Studies On Arabidopsis Protein-Protein Interactions And The Function Of Homologous Gene In Mulberry

Plant research is moving into the post-genomic era. Plant genome sequencing projects have identified a large number of genes for which a function has yet to be assigned. One of the most important technique which has been developed to study the mapping of protein-protein interactions is yeast two-hybrid system. A two-hybrid screen needs a series of yeast and E·Coli transformations, followed by selection of positives in nutrient-deficient media, and became one of the most important method in the studying of protein-protein interactions after it was established in 1989.The Arabidopsis protein, AtCHIP, is structurally, functionally similar to animal such as human and mouse CHIP protein that are E3 ubiquitin ligse involving in ubiqutin-proteasome degradation system. AtCHIP plays an important role in plant response to abiotic stress conditions such as temperature or salt. To understand the molecular mechanism of AtCHIP's involvement in stress response in plants , we identified proteins that physically interact with AtCHIP by using the yeast two-hybrid system technique from the Arabidopsis cDNA prey library. In addition, we also studied the functions of AtCHIP's interacting proteins , ClpP4 and FtSH1. The results are summarized as follows:1. 1036 candidate clones of AtCHIP's interacting proteins were identified in which 111 plasmid DNA of the positive clones were sequenced, and provided to the Arabidopsis gene bank. It showed that 29 different types genes of the 111 cDNA were homologous to the Arabidopsis genes (identities>98%); meanwhile, 12 cDNA were not found in the gene bank, may be the unknown gene in plants. The interacting proteins mainly include in UBC family enzymes, H+transporting ATP synthesizing chain 9, and chloroplast proteases such as ClpP4, FtSH1.2. The evidences have been obtained that AtCHIP interacts with three of the E2 enzymes and two of the ubiquitin molecules specifically.There are about 40 E2 genes in the Arabidopsis genome, yet only three encode proteins, UBC8, UBC9, and UBC10 that were found to interact with AtCHIP in the yeast two-hybride screening, indicating that the interaction between AtCHIP and E2s (i.e.UBC8/UBC9/ UBC10) is very specific; AtCHIP also interacts with two ubiquitin proteins, UBQ1 and UBQ5, there are 15 ubiquitin and ubiquitin-likes in Arabidopsis genome, yet only two were found to encode proteins that interact specifically with AtCHIP.3. Chloroplast proteases, ClpP4 and FtSh are likely the substrate proteins of AtCHIP by the in vitro ubiquitylation experiment of AtCHIP.Firstly, Three proteins of AtCHIP and two of its interacting proteins, UBC8 and ClpP4, were induced, extracted and purified by using the PET system. To test if ClpP4 is AtCHIP's substrate protein, we conducted an in vitro ubiquitylation assey using Arabidopsis AtCHIP as E3, Arabidopsis UBC8 as E2, and we were able to demonstrate that ClpP4 could be ubiquitylatied by AtCHIP in vitro. It appeared that three ubiquitin molecules were added to ClpP4.4. The ClpP4 and FtSh are likely the substrate proteins of AtCHIP using western blot test {in vivo).Because AtCHIP can add three ubiquitin molecules to ClpP4, we thought that the outcome of ClpP4 ubiquitylation might be ClpP4 degradation. Therefore , we conducted Western blot analysis with AtCHIP overexprssion plants. Under normal conditions, the steady-state levels of ClpP4 appear not affected in with AtCHIP overexpression plants when compared to wild type plants; however under heat and high intensity light conditions, the steady-state level of ClpP4 is reduced in most AtCHIP overexpression plants. The data indicate that AtCHIP overexpression leads to destruction of ClpP4 in plant cells under stress condition. Meanwhile, under high-intensity light condition, the steady-state level of FtSHl is also significantly reduced.5. AtCHIP overexpression display necrotic phenotype under high-intensity light condition.High-light light induced cell death is likely due to decrease capacity in repairing the photodamaged Dl protein in the PSII of ^CMP-overexpression plants. We analyzed steady-state level of Dl protein in ^/CWP-overexpression plants, which leads to inefficient removal of the damaged Dl fragment from thylakoid membranes, preventing insertion of newly synthesized Dl protein to replace the damaged Dl protein. Consequently we observed a decrease in the full-length Dl protein, but an increase in the 23-KD fragment of Dl protein in overexpression plants. That indicated that the phenotype was caused by the assimilation of the Dl fragment in the cells.6. C7/?P4-overexpression and ClpP4 anti-sense plants plants display chlorotic phenotype. In order to study the function of ClpP4 in plant cells, we generated the transgenic plants that overexpressoin ClpP4 and ClpP4 anti-sense plants, we found that about 7 independent lines display chlorotic phenotype when the rosette leaves are establishing. We observed a significantly increased amount of ClpP4 transcript in the ClpP4-overexpression plants and ClpP4 anti-sense plants by Northern blot analysis, which leads to corresponding increase in the steady-state level of ClpP4 protein ClpP4-overexpression as demonstrated by western blot analysis. Our data indicate that ClpP4 overexpression is likely responsible for the observed chlorotic phenotype.7. The ClpP4 knockout plants showed different growth phenotypes between wild type and mutants after normal and high temperature treatments.Under normal temperature treatment, wild type plants grew slower than that of mutants; under high temperature treatment, the 25-day growing wild type plants continue to show green and produce a few amount of seeds, whereas ClpP4 mutants show yellow and produce few seeds.8. Our data provides the first example that a nuclear gene (i.e. AtCHIP) regulates chloroplast protein degradation by directly regulating the activity of a chloroplast protease(i.e. ClpP4 and FtSHl), which helps filling the gap in our knowledge about protein degradation in cytoplasm and organelles.9. The mulberry gene mHNXl was cloned from the template cDNA library, which was synthesized by improved RT-PCR technique from the mRNAs of the mulberry buds. As one of the homologue gene of Arabidopsis thaliana, mHNXl encode the vacuolar membrane protein and play the function of vacuolar-type Na+/H+ antiporter (Venema, K., 2002). We created Arabidopsis overexpression mutants by the mulberry gene mNHXl and analysized by PCR, a total of 51 independent transgenic plants were obtained, and among them 18 homologous were chosen for further experiments.10. The results suggest that the products of the novel gene, mNHXl, on the vacuolar membrane function as a Na+/H+ antiporter and may be one of the most important factor determining salt tolerance in mulberry trees. The study established a possibility basic for improving crop quality such as mulberry using the rich mulberry resources in China.