Vladimír Skalický is standing on the left side of the image next to an open machine. He is looking to Ioanna Antoniadi who is standing on the right side of the image pointing with her right hand towards two computer screens.Vladimír Skalický and Ioanna Antoniadi are working at the cell sorting instrument which allows separating differently labelled cell compartments (Photo: Asal Atakhani).

Research teams from Sweden and the Czech Republic have developed a novel method that allows to analyse low concentrated substances on the subcellular level. Using this method, the researchers demonstrated that plant growth substances are distributed unevenly in the different compartments of a plant cell. Although the method was initially established on plant cells, its potential extends to numerous research applications beyond plant science.

The study, published online in September in The Plant Journal Technical Advance, involved researchers from Umeå Plant Science Centre (UPSC), a collaboration between Swedish University of Agricultural Sciences (SLU) and Umeå University, from Palacký University and from the Czech Academy of Sciences.

Plant hormones, which are growth substances found in very low concentrations, regulate diverse developmental processes in plants. Karin Ljung’s and Ondřej Novák’s research groups had previously analysed plant hormones at the tissue and cellular levels, but the resolution of the analytical methods had not been sufficient to measure plant hormone concentrations inside a cell and in its compartments, the organelles.

Plant hormones are unevenly distributed within plant cells

“Based on the distribution of proteins responsible for transporting plant hormones within and between cells, we assumed that plant hormones themselves were also distributed unevenly within plant cells. However, this remained unproven until now”, says Karin Ljung who is professor at SLU and group leader at UPSC and has a longstanding collaboration with professor Ondřej Novák, who leads the Laboratory of Growth Regulators at Palacký University Olomouc and the Institute of Experimental Botany at the Czech Academy of Sciences.

The researchers named their new method “Fluorescence-Activated multi-Organelle Sorting” or FAmOS for short. They labelled four different organelles in the cell with distinct fluorophores - chemical compounds that emit specific colours of light when they are excited by light. Every of these organelles received a different label, and the researchers then sorted them according to the colour that the respective label emitted.

Simultaneous isolation of four different organelles

“We chose this approach because previous fractionation techniques were both too time-consuming and lacked common features. When using different methods to isolate, for example, the nucleus and the chloroplast, concentrations of plant hormones in the two separately isolated organelles were not comparable with each other”, says Ioanna Antoniadi who is working in Karin Ljung’s group. “It was very challenging to find the right conditions to keep the sorting time short while ensuring the stability of plant hormones, but finally, we managed to get it to work.”

On the left side, a graphical overviews of the compartments of a plant cell that are labeled in different colours are shown. On the upper right corner the concentration of the plant hormone auxin is shown in different blue coulors in the different cell compartments illustrating that the highest concentration. Similarly, the concentration of the plant hormoone cytokinin in the different cell compartments is shown on the lower right corner in different red colours.  The researchers labelled four different organelles (nucleus (Nu), Endoplasmic reticulum (ER), Chloroplast (Chl) and Mitochondrion (Mi); illustrated left) and separated them from the rest of the cell which consists mainly of the vacuole. Then, they measured the concentration of the plant hormones auxin (top right) and cytokinin (bottom right) in the different organelles. The colour range illustrates the amount of cytokinins in the different organelles: a dark colour corresponds to a high concentration, a light colour to a low concentration. Blue is used for Auxin concentration and red for Cytokinin concentrations. Illustration: Ondřej Novák

Existing methods were optimised to measure minute plant hormone amounts

Another challenge the researchers encountered was the low concentration of the plant hormones in plant cells. After separating the organelles, the plant hormone concentration was measured within them using liquid chromatography coupled to tandem mass spectrometry. This technique is typically used to separate various compounds in a mixture based on their mass and then identify and quantify them based on their mass. While it had been previously used to measure different plant hormones in plant tissues, organs or cells, it had not been employed for minute amounts such as found in 200.000 organelles per sample.

“We were many times close to the detection limit of mass spectrometry. As comparison, one can imagine dissolving one teaspoon of the plant hormone cytokinin in an Olympic size swimming pool and then taking out only ten microlitres of this mixture for the analysis,” explained Vladimír Skalický who developed the method together with Ioanna Antoniadi as part of his PhD thesis in Ondřej Novák’s group at Palacký University, Olomouc.

“The amount of cytokinin in these ten microliters corresponds to the quantity of cytokinin in one ten-day old thale cress seedling, which is just about five centimetres long and served as the starting material for our experiments. We needed several attempts and comprehensive sets of control experiments until we ultimately succeeded.”

The new method has great potential for numerous future applications

The researchers believe that their new method has great potential for numerous future applications. It is not restricted to plant cells but can be applied to mammalian, algae or bacterial cells as well and allows to analyse other substances than plant hormones once the organelles are sorted and separated. This includes for example the measurement of other metabolites, proteins as well as gene activity at the subcellular level.

“It has been extremely important to develop this highly sensitive and robust method. It is efficient in terms of time and enables us monitor plant hormone concentrations and also other metabolites within the cell itself”, says Ondřej Novák. “Such high-resolution mapping of metabolites within isolated organelles will improve our understanding of metabolic and signalling processes within the cell.”

The article

Skalický, V., Antoniadi, I., Pěnčík, A., Chamrád, I., Lenobel, R., Kubeš, M.F., Zatloukal, M., Žukauskaitė, A., Strnad, M., Ljung, K. and Novák, O. (2023). Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution. Plant J. https://doi.org/10.1111/tpj.16456

For questions, please contact:

Karin Ljung
Umeå Plant Science Centre
Department of Forest Genetics and Plant Physiology
Swedish University of Agricultural Sciences
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