Functional Analysis Of PPT1Gene In Regulating Plant Development And Isolation Of A Vein Specific Expression Enhancer

Carbon metabolism in plant not only plays a key role in growth and development, but also provides energy and precursors for synthesis of other important compound. Therefore, metabolism and flux of carbon in plants have been a hot issue for plant and life sciences. The transporters located in plastid play a pivotal role in carbon flow in plants because they control the rates of both primary and secondary metabolisms. PEP (phosphoenolpyruvate) is an important intermediate in plant carbon metabolism. Deficiency or limitation of PEP supply to plastids would be fatal to plants. AtPPT1gene in Arabidopsis was involved in PEP-mediated regulation of several developmental processes. However, there is little understanding of interactions and interdependencies of individual metabolic or signal pathways. Rice is not only an important crop, but also a model plant in studies on cereal plants. In this thesis, the main objectives are to elucidate the mechanism of AtPPT1in Arabidopsis and to investigate functions of OsPPT1h in rice. Firstly, functions and mechanism of AtPPT1gene in the regulation of root development was investigated using Arabidopsis. Then, the interrelationship between phenotype of leaf color and short root mutant caused by AtPPT1gene was investigated. A metabonomics method was adopted to analyze metabolites in roots and leaves of the mutants. Secondly, a homologous gene in rice named as OsPPT1h was obtained and its biological function was analyzed. Thirdly, a VVE enhancer with vascular vein specific expression was isolated from the promoter of an amidase gene to determine gene expression in leaf vein. The main results were summarized as follows:1. AtPPT1regulates the development of primary roots in ArabidopsisIn attempt to investigate the role of AtPPT1in root development, a cue1mutant was obtained from the European Arabidopsis Stock Centre (NASC) with the stock number N3156. cue1seedlings presented obviously short primary roots in comparison with that of wild type (Col-0plants). The phenotype of short primary roots could be completely rescued by over-expression of AtPPT1gene under CaMV35S promoter in the cuel background. On the other hand, several transgenic plants harboring a RNAi structure for AtPPT1gene driven by strong constitutive CaMV35S promoter in wild type showed that primary root lengths of the transgenic plants were highly correlated to transcripts of AtPPT1gene. The results suggested that apart from the roles of regulating aboveground development, AtPPTl gene is also involved in root development and this process is regulated at the transcriptional level. In addition, it was found that zone of cell elongation in the primary roots of cuel mutant was negatively affected and the effects of AtPPT1gene on the activities of root meristem is usually taken as a marker to visualize cell division at G2-M phase of the cell cycle. The region of GUS staining was apparently reduced in the primary roots of cuel mutants when compared with the wild type. The DR5-GUS containing a fusion of a synthetic auxin responsive promoter (DR5) and GUS reporter gene has been used as a marker gene to monitor auxin response at the cellular level. In cuel seedlings, DR5-GUS expression was observed in root apexes of both the primary roots and the lateral roots, but with a discriminable reduction. The results indicated that AtPPT1gene is required for root growth possible through regulating the meristic ability in the root meristem and auxin content in the root apex.2. AtPPT1gene differently regulates root and leaf development.As the AtPPT1gene mutant cuel could alter root length and leaf architecture, it could be speculated that AtPPT1gene in Arabidopsis might function dependency during these two important developmental processes. To test whether these two processes were dependently regulated by AtPPT1gene, specific modulations of AtPPT1gene driven by promoter of AtSuc2gene (AtSuc2P) of suppression through RNAi method and over-expression were adopted. The AtSuc2P promoter drives expression with a pattern of phloem specific expression and is taken for an ideal marker gene for the sink-source transition in leaves. The results showed that both specific suppression in wild type Col-0and over-expression in cuel background of AtPPT1gene only cause changes of the leaf phenotypes, but not of the short root phenotypes. In contrast, specific modulations of suppression or overexpression of AtPPTl gene driven by the promoter of ARSK1gene (ARSK.1P) only caused changes of the root phenotypes but not of the leaf phenotypes. ARSK1P could drive expression of the GUS reporter gene in roots exclusively. The results suggested that the short roots and reticulate leaves were regulated independently by AtPPT1gene.In order to analyze the metabolite changes in the shoots and roots of cue1mutants, metabonomics method using gas charomatography-mass spectrometry (GC-MS) was adopted. It was found that the features of the changed metabolites between cue1mutants and wild type seedlings were different in the roots and aboveground organs. In the mutant leaf tissues, the levels of most metabolites decreased. The results demonstrated that metabolic changes in roots and leaves caused by loss of AtPPTl gene are not the same and that may be regulated by different pathways. The results also indicated that AtPPTl gene independently regulates the developmental processes of roots and leaves in Arabidopsis.3. Plastid targeting signal (PTS) is essential for AtPPTl to fulfill its function.The amino acid sequences of two functional transport proteins for PEP named as AtPPTl and AtPPT2in Arabidopsis thaliana were used to predict the subcellular localization using the online TargetP. The results showed that two proteins could be located in chloroplasts, but the predicting reliability of AtPPT2in chloroplast is relatively low. Transient expression in tobacco leaf cells further showed that AtPPT2could not effectively target toward chloroplast membranes, while AtPPT1could be located in chloroplasts. A sequence encoding the100amino acids at the N-terminal of AtPPTl protein for plastid target signal (PTS) was cloned. The PTS was further fused to eGFP and AtPPT2-eGFP, respectively and allowed to express transiently. The results showed that both eGFP and AtPPT2-eGFP fused with PTS could effectively located in chloroplasts. Moreover, transgenic plants with the chimeric PTS and AtPPT2-eGFV fusion protein in cue1background showed a similar leaf phenotype as that of wild type plants. Moreover, neither the PTS-eGFP nor AtPPT2-eGFP fusion protein could completely rescue the cue1leaf phenotype. Thus, it may be assumed that PTS is an indispensable factor for AtPPT1to fulfill its function. The isolated OsPPT1h gene in rice showed the very similar actions as that of AtPPT2gene, but the sequences encoding target signal peptide are "missing" in the OsPPT1h gene.4. Differences and similarities of PPT1sequences in mono-and di-cotyledons. A rice sequence (Os09g0297400) with72%similarity to AtPPT1protein at the amino acid level was found by BlastP search against the Genbank rice protein database. Based on the nucleotide sequences of Os09g0297400and AtPPT1, degenerating primers were designed to amplify the Os09g0297400cDNA sequence using rice total RNA by RT-PCR. A cDNA sequence with255bp shorter than Os09g0297400was cloned and designated as OsPPT1h for AtPPT1homology in rice. Using this strategy, a cDNA sequence for AtPPT1homology in barley named HvPPT1h was obtained. The barley HvPPT1h sequence was also195bp shorter at the5’end compared with the sequence in database. However, the cloned OsPPT1h cDNA contained a complete protein coding frame. The deduced protein (OsPPT1h) showed a high similarity of98.8%to Os09g0297400at amino acid level, only with a lack of85aa at the N terminal. The similarity between OsPPT1h and AtPPT1protein sequences was74.7%, higher than that between OsPPT1h and AtPPT2(59.9%), and between AtPPT1and Os09g0297400(62.5%). Furthermore, the cloned HvPPT1h cDNA sequence also harbored a complete protein coding frame with a deduced protein (HvPPT1h) lack of the N-terminal65aa compared with the sequence in database. Therefore, either OsPPT1h in rice or HvPPT1h in barley showed a deletion at the N terminal, which corresponded exactly to the PTS region of AtPPT1.5. Conserved and changed functions of OsPPT1h gene in riceMore than20independent transgenic rice lines in Nipponbare background for both over-expression and RNAi constructs of OsPPT1h gene were obtained respectively through agrobacterium-mediated method. In the field, all TO transgenic plants of over-expression for OsPPT1h (named OX) presented similar phenotypes to that of non transgenic control (wt) plants. Similarly, there were4RNAi transgenic lines (designated as DX) showing weak tillerings than wt plants. For T1plants in the field, phenotypes of all OX plants were similar to wt plants. While for DX transgenic lines, except for4lines with tillering phenotype at TO generation, the plants of all other lines also displayed visible phenotypes, including reticulate leaves with pale-green mesophyll cells, dark-green bundle-sheath cells aligning the veins, dwarf shoots, and fewer tillerings. RT-PCR analysis indicated that the phenotypes of the RNAi transgenic plants were attributed to a diminished internal OsPPTlh expression. These results suggested that OsPPT1h gene in rice may play an important role in tiller development, in addition to its functions showed by AtPPTl gene, such as regulating leaf and other tissue development.6. VVE is an enhancer characterized by a leaf vein specific expression patternA regulatory sequence of1500bp, at upstream in translation initiation site of amidase (amidase, At4g34880)(named AmidP) was cloned. Histochemical GUS staining in transgenic Arabidopsis plants indicated that AmidP is a promoter with a vein specific expression pattern. Interestingly, the activity of AmidP resembled the pattern of sink-to-source transition of leaves, reflected by GUS staining being restricted to the vascular veins of cotyledons (source tissues) and expanded source leaves. VVE motif could be applied as a vascular vein specific expression enhancer. Further, fine deletions of VVE motif combined with the-65minimal35S promoter to drive the GUS reporter gene were transformed into Arabidopsis plants respectively. A conservative DOF2-domain was a key element responsible for the vein specific expression. Other elements among the motif also contributed to expression specificity and intensity of VVE. In addition, expression of AtPPTl gene in the cuel mutant could partly recover the leaf phenotype, indicating that WE can act as an enhancer to increase expression of a target gene in leaf veins. Therefore, VVE may be used in regulation of gene expression in the vein system.