The Physiological And Regulation Mechanisms Of Absorbing Phosphorus And Potassium In EdHP1 Transgenic Tabacco

Pyrophosphate (PPi) is hydrolyzed by pyrophosphorylase (H+-PPase) and releases free energy, and then produces proton gradient, which provide driving power for secondary active trans-membrane transportation of solutes such as positive ion, negative ion, ammonium acid, sugar, and etc. As efficient H +pump, H+-PPase plays a key role in responding to salinity and drought stresses in plants. Working together with vacuole-membrane-located H+-ATPase, H+-PPase not only can maintain the balance of ion and osmotic of the cell, but also can reduce the toxic effect to the cytoplasm causing by inorganic ion such as Na+. In pervious work, an H+-PPase gene, EdHP1 had been cloned from Elymus dahuricus. Result of gene functional analysis indicated that the EdHP1 gene could improve the Na+ and K+ uptaking in transgenic tobacco plants. However, the physiological and regulation mechanisms of nutritional absorption of EdHP1 transgenic plants still remain unknown. In the present study, by comparing each physiology index between EdHP1 transgenic and non-transgenic tobacco, we studied the physiological mechanism of P +and K+ uptaking controlled by EdHP1 gene, and those results led to better understanding of respective regulation mechanism. Meanwhile, we studied signaling regulation pathway involving EdHP1underlying drought tolerance. And the results are demonstrated below:1. Using the EdHP1 transgenic tobaccos as the studying materials, under low nutritional condition of low K+ and P+ concentration (0.05 level and 0.016 respectively), we compared parameters difference between transgenic tobacco and wide-type (WT) tobacco in terms of root system development, plant morphology, growth hormone levels in vivo, chlorophyll content, K+ and P+ content of plants. The results indicated that, under low content of K+ (0.05 mmolL-1) condition, the root systems of transgenic tobacco were more healthy than that of WT's, and similarly, the K+ content in vivo was higher than that of WT's. In general, low content of K+ effect on thin root developing specifically, and in this research, thin root of transgenic tobacco were more healthy than wild type tobacco, whereas there were no differences between transgenic and WT tobaccos in medium-thick and thick roots. Facilitated by the Non-Invasive Detection Method, the H+ and K+ flux in the root tip, elongation zone, and hair zone of both transgenic and WT tobaccos were detected under the low potassium condition. The results indicated that there were less K+ efflux and more H+ efflux in transgenic tobaccos compared to WT ones. Similar with above, the result coming from low phosphorus experiment (0.016 mmolL-1) was demonstrated as follows: Root system of transgenic tobaccos were better developed than of WT tobaccos', and growth hormone content of transgenic tobaccos was higher than that of WT tobaccos'. According the result above, it was suggested that the mechanism of EdHP1 gene might increase the H+ gradient in transgenic tobacco's root cells, which can resulted in cells acidic, increasing the IAA distribution among roots, and in turn, improving the roots development. On the other hand, higher H+ gradient can also active the transporter of the potassium and phosphorus on the membrane, which can improve the active transportation in roots. Both two effects resulted in higher the potassium and phosphorus up taking in root eventually.2. Result of drought tolerance experiment (2% PEG) indicated that the seed germination of EdHP1 transgenic tobacco were higher than that of WT plants'. The root systems of transgenic tobaccos were better-developed than that of WT ones', as suggested that overexpression of EdHP1 can confer higher drought tolerance to plants.3. To reveal the regulation mechanisms of EdHP1 in plants, Yeast Two-Hybrid system was used to screen Arabidopsis cDNA library, and AtVP1 from Arabidopsis was used as the bait protein, and finally some positive clones were obtained. On other hand, two key regulation proteins, LKS and SOS2 that are two key regulation proteins involved in signaling pathway underlying nutrition uptaking, were used for this experiment to identify whether they interacted with AtVP1. As a result, AtVP1 didn't interacted with none of them in other words, suggested that AtVP1 was not controlled by LKS or SOS2.The an member of H+-PPase family, EdHP1 can not only increase nutrition up taking, but also improve the tolerance to abiotic stress in plants, which were important for improving crop stress tolerance. Dissection of physiological mechanism provided a theoretical basis to improve the tolerance to abiotic stress in crops, and give better understand on the process of nutrient absorption.