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How is phytochrome B translocated into the cell nucleus?

Plants cannot see, but they can perceive the quantity and quality of light. As they have evolved, plants have developed numerous molecular photodetectors such as phytochromes. Phytochromes can detect changes in the light situation. The undergrowth of forests thus manages to grow towards the few patches of sunlight that the phytochromes can detect. Researchers have long puzzled over how phytochromes transmit information about the light level into the nucleus and enable plants to react to changing light situations by altering the activity of specific genes. Four years ago, a group of researchers led by Prof. em. Dr. Eberhard Schäfer clarified the principle underlying the translocation of phytochrome A into the nucleus. Now, the team led by Schäfer and Dr. Tim Kunkel has achieved the same success with phytochrome B. The researchers may have discovered a new universal principle that is also of importance in the transduction of signals in animal and human cells.

Fighting for light: Plants growing close to the ground sometimes need to twist around in order to gain exposure to life-giving sunlight. © Wikipedia

Plants that grow in the shade of a neighbouring plant cannot just step to the side where more light is available. But they can grow towards the light, even if this means that they have to find some way to “crane their necks”. In order to be able to react to changes in the ambient light level, plants have evolved a number of light receptors that are sensitive to specific light waves. These receptors can react to changing light conditions with changes in their three-dimensional structure. These changes lead to the activation of the receptors, which can then transfer the signals into the cell. Phytochrome is one such photoreceptor; it is sensitive to light in the red and far-red region of the visible spectrum. When activated, it can switch on numerous genetic programmes in the cell nucleus. In situations where plants do not have enough available light, such programmes can induce longitudinal growth in the soil or the development of side shoots. “We already understand some aspects of the molecular signalling of phytochromes. This was not the case a short while ago. Until recently, many researchers were still puzzling over the question as to how information about the light level was transferred from the different phytochrome types in the cytoplasm to the cell nucleus,” said Professor Dr. Eberhard Schäfer from the Department of Molecular Plant Physiology at the Institute of Biology II at the University of Freiburg.

Not a classic nuclear transport mechanism

Schäfer has been dealing with the mechanisms of light perception and photomorphogenesis of plants for around forty years now. The importance of Schäfer’s research becomes quite obvious when one looks at a crop field where plants grow in closely planted rows. The plants block each other’s light and are engaged in a kind of battle for access to the sun’s rays. In order to optimize plant growth, the plants would need to be planted further away from each other, which in turn would reduce the number of plants that can be grown and the yield. “If we understand how changes in the light situation affect a plant’s growth, it will perhaps be possible at some stage in the future to interfere with these molecular processes and breed plants that can grow nearer to each another and still produce high yields,” said Schäfer.

In the meantime, biologists have obtained some reasonably good insights into how phytochromes “perceive” light and the molecules that control this process. “However, there is another aspect that needs to be tackled. Phytochromes modulate gene expression in order to control many physiological progresses. But how can they do this given that they are synthesized in the cytosol and need to be translocated into the nucleus to achieve this control?” asks Dr. Tim Kunkel, group leader in Schäfer’s department.

Four years ago, Schäfer’s team, which at the time also included Dr. Andreas Hiltbrunner who now works at the Tübingen-based Centre for Plant Molecular Biology, succeeded in identifying a molecular shuttle service for phytochrome A. The inactive form of phytochrome absorbs red light and is activated; it then interacts with the molecule FHY1, which transports more and more phytochrome A molecules through the nuclear pores. This mechanism does not apply to phytochrome B. The biologists have been studying the phytochrome B mechanism for many years, without being able to find any solutions. And worse still, at one stage they were even on completely the wrong track: they assumed that light-induced changes in the structure of phytochrome B would lead to the exposure of a nuclear localization sequence (NLS), which would then mediate the translocation of the phytochromes into the cell nucleus. However, such a nuclear localization sequence could not be found in phytochrome B.

Siphonal Acetabularia algae are single celled algae composed of three segments – the “foot” which contains the nucleus, the stalk and the cap from which the gametes are released. © Dr. Tim Kunkel

The hypothesis was discovered to be completely erroneous when Schäfer’s and Kunkel’s team reconstructed a minimal form of the system in a test tube: together with Anne Pfeiffer, who spent both her graduate and doctoral periods in Kunkel’s laboratory, Kunkel developed an in vitro system containing nuclei of Acetabularia, a genus of single celled green algae. These nuclei are veritable giants: up to 200 micrometres in size, they are as big as an Arabidopsis cell. “This system is excellently suited for testing whether phytochrome B translocates into the nucleus on its own when it is exposed to different quantities and qualities of light or whether it requires additional factors to do so,” said Kunkel who, together with the main author of the resulting scientific study, Dr. Anne Pfeiffer, and some graduate and doctoral students has been working on this particular project for the last four years. 

Four photos showing a transparent nucleus. The nuclei on the left are grey; the nucleus in the top right photo glows green; in the bottom right photo only the area around the nucleus glows weakly.
Nuclei of the green alga Acetabularia. Phytochrome B was labelled with a fluorescent dye and glows green. NLS = nuclear localization sequence. ABP = fragment of a phytochrome-interacting factor (PIF) that can bind to phytochrome B. The nucleus appears green (right) only when ABP and NLS are added to the experiment. The control experiment shown in the bottom part of the photo shows that phytochrome B is not translocated into the nucleus in the absence of ABP and NLS. © Dr. Tim Kunkel

A brilliant principle of evolution

Using a fluorescence microscope, the researchers found that fluorescence-labelled phytochrome B only accumulates in the nucleus when the solution also contains phytochrome-interacting factors (PIFs). These molecules are transcription factors that translocate into the nucleus when cells are exposed to light. PIFs have a nuclear localization sequence that enables them to move from the cytosol into the nucleus. This is a revolutionary finding as it implies that phytochrome needs to bind to a PIF in order for it to mediate its effect in the cell nucleus, i.e. enable the expression of genes.

Six images of plant cells. The photos in the bottom row show light nuclei; no nuclei can be seen in the images in the top row.
Images of phytochrome B-YFP (yellow fluorescent protein) in Arabidopsis cells after 30 minutes of red light irradiation. The bean-shaped structures with the light spots are cell nuclei. Top row: mutant lacking the four different phytochrome-interacting factors (PIFs) under investigation. Bottom row: wild-type Arabidopsis cells at the same point in time already have twice as much PHY-YFP in the nucleus. The quantity of PHY-YFP was determined from the grey colour of the nuclei. © Dr. Tim Kunkel

“This is a really brilliant principle that has been brought about by evolution. Phytochrome B is translocated into the nucleus by the very same molecules which it activates in the nucleus. The activated molecules switch on genes and are then degraded,” said Schäfer. “Evolution has ensured that the components that need to interact in the nucleus enter it together.” Schäfer and Kunkel believe that the interest of the system lies in the fact that it might be universal. They assume that it will also be found in the cells of other organisms, including humans, as well as in different signalling systems. 

The researchers from Freiburg have confirmed their in vitro results in experiments carried out in cooperation with researchers from Hungary. The researchers used living Arabidopsis cells that lack four different PIFs and found that photoreceptors did not accumulate in the cell nuclei of these mutants when exposed to light for two hours. The researchers found that YFP-labelled phytochrome B molecules only started to translocate into the nucleus after two hours or more. “This shows that PIFs are also required for transporting phytochrome B into the nucleus in vivo,” said Kunkel. “However, this also shows that other factors are able to partially replace the effect of PIFs.”

Will farmers soon be able to determine how close together their plants can grow? “I believe that this is feasible,” said Schäfer, going on to add “but green genetic engineering is not wanted in Europe, which is why our knowledge will have to be transferred to classical breeding so that we can breed the right mutants naturally. But I am pretty sure that this will take quite a long time.” 

Further information:

Dr. Tim Kunkel
Institute of Biology II
University of Freiburg
79104 Freiburg
Tel.: +49 (0)761/ 203 2662
Fax : +49 (0)761/ 203 2612
E-mail: tim.kunkel(at)biologie.uni-freiburg.de

Website address: https://www.biooekonomie-bw.de/en/articles/news/how-is-phytochrome-b-translocated-into-the-cell-nucleus