Stefanie Neumann

Transport of xenobiotics in higher plants. I: Structural prerequisites for translocation in the phloem

Summary The correlation between the mobility of xenobiotics in the phloem and their chemical structure was investigated using the following substances: phloem-mobile 2,4-D, xylem-mobile 2,4-dichloro-anisole derived from the elimination of the carboxyl group, xylem-mobile defenuron and atrazine, and their ambimobile derivatives N-(p-carboxyphenyl)-N′-methylurea, phenylureidoacetic acid and 2-chloro-4,6-bis-( β -alanino)-l,3,5-triazine. Mobility was characterized quantitatively by the translocation rates and the translocation quotient Q tr . The Q tr was determined by the Sinapis test exhibiting the ratio of mobility in phloem following leaf application and mobility in xylem following root application. Analysis of exudates from detached Yucca inflorescence stalks revealed the presence of phloem- and ambimobile compounds without any metabolic conversions in the phloem sap exclusively. The partition coefficients of substances mobile in the phloem were higher at p H 8 than at p H 5. The corresponding coefficients of xylem-mobile chemicals were of the same value. These results suggest that xenobiotics enter the sieve element-companion cell complex by diffusion. Phloem- and ambimobile compounds are accumulated by an ion trapping mechanism causing their retention in the sieve tube and preventing a complete escape along the path. Consequently, acidic compounds with pK a lower than 7 are expected to be phloem- or ambimobile.

QUANTITATIVE DETERMINATION OF MOBILITY OF XENOBIOTICS AND CRITERIA OF THEIR PHLOEM AND XYLEM MOBILITY

Abstract The general criteria for establishing the relationship between chemical structures of xenobiotics and the properties of mobility in higher plants were investigated. A translocation test with seedlings of Sinapis alba was developed where the ratio of phloem and xylem mobility of exogenously applied compounds can be expressed be means of the translocation quotient Qtr. It represents a physiological constant of the substance under investigation. Phloem as xylem mobility requires substances which are neither extremely lipophilic nor extremely hydrophilic. Favourable prerequisites for phloem mobility are given by polar acidic compounds which have a clearly different degree of dissociation at pH 5 and pH 8. The absorption into the sieve tube-companion cell complex is effected passively by the principle of an ion trap. Xylem mobility is favoured by the apolar character of a compound and - in the case of polar substances - by anions with no difference in dissociation at pH 5 and pH 8. The presence of such a difference in addition to xylem-mobile properties defines ambimobility.

Transport of Xenobiotics in Higher Plants IV. Amobility of the Acidic Compounds Bromoxynil and Pentachlorophenol

Summary In contrast to the conclusion that acidic compounds with p Ka smaller than 7 are phloem- or ambimobile, on the basis of the results of the Sinapis translocation test the phenolic derivatives 2,5-dibromo-4-hydroxybenzonitrile (bromoxynil, p K a = 4.1) and pentachlorophenol (PCP, p K a = 4.5) have to be classified as amobile. Amounts of up to 10 -4 M bromoxynil and 10 -5 M PCP do not have any poisonous effect on phloem transport in Sinapis seedlings. On the other hand sucrose uptake into Cyclamen conducting tissue was affected if the concentrations of phenolic compounds exceeded 10 -6 M. Uptake of both compounds by isolated conducting tissue was investigated in the nontoxic concentration range and was found to be accomplished by diffusion. While PCP absorption was independent of the proton concentration of the external solution, the uptake of bromoxynil exhibits an optimum at p H 4.0 and was inhibited by carbonyl cyanide m-chlorophenylhydrazone and dinitrophenol. Consequently, the retention of bromoxynil in the symplast is determined by ion trapping. From experiments with frozen-thawed conducting tissue and Sinapis cotyledons as well as from efflux studies it was concluded that both compounds are unspecifically bound on several cellular constituents (cutin, lignin, membrane lipids). Binding of PCP is higher than that of bromoxynil. The same applies to the adsorption by Sinapis cotyledons when compared with isolated Cyclamen conducting tissue. Nevertheless, in both cases the adsorption prevents a quantitative transfer to, and the transport in, the long distance streams, although even bromoxynil is assumed to be potentially translocable in the phloem due to its retention in the symplast by ion trapping.

Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive 11C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. 14C-Xenobiotics and [3H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log Kow = — 2 to — 2.4 (Kow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q≥0.7; maleic hydrazide, dalapon); (ii) acidic substances with log Kow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).

Transport of Xenobiotics in Higher Plants III. Absorption of 2,4-D and 2,4-Dichloroanisole by Isolated Conducting Tissue of Cyclamen

Summary Uptake of phloem-mobile 2,4-D and xylem-mobile 2,4-dichloroanisole (2,4-DCA) into isolated conducting tissue was investigated. 2,4-D absorption exhibited a pH optimum at 4.0 and attained sixfold accumulation within 1 h. It was sensitive to metabolic inhibitors. A Q10 value of 1.2 indicates a passive entrance mechanism. These results confirm the hypothesis that ion trapping causes retention in the alkaline sieve tube sap. However the concentration response of 2,4-D uptake tended to saturate even at concentrations higher than 10−4 M. Monitoring sucrose uptake after 2,4-D preincubation revealed an injuring effect on the plasmalemma, which seems to be responsible for the observed pattern of concentration dependent uptake. The absorption of xylem-mobile 2,4-DCA deriving from phloem-mobile 2,4-D by elimination of the carboxyl group was not affected by the external pH or the presence of inhibitors in the medium. The Q10 amounted to 1.2. Although this corresponds to previous investigations of xylemmobile substances, additional results disagree with past experience. 2,4-DCA was accumulated in the conducting tissue. Influx and efflux showed a slow initial phase and a slight escape respectively. The concentration kinetic was hyperbolic. This behaviour seems to be caused by unspecific binding to different cell constituents. The sites involved are still uncertain, but they are obviously not concerned with retention and transport in sieve tubes.

Studies on Cuscuta reflexa Roxb. VI. Is there an autoparasitic withdrawal of nutrients

Summary The phenomenon of autoparasitism in the genus Cuscuta has been reported on a number of occasions. Attempts to prove the transport of radiolabelled sucrose from a host filament of Cuscuta reflexa to a parasitizing shoot of the same species were not successful. The growth of parasitizing Cuscuta stems separated from the stock culture was not stronger than the growth of isolated shoots of the same length. Light and electron microscopic pictures of cross-sections of parasitized Cuscuta stems showed an isolating layer of cells in front of the penetrating haustoria formed in the cortex of the infested shoot. This way the haustorial cells cannot reach the vascular bundles of the parasitized Cuscuta stem. In spite of the presence of haustoria, Cuscuta reflexa cannot absorb food substances autoparasitically.

Untersuchungen zum Aufnahme- und Transportverhalten von stereoisomeren Aminosäuren in Phaseolus vulgaris L.

Summary The stereo-isomeric forms of 14 C labelled leucin and glutamic acid were applied to primary leaves of Phaseolus vulgaris . The uptake of D- or L-glutamic acid was higher than that of leucin isomers during a 3 hours experiment. The content of radioactive substances in the protein fraction of leaves was 2 to 3 times higher after application of the L-isomers of both amino acids compared with the D-isomers. After application of D-Ieucin-1- 14 C the L-isomer was also present in the hydrolysed protein, as demonstrated by an enzymatic test with D-amino-acid-oxidase. The patterns of distribution into the plants were identical by feeding with D- or L- forms of the two amino acids. From the treated leaves only a quantity of 12 to 16 percent was translocated to other parts of the plant. In the alcohol extracts of these parts it was established that translocated D-leucin was partially transformed into the L-isomer. The low content of D-Ieucin in the stem and root compared with that of the treated leaf and the shoot lead us to suppose that the conversion of D- to L- amino acid takes place after translocation in the different tissues.