| The Phosphoinositide (PI) signaling pathway and the phospholipid-derived second messenger inositol (1, 4, 5)-trisphosphate [Ins(1,4,5)P3] are involved in mediating plant responses to many biotic and abiotic stimuli. This pathway has been well studied in plants, mainly in Arabidopsis. With very few exceptions, the key enzymes in the PI signaling pathway have not been well characterized in tomato plants. The initial hypothesis of this dissertation is that altered Ins (1,4,5)P3 level affects phosphate sensing and signaling in tomato plants.;To approach the hypothesis, we first need to have a tentative idea about the composition of the pathway and how many genes may encode enzymes involved in the PI pathway in tomato. Therefore, we carried out similarity based computational predictions of major genes involved in the PI signaling pathway. In Chapter 1, we predicted genes encoding the upstream components of the PI pathway, including Phosphatidylinositol 4- kinases (PtdIns 4-kinases), Phosphatidylinositolbisphosphate 5-kinases (PtdInsP 5-kinases) and phospholipase C (PLC), as well as downstream enzymes of Ins(1,4,5)P3 metabolism including the Inositol 5-phosphatases (Ins 5-ptase) and the Inositol Polyphosphate Kinases IPK2 and IPK1. Our investigation showed that major components of this pathway are conserved between Arabidopsis and tomato. Chapter 1 details the similarities and differences. The transcript expression profiles for major pathway components in tomato roots and shoots are also described.;To examine how PI signaling pathway involved in abiotic stress, specifically phosphate (Pi) limiting conditions in tomato, we generated transgenic tomato plants constitutively expressing the mammalian type I inositol polyphosphate 5-phosphatase. In Chapter 2, we characterized the transgenic tomato plants. Our results showed that the transgenic tomato plants have significantly reduced basal Ins(1,4,5)P3 levels. There is no change in the total mass of hexakisphosphate (InsP6) in transgenic plants. The transgenic tomato plants have little difference in response to drought stress compared to Wt; however, they showed differential regulation of some defense-related genes and less resistance to pathogen infection by higher bacteria growth in the infection area. We also examined the membrane lipid kinases activity and developed a system to carry out reciprocal micro-grafting of transgenic and wild type tomato seedlings under tissue culture conditions, to prepare for future studies on root and shoot communication.;To further investigate the role of PI pathway, especially Ins(1,4,5)P 3, in the Pi sensing and signaling, in Chapter 3, we compared the tissue Pi content in the transgenic and Wt plants. We found that the transgenic plants have less Pi in the shoot, but higher in the roots compared to the Wt plants, especially at the bordering Pi condition, a Pi condition between sufficient and depleted Pi concentrations. To address the hypothesis at the transcriptomic level, we carried out deep-sequencing (RNAseq) of transgenic and Wt tomato shoot and root tissues from seedlings grown under low, bordering and sufficient Pi conditions. Our data indicated a dramatic difference in the molecular responses of roots and of shoots. Particularly at the bordering Pi concentration shoots have more transcripts differentially regulated compared to low Pi, and this trend became even more evident in the transgenic line. These results suggest that the most effect of the transgene could be in the shoot during the signaling coordinating stage in response to Pi availability. We identified transcripts that differentially regulated in transgenic compared to Wt plants at different Pi concentrations. Among these transcripts, cell wall related functions, plant defense related functions and putative Fe/Cu related biological processes were maybe the most affected. We suggested possible models to explain the interactions between Pi availability, PI signaling pathway, plant defense and cell wall synthesis. |
