Transcriptomic Analysis of Rice Suspension Cells under Sucrose Starvation and Lily Pollen Germination, and Functional Assay of Actin Binding Protein in Lily Pollen Tubes

• Main Authors: Huei-Jing Wang, 王暉景
• Other Authors: Guang-Yuh Jauh
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/59516411035469472131

博士 === 國防醫學院 === 生命科學研究所 === 95 === DNA microarray is an efficient technology to detect gene expression level of a lot of targets in parallel. In this dissertation, the microarray analyses are foundational strategies used to investigate the gene expression profiles of rice suspension cells and lily pollen. In chapter 1, we employed commercial rice microarray containing 21495 probes to monitor the gene expression profiling during sucrose starvation. Metabolic pathway analyses illustrate the molecular insights of gene expression profile in sucrose-starved cells: 1) down-regulates gene expression in metabolic pathways such as glycolysis, the citrate cycle, oxidative phosphorylation reaction, and the biosynthetic processes of many macromolecules; 2) up-regulates transcripts for proteins involved in degradation of various molecules such as sucrose, polysaccharides, fatty acids, and branched-chain amino acids; and 3) up-regulates the genes encoding for transcription factor families of bZIP, NAC and WRKY. In addition, a statistical analysis of promoter sequences of sucrose-starved up-regulated genes reveals several putative sucrose starvation responsive elements, such as ABA-responsive element, CACG, ACI, ACII and TATCCA-derived CTTATCC. A particle bombardment-mediated and luciferase activity-based transient promoter activity assay further verified the CTTATCC- and AC-elements as the promising sucrose starvation responsive activators in rice suspension cells. The potential coordinated roles of these transcription factors and their corresponding cis-elements in regulating genes expression under sucrose starvation are discussed. In chapter 2, we employed home-made cDNA microarray to study the changes of expression profiles of lily pollen/pollen tubes induced by stigma exudates. At first, we found that lily pollen grains germinated in medium containing stigma exudates, germination rate, but not growth rate, was significantly enhanced. To understand the molecular events, lily microarray chips were used to identify transcripts that were up-regulated by stigma exudates during pollen germination. Among about 5000 clones presented in our chip, 1,589 and 4 clones showed significantly up-and down-regulated, respectively, by stigma-exudates treatment. After DNA sequencing and deduced amino acids blasting, several clones involving in protein degradation, signal transduction, and cytoskeleton remodeling were identified. In chapter 3, we performed functional assay of an actin binding protein, LlLIM1, identified from lily microarray in chapter 2. LlLIM1 transcript was wildly expression in several organs, such as stem and pistil, but enriched in pollen/pollen tube, only present in mature and desiccated pollen during pollen developmental process. Localization of fluorescence protein-tagged LlLIM1 in elongating lily pollen tubes and in vitro F-actin sedimentation assay showed LlLIM1 bound to F-actin and promoted actin bundling formation. In addition, LlLIM1 could protect actin filament from depolymerization by LatB treatment, a drug depolymerized F-actin. When LlLIM1 highly overexpressed in pollen, the reducing of germination rate and abnormal tube growth were observed and these phenomena were closely associated with the formation of abnormal F-actin and aggregation of endomembrane structures in the clear zone of slowly or non-grown pollen tubes. All these results suggest the impaired pollen tube morphology is caused by formation of over-stabilized F-actin structures to interfere endomembrane trafficking. In pollen tubes overexpressing moderate amount of LlLIM1 showed an oscillatory formation of asterisk-sharp actin structures in clear zone at the period of low growth velocities. Interestingly, F-actin sedimentation assay showed that the actin binding affinity of LlLIM1 was regulated simultaneously by both pH and [Ca2+]; LlLIM1 prefers to bind to actin filaments in lower pH condition and in the presence of low calcium. The potential functions of LlLIM1 as an ABP with a pH- and calcium-sensitive manner in integrating endomembrane trafficking and oscillatory pH, calcium circumstances and growth happened in apical region of pollen tubes are discussed.