List of top thirteen experiments on photosynthesis in plants:- 1. Simple Demonstration 2. Demonstration of Hill Reaction 3. Demonstration of Essentiality of Different Environmental Factors for Photosynthetic Process 4. Effect of CO2 Concentration on Photosynthesis 5. Study of the Effect of Monochromatic Light on Photosynthesis and a few others.
Experiment # 1. Simple Demonstration:
Photosynthesis is a combination of biophysical and biochemical processes during which solar energy is captured and converted into chemical energy which is contained in the molecules of organic compounds. The central role of this process is in the energy cycle of life. Chemically, this process involves the uptake of CO2 which, in turn, gets converted into organic compounds, and oxygen is evolved.
The process can be measured usually by measuring the volume of CO2 consumed, or influence of CO2 conc. on the rate of photosynthesis, or volume of O2 evolved, or total amount of dry mass/grain formed. The whole process is principally dependent on light, O2, temperature and water supply from the surrounding environment.
(a) By Simple Glass Apparatus (Fig. 3.20):
Materials and Equipments Required:
1. Beaker (1 lit), graduated test tube, funnel fitted with jet etc.
2. 0.1% KHCO3 soln., dist. water
3. Hydrilla plants
4. Thread, blade, stand with clamp, etc.
1. Fill the beaker with distilled water up to 2/3 mark.
2. Take some fresh and healthy Hydrilla plants and cut their ends and tie them loosely with a thread.
3. Insert the cut ends inside the neck of the funnel with a jet.
4. Place the funnel inside the beaker in such a manner that all plants remain inside the funnel.
5. Add a few ml of 0.1% KHCO3 soln. for dissolved CO2 source.
6. Invert a graduated test tube filled with water over the neck of the funnel so that the jet of the funnel remains inside the tube in vertical position.
7. Place the whole set-up under bright light and keep the graduated tube erect with stand and clamp, if necessary.
8. Allow the experiment to continue for about 20 minutes. Then record the evolution of air bubble (O2 gas) inside the tube after passing through jet for 5 or 10 mins.
It is observed that evolution of bubbles from the cut ends of the plants takes place in the set-up exposed to light.
Rate of photosynthesis = No. of bubbles formed per min per fresh plant.
(b) By Wilmott’s Bubbler (Fig. 3.21):
Materials and Equipments Required:
1. Wilmott’s bubbler:
The apparatus consists of a flask of 500 ml capacity fitted with a rubber cork having a central hole through which passes a glass tube. The lower end of the tube reaches the middle of the flask while its upper end forms a jet within a cylindrical cup. A graduated tube having a stopcock at one end remains inverted over the jet.
2. Hydrilla plants
3. 0.1% KHCO3 soln., distilled water
1. Take some fresh Hydrilla plants and insert their cut ends inside the lower end of the glass tube.
2. Fill the flask with distilled water and add a few ml of 0.1% KHCO3 soln.
3. Fill the cylindrical cup of the glass tube with dist. water and insert a graduated water-filled glass tube over the jet.
4. Clamp the whole set with a stand and place under bright sunlight.
5. Record the release of air bubbles produced by the green plants, passing through the jet per 5 min, when the evolution of bubbles become steady.
Regular steady release of bubbles takes place through the jet fitted with glass tube.
Experiment # 2. Demonstration of Hill Reaction:
Photosynthesis is the complex physico-chemical process by which solar radiation is converted into chemical energy in the form of carbohydrate.
The basic reaction is:
In photosynthesis there are two major phases: Light reaction phase in which light energy is absorbed by chlorophyll and produces ATP and reductant and chemical or dark reaction phase in which CO2 is converted into Carbohydrate. The reducing properties of isolated chloroplast was first reported by Robin Hill (1937), and this phenomenon is termed as Hill Reaction.
There are two major ways for determination of Hill Reaction in isolated chloroplasts:
(a) Reduction of the dye, dichlorophenol indophenol, by chloroplasts measured with colorimeter or spectrophotometer.
(b) Oxygen evolution in the presence of potassium ferricyanide as the electron acceptor measured in Warburg manometer or oxygen electrode.
The dye reduction methods are most conventionally used in the laboratory.
The dye 2-6 DCPIP becomes colourless when reduced as a result of the following oxidation-reduction reaction:
Materials and Equipments Required:
(a) Instruments — Spectrophotometer, Centrifuge
(b) Chemicals: 0.5 M Sucrose, 2, 6 DCPIP, Soln. 0.1 M phosphate buffer (pH 6.5), etc.
(c) Glass wares: Test tubes, beakers etc.
(d) Plant materials: Freshly collected leaves of spinach or cucurbits
(a) Preparation of chloroplasts:
About 50 gms of leaves are homogenized in presence of 20 ml of phosphate buffer and then suspended in 30 ml of 0.5 M sucrose solution. The suspension is then filtered and finally centrifuged (10 min at 10,000 g) to remove the supernatant fluid.
The precipitated substance was re-suspended in sucrose solution in presence of phosphate buffer and centrifuged once again to get washed samples of chloroplast and, finally, re-suspended in 30 ml of sucrose solution and kept in darkness in ice-bag.
(b) Determination of dye reduction by isolated chloroplasts:
1. Take 9 ml phosphate buffer, 1 ml of chloroplast suspension and 1 ml dye soln. in a tube. Then record the initial O. D. value by Spectrophotometer. A replica of three separate sets were done.
2. Prepare a control set without chloroplast suspension or with denatured suspension (prepared by keeping the suspension in boiling water for 5 mins.), and record the initial O.D. value.
3. Keep all the experimental sets in light for about an hour and then record the O.D. values by Spectrophotometer separately.
4. The amount of dye reduced can be determined by standard curve prepared (by using known concentration of dye solns.).
The experimental sets with chloroplast suspensions showed reduction of dye due to Hill reaction.
Experiment # 3. Demonstration of Essentiality of Different Environmental Factors for Photosynthetic Process:
(a) Essentiality of Light by using Ganong’s Light Screen:
Light is one of the major requirements of photosynthetic process. Actually in photosynthesis light energy is initially trapped by the green plants using photosynthetic pigments and then photosynthetic electron flow starts.
Finally energy and reductant thus formed from electron flow path (Light reaction) is used for chemical reaction i.e. CO2 to carbohydrate conversion. Thus, without light, photosynthesis cannot take place.
(i) Ganong’s light screen apparatus
(ii) Potted plants (Dicot)
In all experiments on photosynthesis, where iodine test is performed to detect the presence of starch in leaves, the plant taken must be a dicot, wherein assimilatory starch is formed. This does not occur in monocot leaves.
(iii) 95% Alcohol, 1% Iodine soln.
(i) Keep a potted plant in darkness for 2 days to remove the accumulated starch of the leaves.
(ii) Cover some leaves with Ganong’s light screens (or cover some portion of leaf by black paper) and then place the plant in sunlight for several hours (Fig. 3.22).
(iii) Then remove the leaves to which light screens were fixed and bleach them by using 95% boiling alcohol. A few drops of lactic acid may be added for better bleaching.
(iv) Treat the bleached leaves with 1% Iodine solution for 1-2 minutes.
The exposed parts of the leaves turn blue whereas the covered portions (where light could not penetrate) remain yellowish-red.
The blue colour indicates the formation of starch — a product of photosynthesis. Yellowish portion shows absence of starch. Thus the experiment proves the necessity of light in photosynthesis.
(b) Essentiality of CO2 — by Moll’s Experiment:
In photosynthesis, during dark or chemical reaction, CO2 is reduced to carbohydrate by a series of enzymatic reactions. In CO2 free environment, thus, photosynthesis is not possible.
(i) A wide-mouthed bottle fitted with a split cork
(ii) A potted plant
(iii) 20% KOH solution, 95% Ethyl alcohol, 1% Iodine solution etc.
(iv) Stand with clamp
(i) Take a wide-mouthed bottle fitted with a split cork and then pour a small quantity of KOH solution (20%) in the bottle and finally clamp it horizontally on a stand.
(ii) Take a suitable starch-free potted plant (the plant is kept in darkness for 2 days), and place one of its leaves inside the bottle through the split cork in such a way that half of the leaf remains inside the bottle and the other half outside it (Care should be taken that the portion of the leaf inside the bottle does not come in contact with KOH solution.) (Fig. 3.23).
(iii) Make all connections air-tight with vase-line and place the whole set-up in sunlight for 3-4 hours.
(iv) Then carefully remove the leaf from the bottle and bleach it by alcohol treatment.
(v) Treat the bleached leaf with 1% Iodine solution.
The portion of the leaf remaining outside the bottle is coloured blue with iodine while the portion remaining inside the bottle turns yellowish-red.
The portion of the leaf remaining outside the bottle receives all essential components of photosynthesis (CO2, light, chlorophyll, H2O) and so starch-formation takes place normally which is evident from the blue colour following iodine treatment.
The other half of the leaf which remains inside the bottle and where CO2 is not available due to absorption by KOH solution, remains yellowish-red showing no starch formation. This proves the fact that photosynthesis cannot occur in a CO2 free environment.
Experiment # 4. Effect of CO2 Concentration on Photosynthesis:
The concentration of CO2 of the atmosphere is one of the major determinants of photosynthesis. The photosynthetic efficiency of plants increase in proportionate manner with the rise of CO2 concentration up to a certain limit at a particular light intensity and temperature.
Materials and Equipments Required:
1. Beaker (1 lit capacity), graduated tube, funnel fitted with jet
2. KHCO3 salt
3. Distilled water
4. Hydrilla plants
5. Thread, blade, stand with clamp etc.
1. Set up an experiment using distilled water and Hydrilla plants in the beaker and a funnel fitted with jet-glass tube.
2. Place the experimental set-up in sunlight for 30 min and record the volume of oxygen collected, if any, in the graduated tube. If graduated tube is not available fit a nozzle at the tip of the funnel and count the no. of bubbles evolved per minute.
3. Put 200 mg of KHCO3 salt into the water of the beaker, stir and wait for 15 to 20 minutes and then record the volume of oxygen collected in the graduated tube.
4. In the same manner, at an interval of 30 minutes, add another 200 mg of KHCO3 salt to the beaker and record the photosynthetic rate in terms of the volume of oxygen generated up to a considerable length of time and concentration of KHCO3 salt.
5. Graphically plot the data: volume of O2 evolved against the concentration of KHCO3.
The photosynthetic rate increases with increase in CO2 concentration (= KHCO3 concentration) up to a certain extent at a particular light intensity and temperature. Even at high CO2 concentration, initially there is saturation effect followed by gradual decrease of photosynthesis.
Initially the rise of photosynthetic rate due to CO2 is maintained for a considerable length of time at a particular light intensity and temperature. Then saturation of photosynthetic rate is obtained.
Finally, at high CO2 concentration, photosynthetic rate decreases due to:
(i) Increased acidity of the mesophyll cells,
(ii) A narcotic effect on the metabolic function of the cells,
(iii) Closing of stomata, and
(iv) Limitation of light for consumption of available CO2.
Experiment # 5. Study of the Effect of Monochromatic Light on Photosynthesis:
The quality, intensity and duration of exposure to light are some of the principal limiting factors of photosynthetic process in green plants. A very small fraction of light wavelength can be trapped by the green plants and utilized in photosynthesis. Further, the efficiency of photosynthesis (as measured by O2 evolution) by monochromatic light (as a function of the wavelength of light) is termed ‘action spectrum’.
Thus the action spectrum varies widely with reference to the variation of monochromatic light. However, the photosynthetic efficiency decreases very dramatically with increasing wavelength beyond 685 nm even though chlorophyll still absorbs light at these wavelengths. This fact is the so-called red drop in photosynthesis.
Materials and Equipments:
1. Beakers (1 lit capacity), funnel fitted with jets, graduated glass tubes, glass rods
2. Hydrilla plants
3. Dist. water
4. Illuminated chambers (Blue, Green, Yellow, Red)
5. Thread, blade, graph paper, pencil, stop-watch, etc.
1. Prepare 4 photosynthetic experimental sets using Hydrilla plants in beakers in the usual manner. Add traces of KHCO3 salt in each set.
2. Place the experimental sets in separate illuminated chambers (Blue, Green, Yellow, Red) for artificial exposure to light for an hour.
3. Record the evolution of O2 as a result of photosynthesis separately in each experimental set and graphically plot the data against the respective light wavelengths for comparative analysis of the effect of monochromatic light.
For individual wavelength of light, the photosynthesis rate varies. The higher rate is seen in red light zone (600-660 nm).
The relative efficiency of light absorption and its subsequent utilization in photosynthesis depends on a number of factors: available photosynthetic pigment system, efficiency of PS I & PS II, photophosphorylation; and also efficiency of CO2 fixation.
Experiment # 6. Effect of Temperature on Photosynthesis:
As in all life processes, photosynthesis is restricted to a temperature range that roughly corresponds to that tolerated by protein compounds, which are generally active at temperatures above 0°C and below 60°C.
Although the photochemical part of photosynthesis is independent of temperature, the biochemical part — which is controlled by enzyme activity — is strictly temperature-dependent. However, there appears to be wide variance and adaptability among plants in their ability to tolerate temperature extremes.
Materials and Equipments Required:
1. Beakers (1 lit capacity), funnels fitted with jets, graduated test tubes, glass rods
2. Distilled water, 1% KHCO3 soln.
3. Hydrilla plants
4. Blade, thread, graph paper, thermometer, stop-watch
5. Ice water, hot water, etc.
1. Prepare three separate photosynthetic experimental sets (almost identical to each other using the same amount of plant materials) in the usual manner.
2. Place the experimental sets in three different water-baths maintained at temperature — 18 ±, 28±, 38±2 °C. For maintenance of water temperature of experimental sets ice water or hot water can be used.
3. Illuminated by artificial light for 1 hour to induce photosystem in the experimental sets and record the photosynthetic rate in terms of the volume of 02 evolved.
4. Graphically plot the 02 evolution (mm/1 gm. of plant tissue/1 hr) against each treatment temperature.
The rise of photosynthetic rate is noticed with the rise of temperature. Q10 may be calculated from the rise of the rate of photosynthesis at every 10°C rise of temperature from the graph.
The rate of photosynthesis is retarded — both directly and indirectly — by cold or high temperature, either through the inhibition of enzyme activity or other associated physiological processes.
Experiment # 7. Determination of Interaction of CO2 Concentration, Light Intensity and Temperature on the Rate of Photosynthesis:
Photosynthesis, like any other physico-chemical process, is affected by the conditions of the environment in which it occurs. There are three major factors, viz. CO2, light and temperature, that affect highly the process of photosynthesis. Blackman proposed the term ‘Principle of limiting factors’ to these interacting phenomena.
Materials and Equipments Required:
1. Audus micro-burette (Fig. 3.24A), beaker
2. Hydrilla Sp
3. NaHCO3, water
4. Light source, heater, light meter
5. Balance, weight box, stand etc.
1. Arrange the apparatus (Audus micro-burette) in vertical position after insertion of Hydrilla plants and change the tubes containing different concentrations of NaHCO3 under different light intensities and under different temperature conditions.
2. Within a short time O2 will accumulate at the upper poles of the tubes fitted with capillary tubes.
3. Release the water from the main apparatus with the help of the stop-cock drop by drop to facilitate the sucking of air in the capillary tubes.
4. Measure the length of the air bubble in the tubes after 30 minutes from the onset of experiment.
5. Repeat the experiment thrice under different conditions.
6. Express the results in tabular form and also graphically.
Tabulate the data relating the interaction of CO2, temperature and light on the rate of photosynthesis.
[Here C1 C2 C3 — Different CO2 concentrations; L1, L2, L3 — Different light intensities; T1, T2, T3 — Different temp, conditions.]
Experiment # 8. Relationship between Action Spectrum and Absorption Spectrum in Photosynthesis:
The efficiency of photosynthesis with monochromatic light as a function of the wave length of light is known as the action spectrum of photosynthesis.
On the contrary, the absorption of light by the pigments is termed as absorption spectrum, which varies from pigment to pigment. For photochemical reactions involving a single pigment, the action spectrum has the same general shape as the absorption spectrum of the pigment; otherwise both are quite distinct.
Materials and Equipments Required:
1. Beakers (1 lit. capacity), funnels fitted with jet, graduated glass tubes, measuring cylinder, test tubes, filter papers etc.
2. 1 M KHCO3 soln., distilled water, 80% acetone
3. Hydrilla Plants
4. Artificially illuminated chambers (Red, Yellow, Green, Blue)
(A) Determination of Action Spectrum:
1. Prepare photosynthesis experiment sets in the usual manner, using 2 gms of healthy Hydrilla plants and 2 ml of 1 M KHCO3 soln.
2. Place the experimental sets separately in artificially illuminated chambers for an hour.
3. Record this volume of O2 evolved from each experimental set and calculate the rate of photosynthesis (O2 vol./hr/1 gm. of plant material).
(B) Determination of Absorption Spectrum:
1. Extract the chlorophyll from 2 gms of Hydrilla plants using 80% acetone.
2. Dilute the extract 10 times with 80% acetone and record the absorbance by colorimeter using Red, Yellow, Blue and Green filters.
Both the results are plotted on graph paper and the relationship between absorption spectrum and action spectrum determined.
There are two major peaks, one at blue-violet region and the other at orange-red region, whore the action spectrum and absorption spectrum have positive correlation.
Experiment # 9. Determination of the Rate of Photosynthesis in Terrestrial Plant:
In most of the photosynthetic experiments, the rate of photosynthesis is determined either by measuring the volume of OI2 evolved per unit time or mg or ml of O2 evolved as estimated by titration. But there are some simple photosynthometers by which one can easily determine the amount of CO2 absorbed at a given time during photosynthesis by green plants.
Materials and Equipments Required:
1. Ganong’s photosynthometer:
The apparatus essentially consists of three parts:
(a) A glass bulb fitted on a wooden base,
(b) A graduated tube with a stopcock and
(c) A connecting link (rubber tubing) fitted with a stopcock which connects the graduated tube with the glass tube. The volume of the whole set when fitted is 103 ml (Fig. 3.24B).
2. A potted plant
3. 30% KOH solution
4. Measuring cylinder
1. Place about 3 ml (measured by displacement of water) of suitable fresh green leaf in the glass bulb. The volume of the apparatus now becomes 100 ml.
2. Invert the graduated tube and close the stopcock (b) and fill the graduated tube with water up to a desired mark.
3. Fit a connecting link (rubber tubing) with the graduated tube and now close the stopcock (c) of connecting link. Fill the hollow end of the connecting link with water and, finally, invert into a suitable measuring cylinder containing water closing the hollow end with the palm.
4. Clamp the set, keeping the level of water in the measuring cylinder up to the hole of the connecting link (h).
5. Connect the top of the graduated tube to a CO2, generator or Kipp’s apparatus and open both the stopcocks of the graduated tube and the connecting link for diffusion of CO2 into it.
6. Allow CO2 to flow down the water in the graduated tube and fill the vacuum space by CO2. Close the stopcock of the graduated tube when the level of water in the graduated tube falls to the level of water outside. The tube now contains the desired amount of CO2.
7. Fill the whole set at the top of the glass bulb (a) so that the hole of the connecting link coincides with that of the glass tube. This ensures that the pressure inside the bulb and the hollow end of the connecting link is at atmospheric pressure.
8. Then slightly twist the connecting tube to cut-off the connection with the atmosphere. Make all connections air-tight thereafter.
9. Now open the stopcock of the connecting link to allow CO2 to diffuse into the bulb.
10. Place the whole apparatus in bright sunlight for about three hours.
11. Close the stopcock (e) of the connecting link after recording the time.
12. Remove the graduated tube from the bulb and put it in a basin of water and also remove the connecting link under water.
13. Fix the graduated tube with the zero mark exactly on the surface of the water and open the stopcock of the graduated tube to allow water to rise inside the tube up to the zero mark.
14. Fill one test tube completely with 30% KOH solution and fit it to the top of the graduated tube with the help of a rubber tubing which is fitted with a pinch-cock.
15. Remove the tube from the water and open the pinch-cock and allow KOH solution to flow into the graduated tube.
16. Drain back KOH solution into a test tube after shaking thoroughly, and close the pinch-cock.
17. Place the end of the graduated tube underwater with its zero mark at the surface level and disconnect the test tube after closing the stopcock of the graduated tube. The water now runs up to a mark corresponding to the volume of CO2 absorbed by KOH which equals the volume of CO2 left unutilized by the photosynthetic experimental material after the desired time.
18. Now fill the test tube with alkaline pyrogallate solution; and then repeat the procedure to measure the volume of O2 evolved.
19. The difference between the actual amount of CO2 present at the beginning of the experiment and the amount of unused CO2 gives the actual amount of CO2 utilized in photosynthesis.
20. The photosynthetic quotient (O2/CO2) may be calculated from the results.
21. Express the rate of photosynthesis as ml of CO2 absorbed, or O2 evolved per gram weight of the experimental material per hour.
Rate of photosynthesis = Amount of CO2 consumed/Fresh wt. of plant materials x Period of Photosynthesis
= X/3 × 3 ml/gms of fresh wt. of plant – material/hr.
Experiment # 10. Effect of an Inhibitor and an Un-Coupler on the Rate of Photosynthesis:
In the photosynthetic light reaction process, the electron flow and subsequent photophosphorylation is affected by a number of un-couplers and inhibitors. An un-coupler is a compound which permits the flow of the electrons at a faster rate as it uncouples electron transport from phosphorylation.
For instance, ammonium ion (NH+4) is an un-coupler of phosphorylation. On the other hand, an inhibitor is the compound that inhibits the flow of electron through electron carriers. The compound “Dichlorophenyl dimethyl urea” (DCMU) is a potent inhibitor of PS II electron flow path.
1. Plant material:
Freshly collected healthy plant twigs of Hydrilla.
2. Chemicals & reagents:
(i) 10-6 M ammonium chloride solution as un-coupler.
(ii) 10-6 M DCMU solution as inhibitor.
Beakers, funnels fitted with jet, measuring cylinder, pipette etc.
1. Three separate photosynthesis sets were prepared using Hydrilla plants in usual ways. The volume of each set was 1,000 ml.
2. One of the sets is used as control set, where the set is filled with 1,000 ml tap water. In other two sets, DCMU solution and NH4Cl solution were separately poured and the volume made up to 1,000 ml.
3. All the sets were placed in sunlight or artificial light for 30 minutes. Then evolution of gas bubbles thus developed from each set were measured separately from graduated tube as a measure of photosynthesis at different time intervals.
4. The rate of photosynthesis thus, calculated as:
Photosynthetic rate = O2 volume evolved at different time intervals.
The data thus obtained is tabulated and graphically expressed as:
From the above result, it appears that NH4C1 acting as un-coupler and thus accelerate the photosynthetic reaction rates, while DCMU inhibit the photosynthetic processes.
Experiment # 11. Determinations of Dissolved Oxygen by Titrimetric Method:
It is well-known that, during photosynthetic light reaction, phase hydrolysis of water — resulting in the production of O2 acid — consequently enable to run the photosynthetic electron flow path.
In usual cases, the evolution of O2 is thus measured by volume through water replacement method. But through a suitable titrimetric method dissolved oxygen produced by aquatic plant during photosynthesis could be measured. This is modified version of Winkler’s D.O. measuring method.
1. Plant materials:
Freshly collected Hydrilla plants
2. Chemicals & Reagents : 40% MnCl2 solution (40 gm. MnCl2, dissolved in 100 ml distilled water); KI and KOH solution mixture (1.75 gm. KOH and 37.5 gm. KI dissolved in 250 ml distilled water) concentrated HC1; 0.009 (N) Na2S2O3, solution; 1% starch indicator solution; 0.5% KHCO3 solution; distilled water
Conical flasks, burette, pipette, measuring cylinder, beaker etc.
Balance with weight box, blotting paper etc.
1. The conical flasks of almost 250 ml capacity were filled with 200 ml tap water. Then in each flask a pinch of KHC03 solution was added.
2. About 5 gm. of freshly collected Hydrilla plants were then placed in each conical flask and the sets were kept in bright sunlight or under artificial light.
3. After an hour, 10 ml of water of each flask were taken separately in 100 ml flask, and then to each flask the following reagents were added:
0.5 ml of 40% MnCl2; 1 ml of KI & KOH solution mixture.
4. The flasks were then stoppered quickly and shaken thoroughly. After 2 min 2 ml of conc. HCl was added and stoppered again. The precipitate of MnCI2 was then re-dissolved.
5. Dissolved O2 now liberates free I2 in the reaction mixture, which is then titrated by sodium thiosulphate solution using starch indicator.
6. One control set also maintained for similar titration of dissolved oxygen.
The volume of thiosulphate required for titration is tabulated in next page.
Suppose the excess thiosulphate required for D. O. produced due to photosynthesis was x ml. Thus amount of standard thiosulphate requirement can be computed by the formula:
V1S1 = V2S2
where, V1 = x ml
S1 = strength Na2S2O3 used [0.009 (N)]
S2 = Strength of standard Na2S2O3 [0.01 (N)]
It is also known that 0.01 (N) Na2S2O3 = 0.08 mg O2
From this amount, dissolved O2 thus produced by 5 gm. of plants within 60 min can also be computed.
Finally, rate of increment of D. O. by the photosynthesis can be expressed as µg of O2 evolved/ gm./ min/ml of water bodies.
Experiment # 12. Study of Hill Activity in Isolated Chloroplast:
Photosynthesis is the process by which light energy is used to synthesize carbohydrate in green plants from CO2 and H2O. The synthesis of carbohydrate takes place in two stages known as the light and dark reactions.
The light reaction consists essentially of the removal of electrons from water and these are then used to reduce NHDP+ and generate ATP. There are two light-driven reactions PS I & PS II which operate in a series. The dark reaction, on the other hand, involves the utilization of NADPH and ATP generated by the light reaction to fix CO2.
Electron flowing from PS II towards PS I or from ferredoxin towards NADP+ can be intercepted by artificial electron acceptors.
This process is known as the Hill reaction and can be used to measure the photochemical activity of PS I and PS I acting in series or of PS II alone. The photochemical activity of PS I can be measured if the PS II activity is blocked by the powerful herbicide di-chloro-di-nethyl-urea (DCMU) and the electron flow provided by an artificial electron donor.
1. Fresh spinach leaves
2. Isolated medium (0.3 mol/lit NaCl, 3 m mol/liter MgCl2 0.2 mol/lit tricine, pH 7.6)
3. Double strength assay medium (0.2 mol/lit sorbitol, 6 m mol/lit MgCl2, 0.4 mol/lit tricine, pH7.6)
4. Warring blender and muslin
5. Centrifuge in cold room
6. Potassium ferricyanide (10 m mol/lit)
7. Dichlorophenol indophenol (1 m mol/lit)
8. Di-chloro-phenyl-dimethyl urea (250 µml/lit)
9. Sodium dithionite (solid)
10. Oxygen electrode
(A) Isolation of Chloroplasts:
100 gm of spinach leaves are taken and their midrib is removed. Then the leaves are chopped by knife into small sections. The leaf pieces are finally homogenated with 100 ml of isolation medium in the Waring blender. The suspension is then filtered through muslin and centrifuged in cold room as 2,500 rpm for 5 min.
The supernatant is discarded and the pellet is washed carefully with single strength assay medium spinning at maximum speed for 1 min. The pellet is re-suspended in 2-3 ml of assay medium and stored on ice until required.
(B) Oxygen electrode assays for determination of Hill activity:
The oxygen electrode is widely used in biochemical laboratories to monitor processes or reactions involving oxygen exchange. The evolution of oxygen by illuminated chloroplasts can be readily followed with this equipment.
The oxygen electrode consists of a platinum cathode and silver anode in saturated potassium chloride solution. When a potential is applied across the ‘cell’ formed by these electrodes dipping in the test solution, oxygen is electrolytically reduced.
Four electrons are generated at the anode which are then used to reduce a molecule of oxygen at the cathode:
If the polarizing voltage is in the range 0.5-0.8V, then the current generated is proportional to the oxygen concentration in the medium. The amplified current is fed to a chart recorder which gives a trace of the change in oxygen concentration with time.
Zero oxygen concentration is obtained by adding a crystal of sodium dithionite to the test solution and adjusting the pen to the baseline. The air-saturated buffer is taken to be 100% O2 and the pen adjusted accordingly. In practice, 100% O2 is assumed to be 240 µ mol of dissolved O2 per lit, which is the solubility of O2 in an aqueous solution at 26°C.
If 4 ml is present in the reaction vessel then the total 02 content is 4 × 240 = 960 n mol when saturated. For this reason, it is best to adjust the pen on the recorder to 96 rather than 100. In these circumstances, one division on the chart recorder is equivalent to 10 n mol of O2.
The oxygen electrode is set up and calibrated in the usual way and, after calibration, move the pen to the centre of the chart paper with the zero control as O2 evolution is being measured.
Each experiment is started by adding 2 ml of double strength assay medium to the electrode and sufficient chloroplasts is given in an apple-green suspension. Finally, sufficient distilled water is added to the mixture so that the final volume will be 4 mi of single-strength assay medium.
The assays should be set up in the dark by surrounding the electrode with aluminium foil; the rate of oxygen evolution is monitored in the light following the addition of co-factors, inhibitors etc. The chloroplast suspension can be conveniently illuminated by a 100 watt bulb.
To test the effectiveness of ferricyanide and DCP1P as Hill oxidants, an increasing amount of oxidants is added to the chloroplast suspension and the rate and total amount of O2 evolved at each concentration is recorded and plotted on graph.
Experiment # 13. Spectrophotometric Assay of the Hill Reaction and Estimation of Chlorophyll:
The Hill reaction is assayed as DCPLP loses in blue colour on reduction and this can be followed in a spectrophotometer at 600 nm. On the contrary, chloroplast is extracted from the chloroplast suspension by shaking with acetone and the absorption of the solution is plotted in the visible region of the spectrum.
1. All the materials of the previous experiments
2. Acetone (80% v/v)
Assay of Hill reaction:
Instead of the oxygen electrode, the sample is prepared for spectrophotometric assay and the rate of DCPIP reduction is measured in expt. sets where the chloroplast suspension mixed with DCPIP solution is expo d to light for 15 s or 30 s period. The chloroplast suspension concentrations and DCPIP concentration are conveniently adjusted so that the rate of reduction could be measured spectrophotometrically.
Assay of chlorophyll content:
A suitable volume of chloroplast suspension (0.2 ml) is added to the 10 ml of solvent solution and then shaken thoroughly and filtered through Whatman No. 1 filter paper into a 25 ml volumetric flask.
Then rinse out the test tube with a further 5 ml of the aqueous acetone and use this to wash the filter paper. The working is repeated and final volume of the solvent extract is made up to 25 ml. Finally the chlorophyll content is measured at 625 nm against a solvent blank.
Chlorophyll (mg/ml) = Extinction at 625 nm × 5.8