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The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato (Solanum tuberosum L.)
]. Plant PhysIOI. Vol. 132. pp. 540- 544 (1988)
The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato (Solanum tuberosum L.) H. V.
DAVIES,
H. A. Ross, and R.
THOMPSON
Scottish Crop Research Institute, Invergowrie, Dundee DD25DA, United Kingdom Received September 11, 1987 . Accepted October 28, 1987
Summary Changes in nitrate reductase activity in the youngest expanded leaves of the potato plant have been monitored in the field under a range of N regimes. Highest activity (in vivo assay, substrate non-limiting) occurred with the highest rate of N application, as did the highest leaf protein content. In high-N leaves the onset of the decline in nitrate reductase activity and protein content, which commenced around 80 days after planting, was not prevented by the application of high levels of nitrogen. This could not be explained by a fall in the concentration of nitrate in the petiole xylem sap supplying the leaves in question. The ratio of nitrate-N to amino acid-N in petiole xylem sap remained relatively constant throughout the growing period in plants receiving 8 g N m -2 at 14 day intervals, but declined under lower N inputs. Only in the highest N treatment did the ratio remain greater than unity. The reasons for these observations are discussed. Nitrate reductase activity was also determined in leaves of field-grown plants with all of the N (20gm- 2) applied pre-planting. A comparison of in vivo assays carried out with and without nitrate in the assay medium showed that nitrate reductase was likely to limit nitrate assimilation only during the earliest stages of post-emergence growth. Abbreviations: DAP - days after planting; NR - nitrate reductase.
Introduction Nitrate reductase activity in the potato plant is located principally in the leaves (Kapoor and Li, 1982). Recent investigations (Davies and Ross, 1985) have demonstrated a distinctive pattern of change in nitrate reductase activity in leaves of field-grown crops over the course of a growing season. Enzyme activity increased to a peak around mid-July and declined substantially thereafter, peaks in leaf chlorophyll and protein content occurring at approximately the same time. The onset of chlorophyll and protein degradation in leaves from the unshaded, photosynthetically active region of the canopy, evidently signals the initiation of processes which will ultimately affect leaf and canopy photosynthetic performance and hence yield. Although plant senescence has, in several instances, been delayed or prevented by nitrogen supplied exogenously (Hill, 1980), the © 1988 by Gustav fischer Verlag, Stuttgart
nutrient theory of senescence is sometimes criticised (Thomas and Stoddart, 1980; Neumann and Stein, 1986). Nevertheless, the response of ageing leaves to modifications in the supply of essential elements such as nitrogen may ultimately determine the plant's capacity to modify its pattern of senescence. The present study examines the possibility that frequent applications of very high levels of nitrogen to field-grown potato plants might modify the pattern of senescence. The study also examines further the limitation to the rate of nitrate reduction set by the activity of nitrate reductase.
Materials and Methods Seed tubers of potato (Solanum tuberosum L.) cv. Maris Piper were planted in flat beds in the field at SCRI on 30 April 1985 in
The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato 75 em rows and at a density of 6 plants m -2. P was applied at 8 g m - 2 prior to planting and K at 20 g m - 2. N (as ammonium nitrate) was applied through trickle irrigation at 14 d intervals from planting. Four N treatments were used; at each application NOno N applied, N1- 0.8gNm-2, N2- 3.2gNm- 2, N38gNm-2. This resulted in total application rates for the season of 0, 7.2, 28.8 and 72.0 g N m - 2, respectively. Plots, each 12 m x 7 m and arranged in two randomised blocks, were irrigated when soil moisture deficit reached 25 KPa. Between 11.00 and 12.00 h at each harvest date four plants were taken from each plot. Two plants from each plot were combined for analysis. The youngest expanded leaves from the top of the canopy (commonly leaves 4 and 5 from the apex) were excised at their point of attachment to the stem and combined to give sufficient leaf material for analyses. With the exception of nitrate reductase determinations leaf analyses were performed on freeze-dried and finely-ground material. All analyses were carried out in triplicate. Determinations of In VIVO nitrate reductase activity, with and without nitrate in the assay medium, were carried out on a crop grown in 1986. These plots were prepared as outlined by Davies et al. (1987) with all of the N applied prior to planting (20 g N m - 2). Nitrate reductase activity was determined by the In vivo method described by Davies and Ross (1985). Protein, amino acids, and nitrate were quantified by the methods described by Davies et al. (1987). Petiole xylem sap was exuded with a pressure bomb and stored immediately at - 20°C until analysed. Remaining petiole sap was squeezed from the cut surface with a glass rod and immediately frozen.
were not determined in our study the nitrate concentration in the petiole xylem sap of treatment N3 was maintained at a high level (Fig. 2 a). There was no indication that the decline in leaf NR activity in this treatment was related to a consistent decline in petiole xylem sap nitrate concentration. Similarly, total petiole sap nitrate and leaf nitrate contents were maintained at relatively constant levels (Figs. 2 b, c). Compare this with the declining nitrate status of sap and leaf in treatments NO, N1 and N2. The evidence suggests, therefore, that the onset of leaf senescence, defined here as the onset of net protein degradation, was not modified through N nutrition. However, the accumulation of larger quantities of protein in leaves of plants receiving higher inputs of N does provide a substantial buffer against the possible deleterious effects of reduced protein content and in particular the degradation of RuBP carboxylase (Lauriere, 1983). Only when a critical N concentration is reached is photosynthetic performance likely to be adversely affected. The decline in NR activity and protein content may be initiated by the increased rates of proteolysis and/or the reduced rates of protein synthesis which generally accompany senescence (Lamattina et al., 1985; Feller, 1986). If reduced rates of protein synthesis were the main reason this could not be explained by substantial decreases in the availability of leaf amino acidN (Fig. 3 a). Furthermore, with the exception of the final harvest from treatment NO, proteolysis was not accompanied by considerable increases in leaf amino acid content. This is perhaps not surprising as low-molecular-weight products of proteolysis can be rapidly exported rather than accumulate within the leaf (Feller, 1986). Fig. 3 b shows the amino acid concentration in petiole xylem sap. Again substantial increases occurred at the end of the season in plants receiving low N inputs. This may be explained by xylem transport of amino acids remobilised from roots, lower leaves and stems and not, apparently, by transfer of significant amounts of amino acids stored in the surrounding petiole tissue (Fig. 3 c). Alternatively, phloem to xylem transport within the petiole could have contributed to the increase in sap amino acid content (see Pate, 1973; 1976 and references within). Although some degree of contamination
Results and Discussion Nitrate reductase activity showed a similar pattern of development in all four N treatments, increasing to a peak value at around 80 DAP (Fig. 1 a). Highest activity occurred in N4 plants, as did the highest protein content (Fig. 1 b). However, despite the application of up to 72 g N m -2 neither the decline in NR activity nor the onset of protein degradation could be prevented. Nitrate reductase has, in many instances, been shown to be a substrate inducible enzyme, its activity related more to the flux of nitrate into an organ than the nitrate content of the organ per se (Shaner and Boyer, 1976). Although absolute rates of nitrate flux into the leaf
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of xylem sap with sieve tube or parenchyma exudate could have contributed to these observations, the sucrose content of collected sap was generally less than 0.3 mg cm - 3 (data not presented). This is similar to the concentration reported in xylem vessel bleeding sap of Quercus rubra (Die and Wil-
lemse, 1975) and approximately three orders of magnitude lower than the concentration in the phloem exudate of other species (Pate, 1976 and data therein). Furthermore, for most harvests the concentration of amino acids in total petiole sap was almost an order of magnitude higher than in xylem ex-
The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato Table 1: Changes in the ratio of nitrate-N : amino acid-N in petiole xylem sap during the 1985 growing season. Petiole Xylem Sap N0 3-N: Amino Acid N Days After Planting 100 120 80 86 N treatment 50 70 0.08 0.04 0 NO 0.17 0.03 0 0.01 N1 0.5 0.12 0.26 0.67 N2 1.28 1.20 0.97 N3 3.57 1.56 2.36 2.41 2.16 1.93
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udate. Contamination problems therefore appear to have been minimised. Table 1 shows the nitrate-N : amino acid-N ratio for petiole xylem sap. Over the time course studied the ratio remains consistently above one only in treatment N3. Reductions in N supply result in (a) an increased predominance of amino acid-N over nitrate-N in earlier harvests and (b) a more rapid decline in the ratio as the season progressed. These results indicate that in these leaf positions the potential for nitrate assimilation is more important during the early stages of growth than later on, when reduced N appears to supply much of the leaf's N requirements. However, by comparing nitrate-N and protein N contents it is evident that, even at the first harvest, more than 98 % of the leaf N was in a reduced form. Even the lower activity of NR recorded at this stage was sufficient to prevent the accumulation of substantial quantities of nitrate. Recent experiments have shown that the ratio of nitrate-N: amino-N in xylem sap bleeding from stems cut at ground level is considerably higher than in sap exuded from petioles from the top of the canopy (Davies, unpublished data). This suggests that a substantial proportion of nitrate ascending in the xylem stream is diverted into lower leaves and stem tissue. In this respect it is worthwhile pointing out that a considerable proportion of the nitrate in a potato plant is located in the stem (Davies et al., 1987) and that the concentration of nitrate in leaves at the centre and base of the canopy substantially exceeds that in young expanded leaves from the top (Millard and MacKerron, 1986). This may be due to lower rates of reduction in shaded leaves, higher rates of nitrate influx or a combination of both processes. Fig.4 shows the pattern of NR development in a fieldgrown crop where all of the fertiliser N was applied preplanting. The figure compares in vivo activities with [NR( + )] and without [NR( -)] an exogenous supply of nitrate in the assay medium. The NR( + )assay is considered to be an indicator of nitrate reduction in the tissue when nitrate is not limiting (Andrews et al., 1984 and references therein). The NR( - ) assay is considered by many workers to be a better estimate of NR activity in situ (Radin, 1978; Santoro and Magalhaes, 1983). Furthermore, NR( -) activity has been shown to be directly related to the internal nitrate content of the organ (Aslam, 1981; Baer and Collett, 1981). Data presented in Fig.4 appears to confirm a relationship between NR( - ) activity and the nitrate concentration in both the leaf lamina and petiole xylem sap. The data also indicates that for most of the season the rate of nitrate assimilation in these leaves is limited more by the availability of nitrate than the potential activity of NR. Similar conclusions have been drawn for other species (Sekhon et al., 1986; Claussen, 1986).
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Fig. 4: Changes in leaf nitrate reductase activity with (e) and without (- - -0 - - -) nitrate in the assay medium during the 1986 growing season. Nitrate concentration in leaf lamina (_. -e- - -) and petiole xylem sap (.6) are also shown. Nitrogen at 20 g m-2 was applied preplanting. Bars indicate standard errors of the mean. However, the indications are that in the potato plant the potential may be realised at stages earlier than our first harvest date. It would appear, therefore, that the capacity for nitrate reduction in these leaves may be limited by NR activity only for a short period following shoot emergence, if at all. Preliminary studies with a flowing nutrient culture system (10molm - 3 N0 3-N, 20Imin - 1) have shown that it is possible to attain 90 % of NR( + )activity with the NR( - ) assay, even with large plants. The enzyme assay technique does not appear to have limited NR( -) activity relative to NR( +) activity in our experiments. Rapid leaf and hence canopy expansion is one pre-requisite of an extended period of light interception (Millard and MacKerron, 1986 and references within). An increase in potential NR activity [NR( + )] could serve to improve leaf expansion growth in the early postemergence period. Improved NR activity may also benefit pre-emergence growth. Davies and Ross (1985), using nutrient culture techniques, reported a positive effect of nitrate application on sprout elongation growth and on dry weight accumulation by both roots and sprouts. This occurs despite the considerable reserves of free amino acids and proteins in the mother tuber (Davies, 1984). Root and sprout NR( +) activities are considerably lower than in leaves, however (Kapoor and Li, 1982). While there may be energetic gains from reducing nitrate in leaves compared with roots (Smirnoff and Stewart, 1985) increasing the nitrate assimilating capacity of potato roots and sprouts may still prove beneficial to the rate of plant establishment. It should also be borne in mind that the contribution of the rooting system to the plants nitrate assimilating capacity is likely to vary depending on the nature of the cation which accompanies nitrate during the uptake process. The indications are that potassium increases nitrate uptake but lowers the percentage of nitrate reduced in the root (Rufty et al., 1981; Barneix and Breteler, 1985). By comparison, calcium ions reduce nitrate uptake but in-
544
H. V. DAVIES, H. A. Ross, and R. THOMPSON
crease the proportion of nitrate reduced in the roots (Rufty et at, 1981). The composition of the soil solution, and hence the relationship between nitrate uptake and reduction by roots may, therefore, be manipulable through the type of nitrogen fertiliser applied. References ANDREws, M., J. M. SUnlERLAND, R. J THOMAS, and J I. SPRENT: Distribution of nitrate reductase activity in six legumes: the importance of the stem. New Phytol., 98, 301-310 (1984). ASLAM, M.: Re-evaluation of anaerobic nitrate production as an index for the measurement of the me~abolic pool of nitrate. Plant Physiol. 68, 305-308 (1981). BAER, G. R. and G. F. COLLET: In vwo determination of parameters of nitrate utilisation in wheat (Triticum aestivum L.) seedlings grown with a low concentration of nitrate in the nutrient solution. Plant Physiol. 68, 1237 -1243 (1981). BARNEIX, A. J and H. BRETELER: Effect of cations on uptake, translocation and reduction of nitrate in wheat seedlings. New Phytol. 99,367 -379 (1985). CLAUSSEN, W.: Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena). Physiol. Plant. 61, 73-80 (1986). DAVIES, H. V.: Mother tuber reserves as factors limiting sprout growth. Pot. Res. 21, 373-381 (1984). DAVIES, H. V. andH. A. Ross: The effects of mineral nutrition on intersprout competition in cv. Maris Piper. Pot. Res. 28, 43 - 53 (1985). DAVIES, H. V., H. A. Ross, and K. J. OPARKA: Nitrate reduction in Solanum tuberosum L.: Development of nitrate reductase activity in field-grown plants. Annals. Bot. 59, 301-309 (1987). DIE, J VAN and P. C. M. WILLEMSE: Mineral and organic nutrients in sieve tube exudate and xylem vessel sap of Quercus rubra L. Acta. Bot. Neerl. 24, 237 -239 (1975). FEllER, U.: Proteolytic enzymes in relation to leaf senescence. In: M. J DALLING (ed.), Plant Proteolytic Enzymes, Vol. 2. CRC Press Inc. Florida (1986). HILL, J: The remobilisation of nutrients from leaves. J Plant Nutrition,2, 407 -444 (1980).
KApOOR, A. C. and P. H. LI: Effect of age and variety on nitrate reductase and nitrogen fraction in potato plants. J Sci. Food Agr. 33,401-406 (1982). LAMATTINA, L., R. P. LEZICA, and R. D. CONDE: Protein metabolism in senescing wheat leaves. Determination of synthesis and degradation rates and their effects on protein loss. Plant Physiol. 77, 587 - 590 (1985). LAURlERE, Enzymes and leaf senescence. Physiol. Veg. 21, 1159-1177 (1983). MILLARD, P. and D . K. L. MACKERRON: The effects of nitrogen application on the growth and nitrogen distribution within the potato canopy. Ann. appl. BioI. 109, 427-437 (1986). NEUMANN, P. M. and Z. STEIN: Ion supply capacity of roots in relation to rejuvenation of primary leaves in vivo. Physiol. Plant 67, 97 -101 (1986). PATE, J S.: Uptake, assimilation and transport of nitrogen compounds by plants. Soil BioI. Biochem. 5, 109-119 (1973). - Nutrients and metabolites of fluids recovered from xylem and phloem: significance in relation to long-distance transport in plants. In: WARDLAW, I. F. and J. B. PASSJOURA (eds.), Transport and Transfer Processes in Plants. Academic Press, 253 -281 (1976). RADIN, J. W.: A physiological basis for the diversion of nitrate assimilation between roots and leaves. Plant Sci. Letts. 13, 21- 25 (1978). RufTY, T . W., W. A. JACKSON, and C. D. RAPER JR.: Nitrate reduction in roots as affected by the presence of potassium and by flux of nitrate through the roots. Plant Physiol. 68, 605-609 (1981). SANTORO, L. G. and C. N. MAGALHAES: Changes in nitrate reductase activity during the development of soybean leaf. Z. Pflanzenphysiol. 112, 113 - 121 (1983). SEKHON, B. S., S. KUMAR, K. S. DHILLON, and R. SINGH: Effect of nitrogen on nitrate reductase activity in the nodules and leaves of summer moong(Vigna radiata). Annals. Bot. 58, 515-521 (1986). SHANER, D. L. and J. S. BoYER: Nitrate reductase activity in maize (Zea mays L.) leaves. Plant Physiol. 58, 499 -504 (1976). SMIRNOFF, N . and G. R. STEWART: Nitrogen assimilation and translocation by higher plants: comparative physiology and ecological consequences. Physiol. Plant 53, 133 -140 (1985). THOMAS, H. and J L. STODDART: Leaf senescence. Ann. Rev. Plant Physiol. 31,83-111 (1980).
c.:
The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato (Solanum tuberosum L.) H. V.
DAVIES,
H. A. Ross, and R.
THOMPSON
Scottish Crop Research Institute, Invergowrie, Dundee DD25DA, United Kingdom Received September 11, 1987 . Accepted October 28, 1987
Summary Changes in nitrate reductase activity in the youngest expanded leaves of the potato plant have been monitored in the field under a range of N regimes. Highest activity (in vivo assay, substrate non-limiting) occurred with the highest rate of N application, as did the highest leaf protein content. In high-N leaves the onset of the decline in nitrate reductase activity and protein content, which commenced around 80 days after planting, was not prevented by the application of high levels of nitrogen. This could not be explained by a fall in the concentration of nitrate in the petiole xylem sap supplying the leaves in question. The ratio of nitrate-N to amino acid-N in petiole xylem sap remained relatively constant throughout the growing period in plants receiving 8 g N m -2 at 14 day intervals, but declined under lower N inputs. Only in the highest N treatment did the ratio remain greater than unity. The reasons for these observations are discussed. Nitrate reductase activity was also determined in leaves of field-grown plants with all of the N (20gm- 2) applied pre-planting. A comparison of in vivo assays carried out with and without nitrate in the assay medium showed that nitrate reductase was likely to limit nitrate assimilation only during the earliest stages of post-emergence growth. Abbreviations: DAP - days after planting; NR - nitrate reductase.
Introduction Nitrate reductase activity in the potato plant is located principally in the leaves (Kapoor and Li, 1982). Recent investigations (Davies and Ross, 1985) have demonstrated a distinctive pattern of change in nitrate reductase activity in leaves of field-grown crops over the course of a growing season. Enzyme activity increased to a peak around mid-July and declined substantially thereafter, peaks in leaf chlorophyll and protein content occurring at approximately the same time. The onset of chlorophyll and protein degradation in leaves from the unshaded, photosynthetically active region of the canopy, evidently signals the initiation of processes which will ultimately affect leaf and canopy photosynthetic performance and hence yield. Although plant senescence has, in several instances, been delayed or prevented by nitrogen supplied exogenously (Hill, 1980), the © 1988 by Gustav fischer Verlag, Stuttgart
nutrient theory of senescence is sometimes criticised (Thomas and Stoddart, 1980; Neumann and Stein, 1986). Nevertheless, the response of ageing leaves to modifications in the supply of essential elements such as nitrogen may ultimately determine the plant's capacity to modify its pattern of senescence. The present study examines the possibility that frequent applications of very high levels of nitrogen to field-grown potato plants might modify the pattern of senescence. The study also examines further the limitation to the rate of nitrate reduction set by the activity of nitrate reductase.
Materials and Methods Seed tubers of potato (Solanum tuberosum L.) cv. Maris Piper were planted in flat beds in the field at SCRI on 30 April 1985 in
The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato 75 em rows and at a density of 6 plants m -2. P was applied at 8 g m - 2 prior to planting and K at 20 g m - 2. N (as ammonium nitrate) was applied through trickle irrigation at 14 d intervals from planting. Four N treatments were used; at each application NOno N applied, N1- 0.8gNm-2, N2- 3.2gNm- 2, N38gNm-2. This resulted in total application rates for the season of 0, 7.2, 28.8 and 72.0 g N m - 2, respectively. Plots, each 12 m x 7 m and arranged in two randomised blocks, were irrigated when soil moisture deficit reached 25 KPa. Between 11.00 and 12.00 h at each harvest date four plants were taken from each plot. Two plants from each plot were combined for analysis. The youngest expanded leaves from the top of the canopy (commonly leaves 4 and 5 from the apex) were excised at their point of attachment to the stem and combined to give sufficient leaf material for analyses. With the exception of nitrate reductase determinations leaf analyses were performed on freeze-dried and finely-ground material. All analyses were carried out in triplicate. Determinations of In VIVO nitrate reductase activity, with and without nitrate in the assay medium, were carried out on a crop grown in 1986. These plots were prepared as outlined by Davies et al. (1987) with all of the N applied prior to planting (20 g N m - 2). Nitrate reductase activity was determined by the In vivo method described by Davies and Ross (1985). Protein, amino acids, and nitrate were quantified by the methods described by Davies et al. (1987). Petiole xylem sap was exuded with a pressure bomb and stored immediately at - 20°C until analysed. Remaining petiole sap was squeezed from the cut surface with a glass rod and immediately frozen.
were not determined in our study the nitrate concentration in the petiole xylem sap of treatment N3 was maintained at a high level (Fig. 2 a). There was no indication that the decline in leaf NR activity in this treatment was related to a consistent decline in petiole xylem sap nitrate concentration. Similarly, total petiole sap nitrate and leaf nitrate contents were maintained at relatively constant levels (Figs. 2 b, c). Compare this with the declining nitrate status of sap and leaf in treatments NO, N1 and N2. The evidence suggests, therefore, that the onset of leaf senescence, defined here as the onset of net protein degradation, was not modified through N nutrition. However, the accumulation of larger quantities of protein in leaves of plants receiving higher inputs of N does provide a substantial buffer against the possible deleterious effects of reduced protein content and in particular the degradation of RuBP carboxylase (Lauriere, 1983). Only when a critical N concentration is reached is photosynthetic performance likely to be adversely affected. The decline in NR activity and protein content may be initiated by the increased rates of proteolysis and/or the reduced rates of protein synthesis which generally accompany senescence (Lamattina et al., 1985; Feller, 1986). If reduced rates of protein synthesis were the main reason this could not be explained by substantial decreases in the availability of leaf amino acidN (Fig. 3 a). Furthermore, with the exception of the final harvest from treatment NO, proteolysis was not accompanied by considerable increases in leaf amino acid content. This is perhaps not surprising as low-molecular-weight products of proteolysis can be rapidly exported rather than accumulate within the leaf (Feller, 1986). Fig. 3 b shows the amino acid concentration in petiole xylem sap. Again substantial increases occurred at the end of the season in plants receiving low N inputs. This may be explained by xylem transport of amino acids remobilised from roots, lower leaves and stems and not, apparently, by transfer of significant amounts of amino acids stored in the surrounding petiole tissue (Fig. 3 c). Alternatively, phloem to xylem transport within the petiole could have contributed to the increase in sap amino acid content (see Pate, 1973; 1976 and references within). Although some degree of contamination
Results and Discussion Nitrate reductase activity showed a similar pattern of development in all four N treatments, increasing to a peak value at around 80 DAP (Fig. 1 a). Highest activity occurred in N4 plants, as did the highest protein content (Fig. 1 b). However, despite the application of up to 72 g N m -2 neither the decline in NR activity nor the onset of protein degradation could be prevented. Nitrate reductase has, in many instances, been shown to be a substrate inducible enzyme, its activity related more to the flux of nitrate into an organ than the nitrate content of the organ per se (Shaner and Boyer, 1976). Although absolute rates of nitrate flux into the leaf
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Fig. 2: Nitrate concentration in petiole xylem sap (a) total petiole sap (b) and leaf lamina (c) in plants grown under a range of N regimes. Symbols as in Fig. 1.
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Fig. 3: Amino acid concentration in leaf lamina (a) petiole xylem sap (b) and total petiole sap (c). Symbols as in Fig. 1.
of xylem sap with sieve tube or parenchyma exudate could have contributed to these observations, the sucrose content of collected sap was generally less than 0.3 mg cm - 3 (data not presented). This is similar to the concentration reported in xylem vessel bleeding sap of Quercus rubra (Die and Wil-
lemse, 1975) and approximately three orders of magnitude lower than the concentration in the phloem exudate of other species (Pate, 1976 and data therein). Furthermore, for most harvests the concentration of amino acids in total petiole sap was almost an order of magnitude higher than in xylem ex-
The Influence of Nitrogen Supply on the Development of Nitrate Reductase Activity in Leaves of Potato Table 1: Changes in the ratio of nitrate-N : amino acid-N in petiole xylem sap during the 1985 growing season. Petiole Xylem Sap N0 3-N: Amino Acid N Days After Planting 100 120 80 86 N treatment 50 70 0.08 0.04 0 NO 0.17 0.03 0 0.01 N1 0.5 0.12 0.26 0.67 N2 1.28 1.20 0.97 N3 3.57 1.56 2.36 2.41 2.16 1.93
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udate. Contamination problems therefore appear to have been minimised. Table 1 shows the nitrate-N : amino acid-N ratio for petiole xylem sap. Over the time course studied the ratio remains consistently above one only in treatment N3. Reductions in N supply result in (a) an increased predominance of amino acid-N over nitrate-N in earlier harvests and (b) a more rapid decline in the ratio as the season progressed. These results indicate that in these leaf positions the potential for nitrate assimilation is more important during the early stages of growth than later on, when reduced N appears to supply much of the leaf's N requirements. However, by comparing nitrate-N and protein N contents it is evident that, even at the first harvest, more than 98 % of the leaf N was in a reduced form. Even the lower activity of NR recorded at this stage was sufficient to prevent the accumulation of substantial quantities of nitrate. Recent experiments have shown that the ratio of nitrate-N: amino-N in xylem sap bleeding from stems cut at ground level is considerably higher than in sap exuded from petioles from the top of the canopy (Davies, unpublished data). This suggests that a substantial proportion of nitrate ascending in the xylem stream is diverted into lower leaves and stem tissue. In this respect it is worthwhile pointing out that a considerable proportion of the nitrate in a potato plant is located in the stem (Davies et al., 1987) and that the concentration of nitrate in leaves at the centre and base of the canopy substantially exceeds that in young expanded leaves from the top (Millard and MacKerron, 1986). This may be due to lower rates of reduction in shaded leaves, higher rates of nitrate influx or a combination of both processes. Fig.4 shows the pattern of NR development in a fieldgrown crop where all of the fertiliser N was applied preplanting. The figure compares in vivo activities with [NR( + )] and without [NR( -)] an exogenous supply of nitrate in the assay medium. The NR( + )assay is considered to be an indicator of nitrate reduction in the tissue when nitrate is not limiting (Andrews et al., 1984 and references therein). The NR( - ) assay is considered by many workers to be a better estimate of NR activity in situ (Radin, 1978; Santoro and Magalhaes, 1983). Furthermore, NR( -) activity has been shown to be directly related to the internal nitrate content of the organ (Aslam, 1981; Baer and Collett, 1981). Data presented in Fig.4 appears to confirm a relationship between NR( - ) activity and the nitrate concentration in both the leaf lamina and petiole xylem sap. The data also indicates that for most of the season the rate of nitrate assimilation in these leaves is limited more by the availability of nitrate than the potential activity of NR. Similar conclusions have been drawn for other species (Sekhon et al., 1986; Claussen, 1986).
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Fig. 4: Changes in leaf nitrate reductase activity with (e) and without (- - -0 - - -) nitrate in the assay medium during the 1986 growing season. Nitrate concentration in leaf lamina (_. -e- - -) and petiole xylem sap (.6) are also shown. Nitrogen at 20 g m-2 was applied preplanting. Bars indicate standard errors of the mean. However, the indications are that in the potato plant the potential may be realised at stages earlier than our first harvest date. It would appear, therefore, that the capacity for nitrate reduction in these leaves may be limited by NR activity only for a short period following shoot emergence, if at all. Preliminary studies with a flowing nutrient culture system (10molm - 3 N0 3-N, 20Imin - 1) have shown that it is possible to attain 90 % of NR( + )activity with the NR( - ) assay, even with large plants. The enzyme assay technique does not appear to have limited NR( -) activity relative to NR( +) activity in our experiments. Rapid leaf and hence canopy expansion is one pre-requisite of an extended period of light interception (Millard and MacKerron, 1986 and references within). An increase in potential NR activity [NR( + )] could serve to improve leaf expansion growth in the early postemergence period. Improved NR activity may also benefit pre-emergence growth. Davies and Ross (1985), using nutrient culture techniques, reported a positive effect of nitrate application on sprout elongation growth and on dry weight accumulation by both roots and sprouts. This occurs despite the considerable reserves of free amino acids and proteins in the mother tuber (Davies, 1984). Root and sprout NR( +) activities are considerably lower than in leaves, however (Kapoor and Li, 1982). While there may be energetic gains from reducing nitrate in leaves compared with roots (Smirnoff and Stewart, 1985) increasing the nitrate assimilating capacity of potato roots and sprouts may still prove beneficial to the rate of plant establishment. It should also be borne in mind that the contribution of the rooting system to the plants nitrate assimilating capacity is likely to vary depending on the nature of the cation which accompanies nitrate during the uptake process. The indications are that potassium increases nitrate uptake but lowers the percentage of nitrate reduced in the root (Rufty et al., 1981; Barneix and Breteler, 1985). By comparison, calcium ions reduce nitrate uptake but in-
544
H. V. DAVIES, H. A. Ross, and R. THOMPSON
crease the proportion of nitrate reduced in the roots (Rufty et at, 1981). The composition of the soil solution, and hence the relationship between nitrate uptake and reduction by roots may, therefore, be manipulable through the type of nitrogen fertiliser applied. References ANDREws, M., J. M. SUnlERLAND, R. J THOMAS, and J I. SPRENT: Distribution of nitrate reductase activity in six legumes: the importance of the stem. New Phytol., 98, 301-310 (1984). ASLAM, M.: Re-evaluation of anaerobic nitrate production as an index for the measurement of the me~abolic pool of nitrate. Plant Physiol. 68, 305-308 (1981). BAER, G. R. and G. F. COLLET: In vwo determination of parameters of nitrate utilisation in wheat (Triticum aestivum L.) seedlings grown with a low concentration of nitrate in the nutrient solution. Plant Physiol. 68, 1237 -1243 (1981). BARNEIX, A. J and H. BRETELER: Effect of cations on uptake, translocation and reduction of nitrate in wheat seedlings. New Phytol. 99,367 -379 (1985). CLAUSSEN, W.: Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena). Physiol. Plant. 61, 73-80 (1986). DAVIES, H. V.: Mother tuber reserves as factors limiting sprout growth. Pot. Res. 21, 373-381 (1984). DAVIES, H. V. andH. A. Ross: The effects of mineral nutrition on intersprout competition in cv. Maris Piper. Pot. Res. 28, 43 - 53 (1985). DAVIES, H. V., H. A. Ross, and K. J. OPARKA: Nitrate reduction in Solanum tuberosum L.: Development of nitrate reductase activity in field-grown plants. Annals. Bot. 59, 301-309 (1987). DIE, J VAN and P. C. M. WILLEMSE: Mineral and organic nutrients in sieve tube exudate and xylem vessel sap of Quercus rubra L. Acta. Bot. Neerl. 24, 237 -239 (1975). FEllER, U.: Proteolytic enzymes in relation to leaf senescence. In: M. J DALLING (ed.), Plant Proteolytic Enzymes, Vol. 2. CRC Press Inc. Florida (1986). HILL, J: The remobilisation of nutrients from leaves. J Plant Nutrition,2, 407 -444 (1980).
KApOOR, A. C. and P. H. LI: Effect of age and variety on nitrate reductase and nitrogen fraction in potato plants. J Sci. Food Agr. 33,401-406 (1982). LAMATTINA, L., R. P. LEZICA, and R. D. CONDE: Protein metabolism in senescing wheat leaves. Determination of synthesis and degradation rates and their effects on protein loss. Plant Physiol. 77, 587 - 590 (1985). LAURlERE, Enzymes and leaf senescence. Physiol. Veg. 21, 1159-1177 (1983). MILLARD, P. and D . K. L. MACKERRON: The effects of nitrogen application on the growth and nitrogen distribution within the potato canopy. Ann. appl. BioI. 109, 427-437 (1986). NEUMANN, P. M. and Z. STEIN: Ion supply capacity of roots in relation to rejuvenation of primary leaves in vivo. Physiol. Plant 67, 97 -101 (1986). PATE, J S.: Uptake, assimilation and transport of nitrogen compounds by plants. Soil BioI. Biochem. 5, 109-119 (1973). - Nutrients and metabolites of fluids recovered from xylem and phloem: significance in relation to long-distance transport in plants. In: WARDLAW, I. F. and J. B. PASSJOURA (eds.), Transport and Transfer Processes in Plants. Academic Press, 253 -281 (1976). RADIN, J. W.: A physiological basis for the diversion of nitrate assimilation between roots and leaves. Plant Sci. Letts. 13, 21- 25 (1978). RufTY, T . W., W. A. JACKSON, and C. D. RAPER JR.: Nitrate reduction in roots as affected by the presence of potassium and by flux of nitrate through the roots. Plant Physiol. 68, 605-609 (1981). SANTORO, L. G. and C. N. MAGALHAES: Changes in nitrate reductase activity during the development of soybean leaf. Z. Pflanzenphysiol. 112, 113 - 121 (1983). SEKHON, B. S., S. KUMAR, K. S. DHILLON, and R. SINGH: Effect of nitrogen on nitrate reductase activity in the nodules and leaves of summer moong(Vigna radiata). Annals. Bot. 58, 515-521 (1986). SHANER, D. L. and J. S. BoYER: Nitrate reductase activity in maize (Zea mays L.) leaves. Plant Physiol. 58, 499 -504 (1976). SMIRNOFF, N . and G. R. STEWART: Nitrogen assimilation and translocation by higher plants: comparative physiology and ecological consequences. Physiol. Plant 53, 133 -140 (1985). THOMAS, H. and J L. STODDART: Leaf senescence. Ann. Rev. Plant Physiol. 31,83-111 (1980).
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