Effects of Methyl Jasmonate on Anthocyanin Accumulation, Ethylene Production, and CO2 Evolution in Uncooled and Cooled Tulip Bulbs

Effects of methyl jasmonate (JA-Me) on anthocyanin accumulation, ethylene production, and CO₂ evolution in uncooled and cooled tulips (Tulipa gesneriana L. cvs. Apeldoorn and Gudoshnik) were studied. JA-Me stimulated anthocyanin accumulation in stems and leaves from uncooled and cooled bulbs of both cultivars. The highest level of anthocyanin accumulation was observed in leaves from cooled bulbs treated with 200 μL/liter JA-Me. In sprouting bulbs treated with 100 μL/liter and higher concentrations of JA-Me, the ethylene production began to increase at 3 days after treatment, being extremely greater in uncooled bulbs than in cooled ones. JA-Me also stimulated CO₂ evolution in both cultivars, depending on its concentrations. CO₂ evolution in sprouting bulbs was not affected by cooling treatment. These results suggest that anthocyanin accumulation by JA-Me in tulip leaves is not related to ethylene production stimulated by JA-Me. Received October 10, 1997; accepted November 17, 1997     

Growth and tissue senescence inPrunus avium shoots grownin vitro at different CO3/O2 ratios

Summary The rate of metabolism and biosynthetic processes makein vitro cultures very sensitive to environmental changes, and therefore subject to physiological and morphological alterations leading to senescence in the short term. The effect of three different calibrated atmospheric compositions were studied duringin vitro culture ofPrunus avium shoots. At 0.034% CO₂-21% O₂ (vol/vol), which stimulate the natural atmosphere, the highest growth rate and chlorophyll content were recorded. When grown at 0.09% CO₂-8% O₂ (vol/vol), a favorable condition for photosynthesis and growth, cultures showed a higher percentage of dry matter and elevated ethylene production, but total chlorophyll was lower. These shoots were also highly lignified and fibrous with red pigmentation along the leaves and stems. At 0% CO₂-21% O₂ (vol/vol), in contrast, growth and ethylene formation were inhibited; chlorophyll content was lowest in comparison with the other two environmental conditions, but regreening of tissues was observed after the first half of the culture period. Senescence symptoms, as indicated by decreased chlorophyll, appeared after about 18 d of culture for tissues grown in CO₂-containing atmospheres. These experiments provided evidence that in CO₂-enriched cultures biomass production steadily increased even when chlorophyll decreased. A possible role of CO₂ in promoting tissue-senescence through activation of photooxidative events and ethylene synthesis is discussed.     

Growth and nitrogen fixation inAlnus glutinosa (L.) Gaertn. under carbon dioxide enrichment of the root atmosphere

The effects of aeration of the N-free rooting medium with elevated CO₂ on (a) acetylene reduction by perlite-grown plants and (b) N₂-fixation and long-term growth of nutrient solution-grown plants were determined for nodulatedAlnus glutinosa (L.) Gaertn. In the former experiments, roots of intact plants were incubated in acetylene in air in darkened glass jars for 3 hr, followed by a further 3 hr incubation period in air enriched with CO₂ (0–5%). During incubation, the CO₂ content of the jars increased by 0.17% per hour due to respiration of the root system, so that the CO₂ content at 3 hr was 0.5%. Additional enrichment of the rooting medium gas-phase with CO₂ equivalent to 1.1% and 1.75% CO₂ of the gas volume significantly increased nitrogenase activity (ethylene production) by 55% and 50% respectively, while enrichment with greater than 2.5% CO₂ decreased activity. In contrast, ethylene production by control plants, where CO₂ was not added to the assay jars, decreased by 8% over the assay period. In long-term growth experiments, nodulated roots of intactAlnus glutinosa plants were sealed into jars containing N-free nutrient solution (pH 6.3) and aerated with air, or air containing elevated levels of CO₂ (1.5% and 5%). Comparison of the appearance of CO₂-treated with air treated plants suggested that 1.5% CO₂ stimulated plant growth. However, at harvest after 5 or 6 weeks variability between plants masked the significance of differences in plant dry weight. A significant increase of 33% in total nitrogen of plants aerated with 1.5% CO₂, compared with air-treated plants, was demonstrated, broadly in line with the short-term increase in acetylene reducing activity observed following incubations with similar CO₂ concentrations. Shoot dry weight was not affected significantly by long-term exposure to 5% CO₂, the main effect on growth being a 20% reduction in dry weight of the root system, possibly through inhibition of root system respiration. However, in contrast to the inhibitory effects of high CO₂ on acetylene reduction there was no significant effect on the amounts of N₂ fixed.     

Interactions between Ethylene, CO(2), and ABA on GA(3)-Induced Amylase Synthesis in Barley Aleurone Tissue

Gibberellic acid-induced synthesis and release of α-amylase in barley aleurone tissue was inhibited by abscisic acid. This inhibition was relieved by simultaneous application of ethylene ranging in concentration from 0.1 to 100 microliters per liter. When CO₂ was applied, it eliminated the effect of 0.1 microliter per liter ethylene and reimposed the abscisic acid inhibition. All concentrations of CO₂ tested from 400 to 10⁵ microliters per liter counteracted the effect of 0.1 microliter per liter ethylene, but had no observable effect on any higher concentration of ethylene. The results indicate that some processes necessary for embryo growth may be subject to regulation by ethylene and carbon dioxide at naturally occurring concentrations of the gases.     

Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism

The relationship between ethylene action and metabolism was investigated in the etiolated pea seedling (Pisum sativum L. cv. Alaska) by inhibiting ethylene action with Ag⁺, high CO₂, and low O₂ and then determining if ethylene metabolism was inhibited in a similar manner. Ag⁺ (100 milligrams per liter) was clearly the most potent antiethylene treatment. Ag⁺ pretreatment inhibited the growth retarding action of 0.2 microliters per liter ethylene by 48% and it also inhibited the incorporation of 0.2 microliters per liter ¹⁴C₂H₄ into pea tips by the same amount. As the ethylene concentration was increased from 0.2 to 30 microliters per liter, the effectiveness of Ag⁺ in reducing ethylene action and metabolism declined in a similar fashion. Although Ag⁺ significantly inhibited the incorporation of ¹⁴C₂H₄ into tissue metabolites, the oxidation of ¹⁴C₂H₄ to ¹⁴CO₂ was unaffected in the same tissue.     

Effects of atmospheric CO2 enrichment on regrowth of sour orange trees (Citrus aurantium; Rutaceae) after coppicing

Sixteen sour orange tree (Citrus aurantium L.) seedlings were grown out-of-doors at Phoenix, Arizona, in eight clear-plastic-wall open-top enclosures maintained at four different atmospheric CO₂ concentrations for a period of 2 years. Over the last year of this period, the trees were coppiced five times. The amount of dry matter harvested at each of these cuttings was a linear function of the atmospheric CO₂ concentration to which the trees were exposed. For a 75% increase in atmospheric CO, from 400 to 700 microliter per liter (μL liter ¹), total aboveground biomass rose, in the mean, by a factor of 3.19; while for a 400 to 800 μL liter ¹ doubling of the air's CO₂ content, it rose by a factor of 3.92. The relative summer (mean air temperature of 32.8 C) response to CO₂ was about 20% greater than the relative winter (mean air temperature of 16.4 C) response.     

Prior illumination and the respiration of maize leaves in the dark

The course of respiration of attached maize (Zea mays L.) leaves was measured by infrared gas analysis of CO₂ efflux in the dark following illumination in atmospheres of 300 microliters of CO₂ per liter of air, CO₂-free air, and CO₂-free N₂ containing 400 microliters of O₂ per liter. CO₂ efflux from control leaves started 3 to 4 minutes after darkening, increased to a maximum after about 20 minutes, and returned to a steady minimum after 2 to 3 hours. Respiration was quantitatively related to prior illumination, independent of net CO₂ fixation in the light, and depressed by N₂. Light, but not air, was required to produce a substrate for respiration in the subsequent dark period; air was required for oxidation of the substrate to CO₂. The stimulation of respiration by prior illumination in maize leaves differs in its slower onset and greater duration from the postillumination burst of photorespiration.     

Abscission: the phytogerontological effects of ethylene

The role of ethylene in the aging of bean (Phaseolus vulgaris L. cv. Red Kidney) petiole abscission zone explants was examined. The data indicate that ethylene does accelerate aging in addition to inducing changes in break strength. Application of ethylene during the aging stage (stage 1) promoted abscission when followed by a second ethylene treatment during the cell separating stage (stage 2). The half-maximal effective concentration of ethylene to induce aging was around 0.3 microliter per liter; 10 microliters per liter was a saturating dose. CO₂ reversal of ethylene action during stage 1 was incomplete and gave ambiguous results. CO₂ (10%) reversed the effect of 10 microliters per liter ethylene but not 1 microliter per liter ethylene. The possibility that ethylene not only accelerated aging but was also a requirement for it was tested, and experimental evidence in favor of this idea was obtained. It was concluded that ethylene plays a dual role in the abscission of bean petiole explants: a phytogerontological effect and a cellulase-inducing effect.     

Acclimation to High CO(2) in Monoecious Cucumbers : II. Carbon Exchange Rates, Enzyme Activities, and Starch and Nutrient Concentrations

Carbon exchange capacity of cucumber (Cucumis sativus L.) germinated and grown in controlled environment chambers at 1000 microliters per liter CO₂ decreased from the vegetative growth stage to the fruiting stage, during which time capacity of plants grown at 350 microliters per liter increased. Carbon exchange rates (CERs) measured under growth conditions during the fruiting period were, in fact, lower in plants grown at 1000 microliters per liter CO₂ than those grown at 350. Progressive decreases in CERs in 1000 microliters per liter plants were associated with decreasing stomatal conductances and activities of ribulose bisphosphate carboxylase and carbonic anhydrase. Leaf starch concentrations were higher in 1000 microliters per liter CO₂ grown-plants than in 350 microliters per liter grown plants but calcium and nitrogen concentrations were lower, the greatest difference occurring at flowering. Sucrose synthase and sucrose-P-synthase activities were similar in 1000 microliters per liter compared to 350 microliters per liter plants during vegetative growth and flowering but higher in 350 microliters per liter plants at fruiting. The decreased carbon exchange rates observed in this cultivar at 1000 microliters per liter CO₂ could explain the lack of any yield increase (MM Peet 1986 Plant Physiol 80: 59-62) when compared with plants grown at 350 microliters per liter.     

Requirement for Ethylene Synthesis and Action during Relief of Thermoinhibition of Lettuce Seed Germination by Combinations of Gibberellic Acid, Kinetin, and Carbon Dioxide

Application of exogenous ethylene in combination with gibberellic acid (GA₃), kinetin (KIN), and/or CO₂ has been reported to induce germination of lettuce seeds at supraoptimal temperatures. However, it is not clear whether endogenous ethylene also plays a mediatory role when germination under these conditions is induced by treatment regimes that do not include ethylene. Therefore, possible involvement of endogenous ethylene during the relief of thermoinhibition of lettuce (Lactuca sativa L. cv Grand Rapids) seed germination at 32°C was investigated. Combinations of GA₃ (0.5 millimolar), KIN (0.05 millimolar), and CO₂ (10%) were used to induce germination. Little germination occurred in controls or upon treatment with ethylene, KIN, or CO₂. Neither KIN nor CO₂ affected the rate of ethylene production by seeds. Both germination and ethylene production were slightly promoted by GA₃. Treatments with GA₃+CO₂, GA₃+KIN, or GA₃+CO₂+KIN resulted in approximately 10-to 40-fold increases in ethylene production and 50 to 100% promotion of germination as compared to controls. Initial ethylene evolution from the treated seeds was greater than from the controls and a major surge in ethylene evolution occurred at the time of visible germination. Application of 1 millimolar 2-aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene synthesis, in combination with any of above three treatments inhibited the ethylene production to below control levels. This was accompanied by a marked decline in germination percentage. Germination was also inhibited by 2,5-norbornadiene (0.25-2 milliliters per liter), a competitive inhibitor of ethylene action. Application of exogenous ethylene (1-100 microliters per liter) overcame the inhibitory effects of AVG and 2,5-norbornadiene on germination. The results demonstrate that endogenous ethylene synthesis and action are essential for the alleviation of thermoinhibition of lettuce seeds by combinations of GA₃, KIN, and CO₂. It also appears that these treatment combinations do not act exclusively via promotion of ethylene evolution as the application of exogenous ethylene alone did not promote germination.     

EFFECTS OF CO2 ENRICHMENT ON THE GROWTH AND MORPHOLOGY OF A NATIVE AND AN INTRODUCED HONEYSUCKLE VINE

Japanese honeysuckle (Lonicera japonica Thunb.), introduced to the United States, and the native coral honeysuckle (Lonicera sempervirens L.) were compared to determine how intrinsic differences in their growth characteristics would affect their response to atmospheric carbon dioxide enrichment. Plants of both species grown from cuttings were harvested after 54 days of growth in controlled environment growth chambers at 350, 675, or 1,000 μl/liter CO₂. The biomass of Japanese honeysuckle was increased 135% at 675 μ∗∗∗l/liter CO₂ and 76% at 1,000 μl/liter CO₂ after 54 days. Morphologically, the main effect of CO₂ enrichment was to triple the number of branches and to increase total branch length six times. Enhanced and accelerated branchingalso increased total leaf area 50% at elevated CO₂ concentrations. In coral honeysuckle, total biomass was only 40% greater in the elevated CO₂ treatments. Branching was quadrupled but had not proceeded long enough to affect total leaf area. Main stem height was increased 36% at 1,000 μl/liter CO₂. The much less significant height response of other woody erect growth forms suggests that vines may increase in importance during competition if atmospheric CO₂ concentrations increase as predicted. The impact of Japanese honeysuckle in the United States may become more serious.     

Effect of changes in shoot carbon-exchange rate on soybean root nodule activity

The effect of short- and long-term changes in shoot carbon-exchange rate (CER) on soybean (Glycine max [L.] Merr.) root nodule activity was assessed to determine whether increases in photosynthate production produce a direct enhancement of symbiotic N₂ fixation. Shoot CER, root + nodule respiration, and apparent N₂ fixation (acetylene reduction) were measured on intact soybean plants grown at 700 microeinsteins per meter per second, with constant root temperature and a 14/10-hour light/dark cycle. There was no diurnal variation of root + nodule respiration or apparent N₂ fixation in plants assayed weekly from 14 to 43 days after planting. However, if plants remained in darkness following their normal dark period, a significant decline in apparent N₂ fixation was measured within 4 hours, and decreasing CO₂ concentration from 320 to 90 microliters CO₂ per liter produced diurnal changes in root nodule activity. Increasing shoot CER by 87, 84, and 76% in 2-, 3-, and 4-week-old plants, respectively, by raising the CO₂ concentration around the shoot from 320 to 1,000 microliters CO₂ per liter, had no effect on root + nodule respiration or acetylene-reduction rates during the first 10 hours of the increased CER treatment. When the CO₂-enrichment treatment was extended in 3-week-old plants, the only measured parameter that differed significantly after 3 days was shoot CER. After 5 days of continuous CO₂ enrichment, root + nodule respiration and acetylene reduction increased, but such changes reflected an increase in root nodule mass rather than greater specific root nodule activity. The results show that on a 24-hour basis the process of symbiotic N₂ fixation in soybean plants grown under controlled environmental conditions functioned at maximum capacity and was not limited by shoot CER. Whether N₂-fixation capacity was limited by photosynthate movement to root nodules or by saturation of metabolic processes in root nodules is not known.     

Excretion of l-Tryptophan by Analogue-resistant Mutants of Pseudomonas hydrogenothermophila TH-1 in Autotrophic Cultures

Cells of a thermophilic hydrogen bacterium, Pseudomonas hydrogenothermophila TH-1 were treated with N-methyl-N′-nitro-N-nitrosoguanidine and resulting mutants resistant to tryptophan analogues were selected under autotrophic culture conditions (energy source, H₂; carbon source, CO₂). A mutant strain, 7922, which was resistant to 2000 µg/ml of 5-methyltryptophan and 200–500 µg/ml of 5-fluorotryptophan, was obtained by two step mutations. This mutant excreted 38–70 µg/ml of tryptophan into flask culture broth and a maximum of 200 µg/ml into jar fermentor broth.     

Microbial Methanogenesis and Acetate Metabolism in a Meromictic Lake

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 μmol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of ¹⁴CH₄ from ¹⁴C-labeled HCOOH, HCO₃⁻, and CH₃OH and [2-¹⁴C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H¹⁴CO₃⁻ by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH₄ and CO₂ in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 μg/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO₂ production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 μg/liter) completely inhibited methanogenesis and stimulated CO₂ formation.     

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO₂, 6% O₂, and 88% N₂ for 14 weeks at 12°C, exhibited a temporal separation of certain biochemical events associated with ripening.

The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.

When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.

To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22°C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.

     

Measurement of ethylene binding in plant tissue

Tobacco leaves were exposed to ¹⁴C-labeled ethylene (3.7 × 10⁻² microliters per liter) in the presence and absence of unlabeled ethylene and other compounds. Most of the [¹⁴C]ethylene appears to be bound to displaceable sites. Lineweaver-Burk plots for a one-half maximum response in a tobacco leaf respiration test gave a value of 0.3 microliter per liter for ethylene, 50 microliters per liter for propylene, and 266 microliters per liter for carbon monoxide. Scatchard plots for displacement of [¹⁴C]ethylene from the site gave 0.27 microliters per liter for ethylene, 42 microliters per liter for propylene, and 746 microliters per liter for carbon monoxide. At 2%, CO₂ displaces about 35% of the bound ethylene, but increasing the concentration to 10% does not displace the remaining [¹⁴C]ethylene. A value of 3.5 nanomolar was calculated for the concentration of ethylene-binding sites available to exogenous ethylene. This does not account for the sites occupied by endogenous ethylene, and the total number of binding sites is probably somewhat higher. Using tissue culture material, the system was shown to be stable to freezing and thawing; and the π-acceptors, carbon monoxide, cyanide, n-butyl isocyanide, phosphorous trifluoride, and tetrafluoroethylene, were shown to compete with ethylene for binding.     

Gas Exchange Characteristics of the Submerged Aquatic Crassulacean Acid Metabolism Plant, Isoetes howellii

The submerged aquatic plant Isoetes howellii Engelmann possesses Crassulacean acid metabolism (CAM) comparable to that known from terrestrial CAM plants. Infrared gas analysis of submerged leaves showed Isoetes was capable of net CO₂ uptake in both light and dark. CO₂ uptake rates were a function of CO₂ levels in the medium. At 2,500 microliters CO₂ per liter (gas phase, equivalent to 1.79 milligrams per liter aqueous phase), Isoetes leaves showed continuous uptake in both the light and dark. At this CO₂ level, photosynthetic rates were light saturated at about 10% full sunlight and were about 3-fold greater than dark CO₂ uptake rates. In the dark, CO₂ uptake rates were also a function of length of time in the night period. Measurements of dark CO₂ uptake showed that, at both 2,500 and 500 microliters CO₂ per liter, rates declined during the night period. At the higher CO₂ level, dark CO₂ uptake rates at 0600 h were 75% less than at 1800 h. At 500 microliters CO₂ per liter, net CO₂ uptake in the dark at 1800 h was replaced by net CO₂ evolution in the dark at 0600 h. At both CO₂ levels, the overnight decline in net CO₂ uptake was marked by periodic bursts of accelerated CO₂ uptake. CO₂ uptake in the light was similar at 1% and 21% O₂, and this held for leaves intact as well as leaves split longitudinally. Estimating the contribution of light versus dark CO₂ uptake to the total carbon gain is complicated by the diurnal flux in CO₂ availability under field conditions.     

Role of Ethylene Metabolism in Amaranthus retroflexus

¹⁴C-Ethylene was metabolized by etiolated pigweed seedlings (Amaranthus retroflexus L.) in the manner similar to that observed in other plants. The hormone was oxidized to ¹⁴CO₂ and incorporated into ¹⁴C-tissue components. Selected cyclic olefins with differing abilities to block ethylene action were used to determine if ethylene metabolism in pigweed is necessary for ethylene action. 2,5-Norbornadiene and 1,3-cyclohexadiene were effective inhibitors of ethylene action at 800 and 6400 microliters per liter, respectively, in the gas phase, while 1,4-cyclohexadiene and cyclohexene were not. However, all four cyclic olefins inhibited the incorporation and conversion of ¹⁴C-ethylene to ¹⁴CO₂ by 95% with I₅₀ values below 100 microliters per liter. The results indicate that total ethylene metabolism does not directly correlate with changes in ethylene action. Additionally, the fact that inhibition of ethylene metabolism by the cyclic olefins did not result in a corresponding increase in ethylene evolution indicates that ethylene metabolism does not serve to significantly reduce endogenous ethylene levels.     

Ethylene-promoted conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in peel of apple at various stages of fruit development

Internal ethylene concentration, ability to convert 1-amino-cyclopropane-1-carboxylic acid (ACC) to ethylene (ethylene-forming enzyme [EFE] activity) and ACC content in the peel of apples (Malus domestica Borkh., cv Golden Delicious) increased only slightly during fruit maturation on the tree. Treatment of immature apples with 100 microliters ethylene per liter for 24 hours increased EFE activity in the peel tissue, but did not induce an increase in ethylene production. This ability of apple peel tissue to respond to ethylene with elevated EFE activity increased exponentially during maturation on the tree. After harvest of mature preclimacteric apples previously treated with aminoethoxyvinyl-glycine, 0.05 microliter per liter ethylene did not immediately cause a rapid increase of development in EFE activity in peel tissue. However, 0.5 microliter per liter ethylene and higher concentrations did. The ethylene concentration for half-maximal promotion of EFE development was estimated to be approximately 0.9 microliter per liter. CO₂ partially inhibited the rapid increase of ethylene-promoted development of EFE activity. It is suggested that ethylene-promoted CO₂ production is involved in the regulation of autocatalytic ethylene production in apples.     

Carbon and nitrogen limitations on soybean seedling development

Carbon and nitrogen limitations on symbiotically grown soybean seedlings (Glycine max [L.] Merr.) were assessed by providing 0.0, 1.0, or 8.0 millimolar NH₄NO₃ and 320 or 1,000 microliters CO₂/liter for 22 days after planting. Maximum development of the Rhizobium-soybean symbiosis, as determined by acetylene reduction, was measured in the presence of 1.0 millimolar NH₄NO₃ under both levels of CO₂. Raising NH₄NO₃ from 0.0 to 8.0 millimolar under 320 microliters CO₂/liter increased plant dry weight by 251% and Kjeldahl N content by 287% at 22 days after planting. Increasing NH₄NO₃ from 1.0 to 8.0 millimolar under 320 microliters CO₂/liter increased total dry weight and Kjeldahl N by 100 and 168%, respectively, on day 22. Raising CO₂ from 320 to 1,000 microliters CO₂/liter during the same period had no significant effect on Kjeldahl N content of plants grown with 0.0 or 1.0 millimolar NH₄NO₃. The maximum CO₂ treatment effects were observed in plants supplied with 8.0 millimolar NH₄NO₃, where dry weight and Kjeldahl N content were increased 64% and 20%, respectively. An increase in shoot CO₂-exchange rate associated with the CO₂-enrichment treatment was reflected in a significant increase in leaf dry weight and starch content for plants grown with 1,000 microliters CO₂/liter under all combined N treatments. These data show directly that seedling growth in symbiotically grown soybeans was limited primarily by N availability. The failure of the CO₂-enrichment treatment to increase total plant N significantly in Rhizobium-dependent plants indicates that root nodule development and functioning in such plants was not limited by photosynthate production.