Plant Physiol. (1999) 120: 283-292

Inhibition of Nitrate Uptake by Ammonium in Barley. Analysis of Component Fluxes¹

Herbert J. Kronzucker^*, Anthony D.M. Glass, and M. Yaeesh Siddiqi

Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 5B7 (H.J.K.); and Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.D.M.G., M.Y.S.)

	ABSTRACT

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NO₃ uptake by plant roots is rapidly inhibited by exposure to NH₄⁺. The rapidity of the effect has led to the presumption that the inhibition results from the direct effects of NH₄⁺ at the plasma membrane. The mechanism of this inhibition, however, has been in contention. In the present study we used the radiotracer N to determine the relative effects of short-term exposures to NH₄⁺ on the ¹³NO₃ influx, efflux, and partitioning of absorbed ¹³N in barley (Hordeum vulgare) roots. Plants were grown without NO₃ or NO₂ (uninduced for NO₃ uptake), or with 0.1, 1.0, 10 mM NO₃, or 0.1 mM NO₂ (to generate plant roots induced for NO₃ uptake). Exposure to 1 mM NH₄⁺ strongly reduced influx; the effect was most pronounced in plants induced for NO₃ uptake when NO₃ absorption was measured at low external NO₃. At higher [NO₃] and in uninduced plants the inhibitory effect was much diminished, indicating that NH₄⁺ inhibition of influx was mediated via effects on the inducible high-affinity transport system rather than on the constitutive high-affinity transport system or the low-affinity transport system. Exposure to NH₄⁺ also caused increased NO₃ efflux; the largest effect was at low external [NO₃] in uninduced plants. In absolute terms, the reduction of influx made the dominant contribution to the observed reduction of net uptake of NO₃. Differences in response between plants induced with NO₃ and those induced with NO₂ indicate that NO₂ may not be an appropriate analog for NO₃ under all conditions.

	INTRODUCTION

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The inhibitory effects exerted by the NH₄⁺ ion upon NO₃ uptake by the roots of higher plants have been studied extensively (Weissman, 1950; Lycklama, 1963; Fried et al., 1965; Minotti et al., 1969; Jackson et al., 1976; Rao and Rains, 1976; Doddema and Telkamp, 1979; MacKown et al., 1982a; Deane-Drummond and Glass, 1983; Rufty et al., 1983; Breteler and Siegerist, 1984; Glass et al., 1985; Ingemarsson et al., 1987; Oscarson et al., 1987; Lee and Drew, 1989; Warner and Huffaker, 1989; de la Haba et al., 1990; Ayling, 1993; Aslam et al., 1994, 1997; Chaillou et al., 1994). It is evident that there are short-term effects of NH₄⁺ on NO₃ uptake that are presumed to result from the direct effects of NH₄⁺ on the plasma membrane; these short-term effects are apparent within minutes of exposure to NH₄⁺. Moreover, longer-term effects due to NH₄⁺ and/or assimilation products of NH₄⁺ are thought to operate at the transcriptional level (Glass and Siddiqi, 1995; Krapp et al., 1998; Zhuo et al., 1999).

Despite the efforts of many investigators, a lack of consensus persists concerning the mechanism(s) responsible for the short-term inhibition of NO₃ uptake by NH₄⁺; specifically, whether the NH₄⁺ effect is achieved by the direct inhibition of influx or by stimulating efflux. Although early reports suggested that NH₄⁺ enhanced NO₃ efflux (Jackson et al., 1976; Doddema and Telkamp, 1979; MacKown et al., 1982a; Deane-Drummond and Glass, 1983; Deane-Drummond, 1985, 1986), later studies using ¹³NO₃ clearly documented an inhibition of influx (Glass et al., 1985; Lee and Clarkson, 1986; Ingemarsson et al., 1987; Oscarson et al., 1987; Lee and Drew, 1989; Ayling, 1993; King et al., 1993).

The debate has recently been revived by Aslam and coworkers (1994, 1997), who concluded that the main effect of NH₄⁺ on net NO₃ uptake was through stimulation of NO₃ efflux; they discounted the significance of influx inhibition. A resolution of this controversy has proved difficult because the experiments have used different species or cultivars; more importantly, different techniques were used to determine NO₃ fluxes. Furthermore, in none of the above studies were both influx and efflux of NO₃ measured directly and simultaneously; rather, conclusions were based upon measurements of net flux or influx or, where efflux was determined, upon the use of NO₃ analogs such as ClO₃ (Deane-Drummond and Glass, 1983; Deane-Drummond, 1985, 1986) and NO₂ (Aslam et al., 1994). Glass et al. (1985) and Siddiqi et al. (1992) have questioned whether analogs of this sort are appropriate. In addition, Glass et al. (1985) and Ingemarsson et al. (1987) have argued that design features of experiments may have caused perturbations from a steady state, which might explain the sometimes large increases in NO₃ efflux that were observed in some of the above studies.

We have designed the present study to address such issues. To eliminate problems associated with the choice of plant material, we used the same barley cultivar (CM-72) used by Aslam and coworkers (1994, 1997), whose studies led to the conclusion that the short-term inhibition of NO₃ uptake by NH₄⁺ resulted exclusively from the stimulation of efflux. In this study we have determined the influx and efflux of NO₃ by using ¹³NO₃ under both steady-state and perturbation protocols, by directly measuring influx and efflux, and by estimating influx calculated from CAE. To investigate possible differential effects of NH₄⁺ on the CHATS, IHATS, and LATS for NO₃ transport, we measured NO₃ uptake at [NO₃] that characterize these transport systems and we used plants induced or uninduced for NO₃ transport. In addition, the experiments were designed to compare the effect of NH₄⁺ on NO₃ influx and efflux in plants induced for NO₃ uptake by prior exposure to either NO₃ or NO₂.

	MATERIALS AND METHODS

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Plant Growth Conditions

2

1

Nutrient Solutions

Influx Analysis

The chemical composition of the uptake, prewash, and desorption solutions was identical to the growth solution in the hydroponic tanks (see above) and contained 0.1, 1.0 or 10 mM NO₃. When NH₄⁺ was present in uptake solutions it was provided at a concentration of 1 mM. In experiments where NO₂ was used to induce NO₃ transport (King et al., 1992; Aslam et al., 1997), NO₂ was not present during loading with ¹³NO₃, but only during the induction period (24 h); it was replaced by NO₃ during ¹³NO₃ loading and flux measurement. Uninduced plants received no N during growth but were exposed to 0.1 mM NO₃ for flux determinations.

To minimize plant perturbation during experiments, a syringe was used to add tracer to the individual uptake vessels. At the start of the influx experiments, barley seedlings were transferred from the hydroponic growth tanks to prewash solutions in 1-L vessels for 5 min prior to addition of radioisotope to the uptake solutions. This protocol minimized plant perturbation and allowed the roots to equilibrate to the exact solution temperature and composition used during flux analysis. The roots were then exposed to tracer for 5 min. Immediately after loading with isotope, roots were dipped into nonlabeled solutions for 5 s to minimize the carryover of label by the root surface to the desorption solution. Roots were then desorbed for 2 min in unlabeled solution, which was otherwise chemically identical to the influx solution, to remove the ¹³NO₃ contained in the cell-wall free space. The duration of these steps was based on the half-lives of exchange of NO₃ for the root surface, the free space, and the cytoplasm as determined by efflux analysis (see below; Siddiqi et al., 1991; Kronzucker et al., 1995a, 1995b, 1995e).

We chose exposure times of 5 min for ¹³NO₃ influx, because we expected the contribution of tracer efflux from the cytoplasm to be negligible during this time (Lee and Clarkson, 1986; Siddiqi et al., 1991; Kronzucker et al., 1995a, 1995b, 1995d, and 1995e). After desorption, seedling roots were excised from the shoots; the roots were spun in a low-speed centrifuge for 30 s to remove any surface liquid; and the fresh weights of the roots and shoots were measured. The plant organs were then introduced into 20-mL scintillation vials, and a -counter (Minaxi , Auto- 5000, Packard Instruments, Meriden, CT) determined the radioactivities of the roots and shoots, measuring the 511-keV positron-electron annihilation radiation generated by the recombination of ambient electrons and ⁺ particles emitted from ¹³N. Using the specific activity (¹³N/[¹³N + ¹⁴N] cpm µmol¹) of the loading solution and the total root fresh weight of each seedling, we calculated the NO₃ fluxes and expressed them in micromoles per gram root fresh weight per hour.

In addition to direct influx determinations by ¹³N count accumulation over the 5-min loading periods (designated as _oc*), influx was also determined by CAE (_oc) and net flux was determined by N depletion over a period of up to 2 h (_net*). We repeated all experiments at least three times. Each experimental treatment consisted of three to four replicates (n  9).

CAE

View larger version (19K): [in this window] [in a new window]   Figure 2. 13NO3-efflux plot for intact seedlings of cv CM-72 maintained at 0.1 mM [NO3]o with a one-time addition (and continued presence) of 1.0 mM NH4+ during cytoplasmic efflux (at 12 min during tracer elution). Inset shows magnified cytoplasmic exchange.

View larger version (20K): [in this window] [in a new window]   Figure 3. 13NO3-efflux plot for intact seedlings of cv CM-72 maintained at 0.1 mM [NO3]o with two-time addition and subsequent withdrawal of 1.0 mM NH4+ during cytoplasmic efflux (additions at 5 and 16 min; withdrawals at 12 and 21 min during tracer elution). Inset shows magnified cytoplasmic exchange.

We maintained steady-state conditions during loading and elution. We chose the duration of the loading period on the basis of the half-lives of exchange for the cytoplasmic compartment for NO₃ in barley (compare below with Siddiqi et al., 1991). Therefore, 60 min of exposure to tracer ensured that cytoplasmic specific activity was 95% of that in the loading solution (Kronzucker et al., 1995e). After loading with ¹³N, seedlings were transferred to efflux funnels (Siddiqi et al., 1991; Kronzucker et al., 1995b) and the roots were eluted with 20-mL aliquots of nonradioactive solution after varying time intervals. Using an elution protocol lasting 22 min, these time intervals ranged from 5 s to 2 min, as described by Kronzucker et al. (1995b), except when we monitored the response of the NO₃ efflux to the NH₄⁺ addition during elution (Figs. 2 and 3); we used 1-min intervals in those cases to ensure appropriate time resolution.

Eluates from a total of 25 time intervals were collected, and the -counter (see above) determined the radioactivities of 20-mL subsamples from each eluate. After each final elution, we excised the seedling roots from the shoots, spun the roots in a low-speed centrifuge for 30 s to remove surface liquid, and determined the fresh weights of the roots and shoots. We then introduced the plant organs into 20-mL scintillation vials and determined the radioactivities of the roots and shoots as described previously for the influx experiments. We repeated the experiments three times with two replicates per experiment. We pooled the data from several experiments (n  6) to calculate means and SE. Symbols and calculation of fluxes in CAE were as follows: _co, efflux from the cytoplasmic compartment at time 0 divided by the specific activity of ¹³N in the loading solution; _net, net flux, obtained from the accumulation of ¹³N in the plants at the end of the loading period (60 min); _oc, unidirectional influx, calculated from _net + _co; _xylem, flux of ¹³N to the shoot at the end of the elution period; and _red/vac, combined flux to reduced N and the vacuole, resulting in _{net  xylem}. Calculations of half-lives of exchange and cytosolic concentrations were done as described in detail elsewhere (Lee and Clarkson, 1986; Siddiqi et al., 1991; Kronzucker et al., 1995a, 1995b, 1995c, 1995e).

	RESULTS AND DISCUSSION

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Effect of NH₄⁺ on Half-Lives of Cellular NO₃ Exchange

View larger version (18K): [in this window] [in a new window]   Figure 1. Representative semilogarithmic plots for the rate of release of 13NO3 versus time of elution for roots of intact cv CM-72 seedlings maintained under steady-state conditions of 0.1 mM [NO3]o without NH4+ () and following the addition of 1 mM NH4+ (). Plots include linear regression lines for the three phases of efflux: I, surface film; II, cell wall; and III, cytoplasm. Regression lines are dashed for the plus-NH4+ treatment and solid for the control (phases I and II overlapped). For derivation of compartmental parameters, see text.

View this table: [in this window] [in a new window]   Table I. Half-lives of NO3 exchange for the cytoplasmic compartment and cytoplasmic [NO3] ([NO3]cyt) in roots of intact cv CM-72 plants, determined by compartmental analysis Plants were exposed to and labeled at the indicated concentrations of NO3 (steady state). Uninduced plants were grown in N-free solution but were exposed to 0.1 mM NO3 during labeling and elution. NO2-induced plants were exposed to 0.1 mM NO2 for 24 h prior to labeling and elution at 0.1 mM NO3. NH4+ was present at 1.0 mM during labeling and elution in + treatments. Data are ±SE (n  6).

In general, half-life values for the cytoplasm were longer in the cv CM-72 used in the present study compared with the Klondike variety of barley used in previous studies (Siddiqi et al., 1991; Devienne et al., 1994; Kronzucker et al., 1995a, 1995b), indicating a larger relative accumulation capacity for NO₃ (at comparable fluxes) in the former variety. The addition of NH₄⁺ significantly affected cytoplasmic exchange kinetics in NO₂-induced plants as well as in plants grown at 0.1 and at 1 mM [NO₃]_o: Half-lives increased in all of these cases (Fig. 1), whereas they remained unaffected in uninduced plants and at 10 mM [NO₃]_o (Table I). These differences in half-life values are direct reflections of differences in flux partitioning, as discussed below.

Effect of NH₄⁺ on NO₃ Uptake and Subcellular N-Flux Partitioning

View this table: [in this window] [in a new window]   Table II. Component fluxes of NO3 as determined by compartmental analysis Barley plants were exposed to and labeled at the indicated concentrations of NO3 for 24 h prior to experiments. Uninduced plants were grown in N-free solution but exposed to 0.1 mM NO3 during labeling and elution. "NO2-induced" plants were exposed to 0.1 mM NO2 for 24 h prior to labeling and elution at 0.1 mM NO3. NH4+ was present at 1.0 mM during labeling and elution in + treatments. Data are ±SE (n  6).

View this table: [in this window] [in a new window]   Table III. Estimates of NO3 influx and net flux into roots of intact cv CM-72 plants by methods independent of compartmental analysis Plants were exposed to, and fluxes were measured at, the indicated concentrations of NO3 (steady state). Uninduced plants were grown in N-free solution prior to flux measurement at 0.1 mM NO3. NO2-induced plants were exposed to 0.1 mM NO2 for 24 h prior to flux measurement at 0.1 mM NO3. NH4+ was present at 1.0 mM during uptake in + treatments. Data are ±SE (n  9).

NH₄⁺ had a distinct and more potent effect on NO₃ uptake in the high-affinity transport range (i.e. on the IHATS), which is evident below 1 mM [NO₃]_o, than in the range of the LATS, which operates at [NO₃]_o  1 mM (Siddiqi et al., 1989; King et al., 1993; Kronzucker et al., 1995d). This provides additional support for the argument that high- and low-affinity transport systems are biochemically distinct modes of transport (for review, see Glass and Siddiqi, 1995). NO₂-induced plants were substantially less sensitive to NH₄⁺ inhibition of NO₃ uptake than NO₃-induced plants. Aslam and coworkers (1994, 1997) used plants induced by and "labeled" with NO₂ as model systems for NO₃-induced plants. Although NO₂ and NO₃ have been shown to act competitively at the level of uptake (Aslam et al., 1992; Siddiqi et al., 1992), the present findings suggest that NO₂ may not serve as a satisfactory quantitative analog for NO₃. This is consistent with the finding by Siddiqi et al. (1992) that NO₂ was not capable of inducing NO₃ reductase activity in barley. Aslam et al. (1987, 1993, 1997), however, reached the opposite conclusion.

The effect of 1 mM NH₄⁺ on ¹³NO₃ influx followed the same pattern (with one exception) as that observed for net uptake, i.e. the extent of inhibition declined with increasing [NO₃]_o, with 33% inhibition at 0.1 mM, 23% at 1.0 mM, and no effect at 10 mM. Inhibition was much smaller in NO₂-induced plants (11.6%). This was true when influx was determined by CAE and count accumulation after a 5-min exposure to tracer (Tables II and III). The one exception was that influx in uninduced plants (determined by CAE) was unaffected by NH₄⁺, in contrast to the situation for net uptake by uninduced plants. We interpret this result to indicate that, like LATS, the NO₃ influx via CHATS (King et al., 1992; Kronzucker et al., 1995d) is unaffected by NH₄⁺. That exposure to NH₄⁺ diminished net uptake in uninduced plants indicates an effect on efflux of NO₃ in uninduced plants.

We measured CAE on uninduced plants by exposing roots to 0.1 mM [NO₃]_o for the duration of the efflux analyses (the labeling and elution procedures lasted 82 min); by this time, the physiological induction of the IHATS and NO₃ reductase would be relatively small (Friemann et al., 1992; Glass and Siddiqi, 1995; Kronzucker et al., 1995a, 1995b). In contrast to its lack of effect on constitutive influx, NH₄⁺ stimulated NO₃ efflux by as much as 86% (Table II) in uninduced plants. Although not measured directly, efflux was probably even larger during the shorter 5-min exposures to NH₄⁺ (see discussion of short-term efflux enhancements below), which may explain the apparent depression of influx that we saw in the short-term determinations after the addition of NH₄⁺ (Table III), but not in the CAE determinations (Table II).

Efflux stimulation was also substantial in NO₂-induced plants (approximately 62%), whereas in plants grown at 0.1 mM [NO₃]_o, efflux was enhanced by less than 50% and no efflux stimulation at all was seen in plants grown at 1.0 and 10 mM [NO₃]_o (Table II; Fig. 1). NO₃ efflux (expressed as a percentage of influx) also increased as [NO₃]_o was raised from 0.1 to 10 mM even in the absence of NH₄⁺ (Table II), as previously reported (Siddiqi et al., 1991; Kronzucker et al., 1995b; Volk, 1997). Our results confirm a distinct difference between inducible high-affinity transport and low-affinity transport; they also confirm the fact that NO₂ induction of NO₃ transport cannot be used as a quantitative model for NO₃ induction and provision under steady-state conditions. The latter point is particularly important, because experiments on plants pretreated in this way have led to the conclusion that the NH₄⁺ inhibition of net NO₃ uptake results exclusively from the effects on NO₃ efflux and that influx is unaffected (Aslam et al., 1994). Furthermore, the relative effect of NH₄⁺ was high in the present study (>50% stimulation) because efflux is typically low under control conditions, and the absolute contribution to reduced net uptake was still small in comparison with the contribution arising from reduced influx.

Our experiments show that in NO₂-induced plants, the combined flux of ¹³NO₃ and ¹³N-assimilation products to the shoot (*_xylem) was unaffected by the addition of NH₄⁺, whereas it was reduced in all other treatments (Table II). Also, *_xylem in NO₂-induced plants was substantially lower than in NO₃-induced plants, and approximated that of plants induced at 0.1 mM [NO₃]_o after the application of 1 mM NH₄⁺. Because significant suppression of NO₃ reductase activity by NH₄⁺ is well documented (MacKown et al., 1982a, 1982b; Breteler and Siegerist, 1984; Pan et al., 1985; de la Haba et al., 1990; Aslam et al., 1997; however, see Oaks et al., 1979), *_xylem under these conditions will be mostly in the form of ¹³NO₃ after NH₄⁺ addition. Therefore, the lack of an effect of NH₄⁺ on *_xylem in NO₂-induced plants (measured at 0.1 mM [NO₃]_o) appears to support the biochemically based conclusion arrived at by Siddiqi et al. (1992) and King et al. (1993) that NO₃ reductase activity is not induced to any significant extent by NO₂ in barley roots, in contrast to its full induction by NO₃. Claims to the contrary have been made by Aslam et al. (1987, 1993; see Glass and Siddiqi, 1995).

Our CAE analyses do not allow a separation of the reduced and unreduced components of the xylary ¹³N-translocation term, nor do they permit separation of the components of _red/vac, namely the biochemical N flux to reduced N, and the N flux to the vacuole (Lee and Clarkson, 1986; Siddiqi et al., 1991; Kronzucker et al., 1995a, 1995b). However, given that the cytoplasmic [NO₃] values in NO₂- and NO₃-induced plants were indistinguishable after ¹³N loading (Table I), and thus similar values of *_xylem for NO₃ are to be expected, we must conclude that NO₂ induction leads to a relatively greater stimulation of the NO₃ flux to the vacuole (in both the control and NH₄⁺-treated plants). The reduction in _red/vac after NH₄⁺ addition to NO₂-induced plants (Table II) appears to be a direct result of the inhibition of influx and the stimulation of efflux rather than the effect upon NO₃ reductase activity.

The clear differences in flux partitioning between NO₃- and NO₂-induced plants revealed by CAE lead us to caution against the use of NO₂ as an "analog" for NO₃. Similar concerns regarding the use of ³⁶ClO₃ as an analog for NO₃ were expressed in earlier work (Deane-Drummond and Glass, 1983; Dean-Drummond, 1985, 1986), although the basis of the failure to faithfully trace NO₃ in the case of ClO₃ was more straightforward (Glass et al., 1985; Lee and Drew, 1989; Siddiqi et al., 1992; Aslam et al., 1994; Glass and Siddiqi, 1995). Doddema and Telkamp (1979) also observed a significant rise in NO₃ efflux upon the addition of NH₄⁺; however, this response was transient and restricted to the perturbational condition (see below). Our present analyses demonstrate that an enhancement of the efflux component makes only a small contribution to the reduction of net uptake, whereas the principal effect of NH₄⁺ on NO₃ uptake comes through the inhibition of influx (except in uninduced plants and in plants pretreated with NO₂, where the efflux contribution is magnified).

Kinetics of the Response

The rapidity of the response to NH₄⁺ on both the influx and efflux components of NO₃ uptake provides a compelling argument that the NH₄⁺ effect occurs directly at the plasma membrane. Lee and Drew (1989) demonstrated a logarithmic relationship between the inhibition of NO₃ influx by NH₄⁺ and external [NH₄⁺], which led to the suggestion that membrane depolarization by NH₄⁺ may inhibit the NO₃/2H⁺ cotransport system due to effects on the proton motive force (Ullrich et al., 1984; Ayling, 1993). However, the provision of K⁺, which also depolarizes the plasma membrane to an extent similar to that of NH₄⁺, fails to inhibit NO₃ uptake (Glass and Siddiqi, 1995; Wang et al., 1996), arguing for a more specific effect of NH₄⁺. Given the rapidity of the response, the inhibition probably occurs allosterically, rather than by involving the products of NO₃ reduction and N assimilation, or possibly the effects of transcription or translation.

Similar conclusions have been reached by others (Deane-Drummond and Glass, 1983; Ingemarsson et al., 1987; Lee and Drew, 1989; Warner and Huffaker, 1989; Aslam et al., 1994). In agreement with de la Haba et al. (1990) and Aslam et al. (1994), we found that pretreatment of barley and rice plants with the Gln synthetase inhibitor Met sulfoximine for 6 h at 1 mM did not alleviate the inhibitory effect exerted by externally added NH₄⁺ (data not shown). Thus it seems unlikely that N assimilates downstream of NH₄⁺ are involved in the inhibition of NO₃ uptake. We must stress that these conclusions apply only to the rapid effects of NH₄⁺ on NO₃ uptake; some (Krapp et al., 1998; Zhuo et al., 1999) have suggested that there may be long-term effects of Gln and other amino acids at the transcription level.

	Summary

Our analyses provide evidence that the inhibitory effect of NH₄⁺ upon NO₃ uptake is mediated primarily by inhibiting NO₃ influx, with only a small contribution from the enhancement of NO₃ efflux, which: (a) is both transient and reversible, (b) is associated with a large efflux only in uninduced plants and plants induced by NO₂ (i.e. under conditions where influx is very low), (c) is dependent on [NO₃]_o, (d) is strong for IHATS but small for CHATS and LATS, (e) occurs directly at the plasma membrane (i.e. it does not involve NO₃ reduction or N-assimilation products in the short term, although it may in the long-term); and (f) cannot be modeled quantitatively by the use of NO₂ as an analog of NO₃.

	FOOTNOTES

   Received September 18, 1998; accepted February 10, 1999.

	ABBREVIATIONS

Abbreviations: CAE, compartmental analysis by efflux. CHATS, constitutive high-affinity transport system. IHATS, inducible high-affinity transport system. LATS, low-affinity transport system. [NO₃]_o, external [NO₃] , ionic (N) flux (component fluxes denoted by subscripts, as indicated in text) .

	ACKNOWLEDGMENTS

We thank D.T. Britto, M. Okamoto, D. Zhuo, and the staff at the Tri-University Meson Facility particle accelerator for technical help and discussions. We also thank Prof. R.C. Huffaker for generously providing us with cv CM-72 seeds.

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