- Email: [email protected]
Inhibition of nitrate and nitrite reductase activities by salinity stress in Sorghum vulgare
Phytochemistry,Vol. 29, No. 4, pp. 1047-1049,1990. Printed in Great Britain.
INHIBITION
003l-9422/90 $3.00+ 0.00 0 1990Pergamon Press plc
OF NITRATE AND NITRITE REDUCTASE ACTIVITIES SALINITYSTRESSIN SORGHUM VULGARE
BY
R. KRISHNA RAO*~ and A. GNANAM~ Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India; SUniversity of Madras, Madras, India (Received in
revisedform 22 August 1989)
Key Word Index-Sorghum uulgare; Gramineae; nitrate reductase; nitrite reductase; salinity stress.
early effects of sodium chloride (NaCl) salinization on the activities of nitrate reductase (NR) and nitrite reductase (NIR) have been studied in the leaves of intact seedlings of Sorghum uulgare The activities of both NR and NIR were inhibited, albeit differentially, as a function of NaCl concentration in the growth medium. The inhibition of NR as well as NIR activities was more at much lower NaCl concentrations when the enzyme extract was exposed to the salt in oitro for 15 min. A similar inhibition of NR activity was observed when the enzyme extracts were exposed to sodium sulphate in vitro. The inhibition of NR activity was comparatively more when the enzyme extract was incubated with potassium chloride. The results show that NR is more sensitive than NIR to NaCl stress in uiuo as well as in vitro, and anionic salinity is more toxic to NR activity than is cationic salinity. Abstract-The
INTRODUCTION
RESULTS
The deleterious effects of salinity stress on the growth and metabolism of non-halophytic higher plants have been attributed to osmotic (water deficit) as well as ion-specific toxic effects [l-3]. Nitrate is the predominant form of nitrogen available to crop plants in normal field conditions and productivity of plants is largely influenced by the acquisition and reduction of nitrate nitrogen and its incorporation into amino acids and proteins 143. In view of the decreased productivity of nonhalophytic crop plants in saline soils [3], uptake and reduction of nitrate assume central importance in plants exposed to saline and other stress conditions. Nitrate reductase (NR, EC 1.6.6.1), localized in the cytoplasm, is the first enzyme in the pathway of nitrate assimilation and catalyses the reduction of nitrate to nitrite which is further reduced to ammonium by nitrite reductase (NIR, EC 1.7.7.1) in the chloroplast [4]. The effects of salinity on nitrogen assimilation are controversial. Whereas impaired uptake of nitrogen has b en reported in barley plants salinized with sodium cB loride (NaCI) [S], increase in protein content due to NaCl salinity has been observed in stargrass [6]. Though specific ion effects of K+ on proline accumulation [7] and leaf growth [S] have been observed in Sorghum bicolor stressed with sodium and potassium salts, relatively less is known about the effects of NaCl stress on the activities of cytoplasmic NR and chloroplast-localized NIR in sorghum. To separate the involvement of water stress effects from salt effects, we have chosen a droughttolerant variety, C024, of sorghum and examined the effects of salts on the enzyme activities in extracts of unstressed sorghum leaves.
When eight-day-old Sorghum uulgare seedlings were exposed to varying amounts of NaCl given in four consecutive days in the growth medium, no visible leaf damage occurred at external NaCl concentration of 0.25 M (- 12 bar) and 0.5 M (-24 bar). Only at 1 M NaCl (-48 bar) was necrosis of the leaf tip observed. On the other hand, at all the aforementioned NaCl concentrations in the growth medium NR as well as NIR activities were inhibited, the inhibition of NR activity being comparatively more than that of NIR (Fig. 1). The I,, for NR activity was 0.7 M NaCl. In order to separate the effects of reduced leaf water potential due to NaCl in the growth medium from those of NaCl per se on NR and NIR activities, crude enzyme extracts from leaves of unstressed seedlings were incubated with varying amounts of NaCl for 15 min. NR and NIR activities were inhibited progressively with increase in NaCl concentration in vitro, inhibition of NR activity being more than that of NIR at all NaCl concentrations used (Fig. 2). Interestingly, the I,, values for NR and NIR activities decreased to 110 and 200 mM, respectively. Recent evidence indicates that Cl- and SO:- salts differentially affect NO; uptake in barley seedlings [S]. In order to check whether the inhibitory effect of NaCl on NR activity in uitro was confined to NaCl alone, crude enzyme extracts were incubated for 15 min. with sodium sulphate (Na,SO,) and potassium chloride (KCl). Results in Fig. 3 show that NR activity was inhibited progressively with increased concentration of Na,SO, and KCl in the assay medium. The magnitude of inhibition of NR activity was more in presence of KC1 compared to NazSO,, the I,, of the former (22 mM) being nearly fourfold lower than that of the latter. A comparison of the data presented in Figs 2 and 3 shows less marked differences in the level of inhibition of enzyme activity, even at twice Na+ concentration. Further, in the presence of the corresponding concentrations of KCl, inhibition of
*Author to whom correspondence should be addressed. tPresent address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, U.S.A.
1047
R. KRISHNA RAO and A. GNANAM
/
1
0
0.25
1.0
0.5
Fig. 1. Effect of NaCl stress on the activities of NR and NIR_in sorghum leaves. The seedlings were salinized through the roots (see Experimental). The control (= 100) rates for in uitro NR and NIR activities were 1.4411mol NO; formed/‘hr/g fr. wt and 43.2 pmol NO; reducedlhr,‘g fr. wt respectively.
25
50
200
loo NaCl ( mM
1
1
25
SO
Salt
NaCl ( M )
0
0
)
Fig. 2. Effect of NaCl in the assay mixture on the in uirro NR and NIR activities of sorghum leaves. Control activities (= 100) for NR and NIR activities were respectively 2.55 pmol NO, formedjhr,‘g fr. wt and 42.0 !cmol NO; reducedjhrlg fr. wt.
NR activity was 55,62 and 75% (Fig. 3), showing that K’ is more inhibitory to NR activity than is Na+.
DISCUSSION
Plaut has attributed the decrease in NR activity in leaves of wheat seedlings subjected to an osmotic potential of -6 bar caused by NaCl supplied to the roots through the nutrient medium to a decline in osmotic potential and not to a direct inhibition of enzyme reaction [9]. Though the present results with intact sorghum seedlings do not rule out the contribution of a reduction in leaf water potential to the decline in NR and NIR
loo
( mM )
Fig. 3. Effect of Na,SO, and KC1 in the assay mixture on the in vitro NR activity. The control (= 100) rate was 2.0 jtmol NO; formed’hr g fr. wt.
activities (Fig. l), the inhibition of both enzyme activities in vitro by much lower concentrations of NaCl per se (Fig. 2) the drought-tolerant nature of sorghum and its reported ability to exclude Na’ [lo] lead us to speculate that the inhibition of NR and NIR activities in leaves of intact Sorghum vulgare seedlings was more due to the salt per se. This is in agreement with a widely accepted view that salinity-induced derangement of plant metabolism is due to a condition of ion excess [Z]. Further. the requirement of 0.7 M NaCl in the growth medium to cause 50% inhibition of leaf NR activity compared to 0.1 M NaCl for a similar magnitude of inhibition of the enzyme activity in vitro clearly demonstrates that NR activity is more sensitive to the salt in vitro. Despite twice the cation concentration in extracts incubated in Na,SO,, the magnitude of inhibition of NR activity in enzyme extracts exposed to NaCl (t&l00 mM; Fig. 2) and Na,SO, (Fig. 3) was similar, indicating that NR activity is more sensitive to anionic salinity than cationic salinity. A recent observation by Barber et al. shows that in vitro inhibition of NR occurs by chloride [ 111, supporting our present observations. Although Cl and SO, have been shown to affect differentially NO; uptake rate in barley seedlings [5] they do not appear to affect NR activity similarly in vitro. A greater inhibition of NR activity by KC1 in rirro compared with that by Na,SO, (Fig. 3) and equal concentrations (&IO0 mM) of NaCl (Fig. 2) indicates that K + is toxic to sorghum leaf NR. Though many cytoplasmic enzymes have been reported to respond similarly to Na+ and K + [ 12, 133, the present results show that the cytoplasmic NR in sorghum responds differently to Na ’ and K ‘. This agrees with greater inhibition of phosphophenol pyruvate carboxylase activity by KC1 relative to Na,SO, [13] and also supports similar observations of Schrader [ 141. The greater decline in NR activity relative to NIR activity in seedlings stressed with NaCl as well as in enzyme extracts incubated with the salt (Fig. 2) is in agreement with the reported lesser sensitivity of NIR to stresses caused by water deficit [ 15, 161 and bleaching and non-bleaching herbicides [17, 181.
Nitrate
and nitrite reductases
EXPERIMENTAL P/ant material. Seeds of Sorghum uulgare L. (CO24), a drought-tolerant variety, were surface-sterilized in 5% NaCIO for 5 min, thoroughly washed several times in tap H,O and soaked overnight in running tap H,O. Seedlings were grown in a mixture of washed vermiculite and sand (1: 1) in fiat plastic trays with drainage holes and irrigated daily after shoot emergence with Hoagland’s medium containing 15 mM nitrate [5 mM KN03, 5 mM Ca(NO,),]. Seedlings were raised in natural conditions (lGOOO0 lx, 12 hr day/l2 hr night). Salinization. 7-day-old seedlings raised in dist. H,O in vermiculite and sand mixture (1: 1) were fertilized once in 2 days with Hoagland’s medium containing 15 mM KNO, k NaCl (pH 5.8) for 5 days. Seedlings, (15-day-old) were harvested after the last exposure to salinization. Extraction and assay of NR and NIR activities. The time of leaf harvest, extraction of NR and NIR and the in oitro assay methods of NR and NIR were essentially as described in ref. [17] except that the enzyme extraction medium was composed of 100 mM Tris-HCI, pH 8.0, 4 mM NiCI,, 5 mM dithiothreitol and 1 mM EDTA. Unless otherwise stated, the results are an average of 2 experiments. REFERENCES
1. Jennings, D. H. (1976) Biol. Rev. 51, 453. 2. Greenway, H. and Munns, R. (1980) Ann. Rev. Plant Physiol. 31, 149. 3. Levitt, J. (1980) Responses of Plants to Environmental Stresses Vol. II. Academic Press, New York.
in Sorghum
1049
4. Beevers, L. and Hageman, R. H. (1983) in Encyclopedia of Plant Physiology New Series Vol. 15A (Lauchli, A. and Bieleski, R. L., eds), pp. 151-197. Springer, Berlin. 5. Aslam, M., Huffaker, R. C. and Rains, D. W. (1984) Plant Physiol. 76, 321. 6. Langdale, G. W., Thomas, J. R. and Littleton, T. G. (1973) Agron. J. 65, 468. 7. Weimberg, R., Lerner, H. R. and Poljakoll-Mayber, A. (1982) Physiol. Plant. 55, 5. 8. Weimberg, R., Lerner, H. R. and Poljakoff-Mayber. A. (1984) Physiol. Plant. 62, 472. 9. Plaut, Z. (1974) Physiol. Plant. 30, 212. 10. Flowers, T. J. and Yeo, A. R. (1981) New Phytol. 88, 363. M. J.. Notton, B. A., Kay, C. J. and 11. Barber, Solomonson, L. P. (1989) Plant Physiol. 90, 70. 12. Greenway, H. and Osmond, C. B. (1972) Plant Physiof. 49, 256. 13. Osmond, C. B. and Greenway, H. (1972) Plant Physiol. 49, 200. 14. Schrader, L. E. (1978) Nitrogen in the Environment. (Nielsen, D. R. and McDonald, J. G., eds), pp. 101-141. Academic Press, New York. 15. Heuer, B., Plaut, Z. and Federman, E. (1979). Plant. Physiol. 46, 318. 16. Hanson, A. D. and Hitz, W. D. (1982) Ann. Rev. Pfant Physiol. 33, 163. 17. Rao, R. K., Mannan, R. M., Gnanam, A. and Bose, S. (1988) Phytochemistry 27, 685. 18. Rao, R. K., Mannan, R. M., Gnanam, A. and Bose, S. (1988) Biochem. Int. 17, 691.
INHIBITION
003l-9422/90 $3.00+ 0.00 0 1990Pergamon Press plc
OF NITRATE AND NITRITE REDUCTASE ACTIVITIES SALINITYSTRESSIN SORGHUM VULGARE
BY
R. KRISHNA RAO*~ and A. GNANAM~ Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India; SUniversity of Madras, Madras, India (Received in
revisedform 22 August 1989)
Key Word Index-Sorghum uulgare; Gramineae; nitrate reductase; nitrite reductase; salinity stress.
early effects of sodium chloride (NaCl) salinization on the activities of nitrate reductase (NR) and nitrite reductase (NIR) have been studied in the leaves of intact seedlings of Sorghum uulgare The activities of both NR and NIR were inhibited, albeit differentially, as a function of NaCl concentration in the growth medium. The inhibition of NR as well as NIR activities was more at much lower NaCl concentrations when the enzyme extract was exposed to the salt in oitro for 15 min. A similar inhibition of NR activity was observed when the enzyme extracts were exposed to sodium sulphate in vitro. The inhibition of NR activity was comparatively more when the enzyme extract was incubated with potassium chloride. The results show that NR is more sensitive than NIR to NaCl stress in uiuo as well as in vitro, and anionic salinity is more toxic to NR activity than is cationic salinity. Abstract-The
INTRODUCTION
RESULTS
The deleterious effects of salinity stress on the growth and metabolism of non-halophytic higher plants have been attributed to osmotic (water deficit) as well as ion-specific toxic effects [l-3]. Nitrate is the predominant form of nitrogen available to crop plants in normal field conditions and productivity of plants is largely influenced by the acquisition and reduction of nitrate nitrogen and its incorporation into amino acids and proteins 143. In view of the decreased productivity of nonhalophytic crop plants in saline soils [3], uptake and reduction of nitrate assume central importance in plants exposed to saline and other stress conditions. Nitrate reductase (NR, EC 1.6.6.1), localized in the cytoplasm, is the first enzyme in the pathway of nitrate assimilation and catalyses the reduction of nitrate to nitrite which is further reduced to ammonium by nitrite reductase (NIR, EC 1.7.7.1) in the chloroplast [4]. The effects of salinity on nitrogen assimilation are controversial. Whereas impaired uptake of nitrogen has b en reported in barley plants salinized with sodium cB loride (NaCI) [S], increase in protein content due to NaCl salinity has been observed in stargrass [6]. Though specific ion effects of K+ on proline accumulation [7] and leaf growth [S] have been observed in Sorghum bicolor stressed with sodium and potassium salts, relatively less is known about the effects of NaCl stress on the activities of cytoplasmic NR and chloroplast-localized NIR in sorghum. To separate the involvement of water stress effects from salt effects, we have chosen a droughttolerant variety, C024, of sorghum and examined the effects of salts on the enzyme activities in extracts of unstressed sorghum leaves.
When eight-day-old Sorghum uulgare seedlings were exposed to varying amounts of NaCl given in four consecutive days in the growth medium, no visible leaf damage occurred at external NaCl concentration of 0.25 M (- 12 bar) and 0.5 M (-24 bar). Only at 1 M NaCl (-48 bar) was necrosis of the leaf tip observed. On the other hand, at all the aforementioned NaCl concentrations in the growth medium NR as well as NIR activities were inhibited, the inhibition of NR activity being comparatively more than that of NIR (Fig. 1). The I,, for NR activity was 0.7 M NaCl. In order to separate the effects of reduced leaf water potential due to NaCl in the growth medium from those of NaCl per se on NR and NIR activities, crude enzyme extracts from leaves of unstressed seedlings were incubated with varying amounts of NaCl for 15 min. NR and NIR activities were inhibited progressively with increase in NaCl concentration in vitro, inhibition of NR activity being more than that of NIR at all NaCl concentrations used (Fig. 2). Interestingly, the I,, values for NR and NIR activities decreased to 110 and 200 mM, respectively. Recent evidence indicates that Cl- and SO:- salts differentially affect NO; uptake in barley seedlings [S]. In order to check whether the inhibitory effect of NaCl on NR activity in uitro was confined to NaCl alone, crude enzyme extracts were incubated for 15 min. with sodium sulphate (Na,SO,) and potassium chloride (KCl). Results in Fig. 3 show that NR activity was inhibited progressively with increased concentration of Na,SO, and KCl in the assay medium. The magnitude of inhibition of NR activity was more in presence of KC1 compared to NazSO,, the I,, of the former (22 mM) being nearly fourfold lower than that of the latter. A comparison of the data presented in Figs 2 and 3 shows less marked differences in the level of inhibition of enzyme activity, even at twice Na+ concentration. Further, in the presence of the corresponding concentrations of KCl, inhibition of
*Author to whom correspondence should be addressed. tPresent address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, U.S.A.
1047
R. KRISHNA RAO and A. GNANAM
/
1
0
0.25
1.0
0.5
Fig. 1. Effect of NaCl stress on the activities of NR and NIR_in sorghum leaves. The seedlings were salinized through the roots (see Experimental). The control (= 100) rates for in uitro NR and NIR activities were 1.4411mol NO; formed/‘hr/g fr. wt and 43.2 pmol NO; reducedlhr,‘g fr. wt respectively.
25
50
200
loo NaCl ( mM
1
1
25
SO
Salt
NaCl ( M )
0
0
)
Fig. 2. Effect of NaCl in the assay mixture on the in uirro NR and NIR activities of sorghum leaves. Control activities (= 100) for NR and NIR activities were respectively 2.55 pmol NO, formedjhr,‘g fr. wt and 42.0 !cmol NO; reducedjhrlg fr. wt.
NR activity was 55,62 and 75% (Fig. 3), showing that K’ is more inhibitory to NR activity than is Na+.
DISCUSSION
Plaut has attributed the decrease in NR activity in leaves of wheat seedlings subjected to an osmotic potential of -6 bar caused by NaCl supplied to the roots through the nutrient medium to a decline in osmotic potential and not to a direct inhibition of enzyme reaction [9]. Though the present results with intact sorghum seedlings do not rule out the contribution of a reduction in leaf water potential to the decline in NR and NIR
loo
( mM )
Fig. 3. Effect of Na,SO, and KC1 in the assay mixture on the in vitro NR activity. The control (= 100) rate was 2.0 jtmol NO; formed’hr g fr. wt.
activities (Fig. l), the inhibition of both enzyme activities in vitro by much lower concentrations of NaCl per se (Fig. 2) the drought-tolerant nature of sorghum and its reported ability to exclude Na’ [lo] lead us to speculate that the inhibition of NR and NIR activities in leaves of intact Sorghum vulgare seedlings was more due to the salt per se. This is in agreement with a widely accepted view that salinity-induced derangement of plant metabolism is due to a condition of ion excess [Z]. Further. the requirement of 0.7 M NaCl in the growth medium to cause 50% inhibition of leaf NR activity compared to 0.1 M NaCl for a similar magnitude of inhibition of the enzyme activity in vitro clearly demonstrates that NR activity is more sensitive to the salt in vitro. Despite twice the cation concentration in extracts incubated in Na,SO,, the magnitude of inhibition of NR activity in enzyme extracts exposed to NaCl (t&l00 mM; Fig. 2) and Na,SO, (Fig. 3) was similar, indicating that NR activity is more sensitive to anionic salinity than cationic salinity. A recent observation by Barber et al. shows that in vitro inhibition of NR occurs by chloride [ 111, supporting our present observations. Although Cl and SO, have been shown to affect differentially NO; uptake rate in barley seedlings [5] they do not appear to affect NR activity similarly in vitro. A greater inhibition of NR activity by KC1 in rirro compared with that by Na,SO, (Fig. 3) and equal concentrations (&IO0 mM) of NaCl (Fig. 2) indicates that K + is toxic to sorghum leaf NR. Though many cytoplasmic enzymes have been reported to respond similarly to Na+ and K + [ 12, 133, the present results show that the cytoplasmic NR in sorghum responds differently to Na ’ and K ‘. This agrees with greater inhibition of phosphophenol pyruvate carboxylase activity by KC1 relative to Na,SO, [13] and also supports similar observations of Schrader [ 141. The greater decline in NR activity relative to NIR activity in seedlings stressed with NaCl as well as in enzyme extracts incubated with the salt (Fig. 2) is in agreement with the reported lesser sensitivity of NIR to stresses caused by water deficit [ 15, 161 and bleaching and non-bleaching herbicides [17, 181.
Nitrate
and nitrite reductases
EXPERIMENTAL P/ant material. Seeds of Sorghum uulgare L. (CO24), a drought-tolerant variety, were surface-sterilized in 5% NaCIO for 5 min, thoroughly washed several times in tap H,O and soaked overnight in running tap H,O. Seedlings were grown in a mixture of washed vermiculite and sand (1: 1) in fiat plastic trays with drainage holes and irrigated daily after shoot emergence with Hoagland’s medium containing 15 mM nitrate [5 mM KN03, 5 mM Ca(NO,),]. Seedlings were raised in natural conditions (lGOOO0 lx, 12 hr day/l2 hr night). Salinization. 7-day-old seedlings raised in dist. H,O in vermiculite and sand mixture (1: 1) were fertilized once in 2 days with Hoagland’s medium containing 15 mM KNO, k NaCl (pH 5.8) for 5 days. Seedlings, (15-day-old) were harvested after the last exposure to salinization. Extraction and assay of NR and NIR activities. The time of leaf harvest, extraction of NR and NIR and the in oitro assay methods of NR and NIR were essentially as described in ref. [17] except that the enzyme extraction medium was composed of 100 mM Tris-HCI, pH 8.0, 4 mM NiCI,, 5 mM dithiothreitol and 1 mM EDTA. Unless otherwise stated, the results are an average of 2 experiments. REFERENCES
1. Jennings, D. H. (1976) Biol. Rev. 51, 453. 2. Greenway, H. and Munns, R. (1980) Ann. Rev. Plant Physiol. 31, 149. 3. Levitt, J. (1980) Responses of Plants to Environmental Stresses Vol. II. Academic Press, New York.
in Sorghum
1049
4. Beevers, L. and Hageman, R. H. (1983) in Encyclopedia of Plant Physiology New Series Vol. 15A (Lauchli, A. and Bieleski, R. L., eds), pp. 151-197. Springer, Berlin. 5. Aslam, M., Huffaker, R. C. and Rains, D. W. (1984) Plant Physiol. 76, 321. 6. Langdale, G. W., Thomas, J. R. and Littleton, T. G. (1973) Agron. J. 65, 468. 7. Weimberg, R., Lerner, H. R. and Poljakoll-Mayber, A. (1982) Physiol. Plant. 55, 5. 8. Weimberg, R., Lerner, H. R. and Poljakoff-Mayber. A. (1984) Physiol. Plant. 62, 472. 9. Plaut, Z. (1974) Physiol. Plant. 30, 212. 10. Flowers, T. J. and Yeo, A. R. (1981) New Phytol. 88, 363. M. J.. Notton, B. A., Kay, C. J. and 11. Barber, Solomonson, L. P. (1989) Plant Physiol. 90, 70. 12. Greenway, H. and Osmond, C. B. (1972) Plant Physiof. 49, 256. 13. Osmond, C. B. and Greenway, H. (1972) Plant Physiol. 49, 200. 14. Schrader, L. E. (1978) Nitrogen in the Environment. (Nielsen, D. R. and McDonald, J. G., eds), pp. 101-141. Academic Press, New York. 15. Heuer, B., Plaut, Z. and Federman, E. (1979). Plant. Physiol. 46, 318. 16. Hanson, A. D. and Hitz, W. D. (1982) Ann. Rev. Pfant Physiol. 33, 163. 17. Rao, R. K., Mannan, R. M., Gnanam, A. and Bose, S. (1988) Phytochemistry 27, 685. 18. Rao, R. K., Mannan, R. M., Gnanam, A. and Bose, S. (1988) Biochem. Int. 17, 691.