Cloning And Analysis Of Genes Associated With The Release Of Dormant Floral Buds In Tree Peony (Paeonia Suffruticosa Andr.)

Tree peony (Paeonia suffruticosa Andr.) is one of ten famous traditional landscape flowers in China. Like many perennial woody plants, the floral buds will enter the"dormant"state every winter and must go through a period of chilling treatment to break dormancy before flowering well and developing normal leaves next year. The dormancy of tree peony floral buds is endo-dormancy. Until now, the molecular mechanism of endo-dormancy is not clear. It is helpful to explain the mechanism of endo-dormancy and further control the flowering time by isolating different expression genes from tree peony floral buds and studying their molecular functions. These kinds of studies have important academic significances and practical values.In our study, tree peony (Paeonia suffruticosa)'Lu He Hong'was used as plant materials. By observing the floral buds anatomical changes under natural chilling treatment in winter to determine the progress of dormancy releasing, we regarded that about 30-35 d under the average temperature of about 0-10℃is required to break floral buds dormancy completely. A subtractive cDNA library was developed to study genes associated with the release of dormant floral buds in tree peony by using the RNA from burst floral buds as"tester"and that from dormant floral buds as"driver". By differential screening, 31 genes which were associated with bud dormancy release were obtained. According to their blast results, the molecular functions of them were presumed as following: (1) structural molecular activity group which included PsRPS7A, PsRPSL21, PsRPS, PsRPSL10, PsRPS7B, PsEDR, PsRPSL37, PsRPSL19, PsRPSL1, PsRPSL15, PsTUB. (2) PsSERK1 was in kinase activity group. (3) PsXRCC1 and PsPOB in protein binding group. (4) PsMPT in other binding group. (5) PsAEP in transporter activity group. (6) PsGA20 and PsSAMT in other enzyme activity group. (7) PsPII in other molecular functions group. (8) PsBDR1, PsBDR2, PsARP, PsFB2, PsCXE, PsDHN in unknown molecular functions group. (9) The other six unmatched sequences (PsBDR 3-8) were classified into'no matches'group.According to their putative function and literatures previously reporting an association with dormancy changes, eight cDNA clones, PsPII, PsDHN, PsGA20, PsARP, PsMPT, PsCXE, PsSERK1 and PsPOB were chosen from different molecular functional groups for further analysis by northern blot and semi-quantitative RT-PCR. The expression profiles of them during floral bud dormancy release indicated that bud breaking is a high energy and nutrition-consuming process. The transcriptional expressions of the isolated genes are related to growth regulation and stress response. Moreover, low-temperature-induced ribosomal proteins may be involved in floral bud breaking.In order to determine the molecular roles of auxin-repressed protein (PsARP) in bud dormancy release of tree peony, the full-length cDNA sequence was amplified by RACE-PCR and the deduced amino acid sequence were analyzed with the ExPASy Molecular Biology Sever (http://expasy.org/tools/). The results suggested that PsARP belongs to ARP/DRM gene family. And the putative protein contains two conserve domains without signal peptide and transmembrane domains. The subcellular localization of PsARP was further proved that putative PsARP is cytosolic protein. The expression pattern of PsARP under different chilling treatments showed up-regulated PsARP mRNA would accelerate bud dormancy release but not control bud development under normal conditions. So we presumed that the expression level of PsARP maybe relate to the interconversion between conjugated IAA and free IAA in floral buds. The free IAA probably affects floral bud dormancy process on post-transcriptal level by reacting with auxin-repressed protein (PsARP), implying indirectly that IAA also play a certain role in controlling floral bud dormancy release in tree peony. The characteristics of PsMPT-overexpression Arabidopsis plants by comparing to wild-type ones further indicated that PsARP maybe not control the normal growth.A putative mitochondrial phosphate transporter (PsMPT) gene is another clone from the subtractive cDNA library, which showed higher expression by chilling treatment during floral bud dormancy. By RACE-PCR, the full-length cDNA of PsMPT was obtained and the putative protein was also analyzed. The deduced amino acid sequence of PsMPT showed high identities with mitochondrial phosphate transporters in various organisms. The PsMPT protein also has the common six transmembrane domains while the conserved Cys residue exists in catalytic center. Northern bolt results and the characteristics of PsMPT-overexpression Arabidopsis plants indicated that the changes of the expression profile of PsMPT were correlated well with the process of floral bud dormancy release and the changes of the ATP content in floral buds under different chilling treatments, suggesting that PsMPT is a cold-responsive gene in tree peony and higher expressed PsMPT would promote the ATP synthesis and then directly increase the ATP/ADP ratio which regulates significant proteins synthesis to break floral bud dormancy release completely. To our knowledge, it is the first time to show the involvement of MPT in bud dormancy release and elucidate primarily the molecular mechanism of ATP content increasing in dormancy release.