Evolution and Development of Leaf Shape

Leaves come in a dizzying array of shapes, from simple, single bladed leaves to more complex shapes made up of smaller leaflets. How is such a diversity of forms generated by the underlying genetic machinery? While we still don't have a complete answer, research recently published in Science has brought us a step closer by identifying the gene responsible for the formation of leaflets in complex leaves.

To find out what gene controls whether leaves become simple or complex, a team of researchers treated Caradmine hirsuta seeds with a chemical that causes mutations and then looked for changes in the leaves of plants grown from that seed. C. hirsuta normally has complex leaves, while its close relative, the model plant Arabidopsis thaliana, has simple leaves. By looking for mutated C. hirsuta plants with leaves that looked like those of A. thaliana, the team hoped to discover the gene responsible for the difference.

The strategy worked wonderfully. After finding C. hirsuta plants with simple leaves, the team was able to identify the gene responsible. The gene, which they named REDUCED COMPLEXITY (RCO), wasn't present in the A. thaliana genome, highlighting the value of working with different species instead of sticking to model organisms. “If we had not compared the two plants, we would never have discovered this difference, as it is impossible to find a gene where none exists,” said Professor Miltos Tsiantis of the Max Planck Institute for Plant Breeding Research, who led the study.

RCO arose through the duplication of a single gene in the ancestor of C. hirsuta and A. thaliana to produce a cluster of three genes. After the duplication, the three genes diverged, following their own evolutionary course; for example, LMI1 (one of the other genes in the cluster) and RCO are active in different places in the leaf, showing complementary expression patterns. Although its ancestors had all three genes, A. thaliana lost two of them during its evolution, leaving it with only LMI1.

RCO is active in the margin of developing leaves between the sites where leaflets will grow out. By restricting growth in these areas, it changes the shape of the leaf, breaking the margin and transforming a simple leaf with a single blade into a complex leaf made up of leaflets. This doesn't happen in plants lacking a working copy of RCO, like A. thaliana or the C. hirsuta mutants the team created. “The leaves of Arabidopsis are simple and entire because growth is not inhibited by the RCO gene,” explained Tsiantis.

An A. thaliana (left) and C. hirsuta (right) plant

But Arabidopsis still has one of the genes from the cluster — LMI1 — so why doesn't that gene reshape the leaves? Is RCO somehow different from the other genes in the cluster? The major difference, the team discovered, is in how and where the genes are expressed. Trangenic C. hirsuta plants with a mutated version of RCO and a normal copy of LMI1 still produced simple leaves, but when the team engineered RCO mutants with a copy of LMI1 that was expressed where RCO normally is, the plants regained their complex leaves. Likewise, expressing LMI1 in the RCO domain in A. thaliana transformed its simple leaves into complex leaves, even though this species doesn't have a copy of the RCO gene. In other words, even though the genes aren't identical — LMI1 normally does something slightly different in another part of the leaf -- they're similar enough that LMI1 can fill in for RCO if it's expressed in the right place.

So that tells us how these little plants in their corner of life evolved different leaf shapes, but can we really generalize to other species? To find out, the researchers selected LMI/RCO-like genes from evolutionarily diverse plants (a rosid and a ranunculid) and tested their effect when expressed in the RCO domain in A. thaliana. In every case except one, the resulting plants had complex leaves, suggesting that the these genes' ability to repress growth probably evolved before the split between eudicots and other seed plants.

The researchers also showed that RCO doesn't seem to affect the distribution of auxin in the leaf, a plant hormone that plays a central role in development and patterning, including leaf serration. Instead of controlling leaf shape by changing the auxin pattern, it accomplishes its task simply by regulating the rate of growth. The separation between patterning (controlled by auxin) and growth (controlled by RCO) offers interesting possibilities for the evolution of development in plants and will likely prove a valuable insight for future research in the field.

Reference
Vlad et al. Leaf Shape Evolution Through Duplication, Regulatory Diversification, and Loss of a Homeobox Gene. Science 343(6172): 780-783. (2014) doi:10.1126/science.1248384

Image credits
The leaf image is by user Debivort on Wikimedia Commons.
The A. thaliana & C. hirusta picture was included in an MPI press release and is © MPI f. Plant Breeding Research/ Lempe.