No — not reliably. The differences between C₃ and C₄ plants are internal: anatomical and biochemical, not external. A C₄ plant does not carry any distinguishing feature on the outside of its leaf, stem or flower that a C₃ plant cannot also have.

Why the external view fails. The defining C₄ features are the presence of Kranz anatomy (large bundle sheath cells with many chloroplasts, thick gas-impervious walls and no intercellular spaces), the presence of PEPcase in the mesophyll, and the restriction of the Calvin cycle to the bundle sheath. All three can only be seen or assayed inside the leaf.

What external observation can and cannot give you. At best you may form a suspicion. C₄ plants are largely tropical grasses and plants adapted to dry tropical regions — maize, sorghum, sugarcane, pearl millet, Amaranthus — and such plants often grow vigorously in hot bright conditions where neighbouring C₃ plants look stressed at mid-day. But this is habitat-based guesswork, not identification: many tropical plants are C₃, and the suspicion must always be confirmed by cutting a section.

By looking for Kranz anatomy in a vertical section of the leaf — specifically, for the bundle sheath around the vascular bundles.

What to look for. In a C₄ leaf the vascular bundles are surrounded by particularly large bundle sheath cells, which may form several layers. These cells are characterised by:

• a large number of chloroplasts,
• thick walls impervious to gaseous exchange,
• no intercellular spaces.

The arrangement resembles a wreath, which is what ‘Kranz’ means. In a C₃ leaf there is no such conspicuous wreath: the mesophyll is differentiated into ordinary palisade and spongy tissue with abundant intercellular air spaces, and the sheath around the bundle is thin and inconspicuous.

Why this structure is diagnostic. The anatomy exists to serve the biochemistry. The bundle sheath is where the C₄ acid is decarboxylated and where the Calvin cycle runs, so it must be packed with chloroplasts containing RuBisCO; and the released CO₂ must not escape, so the walls are sealed and there are no air spaces. Because the structure is inseparable from the pathway, its presence identifies a C₄ plant. Maize and sorghum are the standard specimens for this observation.

Because in those few cells, almost no fixed carbon is wasted, and they are continuously fed with concentrated CO₂ by the whole mesophyll acting as a pump. Productivity depends on how efficiently each Calvin-cycle site works, not on how many sites there are.

1. No photorespiratory loss. The C₄ acid arriving from the mesophyll is broken down in the bundle sheath cells to release CO₂, which increases the intracellular concentration of CO₂. Since RuBisCO's binding of CO₂ and O₂ is competitive and determined by their relative concentrations, this ensures RuBisCO functions as a carboxylase, minimising the oxygenase activity. So photorespiration does not occur, and none of the RuBP is diverted into a pathway that yields neither sugars nor ATP and in fact releases CO₂ while consuming ATP. In a C₃ plant, by contrast, every mesophyll cell loses some of its RuBP this way, and the loss is worst exactly when light and temperature are high.

2. The mesophyll is a CO₂ delivery system. The mesophyll cells, which lack RuBisCO, are given over entirely to trapping CO₂ with PEPcase — an enzyme that does not bind O₂ at all — and shuttling it inward as a 4-carbon acid. Many cells therefore serve the few, keeping them saturated with substrate.

3. The sealed chamber holds the gain. Thick walls impervious to gaseous exchange and no intercellular spaces prevent the concentrated CO₂ from leaking back out.

4. Tolerance of the conditions that limit C₃ plants. C₄ plants tolerate higher temperatures (optimum 30–40°C) and respond to high light intensities, so they keep working through hot bright afternoons, and they can afford partial stomatal closure in dry conditions because their CO₂ concentrating mechanism still supplies RuBisCO.

Taken together, these give greater productivity of biomass despite the small number of Calvin-cycle cells.

Because in C₄ plants the enzyme is deliberately placed in an environment of high CO₂ concentration, and the choice between the two activities is decided by the relative concentrations of CO₂ and O₂ at the active site.

The principle. RuBisCO's active site can bind both CO₂ and O₂, and this binding is competitive. It is the relative concentration of O₂ and CO₂ that determines which of the two will bind to the enzyme. RuBisCO has a much greater affinity for CO₂ when the CO₂ : O₂ ratio is nearly equal — so raising the CO₂ share of that ratio pushes the enzyme decisively towards carboxylation.

How C₄ plants raise the ratio. The C₄ acid formed in the mesophyll is transported to the bundle sheath, where it is broken down to release CO₂. This increases the intracellular concentration of CO₂ well above the atmospheric level. The bundle sheath cells' thick walls impervious to gaseous exchange and absence of intercellular spaces stop that CO₂ escaping. The result is a pocket of CO₂-rich cytoplasm around RuBisCO.

Consequence. With CO₂ abundant and O₂ unable to compete, RuBisCO functions as a carboxylase and its oxygenase activity is minimised. Hence photorespiration does not occur in C₄ plants, and nearly all the enzyme's activity goes into productive carbon fixation. In a C₃ plant the enzyme sits in mesophyll cells open to the intercellular spaces, where CO₂ is only 0.03–0.04 per cent while O₂ is about 21 per cent and is additionally being generated by the light reaction — so a share of the enzyme's work is inevitably oxygenation.

No, such a plant could not carry out photosynthesis.

Why not. Chlorophyll a is the chief pigment associated with photosynthesis, and crucially it is the reaction centre pigment. In each photosystem, all the pigments except one molecule of chlorophyll a form the light harvesting antenna; that single excepted chlorophyll a molecule forms the reaction centre — P700 in PS I and P680 in PS II. The reaction centre is where the photochemistry actually happens: where an electron is excited and handed to the primary acceptor, starting the electron transport that makes ATP and NADPH. Chlorophyll b and the other accessory pigments cannot perform this step. They can absorb light and pass energy on, but they have nowhere to pass it to. A plant with abundant chlorophyll b and no chlorophyll a would absorb light and simply dissipate it.

Then why have accessory pigments at all? Chlorophyll b, xanthophylls and carotenoids serve two functions:

• They widen the range of wavelengths that can be used. They absorb light at wavelengths where chlorophyll a absorbs poorly and transfer the energy to chlorophyll a. This is why the action spectrum of photosynthesis is broader than, and does not overlap exactly with, the absorption spectrum of chlorophyll a — photosynthesis proceeds even in the intermediate wavelengths. More of the incident sunlight is harvested, so photosynthesis is more efficient.
• They protect chlorophyll a from photo-oxidation. In bright light the reaction centre pigment is at risk of oxidative damage, and the accessory pigments — carotenoids in particular — shield it.

The summary sentence: chlorophyll a is indispensable because only it can be a reaction centre; the accessory pigments are enhancing and protective, not substitutes.

Because in the dark the chlorophyll is gradually lost and is not replaced, while the yellow carotenoids and xanthophylls persist and are then no longer masked. The colour that remains is the colour of the pigments that survived.

The reasoning. A green leaf contains four pigments — chlorophyll a (bright or blue green), chlorophyll b (yellow green), xanthophylls (yellow) and carotenoids (yellow to yellow-orange). In a healthy illuminated leaf the chlorophylls are present in far greater quantity and mask the yellow pigments completely, so the leaf looks green. Chlorophyll synthesis requires light, and chlorophyll is continually broken down and re-made. In darkness the breakdown continues but the synthesis stops, so the chlorophyll content falls. As it falls, the masking is removed and the underlying yellow pigments show through — giving first a pale green and then a yellow leaf.

Which pigment is more stable? The carotenoids (and xanthophylls) are the more stable pigments. Chlorophyll is the more labile: it degrades readily in the dark, and also breaks down under excessive light. You see the same phenomenon in autumn leaves and in etiolated seedlings raised without light.

The leaves on the shady side — and the plants kept in the shade — are the darker green.

Why. A leaf in weak light is short of the one resource it cannot do without, so it compensates by making more chlorophyll per unit leaf area. A higher chlorophyll concentration means a higher chance of intercepting each scarce photon, and it is also what makes the leaf look a deeper green. Such shade leaves are typically broader and thinner as well, spreading the same investment over a larger light-catching surface.

Why the sunlit leaves are lighter. Two reasons work together. First, they simply do not need as much chlorophyll: light saturation occurs at 10 per cent of full sunlight, so a leaf in the open is receiving far more light than it can use, and extra chlorophyll would buy nothing. Second, an increase in incident light beyond a point causes the breakdown of chlorophyll, so strong sunlight actively degrades the pigment. The result is a paler, often thicker leaf.

A caution worth adding. Darker green does not mean the shade leaf photosynthesises more. It is making the best of a limiting factor. By Blackman's Law of Limiting Factors, the shade leaf's rate is still limited by light, and the chapter notes that plants in shade or in dense forests are precisely the exception where light is the limiting factor in nature.

(a) Light is a limiting factor at point A — and only there among the three. Point A lies on the initial, steeply rising, linear part of the curve, where there is a linear relationship between incident light and CO₂ fixation rates at low light intensities. Every increment of light produces a proportional increment of rate, which is the definition of a limiting factor. At B the curve is bending over, so light is becoming less limiting and is only partly so; at C the curve has flattened completely, so light is not limiting at all.

(b) In region A the limiting factor is light itself — specifically the light intensity. Because the rate rises in direct proportion to the light supplied, light must be the factor nearest its minimal value, exactly as Blackman's Law of Limiting Factors predicts. All other factors — CO₂, temperature, water, chlorophyll content — are present at adequate levels in this region, which is why changing them would not shift the rate while light stays low.

(c) C represents the plateau of the curve — the light-saturated region, where light is no longer limiting and the rate is held down by some other factor that has now become limiting, most commonly the CO₂ concentration, or temperature. D represents the point on the light-intensity axis at which this saturation is reached — the saturating light intensity, beyond which further increase in incident light produces no further increase in rate. The chapter notes that this saturation is reached at about 10 per cent of full sunlight. (E, marked on the vertical axis, correspondingly represents the maximum or light-saturated rate of photosynthesis.)

One further inference the graph invites. If the light intensity were pushed far beyond D, the rate would eventually fall rather than stay flat, because increase in incident light beyond a point causes the breakdown of chlorophyll and a decrease in photosynthesis.

(a) C₃ and C₄ pathways

Note carefully: the C₄ pathway does not replace the C₃ pathway. The Calvin pathway occurs in all photosynthetic plants; in C₄ plants it is simply preceded by the extra PEP cycle and confined to the bundle sheath.

(b) Cyclic and non-cyclic photophosphorylation

(c) Anatomy of leaf in C₃ and C₄ plants

The functional link: every one of the C₄ anatomical features exists to serve one purpose — to build and hold a high concentration of CO₂ around RuBisCO, so that the enzyme carboxylates instead of oxygenating.