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Title: Engineering of tomato Proline transporter (LePROT1) and Fatty acid desaturase (FAD7) genes against abiotic stress in tomato (Solanum lycopersicum L.)
Authors: Sanampudi, Venkata Rami Reddy
Keywords: Proline transporter;Fatty acid;Desaturase;Abiotic stress;Solanum lycopersicum;AGR/07
Issue Date: 21-Mar-2011
Publisher: Università degli studi della Tuscia - Viterbo
Series/Report no.: Tesi di dottorato di ricerca. 22. ciclo
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
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
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.
Dottorato di ricerca in Biotecnologie vegetali
Appears in Collections:Archivio delle tesi di dottorato di ricerca

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