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You Shall Not Pass! Time-gated Detection Takes Care of Endogenous Fluorescence in Plant Research.

Interview with Prof. Yutaka Kodama from Utsunomiya University in Japan

Yutaka Kodama  is Associate Professor at the Biosciences Education Research Center, Utsunomiya University, Japan. He graduated from Saga University, Faculty of Agriculture, in 2004 and received his PhD from the Graduate School of Biological Science at the Nara Institute of Science and Technology in 2007.

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Prof. Kodama, what is the focus of your lab?

My laboratory is at a leading institution for plant cell biology. We investigate the arrangement of organelles within cells and the mechanisms of their movement. The simplest example is the behavior of chloroplasts, which change their intracellular positions to optimize photosynthetic activity and/or reduce photodamage upon light irradiation. We are interested in elucidating the mechanisms that govern this repositioning.

Why is endogenous fluorescence an issue in your field of research?

Dealing with endogenous fluorescence has been a long-standing issue in plant research. For example, it may appear as a fluorescent protein signal in chloroplasts and, therefore, lead to false conclusions. Endogenous fluorescence is very difficult to eliminate using chemical treatments of the sample (they often destroy the sample). Conventional spectral unmixing methods also fail.

How has time-gated detection changed your experiments?

The first time we applied time gated imaging on one of our samples, the endogenous fluorescence “disappeared”. My students were very excited. I remember saying “This is amazing!”, when I saw the results. Now, we use time-gated imaging routinely and can image any protein without worrying at all about endogenous contributions.

For example, we study the light-induced cellular response of phototropin, a blue-light photoreceptor localized at the chloroplast periphery protein fused with YFP fluorescent protein. When performing imaging with such fluorescent constructs, it is important to account for the contribution coming from the endogenous chlorophyll fluorescence. We showed that this was possible thanks to time-gated detection in an early publication from our lab [1].

Chloroplasts contain green grains that are important for photosynthesis. Under low light conditions, they are positioned near the surface of the cell to optimize the light yield. If they are exposed to higher irradiation conditions, they move towards the center of the cell to avoid damage. Chloroplasts are known to show a similar response to temperature and we recently discovered that phototropin is a temperature acceptor [2]. We posted a short video clip featuring this effect of moving chloroplasts on our homepage. It shows their reaction at low temperature. However, chloroplasts decrease their ability to use light for the photosynthesis with dropping temperature. At low temperature, chloroplasts seem to misinterpret this stimulus and move to the center of the cell as if hit by strong irradiation. The effects of light and temperature are very closely related. Because the position of chloroplasts in cells changes dynamically, depending on the light intensity, it is possible to diagnose whether the current state is a low light or a strong light environment by looking at the plant cells. When we analyzed plant cells kept in an artificial environment, we found that the placement of chloroplasts in cells affects the growth of the plants.

How can these findings change our daily life?

I believe we are heading towards a change in the way we grow plants. For example, a lot of farms depended on rebuilding efforts after the Great East Japan Earthquake. I visited one of these farms and I realized there is a lot of potential to use plant physiology to optimize growth factors in this very concrete situation. The people who had been farmers until then raised vegetables suitable for the land, so there was no need to adapt the environment. However, growth-influencing factors, such as light intensity and temperature, play a critical role when the same vegetables must grow indoors. In my lab, we saw this as a chance to provide the technology to grow plants better adapted to the environment, using chloroplasts as growth sensors [3].

Why do you continue to use imaging methods for experimental plant research?

Investigation with a microscope provides the ability to reveal novel discoveries. In the cell, there are many things we do not know yet as there are many things that we do not see. One can find actin and microtubule filaments, chloroplasts, mitochondria, only to cite some examples. Finding them is extremely complicated, as the organelles move. Microscopy provides a unique insight into this world and nothing compares with the excitement of a new discovery observed through the eyepieces for the first time.

This interview has been translated from the Japanese webpage.