




The ornate leaves of a humble sprig of cilantro are produced by the action of the plant hormone auxin.



In recent years, as more and more of these proteins were discovered, the auxin signaling machinery began to seem baroque to the point of being unintelligible.



We have some evidence that proteins chain in plant cells as well as in solution, said senior author Lucia Strader, PhD, assistant professor of biology, in Arts & Sciences, and an auxin expert. By varying the length of these chains, plants may fine-tune the response of individual cells to auxin to produce detailed patterns such as the toothed lobes of the cilantro leaf.

Combinatorial explosion
Sculpting leaves is just one of many roles auxin plays in plants. Among other things, the hormone helps make plants bend toward the light, roots grow down and shoots grow up, fruits develop and fruits fall off.


The backstory here is that whole families of proteins intervene between auxin and genes that respond to auxin by making proteins. In the model plant Aribidopsis thaliana, these include five transcription factors that activate genes when auxin is present (called ARFs) and 29 repressor proteins that block the transcription factors by binding to them (Aux/IAA proteins). A third family marks repressors for destruction.

“Different combinations of these proteins are present in each cell,” Strader said. “On top of that, some combinations interact more strongly than others, and some of the transcription factors also interact with one another.”

In an idle moment, David Korasick, a graduate fellow in the Strader and Jez labs and first author on the PNAS article, did a back-of-the-envelope calculation to put a number on the complexity of the system they were trying to understand. From a strictly mathematical point of view, there are 3,828 possible combinations of the auxin-related Arabidopsis proteins. That is assuming interactions involve only one of each type of protein; if multiples are possible, the number, of course, explodes.



Korasick had the job of crystallizing ARF7, a transcription factor that helps Arabidopsis bend toward the light. With the help of Joseph Jez, PhD, associate professor of biology, and postdoctoral research associates Corey Westfall and Soon Goo Lee, Korasick cut “floppy bits” off the protein that might have made it hard to crystallize, leaving just the part of the protein where it interacts with repressor molecules.

After he had that construct, crystallization was remarkably fast. He set up his first drops in solution wells on the Fourth of July. The protein crystallized with a fuss, and he ran the crystals up to the Advanced Photon Source at the Argonne National Laboratory outside Chicago. By Aug. 1, he had the diffraction data he needed to solve the protein’s structure.

Surprise, surprise


The transcription factor ARF7 turned out to have a magnet-like interaction region, called a PB1 domain, with positively (blue) and negatively (red) charged faces. (Credit: Strader, Jez et al.)






The double-sided interaction domain may allow multi-protein chains to form. In Korasick’s crystal, five of the ARF7 PB1 domains stuck to one another, forming a pentamer. “It was really beautiful to look at in the software, because you could actually see its spirals and turns,” said Korasick. (Credit: Strader, Jez et al.)




“The difference between plants and animals,” said Strader, “is that plants have rigid cell walls. So when a plant cell decides to divide itself or lengthen itself, that’s a permanent decision, which is why it’s so tightly controlled.“