The Light-Independent Reaction (Calvin Cycle)

Now for the second half of photosynthesis - the light-independent reaction or Calvin cycle. This happens in the stroma and uses all that lovely ATP and reduced NADP from the light-dependent reactions to actually make glucose.

Carbon dioxide enters through the stomata and diffuses into the stroma, where it meets RuBP (ribulose bisphosphate). The enzyme RuBisCO combines CO₂ with RuBP to form two molecules of G-3-P $glycerate 3-phosphate$. This is called carbon fixation.

Next comes the reduction stage - ATP and reduced NADP convert G-3-P into triose phosphate (TP). Some of this TP gets converted into useful molecules like glucose (you need 6 turns of the cycle to make one glucose molecule), whilst the rest gets recycled back into RuBP using more ATP.

It's basically a recycling system that keeps going round and round, fixing carbon dioxide and turning it into useful organic molecules. The cycle regenerates itself, so it can keep running as long as there's CO₂, ATP, and reduced NADP available.

Key Insight: This reaction doesn't directly need light, but it depends entirely on the ATP and reduced NADP from the light-dependent reactions!

Required Practical 7: Chromatography of Plant Pigments

Ever wondered why leaves change colour in autumn? It's because plants contain several different photosynthetic pigments, not just the green chlorophyll you can see. Chromatography lets you separate and identify these hidden pigments.

The main pigments include chlorophyll a $blue-green$, chlorophyll b $yellow-green$, carotene (orange), and xanthophyll (yellow). Each absorbs different wavelengths of light, so plants can maximise their energy absorption by having different proportions of each pigment.

Here's the method: crush leaf pigments onto chromatography paper, draw a pencil line 5mm above the solvent level, and add your pigment sample. Let it dry, then place the paper in solvent and watch the pigments separate as they travel up the paper. You'll calculate Rf values to identify each pigment.

The key thing is that plants from different environments will have different pigment combinations - shade-tolerant plants might have more chlorophyll b, whilst sun-loving plants might have more protective carotenes.

Pro Tip: Always use pencil, not pen, for your origin line - ink will dissolve and mess up your results!

The Light-Dependent Reaction

Think of this as the "energy capture" stage of photosynthesis - it's all about converting light into chemical energy you can actually use. The light-dependent reaction takes place in the thylakoid membranes inside chloroplasts, where special pigments absorb light energy.

Here's what happens: light energy gets absorbed by photosynthetic pigments in structures called photosystems. This energy then powers two crucial processes - making ATP from ADP and turning NADP into reduced NADP. These are like the plant's energy currency and will be essential for the next stage.

During this whole process, water molecules get split (oxidised), which produces oxygen as a waste product. That's right - the oxygen we breathe is basically plant waste! The ATP and reduced NADP then head off to fuel the light-independent reactions.

Key Point: Without light, none of this happens - that's why it's called the light-dependent reaction!

Required Practical 8: Investigating Dehydrogenase Activity

This practical might seem complicated, but you're essentially investigating how dehydrogenase enzymes in chloroplasts work during photosynthesis. These enzymes help transfer electrons during the light-dependent reactions, and you can measure their activity using a clever indicator called DCPIP.

DCPIP is a redox indicator that starts blue when oxidised but turns colourless when it picks up electrons (gets reduced). Normally, NADP would accept these electrons, but DCPIP can steal them instead, giving you a visible way to measure the reaction rate.

Your hypothesis is that ammonium hydroxide will slow down the reaction because it's alkaline and can denature the enzyme. You'll set up five test tubes: a green standard $chloroplasts + water$, controls to prove you need light and chloroplasts, and experimental tubes with and without ammonium hydroxide.

You'll time how long it takes for the blue-green DCPIP to turn the same colour as your green standard. The results should show that ammonium hydroxide does indeed slow down or stop the dehydrogenase activity.

Remember: All solutions must be ice-cold and isotonic to keep the chloroplasts alive and functioning!

Photosynthesis Introduction & Chloroplast Structure

The overall equation for photosynthesis might look scary, but it's actually quite simple: 6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂. Basically, carbon dioxide plus water makes glucose plus oxygen when you add light energy.

Chloroplasts are the green powerhouses where all this magic happens. They've got a clever structure with outer and inner membranes, plus stacks of thylakoids called grana. The thylakoid membranes contain all those photosynthetic pigments we mentioned, whilst the fluid-filled stroma contains the enzymes needed for making glucose.

Everything's designed for maximum efficiency. Thylakoids have a massive surface area to absorb as much light as possible, and ATP synthase molecules are embedded right in the membranes to pump out ATP. The stroma is packed with enzymes, sugars, and organic acids - basically everything needed for the light-independent reactions.

Remember: Structure equals function in biology - every part of the chloroplast is perfectly designed for its job!

Non-Cyclic Photophosphorylation

Non-cyclic photophosphorylation is the fancy name for the main pathway of the light-dependent reactions. It starts with photoionisation at photosystem II, where light energy excites electrons in chlorophyll so much that they actually leave the molecule entirely.

When electrons get this excited, they're picked up by electron carriers, leaving the chlorophyll oxidised (missing electrons) and the carrier reduced (gained electrons). This is the first step in a chain reaction that will eventually produce both ATP and reduced NADP.

The process is called "non-cyclic" because the electrons don't return to where they started - instead, they follow a linear pathway from photosystem II through various carriers to photosystem I, and finally end up in NADP. This is different from cyclic photophosphorylation, where electrons do return to their starting point.

Key Concept: Photoionisation is the crucial first step - without light energy exciting those electrons, the whole process grinds to a halt!

Practical Controls & Results Analysis

Understanding the control experiments in the dehydrogenase practical is essential for interpreting your results correctly. Test tube 3 $water + DCPIP + isolation medium, no chloroplasts$ proves that you actually need chloroplasts for the reaction - without them, the blue DCPIP stays blue because there's no dehydrogenase enzyme present.

The experimental comparison is between test tube 4 (normal conditions) and test tube 5 (with ammonium hydroxide). Test tube 4 should change from teal to green as DCPIP gets reduced, whilst test tube 5 should show little or no colour change because the ammonium hydroxide interferes with enzyme activity.

Your results confirm that ammonium hydroxide decreases dehydrogenase activity - it can both denature the enzyme (being alkaline) and compete for electrons that would normally reduce DCPIP. You calculate reaction rates as 1/time, so no colour change gives you a rate of 0 s⁻¹.

Key practical points: use ice-cold solutions to protect the chloroplasts, ensure the isotonic medium prevents chloroplast damage, and blend the spinach leaves to release chloroplasts from broken cells.

Method Insight: Every control has a purpose - they eliminate alternative explanations for your results!

Exam Questions & Key Concepts

When tackling photosynthesis exam questions, focus on the key processes and connections between the two main reactions. For light-independent reactions, remember that RuBP combines with CO₂ to form 3-carbon compounds using RuBisCO, then ATP and reduced NADP convert these into hexose sugars.

For electron transport chains in light-dependent reactions, explain how excited electrons lose energy as they move along the chain, with this energy used to make ATP from ADP and Pi. Don't forget that NADPH forms when electrons and H⁺ ions (from photolysis) combine with NADP.

A tricky concept is why a plant's dry mass increase is less than the hexose it produces. Think about it - some hexose gets used in respiration (producing CO₂), and plants lose parts through leaf fall, being eaten, or decomposition. So not all that lovely glucose stays in the plant!

Exam Tip: Always link the two reactions together - the light-dependent reactions provide the ATP and reduced NADP that power the Calvin cycle!

Leaf Structure for Photosynthesis

Leaf structure is perfectly designed for efficient photosynthesis - every feature has a specific function. The palisade mesophyll is packed with chloroplasts to maximise light absorption, whilst the spongy mesophyll has air spaces for gas exchange.

Stomata (controlled by guard cells) allow CO₂ to enter and O₂ to leave, whilst the waxy cuticle is both waterproof (preventing water loss) and transparent (letting light through). The leaf is thin with a large surface area to absorb maximum light energy.

Inside each chloroplast, the structure continues this efficiency theme. Grana (stacks of thylakoids) provide huge surface areas for light absorption, whilst the stroma contains all the enzymes needed for the Calvin cycle. Intergranal lamellae connect different grana together.

The vascular system (xylem and phloem) ensures reactants like water and CO₂ can reach the photosynthetic cells, whilst products like glucose can be transported away to where they're needed.

Structure-Function Link: Every part of a leaf, from the transparent cuticle to the air spaces, is perfectly adapted for photosynthesis!

Chromatography Method & Analysis

Getting your chromatography technique right is crucial for reliable results. Once your pigment sample is dry, place the paper vertically in the solvent container, making sure the solvent level stays below your origin line. Keep the paper straight so pigments travel directly upwards rather than sideways.

Remove the paper when the solvent reaches about 2mm from the top, then immediately mark the solvent front with pencil (it evaporates quickly!). Circle each pigment spot and calculate Rf values using the formula: distance moved by pigment ÷ distance moved by solvent.

Rf values are constant for each pigment in a particular solvent, so you can identify unknown pigments by comparing your values to known standards. Always measure from the centre of each pigment spot to standardise your measurements and allow proper comparisons.

The beauty of this practical is comparing chromatograms from different plant species - you'll see how plants adapt to their environments by having different pigment combinations for maximum light absorption efficiency.

Technical Tip: Measure Rf values from the middle of pigment marks because the spots spread out during separation!