Molecular Mechanism Of ARF7 Protein Integrates Auxin And Gibberellin Signaling To Regulate Cambium Activity In Poplar

Wood is an important renewable resource whose formation depends on the development of secondary xylem in trees.The secondary xylem is differentiated from the inward division of the vascular cambium,a type of lateral stem cells distributed in a ring shape in stem of the tree.Therefore,the continuous division and differentiation activity of the cambium is a key factor in determining wood development.The regulation mechanism of cambium stem cell activity maintenance is a very characteristic and urgent scientific question in forest biology research.Previous studies have shown that a variety of hormones,small peptide signals and a series of transcription factors are involved in the regulation of secondary plant growth.The small mobile peptide TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR(TDIF)encoded by the phloem-specific expression gene CLE41/44 is sensed by the receptor kinase TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR/PHLOEM INTERCALATED WITH XYLEM(TDR/PXY)receptors in the cambium region and promotes the expression of WOX4 transcription factor to maintain cambium stem cell activity.As two key hormones,auxin and gibberellin(GA)affect the entire secondary growth process from the maintenance of cambium activity to xylem differentiation.Auxin was mainly distributed in the vascular cambium region.In the absence of auxin or compromised auxin signaling,the cambium activity was significantly inhibited.GA are mainly distributed in the developing xylem region,altering the level or signaling of GA and affects the homeostasis of cambium stem cell activity.In the experiment of exogenous hormone treatment on poplar showed that,compared to the separate treatment of auxin or GA a wider cambium area and more layers of xylem cells was detected by the treatment of both hormones.These results implied there was the crosstalk regulation of auxin and GA,however the molecular mechanism of their synergistic regulation is unknown.The signal transduction pathways of gibberellin and auxin have been clearly reported.GA induces a conformational change of the receptor GID1 results in its interact with DELLA protein,leading to DELLA protein ubiquitination and degradation by 26 S proteasome.DELLA proteins interact with other transcription factors to achieve transcriptional regulation of GA-responsive genes.The auxin response factor(ARF)acts as a core directly linking auxin signaling and transcriptional regulation of downstream genes.Whether DELLA proteins and ARF transcription factors are directly involved in the regulation of cambium activity remains unclear.In response to the above problems,this study took Populus tomentosa as material,analysis of poplar vascular cambial activity through sectioning,protein interaction screening and verification,transcriptome analysis and downstream target gene verification,in-depth analysis the molecular mechanism of the synergistic regulation in cambium activity by GA and auxin.The main findings are as follows:1)GA regulates poplar cambium activity and wood formationTo accurately characterize the role of GA in the secondary growth of plants,the active GA synthase GA3 ox or the inactivating enzyme GA2 ox were specifically expressed in the developing xylem tissue to up-regulate or down-regulate the content of endogenous active GA.Through slice analysis,the number and proportion of xylem layers were counted,and it was found that GA promoted the development of secondary xylem.Statistical analysis of the number of cambium layers and the frequency of periapical division shows that GA promotes and stimulates cambium cell division,thereby maintaining cambium stem cell activity.2)GA promotes cambium activity through its local signalingIn order to further study the molecular mechanism of GA regulating cambium activity,the gene expression of GA receptor GID1 and key repressor DELLA in the cambium region was analyzed,indicating that there is a complete GA signal transduction pathway in the vascular cambium region,and It is speculated that the DELLA protein RGL1 may be involved in the regulation of cambium activity.Protein interaction analysis showed that GID1 and DELLA could interact physically.The DELLA tobacco transiently transformed lines fused with fluorescent tags were treated with exogenous GA,and the fluorescence quantity and fluorescence intensity were significantly reduced/attenuated,indicating that the stability of poplar DELLA protein was negatively regulated by GA,which was similar to that in the model plant Arabidopsis thaliana.The reports were consistent.Mutating the DELLA domain of RGL1 was found to inhibit the degradation of RGL1 by GA,and based on this,a transgenic line with cambium-specific blocking of GA signaling was constructed.It was found that its cambium activity was significantly inhibited.The results suggest that GA promotes cambium activity through local signaling in the cambium region.3)GA and auxin synergistically regulate cambium activity through the interaction of their signaling pathwaysTo explore how GA and auxin synergistically regulate cambium activity,exogenous auxin treatment of GA-downregulated transgenic lines restored cambium activity;cambium-specifically upregulated GA levels blocked auxin signaling in transgenic lines,cambium stem cell activity was inhibited,suggesting that the regulation of cambium activity by GA is dependent on auxin signaling.The transgenic lines that specifically blocked GA signaling in the cambium region were treated with GA and auxinrespectively,and found that their response to GA disappeared and the response to auxin was significantly weakened,indicating that the regulation of auxin on the cambium is also partially dependent on GA signaling.These results suggest that the regulation of cambium activity by GA and auxin is dependent on each other’s signaling.4)ARF7 interacts with DELLA and Aux/IAA proteins to form a ternary complexProtein interaction screening found that DELLA can interact with the key components of auxin signaling,ARF6,ARF7 and ARF8 members.Analysis of the expression levels of ARF genes showed that all members of ARF6 and ARF8,ARF7.1,and ARF7.4 decreased in expression with the transition from primary growth to secondary growth,while ARF7.2 and ARF7.3 may be involved in the regulation of poplar and the expression of secondary growth showed an upward trend.The interaction between ARF7.2 and DELLA protein was further verified using bimolecular fluorescence complementation and co-immunoprecipitation.The domain of ARF7.2was truncated and replaced with the ARF5 domain that does not interact with DELLA protein.Yeast two-hybrid showed that the MR domain of ARF7 mediates the interaction between DELLA protein and ARF7,which is different from the PB1 domain that mediates the interaction between Aux/IAA and ARF.It is speculated that the three may form a complex.Yeast three-hybrid,luciferase bimolecular complementation and coimmunoprecipitation indicated that ARF7 acts as a molecular bridge to interact with DELLA and Aux/IAA proteins to form a ternary complex that integrates GA and auxin signaling.5)ARF7 mediates GA and auxin signaling to regulate cambium activityTransgenic overexpression lines of ARF7.2 and co-knockout lines of ARF7.2 and ARF7.3 were constructed,and the cambium phenotype was analyzed by section,confirming that ARF7 is a positive regulator of cambium activity.The arf7.2 arf7.3 coknockout line was treated with GA and auxin and found that its cambium response to GA and auxin disappeared.In the context of overexpressing RGL1,overexpressing a variant of ARF7.2 that does not interact with the DELLA protein,the inhibitory effect of cambium activity was restored.These results suggest that ARF7 is involved in mediating GA and auxin signaling to regulate cambium activity.6)ARF7 directly activate WOX4 expression to promote cambium activityThe downstream genes induced by GA and auxin were identified by RNA-seq data analysis.And the Aspwood database were used to screen genes expressed in cambium and WOX4,the key factor regulating cambium activity,responds to GA and auxin induction was found.q RT-PCR analysis confirmed that WOX4 is downstream of GA and auxin signaling and regulated by DELLA proteins RGL1 and ARF7.The analysis of promoter binding sites showed that there are multiple ARF binding sites in the promoter of WOX4.Ch-IP indicated that ARF7 was able to directly bind the WOX4 promoter.Yeast one-hybrid and EMSA confirmed this result.The dual luciferase reporter system and exogenous hormone treatment showed that ARF7 can directly activate the expression of WOX4.The addition of DELLA protein RGL1 and Aux/IAA protein IAA9,respectively,can significantly inhibit the activation of ARF7 on WOX4.Adding RGL1 and Aux/IAA9 at the same time enhanced the inhibitory effect.Treatment with GA and NAA degraded DELLA protein and Aux/IAA9,released the activation activity of ARF7,and promoted the expression of WOX4.It indicated that ARF7 interacted with DELLA protein and Aux/IAA9 to integrate GA and auxin signals to activate WOX4 expression.7)ARF7 mediates GA signaling to directly regulate PIN1 expression and promote auxin accumulation in the cambiumPrevious studies have shown that auxin has a positive self-feedback,and the auxin efflux transporter PIN1 is also positively regulated by GA.To investigate whether GA affects auxin polar transport in the regulation of cambium activity,the expression of PIN1 in transgenic lines with altered GA levels and in lines treated with exogenous GA was analyzed,indicating that GA can promote PIN1 expression.The fluorescently labeled lines of PIN1 protein were further treated with exogenous GA,and it was found that GA promoted the accumulation of PIN1 protein.The content of auxin IAA in the stems of transgenic lines with altered GA levels was detected by immunofluorescence and LC-MS/MS,indicating that GA promotes the accumulation of auxin in the cambium.Analysis of the expression levels of transgenic lines that specifically blocked GA signaling in the cambium,ARF7-overexpressing lines and knockout lines showed that PIN1-mediated auxin accumulation was regulated by both GA signaling and ARF7.The PIN1 promoter contains multiple ARF7-binding elements,and Ch-IP,yeast onehybrid and EMSA showed that ARF7 could directly bind to the PIN1 promoter.The dual-luciferase reporter system was used to verify that the regulation of PIN1 by ARF7 depends on the binding site of ARF on the promoter.The above results suggest that ARF7 mediates GA signaling to directly regulate PIN1 expression and promote the accumulation of auxin in the cambium.In conclusion,ARF7 regulates vascular cambium activity in poplar by integrating GA signaling and auxin signaling.ARF7 forms a ternary complex with DELLA protein and Aux/IAA in response to GA and auxin signaling,and by directly binding to the promoters of WOX4 and PIN1,activates the expression of these genes,and regulates cambium activity and auxin accumulation.