C ADMIUM STRESS

For further investigation of the genes of interests the plants were cultivated in hydroponics (4.2.3). The plants were cultivated for up to 18 days in a nutrition media including cadmium. After certain time morphological differences could be monitored in comparison to the non-treated plants (figure 17).

Figure 17: Cultivated maize plants grown in hydroponics with and without a cadmium [Cd(NO)3 ·4H2O] concentration of 15 µM. A) Shoot of control plant; B) Shoot of cadmium stressed plant; C) Root of control plant; D) Root of cadmium stressed plant. Morphological differences are displayed.

The cadmium treated plants showed morphological changes in root and shoot. The development was decelerated in comparison to the control plants. The leaf tips showed necrosis and chlorosis. The root was affected as well. Lateral roots were significantly shorter than the lateral roots of the control. The root surface was reduced, thus the plant

4 cm

A B

C D

4 cm 4 cm

was restricted in nutrient uptake. Cadmium had a severe impact on maize roots and shoots.

To proof that the morphological changes were caused by cadmium and not by nitrate another experiment using cadmium chloride was done. The morphological changes were the same, after the same period of time.

5.3.1 Cadmium measurements

The cadmium concentration within the hydroponics was measured. 10 mL of the solution were sampled from fresh and used (seven days old) hydroponics. This was performed to get information about how the cadmium concentration varies. The measurements were performed externally by Eurofins WEJ Contaminants.

Table 5: Overview of cadmium measurements

variant [mg/L]

treatment

day zero 186.102 day seven 182.799

control A day zero 0.00126

day seven 0.298

control B day zero 0.00014

day seven 0.00303

By the measurements the concentration of cadmium could be monitored. For the treatment the changes in the cadmium concentration are not significant. In the controls low concentration could be detected due to a normal pollution (acceptable level) of the water source. The table shows results of preliminary work. It was observed that the amount of cadmium was higher after 7 days in the control. This could be explained by the pots used were not clean. Residues of cadmium could solute into the nutrition solution.

For the following experiments always the same pots for control and treatment were used.

The accumulation of cadmium in the control is still under the critical value for drinking water. The permitted cadmium concentration in drinking water is less than 0.005 mg/L (ifau.org, 2015).

5.3.2 Short-term cadmium exposure

To analyse the implication of cadmium to the expression of zmprx01, zmprx66 and zmprx70 three days old etiolated maize seedlings were exposed to a nutrition solution including cadmium nitrate in a final concentration of 15 µM (4.2.5). To detect the short term impact, the plants were cultivated on the contaminated solution for 60 min and sampling was performed at time: 0 min, 10 min, 15 min, 30 min, 45 min and 60 min. A control experiment was performed in parallel without cadmium.

Figure 18: Expression profile of the membrane-bound PRX A) zmprx01, B) zmprx66 and C) zmprx70 in maize roots at short term cadmium exposure. The graphic shows the allocated expression in comparison to the control in percentage. 100 % in this case was the mean value of the expression of the control plants. Expression values are indicated in percentage and standard deviations are shown, n ≈ 18.

The short term cadmium exposure experiment showed which of the three peroxidases analysed was responding quickly to cadmium stress. zmprx01 responded to cadmium

treatment with weak changes in the expression within 60 min. After 15 min the expression increased up to 117 % and then decreased down to 89 % after 30 min and 87 % after 45 min in comparison to the control. The expression value normalises back to 100 % after 60 min.

In contrast, the expression profile of zmprx66 decreased down to 78 % after 10 min and then the expression increased to a peak with 190 % after 15 min. After that the expression decreased back to nearly 100 %.

For zmprx70 the expression decreased at first to 82 % and then increased to 172 %. Until 30 min the expression stayed on that increased level and decreased after 45 min to 60 min back to 100 %. Both, zmprx66 and zmprx70 showed a quick response to cadmium stress within 15 min. In this experimental set-up zmprx01 showed a weak response to cadmium treatment, in comparison.

5.3.3 Long-term cadmium exposure

For detection of a long-term impact of cadmium the plants were cultivated on previously named hydroponics (4.2.4) for 18 days. Sampling was performed at the day one, three, five, 10 and 18. A control experiment was performed in parallel without cadmium.

Figure 19: Expression profile of the membrane-bound peroxidases A) zmprx01, B) zmprx66 and C) zmprx70 in maize roots at long-term cadmium exposure. Analyses of the transcription level of zmprx01, zmprx66 and zmprx70 via qPCR. The graphic shows the allocated expression in comparison to the control in percentage. 100 % in this case was the mean value of the expression of the control plants. For each value standard deviation and significances are shown (*<0.05; **<0.005; ***<0.001), n ≈ 10.

The expression of each peroxidase was decreasing after ten days of cadmium exposure.

For zmprx01 a decrease of the expression was measured after five days. At day ten the lowest expression for zmprx01 was measured. The value decreased down to 46 % in comparison to the control. An increase of the expression up to 73 % could be detected

after 18 days in comparison to the control. Within five to 18 days the expression level was below the expression level of the control plants.

For zmprx66 a continuous decrease of the expression was detected within five to 18 days.

The expression decreased down to 65 % at day ten and finally decreased down to 51 % in comparison to the control, at day 18. Within day one to day three no impact in comparison to the control could be detected.

zmprx70 showed a different expression level due to cadmium exposure in comparison to zmprx01 and zmprx66. At day one the expression was decreased down about 80 %. At day three the expression level increased up to 100 % (same value as the control plants). On the fifth day of cadmium exposure the expression increased up to 140 % in comparison to the control and then decreased down to 57 % at day ten. Until day 18 the expression stayed that downregulated (66 %). This gained data proofs an implication between cadmium and zmprx01, zmprx66 and zmprx70 during a long-term exposure.