compared to WT tobacco,as well as disruption of metalloid homeostasis.Results of comparative transcriptomics suggest that the miR156-SPL module may be involved in multiple signaling pathways in response to environmental heavy metal stress.Overexpression of Nta-miR156a in Arabidopsis confers enhanced Cd tolerance and accumulation in transgenic Arabidopsis,implying that miR156 has potential applications in phytoremediation of Cd pollution.This study provides important evidence for an in-depth understanding of the biological functions of miR156 under Cd stress and lays the theoretical foundation for the development of novel germplasm resources of tobacco with strong resistance to stress.