Functional Studies On Populus Euphratica BRs Biosynthesis Enzyme Gene PeCPD

Brassinosteroids (BRs) are a new class of steroid phytohormones that are essential for many plant growth and development processes, such as cell division, differentiation and expantion, stem elongation, vascular tissue development, tolerance to stress, photomorphogenesis. CPD is a gene encoding key BR biosynthetic enzyme, which was first identified in Arabidopsis. Currently, the studies on genetic mechanism of BR biosynthesis has made significant progress, but most of them are focused mainly on the annual herbaceous plants, such as Arabidopsis thaliana, soybean, rice and so on. There is little in the perennial woody plants, especially tree. A full-length cDNA sequence homologous to Arabidopsis CPD (AtCPD) was cloned from Populus euphratica, named PeCPD, by our lab several years ago. In the present research, using this PeCPD, we constructed an expression vector driven by the promoter of AtCPD, named AP::PeCPD. Then this vector was inserted into the genome of cpd mutant of Arabidopsis, and two cpd mutant-PeCPD transgenic lines, named CAPC1and3, were obtained. The results of morphology, genetics, anatomy, cytology and molecular biology analysis of the CAPC lines are as follows:1. Due to heterologous expression of PePCD, the phenotype of the cpd mutant is basically restored to the level of wild type. The CAPC lines showed increased plant size and height, expanded rosette leaves, increased leaves area and fertil flowers.2. Although the CAPC lines showed restoration of different extent in phenotype relative to cpd mutant, but compared with Col, they still showed significant differences. For example, in leaf width and area, numbers of flower and siliques and inflorescence stem height, they showed obvious increase, and in the spaces between siliques, silique length and seeds per silique, they displayed obvious reduction.3. Scanning electron microscope and paraffin section observation showed that the shoot apical meristem(SAM) of the CAPC lines enlarged and projected with increased cell number.4. RT-PCR analysis of expression level of SAM and flower development-related genes in the transgenic lines revealed that the expression levels of WUS, CUC2, LFY and API increased and that of CLV3and STMdecreased compared with cpd mutant and Col plants.These results above suggest that the heterologous expression of PeCPD could repare the phenotype deffect of Arabidpopsis caused by the inactived AtCPD through reatoring BR biosynthesis in the cpd mutant. These results also imply that PeCPD and AtCPD share similar function in the regulation of both BR biosynthesis and plant growth and development. On the other hand, it also suggests that PeCPD may have other functions in the growth and development of plant besides participating in BR biosynthesis.