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.     

Gas-exchange activity,carbohydrate status,and protein turnover in root nodule subpopulations of field pea (Pisum sativum L. cv. Century)

Root nodule ontogeny was followed in different parts of the root system of field peas (Pisum sativum L. cv. Century) to investigate the contribution to total nitrogen fixation by different nodule subpopulations. Seed-inoculated plants were grown to maturity in controlled-environment growth chambers. In a flow-through system nitrogenase activity (H₂-evolution in air) and nodulated-root respiration (net CO₂-evolution) were measured weekly or biweekly in different parts (top and mid) of the root system. Root nodule extracts were assayed for total soluble cytosolic protein, total heme, proteolytic capacity (at pH 7.0), soluble carbohydrates and starch. Total nitrogenase activity and nodule respiration were higher in the top zone, which was explained by differences in root and nodule mass. Nodule specific nitrogenase activity was similar in both zones, and gradually declined throughout the experiment. No differences were found between nodule subpopulations in the dry-matter specific concentrations of glucose, fructose, sucrose or starch. Neither did nodule concentrations of protein or leghemoglobin differ between the zones. Throughout reproductive growth, no decline was found in total or nodule specific nitrogenase activity, in any of the nodule subpopulations. Growth of the root nodules continued throughout the experiment, though growth of shoot and roots had ceased. The data gives no support for carbohydrate limitation in root nodules during pod-filling, since nodule respiration remained high, the concentration of soluble carbohydrates increased significantly, and the amount of starch was not reduced. We conclude that when this symbiosis is grown under controlled conditions, nitrogenase activity in nodules sub-sampled from the crown part of the root system is representative for the whole nodule population.     

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.     

Effect of low rhizosphere oxygen on growth, nitrogen fixation and nodule morphology in lucerne

Oxygen shortage in soils can occur following a wide range of natural circumstances, affecting the plant's physiology. In this paper the performance of nodulated lucerne plants under severe hypoxia is examined and the mechanisms involved to achieve this adaptation are discussed. Nodulated lucerne plants ( Medicago sativa L.) were grown with their rooting medium exposed to 1 or 21 kPa oxygen. Final yield, as expressed on a shoot dry weight basis, was unaffected but root and nodule dry weights were reduced by 50%. Water content in roots and nodules was higher at 1 kPa as a result of the formation of aerenchyma. Specific acetylene reduction activity was higher in hypoxic nodules as a consequence of modified nodule structure, although they were more sensitive to the presence of acetylene or nitrate. Root respiration was insensitive to changes in external oxygen supply, therefore providing adequate support for mineral uptake. Nodule respiration rates were 5 times higher in control plants when measured as CO₂ evolution, whereas no differences were observed in O₂ uptake. It is suggested that adaptation of nodulated lucerne to low oxygen concentrations involves changes in photosynthate allocation and nodule morphology, which provide a more efficient nitrogen fixation.     

Photoassimilate partitioning in nodulated soybean II. The effect of changes in photoassimilate availability shows that nodule permeability to gases is not linked to the supply of solutes or water

It is concluded that the permeability of the soybean nodule to gases is not linked to the supply of solutes or water via the phloem to the nodule. Nodule respiration and nitrogenase activity were less affected by diel variation and shading treatments than partitioning to the nodule, as assessed using a non-invasive ¹¹C-based technique. Thus C import to the nodule was not matched to C requirement by the nodule. Transit times of tracer to, and within, the nodulated root increased under conditions of reduced photosynthetic rate. The increase in transit time was interpreted as a reduction in the flux of phloem sap. Thus the fluxes of both water and C to the nodule decreased following a reduction in photosynthetic rate. The change in partitioning of recent photosynthate to soybean roots and nodules in response to changes in photoassimilate availability was also used to assess the 'priority' of these sinks. Partitioning from the leaf to the root system was greatly decreased when photoassimilate availability was limited, indicating that root system priority is lower than that of the shoot, as reported for other systems. However, partitioning of tracer arriving in the root system between the nodulated and non-nodulated zones of the root was not affected by changes in photoassimilate availability, as caused by diel change, shading, or steaming of branch roots. Thus although nodules are sinks of high sink 'activity', they have 'priority' equal to that of other root sinks. It is suggested that there are similar phloem unloading kinetics, despite the very different metabolic destiny of the carbohydrate within the two organs.     

Carbon and nitrogen assimilation and partitioning in soybeans exposed to low root temperatures

Low root temperature effects on vegetative growth of soybean (Harosoy 63 × Rhizobium japonicum USDA 16) were examined in 35 day old plants exposed to temperatures of 15°C (shoots at 25°C) for an 11 day period. Duing this period various aspects of C and N assimilation and partitioning were monitored including shoot night and nodulated root respiration, C and N partitioning to six plant parts, C₂H₂ reduction, H₂ evolution, leaf area, transpiration, net photosynthesis, and N₂ fixation. The low temperature treatment resulted in a decrease in the net rate of N₂ fixation but nitrogenase relative efficiency increased. In response, the plant retained N in the tissues of the nodulated root and decreased N partitioning to young shoot tissues, thereby inducing the remobilization of N from older leaves, and reducing leaf area development. The leaf area specific rate of net photosynthesis was not affected over the study period; however, shoot and nodulated root respiration declined. Consequently, C accumulated in mature leaves and stems, partly in the form of increased starch reserves. Three possibilities were considered for increasing low temperature tolerance in nodulated soybeans: (a) decrease in temperature optima for nitrogenase, (b) increased development of nodules and N₂ fixation capacity at low temperature, and (c) alterations in the pattern of C and N partitioning in response to low temperature conditions.     

Comparisons of the Respiratory Effluxes of Nodules and Roots in Six Temperate Legumes

The specific respiration rates of nodulated root systems, ofnodules and of roots were determined during active nitrogenfixation in soya bean, navy bean, pea, lucerne, red clover andwhite clover, by measurements on whole plants before and afterthe removal of nodule populations. Similar measurements weremade on comparable populations of the six legumes, lacking nodulesbut receiving abundant nitrate-nitrogen, to determine the specificrespiration of their roots. All plants were grown in a controlled-environmentclimate which fostered rapid growth. The specific respiration rates of nodulated root systems ofthe three grain and three forage legumes during a 7–14-dayperiod of vegetative growth varied between 10 and 17 mg CO₂g^–1 (dry weight) h^–1. This mean value consistedof two components: a specific root respiration rate of 6–9mg CO₂ g^–1 h^–1 and a specific nodule respirationrate of 22–46 mg CO₂ g^–1 h^–1. Nodule respirationaccounted for 42–70 per cent of nodulated root respiration;nodule weight accounted for 12–40 per cent of nodulatedroot weight. The specific respiration rates of roots lackingnodules and utilizing nitrate nitrogen were generally 20–30per cent greater than the equivalent rates of roots from nodulatedplants. The measured respiratory effluxes are discussed in thecontext of nitrogen nitrogen fixation, nitrate assimilation. Glycine max, Phaseolus vulgaris, Pisum sativum, Medicago sativa, Trifolium pratense, Trifolium repens, soya bean, navy bean, pea, lucerne, red clover, white clover, nodule respiration, root respiration, fixation, nitrate assimilation     

Compartmental Analysis of the Partitioning of Photo-assimilated Carbon in Nodulated Soybean Plants during the Light Period

Kouchi, H., Yoneyama, T. and Akao, S. 1986. Compartmental analysisof the partitioning of photo-assimilated carbon in nodulatedsoybean plants during the light period.—J. exp. Bot. 37:994–1005. Dynamics of the partitioning of photo-assimilated carbon invegetative nodulated soybean (Glycine max L.) plants in thelight period was investigated by compartmental analysis basedon data from steady-state ¹³CO₂ assimilation experiments. Themodel assumes a total of 18 compartments consisting of activeand temporary storage pools for soluble materials, starch andstructural materials in leaves, stems plus petioles, roots andnodules together with respired carbon from the roots and nodules.Carbon flow between compartments was described by 22 rate parameters.The rate parameters were evaluated by a non-linear least squaresearch method to optimize the fitness of the simulated resultswith the experimental tracer distribution. The compartment model was well applicable to interpret the carbonpartitioning in whole plants. The analysis showed that: (I)The largest carbon flux during the light period was to storagematerials (starch and temporary storage soluble pools) in theabove-ground parts. The total flux to storage pools was considerablylarger than the transporting flux to below-ground parts. (2)The main carbon flux to the nodules was via direct phloem pathwaysfrom the shoot and not via the compartment of root soluble materials.This flux was 72% of the total carbon flux from the shoot tothe nodulated root system. (3) A large amount of carbon wasreturned to the shoot from below-ground parts. The total returnof carbon flux to the shoot (85% from nodules) was equivalentto 54% of the total influx of carbon to below-ground parts.Direct carbon transfers between roots and nodules were relativelysmall. Key words: Compartmental analysis, carbon partitioning, root nodules, Glycine max L., ¹³CO₂, assimilation     

Phosphorus deficiency increases the argon-induced decline of nodule nitrogenase activity in soybean and alfalfa

Open-flow assays of H₂ evolution in Ar:O₂ (80:20, v/v) by nodulated roots were performed in situ with soybean [Glycine max (L.) Merr.] and alfalfa [Medicago sativa L.) grown in sand with orthophosphate (Pi) nutrition either limiting (low-P) or non-limiting (control) for plant growth. Nodule growth was more limited than shoot growth by P deficiency. Phosphorus concentration was less affected in nodules than in other parts of the low-P plants. During assays, nitrogenase activity declined a few minutes after exposure of the nodulated roots to Ar. The magnitude of this argon-induced decline (Ar-ID) was less in alfalfa than in soybean. In both symbioses the magnitude of the Ar-ID was larger in low-P than control plants. Moreover, the minimum H₂ evolution after the Ar-ID, was reached earlier in low-P plants. The Ar-ID was partly reversed by raising the external partial pressure of O₂ in the rhizosphere. The magnitude of the Ar-ID in soybean was correlated negatively to nodule and shoot mass per plant, individual nodule mass, H₂ evolution in air prior to the assay, and nodule N and P concentrations. Possible reasons, including nodule size and nodule O₂ permeability, for the increase in Ar-ID in P-deficient plants are discussed and an interpretation of the P effect on nodule respiration and energetic metabolism is proposed. Received: 17 May 1996 / Accepted: 16 September 1996     

Effect of shoot removal on remobilization of carbon and nitrogen during regrowth of nitrogen‐fixing alfalfa

The contribution of carbon and nitrogen reserves to regrowth following shoot removal has been studied in the past. However, important gaps remain in understanding the effect of shoot cutting on nodule performance and its relevance during regrowth. In this study, isotopic labelling was conducted at root and canopy levels with both ¹⁵N₂ and ¹³C‐depleted CO₂ on exclusively nitrogen‐fixing alfalfa plants. As expected, our results indicate that the roots were the main sink organs before shoots were removed. Seven days after regrowth the carbon and nitrogen stored in the roots was invested in shoot biomass formation and partitioned to the nodules. The large depletion in nodule carbohydrate availability suggests that root‐derived carbon compounds were delivered towards nodules in order to sustain respiratory activity. In addition to the limited carbohydrate availability, the upregulation of nodule peroxidases showed that oxidative stress was also involved during poor nodule performance. Fourteen days after cutting, and as a consequence of the stimulated photosynthetic and N₂‐fixing machinery, availability of C_new and N_new strongly diminished in the plants due to their replacement by C and N assimilated during the post‐labelling period. In summary, our study indicated that during the first week of regrowth, root‐derived C and N remobilization did not overcome C‐ and N‐limitation in nodules and leaves. However, 14 days after cutting, leaf and nodule performance were re‐established.     

Effects of CO(2) Enrichment and Carbohydrate Content on the Dark Respiration of Soybeans

During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO₂ per liter air, was 1.45 milligrams CO₂ evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO₂ per liter air). There was a higher foliar nonstructural carbohydrate level (e.g. sucrose and starch) in the CO₂ enriched compared with CO₂ normal plants. For example, leaves of enriched plants displayed levels of nonstructural carbohydrate equivalent to 174 milligrams glucose per gram dry weight compared to the 84 milligrams glucose per gram dry weight found in control plant leaves. As the leaves of CO₂ enriched plants approached full expansion, both the foliar respiration rate and carbohydrate content of the CO₂ enriched leaves decreased until they were equivalent with those same parameters in the leaves of control plants. A strong positive correlation between respiration rate and carbohydrate content was seen in high CO₂ adapted plants, but not in the control plants.

Mitochondria, isolated simultaneously from the leaves of CO₂ enriched and control plants, showed no difference in NADH or malate-glutamate dependent O₂ uptake, and there were no observed differences in the specific activities of NAD⁺ linked isocitrate dehydrogenase and cytochrome c oxidase. Since the mitochondrial O₂ uptake and total enzyme activities were not greater in young enriched leaves, the increase in leaf respiration rate was not caused by metabolic adaptations in the leaf mitochondria as a response to long term CO₂ enrichment. It was concluded, that the higher respiration rate in the enriched plant's foliage was attributable, in part, to a higher carbohydrate status.

     

Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.

Rates of CO₂ evolution and H₂ evolution from intact, nodulated root systems under Ar:O₂ atmospheres decreased in parallel when plants were maintained in an extended dark period. While H₂ evolution approached zero after 36 hours of darkness at 22°C, CO₂ evolution rate remained at 38° of the rate measured in light. Of the remaining CO₂ evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO₂ (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.

The cost of N₂ fixation in terms of CO₂ evolved per electron pair utilized by nitrogenase was estimated from the slope of H₂ evolution rate versus CO₂ evolution rate. The cost was 2 moles CO₂ evolved per mole H₂ evolved and was independent of temperature.

In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H₂ evolution.

     

Short-term Changes in CO2 Evolution Associated with Nitrogenase Activity in White Clover in Response to Defoliation and Photosynthesis

Direct, continuous measurements of the CO₂ evolution of rootnodules, calibrated by direct measurements of rate of ethyleneproduction, were utilized to determine the short-term responseof nitrogenase activity to defoliation and photosynthesis inwhite clover. Defoliation (removal of all expanded leaflets) generally resultedin a fall in nodulated root respiration within 10 min; mostrespiration associated with nitrogenase activity ceased within1–2 h. Darkening of the shoot also reduced nodulated root respirationwithin 10 min, but the subsequent fall in respiration, althoughof the same magnitude, was slower. The re-illumination of shootslargely reversed these effects. The inhibition of photosynthesisby DCMU largely simulated the effects of darkening the shoots. It is concluded that, in these white clover plants of 100 mgto 2.0 g total weight, current photosynthate provides the primarysource of energy for N₂ fixation. The mobilization of reserveenergy substrate appeared to play only a small role. The minimumtime interval of 10 min between onset of treatment and fallin nodule respiration probably reflects the time taken to exhaustthe assimilate in transit between leaf and nodule. Key words: White clover, N₂ fixation, Defoliation, Photosynthesis     

The role of dark carbon dioxide fixation in root nodules of soybean

The magnitude and role of dark CO₂ fixation were examined in nodules of intact soybean plants (Harosoy 63 × Rhizobium japonicum strain USDA 16). The estimated rate of nodule dark CO₂ fixation, based on a 2 minute pulse-feed with ¹⁴CO₂ under saturating conditions, was 102 micromoles per gram dry weight per hour. This was equivalent to 14% of net nodule respiration. Only 18% of this CO₂ fixation was estimated to be required for organic and amino acid synthesis for growth and export processes. The major portion (75-92%) of fixed label was released as CO₂ within 60 minutes. The labeling pattern during pulse-chase experiments was consistent with CO₂ fixation by phosphoenolpyruvate carboxylase. During the chase, the greatest loss of label occurred in organic acids. Exposure of nodulated roots to Ar:O₂ (80:20) did not affect dark CO₂ fixation, while exposure to O₂:CO₂ (95:5) resulted in 54% inhibition. From these results, it was concluded that at least 66% of dark CO₂ fixation in soybean may be involved with the production of organic acids, which when oxidized would be capable of providing at least 48% of the requirement for ATP equivalents to support nitrogenase activity.     

Economy of Carbon and Nitrogen in Nodulated and Nonnodulated (NO(3)-grown) Cowpea [Vigna unguiculata (L.) Walp.

The response of non-nodulated cowpea (Vigna unguiculata (L.) Walp. cv Caloona) to a wide range of NO₃ levels in the rooting medium was studied 40 days after sowing by in vitro assays of plant organs for NO₃ reductase (EC 1.6.6.1) and analyses of root bleeding (xylem) sap for nitrogenous solutes. Plants fed 1, 5, 10, 20, and 40 millimolar NO₃ showed, respectively, 64, 92, 94, and 91% of their total reductase activity in shoots and 34, 30, 66, 62, and 58% of the total N of their xylem sap as NO₃. These data, and the absence in the plants of significant pools of stored NO₃, indicated that shoots were major organs of NO₃ assimilation, especially at levels of NO₃ (10 to 40 millimolar) that maintained plant growth at near maximum rates. Partitioning and utilization of C and N were studied in nodulated, minus NO₃ plants and non-nodulated plants fed 10 or 20 millimolar NO₃, the levels of NO₃ which gave rates of growth and N assimilation closest to those of the symbiotic plants. The conversion of the C of net photosynthate to dry matter was similar in nodulated plants (67%) and NO₃-grown plants (64%), but greater proportions of photosynthate were translocated to below ground parts of nodulated plants (37%) than of NO₃-fed plants (23 to 26%). Greater photosynthate consumption by nodulated roots was associated with proportionately greater root growth and respiration and 2-fold greater export of C in xylem than in the NO₃-fed plants. Theoretical considerations suggest that the elevated CO₂ output of nodulated roots was due not only to CO₂ loss associated with nodule function, but also to a much greater nonassimilatory component of respiration in the supporting root of the nodulated plant compared to roots of the NO₃-fed plants. Data are compared with previously published information from other legumes.     

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.     

Comparative Response of Nodulated and Nitrogen-Supplied Cowpea (Vigna unguiculata (L.) Walp. var. Tuy) Plants to Infection by Cowpea Mosaic Virus

The effect of infection by the Cowpea Mosaic Virus (CpMV) onseveral parameters relevant to symbiotic nitrogen fixation wasdetermined in cowpea (Vigna unguiculata (L.) Walp. var. Tuy)plants nodulated with two strains of Rhizobium cowpea: IVIC–124and IVIC–38. Plants were virus-infected at the seedlingstage before Rhizobium inoculation. The effect of CpMV infectionon plant growth was analysed in nodulated and nitrogen-suppliedplants at 18, 25 and 35 d after germination. At all developmentalstages of nodulated plants CpMV infection caused a reductionof leaf chlorophyll content, leaf area, dry weight of shootsand roots, total nodule weight and nodule number. Most of thenodules from 18- and 25-d-old CpMV-infected plants did not exhibitleghaemoglobin pigmentation. CpMV infection delayed the onsetof nitrogenase activity in nodules of the rhizobial strain IVIC–124and the enzyme activity measured on a per plant basis was reducedin both strains at the first and second harvests. Significantnitrogenase activity was detected in 35-d-old infected plants.Some of the nodules of the rhizobial strain IVIC-124 and mostof the nodules from plants nodulated with the strain IVIC-38developed leghaemoglobin; however, the nodule-specific nitrogenaseactivity, estimated on a milligram nodule dry weight basis,was always higher in virus-infected plants, particularly in18-d-old CpMV-infected plants harbouring the IVIC–124strain. CpMV-infected nodules had a larger peribacteroidal space,a reduced number of peribacteroid units, a greater number ofbacteroids per unit, a lower number of vesicles and 88% lowertotal reducing sugar content. Starch accumulation was detectedin infected leaves of nodulated plants during the first harvest,while high levels of leaf reducing sugars and protein were presentat the second harvest. In healthy nodulated plants the rhizobialstrain IVIC–124 was shown to be more efficient than IVIC–38in promoting plant growth. However, the results indicate thatnodulation by rhizobial strain IVIC–124 and growth ofplants harbouring this strain were affected to a greater extentby virus infection. The effect of CpMV infection on leaf chlorophyllcontent, leaf area, carbohydrate level, leaf proteins and growthof nitrogen-supplied plants, as well as the symptoms inducedin the leaves, were less conspicuous than in nodulated plants. Key words: Cowpea, Rhizobium, virus infection, nodule untrastructure     

Differential usage of storage carbohydrates in the CAM bromeliad Aechmea 'Maya' during acclimation to drought and recovery from dehydration

CAM requires a substantial investment of resources into storage carbohydrates to account for nocturnal CO₂ uptake, thereby restricting carbohydrate partitioning to other metabolic activities, including dark respiration, growth and acclimation to abiotic stress. Flexible modulation of carbon flow to the different competing sinks under changing environmental conditions is considered a key determinant for the growth, productivity and ecological success of the CAM pathway. The aim of the present study was to examine how shifts in carbohydrate partitioning could assure maintenance of photosynthetic integrity and a positive carbon balance under conditions of increasing water deprivation in CAM species. Measurements of gas exchange, leaf water relations, malate, starch and soluble sugar (glucose, fructose and sucrose) contents were made in leaves of the CAM bromeliad Aechmea ‘Maya’ over a 6‐month period of drought and subsequently over a 2‐month period of recovery from drought. Results indicated that short‐term influences of water stress were minimized by elevating the level of respiratory recycling, and carbohydrate pools were maintained at the expense of export for growth while providing a comparable nocturnal carbon gain to that in well‐watered control plants. Longer term drought resulted in a disproportionate depletion of key carbohydrate reserves. Sucrose, which was of minor importance for providing substrate for the dark reactions under well‐watered conditions, became the major source of carbohydrate for nocturnal carboxylation as drought progressed. Flexibility in terms of the major carbohydrate source used to sustain dark CO₂ uptake is therefore considered a crucial factor in meeting the carbon and energy demands under limiting environmental conditions. Recovery from CAM‐idling was found to be dependent on the restoration of the starch pool, which was used predominantly for provision of substrate for nocturnal carboxylation, while net carbon export was limited. The conservation of starch for the nocturnal reactions might be adaptive with regard to responding efficiently to a return of water stress.     

Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

Assimilation,respiration and allocation of carbon inPlantago major as affected by atmospheric CO2 levels

The response ofPlantago major ssp,pleiosperma plants, grown on nutrient solution in a climate chamber, to a doubling of the ambient atmospheric CO₂ concentration was investigated. Total dry matter production was increased by 30% after 3 weeks of exposure, due to a transient stimulation of the relative growth rate (RGR) during the first 10 days. Thereafter RGR returned to the level of control plants. Photosynthesis, expressed per unit leaf area, was stimulated during the first two weeks of the experiment, thereafter it dropped and nearly reached the level of the control plants. Root respiration was not affected by increased atmospheric CO₂ levels, whereas shoot, dark respiration was stimulated throughout the experimental period. Dry matter allocation over leaves stems and roots was not affected by the CO₂ level. SLA was reduced by 10%, which can partly be explained by an increased dry matter content of the leaves. Both in the early and later stages of the experiment, shoot respiration accounted for a larger part of the carbon budget in plants grown at elevated atmospheric CO₂. Shifts in the total carbon budget were mainly due to the effects on shoot respiration. Leaf growth accounted for nearly 50% of the C budget at all stages of the experiment and in both treatments.Abbreviations LAR leaf area ratio - LWR leaf weight ratio - RGR relative growth rate - R/S root to shoot ratio - RWR root weight ratio - SLA specific leaf area - SWR stem weight ratio