ComBio special: C4 plants and the evolutionary explosion

Around 2.5 million years ago, global climate entered a marked drying phase that drove a major expansion of east Africa's fire-adapted savannah woodlands, forcing the region's ancient rainforests into retreat.

Canadian botanist Professor Rowan Sage, of the University of Toronto, believes it is no coincidence that Homo habilis, the first card-carrying, tool-making ancestor of modern humans, appears in the fossil record at the same time.

Like their Australian and Asian analogues, the mosaic of tall grasses, shrubs and sparse woodlands of the African savannah teems with wildlife and proffers a rich, seasonally predictable smorgasbord of food plants.

The savannah was a challenging environment, but one rich with evolutionary opportunity. Here, a dextrous, sociable higher primate already endowed with a preternaturally large (600 - 700cc) brain might find a profitable ecological niche.

Over the next two million years, selection pressure doubled the volume of the human brain, bands of modern humans began moving out of Africa, and the rest is writ large in prehistory. And, if Sage's hypothesis is correct, modern humans owe a great evolutionary debt to C4 grasses.

Sage, an expert in the C4 photosynthesis pathways who will address the ComBio conference next week, says that understanding why requires an appreciation of the nearly four billion-year history of carbon dioxide in the Earth's atmosphere, its role in the evolution of life, and the selection pressures that saw some flowering plants evolve a more efficient system of photosynthesis around 25 million years ago.

It happened not once, but at least 50 times, in disparate groups of both major flowering plant lineages: the monocots (blade-leafed plants like lilies, grasses and sedges), and dicots (broad-leafed plants like magnolias, beeches and eucalypts).

Atmospheric CO2

Sage says that through most of the Mesozoic era (250-65 million years ago) and into the early Cenozoic, the age of mammals, atmospheric CO2 levels hovered around today's greenhouse-amplified levels of 280ppm to 320ppm.

The first flowering plants evolved from seed fern ancestors around 130 million years ago; they employed the relatively inefficient C3 system of photosynthesis, which works optimally at a CO2 concentration above 200ppm.

The plant enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), the world's most abundant protein, catalyses a carboxylation reaction that captures CO2 from the atmosphere and 'fixes' it in plant cells, in the form of carbon-rich sugars like sucrose.

The Achilles heel of C3 plants is that RuBisCo sporadically captures oxygen instead of carbon dioxide, catalysing an oxygenation reaction that synthesises glycine instead of sucrose - a process called photorespiration.

Photorespiration inhibits photosynthesis, because plants must metabolise glycine back to its photosynthetic intermediates with another enzyme, glycine decarboxylase. The decarboxylation process consumes extra ATP, and results in a loss of carbon dioxide and water vapour through the plant's open stomata.

Sage says that during the early Eocene epoch, around 50 million years ago, CO2 levels in the atmosphere went into a gradual decline, and by the early Miocene, around 25 million years ago, reached 280ppm - around the same concentration as in recent, pre-industrial times.

The first C4 plants appeared around 30 million years ago, and underwent explosive evolutionary radiation around 25 million years ago. Whether the primary selective pressure was increasing aridity, or lowering CO2 levels, is still debated.

But the outcome was that C4 plants were more efficient than their C3 ancestors in drier, low-CO2 conditions because they evolved a novel anatomical solution to the photorespiration problem.

Sage says the fact that it was not a one-off innovation suggests that a common pathway proceeded by a series of intermediate steps to a unitary solution.

Unitary solution

Sage has devoted much of his research career to investigating the anatomical and biochemical progression from C3 to C4 photosynthesis.

Grasses constitute around half of the world's 14,000-odd C4 plants, and 25 per cent are sedges. Within the grass family, the trait has evolved about 15 times; among sedges, four times.

Although dicots account for only 15 per cent of C4 taxa, the trait has evolved up to 35 times - Sage says most C4 dicots are herbs and shrubs, with a small minority of woody trees.

This pattern of multiple evolution of C4 photosynthesis suggests the process is ongoing: that there should be possible to catch plants in the evolutionary act of evolving the trait.

"We determine the [independent] origins of C4 by analysising phylogenetic patterns, and we find it popping up in different lineages," he says.

"In a few lineages, we've gone in and looked at individual species, and found traits indicative of change from C3 to C4 photosynthesis.

"The first stage we tend to see in C3 plants is that the veins grow closer together, which appears to improve water transport to the photosynthetic tissues. When the plant's stomata are open in a hot, dry environment, it loses more water, so it needs a better trafficking system to deliver more water to the photosynthetic apparatus.

"The next thing we see is that bundle sheaths, the layer of cells surrounding the vascular bundles that transport water, become bigger - again, this may be an adaptation that provides an immediate reservoir of water in close proximity to the photosynthesis apparatus. If the plant is hit by gusty winds on a hot day, there's a big increase in transpiration, and local storage of water provides a backup.

"Once you have leaves with high vein densities, you then need a mutation in the glycine decarboxylase gene, which recycles the products of photorespiration.

"But the mutation must knock out expression of glycine decarboxylase only in mesophyll tissues, not in bundle-sheath cells, to minimise the disruption to photosynthesis. You then have a system for recapturing the photosynthetic CO2, without disrupting photosynthesis.

"After that, it's an easy ride to C4 photosynthesis."

Selective advantage

The "kicker" in this tale, says Sage, is that as atmospheric CO2 levels in the atmosphere fell, as they did 25 million years ago, C4 plants were at a selective advantage, and began changing landscapes around the world.

As they displaced C3 plants in the herbaceous layer in ecosystems, their flammable tendencies caused wildfires. C4 plants are well-adapted to survive fire, and became dominant in many environments, at the expense of C3 taxa.

Forests began opening up, and yielding to grasslands and savannah woodlands. Sage says an even more pronounced cool, dry phase that set in around 2.5 million years ago gave further impetus to the spread of savannah grasslands in east Africa, and to evolutionary processes already under way in our own higher primate ancestors.

"The savannah-isation of Africa imposed selection pressure for bipedalism, allowing our ancestors to stand up and look over the tall C4 grasses.

"There was pressure for group activity for successful hunting and gathering of food, and the associated social behaviour that goes with it, including tool-making to hunt and process foods.

"I believe the rise of C4 species in the African savannah created the conditions for the final evolution of rational intelligence. If we were to roll back the clock, and there was no lowering of atmospheric CO2 we wouldn't have been here to talk about it."

In the mid-1990s, Sage advanced a hypothesis that linked rising atmospheric CO2 at the end of the last glacial period 13,000 years ago to the independent invention of agriculture at multiple locations in Europe, Africa, Asia and the Americas between 12,000 and 6000 years ago.

Former hunter-gather cultures in the Fertile Crescent region of the Middle East, China, New Guinea, north-eastern North America, central America and South America, turned to agriculture in geologically and climatically diverse environments.

"Why do we have this seeming coincidence of agriculture emerging in multiple centres, and involving very different plant species, when a crucial technology like writing evolved only once or twice in Eurasia and the Far East, and diffused to other cultures?

"Humans had achieved quite sophisticated, pre-agrarian technological capabilities by 60,000 years ago, yet did not turn to agriculture until after the last glacial period."

Sage says that for much of the past 100,000 years, cold, dry conditions prevailed around the globe, and atmospheric CO2 fell to levels as low as 180ppm during glacial periods.

But at the end of the last glacial period, the release of huge quantities of dissolved CO2 from the warming oceans and melting tundra, saw a rise to around 270ppm - the level prevailing at the time of the industrial revolution.

C3 to C4

Sage's research showed that the C3 ancestors of most modern crop species, including cereals like wheat, rice, barley and oats, would not have been productive enough to support an earlier transition to agriculture during this prolonged phase of acute carbon stress and water deficiency.

With notable exceptions like maize, millet, sorghum and sugar cane, most crop species are C3 photosynthesers - and maize was a relatively late addition to the meso-American crop catalogue, because it did not appear in the archaeological record until around 8,000 years ago, after a mutation in its wild ancestor, the hard-seeded, weedy Teosinte gave rise to the first maize varieties.

C4 plants are predominantly distributed in the tropics and sub-tropics, and the major centres for plant domestication and agriculture in Europe and China are temperate.

Millet and sorgum, both C4 species, were both domesticated in tropical Africa, and millet was also domesticated in sub-tropical Asia. Amaranth, a dual-purpose grain and vegetable crop domesticated in central and South America, and now widely grown in Africa, is also a C4 species.

The boom in plant genomics has provided opportunities to identify and analyse the genetic changes underlying the transition from C3 to C4 photosynthesis - and perhaps to transform C3 crops like wheat and rice into more productive C4 crops that will deliver higher yields and be more sparing of the world's dwindling water supplies.

"There's a lot of interest in developing new C4 crops from C3 precursors," Sage says. "I don't know how we're going to feed the world in 50 years' time without substantial improvements in crop productivity and drought tolerance.

"If we can identify the genetic switches required to effect a rapid transition from C3 to C4 carbon fixation, it would be an important step."

Sage suspects that mutations in as-yet unidentified genes for transcription factors may have been involved, because they have the potential to re-regulate entire genetic pathways, leading to wholesale anatomical changes required for the C3-C4 transition.

He says it may be possible to create and select mutants with some of the early transitional traits like denser leaf venation and then move through a series of engineered steps, including enlarged bundle sheaths and localised glycine decarboxylation to achieve full C4 photosynthesis.

But rice, which grows with its roots in water, is an unpromising candidate for such genetic surgery, says Sage. "Rice anatomy is so specialised for C3 photosynthesis that you'd have to take it back to something much more basic before you could advance towards C4 photosynthesis."

Australia's C3 and C4 grasses

Rowan Sage's interest in the origins and taxonomic relationships of C4 plants have taken him to every continent except Antarctica. He also investigates C4 species whose exceptional tolerance of drought and salinity could be used to advantage.

Only a few dozen woody shrubs and trees are represented among the ranks of C4 plants - the saltbush family Chenopodiaceae, widely represented in Australia, is a notable example. Most of Australia's C4 species are tall tropical grasses.

Sage says the Amaranthaceae family, represented in Australia by ornamental herbs like Ptilotus and Gomphrena, is an example of independent evolution of C4 photosynthesis.

All Ptilotus species, found throughout the Australian semi-arid and arid zones, are C3. All Australian species in the cosmopolitan genus Gomphrena are C4, but there are distinct C3 and C4 lineages among non-Australian species.

Like the native chenopod genera Atriplex (saltbushes), Maireana (bluebushes) and Halosarcia (glassworts), Gomphrena appears to have been a relatively late invader that underwent rapid evolutionary radiation in a drying climate, after the continent separated from Antarctica and drifted north towards Asia some 30 million years ago. Another prehistoric C4 invader, Heliotropium, followed a similar pattern.

Sage is very interested in the woody chenopod genus Haloxylon, which grows throughout arid regions of central Asia, and is one of the few C4 plants that grows to tree stature.

Highly drought and salt-tolerant, Haloxylon produces extremely dense wood that burns long and hot. Sage believes it has great potential as a fuelwood species, and for restoring degraded and salinised environments - including regions of Australia affected by dryland salinity.

"Haloxylon is adapted to severe drought, and it can grow in areas where there is not much else of value," he says.

Haloxylon grows slowly, and is not highly productive, but Sage believes could be used to stabilise the extensive areas of degraded or desertified land in Australia, Africa and Asia, while providing cheap, high-quality fuelwood for local populations for cooking and heating.

For centuries, traders moving down the Silk Route between China and the Middle East used Haloxylon as fuelwood for cooking, and to warm themselves at night.

Another woody C4 shrub, Calygonum (Polygonaceae), which grows widely in the Sahara and Arabian deserts, served a similar purpose for camel drivers in Africa and the Middle East.

Sage says both Haloxylon and Calygonum could benefit from systematic selection and breeding to improve their productivity. If they were grown widely in degraded regions of Australia's interior they could draw down carbon from the atmosphere, and offset the energy cost of imported fossil fuels.