Cloning And Function Analysis Of Populus Euphratica Brassinosteroids Biosynthase Gene CPD(PeCPD)

Brassinosteroids (BRs) are new type of plant growth regulators, and are considered as the sixth class of phytohormone, which play critical roles in cell division and elongation, vascular differentiation, leaf and root development, photomorphogenesis, male fertility, senescence, and stress responsion. The mechanism underlying BR biosynthesis and its regulation are the "hot spot" among the current plant biological researches and many important progresses have been made. However, these studies mainly focused on the annual herbaceous plants, such as Arabidopsis, rice, tomato, pea, and so on, few is known in the perennial woody plants, such as trees. CPD (constitutive photomorphogenesis and dwarf) was initially isolated from an Arabidopsis cpd mutant. In BR biosynthesis pathway, CPD encodes a cytochrome P450enzyme functioning in the conversion of22-OH-CR to22OH-4-en-3-one. In present study, the homologous CPD genes were cloned from Populus euphratica Oliv with extremely high tolerance to drought, saline and alkaline and P. nigra var. thevesitina with relatively poor tolerance to stress. Then the corresponding expression vectors were constructed, with which the cpd mutant and wild type of Arabidopsis thaliana were transformed. Finally, the function of the CPD gene from P. euphratica was explored through morphology, genetics, physiology, anatomy and molecular biological analysis of the obtained transgenic lines. The main results are the follows:1. The full-length cDNA homologous with Arabidopsis CPD, were isolated from P. nigra var. thevesitina and P. euphratica oliv, named PeCPD and PnCPD respectively, by homologous cloning combined with in silico cloning. Bioinformatics regarding PeCPD and PnCPD were analyzed. The results were the follows:1) PeCPD and PnCPD contained equal length of coding regions, the proteins encoded by which shared97%amino acid (aa) homology.2) The proteins deduced from the PeCPD and PnCPD shared67～92%aa identity to CYP90A family, were named PnCYP90A and PnCYP90A, respectively.3) The phylogenctic relationship of PeCYP90A to PnCYP90A was closer than that of PeCYP90A/PnCYP90A to CYP90A6V1of P. trichocarpa.4) The phylogenetic relationship of PeCYP90A/PnCYP90A to the CYP90As of Citrus sinensis, Carica papaya and Camellia japonica, were less close than that of PeCYP90A/PnCYP90A to the CYP90As of Vigna radiata and Pisum sativum.5) The subcellular location predicted according to signal peptide sequences was also distinct from each other among the CYP90As from different plant species.2. The relative expression level of PeCPD in different poplar sections (species) and tissue/organ of P. euphratica were analyzed using Real-Time qRT-PCR.1) The expression levels of PeCPD gene in P. cathayana (Tacamahaca section), P. euphratice(Turanga section), P. nigra var. thevesitina (Aigeiros section), P. tomentosa (Leucoides section) and P. bolleana (Leuce section) were1.0,1.4,1.1,0.8and1.1, respectively. The transcription level was the highest in P. euphratica and the lowest in P. tomentosa among them.2) In P. euphratica, the transcription level of PeCPD in leaves was significantly higher than that in roots and stem cambium, and that in mature leaves was significantly higher than that in juvenile and intermediate leaves. In roots, PeCPD did not express.3. Using PeCPD cDNA and plasmid pMDC43, three different expression vectors35S::PeCPDGFP,35S::PeCPD and AP::PeCPD were constructed,35S and AP represent the promoter of CaM35S and Arabidopsis CPD gene, respectively.4. Using35S::PeCPDGFP,35S::PeCPD and AP::PeCPD to transform Arabidopsis cpd mutant (Atcpd), the following three group of Atcpd-PeCPD transgenic lines, named C35CG (3lines), C35C (2) and CAPC (3), were obtained. The morphology, genetics, physiology and anatomy analysis of the Atcpd-PeCPD lines showed:1) Compared with Atcpd, the T1Atcpd-PeCPD lines showed expanded rosette leaves, increased plant size, ahead flowering as well as increased height, numbers and branches of stem. Compared with Arabidopsis wild type (Atwt), the ratio of leaf length to width, petiole length and plant height are significantly reduced in all of C35CG and C35C lines, but their flowering phase were delayed. In plant size and height as well as flowering phase, CAPC lines were similar with corresponding Atwt, but leave's width and area as well as stem's diameter significantly increased. C35CG1, C35CG2, C35CG3and C35C2lines were male sterile even in the case of illegitimate pollination. The early stage flowers of C35C1, CAPC1and CAPC2lines did not set seeds unless illegitimate pollination, but the late stage flowers normally set seeds. The CAPC3line was completely fertile. CAPC3, CAPC2, CAPC1and C35C1line of T3generation were basically similar with T1lines in phenotype. The degree of phenotypic restoration were Atcpd<C35C1<CAPC2/CAPC1<CAPC3/Atwt in order.2) The sterility of the early stage flowers of C35C1and CAPC1lines were related to the inhibited filament cell elongation, which made their anthers not touch with stigma.3) In C35C1and CAPC lines, the size of epidermic cells as well as the length and number of lobes were significantly increased, whereas stomatal density relatively decreased, compared with Atcpd. In comparison with Atwt, the size of epidermic cells as well as the length and number of lobes of C35C1line were decreased, but stomatal density were increased. CAPC lines did not show visible differences from Atwt.4) For the CAPC lines, the number of sponge cells was decreased, while air spaces increased. The number of xylem cell in leaf vascular bundle (VB) was greater than Atcpd and Atwt lines. And the numbers of stem VB, xylem and procambia cell in VB also increased relative to both Atcpd and Atwt.5) The rate of hypocotyls elongation of C35C1, CAPC1and CAPC3lines was significantly increased in the light and dark compared with Atcpd. Phenotype of their cotyledons was similar to that of Atwt in the dark.5. Using Vectors35S::PeCPDGFP,35S::PeCPD and AP::PeCPD to transform Atwt, the24Atwt-PeCPD transgenic lines, named O35CG (10),O35C (7) and OAPC (7), were obtained. From each group, one most promising line O35CG24, O35C7and OAPC4, was selected for further investigation. The results were as follows:1) Compared to Atwt, the rate of hypocotyls and root elongation of O35CG24, O35C7and OAPC4all increased. However, for the O35CG24line, their leaf area and leaf epidermal cells increased, and stoma density decreased during vegetative growth phase. In mature phase, their plant height, silique mummer and stem diameter were also significantly increased. O35C7and OAPC4lines showed no significant change compared to Atwt. 2) For measurement of auxin activity, we crossed the Atwt-PeCPD lines with the transgenic plants expressing the DR5-GUS reporter. The result showed that highest GUS activity was detected in the O35CG24line with the longest root. The GUS activity was positively correlated to the root length.3) Histology research showed that the leaf thickens, mesophyll cell size and spongy air spaces of O35CG24line were increased significantly, whereas, the number of sponge cells decreased. The numbers of VB, xylem and procambia cell in stem were significantly greater than Atwt lines. The number of xylem cell and procambia cell along the base of VB ring were positively correlated to the diameter of stem.6. Real-Time qRT-PCR analysis of expression level of PeCPD and other BR-related genes in the transgenic lines revealed:1) The degree of phenotypic rescue of C35C1, CAPC1and CAPC3lines relative to Atcpd was positively correlated to the expression level of PeCPD and BAS1genes, and negatively to that of DWF4and BR6OX2gene. The expression level of PeCPD was positively correlated to that of BAS1and negatively to that of DWF4and BR6OX2. The expression level of TCH4and SAUR-AC1were not significantly associated with the degrees of their phenotypic rescue.2) Phenotypic change degree of O35CG24/O35C7/OAPC4lines relative to Atwt was positively correlated to the expression level of PeCPD and BAS1. The expression level of PeCPD was positively correlated to that of BAS1. The expression levels of DWF4in O35CG24/O35C7/OAPC4lines were higher than that in Atwt. The expression level of TCH4and SAUR-AC1were not related to with the phenotypic changes of O35CG24/O35C7/OAPC4lines.7. A analysis of total endogenous BR levels revealed that the endogenous BR level in the all transgenic lines, Atcpd and Atwt from lower to higher was O35CG24<CAPC2<O35C7<Atwt<C35C1<CAPC3<CAPC1<Atcpd<OAPC4in order. It seemed that total endogenous BR level was negatively correlated to the expression level of PeCPD.8. Two-dimensional electrophoresis was used to analyze the protein difference between Atcpd and CAPC3lines. The result showed that there were568protein spots in CAPC3and411protein spots in Atcpd. In the CAPC3, there were eight significantly up-regulated proteins, three down-regulated proteins and nineteen new proteins compared to Atcpd. MALDI TOF/TOF-MS/MS identification of seven of the19new proteins found that five were involved in photosynthesis, one in RNA processing, and one in cell defense. It is reported that the7proteins all were positively regulated by BR.9. The PeCPD-GFP fused protein were transient expression by agroinfiltration in Nicotiana benthamiana leaves. The result showed that the PeCPD-GFP protein mainly distributed on cytomembrane and cytoplasm with typical reticulate structure, which is similar to that of the marker protein for endoplasmic reticulum (ER), suggesting that PeCPD protein is possibly located in ER.Based on the above results, the following conclusions are drawn:1) P. euphratica CPD (PeCPD) and Arabidopsis CPD (AtCPD) possibly share similar function in BR biosynthesis, because the heterogeneous expression of PeCPD could apparently made the phenotype of Atcpd mutants to be recovered, and alter the level of BR-related genes expression and endogenous BR.2) PeCPD may have other functions in the growth and development of plant, since the phenotype of Atcpd could not be completely restored by heterogeneous expression of PeCPD.3) PeCPD may regulate BR-independent the leaf and stem development, because the CAPC1lines that has relative low BL level to Atwt showed broad leaves, much branched stem, increased stem diameter.4) PeCPD possibly regulates photosynthesis because the transcripts of PeCPD in mature leaves of P. euphratica are significantly higher than that in juvenile and intermediate leaves. Moreover, proteomics analysis showed that five proteins were involved in photosynthesis in seven proteins identified by MALDI TOF/TOF-MS/MS.5) The level of PeCPD expression was distinctively different in different poplar sections and tissues/organs of P. euphratice.6) In the Atcpd-PeCPD transgenic lines, the efficiency of CaM35S promoter was significantly less than that of the promoter of AtCPD gene, while in the Atwt-PeCPD line, the efficiency of CaM35S promoter was significantly higher than that of the promoter of AtCPD gene, suggesting that the function of CaM35S promoter might be partly suppressed in Atcpd.7) The phenomenon that rate of root elongation is enhanced in the Atwt-PeCPD transgenic lines indicated that PeCPD regulates the root growth possibly though improving the biosynthesis or distribution of auxin. 8) PeCPD is a promising gene that can be used to transform other crop or tree, since plant height, seed yield, stem diameter and resistance to salt are significantly increased in O35CG24lines due to heterogeneous expression of PeCPD.