| Wood are widely used in commericial areas,such as bio-energy,chemical products,building furniture,and paper-making industries.Wood is also known as secondary xylem,and its formation process is,in biology,the secondary development of stem vascular.The secondary development of stem vascular is regulated by a genetic network that include transcription factors,hormones,miRNAs and other internal factors and external environmental signals such as temperature and humidity.China,is the world’s largest consumer of wood resources and the largest wood importer.So it is imminent problem to increase wood biomass in China.In recent years,a number of researches have revealed the basic molecular mechanism for controling stem vascular secondary development,however,the complete network for fine regulation of it remain largely unknown.MicroRNAs(miRNAs)are endogenous non-coding RNAs with a size of about20～22nt,which are one of the most abundant and conserved regulators for gene expression in multicellular organisms.They play an important role in the growth and development of animals and plants by transcripts cleaving or translational repression through binding specifically to the mRNAs of downstream target genes.Poplar is the genetic model species for forest plants.There have been some studies on the function of poplar-specific miRNAs in the growth and development of poplar,but there is still a lot to be researched.Therefore,studying the regulation of poplar-specific miRNAs on the secondary development of stem vascular tissue can provide an experimental basis for revealing the molecular mechanism of secondary vascular tissue development regulation in trees,and provide a theoretical basis for rapid growth of trees and improvement of wood quality.Our group had screened out 32 species-specific miRNA families expressed in the stems of poplar previously.Combined with the existing regulatory networks between miRNAs and various traits of Populus tomentosa,eleven of them have been identified as potential regulators for stem vascular secondary development.To further explore the functions of these 11 miRNAs,they were cloned and their transgenic plants were obtained for bio-functional studies.This paper focused on the-miR7833 for its function and mechanism studies.The main findings are as follows:1.The expression pattern of miR7833 was analyzed.The results of tissue expression profiles,GUS staining,as well as high-throughput data indicate that miR7833 is expressed in various organs,where the stem is mainly highly expressed in the forming layer,while the developing xylem and xylem are hardly expressed.2.The phenotypes of miR7833-overexpressing and 35S::STTM7833 transgenic poplar were observed.Macroscopically,compared with the wild type,there were no significant differences in plant height,diameter and internodes of miR7833 transgenic plants.After sectioning,it was found that compared with wild type,the number of xylem cells and cambium cells decreased after miR7833 overexpression;35S::STTM7833 transgenic plants had the opposite phenotype to overexpression plants.These results suggest that miR7833 plays an important role in regulating cambium division and differentiation into xylem cells.3.Key target genes of miR7833 in regulating secondary vascular development were identified.Based on the prediction results of the miRNA target gene prediction website,literature research reports,degradome sequencing,the expression level in transgenic plants,and the phenotype of miR7833 transgenic plants,a potentially related target gene,AUX6,a member of the AUX/LAX family,was screened.The target genes were verified by fluorescent cleavage experiments in tobacco transient infection,and the results showed that miR7833 could target the cleavage of AUX6 transcripts.4.The expression pattern of the target gene AUX6 was analyzed.The results of GUS staining and high-throughput data showed that AUX6 was highly expressed in the developing and mature xylem,but was less expressed in the cambium.Its expression pattern is complementary to miR7833,which further confirms that AUX6 is the target gene of miR7833.5.A key role of AUX6 in miR7833 regulation of secondary vascular development is demonstrated.The mAUX6 overexpression vector was constructed after synonymous mutation of the binding site of the target gene AUX6 and miR7833,which was transferred into wild-type plants and transgenic plants with overexpression of miR7833,respectively.After statistics,it was found that overexpression of mAUX6 could restore the xylem and cambium cell layers of miR7833 overexpression plants to wild-type levels.These results indicate that the target gene AUX6 can regulate the activity of the vascular cambium in poplar stems,and prove that miR7833 regulates the secondary development of stem vascular by targeting and inhibiting the expression of AUX6.6.The molecular pathways in which miR7833 affects secondary vascular development by regulating auxin were confirmed.AUX6 is a member of the AUX1/LAX family of auxin influx carriers.Wild-type and 35S::STTM7833 transgenic plants were treated with auxin influx inhibitor 1-NOA.Section analysis and observation showed that 1-NOA treatment reduced the number of cambium and xylem cell layers of them.These results suggest that miR7833 affects auxin transport by targeting AUX6,thereby affecting vascular cambium activity during secondary vascular development in poplar stems.These results indicate that miR7833 targets AUX6 to regulate the secondary development of stem vasculars through auxin influx carriers-mediated auxin signaling pathways.In summary,the results of this paper preliminarily confirm that poplar-specific miR7833 targets the AUX6 gene and inhibits its expression level,thereby affecting the activity of the vascular cambium during the secondary development of stem vascular,resulting in changes in cambium and xylem region width,number of layers and proportion of cells.To provide a certain experimental basis for revealing the molecular mechanism of the development and regulation of secondary vascular tissue in poplar. |
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