Uptake and translocation of a-difluoromethylornithine, a polyamine biosynthesis inhibitor, by barley seedlings: effects on mildew infection

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1 New Phytol. (1990), 114, Uptake and translocation of a-difluoromethylornithine, a polyamine biosynthesis inhibitor, by barley seedlings: effects on mildew infection BY D. R. WALTERS AND G. K I N G H A M Department of Plant Sciences, West of Scotland College, Auchincruive, Nr. Ayr KA6 5HW, UK {Received 18 July 1989; accepted 13 November 1989) SUMMARY Uptake of the polyamine biosynthesis inhibitor, a-difluoromethylornithine (DFMO), by barley seedlings was examined. DFMO was taken up by intact roots and approached saturation at a rate of 780 mol m"^^^ g"^ d.wt. The uptake as a function of DFMO concentration in the external medium was biphasic, with Eadie-Hofstee transformations giving K^ values for system 1 uptake of 1-61 mol m"^ and for system 2 uptake of mol m~^. DFMO uptake was reduced in darkness and was greatly influenced by transpiration rate. The inhibitor was taken up from host cells by the powdery mildew fungus, Erysiphe graminis f.sp. hordei Marchal, and DFMO fed to roots, or to excised leaves at different times after inoculation, gave substantial control of mildew infection. Key words: Polyamine biosynthesis, Hordeum vulgare L. (barley), Erysiphe graminis hordei (powdery mildew), DFMO, uptake. INTRODUCTION ^ Walters, 1988) and recent work by Rajam, Weinstein & Galston (1989) has shown that DFMO a-difluoromethylornithine (DFMO) is an enzyme- is a potent inhibitor of uredospore germination both activated irreversible inhibitor of the polyamine in vivo and in vitro. However, certain fungi appear biosynthetic enzyme, ornithine decarboxylase less sensitive to DFMO than rusts and powdery (ODC) (Metcalf et al., 1978). DFMO has been mildews (see Galston & Weinstein, 1988, and West shown to deplete the intracellular concentrations of & Walters, 1989) and recent reports of biosynthetic putrescine and spermidine in mammalian ceils arginine decarboxylase activity in some phyto(pegg, 1986), trypanosomes (Bacchi & McCann, pathogenic fungi (Khan & Minocha, 1989) may help 1987) and phytopathogenic fungi (Foster & Walters, to explain this observation. Although DFMO has 1990), resulting in a powerful antiproliferative effect. been reported to possess some systemic activity This inhibition of cell growth can be abolished by (Rajam et al., 1985; Walters, 1986), very little is addition of exogenous polyamines to the medium, known about its movement within the plant and suggesting that the antiproliferative effect is due to subsequent effects on fungal infection. In the present polyamine depletion (e.g. Foster & Walters, 1990). work we have examined the uptake and translocation Since putrescine can be formed in higher plants by of DFMO in barley seedlings and have studied the decarboxylation of either arginine or ornithine, while effects of root and foliar absorbed DFMO on most fungi appear to make putrescine only by powdery mildew infection, decarboxylation of ornithine (Walters, 1989), it has been suggested that specific inhibition of fungal....,,,,, 1 r 1 MATERIALS AND METHODS ornithme decarboxylase should control tungal,.,... rr / n growth without atiecting the host plant (e.g. Rajam, Weinstein & Galston, 1985). Indeed, DFMO has been shown to give effective control of rust and powdery mildew infections on several hosts (Rajam et al., 1985; Walters, 1986; Weinstein et al., 1987; 44 riant material Barley seeds {Hordeum vulgare L. cv. Golden Promise) were sown on moist filter paper in plexiglass boxes. The boxes were placed in a controlled environment room with a day temperature ANP 114

2 660 D. R. Walters and G. Kingham of 20 ± 1 C and a night temperature of 17 ± 1 C. A light intensity of 250 /imol photons m"^ s~^ was provided for 16 h per day. After 7 d the young seedlings were carefully removed and transferred to beakers containing 250 cm^ of a Letcombe Laboratory nutrient solution of the following composition, macronutrients (mol m~^): 2O, 1-5; KNO3, 5-0; KH^PO^, 1-0; ), 1-5; NaNO3, 2-0; micronutrients (molm-«): FeEDTA, 9-2; H3BO3, 9-22; ), 0-16; KCl, 14-10; MnSO4.4H2O, 3-6; 324.4H2O, 0-016; ZnSO4.7H2O, Four seedlings were supported in each beaker by carefully inserting them through slits in a foam cutout placed at the top of the beaker. The medium in each beaker was maintained between ph 5-3 and 5-5 by additions of H2SO4 and was continuously aerated by means of a small pump. All experiments were performed under the conditions described above, when the seedlings were 14 d old, unless otherwise stated. Uptake of root-fed \^'^C'\DFMO Four seedlings w^ere transferred to 250 cm^ of fresh nutrient solution and 40 fil of [^^CjDFMO (74 kbq; 2-22 GBq mol m"^, Amersham International pic) was added. The uptake solution was stirred and aerated throughout the experiment. Uptake was allowed to proceed for 90 min (over which period uptake was a linear function of time), after which the seedlings were removed from the solution, the roots rinsed in distilled water and the plants divided into roots and shoots. The roots and shoots of each seedling were then cut transversely into 1-5 mm sections and placed in separate vials. The tissue was bleached as described by Lee (1980). Briefly, this involved addition of 1-5 cm^ of distilled water to each vial, followed by 10 cm^ of scintillant (Emulsifier safe, Packard). This was mixed thoroughly and left to stand in daylight for 2-4 d. Once bleached, radioactivity was counted in a Packard liquid scintillation spectrophotometer. The effect of concentration on uptake of the inhibitor was examined by varying the external concentration of unlabelled D F M O and using 40 fi\ of [^*C]DFMO as above. D F M O uptake in the dark was examined either by performing the experiment at night, in total darkness, or by placing the seedlings under a box of a size (dimensions: 0-61 m X 0-45 m x 0-45 m) large enough to minimize any increase in relative humidity. Both methods gave similar results. The effect of transpiration on D F M O uptake was examined by exposing some seedlings to a flow of cool air from a fan (to increase transpiration rate) or by placing a large clear, plastic bag (0-24 m X 0-17 m), moistened on the inside, over the seedlings (to reduce transpiration). In an attempt to examine uptake of labelled D F M O by powdery mildew growing on the surface of barley leaves, 14 d old seedlings were inoculated with powdery mildew conidia (by brushing conidia onto leaf surfaces with a camel hair brush) and left for 7 d. Infected seedlings were then allowed to take up labelled D F M O as described above (for 90 min) and surface mildew growth (mycelium and conidia) was removed using sellotape. Sellotape strips with mildew were placed in 2 cm^ of soluene (Packard), left for 4 d for solubilization of the mildew, after which time the sellotape was removed and 12 cm^ of scintillant (Hionic Fluor, Packard) was added to the soluene. Radioactivity was counted as described above. Transport of foliar-applied [ In order to examine possible movement of labelled D F M O from shoots to roots and also from lower to upper leaves, the flrst and second leaves of 3-weekold seedlings were sprayed to run-off with the inhibitor (20/il [^*C]DFMO, 0-2 cm^ Tween 20, in 200 cm^ distilled water). Movement of labelled D F M O was monitored at 2, 8 and 24 h intervals: sprayed leaves were rinsed in distilled water, leaves 1 4 were cut into 1-5 mm sections and placed individually into vials, and roots were also cut into sections and placed in vials. These tissues were then bleached and counted for radioactivity as described above. Effect of root-fed DFMO on powdery mildew infection of barley seedlings Healthy 2-week-old barley seedlings were transferred either to fresh nutrient solution or to nutrient solution containing 5 mol m"^ unlabelled D F M O. Plants were left for 24 h, after which time they were inoculated with conidia of powdery mildew as described above. Some DFMO-treated seedlings were then immediately transferred to nutrient solution only, while others were left in nutrient solution containing D F M O. Seven days later, the percentage leaf area covered with mildew was estimated using standard area diagrams. Effect of DFMO, fed to lower leaves, on powdery mildew infection of upper leaves First and second leaves of barley seedlings were sprayed to run-off with a solution of 5 mol m~^ D F M O (with 0-1 % Tween 20), left for 24 h and then the upper third and fourth leaves were inoculated with powdery mildew conidia as described above. Seven days later the percentage leaf area infected was estimated using standard area diagrams.

3 uptake of a-difluoromethylornithine by barley seedlings Effect of DFMO fed to excised, infected leaves on powdery mildew infection In order to determine the effects of D F M O taken up from the plant by the powdery mildew haustoria, leaves were detached 1 and 5 d after inoculation and placed with their basal ends immersed in a solution containing 1 mol m"^ D F M O for 4 d. This solution also contained 0-1 mm kinetin to delay leaf senescence. Kinetin has been shown not to affect mildew growth and development (Liu & Bushnell, 1986; Walters D. R., unpublished results). After the 4 d period, leaves were examined for the percentage leaf area covered with mildew. RESULTS Uptake of root-fed [ D F M O uptake into roots of barley seedlings increased with time and was linear for the first 2 h, after which uptake slowed and approached saturation at a rate of 780 mol m~^^ g~-^ d.wt. of root (Fig. 1 a). In whole seedhngs, D F M O uptake was hnear and showed no signs of saturation by the end of the 1000 r 661 experiment (Fig. 1 b). This was probably the result of the pattern of D F M O translocation to the shoots, which increased only slowly over the first 90 min but increased substantially over the subsequent 90 min (Fig. 1 c). During these experiments, there was no indication of a rapid, reversible initial uptake of DFMO. Uptake of DFMO by roots of barley seedlings as a function of concentration in the external medium was clearly biphasic (Fig. 2): D F M O uptake at both low (0-10 mol m-=^) and high (0-100 mol m'^) external concentrations rose sharply as concentration increased over the lower part of the range, but less sharply over the upper part. Eadie-Hofstee plots of these data also suggest that uptake was biphasic (Fig. 3). In these plots the data could be represented as two straight lines fitted by linear regression analysis. This suggests that in roots of intact barley seedlings, there are two systems for DFMO uptake. The affinity constant, K^ and the maximum rate of uptake, I^ax> ^^^ be determined from the Fadie-Hofstee plot. Table 1 shows that K.^ and V^^^ for system 1 are considerably smaller than the values for system 2 uptake. Uptake of D F M O by roots and translocation to shoot were substantially reduced in darkness (Table 2). Manipulating the transpiration rate of the 100 r c ca Q. O) r o E DFMO concentration (mol m-^ Figure 2. DFMO uptake by roots of intact barley seedlings as a function of DFMO concentration in the external medium, with the curves fitted by eye. Each point represents the mean of 4 replicates Time (min) Figure 1. Time course for DFMO uptake by roots of intact barley seedlings (a), whole seedlings (Jb) and distribution of radioactivity between roots ( # ) and shoots (A) with respect to time (c). Each point represents the mean of 4 replicates DFMO uptake/dfmo concentration (mol m"^) Figure 3. Eadie-Hofstee plot for DFMO uptake by barley seedlings; DFMO uptake versus DFMO uptake/dfmo concentration over the concentration range mol m~^. Lines were fitted by linear regression. 44-2

4 662 D. R. Walters and G. Kingham Table 1. Values for K^ and V^^^for DFMO uptake by systems 1 and 2 into intact barley seedlings. Values calculated by linear regression from Eadie-Hofstee plots max (mol m (mol d.wt. Material System 1 System 2 System 1 System 2 Intact barley seedlings Table 2. Uptake of {^^C]DFMO by barley roots and Table 3. Transport of foliar-applied [^^C]DFMO, (a) translocation to shoots, (a) effects of light and dark to roots and (b) from lower to upper leaves, 2 and 8 h treatments, (b) effects of manipulating transpiration after treatment. Values are the means of 5 replicates rate. Values are the means of 5 replicates ± standard + standard error error. Significant differences are shown at P = 0-05* \ ["CJDFMO in tissue (dpm) p = 0-01** Uptake/translocation of ["C]DFMO (mol m"i2 g'^ d. wt. h"^) Treatment (a) Light Dark (b) Control Transpiration increased Transpiration decreased Root Shoot ± * ± * ± *± ±l * ± ** * Shoot Root {b) Leaf r Leaf 2^ Leaf 3 Leaf 4 2h 8h (a) Transport to roots ± ± ± ±5-70 Transport from lower to upper leaves ±18-l ± ± ± ±6-l 12O-5O± ± ±6-5 + ["C]DFMO applied to these leaves. seedlings also affected D F M O uptake: uptake and translocation were greatly increased by artificially increasing transpiration, whereas decreasing transpiration substantially lowered uptake and translocation of the inhibitor (Table 2). Accurate determination of uptake of D F M O by the powdery mildew fungus from the host is difficult, mainly due to the intimacy of contact between the two partners. However, it is possible to examine the movement of a labelled compound into the mildew by careful removal of surface fungal structures. In this way, it was shown that root-fed D F M O did eventually move into the fungus, at an uptake rate of 9-05 mol m""^^ g"^ d.wt. h^^ Transport of foliar-applied Material [^^C]DFMO Although most of the labelled-dfmo applied to the shoots of whole seedlings remained in them, a substantial proportion (14%) of the D F M O appeared in the roots after 2 h (Table 3). Although the amount appearing in the roots increased after 8 h, the proportions of labelled D F M O in roots to total plant D F M O remained at approximately 14%. Radiolabelled D F M O applied to the first and second leaves of seedlings moved into the upper third and fourth leaves. Thus, after 2 h, 16-6 % of the total D F M O in the shoot was detected in third leaves, compared with 10-7 % in fourth leaves (Table 3). The amount of D F M O appearing in these upper leaves increased slightly at 8 h, as did the proportion of labelled inhibitor compared to the total found in the plant (Table 3). Effect of root-fed DFMO on powdery mildew infection In seedlings allowed to take up unlabelled D F M O from a 5 mol m~^ solution for 1 d prior to mildew inoculation, there was no significant reduction in the percentage leaf area infected, compared to controls (Table A a). However, a substantial reduction in mildew infection was observed in seedlings allowed to take up D F M O over a 7 d period (Table Aa). Effect of DFMO, fed to lower leaves, on mildew infection of upper leaves Application of 5 mol m~^ D F M O to leaves 1 and 2 of barley seedlings, resulted in significant reductions in mildew infection on leaves 3 and 4. Thus, infection of leaves 3 and 4 was reduced by 6 8 % and 52 0//o respectively, compared with controls (Table Ab).

5 uptake of a-difluoromethylornithine by barley seedlings Table 4. Effects of DFMO on the percentage of leaf area infected with powdery mildew, (a) Effects of 5 mol m"" DFMO fed via roots for 1 or 7 d, (b) effects of 5 mol m"^ DFMO applied to lower leaves, on infection of upper leaves, (c) effects of 1 mol m'^ DFMO fed to cut ends of leaves for 4 d {leaves were excised 1 or 5 d after inoculation with mildew). Values are means of 5 replicates + standard error. Significant differences are shown at P 0-05*, P = 0-01**. Leaf area infected (%) (a) Treatment 1d 7d ± * Treatment leaf 3 leaf 4 Control 5 mol m~3 DFMO (applied to leaves 1 and 2) ** 45 ± ** Treatment 1 daif Control 1 mol m-3 DFMO (fed to excised leaves) ** Control 5 mol m-3 DFMO (root-fed) ib) 5 dai ±3* f dai, days after inoculation Effect of DFMO fed to excised, infected barley leaves In barley leaves detached 1 d after inoculation with mildew and placed in a solution containing 1 mol m"^ D F M O for 4 d, there was a 55 % reduction in fungal infection (Table 4). Similarly, in leaves detached 5 d after inoculation and placed in 1 mol m"^ D F M O for 4 d, mildew infection was reduced by 3 7 % (Table 4). DISCUSSION This study shows, for the first time, that D F M O can be taken up by roots of intact barley seedlings and transported to the shoots. D F M O uptake appears to be similar to the uptake of amino acids in several systems, e.g. Vicia faba leaf tissue (Despeghel & Deirot, 1983) and tobacco cells (Harrington & Henke, 1981). Thus, uptake of D F M O as a function of external concentration was biphasic, with K^ values for system 1 uptake of 1-61 mol m"^ and for system 2 uptake of mol m"^. These K^ values are of the same order of magnitude as those reported for amino acid uptake in a wide variety of tissues (Reinhold & Kaplan, 1984) and for putrescine uptake in Saintpaulia petals (Bagni & Pistocchi, 1985). D F M O uptake has been examined in Trypanosoma brucei brucei (Bitonti et al., 1986) and in mouse 663 fibroblasts (Erwin & Pegg, 1982). In both of these systems D F M O uptake was non-saturable, not antagonized by basic amino acids and probably occurred via a passive diffusion mechanism. On the other hand, D F M O uptake by intact barley roots was saturable and was substantially reduced by ornithine in the external medium (data not shown). The latter result is interesting in view of the speculation by Slocum & Galston (1987) that D F M O might utilize amino acid transport systems in plants. Indeed, although there is much evidence to support a common carrier for all amino acids (Reinhold & Kaplan, 1984), there is some evidence for a system 2 (basic) amino acid transporter in plants (Johannes & Felle, 1985), which might function in D F M O uptake. Certainly, this aspect of D F M O uptake into plant roots is worthy of further investigation. Light is known to stimulate amino acid uptake in several plant species (Ullrich-Eberius, Novacky & Luttge, 1978; Guy, Reinhold & Rahat, 1980) and, conversely, uptake is much reduced in the dark (Rubenstein & Tattar, 1980; Gepstein, 1982). Indeed, D F M O uptake was greatly reduced when barley seedlings were placed in darkness, suggesting an energy requirement for the process. However, in view of the similar reduction in D F M O uptake when transpiration was reduced, further work using respiratory inhibitors, for example, would be required to clarify this point. It was found that D F M O uptake into barley seedlings was greatly influenced by transpiration. Thus, uptake of the inhibitor was increased when transpiration rate was increased and DFMO uptake was substantially reduced, but not eliminated, under conditions designed to reduce transpiration. Similarly, Bagni & Pistocchi (1988) have recently reported that polyamine uptake into a variety of different seedlings was greatly influenced by transpiration but was not prevented by 100% relative humidity, suggesting the involvement of active processes in the root. As well as transport of D F M O in the xylem stream, it is apparent that there can also be movement in the phloem. Thus, of the total amount of labelled D F M O found in seedlings 2 h after foliar application, 1 3 % was found in roots. Moreover, there was also substantial movement of D F M O from lower to upper leaves of barley seedlings. These results support the findings of other work which showed that D F M O applied to one half of a bean leaf (the two halves separated by the midrib), could protect the other half against rust infection (Rajam et al., 1985; Walters, 1986). In those experiments the inhibitor would have to be taken up from the leaf by the rust fungus. In mildewed barley seedlings, D F M O fed to roots ended up in the fungus and since the only intracellular contact between the mildew and the host would be mildew haustoria, this provides some evidence for haustorial uptake of D F M O. Fungal uptake of D F M O was calculated at

6 664 D. R. Walters and G. Kingham 905 mol m " g ' d.wt. h ', which would probably result in femtomolar concentrations within the pathogen. It is difficult to imagine effective inhibition of fungal ODC in the presence of such low intracellular DFMO concentrations. Moreover, it is unlikely that DFMO uptake from the plant could give lasting control for two reasons. First, DFMO binds covalently to ODC and this, together with other non-specific attachment of the inhibitor to protein, is likely to reduce the amount of available DFMO within the plant tissue. Secondly, it is reasonable to assume that the mildew (or other fungi) would take up polyamines and polyamine precursors from the host. This would replenish fungal polyamine pools in spite of reduced polyamine biosynthesis. Nevertheless, root-fed DFMO (5 mol m"' over a 7 d period) gave reasonable control of mildew infection on barlej' seedlings and DFMO applied to the lower leaves, resulted in substantial reductions in mildew infection on the upper leaves. Moreover, when DFMO was fed to excised barley leaves 1 d after inoculation (i.e. about 6 h after formation of prim.ary haustoria), there was a 55' % reduction in mildew infection. When excised leaves were fed DFMO 5 d after inoculation (i.e. about 48 h after formation of secondary haustoria and with some mycelial growth on the leaf surface), there was still a 37 % reduction in mildew infection. These results support previous work which showed that 1 mol m~' DFMO gave very good control of mildew infection when applied to leaves 3 d after inoculation with very little loss of control if applied 5 d after inoculation (West & Walters, 1988). The discussion so far has assumed that the DFMO taken up by the plant (and the mildew) was not metabolized. Ornithine can be metabolized by the enzyme ornithine transcarbamoylase (E.C ) to yield citrulline and eventually arginine. Although it is possible that DFMO might act as a substrate for this enzyme, Slocum et al. (1988) reported that they were unable to detect such a conversion in tobacco ovary tissues. This does not rule out the possibility of DFMO metabolism and, indeed, conversion of DFMO to the arginine decarboxylase inhibitor, otdifiuoromethylarginine (DFMA) in Phaseolus vulgaris tissue has apparently been detected (see Galston & Weinstein,, 1988). DFMA is known to reduce mildew infection of barley, although it is probably after arginase-mediated conversion to DFMO in the fungus (Slocum et al., 1988), since the mildew fungus does not appear to possess ADC activity (see West & Walters, 1988). It has also been suggested that treatment with DFMO might stimulate plant defences, e.g. phytoalexin biosynthesis (Galston & Weinstein, 1988), which might allow for previously reported systemic activity of DFMO (Rajam et al., 1985; Walters, 1986). For plant defences to be effective, then, the timing of the initiation of resistance responses, e.g. phytoalexin accumulation, is crucial (see Mansfield, 1983). The results described above show that in leaves excised 1 or 5 d after inoculation with mildew, and then fed DFMO, a similar reduction in mildew infection was obtained. In this situation, any DFMO-induced resistance responses would probably be too late at 5 d after inoculation to substantially aftect fungal infection. Indeed, preliminary investigations have failed to detect any stimulation of host defences in barley treated with DFMO' (Walters, D. R., unpubhshed results). The fact that sptay application of putrescine or spermidine with DFMO can partially restore mildew growth on barley leaves may provide some evidence that DFMO-induced reductions in mildew infection are the result of inhibition of polyamine biosynthesis. These possibilities will be the subject of detailed study in this laboratory. In conclusion, DFMO can be taken up by plant roots and translocated in the transpiration stream to the shoot. DFMO (or a DFMO metabolite) also appears to possess some phloem mobility and can be transported from shoot to root and frotn lower to upper leax'es. Further, the powdery tnildew fungus can take up DFMO from barley leaves and substantial reductions in mildew infection can be achieved with DFMO applied via the root system or to excised leaves. In view of the greater efficacy of several other inhibitors of polyamine biosynthesis against a range of plant pathogens (Foster, S. A., West, H. M. & Walters, D. 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