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Using super resolution microscopes to study drought resistance in plants

New super resolution microscope technologies have allowed researchers to measure the movement of individual molecules of membrane proteins, known as aquaporins, involved in water transport in plants. Aquaporin mobility may play an important role in drought and salinity tolerance mechanisms.

View of individual membrane protein molecule (LTi6a) trajectories on the epidermal cells of an Arabidopsis thaliana root. They are marked with fluorescent dye and seen under a super resolution microscope.. © INRA, Doan Luu
By Pascale Mollier, translated by Daniel McKinnon
Updated on 04/16/2015
Published on 03/25/2015

Deeper into the darkness of cells

At the close of the nineteenth century, German physicist Ernst Abbe, a microscopy pioneer, theorised that it would be impossible to see objects smaller than 200 nm because of the diffraction of light. According to Abbe, we should never have been able to see viruses or proteins (see Box Text 3). Three animal science researchers were able to refute this theory by bringing together their respective techniques, and were awarded the Nobel Prize in Chemistry 2014 for their discovery.

Use in plants

Researchers at INRA Montpellier, working with their colleagues in Bordeaux, were able to apply this “super resolution” microscope technology to plants. In looking at the model plant species Arabidopsis thaliana, they were able to see how each protein molecule moves in the cellular membrane. “We were interested in aquaporins, which are proteins found on the epidermal membrane of Arabidopsis roots. These proteins act as channels allowing water exchange between a plant and its external environment. They therefore play an important role during periods of drought or increased salinity, which are referred to as osmotic stresses” says Doan Luu. When the soil is dry, or when the plant lacks water and is retaining salt, water moves from the plant to the external environment by way of osmosis (1), thereby exacerbating a plant’s water loss.

Tracking proteins

To see how the aquaporins behaved, researchers used the techniques developed by the three Nobel Prize winners that made it possible to track the movement of each aquaporin molecule in a membrane (see Box Text 2). “We noticed that these molecules move very little under a plant’s normal growing conditions, which somewhat contradicts the “fluid mosaic” model traditionally used to describe cellular membranes. Importantly, we demonstrated that aquaporin mobility increases during periods of osmotic stress, thereby allowing the internalisation of aquaporins through endocytosis. The removal of aquaporins from the membrane surface reduces the flow of water and, in all likelihood, allows the plant to combat drought and excess salt” says Luu.

Aiming to improving drought resistance

Researchers are currently trying to understand the mechanisms that modify aquaporin mobility during periods of osmotic stress. Research has shown that there is a network of actin cables directly below the membrane and that membrane proteins run up against this network when moving laterally. It could be that the actin network is disrupted during periods of osmotic stress, thereby facilitating protein movement. Another possible hypothesis involves the modification of membrane lipids. Researchers are studying the components of endocytosis, drawing inspiration from findings in the field of animal science and by using a variety of plants to study the individual components.

“Our ultimate aim is to develop mechanisms that would allow us to select plants that have better drought resistance because their membrane aquaporins are more mobile and act more quickly in endocytosis mechanisms. We will apply this research to major crop plants, rice in particular” says Luu.

(1)  Water moves from a region of higher salt concentration to a region of lower concentration.
(2)  In the “fluid mosaic” model, proteins “float” fairly freely in the lipids that constitute the membrane.

Reference

Hosy E, Martinière A, Choquet D, Maurel C, Luu DT. 2014. Super-resolved and dynamic imaging of membrane proteins in plant cells reveal contrasting kinetic profiles and multiple confinement mechanisms. Molecular Plant 8(2): 339-342.

Each molecule is tracked individually

The membrane proteins of the epidermal cells of an Arabidopsis root (not an aquaporin in this case but rather a more mobile protein known as LTi6a) are marked with a fluorescent protein, mEos, derived from coral. Once they are marked, the proteins emit red light when they are activated with a laser. The trick is to use the laser with very low power. In this way, only a single molecule is randomly activated, allowing its trajectory to be visualised with the help of false colours. This technique is known as sptPALM (single particle tracking photoactivated localisation microscopy).

Breaking the 200nm barrier!

Scale of organisms, from German physicist Ernst Abbe. © Wikipedia
Scale of organisms, from German physicist Ernst Abbe © Wikipedia

Until the 2000s, it was not possible to distinguish shapes that were less than 200 nanometres apart, particularly because of phenomena associated with the diffraction of light. The use of a number of new technologies in combination made it possible to obtain a resolution of 40 nm, thereby allowing protein molecules to be distinguished. In this case, the microscope should more correctly be called a “nanoscope”. For this new advance in technology, the Nobel Prize in Chemistry 2014 was awarded to German Stefan W. Hell and Americans Eric Betzig and William E. Moerner, whose respective work focused on the synapses between neurons, cellular division within embryos, and proteins associated with Huntington’s disease.