Raffinose oligosaccharides in seed germination

Apart from sucrose, raffinose family oligosaccharides are the major soluble sugars in plant seeds. They are regarded as anti-nutritional factors, because humans and many animals lack enzymes to digest them. Therefore, they are attractive targets for plant breeders to improve the nutritional characteristics of crop seeds. On the other hand, their importance for the plant is not yet clear. Raffinose oligosaccharides are stored in virtually all plant seeds, while they are only occasioally found in leaves or roots. In contrast to these vegetative tissues, seeds are able to withstand drying to very low water contents, and some of them survive in this state for years, retaining the ability to germinate. The preference of plant seeds to store raffinose oligosaccharides suggests that these sugars may be important components for seed survival or subsequent germination. Despite intense research efforts, a protective role of raffinose oligosaccharides for cellular structures during desiccation or storage remains contentious. The role of raffinose oligosaccharides as reserves fuelling germination, on the other hand, has been scarcely studied. During germination, raffinose oligosaccharides are mobilized very early and are depleted before the major storage reserves of plant seeds (such as starch, protein and oil) are mobilized. The molecular events which lead to their breakdown are not known, and it is unclear how the breakdown products of raffinose oligosaccharides are utilized. This research project aims at clarifying these important questions in seed physiology. To achieve this goal, we will characterize the enzymes and the corresponding plant genes that are responsible for the mobilization of raffinose oligosaccharides. We will analyze where in the cells this breakdown takes place, and we will investigate how this process is regulated. We will use inhibitors to block the mobilization of raffinose oligosaccharides, and analyze how seeds germinate if they can not utilize them as reserves. Finally, we will study whether seeds have molecular sensors for the breakdown products of raffinose oligosaccharides, which could regulate their utilization for growth or respiration. Thereby, this project will not only provide informations to value the importance of raffinose oligosaccharides storage for plant seeds, but will also provide insights into very early events of germination, which is still one of the most mysterious periods in a plant`s life.

Raffinose Family Oligosaccharides (RFOs) are ubiquitous in plant seeds. They are rapidly mobilized during germination and are thought to play critical roles for seed desiccation tolerance and longevity. The aim of this project was to understand the significance of RFO breakdown for seed germination and clarify the role of the enzymes that mobilize RFOs, namely a-galactosidases. In the first part of the project we used a specific a-galactosidase inhibitor to show, for the first time, that RFOs are indeed the primary energy and carbon source for germination. Pea seeds treated with the inhibitor showed significantly lower germination rates than control seeds. To prove that the lack of energy caused by the blocking of RFO breakdown is responsible for the drop of germination rates, we added sucrose or galactose, both substrates of RFO breakdown, to inhibitor treated seeds during germination. Surprisingly, germination rates were not affected by sucrose addition, whereas galactose could completely relieve the inhibition. These results therefore show that while RFOs act as the primary energy source during germination it`s the galactose released from the RFOs that play an important role in the regulation of germination. Another part of the project dealt with cloning and characterizing two classes of a-galactosidases from pea seeds and developing an integrated model for RFO breakdown in germinating pea seeds. We were able to demonstrate that acidic a-galactosidases are already synthesized in the developing seed and stored in vacuoles, whereas RFOs, which are building up during seed development, are mainly found in the cytosol, where they are thought to protect proteins and membranes during desiccation. During imbibition and germination RFOs are then transported from the cytosol into the vacuoles, where about 50% of them are broken down by the acidic a-galactosidases. Only after this happens, alkaline a-galactosidases are synthesized and then facilitate the breakdown of the remaining RFOs in the cytosol. Thus acidic and alkaline a-galactosidases are not only sequentially active (are active at different stages of germination), but also break-down RFOs in different cellular compartments (are active in different places). In summary we were able, for the first time, to show that two galactosidases, an acidic and an alkaline enzyme are active in one seed source and to show that they are sequentially active in the coordinated breakdown of RFOs.