| Drought is an important and restrictive factor for plants growth and development.As responses to drought stress,multiple adaptive changes in root,stem,and leaf were trigged,including the closure of the leaf stomata,the altered root morphorgenesis and so on.In the stem,the drought resistance can be improved by reducing the vessel lumen and meanwhile increasing the density of vessel.Since the xylem development process of stems in woody plant is more complicated and sustained for much longer period than that of herbs,the regulatory mechanism for stem development that response to drought in woody plants is also more complicated.To reveal the genetic pathway and mechanism for the stem xylem adaptive development in response to drought in trees,may have vital significances.micro RNAs(miRNAs)play crucial regulatory roles on plant development and responses to biotic and abiotic stresses.As a model species of woody plants,poplar has evolved a large number of poplar-specific miRNAs with novel functions.Therefore,to reveal the possible roles of these poplar-specific miRNAs in the adaptive developmental changes of stem xylem in response to drought stress,has important scientific significance and may deepen our understanding about how plants adapt to environmental changes.At the same time,it may also improve the application of wood in manufacture and energy industries.In this study,we first screened out 100 poplar-specific miRNAs based on the miRbase database.Next analyzed the expression levels of these poplar-specific miRNAs based on transcriptome data,and further screened out 38 miRNAs that are expressed in stems.And then,according to the q PCR detection of miRNAs precursor levels,we screened out the miRNAs that were up-regulated in stem in response to drought.In order to study whether these miRNAs can improve the drought resistance of plants by affecting the xylem structure and function of stems,we cloned and obtained transgenic plants of these miRNAs.In this study,we fouce on one of them,the miR6425.The main research results are as follows:(1)The expression pattern of miR6425 was analyzed.The results of GUS staining and in situ hybridization showed that miR6425 was highly expressed in phloem,cambium and primary vascular.(2)In transgenic plants,overexpression of miR6425 increased the number of xylem cell layers and reduced the diameter of xylem vessels and fiber.Inhibition of the endougenous miR6425-3p resulted in an opposite phenotypes to the overexpression plants in both of the cell layer number and vascular lumen,while there was no significant difference in xylem when the miR6425-5p was inhibited.The results showed that miR6425-3p simultaneously regulates cambium division and cell expansion.(3)According to the target prediction results of miR6425-3p,coding genes forα-Expansin(EXPA)and JACKDAWs(JKD),which are closely related to the development of xylem,were screened out.In order to analyze whether miR6425 targeted and cleaved target genes,degradome sequencing and 5’RACE(5’rapid-amplification of c DNA ends)showed that α-Expansin 7(EXPA7)and Expansin 15(EXPA15)were cleaved at the binding sites of miR6425.Similarly,degradome sequencing data also showed that miR6425 could directly targeted to and cleaved transcripts of two JKD genes,JACKDAW 1(JKD1)and JACKDAW 2(JKD2).The expression levels of EXPA7,EXPA15,JKD1 and JKD2 are suppressed in transgenic plants of miR6425.The inflorencense of GFP fusion with EXPA7,EXPA15,JKD1 or JKD2 are also largely repressed by miR6425 in the tobacoo leaf cells.Disruption of their recognize sites of miR6425 also lead to a steady inflorencense that resistant to miR6425.These data indicated that miR6425 can target to the transcripts of EXPA7,EXPA15,JKD1 and JKD2 and inhibit their expression on the post-transcriptional level.(4)The expression patterns of the target genes EXPA7,EXPA15,JKD1 and JKD2 were analyzed.The results of in situ hybridization and aspwood database indicated that EXPA15 was highly expression in the developing xylem.However,JKDs is mainly expressed in the cambium and phloem.These results suggest that these two kind of target genes of miR6425 may play different roles in xylem development.(5)The miR6425 recognition sites on EXPA15 and JKD2 were subjected to synonymous mutations to obtain the m EXPA15 and m JKD2 which were insensitive to miR6425.Then,m EXPA15 and m JKD2 were introduced into the wild-type and miR6425-overexpressing plants respectively by genetic transformation.In wild-type background,overexpression of m EXPA15 increase the xylem vessel and fiber cell lumen,while overexpressing m JKD2 can reduce the number of xylem cell layers.Expression of m EXPA15 and m JKD2 in miR6425-overexpressing plants could recovered the cell lumen and xylem cell layers,respectively,to a wild-type level.These results demonstrated that EXPA15 and JKD2 controlled the expansion of poplar xylem cells and the division of vascular cambium,respectively,and proved that miR6425 plays two different roles in regulating xylem development by targeting and inhibiting the expression of these two group of genes,respectively.(6)Upon drought treatment,the expression levels of EXPA7,EXPA15,JKD1 and JKD2 were down-regulated in the stem,which is consistent to the inducement of miR6425 in the stem by drought.It suggests that drought may inhibit the levels of EXPA7,EXPA15,JKD1 and JKD2 in secondary vascular tissues through inducing the miR6425.(7)The results of Yeast two-hybrid system(Y2H)and bimolecular fluorescence complementation(Bi FC)show that JKD2 can interact with SHR,a known regualator for cambium cell division,thus indirectly associatied with SCR through SHR and formed a JKD-SHR-SCR transcription factor ternary complex in poplar to coordinately regulate the transcription of downstream genes and the vascular cambium division.In conclusion,the poplar-specific miR6425 affects both of the devision of vascular cambium into xylem cells and the cell expansion of developing xylem cells,respectively,through inhibiting specifically two groups of target genes,the EXPA7,EXPA15 and JKD1,JKD2 in the stem.Overexpression of miR6425 leading to an increased number of xylem layers and a decreased lumen of the xylem vessels and fibers.In addition,upon drought stress,the expression level of miR6425 was up-regulated,while the expression of its target genes was down-regulated.These results suggest that miR6425 may repress EXPA7,EXPA15,JKD1 and JKD2 in the stem in response to drought stress and result in the adoptive structural changes in the stem xylem through modulating the cell devision and differentiation in vascular development,and thus,improve the tolerance of the plant to drought stress. |
