The Function And Mechanism Of BZR1 In Secondary Development Of Stem Vascular Tissues In Populus

The secondary development of vascular tissues is important to the growth of woody plants.The cells and tissuses produced by this process compose the wood in trees,and take great economic value and ecological benefits.Therefore,exploring the genetic mechanism for secondary development of vascular tissues not only is important for scientific significance,but also has a wide industrial prospect.Previous studies have shown that the plant hormone,brassinosteroids(BR),is involved in the secondary development process of plants,and leads to thicker secondary xylem in stem.However,the detail function and mechanism underline that phenotypes are remain unclear.The BZR(BRASSINAZOLE-RESISTANT)proteins are key factors in the brassinosteroid signaling pathway,which are affected by the phosphorylation of upstream kinases and regulate numerous downstream genes.Studying the function of the BZR proteins may be helpful to clarify the function and mechanism of BR in regulating the secondary development of vascular tissues in stem.With the completion of poplar genome sequencing and the improvement of the genetic transformation system,poplar has become the model specie for the genetic reseaches in woody plants.We screened out a key factor in the poplar brassinosteroid signaling pathway through the technology of bioinformatics and molecular biology,and further explored effects of BZR1 on the secondary growth and related molecular mechanisms of it in regulating stem vascular tissues in poplar by genetic and biochemical experiments.The main research results are as follows:(1)BZR1 and BZR2 are capable to respond to brassinosteroids.Through the evolutionary analysis of BZR transcription factors in multiple species,it was found that BZR1 and BZR2 in Populus are homologous proteins of At BZR1 and At BZR2 in Arabidopsis.In Arabidopsis,At BZR1 and At BZR2 are key factors of brassinosteroid signaling pathway,which implies the importance of BZR1 and BZR2 in poplar.By fussion with a GFP inflorensence,we found that the subcellular localization of these two BZR proteins is regualted by the exogenous brassinosteroid and its inhibitor,thereby affecting their regulatory effects on downstream genes.It indicates that BZR1 and BZR2 respond to the change of brassinosteroids and may play key roles in the brassinosteroid signaling pathway,implying their important biological functions in poplar.(2)BZR1 and BZR2 preferentially express in the stem.In order to explore the function of these two BZR proteins,we analyzed the expression pattern of BZR1 and BZR2 in poplar,respectively.The results showed that both BZR1 and BZR2 expressed in all tested tissues and organs,and had almost the same expression pattern.They enriched highest in stem,followed by leaves and roots.However,the expression level of BZR1 was higher than that of BZR2,especially in the stem.It indicated that BZR1 may play a more predominat role in the vascular development in stem.Therefore,BZR1 was selected for further deep researches in this study.The BZR1pro: GUS reporter line was used to determine the expression pattern of BZR1 in poplar,which showed that BZR1 was especially enriched in the cambium and phloem in the stem of poplar.(3)BZR1 positively regulates the cell division in vascular cambium and enhances the cell differentiation in xylem development.In order to explore the biological functions of BZR1,the 35S:BZR1 vector was transferred into Populus tomentosa.Compared with the wild type,overexpression of BZR1 increased the height and the diameter of plants,suggesting a faster secondary development.The phenotypic results of stem showed that the overexpression of BZR1 led to an increase in the division activity of the vascular cambium and the number of cambium cell layers.At the same time,it enhanced the differentiation of the precurser cells into secondary xylem,resulting in increased number and width of secondary xylem cell layers.The expression levels of cyclin gene CYCD3;3,vascular cell division and differentiation related genes WOX4 a,WOX14,ANT,ARK2 and HB7 were significantly increased in BZR1-overexpression plants,which was consistent with its phenotype.These results indicate that BZR1 positively regulates the cell division and differentiation in secondary vascular development in poplar stem.(4)PSK-α,a small peptide phytohomone in plant,is a downstream factor of brassinosteroid signaling pathway in stem development.In order to screen the downstream target genes of BZR1,a small moleculor activator for BZR proteins,Bikinin,was used to treat poplar.The Bikinin-treated stems were subjected to transcriptome sequencing,together with untreated control,and thousands of differentially expressed genes(DEGs)were found.Through the GO analysis,DEGs that were clustered in the groups with functions that related to the observed phenotypes for BZR1,such as the cell division activity,were selected for further analysis.More than one of these genes were found to encoded precursors of Phytosulfokine-α(PSK-α),which indicated that they might respond to BR at the transcriptional level and be potential target genes of BZR1.Therefore,PSK2,which was most abundantly expressed in the stem in these PSK genes,was selected for further studies.(5)BZR1 positively regulates the expression of PSK2 by binding directly to its promoter.Through a quantitative detection,the expression of PSK2 was found to be induced by BR and reduced by BR inhibitor.In the BZR1-overexpression transgenic poplars,the expression level of PSK2 was significantly higher than that in the wild type,suggesting that PSK2 is a downstream gene responsive to BR and BZR1.The effector-reporter experiment in tobacco cells revealed that BZR1 can activate the transcriptional activity of the PSK2 promoter.It also showed that there are multiple BZR protein-binding elements located in the PSK2 promoter region,suggesting that BZR1 may regulate PSK2 expression by directly binding to its promoter.Ch IP-q PCR proved that BZR1 can directly bind to the PSK2 promoter.In summary,BZR1 can directly bind to the PSK2 promoter to promote the transcription of PSK2.(6)The PSK-α can positively regulates the division activity of the vascular cambium in stem.In order to explore the functions of PSK-α peptides in the development of vascular tissue in poplar stem,we treated poplars with PSK-α.It was found that PSK-αcould enhance the division activity of vascular cambium cells in the stem,leading to an increase in the number of cambium and xylem cell layers.This phenotype is similar to that resulted by BZR1-overexpression,which further implies that PSK-α is a key downstream factor of BZR1 and is responsible for the BZR1-directed regulation of secondary vascular tissues during stem development in poplar.