Light-Dependent Electron Transfer

Chlorophyll absorbs light energy, causing some of its electrons to gain energy and leave the chlorophyll. This loss of electrons is called photoionisation.

The released electrons pass along a series of electron carriers. As they pass between carriers, they transfer energy that contributes to the production of ATP from ADP and inorganic phosphate.

The electrons also provide reducing power: they are used to reduce NADP. NADP accepts electrons and a proton to form reduced NADP.

Photolysis replaces electrons

Light energy drives the splitting of water into electrons, protons and oxygen. This splitting is called photolysis.

The electrons replace those lost from chlorophyll by photoionisation, allowing electron transfer to continue. The protons contribute to processes including the reduction of NADP. Oxygen is released as a product.

Chemiosmotic ATP Synthesis in Chloroplasts

The thylakoid membrane surrounds an internal space inside a chloroplast. As electrons pass along an electron transfer chain in this membrane, they transfer energy. This energy drives the accumulation of protons ($$\ce{H+}$$) inside the thylakoid.

The result is a proton concentration gradient: the proton concentration is higher inside the thylakoid than in the surrounding fluid, called the stroma. The thylakoid membrane restricts the passage of protons, so the gradient can be maintained.

Protons move down this gradient from the thylakoid interior to the stroma through ATP synthase, a protein embedded in the membrane. Their flow supplies energy for ATP synthase to catalyse the formation of ATP from ADP and inorganic phosphate.

This coupling of electron transfer, a proton gradient and ATP synthesis is called chemiosmosis.

Calvin Cycle

The Calvin cycle uses carbon dioxide to produce triose phosphate, a three-carbon compound. It requires ATP and reduced NADP, which are supplied by the light-dependent reactions of photosynthesis. The cycle does not require light directly, so it is part of the light-independent reactions.

Carbon fixation and triose phosphate production

Carbon dioxide, $$\ce{CO2}$$, combines with a five-carbon compound called ribulose bisphosphate (RuBP). The enzyme rubisco catalyses this reaction. The resulting six-carbon compound is unstable and splits into two molecules of the three-carbon compound glycerate 3-phosphate (GP).

GP is reduced to triose phosphate using reduced NADP. This conversion also requires energy released by the hydrolysis of ATP.

Regeneration of RuBP

Some of the triose phosphate produced is used to regenerate RuBP. This regeneration also requires ATP. Once RuBP has been regenerated, it can combine with more carbon dioxide, allowing the cycle to continue.

The remaining triose phosphate can be used to form simple sugars and other useful organic substances.

Limiting Factors of Photosynthesis

The rate of photosynthesis depends on the conditions around a plant. A factor is limiting under the prevailing conditions if increasing it increases the rate of photosynthesis. If that factor is increased enough, the rate may stop rising because a different factor is then limiting.

For example, a plant in dim light may photosynthesise faster when given more light. Once the light is sufficiently bright, adding more may have little effect if carbon dioxide concentration or temperature limits the rate instead.

Factors that limit photosynthesis

• Light intensity: Increasing light intensity can increase the rate because more light is available for photosynthesis. The rate eventually levels off if another factor limits it.
• Carbon dioxide concentration: Increasing the concentration can increase the rate because more carbon dioxide is available for photosynthesis. Again, the rate may level off when another factor becomes limiting.
• Temperature: At low temperatures, increasing temperature can increase the rate because the enzyme-controlled reactions of photosynthesis proceed faster. At excessively high temperatures, enzyme activity can decline, causing the rate of photosynthesis to fall.

Reducing limitations in agriculture

Growers can use supplementary lighting, carbon dioxide enrichment or temperature control to reduce limitations on photosynthesis. The effect of each treatment depends on the other conditions. For example, extra light may have little effect if carbon dioxide concentration remains limiting.

Data on photosynthetic rate or crop yield can help assess a treatment. A comparison between treated and untreated plants under otherwise similar conditions shows whether the treatment produced a benefit under the conditions measured. A higher photosynthetic rate or yield alone, however, does not show whether the treatment is worthwhile financially.

To judge whether the benefit justifies the cost, a grower can compare the value of any additional crop yield with the cost of the treatment. A treatment may increase yield but still cost more than the extra yield is worth. The result applies to the conditions measured: a treatment that is effective under one set of conditions may have little effect when a different factor is limiting.

Chromatography of Leaf Pigments

A leaf extract can contain several pigments. Chromatography separates them because each pigment moves differently between a material that stays in place and a solvent that moves through it.

For example, chromatography paper acts as the stationary phase. A small spot of leaf extract is placed on a pencil baseline near the bottom of the paper. The paper is then placed in solvent, with the baseline above the solvent level. This prevents the extract from dissolving directly into the solvent at the bottom.

The solvent moves up the paper and carries dissolved pigments with it. Pigments that dissolve more readily in the solvent and are less strongly attracted to the paper tend to travel farther. Those that are more strongly attracted to the paper tend to travel more slowly. As a result, the pigments form separate spots or bands on the paper, producing a chromatogram.

The furthest point reached by the solvent is the solvent front. It is marked so that the distance the solvent travelled can be measured.

Chromatography of leaf pigments: the $$R_f$$ value

A pigment’s $$R_f$$ value expresses how far it travelled relative to the solvent front:

Measure both distances from the baseline, in the direction the solvent moved. For a pigment spot, measure to its centre. For example, if a pigment travels $$4\text{ cm}$$ and the solvent front travels $$8\text{ cm}$$, the pigment’s $$R_f$$ value is $$0.5$$.

Comparing leaf pigments

Chromatograms can be used to compare extracts from leaves of different colours, or from shade-tolerant and shade-intolerant plants. Compare the colours and $$R_f$$ values of their spots or bands to look for pigments that may be shared or different.

The chromatograms must be run under comparable conditions, including the same solvent and stationary phase, because these affect how far pigments travel. Matching spots suggest that the same pigment may be present, but a matching $$R_f$$ value alone does not prove its identity.

Investigating Chloroplast Dehydrogenase Activity

Electron transfer in a chloroplast extract can be followed using DCPIP, an artificial electron acceptor. Oxidised DCPIP is blue. When it accepts electrons, it is reduced and becomes colourless.

As DCPIP is reduced, the mixture absorbs less light at a suitable wavelength. A colorimeter measures this absorbance. The rate at which absorbance decreases is therefore an indicator of the rate of electron transfer associated with dehydrogenase activity in the extract.

Testing the effect of light intensity

Prepare several mixtures with the same amounts of chloroplast extract and DCPIP. Expose each mixture to a different light intensity, for example by placing it at a different distance from the same lamp. Measure the light intensity at each position if a light meter is available.

Keep other relevant conditions constant, including the concentrations and volumes of the extract and DCPIP, temperature, pH and the colour of light. Use the same colorimeter wavelength throughout. Keeping temperature constant is important because moving a mixture closer to a lamp may also warm it.

For each mixture, start timing when the extract and DCPIP are mixed, then record absorbance at regular intervals for the same length of time. Use a suitable blank to account for background absorbance, including that of the chloroplast extract.

Comparing rates of DCPIP reduction

For each light intensity, calculate the decrease in absorbance per unit time over the same time interval:

A larger decrease per unit time indicates faster DCPIP reduction and therefore a faster rate of electron transfer under the test conditions. If absorbance does not decrease at a constant rate, compare rates over the same initial interval.

Repeat each light-intensity test using fresh mixtures. The spread of the results indicates how consistent they are; a mean rate can be used to compare the intensities.

Controls for chloroplast extract activity

A control containing DCPIP but no chloroplast extract, with the extract replaced by the same volume of buffer, helps show whether DCPIP changes colour without the extract. A mixture kept in darkness provides a comparison with illuminated mixtures.

Together, these controls help assess whether the observed decrease in absorbance is associated with activity in the chloroplast extract and with illumination. The colorimeter reading is an indirect indicator: it measures DCPIP reduction, rather than dehydrogenase activity directly.