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

UPTAKE AND REDUCTION, the initial processes by which [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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; Cardenas-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, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 (Steingrover 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake and its subsequent availability at the site of enzyme synthesis and/or [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] reduction. Shaner and Borer (1976) reported that a decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] flux to corn (Zea mays L.) leaves resulted in a rapid loss of NRA, although leaf [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] content was unchanged. They concluded that NRA is regulated in shoots by [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] flux rather than [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] content of the tissue. Likewise, Gojon et al. (1991) reported that the xylem flux of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in light is the main determinant of the actual rate of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] reduction in soybean [Glycine max (L.) Merr.] leaves. These reports suggest that the diurnal fluctuation in NRA may be controlled by [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration, or the addition of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 vulgate L.) (Lillo, 1983) leaves. This supports the hypothesis that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] best approximated [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] assimilation rates in situ in cotton (G. hirsutum) (Radin et al., 1975). Consequently, this assay was used to study regulation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] assimilation in cotton (Radin et al., 1975; Radin, 1977) and to determine the partitioning of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake ability at 0.05 mM ambient [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration with Pima being a more efficient user of N than Acala (Aslam et al., 1997).

MATERIALS AND METHODS

Plant Growth

Acid-delinted seeds of Acala, cv. Maxxa and Pima, cv. S-7 cotton were germinated in the dark at 25 [degrees] 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 [degrees] C/20 [degrees] 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 [micro]mol [m.sup.-2] [s.sup.-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 [KNO.sub.3] in aerated quarter-strength Hoagland's solution. Nitrate concentration in the nutrient solutions was assayed twice daily and maintained by adding [KNO.sub.3] solution. Nutrient solution pH was adjusted to 6.0 with [H.sub.2][SO.sub.4] as needed. The seedlings were grown for 25 d after transplanting.

Measurement of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [KNO.sub.3] 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 Ca[SO.sub.4] and 0.1 mM [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in 2.0 mM MES [(N-morpholino) ethanesulfonic acid] (pH 6.0). During this period, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration in the uptake solutions was maintained by adding [KNO.sub.3] 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 [degrees] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] depletion curves as described by Goyal and Huffaker (1986). Net uptake rates were then calculated by linear regression analysis of the cumulative uptake curves.

[ILLUSTRATIONS OMITTED]

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 [KNO.sub.3] under anaerobic conditions. The minus [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] in vivo assay measures the amount of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] being assimilated (Radin et al., 1975) and provides a reasonable estimate of the size of the metabolic [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] pool (Aslam et al., 1976). When [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] is added to the assay medium, the maximum capacity for [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [degrees] C in darkness for 30 min. Propanol was omitted from the assay medium because it decreases the production of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. NRA was calculated from the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration in the incubation medium and is reported as [micro]mol [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] [h.sup.-1] [g.sup.-1] fresh weight. In some experiments, leaf discs were also analyzed for [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. The [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] remaining in the discs after incubation was less than 5% of the total produced. Thus, NRA levels were not corrected for the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] remaining in the discs.

Extraction of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [H.sub.2]0 per gram of tissue and a small amount of acid-washed sand. The extracts were centrifuged at 30 000 x g at 4 [degrees] C for 15 min and the supernatants were used to determine [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration.

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] Determination

Nitrate concentration was determined spectrophotometrically by measuring [A.sub.210] 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 [A.sub.540] after color development for 15 min with a 1:1 mixture of 10 g [kg.sup.-1] sulfanilamide in 1.5 M HCl and 0.2 g [kg.sup.-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.

[ILLUSTRATIONS OMITTED]

RESULTS AND DISCUSSION

Diurnal Fluctuations of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] Uptake

Nitrate uptake rates, measured at 30 [degrees] C, for both Pima and Acala cotton cultivars grown in 0.1 mM [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] under a 14 h/30 [degrees] C light and 10 h/20 [degrees] 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 [degrees] /30 [degrees] C light/dark temperature prior to measurements, initial [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake rates, measured at 30 [degrees] C, were higher and then increased only slightly (10-15%) upon illumination (Fig. 1B). When the uptake rates of the seedlings, grown at 30 [degrees] / 20 [degrees] C light/dark temperature, were measured in darkness at the end of light period at 20 [degrees] 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 [degrees] C in the dark, however, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake rates did not fluctuate (Fig. 2A, B) In fact, the uptake rates measured in the dark at 30 [degrees] C were similar to those observed in light (compare Fig. 2 with Fig. 1B).

These results suggest that the diurnal rhythm of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [micro]mol [m.sup.-2] [s.sup.-1] PPFD) and at constant temperature (20 [degrees] /20 [degrees] C), reported that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [micro]mol [m.sup.-2] [s.sup.-1] PPFD) and constant temperature (20 [degrees] /20 [degrees] C), [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake increased continuously during light cycle and then decreased continuously during the subsequent dark cycle (Cardenas-Navarro et al., 1998).

Since [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake is an energy-consuming process and involves transport protein(s) (Rufty et al., 1989; Glass et al., 1990), light/dark fluctuations in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake (Hansen, 1980; Pearson et al., 1981). While in light, photosynthesis may supply energy for [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake system for several hours. Since no decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake rates occured when the plants were maintained at 30 [degrees] C in darkness (Fig. 2), it is likely that adequate energy was available from stored carbohydrates to support uptake. The lag in achieving optimum [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake rates (Fig. 1A) could be affected not just by temperature but by light deficiency and slowed transpiration rates. At 700 [micro]mol [m.sup.-2] [s.sup.-1] PPFD stomatal opening is slower and there is a lag in achieving full transpiration rate (Krizek, 1986).

Alternatively, the diurnal fluctuations of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake and tissue [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration (Breteler and Nissen, 1982; Siddiqi et el., 1989) under some physiological conditions other than light/dark transitions. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] assimilation products such as [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and certain free amino acids (especially Glu, Asp, Gin, 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake through specific feedback controls. That proposal was based on the accumulation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and Asn in soybean roots in the dark. In a follow-up study, however, the same authors reported that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake was not coupled to [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] translocation, and diurnal fluctuations in uptake were not related to root [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and Asn levels (Delhon et al., 1995b). Thus, they concluded that the effect of light/ dark transitions on [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake system.

Diurnal Fluctuations of In Vivo NRA

Diurnal variations of in vivo NRA over a 14 h/30 [degrees] C day and 10 h/20 [degrees] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], was lower relative to that assayed in the presence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (Fig. 3). Thereafter, the NRA increased during the first hour of illumination in both species and the increase was more rapid in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] increased the enzyme activity about 75% (Fig. 4A). When glucose and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] were added together, however, NRA was increased by only 30% as compared with that with [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] at the reduction site at the onset of light, rather than by the supply of reductant, even though the leaves contained high concentrations of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (Table 1). The addition of glucose or [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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.
Table 1. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
concentration in leaves of Pima (S-7) and Acala (Maxxa) cotton
in light grown in 0.05, 0.1 or 1 mM [MATHEMATICAL EXPRESSION
NOT REPRODUCIBLE IN ASCII] at 30/20 [degrees] C light/dark
temperatures.

            Growth [MATHEMATICAL EXPRESSION NOT
                REPRODUCIBLE IN ASCII] (mM)

                                0.05
Hours in
light            Pima                          Acala

 0         18.2 [+ or -] 0.8([dagger])    9.9 [+ or -] 0.5
 1         26.6 [+ or -] 0.9             16.6 [+ or -] 0.6
 4         34.5 [+ or -] 1.4             29.2 [+ or -] 0.4
 7         43.8 [+ or -] 1.5             26.5 [+ or -] 0.5
10         39.4 [+ or -] 1.2             18.2 [+ or -] 0.5
13         20.2 [+ or -] 0.2              9.8 [+ or -] 0.6

            Growth [MATHEMATICAL EXPRESSION NOT
                REPRODUCIBLE IN ASCII] (mM)

                            0.10
Hours in
light            Pima                Acala

            [micro]mol [g.sup.-1] Fresh Weight

 0         25.5 [+ or -] 0.7   15.2 [+ or -] 0.6
 1         35.7 [+ or -] 0.6   21.7 [+ or -] 0.7
 4         44.8 [+ or -] 1.0   24.3 [+ or -] 0.9
 7         54.7 [+ or -] 1.2   29.3 [+ or -] 1.0
10         51.4 [+ or -] 1.8   25.9 [+ or -] 0.8
13         34.1 [+ or -] 0.7   20.1 [+ or -] 0.5

            Growth [MATHEMATICAL EXPRESSION NOT
                REPRODUCIBLE IN ASCII] (mM)

                            1.00
Hours in
light            Pima                Acala

 0         62.1 [+ or -] 1.8   35.9 [+ or -] 1.3
 1         72.7 [+ or -] 3.2   39.2 [+ or -] 1.3
 4         87.2 [+ or -] 1.6   53.8 [+ or -] 1.3
 7         72.6 [+ or -] 1.5   59.0 [+ or -] 1.1
10         67.9 [+ or -] 1.1   56.8 [+ or -] 1.5
13         63.7 [+ or -] 1.5   43.5 [+ or -] 1.1

([dagger]) Data are mean [+ or -] SE (n = 3).


Leaf NR reached maximum activity after 1 h of illumination in seedlings grown in 0.1 or 1.0 mM [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] were assayed in the absence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (Fig. 3A). Thereafter, enzyme activity decreased rapidly. There was greater variation in the enzyme activity among [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] assays in seedlings grown at 0.05 mM [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (Fig. 3A), perhaps reflecting a smaller metabolic pool of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. That the diurnal fluctuations in NRA were reduced when [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], the enzyme activities, after 4 h of illumination, were similar regardless of the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration in the growth medium (Fig. 3). This result suggests that optimum expression of NR occurred even at the lowest [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] growth concentration.

The initial increase in leaf NRA upon illumination of seedlings grown at 30 [degrees] C/20 [degrees] C light/dark temperature regime may be due to increased [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake (Fig. 1A) and/or translocation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] from roots to shoots (Rufty et al., 1987). When the seedlings were grown at 30 [degrees] C/ 30 [degrees] C light/dark temperature, however, little fluctuation in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake occurred (Fig. 1B); yet in vivo NRA still increased upon illumination (Fig. 5). These results suggest that either [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] absorbed in the dark accumulated in the storage pool, or [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] absorbed in the dark was not translocated into the shoot. Delhon et al. (1995a) reported that darkness adversely affected [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] translocation more than uptake in soybean. In barley leaves [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] flux into the root. Transport of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] from roots to shoots and alterations in intercellular compartmentation, however, may be affected by prolonged illumination. Likewise the decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration in the leaves during the latter portion of the light period (Table 1) could be due to restricted translocation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] from the roots to the leaves or to increased in situ [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] reduction. Less translocation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], however, was 50 to 75% higher, compared with that assayed in the absence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (Fig. 6). These results might suggest that the in vivo NRA was also limited by [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] availability in darkness. Although, the leaf [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentrations were high, especially in Pima (Table 2), this [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], however, the addition of glucose to the assay medium had no effect on NRA, whereas it enhanced enzyme activity by about 35% when [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was present (Fig. 4A). In contrast, the increase of NRA level in the presence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] alone was about 80% greater than in its absence, suggesting that even in darkness NRA was limited more by the availability of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] than by energy supply.
Table 2. [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]
concentration in leaves of Pima (S-7) and Acala (Maxxa) cotton
in the dark grown in 0.1 mM [MATHEMATICAL EXPRESSION NOT
REPRODUCIBLE IN ASCII] at 30/20 [degrees] C light/dark
temperatures.

                                 Cotton species

Hours in dark              Pima                     Acala

                     [micro]mol [g.sup.-1] Fresh weight

 0              29.8 [+ or -] 1.1([dagger])   18.3 [+ or -] 1.1
 3              26.9 [+ or -] 0.9             16.5 [+ or -] 0.8
 6              24.6 [+ or -] 0.8             15.6 [+ or -] 1.0
 8              21.9 [+ or -] 1.0             14.1 [+ or -] 0.6
10              22.8 [+ or -] 1.0             14.8 [+ or -] 0.8

([dagger]) Data are mean [+ or -] SE (n = 3).


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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] was added to the assay medium suggests, that under these growth conditions, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] flux into the leaves regardless of the total [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] content of the leaves. It is known that much of the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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

The results show that the diurnal fluctuations of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake by cotton roots grown at 30/20 [degrees] C light/ dark regimen are temperature related, since no fluctuation occurred when [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake rates were measured at constant temperature. This indicates that light/dark transitions are not the cause of rhythmicity in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. The initial increase in NRA in vivo upon illumination was the result of increased [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake which, in turn, led to an increase in leaf [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration. In contrast, the decline in in vivo NRA upon prolonged exposure to light was not due to a decrease in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake since [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake rates remained constant. During this period, however, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentration in the leaves decreased, suggesting that distribution and/or translocation of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] were 50 to 80% higher than those assayed in the absence of additional [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]; however, when [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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), [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] availability at the enzyme site appears to limit the rate of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] reduction. Furthermore, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] translocation and/or partitioning, rather than [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake and NRA, may be under diurnal control in the cotton plant.

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

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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. This research was supported in part by grants from Cotton Incorporated and the California Crop Improvement Association. Received 24 Nov. 1999. (*) Corresponding author (rltravis@ucdavis.edu).
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Author:Aslam, M.; Travis, R. L.; Rains, D. W.
Publication:Crop Science
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Geographic Code:1USA
Date:Mar 1, 2001
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