Wednesday, May 31, 2006


Estimation of magnitude of oxidative stress or lipid peroxidation or MDA, proline and assays of plant non-enzymatic and enzymatic antioxidants

Estimation of thiobarbituric acid reactive substances

Thiobarbituric acid reactive substances (TBARS), considered as oxidative damage products, may be determined in leaf samples by the method of (Heath and Packer 1968) modified slightly by our research group. Homogenize one gram of fresh tissue in 10 cm3 (ml) 0.1% TCA (trichloroacetic acid) and centrifuged at 7826 g 5 min. Add one cm3 of supernatant with 4.0 cm3 of 0.5% thiobarbituric acid (TBA), which was prepared in 20% TCA. The mixture was heated at 95 0C for 30 min, cooled and centrifuged at 1957 g for 5 min. Read the absorbance of the supernatant at 532 nm and corrected for unspecific turbidity after subtraction from the value obtained at 600 nm. Express TBARS in nmol g–1 fw, quantified using an extinction coefficient of 155/mM-1 cm-1.

Estimation of proline content

Proline content in leaf samples may be estimated by the method of Bates et al. (1973). Homogenize half gram of fresh leaf in 10 cm3 of 3% sulphosalicylic acid and centrifuged at 7826 g for 10 min. Add two cm3 of supernatant with 2 cm3 of acid ninhydrin reagent (1.25% ninhydrin, 30% glacial acetic acid and 20% 6 N o-phosphoric acid) and 2 cm3 of glacial acetic acid. Boil the resultant mixture at 100 0C in a water bath for 30 min, stop reaction in an ice bath and add 4 cm3 of toluene to each sample followed by vigorous cyclomixing for 10-15 s. Lift the toluene (upper) layer to read at 520 nm using UV-Vis Spectrophotometer. The corresponding concentration of proline should be determined against the standard curve of L-proline, express as μ g-1 fw.

Esimation of Asc and Dasc

Ascorbate (Asc), dehydroascorbate (DAsc) and total ascorbate (Asc + DAsc) should be estimated by a modified method of Law et al. (1983). Grind half gram of fresh leaf tissue in two cm3 of Na-phosphate buffer (0.1 M, pH 7, 1 mM EDTA) and centrifuge at 7826 g for 10 min. Distribute the supernatant in two separate sets of microcentrifuge tubes (400 μl in each) for the assay of total ascorbate (Asc + DAsc) and ascorbate (Asc) in first and second set, respectively. To each sample, add 200 μl of 10% TCA. After 5 min, add 10 μl of NaOH (5 M) solution, mix and centrifuge for 2 min in a microfuge. To 200 μl of the supernatant, add 200 μl of Na-phosphate buffer (150 mM, pH 7.4) and 200 μl of DDW. To another 200 μl of supernatant add 200 μl of Na-phosphate buffer (150 mM, pH 7.4) and 100 μl of 10 mM dithiothritol (DTT) and, after a thorough mixing, was leave at room temperature for 15 min. Add 100 μl of 0.5 % N-ethylmaleimide to each of the tubes and incubate at 24 0C for >30 s. Further, add 400 μl of 10% TCA, 400 μl of 44% H3PO4, 400 μl of 4% bipyridyl and 200 μl of 3% FeCl3. After being vortex-mixed, incubated the samples at 37 0C for 60 min record absorbance at 525 nm on a UV-Vis Spectrophotometer. A standard curve in the range of 0-100 nmol of Asc should be used for calibration. Compute dehydroascorbate (DAsc) concentrations from the difference (total ascorbate – ascorbate). Estimated the amounts in nmol g–1 fw.

Estimation of GSH and GSSG

Reduced (GSH), oxidised (GSSG) and total glutathione (GSH + GSSG) may be determined by the glutathione recycling method of Anderson (1985). Homogenize half gram of fresh leaf in 2 cm3 of 5% sulphosalicylic acid at 4 0C. Centrifuge the homogenate at 7826 g for 10 min. To a 0.5 cm3 of supernatant, add 600 µl of reaction buffer (0.1 M Na-phosphate, pH 7, 1 mM EDTA) and 40 μl of 0.15% 5,5-Dithiolbis 2-nitrobenzoic acid (DTNB), and read at 412 nm after 2 min. To the same, add 50 μl of 0.4% NADPH and 2 μl of GR (glutathione reductase; 0.5 enzyme unit) and let the reaction run for 30 min at 25 0C. Read the samples again at 412 nm to determine the total glutathione. Actual values of glutathione content may be determined against GSH standard curve (10-100 nmol). By subtracting GSH from total glutathione (GSH+GSSG), estimate GSSG. The amounts of GSH, GSSH and (GSH + GSSG) should be expressed in nmol g-1 fw.

Enzymatic assays:

Superoxide dismutase (SOD)

Follow the method of Dhindsa et al. (1981) with our slight modification for estimating SOD activity. Homogenized fresh leaf material (0.2 g) in 2.0 cm3 of extraction mixture containing 0.5 M Na-phosphate buffer, pH 7.3, 3 mM EDTA, 1% PVP, 1% Triton X 100 and centrifuge at 11269 g at 4 0C. Assay SOD activity in the supernatant by its ability to inhibit photochemical reduction of nitroblue tetrazolium (NBT). The assay mixture should consist of 1.5 cm3 reaction buffer (0.1 M Na-phosphate buffer, pH 7.5, 1% PVP), 0.2 cm3 of L-methionine (200 mM), 0.1 cm3 enzyme extract with equal amount of 1M NaHCO3, 2.25 mM NBT solution, 3 mM EDTA, 60 µM riboflavin and 1.0 cm3 of DDW in test tubes to incubated under illumination of 15W inflorescent-light lamp for 10 min at 28 0C. Blank A, containing all substances of the reaction mixture, along with enzyme extract, must be placed in the dark. Blank B, containing all substances of reaction mixture without enzyme must be placed in light along with the sample. Terminate the reaction after 10 min by switching off light and cover the tubes with the black cloth. The non-irradiated reaction mixture containing the enzyme extract will not develop bluish-gray color. Read absorbance of samples along with blank B at 560 nm against blank A. The difference of % reduction in colour between blank B and the sample should be calculated. 50% reduction in colour is considered as one unit of enzyme activity expressed in enzyme unit (EU) mg-1 protein h-1.

SOD activity (EU mg-1 protein h-1) = % Reduction in color between blank and sample x Dilution factor x 60 / 50 x Incubation time x mg protein in sample*

*For the estimation of protein content in the sample, use Bradford reagent and quantify the amount of protein against the standard curve of BSA (Bovine Serum Albumin)

Ascorbate Peroxidase (APX)

Ascorbate peroxidase (APX) activity may be estimated by the method of Nakano and Asada (1981). Grind one gram of the fresh leaf material in 5 cm3 of extraction buffer (0.1 M K-phosphate, pH 7, 3 mM EDTA, 1% PVP, 1% Triton X 100), centrifuge at 7826 g for 10 min at 4 0C. Determine APX activity in supernatant by the decrease in absorbance of ascorbate at 290 nm, due to its enzymatic breakdown. One cm3 of reaction buffer should contain 0.5 mM ascorbate, 0.1 mM H2O2, 0.1 mM EDTA and 50 µl extract containing enzyme. Let the reaction run for 5 min at 25 0C. APX activity may be calculated by using extinction coefficient 2.8 mM–1 cm–1 and expressed in enzyme units (EU) mg-1 protein. One unit of enzyme determines the amount necessary to decompose 1 μmol of ascorbate consumed per min at 25 0C.

Glutathione reductase (GR)

Glutathione reductase (GR) activity may be determined by the method of Foyer and Halliwell (1976) modified by Rao (1992). Grind half gram fresh leaf material in 2 cm3 of extraction buffer (0.1 M Na-phosphate, pH 7.0, 3 mM EDTA, 1% PVP, 1% Triton X 100) and centrifuge at 7826 g for 10 min. The supernatant must be immediately assayed for GR activity through glutathione-dependent oxidation of NADPH at 340 nm. One cm3 reaction mixture containing 0.2 mM NADPH, 0.5 mM GSSG and 50 μl of enzyme extract is kept for 5 min at 25 0C. Corrections should be made for any GSSG oxidation in the absence of NADPH. The activity may be calculated by using extinction coefficient 6.2 mM–1 cm–1 and expressed in enzyme units (EU) mg-1 protein. One unit of enzyme determines its amount necessary to decompose 1 μmol of NADPH per min at 25 0C.

Catalase (CAT)

Catalase (CAT) activity may be determined by the method of Aebi (1984). Half gram of the fresh leaf material, ground in 5 cm3 of extraction buffer (0.5 M Na-phosphate, pH 7.3, 3 mM EDTA, 1% PVP, 1% Triton X 100) is centrifuged at 7826 g for 20 min at 4 0 C. Assay the catalase activity in supernatant by monitoring the disappearance of H2O2, measuring a decrease in absorbance at 240 nm. Reaction should be run in a final volume of 2 cm3 of reaction buffer (0.5 M Na-phosphate, pH 7.3) containing 0.1 cm3 3 mM EDTA, 0.1 cm3 of enzyme extract and 0.1 cm3 of 3 mM H2O2 for 5 min. CAT activity should be calculated using an extinction coefficient (ζ = 0.036 mM –1 cm-1) and is expressed in enzyme units (EU) mg-1 protein. One unit of enzyme determines the amount necessary to decompose 1 μmol of H2O2 per min at 25 0C.

Tuesday, May 30, 2006

Advance perfect clap switch

Here is a clap switch free from false triggering. To turn on/off any ap pliance, you just have to clap twice. The cir-cuit changes its output state only when you clap twice within the set time period. Here, you’ve to clap within 3 seconds. The clap sound sensed by condenser microphone is amplified by transistor T1. The amplified signal provides negative pulse to pin 2 of IC1 and IC2, triggering both the ICs. IC1, commonly used as a timer, is wired here as a monostable multivibrator. Trigging of IC1 causes pin 3 to go high and it remains high for a certain time period depending on the selected values of R7 and C3. This ‘on’ time (T) of IC1 can be calculated using the following relationship: T=1.1R7.C3 seconds where R7 is in ohms and C3 in microfarads. On first clap, output pin 3 of IC1 goes high and remains in this standby position for the preset time. Also, LED1 glows for this period. The output of IC1 provides supply voltage to IC2 at its pins 8 and 4. Now IC2 is ready to receive the triggering signal. Resistor R10 and capacitor C7 connected to pin 4 of IC2 prevent false triggering when IC1 provides the supply voltage to IC2 at first clap. On second clap, a negative pulse triggers IC2 and its output pin 3 goes high for a time period depending on R9 and C5. This provides a positive pulse at clock pin 14 of decade counter IC 4017 (IC3). Decade counter IC3 is wired here as a bistable. Each pulse applied at clock pin 14 changes the output state at pin 2 (Q1) of IC3 because Q2 is connected to reset pin 15. The high output at pin 2 drives transis-tor T2 and also energises relay RL1. LED2 indicates activation of relay RL1 and on/off status of the appliance. A free-wheeling diode (D1) prevents damage of T2 when relay de-energises. This circuit costs around Rs 100 *Indian rupees.

Saturday, May 27, 2006

Great fun at Rome/Roma

Tour of Rome/Roma

Rome/Roma etc.

Artemisia annua and Proteomics


Abiotic stresses such as temperature, drought and salinity puts the major hurdles in yield and production of the crops. As a matter of fact, A. annua L. has to face a definite phase of a season of very low temperature (below 10 0C) in the month of December/January mainly in northern and associated parts of India. Further, much (32-84%) of groundwater surveyed in different Indian states is rated either saline or alkali and found to substantially decrease the yield of crops including Artemisia annua L. (Qureshi et al., 2005), affecting plant growth and production of artemisinin. Crop yields start declining when pH of the soil solution exceeds 8.5 or ECe value goes above 4 dS m-1. At higher ECe values the crop yields are reduced so drastically that crop cultivation is not economical without soil amendments. Addition of salts to water lowers its osmotic potential, resulting in decreased availability of water to root cells. Salt stress thus exposes the plant to secondary osmotic stress, which implies that all the physiological responses, which are invoked by drought stress, can also be observed in salt stress. Fogle and Munns (1973) reported that osmotic restriction of root growth decreased the ability of wheat seedling to obtain nutrients from root medium. In addition to its injurious osmotic effects, a salt may injure plant by way of specific toxic effect. Salt stress causes inhibition of growth and development, reduction in photosynthesis, respiration, and protein synthesis and disturbs nucleic acid metabolism (Kaiser, 1987). Decrease in uptake of K+, Mg2+, Ca2+, and thereby decrease in growth at higher sodium concentration have also been reported.

Thus, the productivity and therapeutic properties of medicinal plants is subjected to the environmental and nutrient factors and hence, the ratio of demand and supply too.

Plants exposed to abiotic stress invariably showed marked alteration in the electron transport in both chloroplast and mitochondria. This results in the production of toxic oxygen species such as 1O2, OH and H2O2 (Foyer et al., 1997). Due to the multiplicity of the processes that produce excessive reactive oxygen species (ROS) during metabolism, including photosynthesis and photorespiration (Nocter and Foyer, 1998), fatty acid oxidation, response to pathogen attack and senescence; plants have developed a series of detoxification reaction mechanisms (Vitoria et al., 2001).

The impressive achievements in genome and expressed sequence tag (EST) sequencing have yielded a wealth of information for many model organisms, including the plants Arabidopsis thaliana and Medicago truncatula. Unfortunately, sequence information alone is insufficient to answer questions concerning gene function, developmental/regulatory biology, and the biochemical kinetics of life. To address these questions, more comprehensive approaches that include quantitative and qualitative analyses of gene expression products are necessary at the transcriptome, proteome, and metabolome levels. Transcriptome, approaches using microarray and serial analysis of gene expression technologies are powerful tools; however, mRNA and protein levels (Futcher et al., 1999; Gygi et al., 1999) cannot be correlated due inability of translation of total mRNA into protein. In contrast, proteomics provides a more direct assessment of biochemical processes of monitoring the actual proteins performing the enzymatic, regulatory, structural functions encoded by the genome and transcriptome. Recent improvement in high resolution two dimensional PAGE (2-DE; Klose and Kobalz, 1995; Gorge et al., 1999), increased content of protein and nucleotide databases, and increased capabilities for protein identification utilizing modern mass spectrometry methods such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS; Pappin et al., 1993; Yates, 1998a, 1998b; Corthals et al., 2000) have made the large-scale profiling and identification of proteins a dynamic new area of research in plant biology.

Although there is a substantial amount of work in literature on bacterial (Guerreiro et al., 1999; Morris and Djordevic, 2001), there is relatively less information on plant proteomes (Wan Wijk, 2001). Costa and co-workers have identified proteins from xylem and needles of maritime pine (Pinus pinaster; Costa et al., 1998, 1999), and Tsugita and co-workers have worked on the rice (Oriza sativa) proteome with some success (Tsugita et al., 1994). Both of these groups have relied heavily on Edman sequencing, which suffers due to the inability to sequence proteins blocked at the N terminus. More recently, researchers have reported on subcellular proteomes such as the chloroplast membrane (Peltier et al., 2000; 2002) whereas other have focused on single tissues including Arabidopsis seeds (Gallardo, et al., 2001), Arabidopsis mitochondria (Kruft et al., 2001; Millar et al., 2001), maize (Zea mays) root tips (Cheng et al., 2000), and barrel medic roots (Mathesius et al., 2001, 2002). To date, there has been no large-scale project to identify proteins from multiple tissues of the same plant species.

Being an important medicinal plant, Artemisia annua L. has been used as an experimental material in these studies.


Taking into consideration the above facts, this project has been planned with the following objectives:

*To grow cultures of Artemisia annua L. under in vitro conditions.

*To study the enzymatic and non-enzymatic antioxidant in Artemisia annua L. under salinity and heavy metal stress.

*To analyze the magnitude of expression of different proteins in shoot and their possible role in imparting the abiotic stress tolerance to Artemisia annua L.

*To sequence the amino acids of the stress-induced proteins.

Identify the stress-inducible genes under different abiotic stresses imparting tolerance against abiotic stresses.


Plant material and treatments

In vitro raised Artemisia annua L. plants will be exposed to NaCl and heavy metal with MS media in separate sets. Moderate salinity will be created by the application of NaCl in the nutrient media; drought by removal of the nutrient media for a standard set time; chilling and heat stress will be given by reducing and increasing the temperature of the growth chamber, respectively.

Protein extraction

Total protein of the tissue will be extracted according to the method of Tsugata et al., 1994. In brief, plant tissues will be homogenized in liquid N2 and proteins precipitated at –20 0C with 10% (w/v) TCA in acetone containing 0.07%(w/v) 2-mercaptoethanol for at least 45 min. The mixture will be centrifuged at 35,000g at 4 0Cfor 15 min, and the precipitates will be washed with acetone containing 0.07% (w/v) 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA. Pellets will be dried by vacuum centrifugation and solubilized in 8 M urea, 4% (w/v) CHAPS, 20 mM DTT, 0.1% (v/v) Biolytes (pH 3-10; Bio-Rad laboratories, Hercules, CA; Molloy et al., 1998).

Protein quantification and electrophoresis

Protein concentration of all tissue extracts will be quantified using the Bradford method (Bradford, 1976) with bovine serum albumin as a standard isoelectric focusing and acrylamide gel electrophoresis of will be done according to the method of Asirvatham et al., 2002. Gels will be stained over-night with Coomassie Brilliant Blue R-250 and destained the next day. Gel images will be digitized with UVI-tech gel documentation system equipped with image camera. Experimental molecular mass and Rf/pI will be calculated from digitized 2-DE images using standard molecular mass marker proteins.

Digestion and MALDI-TOFMS

Protein spots will be excised from the gel, washed twice with water for 15 min, and destained with a 1:1 (v/v) solution of acetonitrile and 50mM ammonium bicarbonate while changing solutions every 30 min until the blue colour of Coomassie was removed. SDS-PAGE/2-DE gel bands/spots will be then dehydrated by washing with 100% acetonitrile and dried by vacuum centrifugation. Further processing for sequencing of amino acids of the novel proteins will be done according the method of Watson et al., 2003.

Database queries and protein identifications

The peptide mass fingerprints will be compared with sequences in different databases such as NCBInr, SwissPort and/or dbESTothers, and queried using MS-Fit (