Further experimental approaches

3.8.1 Collection of Guttation droplets

In order to collect the Guttation droplets for further investigation, the seedlings of barley and garden cress were grown in a hydroponic system as described in (section 3.3; method 2). Two weeks after germination, when the plants are well grown, the jars of control and treated plants were placed in a big plastic box with a lid. The box is filled by one-tenth of its volume with water and closed. Then, the closed box was wrapped with a blanket during the night, to increase the humidity

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in the surrounding atmosphere of the plants. Early in the next morning, the small droplets that formed on the leave tips were collected carefully by a pasture pipette from both the control and treated plants. This procedure was repeated daily for four days to collect adequate amounts, and they were stored in the fridge until analyzed. The collected droplets were then evaporated using a gentle stream of air (Zymark Turbo Vap evaporator), and the residue was redissolved in 1 mL of 80%

methanol to be ready for the HPLC analysis.

3.8.2 Application of some enzyme inhibitors

For certain experimental approaches, besides coumarins, the following enzyme inhibitors were also added to the culture medium, as outlined below.

A)-Naproxen (a putative inhibitor of cytochrome P450 enzymes): Seeds of barley and Lepidium were germinated and grown, as described in method 2 (section 3.3).

When the plants are well grown (two weeks after germination), naproxen was applied in a final concentration of 200 μg/mL to the culture medium simultaneously with umbelliferone. To assess accurately the activity of the inhibitor, an experiment employing seedlings treated only with umbelliferone was also performed at the same time. After five days, the shoots of both plant species were harvested, dried, ground, and extracted to be analyzed by HPLC as described in sections (3.5; 3.6).

B)-N-D-Glucosyl piperidine and N-D-Galactoosyl piperidine (inhibitors of glucosidases): Seeds of barley and Raphanus were germinated and grown, as described in method 2 (section 3.3). When the plants are well grown (two weeks after germination), the inhibitors were applied in a final concentration of 200 μg/mL to the media without the addition of coumarin glucoside, i.e., esculin. After several hours, esculin was added to the culture medium of both plants. To assess accurately the activity of the inhibitors, an experiment employing plants treated

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only with esculin was done also at the same time. The shoots of all plant species were harvested after five days, dried, ground, and extracted to be analyzed by HPLC as described in sections (3.5; 3.6).

It is worth mentioning that a simple approach was performed to confirm the activity of apoplastic glucosidases. Barley seedlings were cultivated as described in method 2 (section 3.3). After two weeks of germination, when the seedlings are well grown, a tiny amount (~ 10 mg) of 4-Methylumbelliferyl-β-D- glucoside was added to the culture medium. The appearance of a blue fluorescence through the medium is considered as proof for the glucosidase activity in the medium.

3.8.3 Incubation of excised leaves and roots with umbelliferone

In the following experiment, barley and Lepidium were cultivated in a hydroponic system as described in method 2 (section 3.3). After two weeks of germination, when the plants are well grown, their leaves and roots were excised.

The cut leaves and roots were incubated separately inside Petri dishes containing 20 mL of umbelliferone (Figure 3-4A; B, respectively). After five days, these roots and leaves were washed twice with distilled water to remove any adhered residues of umbelliferone, dried by paper towel and dipped in liquid nitrogen, then in the freeze dryer, ground, and extracted to be analyzed by HPLC as described in sections (3.5; 3.6). As a control, the cut leaves and roots of the same plant species were handled in the same manner, however, they were incubated only in a culture medium without umbelliferone.

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Figure 3-4: Excised leaves (A) and roots (B) are incubated with umbelliferone in Petri dishes.

In another experimental approach, only excised barley leaves were employed, however in a slightly different setup: The cut barley leaves were incubated in umbelliferone-containing medium into two different positions; an upright position where the cut ends of the leaves were immersed in the medium (Figure 3-5A), and upside-down position where just the tips of the leaves were soaked in the medium (Figure 3-5B). After five days, these leaves were washed twice with distilled water to remove any adhered residues of umbelliferone, dried by paper towel, and dipped in liquid nitrogen, then in the freeze dryer, ground, and extracted to be analyzed by HPLC as described in sections (3.5; 3.6). As a control, the cut leaves of barley were handled in the same manner, however, they were incubated only in a culture medium without umbelliferone.

Figure 3-5: Excised barley leaves are incubated with umbelliferone, either in an upright position (A) or in an upside-down position (B).

35 Chapter 4: Results

4.1 Establishing and optimization of a suitable hydroponic system

In order to thoroughly investigate the uptake of a certain compound by plants, a suitable and efficient system that allows the required variation of experimental parameters has to be established and optimized. Therefore, a convenient hydroponic system was developed and optimized to finally achieve the requirements.

In the first trial, various sizes and shapes of test tubes were used to host a single seedling (Figure 4-1). However, this approach was time-consuming, since the second replica of the same experiment is done after more than one week when the first one is finished. The necessary aeration required was realized by bubbling air through small tubes. Unfortunately, the airflow could not be regulated properly to ensure identical aeration in all tubes.

Figure 4-1: Barley seedlings wrapped by sponge and cultivated in test tubes containing Hogland’s medium. Aeration was performed by the small pipes (the first trial in establishing the hydroponic system).

Consequently, the conditions for the seedlings of one batch had not been really identical. Therefore, all seedlings of one batch should be cultivated in the same