Seeing the light in the egg of a clawed frog

Plants produce sugar during photosynthesis. Water is released into the environment in the form of water vapor. This is the responsibility of tiny “valves” on the surface of the leaf, which consist of guard cells arranged in pairs. Depending on whether these guard cells are bulging or comparatively empty, they change their shape – in the same way as a swim ring, which is circular when inflated but can be folded tightly when all the air is released from it.

Guard cells regulate the water exchange

In plant terms, this means as follows: two bulging guard cells form a circle, enabling the release of water vapor into the environment. If they go limp, the valve closes, the plant retains the water internally, and in so doing protects itself against drying out. How this process works at molecular level has been examined by Dr. Dietmar Geiger. Geiger works as an assistant to Professor Rainer Hedrich in the Department of Molecular Plant Physiology and Biophysics.

The findings of his work are reported in the current issue of the journal Proceedings of the National Academy of Sciences (PNAS, USA).

“During times of drought, plants create what is known as a drought stress hormone, which causes the pairs of guard cells to go limp through a chain of reactions in which calcium is also involved,” explains Dietmar Geiger. The “valve” closes, thereby reducing the release of water from the leaf. As the biophysicists discovered in earlier experiments, this process involves certain ion channels and enzymes that fine-tune the process. The scientists were able to clarify which ones exactly using a clever technique that Rainer Hedrich established a good ten years ago that allows ion channels to be examined outside plant cells. The key components are: eggs from a clawed frog and a yellow fluorescent protein.

Complicated search for the enzyme responsible

“The earlier work by Dietmar Geiger, which was also published in PNAS, led us to assume that a very specific anion channel is involved in this process,” explains Rainer Hedrich. What was, however, a mystery was which enzyme is responsible for opening this channel to calcium ions. There were, after all, 34 enzymes to choose from.

It was a molecular biology trick that helped them see the light, quite literally: “We coupled the gene for the guard cell anion channel to one half of the gene for the yellow fluorescent protein. We then bonded the other half to each of the 34 possible enzyme genes in turn,” explains Dietmar Geiger.

Traces of light in the egg of a clawed frog

The idea behind this: in this scenario, the yellow fluorescent protein will only illuminate when the proteins of the enzyme and of the anion channel that have been fused to the two halves are moved to within close proximity to one another. And the eggs of the clawed frog came into play because, firstly, they are sufficiently transparent and, secondly, they work perfectly as a “test tube for loading with foreign genes and translating into active proteins,” says Rainer Hedrich.

The two scientists did indeed succeed in identifying the corresponding calcium-dependent enzyme, a so-called kinase, using this elegant, experimental approach, with the ion channel as bait. The Würzburg “channel workers” then applied the same approach to determine the enzyme that disables the channel again – a protein phosphatase.

Support from Munich

The following questions remained: how do these two switch elements sense the drought stress hormone, and what sensor regulates the activity of the kinase/phosphatase pair? To find this out, the Würzburg researchers collaborated with Professor Erwin Grill's team from the Technical University Munich. The people from Munich had identified a protein that deactivates the phosphatase when it has bonded with the water stress hormone.

This knowledge gave them the final link in the signal chain: “In the presence of the stress hormone, a receptor is stimulated that inhibits the phosphatase. The kinase transfers energy-rich phosphate to the anion channel, thereby activating it. The release of anions triggers a flow of potassium and water, the guard cells release their pressure, and the plant survives the drought with its stomata tightly closed”, explains Dietmar Geiger.

However, not every question has been answered. There is just “one small, but not insignificant detail” remaining, says Rainer Hedrich: “How does the calcium ion get into the cell?” But for this too the Würzburg plant physiologists have already come up with an idea.

The researchers

Dr. Dietmar Geiger received his doctorate at the Department of Molecular Plant Physiology and Biophysics. He then became a post-doctoral student at the Max Planck Institute of Biophysics in Frankfurt. As an assistant to Professor Rainer Hedrich, he applies molecular and biophysical methods in order to understand the structures of ion channels and metabolite carriers that account for the special function of membrane proteins.

Professor Rainer Hedrich was a pioneer in the discovery and deciphering of the special function of ion channels in plants. So far, he has deciphered all the major ion channels of the guard cell – starting with his discovery of the first ion channel in plants, the potassium channel of the guard cell, back in 1984 while working toward his doctorate.

“Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities”; Dietmar Geiger, Sönke Scherzer, Patrick Mumm, Irene Marten, Peter Ache, Susanne Matschi, Anja Liese, Christian Wellmann, Khaled A.S. AL-Rasheid, Erwin Grill, Tina Romeis and Rainer Hedrich. Proc Natl Acad Sci USA. doi/10.1073/pnas.0912030107

Contact:
Prof. Dr. Rainer Hedrich, T: +49 (0)931 31-86100,
e-mail: hedrich@botanik.uni-wuerzburg.de
Dr. Dietmar Geiger, T: +49 (0)931 31-86105,
e-mail: geiger@botanik.uni-wuerzburg.de