Patent Application
20090291476
The invention relates to construction of plant transformation vector for over expression of Protochlorophyllide oxidoreductase C and transformation of plants with the said vector.
Plants are vulnerable to biotic and abiotic stresses and they have a better potential of plant productively and quality than that being currently achieved. Resistance to oxidative stress is also another important aspect.
The gene protochlorophyllide oxidoreductase was cloned in Japan, Switzerland and in the inventor “slab in Indian and have been published. There is no state of art in the over expression of Protochlorophyllide oxidoreductase C.
An object of the invention is to construct plant transformation vector for over expression of protochlorophyllide oxidoreductase C.
Another object is to construct plant transformation vector for over expression of protochlorophyllide oxidoreductase C and transform plant, preferably Arabidopsis by the said transformation vector.
Further object is to construct plant transformation vector for over expression of protochlorophyllide oxidoreductase C and produce plants resistant to oxidative stress, abiotic stress and biotic stress by over expression of protochlorophyllide oxidoreductase C.
Another object is to construct plant transformation vector for over expression of protochlorophyllide oxidoreductase C and produce transgenic plants which under high light, high or low temperatures, high salinity or soil pH, drought, water lagging, UVB radiation, bacterial, fungal or viral infections, become resistant to stress and produce higher biomass, have higher harvest index than their wild type counter parts.
Yet another object to minimize the generation of singlet oxygen by reducing the concentration of the tetrapyrrolic photosensitizer protochlorophyllide.
Still another object to control plant death induced by oxidative stress by regulating the steady state accumulation of chlorophyll biosynthetic intermediates and generation of singlet oxygen.
According to this invention a process for the construction of plant transformation vector comprising:
digesting a binary vector pCAMBIA1304 with EcoRI and Sal I;
subjecting the digested binary vector to the step of end filling by Klenow;
re-ligating the said binary vector by known method;
obtaining CaMV 35 s promoter cassette with Ω enhancer from pSH9;
incorporating the said Camv35S prometer cassette into the Hind III site of the said binary vector to obtain modified pCAMBIA 1304 vetor;
subjecting pore DNA fragment (1206 bp) to the step of amplification using a pair of primers;
introducing EcoRI restriction sites to both the primers;
ligating the said amplified cDNA fragment to pGEM T-easy;
extracting EcoRI digested porC cDNA fragment from cloned pGEM T-easy;
inserting said EcoRI digested porC cDNA fragment into modified pCAMBIA 1304 plant transformation vector to produce recombinant plasmids construct (pCAMBIA 1304-AtporC)
a: shows that omega (Ω) enhancer was used for Agrobacterium mediated plant transformation.
b: shows that Southern blot analysis of different T3 stable Arabidopsis transgenic lines (PORCx).
c: shows that northern and western blot analysis revealed that porC and its gene product were highly over expressed.
d: shows that northern and western blot analysis revealed that porC and its gene product were highly over expressed.
a: shows that the over expression of porC in Arabidopsis plants resulted in greener plants.
b: shows that PORCx plants resulted in efficient photoconversion of Pchlide to Chlide.
c: shows that 1O2 production and its measurement.
a: shows that light-stressed WT plants.
b: shows that accumulated reduced amounts of anthocyanin.
c: shows that presence of excess 1O2 and low at reduced 1O2 in WT and PORCx plants.
d: shows that reduced membrane lipid peroxidation owing to less production of 1O2.
e: shows that in light-stressed PORCx plants the Fv/Fm ratio was not affected demonstrating that due to reduced 1O2 generation and membrane lipid peroxidation their photosynthetic efficiency was intact.
a: shows that both WT and PORCx plants accumulated excess of Pchlide, Proto IX and MP(E) in ALA-treated WT and PORCx plants in dark.
b: shows that both WT and PORCx plants phototransformed Pchlide to Chlide.
c: shows that PORCx plants due to increased abundance of PORC protein, phototransformation of Pchlide to Chlide was much higher.
d: shows that reduced accumulation of the photosensitizer Pchlide resulted in less production of light-mediated-1O2 in PORCx plants.
e: shows that the modulation of 1O2 generation in PORCx plants by manipulation of the level of the photosensitizer Pchlide pool led to reduced MDA production.
f: shows that the Fv/Fm ratio was almost unchanged in ALA-treated and light exposed PORCx plants.
g: shows that the PORCx plants survived and produced flowers even after 10 days of light exposure.
h: shows that light-induced death of WT plants whereas PORCx plants survived.
Oxidative stress is the ultimate manifestation of almost all kinds of biotic and abiotic stresses that affect forest and crop ecosystems. Singlet oxygen, one of the reactive oxygen species is produced in pigment bed of the photosynthetic apparatus and via type II photosensitization reactions of tetrapyrroles3. It causes oxidative cellular damage and plant death. Unlike detoxification of superoxide by superoxide dismutase there is no enzymatic detoxification mechanism available to destroy singlet oxygen produced from chlorphyll biosynthetic intermediates in light stress. The present invention aims to minimize the generation of singlet oxygen by reducing the concentration of the tetrapyrrolic photosensitizer protochlorophyllide, via genetic approach i.e., manipulation of the protochlorophyllide oxidoreductase contents that photo-transforms protochlorophyllide to chlorophyllide. Here we show that in Arabidopsis thaliana overexpression of porC gene that encodes for the light-inducible isoform of protochlorophyllide oxidoreductase C, resulted in reduced accumulation of the photosensitizer protochlorophyllide and minimal production of singlet oxygen in response to light stress and 5-aminolevulinic acid (ALA) treatment. It is demonstrated that it is possible to control plant death induced by oxidative stress by regulating the steady state accumulation of chlorophyll biosynthetic intermediates and generation of singlet oxygen.
Regulation of oxidative stress is extremely important to protect plants from high light and other abiotic and biotic stresses. To minimize the generation of 1O2 I manipulated the steady state accumulation of Chl biosynthetic intermediates by producing protochlorophyllide (Pchlide) oxidoreductase C (porC) over expressing (PORCx) plants.
Protochlorophyllide (Pchlide) oxidoreductase (POR), the first light-dependent enzyme in the Chl biosynthesis pathway has three isoforms i.e., PORA, PORB and PORC. porC is light-inducible and its expression increases in high light. To increase PORC amount for efficient photoconversion of Pchlide to chlorophyllide (Chlide) porC was over expressed in A. thaliana (ecotype Col). The modified Pcambia 1304 binary vector containing the porC cDNA, driven by the control of the cauliflower mosaic virus (CaMV) 35S promoter having omega (Ω) enhancer was used for Agrobacterium mediated plant transformation (
The over expression of porC in Arabidopsis plants resulted in greener plants having enhanced Chl contents (22% more in T12 & 27% more in T13) (
Light-stressed WT plants were purple (
To probe further if manipulation of the level of Chl biosynthetic intermediates would lead to control of oxidative stress induced by 1O2, both WT and PORCx plants were treated with ALA (3 mM), the precursor of Chl end heme and were kept in dark overnight (14 h). As shown in (
Due to oxidative stress there was a severe morphological injury to WT plants. Exposure of ALA-treated (3 mM) and dark incubated (14 h) WT plants to light (100 μmoles photons m−2s−1) caused wilting of leaves followed by appearance of prominent necrotic patches and subsequent death of plants within 24 h (
plants survived (
Results demonstrate that reduction in the steady state accumulation of plant tetrapyrroles would limit 1O2 production and cellular damage in light-stressed plants. In the same vein plant death caused by 1O2 generated from over-accumulated Chl biosynthetic intermediates in ALA-treated plants could be minimized by efficiently converting them to Chl. This approach could be exploited to generate PORCx crop plants that would be resistant to the herbicidal treatment of ALA or other compounds that induce oxidative stress and biotic and abiotic stresses for better crop yield.
I. Construction of Plant Transformation Vector for Over-Expression of pORC in Plants:
A binary vector pCAMBIA 1304 was digested with EcoRI and Sal I, end filled by Klenow and then re-ligated using the standard procedure (Sambrook, et. al 1989). A CaMV 35 S promoter cassette along with the Ω enhancer was taken out from pSH9 and inserted into the Hind III site of the binary vector. For the amplification of a porC cDNA fragment (1206 bp) cDNA library of A. thaliana was used. PCR was carried out with a pair of primers as given in table-4. In both the primers EcoRI restriction sites were introduced. The amplified cDNA fragment was ligated to pGEMT-Easy, and then EcoRI digested porC cDNA fragment was taken out from cloned pGEMT-Easy and inserted into the modified pCAMBIA 1304 plant transformation vector.
Transformation of Agrobacterium: Recombinant plasmid construct (pCAMBIA 1304-AtporC) was transformed into Agrobacterium by freeze thaw method. For the preparation of competent cells, Agrobacterium tumefaciens (GV3101) was grown in 50 ml YEM medium (0.04% yeast extract, 1% mannitol, 0.01 NaCl, 0.02% MgSO4, 7H2O and 0.5% K2HPO4) at 28° C. with vigorous shaking until the O.D600 reached 0.5 to 0.6. The culture was chilled on ice and centrifuged at 3000×g for 5 min at 4° C. The pellet was resuspended in 1 ml of ice cold CaCl2 (20 mM) and 0.1 ml aliquots were dispensed in pre-chilled eppendorf tubes and stored at −80° C.
Transformation of Agrobacterium with pCAMBIA-porC plasmid construct was carried out by mixing 10 μg of DMA with competent ceils followed by immediate freezing in liquid nitrogen. Subsequently cells were thawed, incubating the Eppendorf tubes at 37° C. for 5 min and 1 ml of YEM medium was added to the tube and incubated at 28° C. for 3 hrs. Cells were spread on a YEM agar plate supplemented with 50 μg/ml kanamycin and 25 μg/ml rifampicin and incubated at 28° C. Transformed colonies that appeared after 1-2 days were analysed either by PCR and the positive colonies were confirmed by restriction digestion of the purified recombinant plasmids.
Plant Transformation: One positive colony of Agrobacterium tumefacians from each construct was used to transform Arabidopsis by vacuum infiltration method (Clough and Bent, 1998). A single colony was inoculated into 30 ml of liquid YEM medium with kanamycin plus rifampicin and grown for one day at 28° C. One ml of this bacterial culture was inoculated into fresh medium and grown overnight.
Arabidopsis Transformation of by Vacuum Infiltration: The plastic pots were filled to top with agropeat soil and covered with required size of mosquito net using rubber bands. The vernalised Arabidopsis seeds at 4° C. for 3 days were resuspended in milli-Q water and 20-30 seeds per pot were uniformly distributed over the agropeat soil. The pots were transferred to the growth chamber. Plants were allowed to grow under 16 hrs dark and 8 hrs light till bolts emerged. Bolts were clipped off to produce multiple secondaries.
Vacuum Infiltration: A single colony of Agrobacterium tumefaciens was inoculated into 25 ml of liquid YEM with kanamycin and rifampicin and grown for 48 hrs at 28° C. One ml of this bacterial culture was inoculated into 1L of fresh medium and allowed to grow for overnight at 28° C. The cells were harvested at 5000 g for 10 min at room temperature. The pellet was resuspended in infiltration medium (0.5×MS salts, 1×B5 vitamins, 5% sucrose, 0.04 M BAP, 0.02% silvet, pH 5.7). The plants having multiple secondary bolts were inverted on 250 ml beaker containing Agrobacterium tumefaciens strain with proper construct. The entire set up was kept in bell jar and the vacuum was applied till bubbles were formed on the leaves and stem. The vacuum was rapidly released and plants were left in liquid for 1-2 min. The plants were removed and covered with saran-wrap (to maintain humidity) and left overnight. The plants were uncovered after one day and then grown under green house condition. The seeds were harvested after one month.
Seed Plating in Culture Medium: Seeds (40 mg) of Arabidopsis were surface sterilized with 2 ml of sterilization solution [30% v/v bleach (100% bleach contain 5% hypochlorite) with 1 μl/ml of 20% Triton X-100] and gently mixed by inversion for 15 min. The seeds were allowed to settle for 1 or 2 min before decanting the bleach. The tube with seeds was filled with sterile water and mixed. Washing of seeds was repeated several times till the bleach smell disappeared (it normally takes more than 5 washes). The seeds were finally resuspended in required volume of sterile water and then plated on GM medium containing kanamycin for selection of heterozygous transgene. The sterile water was allowed to evaporate in laminar flow hood and transferred plates in cold under dark for 2 days. After 2 days the plates were transferred to light.
The seedlings, which contain heterozygous transgene, survived on kanamycin plates and all other susceptible seedlings were bleached out. Resistant seedings were transferred to soil pots and grew them for 3 months to collect the seeds. This process was continued for 3 to 4 generations to obtain homozygous lines.
II. Methodology Used in the Whole P:
Plant materials and growth conditions: The Arabidopsis thaliana ecotype Columbia-0 (Col-0) was grown in soil or on GM medium containing 0.8% agar at 22° C.±2° C. with a photoperiod of 16 hrs fight and 6 hrs dark except when specific treatments were given.
General sterilization procedures: Culture media, glassware and tissue culture tools were sterilized by autoclaving at 121° C. and 151-p/inch2pressure for 15 min. Antibiotics and other heat-labile components used were filter sterilized using a syringe filtration unit fitted with an autoclaved cellulose nitrate membrane filter of 0.22 μm pore size (mdi, India).
Nutrient Media: For in vitro experiment, the GM medium (Table 2), Gamborg et al., 1868) was used in case of Arabidopsis. The composition of MS medium (Murashige and Skoog, 1862) was mentioned in (Table 3). The stock solutions of various components as given in (Table 3) were prepared in Milli-Q water, stored at 4° C. end mixed as required just before use. All the phytohormone stocks were prepared in DMSO and stored at 4° C. The medium was prepared from stock solutions and the pH was adjusted to 5.7 using 1 M NaOH prior to the addition of agar and then autoclaved. The filter-sterilized solution of heat labile antibiotics was added to the autoclaved medium pre-cooled to 45° C.
Recombinant DNA techniques for cloning and DNA analysis: Rapid amplification of the cDNA or genomic DNA fragment was carried out using Taq DNA polymerase and a set of convergent primers. Reaction mixture (50 μl) contained 30-50 ng DNA template, 150 ng (or 30 μmoles) of each primer, 8 μl of 1.25 mM dNTPs, 5 μl 10×Taq buffer and 2.5 units of Taq polymerase. The reaction condition of PCR consisted of denaturation (94° C.), annealing (varies from gene to gene) and extension (72° C.) for 1 min per 1 kb pair to be amplified. Reaction was continued for 30 cycles to obtain an amplified product. An aliquot from the mix was electrophoresed on 1% agarose gel to check for amplification.
Purification of DNA fragment from Agarose Gel: PCR amplified product or restriction enzyme digested plasmid was electrophoresed on 1% agarose gel. The desired fragment was identified using standard molecular weight marker (1 kb ladder or Lambda DNA digested with Hind III) and purified using the following technique.
Qiaquick/GFX Gel Extraction Column: To the excised pieces of agarose gel containing DNA fragment 3 volumes of QG/captured buffer (as supplied with Qiaquick/GFX gel extraction kit) was added end dissolved by heating to 55° C. for 10 min. The mixture was loaded onto Qiaquick/GFX spin column and spun briefly. The flow through was discarded and the column was washed with PE/Wash buffer (Qiagen GMBH, Amersham pharmacia). The purified DNA fragment was edited with 50 μl of 10 mM Tris-Cl, pH 8.0.
Preparation of competent cells and transformation: DH5α cells were made competent by following the protocol of Hanahan, 1983. A single colony of DH5α was picked up and inoculated into 5 ml of LB medium and grown overnight at 37° C. One ml of this was inoculated freshly into 100 ml of LB and grown at 37° C. till O.D600 of 0.4-0.5 was reached (2-3 hrs). The cells were harvested by centrifugation at 3000×g for 10 min. The pellet was resuspended in 40 ml of ice cold 100 mM CaCl2 solution, incubated on ice for 1 hr, centrifuged and the pellet was resuspended in a 4 ml of ice cold 100 mM CaCl2 containing 15% glycerol solution. The cell suspension (0.1 ml) was aliquoted into autoclaved un-opened eppendorf tubes and stored at −80° C.
DNA fragments were ligated to the appropriate vectors by using T4 DNA ligase overnight at 4° C. or 16° C. The ligation mixture was added to the competent cells and mixed by tapping and then incubated for 30 min at 4° C. All steps were carried out in a laminar hood under sterile conditions. The cells were subjected to heat shock by incubating at 42° C. for 90 sec, allowed to stand for 2-5 min on ice followed by addition of 0.9 ml of LB and then grown at 37° C. with gentle shaking (185 rpm). Different aliquots of these transformed competent cells were plated onto LB plate containing appropriate antibiotic. For blue white selection, 10 μg IPTG and 1 μg X-gal per plate were spread prior to plating the cells. The plates were incubated overnight at 37° C. Transformed cells containing recombinant plasmid were confirmed by colony PCR and restriction digestion.
Isolation and purification of plasmid DNA: Five milliliters of overnight grown culture of bacterial cells were harvested by centrifugation at a maximum speed for 1 min in a microfuge. The pellet was resuspended in 100 μl of Ice-cold solution I (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 μg/ml RNase A) and vortexed vigorously. Then cells were fysed by adding 200 μl freshly prepared solution II (200 mM NaOH, 1% SDS) and gentry mixed by inverting the tube 5 times and then kept on ice for 5 min.
Following this, 150 μl of chilled solution III (3.0 M potassium acetate, pH 5.5) was added and mixed by inversion. This mixture was incubated on ice for 3-5 min and centrifuged at maximum speed in a microfuge for 5 min at 4° C., The supernatant was extracted with equal volumes of Phenol:Chloroform:Iso amyl alcohol (25:24:1) at maximum speed for 2 min at 4° C. The DNA precipitation was carried out by adding 1/10 volume of 3 M sodium acetate (pH 5.2) and two volumes of ethanol (room temperature). The mixture was incubated at room temperature for 2 min and centrifuged at maximum speed for 5 min to precipitate DNA. The plasmid DNA was washed with 70% ethanol, vacuum dried and dissolved in minimal volume of water or TE buffer.
Spectrophotometric Estimation of Nucleic acid: The quantity and quality of the nucleic acid was determined by measuring the absorbance at 260 nm and 280 nm. The amount was calculated taking 1.0 A260=50 μg/ml for DNA and 1.0 A260=40 μg for RNA. The purity of the nucleic acid was determined by calculating the ratio A250/A260 for each sample.
Pchlide Extraction and Purification: Pchlide was extracted according to Hukamani and Tripathy, 1992. Leaves (Incubated over night with 25 ml of 4.5 mM ALA and 5 mM 2, 2 dipyridil) were homogenized in 200 ml of acetone: 0.1 N NH4OH(9:1). The fully esterified tetrapyrroles were extracted into the hexane while the mono and di-carboxylic tetrapyrroles remained in the hexane extracted acetone residue solvent mixture fraction (HEAR). The pigments were extracted from HEAR fraction to ⅓ volume of diethylether after addition of 1/20 volume of saturated NaCl and 1/40 vol of 0.5 M phosphate buffer (pH 7.0). The ether extracts containing the Pchlide along with other carboxylic tetrapyrroles were concentrated by vacuum drying, dried under N2 gas and were dissolved in minimal volume of methanol:acetone=4:1. The pigments dissolved in methanol:acetone solution were chromatographed on thin layers of silica gel H developed in toluene:ethyl acetate:ethanol (4:1:1) at 4° C. Pchlide, which migrated having Rf value of 0.32 was eluted in methanol:acetone (4:1).
Thin Layer Chromatography: 43 g of silica gel (Type H, sigma chemical Co.) was dissolved in 100 ml of distilled water and mixed in a mixer. This solution was spread uniformly over the clean glass plates as a thin layer. Glass plates were then dried In the over at 50° C.
Spectrophotometry: Absorption spectrum was recorded on a Shimadzu UV 260 spectrophotometer. For the calculation of concentrations of pure samples of Pchlide was dissolved in ether, extinction co-efficient of 3.56×104M−1 cm−1 at 624 nm was used.
Spectrofluorometry: Fluorescence spectra were recorded in ratio mode in a computer-driven SLM AMINCO 8000 spectrofluorometer having a photon-counting device. Rhodamine B was used in the reference channels as a quantum counter. A tetraphenylbutadiene block was used to adjust the voltage in the sample as well as in the reference channels to 20,000 counts per second at excitation and emission wavelengths of 348 and 422 nm, respectively. To reduce the noise level, the sample channel photomultiplier tube was cooled to −20° C. by a thermoelectric cooling device. The emission spectra were recorded from 600 to 700 nm at excitation and emission bandwidths of 4 nm.
Raising of polyclonal Anti body: Polyclonal anti body against ATPORC was raised in rabbit as per the standard procedure (Herlow and Lane 1988). Two-four months of old New Zealand white rabbits were used for raising antibodies. Before the primary injection, pre-immune serum was collected by bleeding the rabbit by puncturing the eye vein. Primary immunization was carried out by making a emulsion of crushed and homogenized gel pieces containing about 150 μg of respective proteins with complete Freund's adjuvant in equal proportions. After four weeks, a secondary immunization was done with an emulsion of incomplete Freund's adjuvant and 100 μg of individual proteins in gel pieces. Test bleeds were done to check the fitter of the antibodies by western analysis. A total number of 5 boosters were given at time interval of 4 weeks and finally blood was collected using capillary from a puncture eye vein. The serum isolated from blood was aliquoted and stored at 70° C.
2-Dimensional Gel Electro phoresis: Protein was estimated by Bradford method (Bradford, 1976) after half hour incubation in ice. Protein sample containing 100 μg of protein was added to rehydration buffer (6M urea, 2 M thiourea, 2% CHAPAS, 0.2% ampholyte, 50 MDTT).
Isoelectric focuring: Protein sample containing 100 μg in 125 μl of rehydration buffer was spread equally in the lain of rehydration tray. 1PG (Immobile pH gradient) strip of 7 cm (pH 4-7) was laid on the sample facing the gel side down. Mineral oil was over laid to prevent any evaporation. It was left for passive rehydration for 16 hour at 20° C. temperature in isoelectric focusing (IEF). After the strips got rehydrated it was moved to IEF focusing tray. Two paper wicks wet with 8 μl of milli Q water were put on two electrodes. Focusing tray was adjusted in isoelectric focusing cell. Mineral oil was overlaid and run was carried out according to program given below. After running got completed 1 pG strips were taken out, put in equilibration buffer 1 for 10 min and subsequently in equilibration buffer II for another 10 min. Then 1 PG strip was kept on stacking gel and 12.5% of SDS-PAGE (sodium dodecyl subfate polyacrylamide gel electrophoresis) was run at constant voltage of 100V. On one side of the gel, medium range molecular weight marker (14.3 kDa tp 97.4 kDa) was loaded. Equilibration buffer I: 6M urea, 2% SDS, 0.375 M Tris-HCL (pH 8.8), 20% glycerol and 2% (w/v) DTT. Equilibration buffer II: 6 M urea, 2% SDS, 0.375 M Tris HCL (pH 8.8), 20% glycerol and 2.5% idoacetamide.
Running Programme for Isoelectric Focusing:
Silver Staining: After completion of SDS-PAGE, gels were soaked in fixative solution (40% ethanol and 12% glacial acetic acid with 75 μl of formaldehyde) for 1 h with 2-3 changes of the same solution with time interval. Then these were washed three times for 20 min each in 50% ethanol. After that gels were treated with 0.02% solution of sodium thiosulphate for 1 min and washed with double distilled Water for 1 min. Then the gels were submerged and shook in silver nitrate solution (0.2%) containing 75 μl of formal dehyde for 20 min. After silver nitrate staining gels were washed for 1 min with distilled water, then transferred to 6% sodium carbonate solution and fermaldehyde was added drop by drop to it till the desired colour was obtained. The reaction was stopped in the 50% methanol and 12% glacial acetic acid solution. Gels were stored in 5% glacial acetic acid solution. Whole of the staining was done with continuous shaking and the timings were maintained very accurately.
III. Analysis of Transgenic Plants:
Gus Histochemical Assay. Putative transgenic plants were screened histochemicalty for verification of the expression of wid. A gene (Jefferson, 1987). Histochemical array for GUS was carried out in the intact tissues (organ or whole seedlings or free hand cut sections). Tissue from the control and transgenic plants were submerged in fixation buffer (2% formaldehyde, 50 mM sodium phosphate pH 7, 0.05% Triton X-100) and Vacuum infiltrated for 4-5 min on ice and kept at room temperature for 10 min. The fixation buffer was removed and the material was washed twice with 50 mM sodium phosphate buffer pH 7 to remove fixative buffer. The tissue sample were stained with 1.5 mM of X-gluc, 50 mM sodium phosphate pH 7 and 0.1 % Triton X-100 by vacuum infiltrating for 5-10 min and then kept the tissue at 37° C. overnight in darkness. After staining, tissue was rinsed extensively in ethanol to remove the chlorophyll.
Preparation of Plant Genomic DNA for PCR Analysis: Small piece of leaf tissue (1 cm×1 cm) was ground with 400 μl of buffer (2% CTAB, 1.4 M Nacl, 20 mM EDTA, 100 mM Tris-cl and 0.1% B Mercap to ethanol) and mixed with another 400 μl buffer and kept at 60° C. in a water bath for 30 min. The aqueous layer was extracted twice with chloroform:isoamyl alcohol (24:1) and genomi cDNA was precipitated with ⅔ volume of isopropanol for 30 min at −20° C. Centrifuged the samples at maximum speed for 3 min at room temperature. The pellet was washed 3 times with 70% ethanol, dried and dissolved in 30 μl TE or sterile water containing 20 μg/ml RNase and incubated at 37° C. for 30 min.
Southern Blotting: Genomic DMA was digested with different restriction enzymes at 37° C. and then electrophoresed overnight on 0.8% agarose gel. Following electrophoresis, DNA was visualized by ethidium bromide staining. DNA from the gel was transferred to nylon membrane using the capillary method as described in Sambrook et al., 1989. Briefly, the gel was treated first with depurination solution (0.2 N HCl) for 20 min if the DNA fragments were more than 10 Kb in size followed by 10 min treatment in denaturation solution (1.5 M NaCl, 0.5 N NaOH) and then 2 washes of 10 min each in neutralizing solution (1 M ammonium acetate). The DNA was transferred to nylon membrane (Hybond-N, Amersham, England) by capillary transfer in 10×SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) or 1 M ammonium acetate for overnight and then the blot was baked for 2 h at 80° C. under vacuum. The membrane was pre-hybridized for 4-6 h at 65° C. in buffer containing 5×SSC, 5×Denhardt reagent (0.5% Ficoll, 0.5% PVP, 0.5% BSA), 0.1% SDS and 100 μg/ml denatured salmon sperm DNA and 10% Dextran sulphate. Thereafter, to the pre-hybridization solution, radiolabeled probe (pre-denatured by boiling for 10 min) was added. The probe was prepared by using the random-labelling Kit (Amersham, England) and purified using Sephadex G-50 spun column (Sambrook et al., 1989). After 16-18 h of incubation at 65° C. in the hybridization solution, membranes were washed once with 2×SSC (room temperature) and twice sequentially in 1×SSC, 0.1% SDS for 15 min each at65° C.
Treatments for RNA Isolation: Rosette Leaves of young Arabidopsis plants grown for 35-40 days under 16 h/6 h photoperiod at 22° C. were used for extraction of total RNA. Four to five days old etiolated Arabidopsis seedlings were exposed to different quality and quantity of light and after a particular period of time of treatments whole seedlings were used to extract RNA. RNA was also isolated from plants grown in low light (LL), high light (HL) and exposed to herbicide i.e; δ-ALA.
Isolation of Total RNA by TRI reagent (Sigma) Method: About 0.1 g of tissue was homogenized in 1 ml of trizol and the insoluble material was separated from the homogenate by centrifugation at 11,000 g for 10 min at 2-8° C. The supernatant was transferred into a fresh Eppendorf tube and to this 0.2 ml chloroform was added and mixed vigorously. The sample was centrifuged at 12,000 rpm for 15 min at 2-8° C. and the supernatant was transferred to a fresh eppendorf tube. The RNA was precipitated from the aqueous phase by mixing with 0.5 ml isopropanol followed by centrifugation at 12,000 rpm for 15 min. The pellet was washed with 80% ethanol, dried and dissolved in minimal volume of Rnase A free sterile water.
RT-PCR: Reverse transcription was carried out using 2 μg of total RNA and oligo-dT as primer. The RNA was treated with RNAse free DNAse (5 unit/μl) to make sure that there is no contamination of DNA. The reaction mixture includes 1 mM dNTPs, 0.5 μM oligo-dT, 20 U Rnase inhibitor, 10 mM DTT, 1×RT buffer and 20-30 unit AMV reverse transcriptase in total volume of 50 μl. The reaction mixture was incubated for one hour at 37° C. Using this reaction mixture as template PGR was carried out with gene specific primers to check the expression of the respective genes. To ensure linearity of the reaction, the minimum number of cycles needed to visualize the transcripts was first determined. When the condition for RT-PGR linearity was established, runs were performed and repeated at least for three times using independently treated samples.
Preparation of Probe by Random Primer Labelling Method: Twenty-five ng DNA, 5 μl of primer was taken in an eppendorf. After denaturation by heating to 95-100° C. for 5 min, brief spin was given. Then dNTPs (4 μl each) were added without dCTP, which was used as label, 5 μl of reaction buffer, 5 μl of α32P dCTP and 2 μl of enzyme and appropriate Volume of water was added to make total volume of 50 μl. The contents were mixed gently by pipetting up and down and capped the tube. The reaction mixture was incubated at 37° C. for 1 hr and stopped by adding of 5 μl of 0.2 EDTA. Before use in a hybridization reaction the labeled DNA was denatured by heating to 100° C. for 5 min and chilled on ice.
Northern Hybridisation: Total RNA was fractionated on 1% formaldehyde denaturing agarose gel according to Sambrook et al., 1989. Gel was electrophoresed for 3-4 hrs and after completion of the run, the gel was rinsed in DEPC treated water and equilibrated with 20×SSC for 10 min. RNA was transferred to nylon membrane (Amersham hybond —N) by capillary transfer in 5×SSC for 16 hrs. Nylon membrane was rinsed in 6×SSC, cross-linked in Stratalinker, soaked in 5% acetic acid and stained with methylene blue. Excess stain was washed with water. The membrane was pre-hybridized for 6-8 hrs at 65° C. in buffer containing 5×SSC, 5×Denhardt reagent (0.5% Ficon, 0.5% PVP, 0.5% BSA) 0.1% SDS and 100 μg/ml denatured salmon sperm DNA and 10% dextran sulphate. Thereafter, to the prehybridization solution radiolabeled probe (pre-denatured by boiling for 10 min) was added. After 16-18 hrs of incubation. at 65° C. in the hybridization solution, the membrane was washed twice with 2×SSC, 0.1% SDS at room temperature for 5 min; twice with 0.5×SSC, 0.1% SDS at 65° C. for 15 min and twice with 2×SSC at room temperature for 5 min. The membrane was then exposed to X-ray film for autoradiography.
Total Protein Isolation from Plants and Estimation: Leaves were homogenized in liquid nitrogen and were grinded in mortar and pestle in extraction buffer consisting of 56 mM Na2CO3, 5 mM DTT, 10 mM isoascorbate, 12% Sucrose, 2 mM EDTA, and 2% SDS. The samples were vortexed thoroughly. They were incubated at 80° C. for 20 min and were x centrifuged at 13000 rpm for 5 min, and supernatant was collected. To the supernatant ice cold acetone (final concentration should be 90%) containing 0.1%v/v b-mercaptoethanol was added, mixed properly and kept in −20° C. for 1 hour. Pellet done by centrifugation (13000 rpm for 10min). The pellet was dissolved in 20 μl of NaOH (0.1M) and protein estimation was done with Bradford's method.
Isolation of Broken Plastids: Isolation of broken plastids were done as described by Tripathy and Mohanty, 1980. Around 5 g of leaves were homogenized in 40 ml of isolation buffer containing 0.4 M sorbitol, 0.05 M Hepes/KOH (pH 7.3), 1 mM MgCl2, 1mM EDTA at 4° C. and in green safe light. Homogenae was passed through 8 layers of cheesecloth and 1 layer of miracloth and centrifuged at 4000 rpm for 7 min. Pellet containing broken plastids was suspended in suspension buffer containing sorbitol 0.4 M, tris 0.05 M pH 7.5, MgCl2 1 mM and EDTA 1 mM.
Polyacrylamide Gel Electrophoresis of Proteins (SDS-PAGE): SDS-PAGE was performed according to Laemmli, 1970. Gets were prepared and electrophoresed under reducing and denaturing conditions in presence of β-ME and SDS. Protein samples were prepared by mixing % volume of 4×sample buffer (200 mM Tris-Cl, pH 6.8, 2% SDS, 40% glycerol, 8% β-ME, 0.04% bromophenol blue). The samples were boiled for 3 min in a water bath and the thylakoid samples were incubated at 50′C for 1 h, and then loaded onto gel. Until the proteins get stacked, the gel was electrophoresed at low voltage (120V). Gels were stained with CBB (0.25% Coomassie brilliant blue R-250, 50% methanol and 10% acetic acid).
Western Blotting: Western blotting was done according to the method described in Sambrook et al., 1989. Mini trans blot Electrophoretic Cell (ATTO Co., Japan) was used to transfer the proteins from the gel onto nitrocellulose membrane. The apparatus for electro blotting was assembled according to the manufacturer's instruction. Electro blotting was performed in the presence of 39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% methanol at a constant voltage (V) for 1 hrs. After the transfer was over, gel-facing side of the membrane was marked and the membrane was stained in ponceau S (0.1% ponceau S in 1% acetic add). Markers were marked and the membrane was rinsed briefly in TBST (10 mM Tris, 150 mM NaCl, pH 7.5 and 0.05% Tween −20) and then incubated in blocking solution (4% BSA in TBST) for 1 hr with gentle shaking. The blocking solution was replaced with primary antibody solution (antibody in TBST containing 1% BSA) and incubation was continued for another 1 hr with gentle shaking at room temperature. Thereafter, the blots were washed thrice with TBST for 5 min each. After Washing, the blots were transferred to alkaline-phosphate conjugated secondary antibody solution (1:15, 000 dilution in TBST containing 1% BSA) and further incubated for 1 hr. The protein antibody complex was developed in 0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5mM MgCl2 containing 150 μg/ml of NBT, and 75 μg/ml BOP. The developing reaction was stopped by adding 10 mM EDTA, pH 8.0.
TRANSMISSION ELECTRON MICROSCOPY: Four-day-old etiolated seedlings of both wild type and porC over expressed Arabidopsis thaliana seedlings were taken and vacuum infiltrated with 2.5% glutaraldehyde solution for 30 minutes. After that the seedlings were kept for overnight in the same solution for fixation (Sabatini et al. 1963). The tissues were spinned down and 0.1 M sodium-phosphate buffer (pH 7.0) was added. After this tissue was kept in secondary fixative containing 1% OsO4. Fixation was done by immersing the tissue in fixative at 4° C. for 2-4 h. After this tissue was washed with 0.1 M PO4 buffer, dehydration was done, which involved following steps:
All these steps were done at room temperature. For clearing tissues of acetone, epoxy propane or xylene was used twice for 30 minutes at room temperature. After this infiltration was done in a resin containing araldite and toluene in following ratios:
For embedding araldite cy212 embedding medium was prepared. After embedding the liquid araldite was polymerized in a gradual process by keeping blocks at 50° C. for 12-24 h and then at 60° C. for 24-48 h. After this, sectioning was done using ultramicrotomes. These ultra thin sections were then stained in uranyl acetate. Saturated solution of uranyl acetate was prepared in 50% ethanol. This was mixed vigorously and centrifuged to allow the excess of uranyl acetate to settle down. For staining small amount of uranyl acetate was taken in a dean watch glass. Grid containing section was placed on to the stain. Staining was done in dark for 10-15 min. Each grid was then washed in 50% ethanol twice and distilled water twice with continuous agitation. This was then dried carefully on filter paper. After this sections were viewed in a Transmission Electron Microscope (Phillips CM-10).
Plant Growth Conditions: Both wild-type (WT) and transgenic sense plants of Arbidopsis thaliana plants were grown in growth chamber (Conviron, Canada) in a 14 h/10 h dark/light cycle. The light intensity was of 100 μmoles m−2 s−1 and the temperature set up was 22° C.±2° C.
Chlorophyll and Carotenoid Estimation: The extraction of Chlorophylls & Carotenoids from leaf tissues, from etiolated seedlings as well as from isolated chloroplasts were done under a dim, green safe light. Leaf tissues (also seedlings) were homogenized in 90% chilled ammonical acetone (10 ml) in a pre-chilled mortar and pestle. Three replicates were taken for each batch. Homogenate was centrifuged at 10,000 rpm for 10 min.
Supernatant was taken for estimating chl and carotenoid. Absorbance was taken at 663 nm, 645 nm and 470 nm. Reference cuvette contained 90% amonical acetone. Chl was calculated as described by Porra et al., 1989 and carotenoids were calculated as described by Welburn and Lichenthaler, 1984.
Chla=(14.21×OD 663−3.01×OD 645) V/W
Chlb=(25.23×OD 645−5.16×OD 663) V/W
Chl(a+b)=9.05×OD 663+22.2×OD 645) V/W
Carotenoids=(1000 ×OD 470−{3.27×Chla−1.04×Chlb}/5) V/227×W.
Extraction and Determination of Tetrapyrroles: Total chlorophyll pigments were isolated as described before by grinding the different tissues with 90% ammonical acetone. Fully esterfied tetrapyrroles were extracted by adding equal volume of hexane to it. While the mono-and dicarboxylic tetrapyrroles remained in the HEAR phase, the fully esterified tetrapyrroles were transferred to hexane phase. Quantitative estimation of Pchlide, Mg-Proto and Mpe were carried out spectrofluorometrically using the HEAR (Hukmani and Tripathy, 1992).
Light and ALA Treatment: The Arabidopsis seedlings grown for four days in complete dark were transferred to different light intensity of 10 μmoles m−2 s−1m 40 μmoles m−2 s−1, 250 μmoles m−2 s−1 and continuous far red (cFR) light (50 μmoles m−2 s−1). Light intensity was measured with a quantum sensor (LI-COR, USA). Both wild type and porC over expressed plants were grown in growth chamber at 22° C. under 100 μmoles m−2 s−1 of light intensity with 14 h/10 h dark/light cycle.
For ALA treatment, both wild type and transgenic plants were grown in pot having agropeat soil for five weeks. Each pot having 4 Arabidopsis plants was sprayed with 15 ml of aqueous ALA solution (3 mM, pH 4.8). The plants along with their pots were immediately wrapped in aluminum foil to exclude light and to prevent the rapid drying of the solution. The control seedlings/plants were sprayed with distilled water (pH 4.8). The pots were then kept in dark room for 14 h before exposing to light (70 μmoles m−2 s−1).
Net synthesis of Pchlide: To empty the endogenous Pchlide pool Arabidopsis plants grown under 100 μmoles m−2 s−1 and 14/8 h light dark photoperiod were exposed to cool-white fluorescent light (70 μmoles m−2 s−1) for 10 minutes and leaves were excised immediately after the light treatment, weighed and homogenized in 90% ammonical acetone. After light exposure, plants were sprayed with ALA and water, kept in dark and leaves were harvested after 3 hours. Three replicates of each sample were hand homogenized in 10 ml of ice cold 90% ammonical acetone at 4° C. and homogenate was centrifuged at 10,000 rpm for 10 min. Supernatant was taken for the HEAR preparation and accumulation of Pchlide was measured spectrofluorometrically (Tewari and Tripathy, 1998). The net synthesis of Pchlide was calculated by deducing the Pchlide value after 10 min of light exposure from those recorded after 3 h dark interval.
Protochlorophyllide Oxidoreductase Activity: Four weeks grown Arabidopsis plants (under 100 μmoles m−2 s−1 and 14/8 h light dark photoperiod) sprayed with ALA and distilled water (without ALA) were transferred to complete dark and incubated for 6 hours. After which leaves were harvested, homogenized in 90% ammonical acetone in dark. Subsequently, plants were exposed to cool-white fluorescent light (70 μmoles m−2 s−1) for 15 minutes and leaves were harvested, weighed and homogenized in 80% ammonical acetone in light. Acetone tissue homogenates were centrifuged at 10,000 rpm in SS-34 rotor for 10 min at 4° C. and from supernatant HEAR was prepared. Pchlide contents of Arabidopsis leaves before and after light exposure, were estimated from HEAR fraction by spectrofluoremetry. Three replicates were taken for each data points. The percent phototransformation was calculated as: [(Pchlide content before phototransformation−pchlide content after phototransformation)/Pchlide content before phototransformation]×100.
Anthocyanin Measurement: The anthocyanin estimation was earned out according to Feinbaum and Ausubel, 1985. Leaves were harvested from both wild and over expressed plants, cut into pieces, their fresh weight measured and anthocyanins extracted overnight in 3 ml 1% acidic methanol (1% HCL in methanol). Phase partitioning was done next day by adding 3 ml chloroform and 2 ml distilled water. Absorbance of aqueous phase was recorded at 530 and 657 nm. The total amount of anthocyanin (A530-A657) was estimated and normalized to per gram fresh weight.
Estimation of lipid peroxide: The level of lipid peroxidation products were estimated using 200 mg fresh tissue according to the method of Hodges et al., 1999 and were expressed as thiobarbituric add reactive substances (TBARS). Plant samples were extracted in 0.25% 2-thiobarbituric add (TBA) in 10% TCA using a mortar and pestle. Contents were heated at 95° C. for 30 minutes and the quickly cooled in an ice bath and centrifuged at 12,000 rpm for 10 min. The absorbance of the supernatant was read at 532 nm and correction for unspecific turbidity was done by subtracting the absorbance of the same at 600 nm. The blank was 0.25% TBA in 10% TCA. The concentration of lipid peroxides together with oxidatively modified proteins were quantified and expressed as total TBARS in terms of nmol g−1 fresh weight using an extinction coefficient of 155 nM−1 cm−1. TBARS are en index of lipid peroxidation.
To increase the accuracy of determining TBA-MDA levels by correcting for compounds other than MDA, which absorb at 532 nm, subtraction of the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA was done. A 1-ml aliquot of appropriately diluted sample was added to a test tube with 1 m of either (i) TBA solution comprised of 20.0% (w/v) trichloroacetic acid (−TBA) or (ii) TBA solution containing the above plus 0.65% TBA (+TBA). Samples were then mixed vigorously, heated at 95° C. in a water bath for 25 min, cooled, and centrifuged at 12,000 rpm for 10 min. Absorbances were read (Shimadzu, Japan) at 440 nm, 532 nm, and 600 nm. Malondialdehyde equivalents were calculated in the following manner: 1) [(Abs 532+TBA)−(Abs 600+TBA)−(Abs 532−TBA)−(ABS600−TBA)]=A; 2) [(Abs 440+TBA−Abs 600+TBA)0.0571]=B; 3) MDA equivalents (nmol.ml−1)=(A−B/157 000)106.
Electrolyte Leakage: Leaf discs (12 in number, 8 mm diameter) from both control and treated plants were taken by using a cork borer and rinsed briefly with distilled water. The discs were dried on filter paper and kept in petriplates containing 20 ml-deionised water. Petriplates were placed on to and fro shaker (80 rpm) for 4 h. After specific interval of time the extent of solute leakage (Initial Conductivity, IC) was monitored with a conductivity meter, which was calibrated against 0.01 N KCL solution. The readings were corrected for the ceil constant C, which was calculated according to the following formula.
where, R=1.412 μmohs cm−2 and t=27° C.
Samples collected at different time periods were then boiled for 10 minutes and allowed to reach the room temperature, while placed on shaker to facilitate total electrolyte leakage from the tissues. Volume was then made to 20 ml with deionised water and shaken for another 5 minutes. Total electrolyte leakage (Final Conductivity, FC) was then recorded. The percentage of electrolyte leakage was calculated by using the formula: (IC/FC)×100.
Reactive Oxygen Species (ROS) Staining: Detection of H2O2 was done by endogenous peroxidase-dependent insitu histochemical staining using, 3,3-diamino benzidine (DAB) (Thordal-Christensen et al., 1997) and detection of super oxide radicals (O2−) was done by imaging of purple formazon deposits (Flohe and Otting, 1984), which result from the reaction of nitrobiue tetrazolium (NBT) with super oxide. Both wild type and porC transgenic Arabidopsis leaves were cut from the petiole, submerged into weds containing 6-8 ml DAB [(1 mg/ml), DAB prepared in 10 mM MES, pH 6.5] and NBT [(1 mg/ml), NBT prepared in 50 mM potassium phosphate buffer, pH 6.4] solution. Then these were vacuum infiltrated for 20 minutes in dark. After infiltration leaves were briefly rinsed in distilled water twice and transferred into falcon tubes containing ethanol (96%), with holes in the cap. Falcon tubes were allowed to float into boiling water until chlorophyll was completely removed from leaves. Leaves were then transferred in fresh ethanol (96%) and kept for 2-3 days at room temperature.
Singlet O2 (1O2) Measurement: The production of 1O2 was determined in terms of RNO bleaching using a spectrophotometric method as described by Joshi and Pathak, 1984. RNO was used for spectrophotometric assay and histidine was used as a trap of 1O2. Chloroplast was isolated in complete dark. Chloroplasts (100 μg Chl ml−1) were incubated with RNO (300 μg ml−1) and histidine (10 mM) In one eppendorf tube in dark and another tube was illuminated in Hansatech oxygen evolution chamber for different time period during the assay. Samples were taken after various time intervals. After centrifugation (12000 rpm for 15 min), the supernatant was decanted and the absorbance was read at 440 nm in a spectrophotometer (Shimadzu, UV160). The dark samples were used as blank.
Fluorescence spectra of Chloroplasts: The room temperature and low temperature (77k) fluorescence emission spectra of chloroplasts were recorded in SLM-AMINCO 8000C spectroflurometer. The slit widths of excitation and emission were 4 nm. For room temperature emission spectra, chloroplasts equivalent to 5 μg Chl ml−1 were suspended in 25 mM hepes-NaoH buffer (pH 7.6). The 77k Chl a emission experiments were done with low temperature accessory and chloroplast were suspended in 25 mM hepes-NaoH buffer (pH 7.6) containing 50% glycerol at a concentration of 5 μg Chl ml−1. The spectra were corrected for photo multiplier sensitivity.
Shibata Shift: To study the Shibata shift the plastids were isolated from Arabidopsis plants grown in complete dark for 4 days. The plastid suspensions were given a saturating pulse of red light (1500 μmoles m−2 s−1, 0.1 Sec) and then incubated for different time periods up to 10 minutes in dark. Low temperature spectra were recorded before red light flash, immediately after red light flash, and after different dark incubation from 600-770 nm. All the operations were carried out in green, safe light.
Pulse Amplitude Modulation Measurement (PAM): F0, Fv/Fm, Electron transport rate (ETR), Photochemical quenching (qP), non-photochemical quenching (qN) and Photo system II quantum yield (ΦH) were measured at room temperature by a PAM-2100 Chl fluorometer (Walz, Germany). Either leave or whole plant was taken as the experimental material. During the time of experiment red actinic illumination (wavelength, 655 nm) provided by five LEDS (H3000) (Stanley, Calif., USA) focused onto the leaf surface (79 mm2). Two other H-3000 LEDS, that emit 650-nm pulses, were used as measuring light Leaf clip holder 2030-B, equipped with a microquantum sensor, monitored photosynthetically active radiation (PAR). An additional spacer was added to the clip in order to avoid pinching of the leaf. Chl fluorescence was detected by a photodiode (BYP 12; Siemens, Munich, Germany) that was shielded by a long-pass far-red filter (RG9; Schott, Southbidge, Mass., USA) and a heat filter. All the PAM-2100 fluorescence data were recorded in a time span of 5 min and 20 s. The rate of acquisition by the DA-2100 software in the preset program used (listed as run 3 in the PAM-2100 handbook) was 10 ms pulse−1. Run 3 measures Chl a fluorescence while calculating values of qN (a measure of NPQ [(Fm−Fm′)/(Fm-F0)]), qP (photochemical quenching of fluorescence [(Fm′−F)/Fm′−F0)]), and Y (the photochemical yield [(Fm′−F)/Fm′]. Recording of data including determination of F0 (minimal fluorescence yield of dark-adapted sample) and Fm (maximal fluorescence of dark adapted sample). (Govindjee and Paul Spilotro, 2002) began 15 s following the onset of run 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1843/DEL/06 | Aug 2006 | IN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IN07/00308 | 7/24/2007 | WO | 00 | 6/15/2009 |
