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Crop Science 41:372-378 (2001)
© 2001 Crop Science Society of America

CROP PHYSIOLOGY & METABOLISM

Diurnal Fluctuations of Nitrate Uptake and In Vivo Nitrate Reductase Activity in Pima and Acala Cotton

M. Aslam, R.L. Travis and D.W. Rains

Dep. of Agronomy and Range Science, Univ. of California, One Shields Avenue, Davis, CA, USA 95616-8515

Corresponding author (rltravis{at}ucdavis.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
This study was conducted to determine whether diurnal fluctuations of nitrate reductase activity in two cotton species, Pima (Gossypium barbadense L.) and Acala (Gossypium hirsutum L.), are regulated by NO-3 uptake or leaf NO-3 concentration. The two species differ greatly in their N use efficiency. The seedlings were grown for 25 d in nutrient solutions containing 0.05, 0.10 or 1.0 mM NO-3 under a 14-h light/10-h dark cycle at 30°C/20°C. Uptake rates were measured by following NO-3 depletion from the uptake solutions at 20°C or 30°C. Nitrate reductase activity (NRA) in fully expanded first true leaves was determined by an anaerobic method in vivo. Upon illumination uptake rates for both species, measured at 30°C, increased until they plateaued in about 5 h and were maintained at that level for the remainder of the light period and the following dark cycle. When measured at 20°C in the dark, however, the uptake rates decreased 25 to 30% for the first 3 h and then remained constant. Leaf NRA increased rapidly during the first hour of illumination, reached a plateau and then decreased after 4 to 6 h of illumination. The decline was more rapid when NRA was assayed in the absence of NO-3. In darkness, NRA levels were low and did not fluctuate. Upon illumination, leaf NO-3 concentration also increased for about 7 h and then decreased gradually. The results indicate that (i) the diurnal rhythm of NO-3 uptake is modulated by temperature rather than by light/dark transitions, and (ii) while the increase in NRA upon illumination may be regulated by NO-3 flux from roots to shoots, the decrease in NRA under prolonged illumination is independent of that flux.

Abbreviations: NR(A), nitrate reductase (activity) • PPFD, photosynthetic photon flux density • SE, standard error


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
UPTAKE AND REDUCTION, the initial processes by which NO-3 is metabolized by higher plants, are modulated by light and dark (Aslam et al., 1976, 1979). Both light-enhanced uptake and decreased uptake in darkness have been reported (Aslam et al., 1979; Rufty et al., 1989; Le Bot and Kirkby, 1992; Delhon et al., 1995a,b; Cárdenas-Navarro et al., 1998). A more complex rhythm consisting of two peaks of uptake activity, one occurring during the day and another during the night, has also been reported for a number of plant species (Hansen, 1980; Le Bot and Kirkby, 1992). On the other hand, in some plant species, under certain growth conditions, NO-3 uptake may not be affected by light/dark transitions (Rufty et al., 1984; Mattsson et al., 1988) or it may even increase in the dark (Steingröver et al., 1986; Scaife and Schloemer, 1994).

Diurnal fluctuations of NRA have been reported in a number of plant species (Nicholas et al., 1976; Lillo, 1983; Huber et al., 1992, 1994). Light stimulates de novo synthesis as well as activation of higher plant nitrate reductase (NR) protein, and the protein is rapidly deactivated in the dark (Huber et al., 1992, 1994). The rapid reversible activation and inactivation by light/dark transitions is strongly correlated with protein phosphorylation and dephosphorylation (Huber et al., 1992, 1994). At the whole-plant level, however, it is not clear whether these diurnal fluctuations are rhythmic responses to light/dark transitions or are the result of fluctuations in NO-3 uptake and its subsequent availability at the site of enzyme synthesis and/or NO-3 reduction. Shaner and Boyer (1976) reported that a decrease in NO-3 flux to corn (Zea mays L.) leaves resulted in a rapid loss of NRA, although leaf NO-3 content was unchanged. They concluded that NRA is regulated in shoots by NO-3 flux rather than NO-3 content of the tissue. Likewise, Gojon et al. (1991) reported that the xylem flux of NO-3 in light is the main determinant of the actual rate of NO-3 reduction in soybean [Glycine max (L.) Merr.] leaves. These reports suggest that the diurnal fluctuation in NRA may be controlled by NO-3 flux in the plant.

If the light/dark transition effect on NRA in vivo, and/or on NR per se, is through the regulation of substrate availability and/or the generation and supply of reductant (Nicholas et al., 1976; Jones and Sheard, 1977), then the use of leaf material with relatively high NO-3 concentration, or the addition of NO-3 to the assay medium in vivo should, at least partially, overcome the dark response. This, in fact, has been reported for soybean (Nicholas et al., 1976) and barley (Hordeum vulgare L.) (Lillo, 1983) leaves. This supports the hypothesis that NO-3 was limiting at the site of reduction during the dark cycle.

While levels of in vitro NRA are generally higher than those in vivo (Nicholas et al., 1976; Jones and Sheard, 1977), diurnal fluctuations in NRA as measured by both assays were similar in soybean leaves (Nicholas et al., 1976). NRA assayed in vivo without additional NO-3 best approximated NO-3 assimilation rates in situ in cotton (G. hirsutum) (Radin et al., 1975). Consequently, this assay was used to study regulation of NO-3 assimilation in cotton (Radin et al., 1975; Radin, 1977) and to determine the partitioning of NO-3 between shoots and roots in most legume (Andrews et al., 1984) and grass (Andrews et al., 1992; Jiang and Hull, 1999) species. Further, the NRA assay in vivo has been suggested for use as a tool in monitoring systems that determine the changes of N concentration in cotton leaves (Chu et al., 1989).

This study was undertaken to determine the dependence (if any) of diurnal fluctuations in vivo of NRA on NO-3 uptake and/or its concentration in Pima (S-7) and Acala (Maxxa) cotton cultivars. These two cotton species were selected because they vary considerably in NO-3 uptake ability at 0.05 mM ambient NO-3 concentration with Pima being a more efficient user of N than Acala (Aslam et al., 1997).


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Plant Growth
Acid-delinted seeds of Acala, cv. Maxxa and Pima, cv. S-7 cotton were germinated in the dark at 25°C as described previously (Aslam et al., 1997). Five-day-old seedlings were placed on a stainless steel screen (mesh size 5 by 5 mm) suspended about 5 cm above 5 L of aerated N-free, quarter-strength Hoagland's solution (Hoagland and Arnon, 1950) in a plastic beaker. The beakers were placed in a growth chamber (Western Environmental, Napa, CA) set for a 14-h light/10-h dark cycle at 30°C/20°C and 60 to 65% relative humidity. Metal halide and incandescent lamps supplied light. Photosynthetic photon flux density (PPFD) measured at the top of the canopy with a LI-COR quantum sensor (Lincoln, NE) was 700 µmol m-2 s-1. After 2 d, the seedlings were transferred to 70-L plastic containers fitted with stainless steel screens (mesh size 9 by 9 mm) and containing 0.05, 0.1, or 1.0 mM KNO3 in aerated quarter-strength Hoagland's solution. Nitrate concentration in the nutrient solutions was assayed twice daily and maintained by adding KNO3 solution. Nutrient solution pH was adjusted to 6.0 with H2SO4 as needed. The seedlings were grown for 25 d after transplanting.

Measurement of NO-3 Uptake Rates
Nitrate uptake was determined by following its depletion from the uptake solution. Measurements were made in a miniature controlled environment chamber described by Goyal and Huffaker (1986). The chamber was designed and constructed in the laboratory. Chamber environmental conditions were the same as described above for plant growth. The seedlings were placed in solutions containing 0.1 mM KNO3 and transferred to the mini-chamber 10 to 12 h prior to beginning the measurement of uptake rates. About 2 h before beginning uptake measurements, three intact plants (about 4 g) were placed in a flat bottom Pyrex glass tube (35-mm diam and 180-mm height) containing 120 mL of an aerated solution of 0.2 mM CaSO4 and 0.1 mM NO-3 in 2.0 mM MES [(N-morpholino)ethanesulfonic acid] (pH 6.0). During this period, NO-3 concentration in the uptake solutions was maintained by adding KNO3 solution. Immediately before the start of the depletion measurements, the uptake solution was gently poured from the tubes and replaced with fresh solution. The solution temperature was 20 or 30°C (see legends, Fig. 1 and 2) . Care was taken that the roots were not disturbed during this process. The uptake solutions were vigorously aerated during the experiments to ensure thorough mixing. The first sample was withdrawn by the automated sampling system about 2 min after transferring the seedlings into the fresh uptake solutions. The automated sampling system was designed and constructed in the laboratory by Goyal and Huffaker (1986). Thereafter, the system automatically removed 0.5-mL aliquots for NO-3 determination at 3-min intervals for a total of 15 min. Measurements were made in light or dark under the same environmental conditions at which the plants were grown. Cumulative uptake was determined from the NO-3 depletion curves as described by Goyal and Huffaker (1986). Net uptake rates were then calculated by linear regression analysis of the cumulative uptake curves.



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  		Fig. 1. Effect of light on net NO-3 uptake by Pima (S-7) and Acala (Maxxa) cotton seedlings grown in 0.1 mM NO-3 at 30/20°C light/dark throughout (A) or 30/30°C light/dark for one cycle (B) prior to measurements. Uptake rates were measured at 30°C. Open and closed symbols represent measurements from different sets of plants during the time course

 


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  		Fig. 2. Effect of darkness on net NO-3 uptake, at 20 and 30°C, by Pima (A) and Acala (B) cotton seedlings grown in 0.1 mM NO-3 at 30/20°C light/dark temperatures. The seedlings at the end of the light cycle were transferred into the uptake solutions and placed in the dark for the measurement of uptake rates at 20°C (circles) or 30°C (triangles). Open and closed symbols represent measurements from different sets of plants during the time course

 
Measurement of In Vivo NRA
Nitrate reductase activity was assayed in vivo by incubating leaf discs in 0.1 M potassium phosphate buffer (pH 7.5) containing 0 or 0.1 M KNO3 under anaerobic conditions. The minus NO-3 in vivo assay measures the amount of NO-3 being assimilated (Radin et al., 1975) and provides a reasonable estimate of the size of the metabolic NO-3 pool (Aslam et al., 1976). When NO-3 is added to the assay medium, the maximum capacity for NO-3 assimilation under nonlimiting conditions is determined (Radin et al., 1975).

Ten to 12 leaf discs (5-mm diam.) weighing 0.12 to 0.14 g fresh weight., were harvested from the first fully expanded true leaves and placed in 10-mL tubes containing 5 mL of the buffer solution. Care was taken to avoid the main veins when cutting the discs. The tubes were then vacuum infiltrated for 2 min with the vacuum released at 20-s intervals. The discs became wetted and sank to the bottom of the solution during infiltration. After vacuum infiltration, the tubes were immediately stoppered and placed in the water bath at 30°C in darkness for 30 min. Propanol was omitted from the assay medium because it decreases the production of NO-2 (M. Aslam, R.L. Travis, and D.W. Rains, 1998, unpublished results). Aliquots (0.5–1.0 mL) were removed from the assay medium for the determination of NO-2. NRA was calculated from the NO-2 concentration in the incubation medium and is reported as µmol NO-2 h-1 g-1 fresh weight. In some experiments, leaf discs were also analyzed for NO-2. The NO-2 remaining in the discs after incubation was less than 5% of the total produced. Thus, NRA levels were not corrected for the NO-2 remaining in the discs.

Extraction of NO-3 from Plant Tissue
Nitrate was extracted from leaves (2–3 g) by homogenizing the tissue in a chilled mortar containing 4 mL of distilled, deionized H2O per gram of tissue and a small amount of acid-washed sand. The extracts were centrifuged at 30 000 x g at 4°C for 15 min and the supernatants were used to determine NO-3 concentration.

NO-3 and NO-2 Determination
Nitrate concentration was determined spectrophotometrically by measuring A210 after separation by High-Performance Liquid Chromatography on a partisil-10 SAX (Phenomenex, Torrance, CA) anion-exchange column (Thayer and Huffaker, 1980). Nitrite was determined by measuring A540 after color development for 15 min with a 1:1 mixture of 10 g kg-1 sulfanilamide in 1.5 M HCl and 0.2 g kg-1 N-naphthylethylenediamine dihydrochloride aqueous solution (Sanderson and Cocking, 1964).

Data Analysis
The experiments were repeated two to three times and the results of representative experiments are reported. For the data in Fig. 3 to 6 , standard errors of the means were calculated. In Fig. 4 and 5, the data were also analyzed statistically by completely randomized design and Duncan's multiple range test was applied. All results are reported on a tissue fresh-weight basis.



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  		Fig. 3. Effect of light duration on in vivo NRA in leaves of Pima (S-7) and Acala (Maxxa) cotton grown in 0.05 (A), 0.1 (B) or 1.0 (C) mM NO-3 at 30/20°C light/dark temperatures. Enzyme assays were performed without (open symbols) or with (closed symbols) 0.1 M KNO3 in the assay medium. Each point is the mean of three replicates, with vertical lines representing ± standard error

 


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  		Fig. 6. Effect of darkness on in vivo NRA in leaves of Pima (S-7) and Acala (Maxxa) cotton grown in 0.10 mM NO-3 at 30/20°C light/dark temperatures. Enzyme assays were performed without (open symbols) or with (closed symbols) 0.1 M KNO3 in the assay medium. Each point is the mean of three replicates, with vertical lines representing ± standard error

 


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  		Fig. 4. Effect of glucose and NO-3 additions on in vivo NRA in leaves of Acala cotton grown in 0.1 mM NO-3 at 30/20°C light/dark temperatures. Leaf samples were harvested either at the beginning of light period (A) or after 1 h exposure to light (B). Vertical lines above the bars show the standard error of means of three replicates. In each panel, bars with the same letter are not significantly different as determined by Duncan's multiple range test (P = 0.05)

 


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  		Fig. 5. Effect of light on in vivo NRA in leaves of Pima (S-7) cotton grown in 1.0 mM NO-3 at 30/20°C throughout (A) or with last cycle of 30/30°C (B) light/dark temperatures. Vertical lines above the bars show the standard error of means of three replicates. In each panel, bars with the same letter are not significantly different as determined by Duncan's multiple range test (P = 0.05)

 

   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Diurnal Fluctuations of NO-3 Uptake
Nitrate uptake rates, measured at 30°C, for both Pima and Acala cotton cultivars grown in 0.1 mM NO-3 under a 14 h/30°C light and 10 h/20°C dark growth cycle increased progressively with the onset of light, reached stable rates in about 5 h and remained at that level for the remainder of the light period (Fig. 1A). Uptake rates for Pima were 30 to 35% higher than those of Acala. The latter finding is in agreement with our earlier report (Aslam et al., 1997). When the same plants were placed under a single cycle of 30°/30°C light/dark temperature prior to measurements, initial NO-3 uptake rates, measured at 30°C, were higher and then increased only slightly (10–15%) upon illumination (Fig. 1B). When the uptake rates of the seedlings, grown at 30°/20°C light/dark temperature, were measured in darkness at the end of light period at 20°C the rates initially decreased by 25 to 30% during the first 3 h before stabilizing for the remainder of the dark period (Fig. 2A, B). When the measurements were made at 30°C in the dark, however, NO-3 uptake rates did not fluctuate (Fig. 2A, B) In fact, the uptake rates measured in the dark at 30°C were similar to those observed in light (compare Fig. 2 with Fig. 1B).

These results suggest that the diurnal rhythm of NO-3 uptake in cotton is modulated by temperature changes rather than by the light/dark cycle. This may not be the case in other species. Delhon et al. (1995a), working with 20-d-old soybean plants grown under a 14-/10-h light/dark cycle (400 µmol m-2 s-1 PPFD) and at constant temperature (20°/20°C), reported that NO-3 influx increased progressively during the first hours of the light period before stabilizing at about 6 h. When the plants were transferred to darkness, influx decreased during the first 2 to 4 h before stabilizing at the lower level (Delhon et al., 1995a). When similar experiments were carried out with 19-d-old tomato (Lycopersicon esculentum Mill.) plants that were grown at 12/12 h light/dark regimen (400 µmol m-2 s-1 PPFD) and constant temperature (20°/20°C), NO-3 uptake increased continuously during light cycle and then decreased continuously during the subsequent dark cycle (Cárdenas-Navarro et al., 1998).

Since NO-3 uptake is an energy-consuming process and involves transport protein(s) (Rufty et al., 1989; Glass et al., 1990), light/dark fluctuations in NO-3 uptake may be due to temperature-related changes in available energy and/or activation of transport protein(s). Fluctuations in carbohydrate status are known to occur during daily light/dark cycles (Kerr et al., 1985); and limitations in carbohydrate supply may likely be, in large part, responsible for decreased rates of NO-3 uptake (Hansen, 1980; Pearson et al., 1981). While in light, photosynthesis may supply energy for NO-3 assimilation (uptake and reduction), in darkness metabolism of stored or externally supplied carbohydrates may sustain both the expression (Liu and Tsay, 1999), and activity (Rufty et al., 1989) of the NO-3 uptake system for several hours. Since no decrease in NO-3 uptake rates occurred when the plants were maintained at 30°C in darkness (Fig. 2), it is likely that adequate energy was available from stored carbohydrates to support uptake. The lag in achieving optimum NO-3 uptake rates (Fig. 1A) could be affected not just by temperature but by light deficiency and slowed transpiration rates. At 700 µmol m-2 s-1 PPFD stomatal opening is slower and there is a lag in achieving full transpiration rate (Krizek, 1986).

Alternatively, the diurnal fluctuations of NO-3 uptake may be due to more specific control in relation to light/dark modifications of N utilization in the plant (Scaife and Schloemer, 1994; Delhon et al., 1995a). This is supported by reports of a negative relationship between NO-3 uptake and tissue NO-3 concentration (Breteler and Nissen, 1982; Siddiqi et al., 1989) under some physiological conditions other than light/dark transitions. NO-3 assimilation products such as NH+4 and certain free amino acids (especially Glu, Asp, Gln, and Asn) may be involved in the regulation of its uptake (Muller and Touraine, 1992; Delhon et al., 1995a, Aslam et al., 1996). Recently Delhon et al. (1995a) proposed that darkness adversely affects NO-3 uptake through specific feedback controls. That proposal was based on the accumulation of NO-3 and Asn in soybean roots in the dark. In a follow-up study, however, the same authors reported that NO-3 uptake was not coupled to NO-3 translocation, and diurnal fluctuations in uptake were not related to root NO-3 and Asn levels (Delhon et al., 1995b). Thus, they concluded that the effect of light/dark transitions on NO-3 uptake is not mediated by changes in translocation and accumulation of N compounds (Delhon et al., 1995b). Jackson et al. (1986) reported that several compounds that are transported by phloem, such as malate and sucrose, may regulate the NO-3 uptake system.

Diurnal Fluctuations of In Vivo NRA
Diurnal variations of in vivo NRA over a 14 h/30°C day and 10 h/20°C night growth cycle were similar for both Pima and Acala cotton (Fig. 3). Leaf enzyme activity, however, was 20 to 30% lower in Acala. At the beginning of the light period, in vivo NRA, assayed in the absence of additional NO-3, was lower relative to that assayed in the presence of NO-3 (Fig. 3). Thereafter, the NRA increased during the first hour of illumination in both species and the increase was more rapid in -NO-3 assay (Fig. 3). In Acala cotton leaves, addition of glucose to the assay medium at the beginning of illumination had little effect on in vivo NRA. Whereas, addition of NO-3 increased the enzyme activity about 75% (Fig. 4A). When glucose and NO-3 were added together, however, NRA was increased by only 30% as compared with that with NO-3 alone (Fig. 4A). A similar response to glucose addition was observed in Pima (M. Aslam, R.L. Travis, and D.W. Rains, 1998, unpublished results). These results suggest that enzyme activity was limited more by insufficient NO-3 at the reduction site at the onset of light, rather than by the supply of reductant, even though the leaves contained high concentrations of NO-3 (Table 1). The addition of glucose or NO-3 to the assay medium had little effect on in vivo NRA in leaves assayed after only 1 h of illumination (Fig. 4B). This result suggests that within 1 h both photosynthate and NO-3 were restored to levels adequate to support maximum in vivo NRA. Huber et al. (1994) reported that light, or metabolites produced by photosynthetic metabolism, rather than an endogenous rhythm, account primarily for diurnal variations in NRA levels in maize leaves.


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  		Table 1. NO-3 concentration in leaves of Pima (S-7) and Acala (Maxxa) cotton in light grown in 0.05, 0.1 or 1 mM NO-3 at 30/20°C light/dark temperatures

 
Leaf NR reached maximum activity after 1 h of illumination in seedlings grown in 0.1 or 1.0 mM NO-3, and was maintained at that level for the next 6 h (Fig. 3B and C). Thereafter, activity decreased gradually until, at the end of the light period, it was about the same as at the beginning of the light period. The same pattern occurred whether or not NO-3 was added to the assay medium (Fig. 3B and C). In contrast, NRA continued to increase for up to 4 h of light exposure when seedlings grown in 0.05 mM NO-3 were assayed in the absence of NO-3 (Fig. 3A). Thereafter, enzyme activity decreased rapidly. There was greater variation in the enzyme activity among -NO-3 and +NO-3 assays in seedlings grown at 0.05 mM NO-3 (Fig. 3A), perhaps reflecting a smaller metabolic pool of NO-3. That the diurnal fluctuations in NRA were reduced when NO-3 was added to the assay medium further indicates that the variations in NRA were likely the result of substrate limitation. When NRA was assayed in the presence of NO-3, the enzyme activities, after 4 h of illumination, were similar regardless of the NO-3 concentration in the growth medium (Fig. 3). This result suggests that optimum expression of NR occurred even at the lowest NO-3 growth concentration.

The initial increase in leaf NRA upon illumination of seedlings grown at 30°C/20°C light/dark temperature regime may be due to increased NO-3 uptake (Fig. 1A) and/or translocation of NO-3 from roots to shoots (Rufty et al., 1987). When the seedlings were grown at 30°C/30°C light/dark temperature, however, little fluctuation in NO-3 uptake occurred (Fig. 1B); yet in vivo NRA still increased upon illumination (Fig. 5). These results suggest that either NO-3 absorbed in the dark accumulated in the storage pool, or NO-3 absorbed in the dark was not translocated into the shoot. Delhon et al. (1995a) reported that darkness adversely affected NO-3 translocation more than uptake in soybean. In barley leaves NO-3 absorbed in the dark accumulated in the storage pool (Aslam et al., 1976). During the latter part of the light cycle, when enzyme activity decreased (Fig. 3), uptake rates remained constant (Fig. 1A), suggesting that the decrease in NRA levels was independent of NO-3 flux into the root. Transport of NO-3 from roots to shoots and alterations in intercellular compartmentation, however, may be affected by prolonged illumination. Likewise the decrease in NO-3 concentration in the leaves during the latter portion of the light period (Table 1) could be due to restricted translocation of NO-3 from the roots to the leaves or to increased in situ NO-3 reduction. Less translocation of NO-3 from the root to the leaves may be due to decrease in transpiration rates during the latter part of illumination. Thus fluctuations in translocation/transpiration may be as important as photosynthate and energy availability in regulating NRA.

Levels of in vivo NRA remained constant throughout the dark period. The enzyme activity assayed in the presence of NO-3, however, was 50 to 75% higher, compared with that assayed in the absence of NO-3 (Fig. 6). These results might suggest that the in vivo NRA was also limited by NO-3 availability in darkness. Although, the leaf NO-3 concentrations were high, especially in Pima (Table 2), this NO-3 apparently was not available for reduction. Since in darkness photosynthate supply to the leaf decreases, low NRA levels may also be due to a lack of reductant availability. In the absence of NO-3, however, the addition of glucose to the assay medium had no effect on NRA, whereas it enhanced enzyme activity by about 35% when NO-3 was present (Fig. 4A). In contrast, the increase of NRA level in the presence of NO-3 alone was about 80% greater than in its absence, suggesting that even in darkness NRA was limited more by the availability of NO-3 than by energy supply.


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  		Table 2. NO-3 concentration in leaves of Pima (S-7) and Acala (Maxxa) cotton in the dark grown in 0.1 mM NO-3 at 30/20°C light/dark temperatures

 
The diurnal fluctuations of in vivo NRA in leaves of cotton seedlings were similar to those observed in soybean leaves grown under similar conditions (Nicholas et al., 1976). The consistent increase in in vivo NRA observed when NO-3 was added to the assay medium suggests, that under these growth conditions, NO-3 may be in limited supply at the site of reduction, especially during the latter part of the day. Shaner and Boyer (1976) observed that NRA in Z. mays leaves appears to be regulated by NO-3 flux into the leaves regardless of the total NO-3 content of the leaves. It is known that much of the NO-3 in plant tissues is sequestered in the vacuole and hence is not immediately available for reduction (Ferrari et al., 1973; Aslam et al., 1976). Sequestration becomes even more important when osmotic balance and stomatal closure are considered, since the stomatal system in cotton is very sensitive to light, temperature and the time of the day (Krizek, 1986).


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The results show that the diurnal fluctuations of NO-3 uptake by cotton roots grown at 30/20°C light/dark regimen are temperature related, since no fluctuation occurred when NO-3 uptake rates were measured at constant temperature. This indicates that light/dark transitions are not the cause of rhythmicity in NO-3 uptake in cotton. The increase in NRA in vivo upon illumination was the result of an increase in substrate availability rather than reductant supply. Similarly, the decline in the enzyme activity during the latter part of the day was due to the decreased availability of NO-3. The initial increase in NRA in vivo upon illumination was the result of increased NO-3 uptake which, in turn, led to an increase in leaf NO-3 concentration. In contrast, the decline in in vivo NRA upon prolonged exposure to light was not due to a decrease in NO-3 uptake since NO-3 uptake rates remained constant. During this period, however, NO-3 concentration in the leaves decreased, suggesting that distribution and/or translocation of NO-3 within the plant was affected. In vivo NRA levels were low and remained constant throughout the dark period. Generally, NRA levels assayed in the presence of NO-3 were 50 to 80% higher than those assayed in the absence of additional NO-3. The addition of an energy source (glucose) to the assay medium at the onset of illumination (end of the dark period) had no effect on in vivo NRA when assayed in the absence of added NO-3; however, when NO-3 was added, NRA increased. In contrast, the addition of glucose after 1 h of illumination had no effect on in vivo NRA whether assayed with or without the addition of NO-3 to the assay medium. The results indicate that under normal energy levels (such as expected in cotton leaves analyzed after only one h of light exposure), NO-3 availability at the enzyme site appears to limit the rate of NO-3 reduction. Furthermore, NO-3 translocation and/or partitioning, rather than NO-3 uptake and NRA, may be under diurnal control in the cotton plant.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
This research was supported in part by grants from Cotton Incorporated and the California Crop Improvement Association.

Received for publication November 24, 1999.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome