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The effect of asulam on water potential and nitrate reduction
Plant Science, 46 (1986) 21--27
21
Elsevier Scientific Publishers Ireland Ltd.
THE EFFECT OF ASULAM ON WATER POTENTIAL AND NITRATE REDUCTION
ALBERTO MUNOZ-RUEDA, CARMEN GONZ.kLEZ-MURUA, JOSE MARfA BECERRIL, CESAREO ARRESEIGOR and MANUEL S.~NCHEZ-DfAZ*
Departamento de Fisiologia Vegetal, Facultad de Ciencias, Universidad del Pais Vasco, Apartado 644, Bilbao and *Departamento de Fisiologia Vegetal, Universidad de Navarra, Apartado 273, Pamplona (Spain) (Received December 24th, 1985) (Revision received May 6th, 1986) (Accepted May 26th, 1986) Nitrate and nitrite reductase activities, stomatal response and water potential were measured in Medicago sativa (cv. Arag6n) and Trifolium pratense (cv. Violeta) subjected to asulam methyl(4-aminobenzenesulphonyl)carbamate concentrations of 0.15 raM, 1.5 mM and 15 raM. The shoots of the asulam-treated plants lost their turgidity since the fresh weight/dry weight ratio decreased and a decay of water potential was observed, and consequently stomatal resistance was increased. Asulam treatment (15 raM) induced 80% stomatal closure in both species after 7 days. The nitrate and nitrite reductase activities appeared to be very sensitive to this herbicide. In general, clover was more sensitive to asulam than lucerne.
Key words: asulam; clover; lucerne; nitrate reduction; stomata; water potential
Introduction The interaction of water potential, respiration and herbicides in nitrate metabolism (nitrate and nitrite reductase activities) is not well understood. The positive effects of glucose in nitrate reductase induction in darkness indicate that respiration may be a key requirement for induction o f nitrate reductase activity in some plants. Respiration may produce the energy and compounds required for driving the induction o f nitrate reductase activity [1]. This hypothesis was supported by results obtained with respiratory inhibitors. Arsenate, cyanide and dinitrophenol which inhibit or uncouple oxidative phosphorylation or the respiratory chain, inhibit induction of nitrate reductase [1--3] ; and it is also known that the absorption of nitrate is decreased by inhibitors of respiration and oxidative phosphorylation. Furthermore, several investigators have presented evidence that herbicides affect nitrate reductase activity in a number of crops [4]. In spite of some contradictory results found in tobacco cells, seedlings of maize, barley
roots, etc. [5], nitrate is an inducer of both nitrate and nitrite reductase enzymes [6--8]. Nitrate uptake appears to play a crucial regulatory role on the control of nitrate reduction [5l. Moreover, nitrate reductase activity is sensitive to changes in the water status of plants and is inhibited when the water potential of plants declines [9--11] ; and so, nitrate reductase activity at low water potential is controlled by the nitrate flux [12] which in turn regulates the rate of synthesis of the enzyme. When water potential decreases the rate of protein synthesis decreases and increases as water potential increases [ 13]. The aim of the present work was to investigate the effect of asulam in water status and the mode in which this effect regulates the nitrate reduction. Materials and methods Plant material and growth conditions Seeds of M. sativa L. (cv. AragSn) and T. pratense L. (cv. Violeta) were placed in a perlite-containing seed-bed, covered with plastic
0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
22
and watered with Hewitt's solution [14]. After emergence, the plants were transplanted into 20-cm plastic pots also containing perlite, covered with a quartz sand layer, watered daily with Hewitt's solution. The plants were grown in a growth chamber (25/20°C day/ night; RH 50--70%; and a photoperiod of 14 h at 330 ~mol m-2 s -1 PAR). The plants used for herbicidal experiments were 4 weeks old. Groups of 10 plants were sprayed with 17 ml of water solutions of asulam at 0.15, 1.5 and 15 mM. The experiments were repeated in five independent series.
the pressure measured was converted to MPa and was used as water potential. The nitrate reductase activity was measured in vivo as described by Ref. 17, and is expressed as ~mole nitrite formed per g dry weight and per h. To determine the in vitro nitrite reductase activity extracts were prepared at 0--4°C. One •gramme leaf tissue was ground in a homogenizer at 2400 Xg for 30 s with 20 ml 50 mM Tris--HC1 (pH 8.0) containing 10 mM 2mercaptoethanol and 1 mM EDTA. The homogenate was squeezed through two layers of Miracloth and then centrifuged at 20 000 X g for 20 min at 0°C. The supernatant was used as the enzyme extract. Nitrite reductase activity was determinated colorimetrically by the diazotization method as in Ref. 18.
Experimental protocol Leaf conductances were measured on mature leaves with a flux porometer, type Alvim [15]. Measurements were made at the break of dawn at 0, 1, 2, 3, 4 and 7 days after asulam application. Predawn leaf water potential was measured with a pressure chamber [16]. The negative of
o o
Results Asulam decreased the fresh weight/dry weight ratio (Fig. 1). Fresh weight/dry weight
7
-4-,-*
3¢:
6
I-,I,,-
I
I
I
I
0
2
4
6
days
1
I
I
I
I
0
2
4
6
days
Fig. 1. Fresh weight/dry weight ratio (%) of M. sativa (open symbols) and T. pratense (closed symbols) shoots: H20 (*), 0.15 mM (o,.), 1.5 mM (v,.) and 15 mM (~,A) asulam.
23
ratio of lucerne was less affected than clover under asulam treatment. The effect of spraying with asulam on predawn leaf water potential is shown in Fig. 2. Control values obtained were about - 0 . 7 4 MPa and - 0 . 9 2 MPa for lucerne and clover, respectively. The asulam application caused a decay o f water status in both species. Values of a b o u t --1.0-- - 1.1 were registered in lucerne 4 days after herbicide application; later these rates remained practically constant. In clover, the water potential was continuously decreasing during the assay, reaching values of about - 1 . 2 5 MPa for 15 mM asulam concentration. Leaf conductance was measured as mole cm-: s -~. The values obtained were approx. 4 for lucerne and approx. 6 for clover. This only indicated that air flow from the lower face to the upper face was faster in clover than in lucerne. Asulam reduced stomatal conductance in leaves {Fig. 3). At the highest herbicide concentration (15 mM} both species closed
-T.
='-
-0.8
.T.
m
their stomata in a 80%, 7 days after asulam application; and above 60% with the lowest herbicide concentration. Nitrate reductase activity of leaves determined in vivo (Fig, 4) showed rates of 7.5 -+ 0.12 pmol NO~ g-1 dry wt. h -~ for lucerne control and 6.3 + 0.05 for clover control throughout the 7 days of study. Leaves of lucerne and clover sprayed with asulam showed a remarkable decrease o f nitrate reductase activity after 24 h of application. The nitrate reductase activity appeared to be very sensitive to this herbicide, so at the end of study the remaining activity was less than 30% of that in the untreated plants, except under 0.15 mM in lucerne where the remaining activity was 45%. The nitrite reductase activity determined in vitro (Fig. 5) showed rates of 1.27 +_ 0.04 mmol NO~ reduced g-1 dry wt. h -~ for lucerne control and 2.22 -+ 0.04 mmol NO~ reduced g-' dry wt. h -~ for clover control during the
m
m,
m . m
e,--
- 1.0
m
¢,,--
,.._.J
-1.2 I 0
I 2
.. I 4
days
I 6
I 0
I 2
I 4
I 6
days
Fig. 2. Leaf water potential of M. sativa (open symbols) and T. pratense (closed symbols): H20 ( t ) , 0.15 rnM (o ,.), 1.5 mM (D ,m ) and 15 mM (~ ,A) asulam.
24
100
8O
60
40
.-
2O
I 0
2
4
2
6
4
6
days
days
Fig. 3. Stomatal conductance (%) of M. sativa (open symbols) and T. pratense (closed symbols): 0.15 mM (c~,e), 1.5 mM (c,.) and 15 mM (~,a). Stomatal conductance of control leaves was constant at 3.95 _+0.09 and 5.97 + 0.06 mol cm-2 s-~ for lucerne and clover, respectively.
,°°t 8O
~.~
.~
60
40
Z
2
0
2
4
days
6
0
2
4
! 6
days
Fig. 4. Nitrate reductase activity (%) of M. sativa (open symbols) and T. pratense (closed symbols): 0.15 mM (o,e), 1.5 mM (o,m) and 15 mM (~,A) asulam. Nitrate reductase activities of leaves control were 7.5 +_ 0.12 and 6.3 _+0.05 ~mol NO~ appeared g-i dry wt. h - ' for lucerne and clover, respectively, along the study.
25
100
m
80
60
~ .
40 m
20 0
I 2
I 4
I 6
days
0
2
4
days
Fig. 5. Nitrite reductase activity (%) of M. sativa (open symbols)and T. pratense (closed symbols): 0.15 mM (o,*) 1.5 mM (o,u) and 15 mM (a,A) asulam. Nitrite reductase activities of leaves control were 1.37 + 0.04 and 2.22 _+ 0.04 mmol NO; reduced g-i dry wt. h -1 for lucerne and clover, respectively, along the study.
7 days of study. The effect of asulam on nitrite reductase activity of lucerne and clover was different; so 24 h post-sprayed the remaining activity in lucerne was still more than 70% whereas in clover it was less than 65%. It is remarkable that 15 mM of asulam decreased nitrite reductase activity up to 30% and 25% in lucerne and clover, respectively. In all treatments T. pratense was more sensitive than M. sativa since the values of fresh weight/dry weight ratio, water potential, leaf conductance and nitrate and nitrite reductase activities were lower in clover than in lucerne. Discussion Asulam inhibits protein metabolism [19] and alters purine synthesis [20]. However, the effect that this herbicide produces in plant water status must be considered, since the shoots of the asulam-treated plants had lost their turgidity, based on a decreasein the fresh weight/dry weight ratio at all concentrations
of asulam tested (Fig. 1). Moreover, asulam application caused a water potential decrease (Fig. 2) in both species, reaching in clover values of a b o u t --1.25 MPa for 15 mM asulam concentration. Boyer and Wu [21] reported that respiration inhibitors and oxygen deficiency probably reduce membrane permeability to water, and so water take [22]. On the other hand, Veerasekaran [23] investigated the effect of asulam on respiration of bracken frond buds. He found that in vitro treatment with 10--1000 ppm asulam inhibited respiration by up to 50%. Likewise we [24] observed that 1.5 mM asulam produced a decrease of oxygen uptake in subclover seedlings. In this way, the decay of water potential could be due to the effect of this c o m p o u n d on respiration. The rapid effect of asulam on stomatal closure (Fig. 3) could be the result of an interference with (a) energy production needed for
26
the operation of the K+ pump because we have previously indicated that this herbicide is an inhibitor of respiration; (b) water status of the plant resulting in an increase in abscisic acid {ABA) which can prevent K ÷ uptake. A n y w a y the effect of asulam in stomatal response is secondary mode of action; consequence of the water potential registered since similar stomata response were obtained in lucerne and clover, subjected to water stress [25], when their water potentials reached values very close to those produced by asulam. Leaves of lucerne and clover sprayed with asulam showed a remarkable decrease of nitrate reductase activity (Fig. 4), so at the end of study the remaining activity was less than 30% of that in the untreated plants. Nitrate reductase is a substrate dependent enzyme [6--8] and it has been shown t h a t the nitrate flux to the leaves from the roots plays a much larger regulatory role than the leaf nitrate content in controlling the level of nitrate reductase activity in intact plants [26--27]. Aparicio-Tejo and S~nchez-D/az [17] showed that water potentials lower than 1.0 MPa cause a lack of nitrate flux in M. sativa, this could explain the low nitrate reductase activity found by us. Moreover, there is a considerable evidence that the rates of protein synthesis decrease as water potential decreases [ 13]. Nitrate reductase activity in maize leaves has been shown to decrease at low water potential due to a decline in the rate o f synthesis of the enzyme rather than an increase rate of degradation or direct effect of water potential on enzyme activity [ 11 ]. On the other hand, several authors [20,28] reported that asulam inhibits purine biosynthesis by interfering with folic acid synthesis; so as a consequence of this, the nitrate reductase synthesis could be inhibited. The decrease of nitrate reductase activity could reasonably be, at least in part, due to the lack of substrate as a consequence of the water potential decrease, or to the inhibition of its synthesis. The nitrite reductase activity, determined in vitro (Fig. 5) also is reduced by asulam.
Since nitrite reductase seems to be a substratedependent enzyme, the inhibition of nitrate reductase caused by asulam (Fig. 4} produces lack of nitrite which is necessary for its synthesis. Moreover, the effect this herbicide produces on nucleic acid biosynthesis [20,28] might contribute to the lack of nitrite reductase activity in asulam-treated plants. Although asulam is regarded as an inhibitor of 7,8-dehydropteroate synthetase causing interference with folate synthesis that inhibits purine biosynthesis during peas germinations [20], we can conclude that in adult plants the effect o f asulam in nitrate metabolism is also a consequence of its action in plant water status. The greater effect that asulam produces in clover than in lucerne could be due to the fact that clover is more pilous, and consequently retains more herbicide.
References 1 M. Aslam, R.C. Huffaker and R.L. Travis, Plant Physiol., 52 (1973) 137. 2 K.P. Rao and D.W. Rains, Plant Physiol., 57 (1976) 55. 3 N. Bent-Salom, R.C. Huffaker and L. Rappaport, Plant Physiol., 71 (1983) 63. 4 P.C. Lolas, Weed Res., 20 (1980) 239. 5 M.G. Guerrero, J.M. Vega and M. Losada, Annu. Rev. Plant Physiol., 32 (1981) 169. 6 L. Beevers and R.H. Hageman, in : A.G. Giese (Ed.), Photophysiology, Vol. 7 (1972) 85. 7 E.J. Hewitt, Annu. Rev. Plant Physiol., 26 (1975) 73. 8 H. Srivastava, Phytochemistry, 19 (1980) 725. 9 J.M. Bardzik, H.V. Marsh, Jr. and J.R. Havis,Plant Physiol., 47 (1971) 828. 10 R.C. Huffaker, T. Radin, G.E. Kleinkopf and E.L. Cox, Crop Sci., 10 (1970) 471. 11 C.A. Morilla, J.S. Boyer and R.H. Hageman, Plant Physiol., 51 (1973) 817. 12 J.S. Boyer, Annu. Rev. Plant Physiol., 36 (1985) 473. 13 T.C. Hsiao, Annu. Rev. Plant Physiol., 24 (1973) 519. 14 E.J. Hewitt, Sand and Water Culture Methods Used in the Study of Plant Nutrition, 1966, p. 547. 15 B. Slavik, Methods of Studying Plant Water Relations (1974) 323. 16 P.F. Scholander, H.T. Hammel, E.D. Badstreet and E.A. Hemminsen, Science, 148 ( 1 9 6 5 ) 3 3 9 .
27 17 P.M. Aparicio-Tejo and M. S~nchez-Dfaz, Plant Physiol., 69 (1982) 479. 18 J.M. Vega, J. C~rdenas and M. Losada, Methods Enzymol., 69 (1980) 479. 19 P.Veerasekaran, R.C.Kirkwoodand W.E. Fletcher, Weed Res., 17 (1977) 85. 20 B.R. Kidd, N.H. Stephen and H.J. Duncan, Plant Sci. Lett., 26 (1982) 211. 21 J.S. Boyer and G. Wu, Planta, 139 (1978) 227. 22 P.J. Kramer, Int. Rev. Cytol., 85 (1983) 253. 23 P. Veerasekaran, Ph.D. Dissertation, Univ. of Strathclyde, Glasgow, 197 5.
24 A. Mufioz-Rueda, F. Hernando, C. Gonz~lezMurua y M. S6nchez-Dfaz, ITEA, 54 (1983) 25. 25 M. S~nchez-Dfaz, P.M. Aparicio-Tejo,C. GonzalezMurua and J.I. Pefia, Physiol. Plant., 54 (1982) 361. 26 D.L. Shaner and J.S. Boyer, Plant Physiol., 58 (1976) 4 9 9 . 27 D.L. Shaner and J.S. Boyer, Plant Physiol., 58 (1976) 505. 28 J.L. Killmer, J.M. Widholm and F. Slife, Plant Sci. Lett., 19 {1980) 203.
21
Elsevier Scientific Publishers Ireland Ltd.
THE EFFECT OF ASULAM ON WATER POTENTIAL AND NITRATE REDUCTION
ALBERTO MUNOZ-RUEDA, CARMEN GONZ.kLEZ-MURUA, JOSE MARfA BECERRIL, CESAREO ARRESEIGOR and MANUEL S.~NCHEZ-DfAZ*
Departamento de Fisiologia Vegetal, Facultad de Ciencias, Universidad del Pais Vasco, Apartado 644, Bilbao and *Departamento de Fisiologia Vegetal, Universidad de Navarra, Apartado 273, Pamplona (Spain) (Received December 24th, 1985) (Revision received May 6th, 1986) (Accepted May 26th, 1986) Nitrate and nitrite reductase activities, stomatal response and water potential were measured in Medicago sativa (cv. Arag6n) and Trifolium pratense (cv. Violeta) subjected to asulam methyl(4-aminobenzenesulphonyl)carbamate concentrations of 0.15 raM, 1.5 mM and 15 raM. The shoots of the asulam-treated plants lost their turgidity since the fresh weight/dry weight ratio decreased and a decay of water potential was observed, and consequently stomatal resistance was increased. Asulam treatment (15 raM) induced 80% stomatal closure in both species after 7 days. The nitrate and nitrite reductase activities appeared to be very sensitive to this herbicide. In general, clover was more sensitive to asulam than lucerne.
Key words: asulam; clover; lucerne; nitrate reduction; stomata; water potential
Introduction The interaction of water potential, respiration and herbicides in nitrate metabolism (nitrate and nitrite reductase activities) is not well understood. The positive effects of glucose in nitrate reductase induction in darkness indicate that respiration may be a key requirement for induction o f nitrate reductase activity in some plants. Respiration may produce the energy and compounds required for driving the induction o f nitrate reductase activity [1]. This hypothesis was supported by results obtained with respiratory inhibitors. Arsenate, cyanide and dinitrophenol which inhibit or uncouple oxidative phosphorylation or the respiratory chain, inhibit induction of nitrate reductase [1--3] ; and it is also known that the absorption of nitrate is decreased by inhibitors of respiration and oxidative phosphorylation. Furthermore, several investigators have presented evidence that herbicides affect nitrate reductase activity in a number of crops [4]. In spite of some contradictory results found in tobacco cells, seedlings of maize, barley
roots, etc. [5], nitrate is an inducer of both nitrate and nitrite reductase enzymes [6--8]. Nitrate uptake appears to play a crucial regulatory role on the control of nitrate reduction [5l. Moreover, nitrate reductase activity is sensitive to changes in the water status of plants and is inhibited when the water potential of plants declines [9--11] ; and so, nitrate reductase activity at low water potential is controlled by the nitrate flux [12] which in turn regulates the rate of synthesis of the enzyme. When water potential decreases the rate of protein synthesis decreases and increases as water potential increases [ 13]. The aim of the present work was to investigate the effect of asulam in water status and the mode in which this effect regulates the nitrate reduction. Materials and methods Plant material and growth conditions Seeds of M. sativa L. (cv. AragSn) and T. pratense L. (cv. Violeta) were placed in a perlite-containing seed-bed, covered with plastic
0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
22
and watered with Hewitt's solution [14]. After emergence, the plants were transplanted into 20-cm plastic pots also containing perlite, covered with a quartz sand layer, watered daily with Hewitt's solution. The plants were grown in a growth chamber (25/20°C day/ night; RH 50--70%; and a photoperiod of 14 h at 330 ~mol m-2 s -1 PAR). The plants used for herbicidal experiments were 4 weeks old. Groups of 10 plants were sprayed with 17 ml of water solutions of asulam at 0.15, 1.5 and 15 mM. The experiments were repeated in five independent series.
the pressure measured was converted to MPa and was used as water potential. The nitrate reductase activity was measured in vivo as described by Ref. 17, and is expressed as ~mole nitrite formed per g dry weight and per h. To determine the in vitro nitrite reductase activity extracts were prepared at 0--4°C. One •gramme leaf tissue was ground in a homogenizer at 2400 Xg for 30 s with 20 ml 50 mM Tris--HC1 (pH 8.0) containing 10 mM 2mercaptoethanol and 1 mM EDTA. The homogenate was squeezed through two layers of Miracloth and then centrifuged at 20 000 X g for 20 min at 0°C. The supernatant was used as the enzyme extract. Nitrite reductase activity was determinated colorimetrically by the diazotization method as in Ref. 18.
Experimental protocol Leaf conductances were measured on mature leaves with a flux porometer, type Alvim [15]. Measurements were made at the break of dawn at 0, 1, 2, 3, 4 and 7 days after asulam application. Predawn leaf water potential was measured with a pressure chamber [16]. The negative of
o o
Results Asulam decreased the fresh weight/dry weight ratio (Fig. 1). Fresh weight/dry weight
7
-4-,-*
3¢:
6
I-,I,,-
I
I
I
I
0
2
4
6
days
1
I
I
I
I
0
2
4
6
days
Fig. 1. Fresh weight/dry weight ratio (%) of M. sativa (open symbols) and T. pratense (closed symbols) shoots: H20 (*), 0.15 mM (o,.), 1.5 mM (v,.) and 15 mM (~,A) asulam.
23
ratio of lucerne was less affected than clover under asulam treatment. The effect of spraying with asulam on predawn leaf water potential is shown in Fig. 2. Control values obtained were about - 0 . 7 4 MPa and - 0 . 9 2 MPa for lucerne and clover, respectively. The asulam application caused a decay o f water status in both species. Values of a b o u t --1.0-- - 1.1 were registered in lucerne 4 days after herbicide application; later these rates remained practically constant. In clover, the water potential was continuously decreasing during the assay, reaching values of about - 1 . 2 5 MPa for 15 mM asulam concentration. Leaf conductance was measured as mole cm-: s -~. The values obtained were approx. 4 for lucerne and approx. 6 for clover. This only indicated that air flow from the lower face to the upper face was faster in clover than in lucerne. Asulam reduced stomatal conductance in leaves {Fig. 3). At the highest herbicide concentration (15 mM} both species closed
-T.
='-
-0.8
.T.
m
their stomata in a 80%, 7 days after asulam application; and above 60% with the lowest herbicide concentration. Nitrate reductase activity of leaves determined in vivo (Fig, 4) showed rates of 7.5 -+ 0.12 pmol NO~ g-1 dry wt. h -~ for lucerne control and 6.3 + 0.05 for clover control throughout the 7 days of study. Leaves of lucerne and clover sprayed with asulam showed a remarkable decrease o f nitrate reductase activity after 24 h of application. The nitrate reductase activity appeared to be very sensitive to this herbicide, so at the end of study the remaining activity was less than 30% of that in the untreated plants, except under 0.15 mM in lucerne where the remaining activity was 45%. The nitrite reductase activity determined in vitro (Fig. 5) showed rates of 1.27 +_ 0.04 mmol NO~ reduced g-1 dry wt. h -~ for lucerne control and 2.22 -+ 0.04 mmol NO~ reduced g-' dry wt. h -~ for clover control during the
m
m,
m . m
e,--
- 1.0
m
¢,,--
,.._.J
-1.2 I 0
I 2
.. I 4
days
I 6
I 0
I 2
I 4
I 6
days
Fig. 2. Leaf water potential of M. sativa (open symbols) and T. pratense (closed symbols): H20 ( t ) , 0.15 rnM (o ,.), 1.5 mM (D ,m ) and 15 mM (~ ,A) asulam.
24
100
8O
60
40
.-
2O
I 0
2
4
2
6
4
6
days
days
Fig. 3. Stomatal conductance (%) of M. sativa (open symbols) and T. pratense (closed symbols): 0.15 mM (c~,e), 1.5 mM (c,.) and 15 mM (~,a). Stomatal conductance of control leaves was constant at 3.95 _+0.09 and 5.97 + 0.06 mol cm-2 s-~ for lucerne and clover, respectively.
,°°t 8O
~.~
.~
60
40
Z
2
0
2
4
days
6
0
2
4
! 6
days
Fig. 4. Nitrate reductase activity (%) of M. sativa (open symbols) and T. pratense (closed symbols): 0.15 mM (o,e), 1.5 mM (o,m) and 15 mM (~,A) asulam. Nitrate reductase activities of leaves control were 7.5 +_ 0.12 and 6.3 _+0.05 ~mol NO~ appeared g-i dry wt. h - ' for lucerne and clover, respectively, along the study.
25
100
m
80
60
~ .
40 m
20 0
I 2
I 4
I 6
days
0
2
4
days
Fig. 5. Nitrite reductase activity (%) of M. sativa (open symbols)and T. pratense (closed symbols): 0.15 mM (o,*) 1.5 mM (o,u) and 15 mM (a,A) asulam. Nitrite reductase activities of leaves control were 1.37 + 0.04 and 2.22 _+ 0.04 mmol NO; reduced g-i dry wt. h -1 for lucerne and clover, respectively, along the study.
7 days of study. The effect of asulam on nitrite reductase activity of lucerne and clover was different; so 24 h post-sprayed the remaining activity in lucerne was still more than 70% whereas in clover it was less than 65%. It is remarkable that 15 mM of asulam decreased nitrite reductase activity up to 30% and 25% in lucerne and clover, respectively. In all treatments T. pratense was more sensitive than M. sativa since the values of fresh weight/dry weight ratio, water potential, leaf conductance and nitrate and nitrite reductase activities were lower in clover than in lucerne. Discussion Asulam inhibits protein metabolism [19] and alters purine synthesis [20]. However, the effect that this herbicide produces in plant water status must be considered, since the shoots of the asulam-treated plants had lost their turgidity, based on a decreasein the fresh weight/dry weight ratio at all concentrations
of asulam tested (Fig. 1). Moreover, asulam application caused a water potential decrease (Fig. 2) in both species, reaching in clover values of a b o u t --1.25 MPa for 15 mM asulam concentration. Boyer and Wu [21] reported that respiration inhibitors and oxygen deficiency probably reduce membrane permeability to water, and so water take [22]. On the other hand, Veerasekaran [23] investigated the effect of asulam on respiration of bracken frond buds. He found that in vitro treatment with 10--1000 ppm asulam inhibited respiration by up to 50%. Likewise we [24] observed that 1.5 mM asulam produced a decrease of oxygen uptake in subclover seedlings. In this way, the decay of water potential could be due to the effect of this c o m p o u n d on respiration. The rapid effect of asulam on stomatal closure (Fig. 3) could be the result of an interference with (a) energy production needed for
26
the operation of the K+ pump because we have previously indicated that this herbicide is an inhibitor of respiration; (b) water status of the plant resulting in an increase in abscisic acid {ABA) which can prevent K ÷ uptake. A n y w a y the effect of asulam in stomatal response is secondary mode of action; consequence of the water potential registered since similar stomata response were obtained in lucerne and clover, subjected to water stress [25], when their water potentials reached values very close to those produced by asulam. Leaves of lucerne and clover sprayed with asulam showed a remarkable decrease of nitrate reductase activity (Fig. 4), so at the end of study the remaining activity was less than 30% of that in the untreated plants. Nitrate reductase is a substrate dependent enzyme [6--8] and it has been shown t h a t the nitrate flux to the leaves from the roots plays a much larger regulatory role than the leaf nitrate content in controlling the level of nitrate reductase activity in intact plants [26--27]. Aparicio-Tejo and S~nchez-D/az [17] showed that water potentials lower than 1.0 MPa cause a lack of nitrate flux in M. sativa, this could explain the low nitrate reductase activity found by us. Moreover, there is a considerable evidence that the rates of protein synthesis decrease as water potential decreases [ 13]. Nitrate reductase activity in maize leaves has been shown to decrease at low water potential due to a decline in the rate o f synthesis of the enzyme rather than an increase rate of degradation or direct effect of water potential on enzyme activity [ 11 ]. On the other hand, several authors [20,28] reported that asulam inhibits purine biosynthesis by interfering with folic acid synthesis; so as a consequence of this, the nitrate reductase synthesis could be inhibited. The decrease of nitrate reductase activity could reasonably be, at least in part, due to the lack of substrate as a consequence of the water potential decrease, or to the inhibition of its synthesis. The nitrite reductase activity, determined in vitro (Fig. 5) also is reduced by asulam.
Since nitrite reductase seems to be a substratedependent enzyme, the inhibition of nitrate reductase caused by asulam (Fig. 4} produces lack of nitrite which is necessary for its synthesis. Moreover, the effect this herbicide produces on nucleic acid biosynthesis [20,28] might contribute to the lack of nitrite reductase activity in asulam-treated plants. Although asulam is regarded as an inhibitor of 7,8-dehydropteroate synthetase causing interference with folate synthesis that inhibits purine biosynthesis during peas germinations [20], we can conclude that in adult plants the effect o f asulam in nitrate metabolism is also a consequence of its action in plant water status. The greater effect that asulam produces in clover than in lucerne could be due to the fact that clover is more pilous, and consequently retains more herbicide.
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