| The secondary vasculature plays a decisive role in the growth and development of terrestrial plants.It is not only the important long-distance material transport channel,but also provides necessary mechanical support for plant roots and stems.The secondary vascular development originates from vascular cambial stem cells.The cells division of vascular cambium is the main cell source of secondary vascular development,and it differentiates into secondary phloem centrifugally and secondary xylem centrifugally,forming the main transportation tissue in the secondary vascular.Therefore,maintaining the homeostasis between cell division and cell differentiation of vascular cambium is crucial to keep vascular development normal and orderly.The vascular cambium development is precisely regulated by complex networks involving transcription factors and phytohormones.Among them,the TDIF/TDR signaling pathway,as the core of the cambium development regulation network,determines the vasculature pattern and maintains cambial stem cells activity by coordinating its downstream WOX4 and GSK-BES branch pathways.However,what signaling factors can enable to regulate TDIF/TDR pathway is still unexplored.Phytohormone is important endogenous signaling factors,and plays an important role in vascular cambial development.The recently study showed that auxin,one of the factors defining the stem cell organizer of vascular cambium,is mainly distributed in the vascular cambium zone maintaining vascular cambial stem cell activity and promoting xylem differentiation via its downstream response factors.In addition,the other phytohormones such as gibberellin,ethylene and brassinolide have been reported to influence vascular cambial development.Cytokinin,as a kind of phytohormones directly involves in cell proliferation,is necessary for maintaining the characteristics of stem cell in meristems.The previous studies in Arabidopsis have been proved that the inhibition of cytokinin biosynthesis will lead to serious vascular development defects,indicating that cytokinin is indispensable in vascular development.In woody plants,cytokinin have also been certified to be involved in the local regulation of vascular cambium activity.However,the function of cytokinin enriched in secondary phloem on the secondary vascular development has not been confirmed.In this study,the poplar was used as the research model to precisely represent the distribution pattern of cytokinin and its receptor in the secondary vasculature by immunofluorescence and in situ hybridization,respectively.Meanwhile,the noncellautonomously regulation of cytokinin signaling located in secondary phloem on vascular cambial homeostasis and its molecular mechanism was further analyzed via molecular biology and genetics methods.The main results are list as follows:1)Precisely characterize the spatial distribution pattern of cytokinin in secondary vasculature.In order to visualize the distribution pattern of cytokinin in secondary vascular tissues,specific antibodies against trans-zeatin(t Z)and its precursor trans-zeatin riboside(t ZR),the main active form of cytokinin,were used to label cytokinin in secondary vasculature.The distribution pattern of cytokinin in secondary vasculature was drawn through the quantitative statistics of fluorescence signals in secondary vasculature,which showed that the concentration peak of cytokinin was located in the secondary developing phloem and decreased along the direction of vascular cambium.2)Maintaining cytokinin distribution pattern is necessary for secondary vascular development.To clarify the biological function of the cytokinin gradient distribution,the cytokinin oxidase gene is activated by the specific promoter in the peak region of cytokinin concentration(secondary phloem),which destroys the distribution pattern of cytokinin in secondary vasculature.By analyzing the phenotypes related to vascular cambial development,it was found that the cell division activity of vascular cambium decreased significantly in transgenic plants,indicating that the gradient distribution pattern of cytokinin is very important for the maintenance of vascular cambial activity during vascular development.3)There is the complete cytokinin signaling transduction pathway in secondary phloem.To explore whether the cytokinin enriched in secondary phloem can carry out local signaling transduction pathway,the distribution of cytokinin receptors in vasculature was detected.The transcripts of cytokinin receptors in secondary phloem and vascular cambium were determined by the RNA in situ hybridization.Meanwhile,the expression level of cytokinin signaling response marker type-A RR genes in the secondary phloem of transgenic plants(pro CLE41b::At CKX2)destroyed the cytokinin distribution pattern was significantly down-regulated,which proved that there was a complete cytokinin signaling transduction pathway in the secondary phloem region.4)Secondary phloem-located cytokinin signaling noncell-autonomously regulates vascular cambial homeostasis.To reveal the regulation of secondary phloem cytokinin signaling on secondary vasculature,the transgenic plants(pro CLE41b::HK-RNAi and pro CLE41b::RR13)with secondary phloem specific promoter mediated interference/enhancement of cytokinin signaling were established.Phenotypic analysis showed that the vascular cambial cell division activity and its differentiation into secondary xylem in transgenic plants changed significantly.Under the background of interfering with phloem cytokinin signaling,changing cytokinin content will not cause more serious phenotype.This indicates that the noncell-autonomous regulation of cytokinin signaling located in secondary phloem on vascular cambial homeostasis depends on local signaling transduction pathway of secondary phloem.5)Preliminary screening of downstream target genes of secondary phloemlocated cytokinin signaling.To analyze the molecular mechanism of secondary phloem-located cytokinin signaling regulating vascular cambial homeostasis,the differentially expressed genes between wild-type and cytokinin signaling change transgenic plants were analyzed by RNA-seq,and screened in combination with the published poplar secondary vasculature RNA-seq database(Asp Wood).The DOF transcription factors family and CLE41/44 gene belonging to CLE peptide family,which are specifically expressed in phloem and induced by cytokinin,were preliminarily determined as potential target genes for cytokinin signaling located in secondary phloem.6)Cytokinin signaling induces the expression of potential target genes DOF and CLE41/44.In order to more accurately verify the induction of cytokinin signaling on the transcription factors of DOF family and CLE41/44 genes,the transcription levels of genes transgenic lines with specifically interfering with secondary phloem-located cytokinin signaling and exogenous cytokinin treated lines were detected in transgenic lines with specifically interfering with secondary phloem-located cytokinin signaling and exogenous cytokinin treated lines.The results of RT-q PCR showed that the potential target genes of cytokinin signaling responded to the change of cytokinin signaling.The cytokinin response factor type-B RR overexpression vector(35s-RR13)was established to determine that the cytokinin signaling can activate the transcriptional activity of DOF family transcription factors and CLE41/44 genes through the Effector-reporter system.7)The protein encoded by DOF and CLE41/44,the potential target genes of cytokinin signaling in secondary phloem,can move between secondary phloem and vascular cambium.To verify whether the proteins encoded by the potential target genes of cytokinin signaling located in secondary phloem can move between secondary vascular cells,transgenic lines(pro DOF35-GUS and pro CLE41b-e YFP-TMC)connecting the reporter genes with the transcription factor DOF35 promoter and CLE41 b promoter were established.It was determined that the transcription initiation position was in the secondary phloem.Furthermore,the DOF35 promoter was constructed to drive the DOF35-e YFP fusion protein,and the TDIF peptide encoded by CLE41/44 in secondary vasculature was labeled by specific antibody.Finally,it was determined that the proteins encoded by the potential target genes of secondary phloem-located cytokinin signaling moved from the secondary phloem to the vascular cambium.8)Cytokinin signaling directly binds to CLE41/44 gene promoters to promote its transcription activity.To determine whether cytokinin directly regulates the transcription of DOF family genes and CLE family CLE41/44 genes,treatment with actinomycin(CHX),a protein synthesis inhibitor,ruled out the possibility of cytokinin regulating downstream target genes with other proteins.After cytokinin treatment,RT-q PCR showed that only CLE41/44 genes were still induced by cytokinin.The chromatin immunoprecipitation(CHIP)analysis also found that there were direct binding sites of cytokinin response factor type-B RR only in the promoter region of CLE41/44 genes.Furthermore,the mutation of the direct binding sites of cytokinin response f in the promoter of CLE41/44 genes was verified again by Luciferase mediated Effector-reporter system,which was no longer induced by type-B RR transcription factor.These results suggest that cytokinin signaling can directly regulate the transcription of CLE41/44 genes through type-B RR transcription factor,while the induction of DOF family transcription factors may be indirect.9)The TDIF peptide encoded by CLE41/44 genes mediates the noncellautonomous regulation of secondary phloem cytokinin-located signaling on vascular cambial homeostasis.The above experiments have determined that cytokinin signaling can directly regulate the transcription of CLE41/44 genes.The distribution of TDIF peptides in transgenic plants(pro CLE41b::HK-RNAi)that specifically interfere with secondary phloem cytokinin signaling has not changed,but its accumulation level has decreased significantly.Meanwhile,the CLE41 b in situ overexpression vector(pro CLE41b::CLE41b)was established and transferred into the transgenic plant interfering with cytokinin signaling(pro CLE41b::HK-RNAi)by genetic transformation technology.The observation of phenotypes related to secondary vascular development found that the overexpression of CLE41 b gene restored the development defect of vascular cambium caused by the interference of cytokinin signaling in secondary phloem,and the expression level of key genes related the maintenance of vascular cambium homeostasis was also up-regulated to the wild-type level.These results suggest that the TDIF peptide encoded by CLE41/44 genes mediates the noncell-autonomous regulation of secondary phloem-located cytokinin signaling on vascular cambial homeostasis.In conclusion,this study revealed that the cytokinin signaling located in the secondary phloem can regulate vascular cambial stem cell homeostasis in a noncellautonomous manner.The cytokinin signaling of secondary phloem directly induces the accumulation of mobile signaling factor TDIF peptide in secondary vasculature,and affects vascular cambial cell division and secondary xylem differentiation through this peptide. |
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