Molecular Mechanism Of Gibberellin And Auxin Synergistic Regulating Vascular Cambial Activity In Populus Tomentosa

The secondary growth of trees determines the output of wood and has important economic value.The secondary growth of wood includes the establishment of vascular tissue,the production and deposition of secondary cell wall,programmed cell death and heart-wood formation.During the process of wood formation,plant hormones,especially gibberellin and auxin,are considered to play a key role in the formation of vascular tissue,especially in the development of vascular cambium.Previous studies have shown that exogenous gibberellin can promote the secondary development of vascular tissue and enhance the division activity of vascular cambium in the decapitated plants of woody plants.Auxin regulates the proliferation of vascular cambial stem cells through signal transduction mediated by ARF response elements.Although great progress has been made in recent years on the synergistic regulation of gibberellin and auxin on wood formation,the specific cellular and molecular basis of the regulation still needs to be further explored.As a model species of forestry research,poplar(Populus trichocarpa)has perfect secondary growth and development,which provides a basis for the molecular regulation of cambium,xylem and phloem.With the completion of poplar genome sequencing and the establishment of genetic transformation and regeneration system,it has greatly promoted the research on the regulation of secondary growth at the level of genetics and molecular biology.This paper focuses on the analysis of the effect of endogenous gibberellin on the activity of vascular cambium division by means of cytology and genetics.It also analyzes the relationship between this regulation and auxin,and preliminarily clarifies the sub mechanism of synergetic regulation of vascular cambium activity between them.The main results are as follows:(1)Endogenous upregulation of gibberellin promotes cell division of vascular cambiumEvolutionary analysis of GA3 ox,a key enzyme gene that regulates gibberellin biosynthesis in Arabidopsis thaliana and all GA3 ox genes in the poplar genome,was performed.Based on the analysis of expression pattern,PtrGA3ox1,which has relatively high expression in stem,was selected as the target gene,and a over-expression vector was constructed and transformed into Populus tomentosa.Compared with the wild type,the content of gibberellin in the stem of the over-expressed PtrGA3ox1 plant increased significantly.Morphological analysis showed that the increase of gibberellin content led to the increase of plant height and stem diameter.Further cytological observation showed that the area of vascular cambium was significantly widened,the number of cambium cells was significantly increased,the number of xylem cells was slightly increased,and the vessel size was significant increased.Real-time quantitative PCR showed that marker gene expression levels regulated by vascular stem cell division,such as WOX4,ANT,CYCD3:3 and other vascular stem cells was significantly up-regulated in the transgenic lines with increased gibberellin content,which further confirmed the results of cytological sections.In order to further analyze the effect of gibberellin mediated regulation of vascular secondary development on auxin,GC-MS was used to detect auxin level in transgenic plants.The results showed that compared with the wild type,the auxin level in the stem of transgenic plants increased significantly.(2)Gibberellin mediated division regulation of vascular cambium partially depends on auxin signalingIn order to find out whether the regulation of gibberellin on vascular cambium activity depends on auxin signal transduction,a point mutant Aux / IAA protein IAA9 m over-expression vector was constructed and transformed into gibberellin up-regulated transgenic material to obtain a successful restorer line.Morphological analysis showed that compared with gibberellin up-regulated lines,the growth height and stem diameter of the recovery lines decreased significantly,but still higher than that of the wild type.Cytological sections and toluidine blue staining showed that the regional range of cambium and the number of cell layers in the recovery lines decreased significantly,but still higher than that of the wild type,and the quantitative series of the number of layers of xylem and the size of vessels.The calculated results are consistent with the variation trend of cambium.The above results indicate that gibberellin positively regulates the division of vascular cambium,which is partially dependent on auxin signaling.(3)Gibberellin affects auxin polar transport-mediated interaction with auxinIn order to elucidate the regulatory mechanism of interaction between gibberellin and auxin,the expression of auxin homeostasis related genes,including synthesis,degradation and transport,was quantitatively detected.The results showed that auxin transporter PIN was up-regulated to a higher degree in transgenic lines compared with wild type.Previous studies have shown that there are 15 PIN proteins in poplar.We use RNA-Seq data to analyze the expression patterns of all PIN proteins in different parts of the stem,and screen out the PIN1 family proteins with high expression in the cambium,suggesting that they play a key role in auxin transport in the stem.The results of tobacco transient transformation showed that PIN1 protein was located on the plasma membrane.The results showed that compared with the control group,gibberellin extended the distribution of PIN1 protein in the cambium and enhanced the signal intensity of PIN1 protein.The above results indicate that gibberellin positively regulates the transcription level expression of auxin transporter,expands its distribution and affects auxin transport.(4)Regulation of auxin transporter PIN on cell division activity of vascular cambiumTo construct a over-expression and interference vector represented by PIN1 a,and to transform Populus tomentosa to obtain the transgenic line.Compared with the wild type,the over-expression line of PIN1 a showed a larger vascular cambium area and a significantly increased number of cell layers.In the interference line of PIN1,the range of vascular cambium was narrowed and the number of cell layers was significantly reduced.This result indicated that the change of auxin transporter affected the division activity of vascular cambium.In conclusion,the change of gibberellin content affects the division activity of vascular cambium.The truncated part of auxin signal restored the cell division activity of vascular cambium mediated by gibberellin.Gibberellin regulates auxin transport by regulating the expression and distribution of auxin transporter at the transcription level,and mediates the coordinated regulation of auxin and auxin on the division activity of vascular cambium.