Fig. 1. Schematic showing competition for Rubisco between oxygen and carbon dioxide.

Fig. 2. Schematic diagram showing an outline of the C₄ pathway

In order to regenerate RuBP from PG, energy is used and CO₂ released in a process known as photorespiration. Competition between O₂ and CO₂ for Rubisco therefore reduces the efficiency of photosynthesis (ie less CO₂ is fixed for a given amount of light energy absorbed by the plant). At high temperatures or low CO₂ concentrations competition from O₂ becomes more severe and efficiency of photosynthesis decreases.

C₄ plants overcome the problem of O₂:CO₂ competition by using a metabolic shuttle to concentrate CO₂ at Rubisco’s active site. This eliminates photorespiration so potentially increasing photosynthetic efficiency. However, concentrating CO₂ requires energy, so overall efficiency of photosynthesis will only increase under conditions where the energetic cost of the C₄ pathway is less than the energy that would be lost through photorespiration without it. Therefore the C₄ pathway is proposed to give plants a selective advantage under conditions where energy loss from photorespiration would be large: high temperatures and low CO₂.

The evolution of the C₄ pathway

a) Ecology:
C₄ plants are proposed to have evolved from C₃ plants about 30 Ma, but did not become widespread until 6-8 Ma. The greater photosynthetic efficiency of C₄ plants under hot, low CO₂ conditions is proposed to have given them a selective advantage over C₃ plants in the tropics and subtropics. However, CO₂ levels are thought to have remained fairly constant during the Miocene (25 to 5 Ma), so can’t account for the rapid expansion of C₄ grasslands toward the end of that epoch. This suggests that other factors, such as changes in temperature, precipitation patterns, fire frequency etc must have played a role in C₄ expansion. So a key aim of this project is to assess how important two of these factors, temperature and soil water availability, are for the growth of C₃ and C₄ plants.

b) Molecular evolution:
Despite the fact that evolution from C₃ to C₄ photosynthesis involves multiple biochemical and anatomical adaptations, it is believed to have happened independently in more than thirty plant lineages. However attempts to artificially engineer or breed C₄ photosynthesis into C₃ grasses have so far failed. This indicates that there may be features of C₄ evolution that we are not yet aware of. So another aim of this project is to understand more about the molecular mechanisms of C₄ evolution.

Fig. 3. The two subspecies of A. semialata side-by-side in an experimental plot.

Alloteropsis semialata
Alloteropsis semialata is the only known species with both C₃ and C₄ subspecies. Both occur in southern Africa and have overlapping distributions although hybrids between them appear to be rare. In addition, it has previously been noted that the C₃ subspecies is always diploid (2n = 18) while the C₄ is polyploid (2n = 54).

These features make A. semialata ideal for studying the mechanism of C₄ evolution and the consequences of evolving C₄ for the ecology of the species.

So this project has two main aims:

1) To investigate the hypothesis that C₄ physiology originated in A. semialata via a hybridisation event between the C₃ subspecies and a closely related C₄ grass.

2) To investigate the hypothesis that C₄ physiology has made the C₄ subspecies better adapted to hot, wet summer climates whilst C₃ physiology makes the C₃ subspecies better adapted to cool, wet winter conditions.

How are these aims being investigated?

Aim 1

• By establishing the phylogenetic relationships between the two A. semialata subspecies and other members of the Alloteropsis genus and Panicoid grasses.

• By determining whether the C₄ subspecies originated from the C₃ one via an auto- or allopolyploidisation event, and to identify candidate parents in the case of allopolyploidisation.

These are being investigated by two molecular biology methods:

• Genomic in-situ hybridisation (GISH) ie is the C₃ genome present in the C₄ subspecies?

• Molecular phylogenetics, ie placing Alloteropsis species in a previously determined molecular phylogeny of Panicoid grasses.

Fig. 4. Schematic diagram to show how the C₄ subspecies could have evolved from hybridisation of the C₃ subspecies. Note that chromosome doubling could also result from fusion of unreduced gametes.

Fig. 5. Common garden experiment at Rhodes University in Grahamstown, South Africa, with plots containing transplanted C₃ and C₄ A.semialata.

Aim 2

• By investigating the temperature response of photosynthesis of the C₃ and C₄ subspecies under controlled laboratory conditions.

• By investigating the seasonal response of photosynthesis on C₃ and C₄ plants grown outdoors in South Africa under well-watered and natural rainfall conditions.

• By investigating the seasonal growth of C₃ and C₄ plants grown outdoors in South Africa under well-watered and natural rainfall patterns and seeing how these correlate with seasonal changes in temperature and rainfall.