A genome diploid species - Analysis of hydroxamic acids in the leaf tissue

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4.3. Analysis of hydroxamic acids in the leaf tissue

4.3.3. A genome diploid species

In the A genome diploids T. monococcum (lines MDR 049, MDR 037, MDR 050, MDR 229) and T. boeoticum (line 8404) two peaks were found for which the retention time was similar to the HAs (Figure 49.). The first peak was tentatively identified as HBOA because the height of the peak significantly increased after co-injection with an HBOA standard, but the other peak showed no similarity to the known HAs compounds (Figure 50.).

Figure 49. HPLC graph of the healthy leaf tissue of MDR 050 (Triticum monococcum)

Figure 50. HPLC graph of the same MDR 050 sample as shows in figure 49. spiked with HBOA standard.

We used the molecular markers for Bx2 and Bx3 genes as a tool to compare the three different seed lots of 8404 (original seed sample from Rothamsted Research to the seed samples multiplied by Rothamsted Research and by KWS UK Ltd) to make sure we are testing the right seed lot and no crosspollination or mistake happened during the handling.

8404 was identified by Nomura et al. (2007) as a type II. mutant, which is deficient for

First peak appeared at 13.4min, running in the same time as the HBOA standard.

Second peak retention time was 13.8min.

First peak significantly increased after adding HBOA standard in the sample.

Second peak retention time was 13.8min.

Bx3 and Bx4 in the shoots and roots, and is not able to produce HBOA. The electrophoresis of the DNA amplification showed that bands corresponding to the size of Bx2 fragment expected were detected at 470-475 bp. This corresponds to the expected band size from Nomura’s genomic PCR analysis result. For Bx2 the expected amplification size with our primers was 471bp as it is indicated for Chinese Spring in the chromosome arm survey data (http://urgi.versailles.inra.fr.). All three seed lots of 8404 show the same amplification band for Bx2 and failed to amplify the Bx3 sequence, confirming their correct seed multiplication and being consistent with this variety being unable to produce HBOA. Two different seed lots of MDR 050 and MDR 002 were also tested to compare the seed multiplied by KWS UK Ltd. with the seed multiplied by Rothamsted Research. For these samples the size of the Bx2 band (470-475bp) was the same as detected for the T. boeoticum (8404). For the Bx3 primer the band was detectable around 700bp which is similar to Nomura’s result. The expected band size for Bx3 was 704bp (http://urgi.versailles.inra.fr.). All results were consistent with correct multiplication. Both Bx2 and Bx3 sequences were amplified for the non-mutant T.

boeoticum MDR 298 and the type I. mutant T. boeoticum (8150). 8150 is not able to produce HAs although all 5 Bx genes are present (Nomura et al. 2007), which we were able to confirm for Bx2 and Bx3. Band sizes were the same as it was previously mentioned (Figure 51.).

Figure 51.Acrylamide electrophoresis gel with the bands for Bx2 (a) and Bx3 (b) primers. Sample numbers 1-3 present the result of the type II. mutant T. boeoticum (8404), number 4-6 are T. monococcum (4=MDR 002; 5-6=MDR 050). Sample number 7 is a non mutant T. boeoticum (MDR 298) and sample number 8 is a

type I. T. boeoticum (8150).

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800

300

The level of the unknown compounds could be interesting because recent relative growth rate experiments on T. monococcum showed MDR 037 is susceptible to R. padi and MDR 049 is less preferred, as seen in the fecundity test.

Summary

In the leaf tissue analyses all hexaploid and tetraploid varieties tested contained DIMBOA-glucoside and the main aglucone DIMBOA (Figure 52.). The DIMBOA (+HDMBOA-glucoside) level was similar in both species, but the DIMBOA-glucoside was about 3 times higher in the 6 day old tetraploid seedlings than in the hexaploids (Figure 53.).

In the leaf tissue of the B genome Ae. speltoides high levels of HAs were detected but other members of the Sitopsis section had none or only a very small amount of the compounds of interest in the leaf, coleoptile or roots. In Ae. searsii, Ae. longissima, Ae.

sharonensis and Ae. bicornis a large unknown peak was found in the HPLC analysis at 13.6-13.7 minutes.

In the A genome T. monococcum and T. boeoticum, no HAs were detected in the leaf tissue of the seedlings, but two unknown compounds (eluting at 13.4 and at 13.8 minutes) were found, which are not related to the known HAs (Figure 52.).

Aegilops sharonensis MDR 229 Tybalt

920/2

Dimboa-glucoside DIMBOA

Hexaploid

Tetraploid

Diploid

MDR 037 MDR 049

2. 1.

8404

Aegilops speltoides BB genome

AA genome

Triticum boeoticum Triticum monococcum

Figure 52. Summary of the peaks in different species. The green arrow shows the DIMBOA –glucoside and the red arrow the combined DIMBOA + HDMBOA – glucoside. Unknown peaks in diploid species: 1.

retention time at 13.4 minutes, 2. retention time at 13.8 minutes and 3. retention time at 13.6-13.7 minutes.

Triticum aestivum AABBDD

DIMBOA-glucoside: 3.11mmol/kg FW

Combined DIMBOA + HDMBOA-glucoside: 3.78mmol/kg FW

Figure 53.

Schematic diagram showing the ancestral origins of Triticum aestivum. Also showing the hydroxamic acid levels in the leaf tissue of the different species. The species in grey have not been included in our

experiments. X= polyploid speciation event.

Figure 53. shows the changes in the levels of HAs in the ancestral species of hexaploid wheat. From the work of Gianoli et al. (1997) and Niemeyer and Jerez (1997) it is known that the A genome donor T. uraurtu contains a small amount of HA which is DIBOA, and

Triticum durum AABB

DIMBOA-glucoside: 8.41mmol/kg FW

Combined DIMBOA + HDMBOA-glucoside: 2.89mmol/kg FW

Aegilops tauschii DD

Hydroxamic acid level has not been tested

Combined DIMBOA + HDMBOA-glucoside: 16.5mmol/kg FW

Triticum monococcum

Small amount of HAs found.

it is not able to accumulate DIMBOA. At least one B genome accession of Ae. speltoides is able to produce large amounts of HAs, but the tetraploid varieties have significantly lower levels of DIMBOA and DIMBOA-glucoside. Niemeyer and Jerez (1997) showed the D genome does not contain any DIMBOA and the incorporation of this genome may inhibit the accumulation of HAs in the hexaploid varieties (Gianoli et al. 1997).

The evidence presented here shows that modern wheat varieties produce less HA than at least some modern representatives of the ancestral diploid species. The evolution of bread wheat involves two polyploidation events where ‘bottle neck’ effects could have occurred.

Our observations suggest that the B genome progenitor of hexaploid wheat was a low HA producer. This may weaken one of the broad spectrum defence mechanisms and make the hexaploid varieties more vulnerable to insects and diseases in the seedling stage.

We investigated the correlation between hydroxamic acid level – aphid settlement and hydroxamic acid level – fecundity in the above mentioned hexaploid and tetraploid varieties. We found no correlation between the number of settled alatae and the DIMBOA-glucoside (Figure 54.) and the DIMBOA (+HDMBOA-DIMBOA-glucoside) level in the leaf tissue (Figure 55.).

Figure 54. The scatter graph shows no correlation between the DIMBOA-glucoside level and aphid settlement.

mmol/kg FW DIMBOA-glucoside in the leaf tissue

Mean difference of settled alatae

Figure 55. The scatter graph shows no correlation between the DIMBOA (+HDMBOA-glucoside) level and aphid settlement.

We recorded a moderate level of correlation (r=0.632) between the DIMBOA-glucoside level and the intrinsic rate of population increase which means 39.9% of the results were affected by the HA level in the plants however the relationship between the two variables was not significant (P=0.127) (Figure 56.). The tetraploid varieties which contained higher level of DIMBOA-glucoside compared to the hexaploids showed reduced reproduction. A very week correlation (r=0.215) was found between the DIMBOA (+HDMBOA-glucoside) level and the number of produced nymphs which means only 4.6% of the results were affected by the aglucone level in the leaf tissue (Figure 57.) but again the relationship was not significant between the variables (P=0.63).

More observation would be necessary to be confident in the level of correlation.

y = 0,1603x + 3,4292 R² = 0,0288

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-2 -1 0 1 2 3 4 5 6

mmol/kg FW DIMBOA (+HDMBOA- glucoside) in the leaf tissue

Mean difference of settled alatae

Figure 56. A moderate level of correlation was recorded between the DIMBOA-glucoside level and the intrinsic rate of population increase.

Figure 57. A scatter graph shows a very week correlation between the DIMBOA (+HDMBOA-glucoside) level and the intrinsic rate of population increase.

y = -104,21x + 43,565 R² = 0,3995

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0,33 0,34 0,35 0,36 0,37 0,38 0,39 0,4

mmol/kg FW DIMBOA-glucoside in the leaf tissue

Intrinsic rate of population increase (r_m)

y = 13,449x - 1,8141 R² = 0,0464

0 1 2 3 4 5 6

0,33 0,34 0,35 0,36 0,37 0,38 0,39 0,4

mmol/kg FW DIMBOA (+HDMBOA- glucoside) in the leaf tissue

Intrinsic rate of population increase (r_m)

4.4. Aphid feeding experiment

In this experiment the changes in the levels of HAs in the plants, triggered by aphid damage, were measured. Three hexaploid (Solstice, Tybalt, Napier) and two B genome diploid (Ae. speltoides, Ae. sharonensis) varieties were tested in this experiment. The aphid feeding resulted in a localised defence reaction in the leaf since there were no significant differences in the HA levels between the test and control plants, either in the base of the first leaf, or in the rest of the plant.

Hexaploid varieties

Across the experiment including both time points DIMBOA (+HDMBOA-glucoside) showed significant changes (P=0.005) due to aphid feeding however no significant differences were detected in the DIMBOA-glucoside level in Solstice. After 24 hours of aphid feeding, the DIMBOA-glucoside level was lower by 40%, 0.3 mmol/kg FW compared to 0.5 mmol/kg FW in the leaf area beneath the clip cage (P=0.0016) while the DIMBOA (+HDMBOA-glucoside) level was higher by 30% in the presence of the aphids, (1 mmol/kg FW in the control compared to 1.3 mmol/kg FW (P=0.12) in the test plant (Figure 58). After 48 hours of aphid feeding the DIMBOA-glucoside level was lower but not significant in the presence of aphids 0.26 mmol/kg FW (P=0.46) compared to the control 0.29 mmol/kg FW. The DIMBOA (+HDMBOA-glucoside) level was 43% higher, 1.21 mmol/kg FW (P=0.008) compared to the control which was only 0.84 mmol/kg FW.

A slight decrease was detected in the overall concentration of the HAs in the control plant, which was probably due to ageing.

Figure 58. HA level changes in the leaf tissue beneath the clip cage in Solstice after 24 and 48 hours aphid feeding. Groupings are used to show the significant treatment differences within timings and compounds.

Another hexaploid variety Tybalt was tested which showed similar evidence of the changes in the HAs in the leaf tissue beneath the clip cage as Solstice.

Across the two Tybalt experiments, including both time points, the HA levels changed significantly (P<0.05). After 24 hour the DIMBOA-glucoside level was lower, at 1.74 mmol/kg FW compared to the control at1.91 mmol/kg FW but the changes were not significant (P=0.37) while the DIMBOA (+HDMBOA-glucoside) level was 0.80 mmol/kg FW higher in the presence of aphids (P=0.39) (Figure 59-60). After 48 hours of aphid feeding the DIMBOA-glucoside level again was lower in the leaf area where the aphids were feeding 1.23 mmol/kg FW compared to the control at 2.25 mmol/kg FW (P=0.01).

The DIMBOA (+HDMBOA-glucoside) level was higher in the presence of aphids 9.50 mmol/kg FW (P=0.09) compared to the undamaged plants where the level was 6.61 mmol/kg FW but the diference was not significant.

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Figure 59. a) HPLC graph of healthy leaf tissue of Tybalt, arrows point out the DIMBOA glucoside) and DIMBOA-glucoside. b) Tybalt leaf sample after aphid feeding, the DIMBOA

(+HDMBOA-glucoside) level increased and the glucoside level decreased.

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Figure 60. HA level changes in the leaf tissue beneath the clip cage in Tybalt after 24 and 48 hours aphid feeding. Groupings are used to show the significant treatment differences within timings and compounds.

A similar reaction was noted in Napier (hexaploid variety) after 24 and 48 hours aphid feeding. We measured a slightly lower DIMBOA-glucoside level in the presence of aphids across the experiment and higher DIMBOA (+HDMBOA-glucoside) level after 48 hours aphid feeding; but in this case there was no significant changes in the HA levels (Figure 61.).

Figure 61. HA level changes in the leaf tissue beneath the clip cage in Napier after 24 and 48 hours aphid feeding. Groupings are used to show the significant treatment differences within timings and compounds.

Diploid varieties

From the Sitopsis section, Ae.sharonensis was tested in this aphid feeding assay, but neither DIMBOA-glucoside nor DIMBOA (+HDMBOA-glucoside) were detected in the control leaf or in the leaf with the feeding aphids. We also did not find HAs in the presence or absence of aphids in the accessions of Triticum monococcum (MDR 050) and Triticum boeticum (8404, MDR 298).

In the case of Ae. speltoides, after 24 hours of aphid feeding the variety showed no significant changes in HA levels, in the leaf tissue sampled from immediately under the clip cage in comparison with control plants (Figures 62 and 63).

Figure 62. Changes in the DIMBOA-glucoside level in Aegilops speltoides after 24 hours aphid feeding.

Groupings are used to show the significant treatment differences within the sampled areas.

Figure 63. Changes in the DIMBOA (+HDMBOA-glucoside) levels after 24 hours of aphid feeding in Aegilops speltoides under the clip cage rest of the plant coleoptile

mmol/kg fresh weight

DIMBOA - glu, 24 hours aphid feeding

a a under the clip cage rest of the plant coleoptile

mmol/kg fresh weight

Combined DIMBOA + HDMBOA-glucoside, 24 hours aphid feeding

a a a a

a a

In the previously analysed DIMBOA producing tetraploid and hexaploid varieties, aphid feeding resulted in a localised HA defence reaction in the leaf. In contrast the diploid species, Ae. speltoides, behaved differently. After 24 hours of aphid feeding, the DIMBOA-glucoside and the DIMBOA (+HDMBOA-glucoside) levels were not significantly different where the aphids were feeding (+aphid: 23.5 mmol/kg FW, control:

20.9 mmol/kg FW) nor in the coleoptile (+aphid: 8.7 mmol/kg FW, control: 7.7 mmol/kg FW). Similarly after 48 hours of aphid feeding, in the leaf beneath the clip cage, we recorded no statistical difference in the level of either compound compared to the control (Figures 64-65). In the rest of the plant there was no significant response to the aphid feeding but in the coleoptile a small but significant decrease in DIMBOA (+HDMBOA-glucoside) was observed in response to aphid feeding under the clip cage (P<0.05).

Figure 64. Changes in the DIMBOA-glucoside level in Aegilops speltoides after 48 hours aphid feeding 0

5 10 15 20 25 30

-aphid +aphid -aphid +aphid -aphid +aphid under the clip cage rest of the plant coleoptile

mmol/kg fresh weight

DIMBOA - glu, 48 hours aphid feeding

a a

a a a

Figure 65. Changes in the DIMBOA (+HDMBOA-glucoside) level after 48 hours of aphid feeding in showed lower and the DIMBOA (+HDMBOA-glucoside) higher levels in the presence of aphids. This may indicate that the transformation into the toxic aglucone was triggered within a single day of aphids starting to feed. The effect was even stronger after 48 hours.

The changes were localised to the area under the clip cage where the aphids were feeding.

In the B genome, Ae. sharonensis had no detectable HAs in the leaf tissue even after stimulation by aphid feeding, but it did produce both compounds in the coleoptile and the roots (chapter 4.3), suggesting that the HA metabolic pathway is working, but is not expressed in the leaf. Aegilops speltoides, differed from the hexaploid varieties, as it contained comparatively large amounts of both DIMBOA-glucoside and aglucone in leaf tissue but showed only a small non-significant response to aphid feeding. It might be speculated that the plant, having a constitutively high level of expression, has lost the response mechanism required to increase the aglucone HA level further.

0 under the clip cage rest of the plant coleoptile

mmol/kg fresh weight

Combined DIMBOA + HDMBOA-glucoside, 48 hours aphid feeding

We investigated further how it is that aphids are able to survive on the HA producing varieties. At this point our hypothesis was that aphids may able to detoxify or excrete the main aglucone. To test this hypothesis the honeydew was collected from the clip cages used in the aphid feeding experiment and analyzed for HA and the break down products of DIMBOA.

4.5. Honeydew collection

Honeydew was collected from the clip cages after the aphid feeding experiment to determine whether any HAs could be detected by HPLC analysis. Givovich et al. (1992 and 1994) and Frébortová (2010) showed that the phloem, from where R. padi is feeding, contains only DIMBOA-glucoside, but aphids can penetrate cell walls and may have also encountered the toxic aglucone during probing behaviour. Where the aphids had fed on diploid, tetraploid and hexaploid varieties which contained DIMBOA-glucoside in the leaf tissue, only DIMBOA-glucoside was identified in the honeydew (Figure 66 and 67.). In T.

monococcum and T. boeoticum, where leaf tissue did not contain any of the known HAs, no HAs were detected in the honeydew (Figure 68.).

Figure 66. DIMBOA-glucoside in the honeydew of aphids feeding on the hexaploid Tybalt

Figure 67. DIMBOA-glucoside in the honeydew of aphids feeding on the B genome diploid Ae. speltoides

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Figure 68. No detectable HA related compounds in the honeydew of aphids feeding on 8404 (Triticum boeoticum)

Summary

This result shows that phloem feeding aphids encounter DIMBOA-glucoside when feeding on a HA producing plant species and that they excrete it in honeydew. Presumably DIMBOA-glucoside is not toxic for R. padi aphids or, because of the high number of generations, they are able to adapt quickly to this compound.

We could not detect DIMBOA or the break down product (MBOA) of DIMBOA or any other HAs in the honeydew of the aphids which were feeding on the HA producer plants.

So it is possible aphids may able to avoid or metabolise the aglucone HA compounds.

In the next step we studied the toxicity of the DIMBOA to find out which level they are able to tolerate in the artificial diet.

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m AU 254nm ,4nm (1.00)

NO HAs

4.6. Artificial diet

Artificial diet was used to assess the toxicity of the main aglucone DIMBOA on aphid survival rate. Our experiment was based on the work of Escobar (1999) and Niemeyer et al. (1992) where they tested the toxicity of different compounds in sucrose solution on Sitobion avenae and R. padi aphids.

4.6.1. Sucrose solution

Initially, 3 different concentrations of sucrose solutions (10, 20 and 30%) were used to determine whether aphid survival relates to the concentration of sucrose. 25 mixed age aptera were placed on each diet in a replicated experiment. After 24 hours of aphid feeding some mortality was recorded, but there was no significant difference in the survival rate on the different concentrations (P>0.66). The survival rate could be affected by the age of the aphids; older adults may not adapt to the new condition as well as the younger nymphs.

After 48 hours the aphid number started to increase as nymphs were produced since the first observation. There was still no significant difference between the 3 concentrations (P>0.28) (Figure 69.).

Because more live aphids were recorded after 48 hours on the 30% sucrose solution and the amount of honeydew produced was higher than on the other two concentrations we choose to use 30% sucrose solution for our experiments.

Figure 69. The average number of survived aphids on the 10, 20 and 30% sucrose solution after 24 and 48 hours.

4.6.2. DIBOA

This initial artificial diet experiment was set up with DIBOA in order to test the method before using it with DIMBOA, which was in short supply. Solutions of 1, 2, 4.83, and 19mM DIBOA in 30% sucrose solution were used in the replicated experiment with 50 L2 nymphs per replicates. A single observation of aphid survival was made after 24 hours and significant differences were found between the control sucrose and all four DIBOA solutions.

On the 1mM solution 48.5% (P<0.0001) and on the 2mM solution 36.8% (P<0.0001) of the aphids survived compared to the control. 100% of the aphids were dead on the 19mM and only 1.8% survived on the 4.83mM DIBOA solution, this being highly significant compared to the control (P<0.000001) (Figure 70.). This result shows that even the low concentration of pure DIBOA is highly toxic to R. padi in artificial diet.

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