| The secondary development of vascular tissues in trees provides the biological basis for wood formation.The vascular cambium with the activity of stem cells divides and differentiates bilaterally in a continuous manner,as a consequence producing the secondary xylem inward and the secondary phloem outward.Xylem cells have abundant secondary cell wall composed of lignin,cellulose and hemicellulose,and are mainly responsible for mechanical support and transporting water from roots to shoots.In contrast,phloem plays an important role in transporting photosynthetic products and signaling molecules.It is known that the differentiation and development of phloem are regulated by transcription factors and plant hormones.APL transcription factors have been shown to play antagonistic roles in promoting phloem differentiation and inhibiting xylem differentiation in Arabidopsis.LBD1 transcription factor promotes secondary phloem development in poplar.Cytokinins are key for the developmental process of vascular tissues via regulating the cell division of vascular cambium.Previous studies indicated that cytokinin is preferentially accumulated in the secondary phloem of poplar stems,and regulate the regeneration of phloem after stem peeling.However,the interactions among cytokinins,LBD1 s and APLs during the regulation of secondary phloem and the underlying molecular mechanisms remain unclear,and need to be further explored.Poplar is an important tree species of fast-growing forest worldwide,and also used as a model tree for the molecular biology research in trees,due to the availability of its whole genome sequences.In this dissertation,we used Populus tomentosa to analyze the distribution pattern of cytokinin,LBD1 s and APLs in secondary vascular tissues of poplar stems and their actions on secondary phloem development,validate their role in phloem regeneration through in vitro secondary vascular tissue regeneration system,and thereby further elucidate their interactions and molecular mechanisms underlying the regulation of secondary phloem development.The main findings are as follows:1.The distribution patterns of cytokinin signaling,LBD1 s and APLs in secondary vascular tissues of poplar stems.To explore the distribution patterns of cytokinin signaling,LBD1 s and APLs,the secondary phloem,cambium and secondary xylem were collected,respectively,from poplar stems via cryosectioning,and subsequently the expression of some A-type RR genes,the markers of cytokinin responses,was investigated by q PCR.We found that the A-type RR genes are preferentially expressed in secondary phloem.Then the YFP reporter line driven by the promoter of an A-type RR gene was also constructed.The fluorescent signals were mainly detected in secondary phloem via confocal microscopy,thereby validating the enrichment of cytokinin in secondary phloem.GUS reporter lines of the LBD1 s and APL1 s revealed their preferential expression in secondary phloem.Therefore,the cytokinin signaling,LBD1 s and APLs exhibited the highly overlapping patterns in the secondary phloem.2.Cytokinin signaling,LBD1 s and APLs affects secondary phloem development.In order to explore the role of cytokinin signaling,LBD1 s and APLs in regulating secondary phloem development,we established the transgenic lines of the RNAi-based knockdown of histidine kinase(HK)encoding cytokinin receptors or overexpressing a type-B RR transcription factor driven by phloem-specific promoter.The knockout lines of LBD1 s and APLs based on CRISPR/Cas9 system were also generated.The cross-sectioning of these transgenic plants combined with toluidine blue staining reveals that the cytokinin signaling,LBD1 s and APLs positively regulates the secondary phloem development.3.The regeneration rate of phloem in the LBD1 s or APLs knockout lines was slower than the wild type.In order to further explore the effect of cytokinin signaling,LBD1 s and APLs on secondary phloem development and their genetic relationship,the LBD1 s or APLs knockout lines were peeled(cambium and phloem removed)and regenerated under the conditions of exogenous cytokinin,as indicated in vitro secondary vascular tissue(SVT)regeneration experimental system established in poplar.We found that the phloem regeneration rate of LBD1 s and APLs knockout lines were slower than the wild type with or without exogenous cytokinin treatment,but their phloem regeneration rate with cytokinin treatment were faster than that without cytokinin treatment.These results suggested that LBD1 s and APLs may regulate the secondary phloem development downstream of cytokinin signaling.4.Cytokinin signaling depends on LBD1 s or APLs to regulate secondary phloem development.To further validate the genetic interactions among cytokinin signaling,LBD1 s and APLs,the vectors of LBD1 s or APLs genes driven by their native promoters were transferred into the transgenic lines that were specifically interfered with phloem cytokinin signaling.We found that the local overexpression of LBD1 s or APLs rescues the secondary phloem defects caused by the compromised cytokinin signaling.These results suggested that LBD1 s and APLs act downstream of cytokinin signaling to regulate secondary phloem development in poplar.5.Cytokinin signaling induces the expression of LBD1 s,but cannot activate the APLs’ transcription.To reveal whether there is a direct regulation between cytokinin signaling and downstream LBD1 s and APLs,we quantitatively detected the gene expression level of LBD1s/APLs in transgenic lines specifically altering cytokinin signaling in secondary phloem and under the cytokinin treatment.The results showed that the expression level of LBD1 s was significantly modulated by the cytokinin signaling,while that of APLs was not.In addition,using the effector-reporter system,we found that the cytokinin-responsive RR transcription factors could activate the expression of LBD1 s instead of that of APLs.6.Cytokinin signaling directly binds to the promoter of LBD1 genes and activates their expression.Chromatin immunoprecipitation(Ch IP-q PCR)assays revealed that the LBD1 s promoters could be bound by cytokinin-responsive B-type RR transcription factors,providing evidence for the direct regulation of cytokinin signaling on LBD1 s.Then the effector-reporter system(luciferase as the reporter)was conducted,and indicated that the B-type RR13 could not activate the expression of LBD1 s when the binding sites were mutated.Taken together,these results suggested that cytokinin signaling can directly bind to the LBD1 s promoters and promoted their transcription.7.The regulation of cytokinin signaling on secondary phloem development depends on the heterodimerization of APLs and LBD1 s.Since APLs act downstream of cytokinin signaling but their expression could not be induced by cytokinin,we hypothesized the possible protein interactions of LBD1 s and APLs.To test it,bimolecular fluorescence complementation(Bi FC)and co-immunoprecipitation(Co IP)were performed,and the results validated the physical interactions of these proteins.These results provided evidence for the dependence of the cytokinin-induced secondary phloem development on LBD1-APL heterodimers.In summary,cytokinin signaling,LBD1 s and APLs combinatorially regulate secondary phloem development.Cytokinin signaling directly activates the expression of LBD1 s while APLs regulate secondary phloem development by interacting with LBD1 s proteins.In conclusion,cytokinin signaling regulates the secondary phloem development through the module composed of LBD1 s and APLs in poplar. |
