Apoplast fluid (AF) analysis

The apoplast fluid (AF) of the plants were analyzed to discover whether aphids come in contact with DIMBOA during probing behavior, owing to cell damage which induces release of the toxic compound, and if this affects the decision making during host plant selection. Because there was a greater response after 48 hours of aphid feeding in the previous experiments (see chapter 4.4.) the plants were analyzed after 48 hours.

In the AF of the tetraploid and hexaploid varieties a small amount of DIMBOA-glucoside and the main aglucone (Figure 75.) was found. The highest amount of AF DIMBOA (which may also have contained HDMBOA-glucoside) was recorded in the hexaploid variety Tasman, with 0.8 mmol/kg FW in the 7 day old plants (GS11, I. timepoint) and in the tetraploid variety Alifen where the level was 0.5 mmol per kg fresh weight in the second time point taken from13 day old plants (GS12-13, II. timepoint). After AF collection the DIMBOA level in the residue leaf tissue in the presence of aphids was higher compared to the control.

Figure 75. Hydroxamic acid levels in the apoplast fluid of the hexaploids Solstice and Tasman and the tetraploid Alifen after 48 hours of aphid feeding at GS 11 (first timepoint) and GS 12-13 (second timepoint).

Groupings are used to show the significant treatment differences within timings and compounds.

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The second experiment included the B genome Ae. speltoides which HA level was extremely high in the whole leaf tissue.

In the AF mainly DIMBOA (+HDMBOA-glucoside) and a small amount of DIMBOA-glucoside was found. In general there was a significant difference in the DIMBOA (+HDMBOA-glucoside) level between the varieties, which clearly responded to the aphid feeding (Figure 76.). The changes were more obvious in the younger plants (GS11;

P<0.001) than at the second sampling point when the plants were at the growth stage 12-13 and the HA levels started to decrease naturally. Across the experiment the DIMBOA level was significantly higher (P<0.001) in the presence of aphids compared to the control.

The highest level of DIMBOA (+HDMBOA-glucoside) in the AF was found in Ae.

speltoides, in the presence of aphids the amount of DIMBOA (+HDMBOA-glucoside) was 3 times higher (4.29 mmol/kg FW) compared to the control (1.45 mmol/kg fresh weight) at GS11. In the control of the hexaploid Tasman only 0.41 mmol/kg FW was found and 0.50 mmol/kg FW was recorded in the test plant after aphid feeding. This was slightly lower than we recorded with the old method. The lowest amount of DIMBOA was found in the control plants of the hexaploid Solstice (0.1 mmol/kg FW) but in the presence of aphids the level reached the 0.44 mmol/kg FW. By the later growth stage the DIMBOA level decreased in all the varieties, but still responded to the aphid damage in the Ae. speltoides.

Across the experiment there was a significant difference between the varieties in the DIMBOA-glucoside level (P<0.01) of the AF but there was no overall response to aphid feeding (P=0.44). Looking at the controls, the highest amount of DIMBOA-glucoside (0.1 mmol/kg FW) was found in Ae. speltoides at the later growth stage which was significantly lower than the level measured after aphid feeding (0.3 mmol/kg FW, P=0.02). The lowest DIMBOA-glucoside level vas measured in the tetraploid Alifen 0.027 mmol/kg FW at GS12-13 which did not show significant changes after aphid feeeding.

Figure 76. HA levels in apoplast fluid of the hexaploids Solstice and Tasman, the tetraploid Alifen and the diploid Ae. speltoides after 48 hours of aphid feeding. Samples containing Proteinase K. Groupings are used to show the significant treatment differences within timings and compounds.

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Figure 77. HAs level in the residue plant tissue after apoplast fluid extraction of the hexaploid Solstice and Tasman, the tetraploid Alifen and the diploid Ae. speltoides after 48 hours aphid feeding. Samples contained Proteinase K. Groupings are used to show the significant treatment differences within timings and compounds.

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The HA levels in the residual plant tissue were analysed (Figure 77.) using HPLC after AF collection. The levels recorded were similar to those previously recorded (see chapter 4.3.

for Solstice and Ae. speltoides). The response of Ae. speltoides to the 48 hours aphid feeding was the same as previously found in the aphid feeding experiment (Chapter 4.5).

In the apoplast fluid of the A genome diploid varieties MDR 049 and MDR 037 only compound I. (peak 1.; Figure 52.) with retention time 13.4 minutes was recorded (Figure 78.). After AF collection and analyzing the remaining tissue samples we were able to detect both unknown compounds (Figure 79.).

Figure 78. HPLC graph of the IF sample of T. monococcum (MDR 037). The peak is slightly delayed because of pressure fluctuation of the HPLC.

Figure 79. HPLC graph of the residue tissue sample of T. monococcum (MDR 037) after IF collection (GS 11). Peaks are slightly delayed because of pressure fluctuation of the HPLC.

In the apoplast fluid of the 7 and 13 day old plants of both varieties compound I. showed no significant changes after aphid feeding (Figure 80.).

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Figure 80. Changes in compound I. in the apoplast fluid of two A genome diploid varieties after 48 hours of aphid feeding at GS 11 (first timepoint) and GS 12 (secound timepoint) . No significant difference were

detected after aphid feeding.

Figure 81. Changes in compound I. (at 13.4 min) and compound II. (at 13.8 min) in the tissue sample after apoplast fluid extraction of two A genome diploid varieties after 48 hours of aphid feeding at GS 11 (first

timepoint) and GS 12 (secound timepoint). No significant difference were detected after aphid feeding.

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After AF extraction, in the residual tissue samples, of the two T. monococcum varieties both unknown compounds were present (Figure 79 and 81.) but we detected no statistical difference in the level of the compounds in the presence and absence of aphids.

Summary

Phloem feeding aphids are able to drive the stylet into the phloem with minimal cell puncture. This ability protects them from coming into contact with toxic compounds such as DIMBOA, which could be released from the damaged cell. Analysis of the apoplast fluid of hexaploid, tetraploid and diploid varieties found DIMBOA (+HDMBOA-glucoside) and a very small amount of DIMBOA-glucoside. The level of DIMBOA (+HDMBOA-glucoside) was higher in response to 48 hours aphid feeding at both growth stages (GS11 and GS13). It is known that DIMBOA is present in the cell in a non-toxic form as DIMBOA-glucoside (Hofman and Hofmanová 1969; Givovich et al. 1994; chapter 2.2.3) which can react with the β-glucosidase enzyme that is released because of the cell damage to produce the aglucone DIMBOA. Our observation is that the AF contains both DIMBOA and DIMBOA-glucoside. One technical explanation for the presence of DIMBOA in the AF could be that the infiltration method is not gentle enough and causes some cell rupture in the tissue, which leads to the conversion of the DIMBOA-glucoside inflating the DIMBOA level during the extraction process. But this does not explain the increased level of DIMBOA after aphid feeding leading to greater confidence in at least these relative studies of AF content. The question is: how much cell damage are aphids able to do during probing behaviour? The punctured cell walls are weakened and the infiltration could have caused further damage and lead to the increase of the DIMBOA level.

These results provide a good lead to follow up in further work with different AF collection methods. This work is very important and could explain why aphids are able to survive on the high HA producing plants, but die on the artificial diet, which contains only a fraction of the amount of the HAs naturally present in the wheat.