Functional Characterization Of Aux/IAA9-ARF5.1 Module In Regulation Of Wood Formation In Populus

Wood,as the most important renewable resource in nature,is the main raw material for paper making,construction industry and bioenergy.To improve the growth rate of trees and the properties of wood has became an important goal of forest breeding.During wood formation,plant hormones,especially auxin,play an essential role in vascular cambium initiation and secondary xylem differentiation.It has been demonstrated that auxin concentration was highest in cambium and decreased along with the differentiation into secondary phloem and xylem.According to the current model of auxin signaling,Aux/IAA(auxin/indole-3-acetic acid)and ARF(auxin response factor)are the two important protein families for mediating auxin response.Previous studies showed that ARF and Aux/IAA genes played vital roles in cambium activity and vascular development.However,the specific regulatory network still needs to be further explored.Populus trichocarpa,(the most widely planted tree in the world has been sequenced in 2006),works as a model plant for studying plant secondary development.In this research,we cloned Aux/IAA and ARF in Populus tomentosa,revealed their significant influence on the secondary development of poplar,clarified the regulatory network of auxin on wood formation.The main results of this thesis are as below:1.Selection of Aux/IAA candidate genes for xylem formation in poplarThe poplar genome harbors 35 loci for Aux/IAA genes.By analyzing the data of microarray and RNA-seq,IAA9 was found to be highly expressed in wood-associated tissues,implying its possible involvement in xylem development regulation.2.IAA9 is highly expressed in vascular cambiumIAA9 is highly expressed in xylem,phloem,stem and root in wild type poplar.GUS staining showed that strong GUS activity driven by the promoter of IAA9 was detected in cambium.RNA in situ hybridization further confirmed the high expression of IAA9 in cambium.3.IAA9 is a canonical Aux/IAA proteinIn wild-type Populus tomentosa,the expression of IAA9 was significantly induced by exogenous auxin.In the transient infection experiment of tobacco,the GFP signal in the leaves disappeared after the addition of auxin(without mutated domain II).However,when expressed mutant IAA9,the GFP signal didn't disappear.These results proved that IAA9 was degraded by auxin at protein level and the degradation depends on domain II.Subcellular localization assay revealed that IAA9 was a nucleus-localized protein.Self-activation test in yeast indicated that it was a transcriptional suppressor.4.Overexpression of IAA9 inhibits xylem formation in poplarBy transforming 35 s overexpression vector into wild-type Populus tomentosa,it found that the plant height,internode number and stem diameter decreased in transgenic poplars.Toluidine blue staining showed that the xylem development was significantly inhibited in the IAA9-overexrepssing plants.Meanwhile the cell layers and relative area of secondary xylem decreased in the transgenic lines.The qPCR analyses showed that the expression levels of lignin biosynthetic genes and cambium-associated genes were down-regulated in the transgenic poplars.5.IAA9 interacts with ARF5Yeast two-hybrid experiments indicated that IAA9 and ARF5.1 /ARF5.2 could interact in vitro.Due to the high similarity in sequence and expression of both poplar ARF5 hoMogs,ARF5.1 was chosen for further analyses.BiFc assays validated the in vivo interaction between IAA9 and ARF5.1.6.ARF5.1 recovers the IAA9-resulting phenotypes of wood formationThe full-length and deletion of domain ?/? ARF5.1 overexpression vectors were transformed into the IAA9 transgenic plants.The full-length ARF5.1 could restore the height of IAA9 almost to the wild-type level.Also the truncated ARF5.1 restored the IAA9 phenotype completely.Yeast self-activation and Yeast two-hybrid experiments showed that truncated ARF5.1 still displayed activation activity whereas failed to interact with IAA9.7.The IAA9-ARF5 module regulates the expression of HB genesThe qPCR assay showed that the expression levels of HB genes were significantly down-regulated in the IAA9 overexpressing lines.It was found that the expression levels of some HB genes were induced by auxin in wild type plant.However,the induction of auxin obviously disappeared in the IAA9 overexpressing lines.The ChIP analysisrevealed that ARF5.1 was able to directly bind to the promoter regions of HB7 and HB8.Thus it was suggested that these HB genes were involved in auxin-mediated secondary regulatory network.Taken together,through the analysis of IAA9,we figured out the interaction of ARF and IAA proteins in vivo.They formed a complex to further regulate the expression of HB family genes and affect the wood formation in poplars.