Separation of the ether soluble

The thin layer chromatograms that were produced suggested much of the ether soluble material present within susceptible plants was 2,4-D because it had an Rf value similar to that measured for 2,4-D (0.8) (Table 9. 1 1 ). In contrast, most of the ether soluble components in resistant plants appeared to be metabolites of 2,4-D as these materials had Rf values that were not 0.8. By combining mean results from Tables 9. 10 and 9. 1 1 , the quantity of active 2,4-D present in the roots of susceptible plants was

estimated to be 1 3.8 times greater than in resistant plants (Table 9. 12). This difference would be large enough to completely explain the mechanism of resistance.

The ether soluble materials with Rf values different from 2,4-D were probably amino acid conjugates of 2,4-D (Feung et al 1973b). The quality of chromatograms did not allow good identification of the compounds present. However significantly more material from the resistant plants was relatively immobile (Rf < 0. 1) on the thin layer plates than from susceptible plants (Table 9. 1 1 ). Investigations by Feung et al (1973a) showed that conjugates of 2,4-D with glycine, lysine, histidine, arginine or

hydroxyproline would all be immobile with the solvent system used for our thin layer plates.

The quality of the paper chromatograms produced was poor due to overloading of the paper with sediment even after the clean-up process using centrifugation. The

radioactivity detected on these chromatograms suggested there was no separation of 2,4-D from metabolites. However Feung et al ( 1 973a) showed that most of the amino acid conjugates listed above have Rf values similar to the Rf value of 2,4-D (0.75) following chromatographic separation on paper using the butanol / ethanol /

Table 9.1 1 : The percentage of radioactive ether soluble compounds isolated from the roots of herbicide resistant and susceptible nodding thistle plants which had Rf values similar to 2,4-D (0.8), or with Rf values below 0.1 , following chromatographic separation on thin layer silica gel G plates using a diethyl ether / petroleum ether / formic acid (70 : 30 : :2) solvent.

Replicate 1 2 3 Mean* % compounds with Rf < 0.1 susceptible plants 21 .8 1 1 .9 6.8 1 3.5 a resistant plants 36.0 72.9 45.3 51 .4 b % compounds with Rf = 0.8 susceptible plants 57. 1 73.8 79.9 70.3 b resistant plants 22.6 6.3 4.4 1 1 .1 a

• Mean values with different letters are significantly different at p = 0.05.

ammonium hydroxide solvent. The arginine conjugate had the closest value at 0.76, and the other four had values between 0.61 and 0.68. The poor quality of the paper chromatograms meant the presence of compounds at any of these Rf values could not be discounted as the resulting radioactive bar merged with that for 2,4-D.

However none of these amino acid conjugates have been identified in species

investigated by other workers. The amino acids most commonly found conjugated to 2,4-D are glutamic acid and aspartic acid (Ashton and Crafts 1 98 1 ). The conjugates of glutamic acid and aspartic acid should have been present at Rf values of 0.2 1 and 0.26 respectively on the thin-layer plates, and at 0.43 and 0.36 respectively on the paper chromatograms (Feung et al 1973a). As explanation of the radioactivity found at

Rf < 0.0 1 is difficult using published results, the possibility exists that radioactivity

may have been immobile on the thin layer plates simply due to poor preparation techniques. However the differences between resistant and susceptible plants then become difficult to explain.

Table 9. 1 2: Estimation of the differences in absolute quantities of active radiolabelled 2,4-0 in the roots of resistant and susceptible nodding thistle plants 7 days after foliar application.

Susceptible Resistant

plants plants

Total radioactivity absorbed

by plants (kBq) 26.5 26.0

Percentage of absorbed

radioactivity found in roots 1 6.8% 1 8.3% Total radioactivity found

in roots (kBq) 4.45 4.76

Percentage of root radioactivity

extracted by ethanol 86.1 % 91 .7%

Total radioactivity extracted

from roots (kBq) 3.83 4.36

Percentage of extracted

radioactivity soluble in ether 37.4% 1 5. 1 % Total radioactivity soluble

in ether (kBq) _{1 .43} 0.66

Percentage of ether-soluble

material with Rf = 0.8 70.3% 1 1 . 1 % Estimate of total active

radiolabelled 2,4-0 (kBq) 1 .01 0.073

Difference between resistant and

susceptible plant roots in

quantity of active 2,4-0 1 3.8 times

If resistance in the nodding thistle plants is to be explained by production of amino acid conjugates, this would require that these conjugates were herbicidally inactive. However Feung et al (1974) showed that all amino acid conjugates of 2,4-D stimulated elongation of oat coleoptiles in a similar manner to 2,4-D, leading them to conclude that amino acid conjugation does not deactivate 2,4-D. These results have since been disputed as these conjugates are readily hydrolysed back to 2,4-D, which could have occurred during the oat coleoptile assay (Pillmoor and Gaunt 1 98 1 ). This ready conversion back to 2,4-D does suggest that amino acid conjugation is a temporary form of deactivation. Davidonis et al (1980) showed in soyabean callus tissue that much of the applied 2,4-D was converted to amino acid conjugates within 24 hours of

application, then conjugates were hydrolysed back to 2,4-0 over subsequent days and also converted to water soluble metabolites by hydroxylation. Although this process eventually leads to more permanent deactivation of 2,4-0 by hydroxylation, it also provided a constant supply of active 2,4-0 within the plant (Pillmoor and Gaunt 1 98 1 ). Unexplained losses of radioactivity during sample preparation may have affected the validity of results with the chromatography of ether soluble compounds. Losses were not measured for the ftrst replicate, but with Replicates 2 and 3, the loss of

radioactivity while preparing the ether solutions for chromatography was 30% and

17% respectively for the susceptible plants, and 55% and 44% respectively for

resistant plants. Further investigation of these losses showed they occurred whenever samples were evaporated to dryness, which occurred once for the ftrst replicate, and twice for the other two replicates with the addition of the centrifugation clean-up step. The radioactivity appeared to be lost into the rotary evaporator as glassware was checked after residues were redissolved to ensure radioactivity was not left behind. These losses were particularly unsatisfactory because they were greater for the resistant plants, suggesting differences between ecotypes in the compounds present were not being measured by the chromatography. An attempt to overcome these losses by not evaporating to dryness during the sample preparation procedure failed as the sample then could not be successfully applied to chromatographic plates.