More Insights with Infrared

New Method Enables Quantitative Visualization of Sucrose Distribution in Plants

  • Quantitative map of sucrose distribution in a developing barley grain recorded by FTIR microspectroscopy. Red (blue) color values correspond to maximum (minimum) concentration of sucrose. The diagram on the left shows how sucrose enters the grain.Quantitative map of sucrose distribution in a developing barley grain recorded by FTIR microspectroscopy. Red (blue) color values correspond to maximum (minimum) concentration of sucrose. The diagram on the left shows how sucrose enters the grain.

Sucrose is the most important form of sugar transport in plants, the main source of energy and a signalling agent in stress. Standard analytical methods can precisely determine the amount of sucrose, but only if the cells and tissues are destroyed in an extraction process. Advances in infrared spectroscopy now allow quantitative visualization of sucrose distribution with microscopic resolution. This new precision tool offers researchers and plant breeders a wide range of possibilities.

 

Sucrose, also known as cane or beet sugar, influences all growth and development processes due to its elementary tasks. Researchers are therefore constantly developing new measuring methods to determine the quantities of this disaccharide in tissues reliably and quickly. However, none of the current methods allows the visualization and simultaneous quantification of the sucrose distribution in tissues. However, this is exactly what would be necessary to make statements on sugar transport, storage activity and yield formation in crops such as barley, wheat or rape.

 

Infrared Fingerprint of Sucrose

Previous imaging methods for the mapping of sucrose are either not quantitative, too unspecific or even require a genetic modification of the plant to be investigated, which would be problematic for the application in cultivated plants. Based on infrared spectroscopy, a new solution has now been developed: the frozen sample is cut into microscopically thin slices (~10 µm) and measured with an infrared microscope [1]. It has long been known that medium infrared radiation leads to specific oscillations in the sucrose molecule. In the resulting IR spectrum, a characteristic “fingerprint” is generated. The art was to filter this fingerprint out of the remaining signals and the very complex Fourier Transform spectra. This was achieved, among other things, by the introduction of an internal standardization, which, analogous to chromatographic procedures, makes the measurement results batch-independent, more comparable and better quantifiable. After baseline correction and comparison with standard spectra, a mathematical model is created which maps the sucrose distribution in the observed tissue section with an accuracy of at least 90%.

The quantifiable range spans 20-1000 mM, i.e. biologically extremely relevant concentrations. The spatial resolution of the method is essentially determined by the objective used in the IR microscope and can be further increased from currently 12 µm. The amount of work required and the sample throughput that can be achieved are of course also important for analytical methods. Depending on the complexity of the tissue and the resolution, the preparation of a quantitative sucrose map takes 2-6 hours - further optimizations are possible.

 

What Can you See now with the New FTIR Method?

First, we wanted to visualize the sucrose distribution in barley leaves for the first time. Our analyses showed that sucrose was much more concentrated in the phloem than in the surrounding leaf tissue (mesophyll). This concentration is mediated by specific transport proteins, and in the case of barley leads to average sucrose amounts of ~0.3 pg/µm2 in the phloem tissue, which corresponds to concentrations of about 45 mM [1]. The systematic analysis of all phloem pathways of leaf and stem showed that this concentration is subject to considerable fluctuations. This was unexpected as it was assumed that all phloem trajectories represent a continuum. Obviously, there are still unknown mechanisms that influence sucrose transport and need to be investigated. The sugar is transported from the leaf and stem via a central vein into the developing grains (see diagram on the left). As the sucrose map (right side) of a grain shows, sucrose accumulates in the ventral part, where the vein is located and discharge processes take place [2]. From here the sucrose flows along a concentration gradient towards the endosperm (this corresponds to the starch storage tissue inside the grain). Our spatially high-resolution sucrose maps show another special feature: in the area of the transfer cell layers, a local accumulation of sucrose occurs. This is probably due to specific transport proteins which - activated by proton pumps - “pump” sucrose into the endosperm. The new FTIR method was able to identify this region consisting of only a few cell layers as a particularly active zone. The visualization of such small-scale transport regions is of particular interest and can hardly be visualized with previous methods.

The FTIR method can also show what happens when specific transport proteins are no longer present. This was demonstrated with the model plant Arabidopsis thaliana: if members of the Sweets gene family are mutated, this leads to characteristic changes in the sucrose distribution in the root area [3]. This has multiple effects on the growth, metabolism and defence of plant pathogens.

 

Conclusion

The new FTIR process offers a wide range of applications for researchers and companies. Plant researchers can now use the standardized method to systematically measure the sucrose concentration in various tissues, including the pathways of the plant. Such measurements are an indicator of the plant’s growth power, which is important not least in the breeding of new, high-yielding varieties. The method is not limited to sucrose, but can be further developed for the analysis of other ingredients. For example, the FTIR method has already been used to map raffinose (a triple sugar) and starch in oil and cereal seeds [1,4]. In principle, many ingredients can be determined by FTIR as long as they show a sufficiently strong interaction with IR radiation. We are therefore optimistic about many new fields of application.

Authors
André Gündel1, Hardy Rolletschek1, Steffen Wagner1, Aleksandra Muszynska1, Ljudmilla Borisjuk1

Affiliation
1 Institute of Plant Genetics and Crop Plant Research (IPK), Seeland-Gatersleben, Germany

Contact
Dr. Hardy Rolletschek

Institute of Plant Genetics and Crop Plant Research (IPK)
Seeland-Gatersleben, Germany
rollet@ipk-gatersleben.de
 

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References

[1] André Guendel, Hardy Rolletschek, Steffen Wagner, Aleksandra Muszynska, Ljudmilla Borisjuk (2018). Micro Imaging Displays the Sucrose Landscape within and along Its Allocation Pathways. Plant Physiology 178 (4): 1448-1460.

[2] Melkus G, Rolletschek H, Fuchs J, Radchuk V, Grafahrend-Belau E, Sreenivasulu N, Rutten T, Weier D, Heinzel N, Schreiber F, Altmann T, Jakob P, Borisjuk L. (2011). Dynamic 13C/1H NMR imaging uncovers sugar allocation in the living seed. Plant Biotechnology Journal 9: 1022-1037.

[3] Walerowski P, Gündel A, Yahaya N, Truman W, Sobczak M, Olszak M, Rolfe SA, Borisjuk L, Malinowski R. (2019) Clubroot disease stimulates early steps of phloem differentiation and recruits SWEET sucrose transporters within developing galls. Plant Cell 30: 3058–3073.

[4] Munz E, Rolletschek H, Oeltze-Jafra S, Fuchs J, Guendel A, Neuberger T, Ortleb S, Jakob PM, Borisjuk L. (2017). A functional imaging study of germinating oilseed rapeseed. New Phytologist 216(4):1181-1190.

Contact

Institute of Plant Genetics and Crop Plant Research (IPK)


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