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Non-competitive inhibition of Rb+ and SO42− uptake caused by Curvularia lunata phytotoxins
Chem-Riot. Interactions, Z6 (1979) 223-226 o Elsevier/North-HoHand Scientific Publishens Ltd.
223
NON-COMPETITIVEINHII3ITIONOF Rb+ AND SO;- UI’TAXECAUSED BY CURVULARIA LUNATA PHYTOTOXINS F. MACROand A. VIANELLO Zslituto di Pato~cgiu Vegetak. Univemitci de Padow W&9
Via Cmdenigo 6, 35100 Padava
(Received Mm& 23rd, 1978) (Revision received July 12th, 1978) (Accepted January 26th, 1979)
In a previous paper it was reported that Curvutati lunr*tu(Wakk.) Boed. in liquid culture ptoduces at least two non-specific toxins of low molecular weight, Dilute solutions of the two phytotoxins inhibit root growth of seedlings and produce necrotic spots on leaves of several plant species E13 . It has been also found that in corn roots C. lunate phytotoxins decrease the
ability of absorbing and retainiig rubidium, sulfate and Lleucine, inhibit the ~co~~tion of ~~oaci~ into promo and sightly affect Oz uptake PI* This communication extends the previous study ZZ] and de& with the effects of the toxin on plasnw membrane to further in~estiga~ some of its potent&l physiological targets. The uptake of rubidium and sulfati in corn roots was tested after a pree ~&~rne~t of 3 h in the ~~n~ of two C. fu~~~ toxiu conceut~tions (20 and 40 pg/ml for rubidium; 30 and 60 (rg/mPfor sulfate) by the method previously described (21. With these two concentrations of tcxin, a 40% and 70% inhibition of sulfate and 30%and SZ%inhibition of rubid.ium uptake were observed, respectively. The data, plotted by the double-reciprocal plot, showed that the toxin ~~~~ the uptake in a ch~ac~~tic pure nom competithe manner, he, it did not affect the affmity of the uptake system = 8 low9 M, unchanged) and for rubidium (K, = both for sulfate e Vmsx,The in~bi~on con$t~~ . 111, ww 10’” M for sulfate tiUvcfinhibi~on indicates that m the t~spo~ s&c in the l
224
inter&, was determined in corn roots pre-treated for 3 h with toxin. The toxin did not affect proton extrusion. A drop in pH was observed within the first 2 h both in the control and in toxin-treated root segments.Therefore, it does not seem to act on the electrogenic pump for H”jK’ exchange 14-7 1. ~thou~h the me~h~rn of ambition of ion untie by tbe toxin at present remains obscure, it seems possible to conclude that the toxin does not interfere with the energy-dependent processes of cell related to ion transport. This is in agreement with the results obtained on oxygen. consumption 121. Figure 1 shows the effect of the toxin on the loss of material absorbing at 260 nm. The toxin caused a rapid loss of such materialwhich was significant@ different from the control after a few minutes This fact might expbjn i;he lag of approx. 30 mm observed iin the effect of the toxin on rubidium and sulfate uptake [2] . The uptake rate of sulfate after ~eatment with toxin at referent incubation temperaturesis shown in Table 1. The effect of the toxin increased
i.I--i__--0
5
Time
15
IQ
of
incubation
[min)
Fig. 1. Loss of material absorbing at 266 nm. Root samples (6.4 g) were placed in 9 mf of toxin solution (20 rglml), pH 3.5 and incubated at 25°C. The leakage was measured as absorbance changes at 266 nm in the incubation medium. The absorbance of the sohrtion without tissue did not change during the experiment. Difference absorbance was considered after substracting the absorbance value due to the toxin. b, control; *, to:~.in-treated. Each value is the mean of three replicates. Vertical bars rearesent standard deviations.
226 TABLE I EFFECT mm
OF TEMPERATURE
ON TOXIN-INDUCED
INHIBITION
OF’ SULFATE
AR amount of 0.4 g of roots was pre-treated with toti (29 &g/ml) for 3 h (pH 3.5) at the specified ~rn~~t~s. Roots were then incubated for sulfate uptake as previously described 1,21 . Inhibition is expressed by considering the values obtained with tire control as 100. Each vaiue is the mean of three replicates. The effect of temperature is significant at P < 9.01. Temperature 2 25 35
(“C)
Controla
Toxin-treateda
4%inbibition
38 37 31
26 16 13
30 50 53
aVaiues expressed as nmollg fresh wt. X h.
linearly with the increasing of the incubation temperature. The toxin was active even at 2°C when all metabolic processes are reduced. Several compounds were tested for a possible protective action when apphed to tissue along with toxin. Cho~e~~~rol,~~itos~rol, sti~~~rol and CaC& were chosen because they are known to be invoked in the stabilization of biological membranes [ES,93. None of the compounds tested &owed a protective action on the membrane towcard the toxin effect. In addition, free sterols did not induce any stimulatory effect on the transport rate of sulfate in controls coning no toxin. Since the toxin is non-specific [I], we suggest that the drastic damage caused in the plasma membranes is the result of a non-specific conformational change of the membrane structure which may impair some functions of the Two experimental findings seem to provide evidence to plasmalemma. support this sugges~c~n. First, the toxin seems to act, as previously suggested 121, at plasmalemma level. Second, the fh&d mosaic of membrane structure is dependent on the type of molecule, temperature, state of hydration and ionic concentration of the medium [lo], and therefore an increment of temperature can lead to an increased membrane fluidity which might favour the contact of the toxin with the sites on the membrane, F. Macri and A. Vianeiio, Rotation and partial characterization of phytotoxins from Cur~ulurior Zunatc; (Wakk.), Boed., Physioi. Pla.nt Pathoi., 9 (1976) 325. A. Vianelio, F. Macri and C. Pas;sera, Effect of Curvularico lunata phytotoxin on membrane permeabiiity of corn roots, Can. J. Bot., 54 (1976) 2913. E. Ma&, P. Lado, F. Rasi Caldogno and R. Colombo, Regulation of pH in the piant ceil wall and celi eniargement. I. Decrease in pII of the rnedi~~ of incubation of pea internode segments treated with fusicocein, Rend. Ace. Naz. Lincei, 53 (1972) 453. II, ubke and U. Luttge, Stoicbiometric correlation Of maiate M!CUU~Uh~i~~ with aUxin-&pen&nt K*++ exchange and growth in avena coieoptile seciments, Plant Physioi., 56 (1976) 696.
226 5 N. Higinbotham and W.P. Anderson, Eiectrogenic pumps in higher plant cells, Can. J. Bot., 52 (1974) 1011. 6 E.A.C. Macrobbii, Puuction of ion transport in plant ceils and tissues, in: D.H. Northcote (Ed.), Plant Biochemistry IT, Vol. 13, University Park Press, Baltimore, 1977, p. 211. 7 E. Marri$, Effect of fusicoccin and hormones on piam ceii membrane activities: observations and hypothesis, in: E. Ma& and 0. Ciferri (&is.), Regulation of Ceil Membrane Activities in Plants, Eisevier/North-HoBand,,Amsterdam, 1977, p. 185. 8 C. Crundwal, Plant sterols, Annu. Rev. Piant Physiol., 26(1975) 209. 9 NC. Marinos. Studies on submicroseopiai aspects of mineral deficiencies. I. Calcium deficiency in the shoot apex of barley, Am. J. Bot., 49 (1972) 834. 10 F-A. Vandenheuvel, Structure of membranes and role of lipids therein, Adv. Lipid Res., 9 (1971) 161.
223
NON-COMPETITIVEINHII3ITIONOF Rb+ AND SO;- UI’TAXECAUSED BY CURVULARIA LUNATA PHYTOTOXINS F. MACROand A. VIANELLO Zslituto di Pato~cgiu Vegetak. Univemitci de Padow W&9
Via Cmdenigo 6, 35100 Padava
(Received Mm& 23rd, 1978) (Revision received July 12th, 1978) (Accepted January 26th, 1979)
In a previous paper it was reported that Curvutati lunr*tu(Wakk.) Boed. in liquid culture ptoduces at least two non-specific toxins of low molecular weight, Dilute solutions of the two phytotoxins inhibit root growth of seedlings and produce necrotic spots on leaves of several plant species E13 . It has been also found that in corn roots C. lunate phytotoxins decrease the
ability of absorbing and retainiig rubidium, sulfate and Lleucine, inhibit the ~co~~tion of ~~oaci~ into promo and sightly affect Oz uptake PI* This communication extends the previous study ZZ] and de& with the effects of the toxin on plasnw membrane to further in~estiga~ some of its potent&l physiological targets. The uptake of rubidium and sulfati in corn roots was tested after a pree ~&~rne~t of 3 h in the ~~n~ of two C. fu~~~ toxiu conceut~tions (20 and 40 pg/ml for rubidium; 30 and 60 (rg/mPfor sulfate) by the method previously described (21. With these two concentrations of tcxin, a 40% and 70% inhibition of sulfate and 30%and SZ%inhibition of rubid.ium uptake were observed, respectively. The data, plotted by the double-reciprocal plot, showed that the toxin ~~~~ the uptake in a ch~ac~~tic pure nom competithe manner, he, it did not affect the affmity of the uptake system = 8 low9 M, unchanged) and for rubidium (K, = both for sulfate e Vmsx,The in~bi~on con$t~~ . 111, ww 10’” M for sulfate tiUvcfinhibi~on indicates that m the t~spo~ s&c in the l
224
inter&, was determined in corn roots pre-treated for 3 h with toxin. The toxin did not affect proton extrusion. A drop in pH was observed within the first 2 h both in the control and in toxin-treated root segments.Therefore, it does not seem to act on the electrogenic pump for H”jK’ exchange 14-7 1. ~thou~h the me~h~rn of ambition of ion untie by tbe toxin at present remains obscure, it seems possible to conclude that the toxin does not interfere with the energy-dependent processes of cell related to ion transport. This is in agreement with the results obtained on oxygen. consumption 121. Figure 1 shows the effect of the toxin on the loss of material absorbing at 260 nm. The toxin caused a rapid loss of such materialwhich was significant@ different from the control after a few minutes This fact might expbjn i;he lag of approx. 30 mm observed iin the effect of the toxin on rubidium and sulfate uptake [2] . The uptake rate of sulfate after ~eatment with toxin at referent incubation temperaturesis shown in Table 1. The effect of the toxin increased
i.I--i__--0
5
Time
15
IQ
of
incubation
[min)
Fig. 1. Loss of material absorbing at 266 nm. Root samples (6.4 g) were placed in 9 mf of toxin solution (20 rglml), pH 3.5 and incubated at 25°C. The leakage was measured as absorbance changes at 266 nm in the incubation medium. The absorbance of the sohrtion without tissue did not change during the experiment. Difference absorbance was considered after substracting the absorbance value due to the toxin. b, control; *, to:~.in-treated. Each value is the mean of three replicates. Vertical bars rearesent standard deviations.
226 TABLE I EFFECT mm
OF TEMPERATURE
ON TOXIN-INDUCED
INHIBITION
OF’ SULFATE
AR amount of 0.4 g of roots was pre-treated with toti (29 &g/ml) for 3 h (pH 3.5) at the specified ~rn~~t~s. Roots were then incubated for sulfate uptake as previously described 1,21 . Inhibition is expressed by considering the values obtained with tire control as 100. Each vaiue is the mean of three replicates. The effect of temperature is significant at P < 9.01. Temperature 2 25 35
(“C)
Controla
Toxin-treateda
4%inbibition
38 37 31
26 16 13
30 50 53
aVaiues expressed as nmollg fresh wt. X h.
linearly with the increasing of the incubation temperature. The toxin was active even at 2°C when all metabolic processes are reduced. Several compounds were tested for a possible protective action when apphed to tissue along with toxin. Cho~e~~~rol,~~itos~rol, sti~~~rol and CaC& were chosen because they are known to be invoked in the stabilization of biological membranes [ES,93. None of the compounds tested &owed a protective action on the membrane towcard the toxin effect. In addition, free sterols did not induce any stimulatory effect on the transport rate of sulfate in controls coning no toxin. Since the toxin is non-specific [I], we suggest that the drastic damage caused in the plasma membranes is the result of a non-specific conformational change of the membrane structure which may impair some functions of the Two experimental findings seem to provide evidence to plasmalemma. support this sugges~c~n. First, the toxin seems to act, as previously suggested 121, at plasmalemma level. Second, the fh&d mosaic of membrane structure is dependent on the type of molecule, temperature, state of hydration and ionic concentration of the medium [lo], and therefore an increment of temperature can lead to an increased membrane fluidity which might favour the contact of the toxin with the sites on the membrane, F. Macri and A. Vianeiio, Rotation and partial characterization of phytotoxins from Cur~ulurior Zunatc; (Wakk.), Boed., Physioi. Pla.nt Pathoi., 9 (1976) 325. A. Vianelio, F. Macri and C. Pas;sera, Effect of Curvularico lunata phytotoxin on membrane permeabiiity of corn roots, Can. J. Bot., 54 (1976) 2913. E. Ma&, P. Lado, F. Rasi Caldogno and R. Colombo, Regulation of pH in the piant ceil wall and celi eniargement. I. Decrease in pII of the rnedi~~ of incubation of pea internode segments treated with fusicocein, Rend. Ace. Naz. Lincei, 53 (1972) 453. II, ubke and U. Luttge, Stoicbiometric correlation Of maiate M!CUU~Uh~i~~ with aUxin-&pen&nt K*++ exchange and growth in avena coieoptile seciments, Plant Physioi., 56 (1976) 696.
226 5 N. Higinbotham and W.P. Anderson, Eiectrogenic pumps in higher plant cells, Can. J. Bot., 52 (1974) 1011. 6 E.A.C. Macrobbii, Puuction of ion transport in plant ceils and tissues, in: D.H. Northcote (Ed.), Plant Biochemistry IT, Vol. 13, University Park Press, Baltimore, 1977, p. 211. 7 E. Marri$, Effect of fusicoccin and hormones on piam ceii membrane activities: observations and hypothesis, in: E. Ma& and 0. Ciferri (&is.), Regulation of Ceil Membrane Activities in Plants, Eisevier/North-HoBand,,Amsterdam, 1977, p. 185. 8 C. Crundwal, Plant sterols, Annu. Rev. Piant Physiol., 26(1975) 209. 9 NC. Marinos. Studies on submicroseopiai aspects of mineral deficiencies. I. Calcium deficiency in the shoot apex of barley, Am. J. Bot., 49 (1972) 834. 10 F-A. Vandenheuvel, Structure of membranes and role of lipids therein, Adv. Lipid Res., 9 (1971) 161.