Engineering of tomato Proline transporter (LePROT1) and Fatty acid desaturase (FAD7) genes against abiotic stress in tomato (Solanum lycopersicum L.)

Abstract

Temperature affects the growth and productivity of plants, but its effect depends on nature of plant species i.e. warm season or cool season plants. Increased temperature, increases rate of photosynthesis and respiration but only up to certain levels of increased temperature; there after it will reduce both photosynthesis and respiration. Increased temperature is not only a problematic factor for vegetative growth but also for reproductive development like flowering, pollen development, fruit set and finally total yield of the crop plants. The negative effect of high temperature is becoming a major problem because of predicted increase of 2°C of earth surface temperature by 2050. In present days due to increasing threat of global warming to horticultural crop production, research on high temperature stress in relation to plant productivity becomes important and urgent. Exposure to high temperature causes reduced yields in tomato (Solanum lycopersicum L.). The level of proline content of anthers plays important roles in acquiring heat tolerance in tomato but this proline transport to anthers is seriously impaired by heat stress leading to increased level of proline accumulation in leaves (source) instead of developing pollen grains (sink) during heat stress. We made an attempt to develop tomato plants expressing the proline transporter to anthers gene LePROT1 under the control of HSP 18.2 promoter from Arabidopsis to increase supply of proline to developing anthers of tomato plants under heat stress. Here we got total six transformed plants after confirmation by PCR. We treated all transformed plants with high temperature at 38°C for 2 hours along with controls at 3 days before anthesis and then grew at normal temperatures after heat treatment. All transformed plants performed far better than control plants; pollen stainability averaged 93.98% in transformed plants and 81.1% in untransformed control plants, pollen germinability averaged 91.55% in transformed plants and 74.6% in untransformed control plants. There was a significant difference in yield between untransformed control plants and plants transformed with LePROT1 gene. All these differences in pollen stainability, germinability and in total yields were positively correlated with proline content in their developing anthers at 3 days before anthesis stage. Proline quantity is significantly different between untransformed control plants (860.87 !M/g FW) and plants transformed with LePROT1 gene (2117.74 !M /g FW). In this same experiment we also did attempt to screen drought and salt tolerance in plants transformed with LePROT1 and untransformed control plants in early seedlings stage by artificial invitro stress II treatments. Seeds from T1 generation plants and control plants were germinated and allowed to grow on artificial medium supplemented with 50g/l PEG to induce drought stress conditions and grown at 38°C for one week. Plants transformed with LePROT1 gene showed higher growth rate than untransformed control plants in terms of root length. Transformed plants had higher root length (9.19 cm) than control plants (6.66 cm) and there was no significant difference between control and transformed plants in hypocotyl length under drought stress. This increased root length in transformed plants than control plants is in accordance with their higher proline accumulation. Untransformed control plants accumulated less proline content (6.82 !g /g FW) and transformed plants accumulated more proline (11.94 !g/g FW) under artificial drought conditions. Seeds from T1 generation plants and control plants were germinated and allowed to grow on artificial salt stressed medium supplemented with 100 mM NaCl to induce salt stress conditions and grown at 38°C for one week. Plants transformed with LePROT1 showed significant differences in both root length (9.42 cm) and hypocotyl length (4.74 cm) than the root length (6.16 cm) and hypocotyl length (1.66 cm) of untransformed control plants. These differences in root and hypocotyl lengths of control and transformed plants was positively correlated with their proline content i.e. control plants accumulated less proline (17.3 !g/g FW) than LePROT1 transformed plants (25.16 !g/g FW). Proline content in all three treatments i.e. heat stress, salt and drought stress is higher in heat stress and then followed by salt and drought stresses. Plants can acquire inducible environmental stress resistance by remodeling of membrane fluidity. Plants ability to adjust membrane lipid fluidity, achieved by changing levels of unsaturated fatty acids through fatty acid desaturase genes, is a feature of stress acclimation. Modification of membrane fluidity results in an environment suitable for the function of critical integral proteins. Membrane fatty acids play main important role in tolerance to high temperature. Plastid omega-3 fatty acid desaturase catalyzes the conversion of dienoic fatty acids (16:2 and 18:2) to trienoic fatty acids (16:3 and 18:3) in glycerolipids which are the main constituents of chloroplast membranes. Exposure to high temperature causes reduced yields in tomato (Solanum lycopersicum L.). We produced transgenic tomato plants that express the transcript of double-stranded RNA (dsRNA) of tobacco plastid omega-3 fatty acid desaturase gene Nt FAD7 to induce post transcriptional gene silencing. The steady state messenger-RNA level of targeted gene was low in transformed plants. Under the heat stress III these transformed plants silenced for this gene showed higher number of viable pollen grains (92.79%) and higher yields (90 fruits/ 6 plants) when compared to the viable pollen (74.6%) and yield (25 fruits) untransformed plants. These results indicated that post transcriptional gene silencing of omega 3 fatty acid desaturase gene is useful to increase tolerance to high temperature in plants. In this experiment trienoic fatty acids quantity in transformed plants was from 10 to 45% less than the trienoic fatty acid composition of control plants and correspondingly increased dienoic fatty acids in transformed plants. Transformed seedlings growing under heat stress at 32°C for one week performed far better than the untransformed seedlings in terms of hypocotyl length (3.6 cm) than the control plant hypocotyl length (1.5 cm). These results indicate that down-regulation of the transcript level in the NtFAD7 by introduction of NtFAD7 dsRNA constructs is useful to decrease the trienoic fatty acid contents of the vegetative tissues in higher plants and give tolerance for higher temperature. Therefore, it is reasonable to conclude that the accumulation of proline through proline transporter in relation to heat stress under the control of heat inducible promoter and decrease of trienoic acid formation through gene silencing could be useful methods to increase heat tolerance in tomato and it could be a solution to increase crop yields under this present arena of global warming.