Source and Magnitude of Ammonium Generation in Maize Roots

Abstract

Studies with ¹⁵N indicate that appreciable generation of NH₄⁺ from endogenous sources accompanies the uptake and assimilation of exogenous NH₄⁺ by roots. To identify the source of NH₄⁺ generation, maize (Zea mays L.) seedlings were grown on ¹⁴NH₄⁺ and then exposed for 3 d to highly labeled ¹⁵NH₄⁺. More of the entering ¹⁵NH₄⁺ was incorporated into the protein-N fraction of roots in darkness (approximately 25%) than in the light (approximately 14%). Although the ¹⁴NH₄⁺ content of roots declined rapidly to less than 1 μmol per plant, efflux of ¹⁴NH₄⁺ continued throughout the 3-d period at an average daily rate of 14 μmol per plant. As a consequence, cumulative ¹⁴NH₄⁺ efflux during the 3-d period accounted for 25% of the total ¹⁴N initially present in the root. Although soluble organic ¹⁴N in roots declined during the 3-d period, insoluble ¹⁴N remained relatively constant. In shoots both soluble organic ¹⁴N and ¹⁴NH₄⁺ declined, but a comparable increase in insoluble ¹⁴N was noted. Thus, total ¹⁴N in shoots remained constant, reflecting little or no net redistribution of ¹⁴N between shoots and roots. Collectively, these observations reveal that catabolism of soluble organic N, not protein N, is the primary source of endogenous NH₄⁺ generation in maize roots.

Short-term studies with ¹³NH₄⁺ have provided estimates of NH₄⁺ influx, efflux, and cytoplasmic concentration in spruce (Kronzuker et al., 1995a, 1995b). As the NH₄⁺ concentration in the external solution increased from 10 to 1500 μm, cytosolic NH₄⁺ levels increased from 2 to 33 mm and efflux increased from 10% to 35% of influx. Similar rates were reported for rice: as external NH₄⁺ levels increased from 2 to 1000 μm, cytosolic NH₄⁺ levels increased from 3 to 38 mm and efflux rose from 11% to 29% of influx (Wang et al., 1993). The half-lives of cytosolic NH₄⁺ were 8 and 15 min for rice and spruce, respectively.

During short-term exposure of cereal seedlings to ¹⁵NH₄⁺ solution, efflux of endogenous ¹⁴NH₄⁺ exceeded the total ¹⁴NH₄⁺ initially present in the root tissues (Morgan and Jackson, 1988a, 1988b). Moreover, after a 5-h pretreatment in ¹⁵NH₄⁺ solution, the subsequent efflux of ¹⁵NH₄⁺ to the ambient ¹⁴NH₄⁺ solution was greater than the initial NH₄⁺ content of the root (Morgan and Jackson, 1989). Thus, appreciable generation of NH₄⁺ from endogenous organic N sources accompanies concurrent uptake and assimilation of NH₄⁺ by roots.

The efflux of NH₄⁺ provides only a minimal estimate of endogenous NH₄⁺ generation because part of the NH₄⁺ is likely reassimilated. In support of this possibility, when NH₄⁺ assimilation via Gln synthetase was blocked with Met sulfoxamine, NH₄⁺ generation in maize roots was estimated to be 50% faster than concurrent NH₄⁺ uptake (Jackson, et al., 1993). The potential for substantial generation, recycling, and efflux of endogenous NH₄⁺ in roots is thus indicated.

A crucial question raised by these observations is whether protein turnover is the source of endogenous NH₄⁺ generation, or if recycling of intermediates of the NH₄⁺ assimilation pathway, such as Gln, is the source. To address this question, maize (Zea mays) seedlings that had been grown on ¹⁴NH₄⁺ were exposed to highly labeled ¹⁵NH₄⁺ for 3 d. It was hypothesized that if protein turnover is the source of NH₄⁺ and if part of this NH₄⁺ is subject to efflux and translocation to the shoot, a decline in endogenous ¹⁴N protein in the root should occur as new protein is synthesized from the entering ¹⁵NH₄⁺.

The fact that ¹⁵NH₄⁺ was applied during six diurnal periods also permitted us to (a) directly measure of the diurnal pattern of NH₄⁺ fluxes into and out of roots, (b) compare ¹⁵NH₄⁺ uptake and assimilation by roots during successive light and dark periods, and (c) determine the relationship of the latter processes to carbohydrate levels in shoots and roots.

MATERIALS AND METHODS

Plant Culture

Maize (Zea mays L. cv Pioneer 3320) caryopses were germinated at 30°C in contact with 0.1 mm CaSO₄. After 30 h, uniform seedlings were selected and their seminal roots excised. Cultures of eight seedlings each were transplanted into 160 L of basal nutrient solution, pH 6.0, containing 0.125 mm (NH₄)₂SO₄, 1.25 mm K₂SO₄, 0.25 mm Ca(H₂PO₄)₂, 1 mm CaSO₄, 46 μm B, 9 μm Mn, 0.8 μm Zn, 0.3 μm Cu, 0.1 μm Mo, and 54 μm Fe as ferric diethylenetriamine pentaacetate. The solution was aerated with compressed air that had been washed with H₂SO₄ and water to remove ambient NH₄⁺. A combination of sodium vapor and metal halide lamps provided 1140 μE m⁻² s⁻¹ illumination at canopy height during a 14-h photoperiod (7 am to 9 pm). The average air temperature during the experiment was 23.4°C ± 1.6°C. Both pH and [NH₄⁺] of the nutrient solution were checked daily and maintained at pH 6.0 and 0.25 mm, respectively, by continuous injection of Ca(OH)₂ and (NH₄)₂SO₄.

Experimental Procedure

At the beginning of the dark period on the 7th d after imbibition, four cultures were harvested and the rest were transferred to fresh basal nutrient solution containing 0.22 mm NH₄⁺ labeled with 96.1 atom % ¹⁵N. An initial sample of ¹⁵NH₄⁺ nutrient solution was taken for subsequent analysis. Four additional cultures were harvested and a solution sample was taken at the beginning (7 am) and end (9 pm) of each light period on the 8th, 9th, and 10th d. The pH and [NH₄⁺] of the nutrient solution were checked daily and maintained as described above by continuous injection of Ca(OH)₂ and (¹⁵NH₄)₂SO₄ containing 99.8 atom % ¹⁵N.

At harvest, the roots were dipped five times in 0.1 mm CaSO₄, excised, blotted lightly, and weighed. After weighing the shoots, which included the remaining seed pieces, all tissue samples were freeze dried, weighed, ground, and mixed thoroughly.

N and ¹⁵N Analysis

Shoots, roots, and nutrient solutions were analyzed for NH₄⁺ and its ¹⁵N enrichment (Jackson et al., 1993). In addition, shoots and roots were analyzed for soluble and insoluble N and their respective ¹⁵N enrichments. These fractions were separated by extraction with acidified (pH 3.0) 80% (v/v) ethanol. Organic N in the extract and residue was converted to NH₄⁺ by Kjeldahl digestion (McKenzie and Wallace, 1954), and the NH₄⁺ was quantified by spectrophotometric analysis (Smith, 1980). NH₄⁺ was recovered by diffusion, converted to dinitrogen gas by a freeze-layer procedure (Volk and Jackson, 1979), and analyzed for ¹⁵N enrichment by MS.

Total N and ¹⁵N analyses were used to calculate the tissue contents of six isotopic N species: ¹⁴NH₄⁺, ¹⁵NH₄⁺, soluble ¹⁴N, soluble ¹⁵N, insoluble ¹⁴N, and insoluble ¹⁵N. Because ¹⁴NH₄⁺ and ¹⁵NH₄⁺ were subtracted from soluble ¹⁴N and soluble ¹⁵N, these fractions represent soluble organic N constituents. Protein is the primary constituent of the insoluble-N fraction.

Carbohydrate Analysis

To obtain comparable tissue samples for nonstructural carbohydrate analysis, the experiment was repeated and the seed pieces were discarded at harvest. Insoluble and soluble carbohydrates of shoots and soluble carbohydrates of roots were assayed by enzymatic and spectrophotometric procedures after separation by extraction with 80% (v/v) ethanol (Jones et al., 1977).

RESULTS

Changes in Endogenous ¹⁴N Fractions

An appreciable increase in insoluble ¹⁴N in shoots (Fig. 1C) was balanced by an equivalent decline in the ¹⁴NH₄⁺ and soluble-¹⁴N fractions (Fig. 1, A and B). Thus, the total endogenous ¹⁴N in shoots remained constant during the 3-d exposure to ¹⁵NH₄⁺ (Fig. 1D). By contrast, the insoluble ¹⁴N of roots remained relatively constant (Fig. 2C), even though ¹⁴NH₄⁺ and soluble ¹⁴N decreased appreciably (Fig. 2, A and B). As a consequence, total ¹⁴N in the root declined during the 3-d exposure to ¹⁵NH₄⁺ (Fig. 2D). On a whole-plant basis, the changes in ¹⁴N (Fig. 3) reflected those in the shoot, which contained more than 75% of the total ¹⁴N in the plant.

Figure 1.

Shoot contents of NH₄⁺ (A), soluble N (B), insoluble N (C), and total N (D) derived from endogenous (¹⁴N) sources and from exogenously supplied NH₄⁺ (¹⁵N) during a 3-d continuous exposure of maize seedlings to highly labeled ¹⁵NH₄⁺. Each value is the mean of four replicates ± se.

Figure 2.

Root contents of NH₄⁺ (A), soluble N (B), insoluble N (C), and total N (D) derived from endogenous (¹⁴N) sources and from exogenously supplied NH₄⁺ (¹⁵N) during a 3-d continuous exposure of maize seedlings to highly labeled ¹⁵NH₄⁺. Each value is the mean of four replicates ± se.

Figure 3.

Whole-plant contents of NH₄⁺ (A), soluble N (B), insoluble N (C), and total N (D) derived from endogenous (¹⁴N) sources and from exogenously supplied NH₄⁺ (¹⁵N) during a 3-d continuous exposure of maize seedlings to highly labeled ¹⁵NH₄⁺. Each value is the mean of four replicates ± se. The diurnal rates of ¹⁵NH₄⁺ uptake (in micromoles per gram fresh weight per hour) are noted in D.

Estimation of NH₄⁺ Fluxes into and out of Roots

During each diurnal period a complete balance sheet of the changes in ¹⁴NH₄⁺ and ¹⁵NH₄⁺ in the uptake solution was compiled. The procedure is illustrated in Table I using data from the initial 10-h dark period. Even though an appreciable quantity of 99.8 atom % ¹⁵N was injected to maintain the [NH₄⁺], the ¹⁵N enrichment of the nutrient solution declined from 96.1 to 93.5 atom % ¹⁵N, reflecting a release of ¹⁴NH₄⁺ from the root. The net rate of release, 4.3 μmol plant⁻¹ h⁻¹, is less than the actual rate because of concurrent uptake of ¹⁴NH₄⁺ from the nutrient solution. The latter can be estimated from the fact that ¹⁴NH₄⁺ and ¹⁵NH₄⁺ are taken up in proportion to their average molar concentrations in the nutrient solution during any given period. The calculation (Table I, line G) reveals that ¹⁴NH₄⁺ was taken up at a rate of 2.6 μmol plant⁻¹ h⁻¹. This rate, when added to the net rate of ¹⁴NH₄⁺ release, provides an estimate of “true” ¹⁴NH₄⁺ release (Table I, line J). Similar calculations were made throughout the experiment to quantify ¹⁴NH₄⁺ release from the root in successive diurnal periods (Table II). Both the uptake and release of NH₄⁺ were greater in light than in darkness. NH₄⁺ release remained appreciable even in the final light period, when it accounted for 4.8% of the total ¹⁴N initially present in roots harvested on the 7th d. As a consequence, cumulative ¹⁴NH₄⁺ release during the 3-d period was equivalent to 25% of the total ¹⁴N initially present in the root.

Table I.

Calculation of NH₄⁺ fluxes into and out of maize roots

Measurement	Volume	[NH4+]	Atom % 15N	I 14NH4+	II 15NH4+	Rate
	L	mm	%	μmol 160 L−1		μmol plant−1 10 h−1
A. Initial (9 pm)	160	0.223	96.10	1392	34,288
B. Injection	0.09756	113	99.80	22	11,002
C. Final (7 am)	160	0.231	93.55	2384	34,576
D. Change (C − A − B)				+970	−10,714
E. Mean concentration (A + C)/2				1888	34,432
F. Net 15NH4+ uptake (DII/227 plants)						47.2
G. 14NH4+ uptakea(F[EI/EII])						2.6
H. Total NH4+ uptake (F + G)						49.8
I. Net 14NH4+ release (DI/227 plants)						4.3
J. “True” 14NH4+ releaseb(I + G)						6.9

Rates of ¹⁵NH₄⁺ uptake, ¹⁴NH₄⁺ uptake, and ¹⁴NH₄⁺ release by 227 maize seedlings were calculated from [¹⁵NH₄⁺] and [¹⁴NH₄⁺] in the nutrient solution at the beginning (9 pm) and end (7 am) of the first 10-h dark period, during which 97.56 mL of 113 mm ¹⁵NH₄⁺ (99.8 atom % ¹⁵N) was injected to maintain the total [NH₄⁺] close to its initial level (0.223 mm).

^a14NH₄⁺ uptake can be estimated from net ¹⁵NH₄⁺ uptake because the two isotopic species are taken up in proportion to their mean molar concentrations in the nutrient solution during the uptake period. 

^bNet ¹⁴NH₄⁺ release = true ¹⁴NH₄⁺ release − ¹⁴NH₄⁺ uptake. Therefore, true ¹⁴NH₄⁺ release = net ¹⁴NH₄⁺ release + ¹⁴NH₄⁺ uptake.  

Table II.

Diurnal rates of ¹⁵NH₄⁺ uptake, ¹⁴NH₄⁺ uptake, and ¹⁴NH₄⁺ release by maize roots

Day	Diurnal Period	Length	Total (15NH4+ + 14NH4+) Uptake	True 14NH4+ Release	True Root 14N Releasea
		h	μmol plant−1	period−1	%
7–8	Dark	10	49.8	6.9	4.0
8	Light	14	103.1	11.6	6.7
8–9	Dark	10	63.9	0.2	0.1
9	Light	14	177.3	11.1	6.4
9–10	Dark	10	90.6	5.2	3.0
10	Light	14	193.3	8.3	4.8
Total			678.0	43.3	25.0

Roots of maize seedlings were exposed continuously to highly labeled ¹⁵NH₄⁺ for 3 d. NH₄⁺ flux rates were calculated from periodic analysis of the uptake solution, as illustrated in Table I.

^a14NH₄⁺ release of the total ¹⁴N present in roots harvested on the 7th d (174 μmol ¹⁴N root⁻¹). 

It is important to note that the data in Table II cannot be compared directly with the data in Figure 2. Table II shows the true release of ¹⁴NH₄⁺ from roots, which consists of measured net release of ¹⁴NH₄⁺ plus the concurrent, calculated uptake of ¹⁴NH₄⁺. By contrast, Figure 2 portrays the net change in root ¹⁴N, which reflects only net release of ¹⁴NH₄⁺ from the root, provided that no interchange of ¹⁴N between roots and shoots occurs.

Uptake and Assimilation of Applied ¹⁵NH₄⁺

The rate of ¹⁵NH₄⁺ uptake by maize seedlings was similar during light and dark periods (Fig. 3D). A small but measurable amount of the entering ¹⁵NH₄⁺ accumulated as NH₄⁺ in the shoot, and the amount increased appreciably as the plants developed (Fig. 1A). By contrast, ¹⁵NH₄⁺ accumulation in the root exhibited a diurnal pattern (Fig. 2A). Except for the initial dark period, root ¹⁵NH₄⁺ increased during illumination and declined in darkness.

Most of the entering ¹⁵NH₄⁺ was assimilated into the soluble-¹⁵N and insoluble-¹⁵N fractions (Fig. 3, B and C). Although the patterns of accumulation in shoots (Fig. 1, B and C) and roots (Fig. 2, B and C) were similar to those in the whole plant, diurnal differences in assimilation and translocation were evident (Fig. 4). During each dark period a greater percentage of the entering ¹⁵NH₄⁺ was retained in the root and incorporated into the insoluble-¹⁵N fraction. Conversely, during each light period a greater percentage of the entering ¹⁵NH₄⁺, or metabolites thereof, was translocated to the shoot and incorporated into the shoot insoluble-¹⁵N fraction.

Figure 4.

Diurnal increments of total ¹⁵N and insoluble ¹⁵N (I ¹⁵N) in shoots and roots of maize seedlings as percentages of ¹⁵N uptake during each of six successive photoperiods. Each value is the mean of four replications. se values are indicated by vertical bars.

Diurnal Changes in Dry Matter and Carbohydrate

The dry weight of maize shoots generally increased during light periods and declined during dark periods (Fig. 5). By contrast, root weight increased at least as rapidly in darkness as in light. Although the carbohydrate content of both shoots and roots exhibited a typical diurnal pattern (Fig. 6), the pattern in roots was attenuated considerably.

Figure 5.

Dry weights of maize shoots and roots harvested at the beginning and end of each photoperiod during a 3-d exposure to highly labeled ¹⁵NH₄⁺. Each value is the mean of four replications. se bars are shown when they are larger than the symbols.

Figure 6.

Soluble and insoluble carbohydrate contents of shoots and soluble carbohydrate contents of roots during a 3-d continuous exposure of maize seedlings to ¹⁴NH₄⁺. Each value is the mean of four replicates ± se.

DISCUSSION

Source of ¹⁴NH₄⁺ Generation in Roots

Morgan and Jackson (1988a, 1988b, 1989) demonstrated that significant generation of NH₄⁺ occurs in roots during uptake and assimilation of exogenously supplied NH₄⁺. They suggested that organic N degradation, NH₄⁺ assimilation, and NH₄⁺ influx and efflux can be modified by environmental and nutritional conditions that alter the pool size of NH₄⁺ in roots.

Throughout the 3-d exposure of maize seedlings to ¹⁵NH₄⁺, during which appreciable synthesis of insoluble ¹⁵N occurred in roots, the endogenous insoluble ¹⁴N of roots remained relatively constant. However, ¹⁴NH₄⁺ and soluble ¹⁴N in roots declined significantly. There was evidence of ¹⁴N-protein synthesis in the shoot, apparently at the expense of the pool of soluble ¹⁴N in the shoot. The ultimate source of ¹⁴N for protein synthesis in the shoot remains tentative, however, because of the likelihood of soluble-¹⁴N interchange between the root and shoot as a consequence of amino acid cycling (Lambers et al., 1982; Cooper and Clarkson, 1989). In spite of this possibility, no measurable net transport of endogenous ¹⁴N from roots to shoots occurred during the 3-d exposure to ¹⁵NH₄⁺. Collectively, these data reveal that soluble organic N, rather than protein, is the primary source of endogenous NH₄⁺ generation in maize roots.

Release of ¹⁴NH₄⁺ from Roots

During the first 24 h of exposure to ¹⁵NH₄⁺ the release of endogenously derived ¹⁴NH₄⁺ from the root into the nutrient solution was equivalent to 10.7% of the initial ¹⁴N content of the root (Table II). A slower but measurable release of ¹⁴NH₄⁺ continued throughout the subsequent 2-d period. This occurred even though the content of ¹⁴NH₄⁺ in the root was less than 1 μmol after 24 h of exposure to exogenous ¹⁵NH₄⁺ (Fig. 2A), indicating a continual generation of ¹⁴NH₄⁺ from endogenous ¹⁴N pools. Thus, it is clear that the source of ¹⁴NH₄⁺, presumably the soluble-¹⁴N pool of the root, was not replaced completely by synthesis of soluble ¹⁵N from entering ¹⁵NH₄⁺. In support of this premise are the observations that the root soluble-¹⁴N pool declined only 22 μmol root⁻¹ (34%) during the first 24 h, and remained relatively constant at 40 μmol root⁻¹ thereafter (Fig. 2B). This occurred even though the root soluble-¹⁵N pool increased from near 0 to more than 200 μmol root⁻¹ during the 3-d period. One interpretation of these observations is that the endogenous soluble-¹⁴N pool, the putative source of ¹⁴NH₄⁺ generation in roots, occupies a different cellular or intracellular “compartment” than that of recently synthesized soluble ¹⁵N. For example, synthesis of soluble ¹⁵N might occur primarily in the root tip (0–2 cm), where NH₄⁺ uptake is maximal (Cruz et al., 1995), whereas NH₄⁺ generation might occur in the more mature regions of the root.

Diurnal Use of Exogenous ¹⁵NH₄⁺

Although effective absorption and assimilation of ¹⁵NH₄⁺ occurred in both dark and light periods, diurnal differences in utilization were observed (Fig. 4). More of the entering ¹⁵N was retained by the root in the dark (33%–56%) than in the light (18%–26%). A similar retention of NH₄⁺ was reported for perennial ryegrass by Ourry et al. (1996). We also observed that more of the entering ¹⁵NH₄⁺ was incorporated into root insoluble ¹⁵N in the dark (approximately 25%) than in the light (approximately 14%). Conversely, the synthesis of shoot insoluble ¹⁵N was enhanced by light.

In contrast to the data reported here, Ourry et al. (1996) found that the rate of NH₄⁺ uptake by perennial ryegrass declined during darkness. However, the plants were exposed to a lower light intensity (500 μmol m⁻² s⁻¹) and a lower [NH₄⁺] (20 μm) than were used in our study. Restricted rates of NH₄⁺ uptake in the dark were also reported for both fescue and timothy grass when grown at 20 μm NH₄⁺ (Macduff et al., 1997). Finally, the rate of NH₄⁺ uptake in the dark by barley grown under light-limited conditions (350 μmol m⁻² s⁻¹) was only 50% of the rate in the light (Rigano et al., 1996), perhaps reflecting a limitation of NH₄⁺ uptake by carbohydrate supply.

Several lines of evidence suggest that both the uptake and assimilation of NH₄⁺ are regulated by carbohydrate supplied from the shoot. First, Massimino et al. (1981) reported that NH₄⁺ uptake by maize declined within 2 h after lowering the light intensity to restrict photosynthesis. Second, after bean plants had been ringed, NH₄⁺ uptake and root soluble carbohydrate content declined concurrently (Michael et al., 1970). When exogenous Suc was supplied to the roots of ringed plants, however, the rate of NH₄⁺ uptake exceeded that of intact plants. Third, Lewis et al. (1987) found that a much higher proportion of ¹⁴C derived from photosynthetic CO₂ fixation was allocated to the root when N was supplied as NH₄⁺ rather than as NO₃⁻.

Based on these observations we hypothesized that diurnal differences in retention and incorporation of NH₄⁺ into macromolecules were related to changes in the diurnal supply of carbohydrate to the root. To examine this possibility, the changes in carbohydrate contents of the shoot and roots in a duplicate experiment were compared with ¹⁵NH₄⁺-assimilation rates in the original experiment (Table III). It was assumed that (a) the carbohydrate changes in the replicate experiment were comparable to those in the original study, (b) assimilation of the entering ¹⁵NH₄⁺ occurred exclusively in the root, (c) the initial product of NH₄⁺ assimilation in the root was Gln, and (d) the assimilation of 1 mol of ¹⁵NH₄⁺ into Gln requires 0.505 mol of Glc equivalents to supply the necessary reductant, ATP, and C skeletons (Schubert, 1980). Using these assumptions, the C required for net ¹⁵NH₄⁺ assimilation during each diurnal period was estimated.

Table III.

Carbohydrate required for ¹⁵NH₄⁺ assimilation by maize roots

Day	Photoperiod	15NH4+ Assimilation	Carbohydrate
			Requirementa	Change in shoot	Change in root	Transport to rootb
		μmol N plant−1		μmol C plant−1
7–8	Dark	39  ± 4	117  ± 13	−319  ± 7	−57  ± 10	60  ± 15
8	Light	102  ± 18	310  ± 53	+556  ± 35	+61  ± 7	371  ± 55
8–9	Dark	91  ± 11	275  ± 34	−550  ± 12	−103  ± 6	172  ± 37
9	Light	110  ± 24	333  ± 74	+590  ± 67	+42  ± 11	375  ± 64
9–10	Dark	120  ± 28	365  ± 84	−501  ± 24	−38  ± 13	327  ± 97
10	Light	288  ± 30	872  ± 91	+1193  ± 147	+118  ± 21	990  ± 108

The diurnal assimilation of ¹⁵NH₄⁺ was measured during a 3-d exposure of maize seedlings to highly labeled ¹⁵NH₄⁺. The minimal transport of C from shoot to root required to support ¹⁵NH₄⁺ assimilation was calculated from the theoretical C requirement for ¹⁵NH₄⁺ assimilation and the changes in tissue carbohydrate. Each value is the mean of four replicates ± se.

^aCalculation of the total C requirement for ¹⁵NH₄⁺ assimilation is based on the assumption that assimilation of entering ¹⁵NH₄⁺ occurs in the root, and that the initial product of NH₄⁺ assimilation is Gln, which requires 0.505 mol of Glc equivalents mol⁻¹ NH₄⁺ to provide the necessary reductant, ATP, and C skeletons (Schubert, 1982). 

^bMinimal C transport to the root required to support ¹⁵NH₄⁺ assimilation is equal to the total C required for ¹⁵NH₄⁺ assimilation plus the change in root C content. 

As plants developed, the C required to support measured ¹⁵NH₄⁺ assimilation increased from 117 μmol plant⁻¹ in the first dark period to 872 μmol plant⁻¹ during the final light period (Table III). Yet the carbohydrate content of the roots at the beginning of each dark period varied from 120 to 200 μmol plant⁻¹ (i.e. 20–33 μmol Glc equivalents plant⁻¹; Fig. 6). These data reveal that with the exception of the first dark period, there were insufficient carbohydrate reserves in the root to support assimilation of the ¹⁵NH₄⁺ absorbed. However, there were adequate reserves in the shoot. Thus, appreciable transport of carbohydrate from the shoot to the root must have occurred during both dark and light periods.

Minimal estimates of the transport of C to the root required to support ¹⁵NH₄⁺ assimilation can be calculated by adding the total C required for ¹⁵NH₄⁺ assimilation to the accumulation of C by the root (Table III). Such estimates ranged from 60 μmol plant⁻¹ during the first dark period to 990 μmol plant⁻¹ during the final light period (Table III). It is of particular interest to note that during the three successive dark periods, ¹⁵NH₄⁺ assimilation accounted for 19%, 31%, and 65%, respectively, of the decline in shoot carbohydrate. This reflects the facts that carbohydrate reserves in the root at the beginning of each dark period were relatively constant (12–19 μmol Glc equivalents plant⁻¹), whereas the rate of ¹⁵NH₄⁺ uptake and assimilation during the three successive dark periods increased from 3.9 to 7.8 μmol g⁻¹ fresh weight root h⁻¹ (Fig. 3D).

The increasing proportion of C reserves required to sustain the assimilation of entering NH₄⁺ supports the concept that carbohydrate availability is involved in the regulation of NH₄⁺ uptake and assimilation by the root. If so, the dark-enhanced assimilation of ¹⁵NH₄⁺ into the insoluble-¹⁵N fraction of the root indicates that C availability is higher during dark periods relative to ¹⁵NH₄⁺ uptake. This possibility is consistent with the diurnal pattern of ¹⁵NH₄⁺ accumulation in roots (Fig. 2A). Although the ¹⁵NH₄⁺ content of roots was low (<5 μmol), fluctuations likely reflect the changing diurnal equilibria between uptake and assimilation of ¹⁵NH₄⁺. With the exception of the first dark period, the ¹⁵NH₄⁺ content of roots declined in darkness and increased during the light. This suggests that in darkness the assimilation of ¹⁵NH₄⁺ usually exceeded its uptake, indicating an adequate supply of carbohydrate. During illumination, however, the ¹⁵NH₄⁺ content of roots increased, suggesting that the supply of carbohydrate was insufficient to assimilate all of the entering ¹⁵NH₄⁺. Yet, measured carbohydrate levels in roots increased during illumination. Additional studies are in progress to examine this apparent anomaly.