TRANSLOCATION OF C14-LABELED PHOTOSYNTHATE IN NODULATED LEGUMES AS INFLUENCED BY NITRATE NITROGEN

Translocation of C¹⁴-labeled photosynthate was studied in nodulated pea and subterranean-clover plants which were grown either continuously without combined nitrogen or exposed to NaNO₃ in the nutrient solution for five days prior to C¹⁴O₂ assimilation. Supplying combined nitrogen decreased the proportion of photosynthate translocated to nodules with a corresponding increase in the proportion going to roots. Nodules on plants grown without combined nitrogen had a higher radioactivity than nodules on plants treated with sodium nitrate.     

Farnesol-like antitranspirant activity and stomatal behaviour in maize and Sorghum lines of differing drought tolerance

Abstract Potted seedlings of four lines of maize and Sorghum of differing drought tolerance were subjected to a single soil drying cycle and were only rewatered when the plants showed the first signs of wilting. Other plants remained well-watered throughout the experimental period. As plant water potentials decreased in the unwatered plants of three of the lines investigated (Sorghum Piper and M35-1, V-4146 and maize Farz 27), endogenous levels of farnesol-like antitranspirants increased. Closure of stomata correlated well with the increase in endogenous antitranspirant. In the fourth line (Sorghum M35-1, V-4184), stomata did not close as the level of plant water stress increased, although leaf diffusion resistance of even the well-watered plants of this line was quite high. In this line, there was no consistent relationship between plant water stress and antitranspirant level or between stomatal behaviour and antitranspirant level. The involvement of farnesol-like antitranspirants in the control of stomatal behaviour in water-stressed plants is discussed.     

Effects of SO(2) and O(3) on Allocation of C-Labeled Photosynthate in Phaseolus vulgaris

A series of laboratory exposures of two varieties of bush bean (Phaseolus vulgaris L., var 274 and var 290) was conducted to determine the sensitivity of [¹⁴C]photosynthate allocation patterns to alteration by SO₂ and O₃. Experiments with the pollution-resistant 274 variety demonstrated short-term changes in both ¹⁴C and biomass allocation to roots of ¹⁴CO₂-labeled plants but no significant effect on yield by up to 40 hours of exposure to SO₂ at 0.50 microliters per liter or 4 hours of O₃ at 0.40 microliters per liter. Subsequent experiments with the more sensitive 290 variety demonstrated significant alteration of photosynthesis, translocation, and partitioning of photosynthate between plant parts including developing pods. Significant increases in foliar retention of photosynthate (+40%) occurred after 8 hours of exposure to SO₂ at 0.75 microliters per liter (6.0 microliters per liter-hour) and 11 hours of exposure to O₃ at 0.30 microliters per liter-hour (3.3 microliters-hours). Time series sampling of labeled tissues after ¹⁴CO₂ uptake showed that the disruption of translocation patterns was persistent for at least 1 week after exposures ceased. Subsequent longer-term exposures at lower concentrations of both O₃ (0.0, 0.10, 0.15, and 0.20 microliters per liter) and SO₂ (0.0, 0.20, and 0.40 microliters per liter) demonstrated that O₃ more effectively altered allocation than SO₂, that primary leaves were generally more sensitive than trifoliates, and that responses of trifoliate leaves varied with plant growth stage. Altered rates of allocation of photosynthate by leaves were generally associated with alterations of similar magnitude and opposite direction in developing pods. Collectively, these experiments suggest that allocation patterns can provide sensitive indices of incipient growth responses of pollution-stressed vegetation.     

Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure

Chitosan (CHT) is a natural compound able to activate the plant own defence machinery against pathogen attacks and to reduce both transpiration and stomatal opening when applied as foliar spray. The data here reported show that CHT-induced antitranspirant activity in bean plants is mediated by ABA, whose level raised over threefold in treated leaves, 24 h after foliar spraying. This is thought to induce partial stomatal closure via a H₂O₂-mediated process, as confirmed by scanning electron microscopy (SEM) and histo-cytochemistry, and, in turn, a decrease of stomatal conductance to water vapor (G_w) and transpiration rate (E), assessed by gas exchange measurements. The relatively high internal CO₂ concentration (C_i) values, suggest the occurrence of a slight decrease in carboxylation efficiency after CHT treatment, which however did not prevail over stomatal limitations. The intrinsic water use efficiency (WUE_i) of CHT treated plants was not statistically different from controls and the maximal photochemical efficiency (F_v/F_m) of PSII was not affected. Moreover, CHT determined a stimulation of the xanthophyll cycle towards de-epoxidation state. On the whole, these results, besides confirming the effectiveness of CHT in reducing plant transpiration, prove that the mechanism underlying this activity differs from that showed by the commercial antitranspirant Vapor Gard^® (VP). In fact, the efficacy of the latter is based on the formation of a thin antitranspirant film over the leaf and not on the reduction of stomatal opening. Finally, suggestions for possible use of the two antitranspirants in different environmental conditions are discussed.     

Partitioning of CO2 Fixation in the Colonial Cyanobacterium Microcystis aeruginosa: Mechanism Promoting Formation of Surface Scums

Constraints on inorganic carbon (C_i) availability stimulated buoyancy in natural, photosynthetically active populations of the colonial blue-green alga (cyanobacterium) Microcystis aeruginosa. In nonmixed eutrophic river water and cultures, O₂ evolution determinations indicated C_i limitation of photosynthesis, which was overcome either by CO₂ additions to the aqueous phase or by exposure of buoyant colonies to atmospheric CO₂. Microautoradiographs of M. aeruginosa colonies revealed partitioning of ¹⁴CO₂ fixation and photosynthate accumulation between peripheral and internal cells, particularly in large colonies. When illuminated colonies were suspended in the aqueous phase, peripheral cells accounted for at least 90% of the ¹⁴CO₂ assimilation, whereas internal cells remained unlabeled. However, when ¹⁴CO₂ was allowed to diffuse into colonies 15 min before illumination, a more uniform distribution of labeling was observed. Resultant differences in labeling patterns were most likely due to peripheral cells more exclusively utilizing CO₂ when ambient C_i concentrations were low. Among colonies located at the air-water interface, internal cells showed an increased share of photosynthate production when atmospheric ¹⁴CO₂ was supplied. This indicated that C_i transport was restricted in large colonies below the water surface, forcing internal cells to maintain a high degree of buoyancy, thus promoting the formation of surface scums. At the surface, C_i restrictions were alleviated. Accordingly, scum formation appears to have an ecological function, allowing cyanobacteria access to atmospheric CO₂ when the C_i concentration is growth limiting in the water column.     

Effects of film-forming and silicone antitranspirants on four herbaceous plant species

Summary Effectiveness of antitranspirants in reducing transpiration was studied in the laboratory in an attempt to evaluate their potential for conservation and reallocation of soil moisture. Film-forming antitranspirants caused about a 50% reduction of transpiration and photosynthesis in all species. Effectiveness decayed slowly over 10–14 days. Effectiveness of the silicone antitranspirant was very short-lived. Water use efficiency was slightly improved following film-forming antitranspirant treatment of three species. Mixing antitranspirants altered their effects.Research supported by the Bureau of Land Management in Cooperation with the Office of Water Research and Technology, United States Department of the Interior (OWRT Grant B-099)     

Assimilate Conversion in Potato Tubers in Relation to Starch Deposition and Cell Growth

The activity of enzymes involved in the conversion of sucrose to starch together with the distribution of ¹⁴C-labelled photosynthate and ⁴C-sucrose was studied in potato tubers showing a range of growth rates and growth patterns. Within a particular tuber the uptake of ¹⁴C from labelled photosynthate and the conversion to ethanol-insoluble ¹⁴C was greatest in the apical tissue where both the rate of production of new storage cells and starch synthesis were likely to be greatest. Uptake and conversion of ¹⁴C was lowest in the older tissue of the tuber base. Pre-treatment of tubers with gibberellic acid reduced the total input of ¹⁴C from labelled photosynthate, reversed the gradient in ¹⁴C uptake between apical and basal tuber tissue, increased the amount of ¹⁴C per g fresh weight in the basal tissue and decreased the conversion of labelled sugars to starch. For tubers with different growth rates both the total uptake of ¹⁴C from labelled photosynthate and the ratio ethanol-insoluble ¹⁴C/ethanol-soluble ¹⁴C appeared to be correlated with growth rate. In contrast when tubers were fed directly with ¹⁴C-sucrose via the tuber surface, total uptake was independent of growth rate but the correlation between growth rate and the ratio ethanol-insoluble ¹⁴C/ethanol-soluble ¹⁴C persisted. Within a particular tuber there was a decreasing gradient in sucrose synthetase activity between youngest tissue of the tuber apex and the older tissue at the tuber base but there was no clear correlation between mean enzyme activity and tuber growth rate. ADPG-pyrophosphorylase and the ratio ADPG-pyrophosphorylase/starch phosphorylase showed some correlation with tuber growth rate. Starch synthase, starch phosphorylase and UDPG-pyro-phosphorylase activities per g fresh weight of tuber tissue appeared to be relatively constant. The results suggest that the transport of sugar from the phloem sieve tubes to the tuber storage parenchyma cells, in particular the phloem unloading step, and the conversion of sugar into starch are subject to separate regulation in the potato tuber.     

MOBILIZATION BY PINUS RESINOSA CONES AND SHOOTS OF C14-PHOTOSYNTHATE FROM NEEDLES OF DIFFERENT AGES

Current-year, 1-year-old, 2-year-old, and 3-year-old needles of 25- to 30-year-old Pinus resinosa trees were separately exposed to C¹⁴O₂ at various times during the growing season. Currently produced C¹⁴-photosynthate was preferentially mobilized in the following order: second-year cones > current needles > current internodes > first-year conelets. A changing seasonal pattern was shown in sources of current photosynthate for growth of cones and shoots. One-year-old needles were the major source of current photosynthate for growth of both cones and shoot internodes. During June the 2- and 3-year-old needles contributed appreciable amounts of current photosynthate to both cones and developing shoots, but after late June their contribution was slight. The supply of carbohydrates to all tissues, except 2nd-year cones, from the three age classes of old needles declined late in the season as tissues mobilized increasingly more carbohydrates from current-year needles. Nevertheless, the bulk of the C¹⁴-photosynthate produced by current-year needles was retained by them. The preferential mobilization of carbohydrates by reproductive tissues over vegetative tissues is emphasized as is the importance of both reserve and currently produced carbohydrate for growth of various tissues.     

UDP-glucose: α-D-galactose-1-phosphate uridylyltransferase activity in cultured human fibroblasts

Galactose-1-phosphate uridyl transferase activity of normal, heterozygous and galactosemic strains is determined throughout the culture cycle of human fibroblasts using a new direct method of assay. The enzyme activities of high-density, stationary-phase cultures define three nonoverlapping classes, which correspond to the genotypes of the donors. During rapid growth, however, galactosemic strains show near-normal transferase activity. The incorporation of ¹⁴C from ¹⁴C₁-galactose by living cells is measured. While heterozygous strains do not appear to differ from normal controls, homozygous mutant cells incorporate ¹⁴C at about one-half the normal rate throughout the culture cycle. Variables affecting the assay are investigated and the implications of our results for further genetic studies of mutations affecting transferase are discussed.Paper # 1105 from the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin. This work was supported by the National Institutes of Health (Grants # GM-08217, # GM-398, and # GM-06983).     

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

A method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of ¹⁴CO₂ fixation both in the light (45 μmol of CO₂ fixed mg^?1 Chl h^?1) and in the dark (20 μmol of CO₂ fixed mg^?1 Chl h^?1). The protoplasts also showed O₂ evolution (40 μmol of O₂ evolved mg^?1 Chl h^?1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O₂ evolution. Analyses of early photosynthetic products in the light showed the formation of both C₃ and C₄ acids. Aspartate was found to be a predominant photosynthate.     

Can a limitation in pholem supply to nodules account for the inhibitory effect of nitrate on nitrogenase activity in soybean?

Within 48 h of exposure of nodulated soybean [Glycine max (L.) Merr. cv. Harosoy 63 x Bradyrhizobium japonicum USDA 16] to 10 mM NO₃, significant decreases were observed in nodule-specific nitrogenase (EC 1.7.99.2) activity and CO₂ evolution and in the proportion of [¹⁴C]-labeled photosynthate partitioned to nodule biomass and respiration. These trends continued over the subsequent 3 days of the study period. Concomitant with these events was an 137% increase in the relative growth rate of the whole plant and a cessation in nodule growth. Although the concentration of total soluble sugar in nodules was not affected by NO₃ treatment, the concentration of starch declined to 13% of the control level after 2 days exposure to NO₃^?. In contrast to the effects of NO₃^?, nodules in which nitrogenase activity was partially inhibited by a 30 min exposure to 100% O₂, showed a 52% increase over control in the starch pool over a 72 h period. The results were compared with recent studies of NO₃^? inhibition of nitrogenase activity in legumes, and in contrast to these studies it was concluded that the inhibitory effects of NO₃^? could be accounted for by alterations in photosynthate partitioning to nodules. A hypothesis is proposed which attempts to account for the recent observation (J. K. Vessey, K. B. Walsh, and D. B. Layzell 1988. Physiol. Plant. 73: 113–121) that nitrogenase activity in phloem-limited and nitrate-inhibited nodules is limited by O₂ diffusion. This hypothesis separates the concepts of photosynthate partitioning and phloem supply from that of carbohydrate deprivation and related effects on the size of the carbohydrate pools in nodules.     

The uptake of carbon dioxide by plant roots

Summary The uptake of C¹⁴O₂ by the roots of intact tomato plants from solution containing Na₂C¹⁴O₃ was studied at different light intensities as well as in darkness.Where plants had previously been starved for CO₂ for 12 hours, a higher rate of C¹⁴ uptake was observed than with plants which had been transferred directly from the soil to the radioactive solution.In general, the C¹⁴ content of the roots was slightly higher than that of the shoots. At light intensities under the compensation point and in darkness the C¹⁴ content of the shoots relative to the roots decreased. This was accompanied by release of C¹⁴O₂ during respiration, indicating that the absorbed C¹⁴ was readily translocated upwards and released as C¹⁴O₂ under these conditions. At light intensities above the compensation point no C¹⁴O₂ was released.     

STUDIES ON STRIGA SENEGALENSIS II. TRANSLOCATION OF C14-LABELLED PHOTOSYNTHATE,UREA-C14 AND SULPHUR-35 BETWEEN HOST AND PARASITE

Using a tracer technique, the nutritional relationship between Striga senegalensis and its host (Sorghum vulgare) has been studied. Preliminary trials with aqueous eosin dye indicated that a mechanism exists for the passage of watery solutions from host to parasite but not vice versa. Use of tracers like C¹⁴O₂, urea-C¹⁴ and sulphur-35 confirmed that minerals as well as organic compounds are translocated from host to parasite. However, when these tracers were applied to the parasites, translocation of labelled products into the host was insignificant. When C¹⁴O₂ was used to label the photosynthate in Sorghum, the bulk of activity appeared in sucrose, glucose and fructose, part of which was presumably obtained by the parasite. The photosynthetic ability of the green tissues of Striga was confirmed. Thus the damaging effect of Striga on the host appears to be due to removal of considerable quantities of water, minerals and organic compounds from the latter. The pattern of translocation of photosynthates and minerals from host to parasite suggests a possible application of selective, systemic weed-killers by aerial spray on the host leaves for control of the parasite.     

Pathway and sink activity for photosynthate translocation in <Emphasis Type="Italic">Pisolithus</Emphasis> extraradical mycelium of ectomycorrhizal <Emphasis Type="Italic">Pinus thunbergii</Emphasis> seedlings

The purpose of this study was to identify the pathway and sink activity of photosynthate translocation in the extraradical mycelium (ERM) of a Pisolithus isolate. We labelled ectomycorrhizal (ECM) Pinus thunbergii seedlings with ¹⁴CO₂ and followed ¹⁴C distribution within the ERM by autoradiography. ¹⁴C photosynthate translocation in the ERM resulted in ¹⁴C distribution in rhizomorphs throughout the ERM, with ¹⁴C accumulation at the front. When most radial mycelial connections between ECM root tips and the ERM front were cut, the whole allocation of ¹⁴C photosynthates to the ERM was reduced. However, the overall pattern of ¹⁴C distribution in the ERM was maintained even in regions immediately above and below the cut, with no local ¹⁴C depletion or accumulation. We inferred from this result that every portion in the ERM has a significant sink activity and a definite sink capacity for photosynthates and that photosynthates detour the cut and reach throughout the ERM by translocation in every direction. Next, we prepared paired ECM seedlings, ERMs of which had been connected with each other by hyphal fusion, alongside, labelled the left seedling with ¹⁴CO₂, and shaded none, one or both of them. ¹⁴C photosynthates were acropetally and basipetally translocated from the left ERM to ECM root tips of the right seedling through rhizomorphs in the left and right ERMs, respectively. With the left seedling illuminated, ¹⁴C translocation from the left to the right ERM increased by shading the right seedling. This result suggests that reduced photosynthate transfer from the host to its ERM increased sink activity of the ERM.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

The Binding of Indole-3-acetic Acid and 3-Methyleneoxindole to Plant Macromolecules

Homogenates of pea (Pisum sativum L., var. Alaska) seedlings exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, an oxidation product of indole-3-acetic acid, were extracted with phenol. In both cases 90% of the bound radioactivity was found associated with the protein fraction and 10% with the water-soluble, ethanol-insoluble fraction. The binding of radioactivity from ¹⁴C-indole-3-acetic acid is greatly reduced by the addition of unlabeled 3-methyleneoxindole as well as by chlorogenic acid, an inhibitor of the oxidation of indole-3-acetic acid to 3-methyleneoxindole. Chlorogenic acid does not inhibit the binding of ¹⁴C-3-methyleneoxindole. The labeled protein and water-soluble, ethanol-insoluble fractions of the phenol extract were treated with an excess of 2-mercaptoethanol. Independently of whether the seedlings had been exposed to ¹⁴C-indole-3-acetic acid or ¹⁴C-3-methyleneoxindole, the radioactivity was recovered from both fractions in the form of a 2-mercaptoethanol-3-methyleneoxindole adduct. These findings indicate that 3-methyleneoxindole is an intermediate in the binding of indole-3-acetic acid to macromolecules.     

Decomposition of diethylstilboestrol in soil

Summary The rate of decomposition of DES-monoethyl-1-C¹⁴ in soil was followed by measurement of C¹⁴O₂ released. From 1.6 to 16% of the added C¹⁴ was recovered as C¹⁴O₂ during 3 months. After six months as much as 12 to 28 per cent was released as C¹⁴O₂.Determination of C¹⁴ in the soil samples after the experiments showed that the amount extractable with benzene decreased to a greater extent than would be expected from the production of C¹⁴O₂ and that the amount extractable with water was increased when compared with that found shortly after the addition of DES.Addition of large amounts of DES (8%) did not inhibit the CO₂ production from the soil.Experiments with -sterilized soil indicated that enzymes present in the soil are able to attack DES.     

C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions

The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of ¹⁴C₂H₄ into the tissue and (b) the conversion of ¹⁴C₂H₄ to ¹⁴CO₂, was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to ¹⁴C₂H₄ markedly reduced both a and b. Increasing the ¹⁴C₂H₄ concentration from 0.14 to over 100 μl/l progressively increased the rate of a and b with tissue incorporation being greater than ¹⁴C₂H₄ to ¹⁴CO₂ conversion only below 0.3 μl/l ¹⁴C₂H₄. Reduction of the O₂ concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO₂ (5%) severely inhibited ¹⁴C₂H₄ to ¹⁴CO₂ conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during ¹⁴C₂H₄ treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b.     

C14 distribution in photosynthate of tomato as influenced by substrate copper and molybdenum level and nitrogen source

Summary The influence of nitrogen source and micronutrient treatment on the apportionment of C¹⁴ in alcohol extracts was studied in tomato. Plants were exposed at several stages of maturity to C¹⁴O₂ and the ethanol-soluble photosynthate fractionated into sugars, organic acids, and amino acids.Ammonium-treated plants had low levels of organic acids at all stages of maturity, suggesting a rapid conversion to amino acid residues. However, the addition of copper in the absence of molybdenum resulted in increased labelling of the organic acid fraction under nitrate nutrition. The amino acid fraction was reduced at some stages of growth under these conditions. This may indicate that copper interferes with the role of molybdenum in the enzymatic reduction of nitrate.Measurement of the total quantity of some individual free amino acids indicated more efficient utilization of these for protein synthesis in the presence of copper. On the other hand, assay of the C¹⁴-activity of individual free amino acids showed that degree of label bore little relation to micro-nutrient treatment.Plant Science Department and Botany Department, University of Connecticut. Scientific Contribution No. 116, Agricultural Experiment Station, University of Connecticut, Storrs. This paper is based on a portion of the senior author's Ph. D. thesis (University of Connecticut).     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.