Molecular Mechanism Of PLETHORA5 Regulating The Secondary Vascular Development In Populus

The secondary development of trees is the biological basis of wood formation.It starts from the continuous cell division of the vascular cambium,which differentiates into phloem on the outside and xylem on the inside.The maturation of the xylem requires a series of cytological processes,including cell expansion,secondary cell wall deposition and programmed cell death,and eventually the formation of highly lignified mature xylem fibers and vessels,which compromise the wood.At present,the multi-level and feedback loop composed of NAC and MYB family transcription factors is the core of the secondary cell wall biosynthesis regulatory network.In this network,SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN(SND)/VASCULAR RELATED NAC DOMAIN(VND)transcription factors act as the master switches to regulate the expression of a series of downstream transcription factors and genes involved in secondary wall biosynthesis.However,the molecular mechanism underlying the upstream regulation of SND/VND is still unknown.Plant development widely depend on the hormonal regulation.Auxin is one of the most important plant hormones known to regulate wood development.The high-level auxin in the cambium of poplar plays an important role in maintaining the activity of cambial cells and regulating cell differentiation.When a certain concentration of auxin is appliedexogenoulsy,the secondary wall deposition of xylem cells would be significantly inhibited,but the underlying mechanism is still unknown.The AIL/PLT transcription factor family is involved in almost all stages of plant growth and development.In the study of root tip development,it has been indicated that the AIL/PLT transcription factor responds to the concentration gradient of auxin,and its regulatory effect is also concentration gradient-dependent.In secondary vascular tissues,ANT,an AIL/PLT family member,is involved in the regulation of cambial cell division and is considered to be a marker gene of cambium.However,the effects of other members of this family on the xylem development,especially the secondary wall deposition,and the regulatory mechanisms have not been reported yet.As a model plant,poplar is widely used in the study of secondary development of trees.In this study,using Populus tomentosa as the experimental material,through gene function analysis of AIL/PLT family genes,we revealed that PLT5 mediates the auxin signaling to regulate downstream SND/VND gene expression and affects secondary cell wall deposition in xylem,thereby improving the secondary growth.The main findings are as follows:(1)Accurate characterization of the auxin distribution pattern in secondary vascular tissuesThrough specific antibody hybridization,the content and distribution pattern of auxin in the secondary vascular tissues of poplar trees was finely detected.The results of immunofluorescence showed that auxin is highly accumulated in cambium,and the gradient decreases towards both sides.This is consistent with the previous report,suggesting that auxin plays an important regulatory role in the secondary developmental process.(2)Screening of the AIL/PLT family genes involved in wood developmentThrough phylogenetic analysis,the AIL/PLT family genes were identified in poplar.Tissue-specific expression analyses showed that most of the AIL/PLT family genes are expressed at a high level in roots,and only ANT and PLT5 are expressed at higher levels in stem.Using the Aspwood database to analyze the expression patterns of the AIL/PLT family genes in secondary vascular tissues of poplar,it was found that ANT and PLT5 exihibited the high expression levels in the cambial zone.Cryosectioning of secondary vascular tissues were further performed in the wild-type Populus tomentosa for detecting the expression levels of ANT and PLT5.It was found that the expression pattern of PLT5 was highly similar to the pattern of auxin.(3)PLT5 is highly expressed in the vascular cambiumA plant expression vector of the fluorescent reporter gene driven by the PLT5promoter(proPLT5-e YFP)was constructed,and the obtained transgenic poplar lines were used to characterize the expression of PLT5 in secondary vascular tissues.Using a confocal microscope,it was found that PLT5 was highly expressed in the vascular cambium region,which was consistent with the region with the high auxin accumulation.However,the PLT5 protein(proPLT5-PLT5-e YFP)was found to be localized in xylem cells.Therefore,there was a difference between the expressionof its transcript and protein,indicating that the PLT protein was mobile.(4)PLT5 is directly regulated by auxin signalingA plant expression vector of proPLT5-GUS was constructed for.transient expression in tobacco leaves in combination with exogenous auxin treatment.The assays of GUS enzyme activity showed that auxin induced the transcription of the PLT5 promoter.The wild-type Populus tomentosa was treated with exogenous application of auxin,and the expression level of PLT5 was found to be significantly up-regulated assayed by q PCR detection.The transgenic plants(ARF5.1-OE)with endogenously increased auxin response were tested by q PCR,and the expression of PLT5 was significantly up-regulated.Furthermore,the results of effector-reporter and CHIP experiments indicated that PLT5 could be directly regulated by the auxin response factor ARF5.1.(5)PLT5 is involved in mediating the regulation of auxin on the development of vascular tissueAuxin regulates many developmental processes of secondary vascular tissues.In order to explain the regulatory effects of PLT5 in response to auxin,the pro WOX4-PLT5 transgenic linse that specifically up-regulate the expression of PLT5 in cambium was established.Cross-sectionging,phloroglucinol staining and scanning electron microscopy results showed that the secondary wall deposition of the secondary xylem in the transgenic plant was significantly inhibited.The overexpression of PLT5 has a significant inhibitory effect on secondary wall synthesis,which was consistent with the regulatory effect of auxin on secondary wall synthesis.It was also found that the development of xylem fiber cells and vessel cells were also affected,indicating that the excessive PLT5 also had an effect on the development of the xylem cells.The above results indicate that PLT5 mediates the regulation of auxin on secondary wall synthesis and vascular tissue development.(6)PLT5 affects secondary wall synthesis by directly regulating the expression of SND1A1 and VND7-1The q PCR assays revealed that in transgenic plants(pro WOX4-PLT5),the expression of secondary wall synthesis-related enzyme genes and the primary regulatory switches SND/VND in the secondary wall regulatory network were significantly down-regulated,and the changes in SND1A1 and VND7-1 were particularly significant.Furthermore,the effector-reporter experiments showed that PLT5 might affect secondary wall synthesis by directly regulating the expression of SND1A1 and VND7-1.In summary,auxin presents a gradient distribution in the secondary vascular of poplar trees and participates in the development of secondary xylem.PLT5 has a gradient distribution similar to that of auxin,and is directly regulated by the auxin response factor ARF5,which in turn affects secondary wall synthesis by regulating downstream SND1A1 and VND7-1.These results revealed the molecular mechanism of PLT5-mediated auxin signaling in poplar to regulate the development of secondary vascular tissues,providing novel insights into the wood improvement.