Nitrogenase activity, nodule respiration, and o(2) permeability following detopping of alfalfa and birdsfoot trefoil

Gas exchange measurements and noninvasive leghemoglobin (Lb) spectrophotometry (nodule oximetry) were used to monitor nodule responses to shoot removal in alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus). In each species, total nitrogenase activity, measured as H₂ evolution in Ar:O₂ (80:20), decreased to <50% of the initial rate within 1 hour after detopping, and net CO₂ production decreased to about 65% of the initial value. In a separate experiment in which nodule oximetry was used, nodule O₂ permeability decreased 50% within 5 hours in each species. A similar decrease in the O₂-saturated respiration rate (V_max) for the nodule central zone occurred within 5 hours in birdsfoot trefoil, but only after 24 hours in alfalfa. Lb concentration, also measured by oximetry, decreased after 48 to 72 hours. The decrease in permeability preceded the decrease in V_max in each species. V_max may depend mainly on carbohydrate availability in the nodule. If so, then the decrease in permeability could not have been triggered by decreasing carbohydrate availability. Both oximetry and gas exchange data were consistent with the hypothesis that, for the cultivars tested, carbohydrate availability decreased more rapidly in birdsfoot trefoil than in alfalfa nodules. Fractional Lb oxygenation (initially about 0.15) decreased during the first 24 hours after detopping but subsequently increased to >0.65 for a majority of nodules of each species. This increase could lead to O₂ inactivation of nitrogenase.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Changes in Nitrogenase Activity and Nodule Diffusion Resistance of Subterranean Clover in Response to pO2

Diffusion resistance to oxygen within nodules was calculatedusing the respiratory quotient (RQ) of nodules from intact plantsof subterranean clover (Trifolium subterraneum L.) cv. SeatonPark nodulated by Rhizobiun trifolii WU95. From 21 to 52% O₂,the RQ remained between 0.94 and 1.04, whereas at 10% O₂, theRQ was 1.65. When nodulated roots of intact plants were exposedto sub-ambient pO₂ in a continuous flow-through system, respirationdeclined immediately, followed by a partial recovery within30 min. The magnitude of the final respiration rate was dependentupon the pO₂ in the gas stream. Initial rates of respirationwere re-established after 24 h at sub-ambient pO₂ as a resultof changes in the resistance of the variable barrier to oxygendiffusion within the nodules. Nitrogenase activity also decreasedlinearly with decreasing pO₂ in the gas stream, but partialrecovery occurred after 24 h incubation at sub-ambient pO₂.Maximum rates of nitrogenase activity occurred at rhizosphereoxygen concentrations between 21% and 36% O₂. Resistance tothe diffusion of oxygen within the nodules increased at supra-ambientpO₂ and at oxygen concentrations above 36% O₂, resulted in adecrease in both nitrogenase activity and nodulated root respiration.The diffusion resistance of nodules to oxygen increased rapidlyin the presence of either supra-ambient pO₂ or saturating pC₂H₂.Reductions in nodule diffusion resistance either during recoveryfrom exposure to 10% acetylene or to sub-ambient pO₂ occurredmore slowly. It is concluded that subterranean clover is welladapted for maximum nitrogen fixation at ambient pO₂. Key words: Nitrogenase activity, oxygen, subterranean clover, diffusion resistance     

Physiological Role of Leghaemoglobin Heterogeneity in Pea Root Nodule Development

Disk-gel-electrophoresis of the leghaemoglobin isolated frompea root nodules revealed two major (Lb I and Lb IV) and threeminor components. The ratio of the two major components (LbI/Lb IV) decreased with increasing age. This ratio was higherin the distal than in proximal region when nodules were cutinto distal and proximal sections. In young nodules, the incorporationof radioactive leucine into Lb I was much higher than into LbIV. In older nodules, the radioactivities were much higher inLb IV than in Lb I. These data suggest that the change in thisratio is due mainly to differences in the rates of biosynthesis. Two major components (Lb I and Lb IV) which were isolated separatelyhad different O₂-binding affinities. The O₂-binding affinityof the component synthesized mainly in older nodules (Lb IV)was higher than that of the component synthesized mainly inyoung nodules (Lb I). These results indicate that changes inthe relative contents of leghaemoglobin during nodule developmentcontribute to more effective nitrogen fixation which is mediatedby changes in the capacities of oxygen transport. (Received July 7, 1981; Accepted November 2, 1981)     

Rhizobium meliloti Mutants Defective in Symbiotic Nitrogen Fixation Affect the Oxygen Gradient in Alfalfa (Medicago sativa) Root Nodules

Rhizobium meliloti bacteroids carrying mutations in either fdxNor fixX isolated from alfalfa root nodules were shown to containthe nitrogenase proteins NifH, NifD and NifK. In contrast toan in vitro system of N₂-fixation based on R. meliloti wild-typebacteroids, nitrogenase activity could not be restored in crudeextracts of these mutant bacteroids by the addition of an artificialelectron donor, indicating that the nitrogenase proteins werepresent but not functional. ESR-studies revealed that both mutantslacked the FeMo-cofactor of nitrogenase. To analyse the roleof free O₂ on the damage of the nitrogenase components and theFeMo-cofactor in these mutant bacteroids, microelectrode studiesof O₂ concentrations and gradients within alfalfa root noduleswere carried out. R. meliloti mutants defective in other genesnecessary for symbiotic N₂-fixation were also included in thisstudy. Four distinct types of O₂ gradients were defined by theapparent presence or absence of an O₂ diffusion barrier andby the minimum internal O₂ concentration. These data clearlydemonstrated the influence of the microsymbiont on the O₂ gradientswithin the nodules. Nodules induced by Rm0540, an R. melilotimutant with altered exopolysaccharide production, which is notable to infect plant cells, did not contain an O₂ diffusionbarrier. In contrast, nodules containing a mutant defectivein dicarboxylate transport (dctA-), produced an O₂ gradientsimilar to the wild-type. Microelectrode measurements revealedH₂ concentrations in alfalfa wild-type nodules comparable tosoyabean, whereas no hydrogen could be detected in nodules harbouringthe dctA mutant or any other mutant strain. Key words: Nitrogen fixation, Rhizobium meliloti bacteroids, ferredoxin-like proteins, microelectrode studies     

Nodulin gene expression in effective root nodules of white sweetclover (Melilotus alba Desr.) and in ineffective nodules elicited by mutant strains of Rhizobium meliloti

Fifteen nodulins and several nodule-stimulated gene productswere expressed in effective, nitrogen-fixing root nodules ofwhite sweetclover (Melilotus alba Desr. cv. U389), as determinedby two-dimensional gel electrophoresis of in vitro translationproducts. The number and gel position of eight leghaemoglobin(Lb) products, as well as a product tentatively identified asnodule-stimulated glutamine synthetase (GS), was similar toprevious reports of alfalfa (Medicago sativa L. cv. Iroquois)nodulins. Three mutants of Rhizobium meliloti, including anexoH mutant, a lipopolysaccharide mutant, and a nifH mutant,elicited ineffective sweetclover nodules blocked at empty (bacteria-free),partially infected, or fully infected stages of nodule development,respectively. In these ineffective nodules, the nodulin Nma30and nodule-stimulated NSTma42 were expressed early in development,while a group of four nodulins and two nodule-stimulated productswere intermediate in order of expression. Lb, GS and the latenodulin Nmal2a were expressed later, following infection. TheexoH mutant, Rm7154, appeared to be a leaky mutant, as a smallpercentage of the plants developed nitrogen-fixing nodules about4 weeks after inoculation. The sequential expression of a largenumber of nodulins and nodule-stimulated products, as well asthe availability of sweetclover nodulation mutants indicatesthat sweetclover is a useful diploid system for analysis ofhost genes essential to the Rhizobium/legume symbiosis. Key words: Nitrogen fixation, nodulation mutants, nodulins     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Metabolic changes in soybean nodules after inhibition of nitrogen fixation by treatment with oxygen

Metabolites that accumulated in soybean [Glycine max (L.) Merr.]nodules after inhibition of nitrogen fixation were analysedto determine what carbon compounds the bacteroids might obtainfrom their host. Exposure of roots of intact soybean plantsto 100% O₂ for 5 min caused a decrease in acetylene reductionactivity within 10 min and then the activity recovered onlyslowly. Analysis of carbohydrates, organic acids, volatile compoundsand amino acids in extracts of nodules revealed that succinate,malate and alanine all accumulated within 10 min after treatmentwith O₂. The concentrations of sucrose, acetone, tyrosine, valine,isoleucine, leucine, and ornithine in the nodules increasedslowly after such treatment. The results are discussed in termsof carbon sources for supporting nitrogen fixation of soybeanbacteroids. Key words: Glycine max, carbon metabolism, nitrogen fixation, nodules     

The Influence of Carbohydrate Reserves on the Response of Nodulated White Clover to Defoliation

A growth-chamber study was carried out to determine whetherthe response of apparent nitrogenase activity (C₂ H₂ reduction)to complete defoliation is influenced by the availability ofcarbohydrate reserves Reserve carbohydrate (TNC) concentrationsof 6-week-old white clover (Trifoliun repens L) plants weremodified by CO₂ pretreatments There was no difference in theresponse of apparent nitrogenase activity to defoliation betweenplants with different TNC concentrations C₂H₂ reduction activitydeclined sharply after defoliation and then recovered similarlyin both high- and low-TNC plants Further experiments were conductedto explain the lack of response of apparent nitrogenase activityto TNC levels Bacteroid degradation was ruled out because invitro nitrogenase activity of crude nodule extracts was stillintact 24 h after defoliation Sufficient carbohydrates appearedto be available to the nodules of defoliated plants becauseadding [¹⁴C]glucose to the nutrient solution did not preventthe decline in apparent nitrogenase activity These conclusionswere supported by the finding that an increase in pO₂ aroundthe nodules of defoliated plants completely restored their C₂H₂reduction activity The comparison of the effects of defoliationand darkness suggested that the decrease in apparent nitrogenaseactivity was not related directly to the interruption of photosynthesisIt appears that lack of photosynthates is not the immediatecause of the decline of nitrogen-fixing activity after defoliation White clover, Trifolium repens L, defoliation, nitrogen fixation, regrowth, reserves, carbohydrates, acetylene reduction, nodule extract     

Inhibition of nitrogenase activity and nodule oxygen permeability by water deficit

Short-term effects of water deficit on nitrogenase activitywere investigated with hydroponically grown soybean plants (Glycinemax L. Merr. cv. Biloxi) by adding polyethylene glycol (PEG)to the hydroponic solution and measuring nitrogenase activity,nodule respiration, and permeability to oxygen diffusion (P_o).These experiments showed a rapid decrease in acetylene reductionactivity (ARA) and nodule respiration. A consequence of thedecreased respiration rate was that P_o calculated by Fick'sLaw also decreased. However, these results following PEG treatmentwere in direct conflict with a previous report of stabilityin P_o determined by using an alternative technique. To resolvethis conflict, an hypothesis describing a sequence of responsesto the initial PEG treatment is presented. An important findingof this study was that the response to water deficit inducedby PEG occurred in two stages. The first stage of decreasednodule activity was O₂-limited and could be reversed by exposingthe nodules to elevated pO₂. The second stage which developedafter 24 h of exposure to PEG resulted in substantial loss innodule activity and this activity could not be recovered withincreased pO₂. Severe water deficit treatments disrupt noduleactivity to such a degree that O₂ is no longer the major limitation. Key words: Glycine max, N₂ fixation, soybean, oxygen permeability, water deficit     

Nitrogen Fixation by Subterranean Clover at Varying Stages of Nodule Dehydration: I. CARBOHYDRATE STATUS AND SHORT-TERM RECOVERY OF NODULATED ROOT RESPIRATION

The nodule water potential (_nod) of subterranean clover (Trifoliumsubterraneum L.) cv. Seaton Park incubated in a flow-throughgas-exchange system was induced to decline independently ofleaf water potential (₁) by passing a continuous dry airstreamover the nodulated roots of intact well-watered plants. Reducedtranspiration by plants whose nodules had become dehydratedwas hypothesized to be related to the decline in nitrogen fixation.Whole-plant and nodule soluble carbohydrates increased as _noddeclined. Throughout an 8 d period of continual nodule dehydration,the gaseous diffusion resistance of nodules increased and theoptimum pO₂ for nitrogenase activity declined from 52 to 28kPa. Following rehydration of the nodulated roots between days4 and 5 and between days 7 and 8, nodulated root respirationincreased to or above pre-stress levels whereas nitrogenaseactivity did not recover. Re-establishment of initial ratesof nodulated root respiration was due to the stimulation ofgrowth and maintenance respiration, not to the respiration coupledto nitrogenase activity. Although no recovery of nitrogenaseactivity occurred, the elapsed time from the introduction ofacetylene into the gas stream flowing past the nodules untilmeasurement of the acetylene-induced decline in nitrogenaseactivity, decreased substantially. This was characteristic ofan increase in the permeability of the nodules to gaseous diffusionupon rehydration. However, calculated values of nodule diffusionresistance after the 24 h periods of rehydration did not indicateany recovery of gaseous diffusion resistance based on measurementsof the respiration coupled to nitrogenase activity. Hence, useof a diffusion analogue (i.e. Fick's Law) in conjunction withnodule respiratory CO₂ efflux was unable to predict changesin permeability of the variable barrier of legume nodules followingnodule dehydration and recovery. Key words: Subterranean clover, gaseous diffusion, respiration, carbohydrates, drought     

Effects of Water Stress on the Respiratory and Nitrogen-fixing Activity of Soybean Root Nodules

Seventy-five per cent of the N₂-fixing activity (measured asthe reduction of C₂H₂ to C₂H₄) and 50 per cent of the respiratoryactivity of detached soybean root nodules was lost when thewater potential () of the nodules was lowered from approximately–1 ? 105 Pa (turgid nodules) to –9 ? 105 Pa (moderatelystressed nodules). Severely stressed nodules ( = –1.8? 10⁶ Pa) showed almost total loss of N₂-fixing activity andup to 80 per cent loss of respiratory activity. Increasing theoxygen partial pressure (PO₂) from 10⁴ to 10⁵ Pa completelyrestored both N₂-fixation and respiration in moderately stressednodules, but only partial recovery was possible in severelystressed nodules. The activity of the stressed nodules was verylow at low PO₂ (5 ? 10³ and 10⁴ Pa). The C₂H₂-reducing activityof nodule slices, nodule breis, and bacteroids from turgid andmoderately stressed nodules was almost identical but some activitywas lost in the breis and bacteroids from severely stressednodules. Calculations showed that at low PO₂ (10⁴ and 2 ? 10⁴Pa), the rate of O₂ diffusion into severely stressed noduleswas ten times lower than that for turgid nodules, but only fourtimes lower at a higher PO₂ (4 ? 10⁴ Pa). Carbon monoxide inhibitionof C₂H₂ reduction was slower in stressed nodules than in turgidnodules. The results are discussed in view of the possible developmentof a physical barrier to gaseous diffusion and/or the possiblealtered affinity of the nodule leghaemoglobin for O₂ in thewater-stressed nodules.     

Effectiveness of Lotus Root Nodules: III. EFFECT OF COMBINED NITROGEN ON NODULE EFFECTIVENESS AND FLAVOLAN SYNTHESIS IN PLANT ROOTS

When grown in a nutrient solution containing combined nitrogen(NH₄NO₃), Lotus pedunculatus and L. tenuis seedlings inoculatedwith a fast-growing strain of Rhizoblum (NZP2037) did neitherdevelop root nodules nor develop flavolans in their roots. Incontrast, the roots of nodulated seedlings growing in a nitrogen-freenutrient solution contained flavolans. Flavolan synthesis coincidedwith root nodule development on these plants. When added as a single dose, high concentrations of NH₄NO₃ (5and 10 mg N per plant) stimulated the growth of L. pedunculatusplants but suppressed nodulation and nitrogen fixation. In contrastthe continued supply of a low concentration of NH₄NO₃ (1?0 mgN d^–1 per plant) stimulated nitrogen fixation by up to500%. This large increase in nitrogen fixation was associatedwith a large increase in nodule fresh weight per plant, a doublingof nodule nitrogenase activity, and a lowering of the flavolancontent of the plant roots. The close relationship between nitrogendeficiency, nodule development, and flavolan synthesis in L.pedunculatus meant that it was not possible (by nitrogen pretreatmentof plants) to alter the ineffective nodule response of a Rhizobiumstrain (NZP2213) sensitive to the flavolan present in the rootsof this plant.     

Ferric Leghemoglobin in Plant-Attached Leguminous Nodules

Leghemoglobin (Lb) is essential for nitrogen fixation by intact leguminous nodules. To determine whether ferric Lb (Lb3+) was detectable in nodules under normal or stressed conditions, we monitored the status of Lb in intact nodules attached to sweet clover (Melilotus officinalis) and soybean (Glycine max [L.] Merr.) roots exposed to various conditions. The effects of N2 and O2 streams and elevated nicotinate levels on root-attached nodules were tested to determine whether the spectrophotometric technique was showing the predicted responses of Lb. The soybean and sweet clover nodules' Lb spectra indicated predominantly ferrous Lb and LbO2 in young (34 d) plants. As the nodule aged beyond 45 d, it was possible to induce Lb3+ with a 100% O2 stream (15 min). At 65 d without inducement, the nodule Lb status indicated the presence of some Lb3+ along with ferrous Lb and oxyferrous Lb. Nicotinate and fluoride were used as ligands to identify Lb3+. Computer-calculated difference spectra were used to demonstrate the changes in Lb spectra under different conditions. Some conditions that increased absorbance in the 626 nm region (indicating Lb3+ accumulation) were root-fed ascorbate and dehydroascorbate, plant exposure to darkness, and nodule water immersion.     

Defoliation-Induced Stress in Nodules of White Clover: I. CHANGES IN PHYSIOLOGICAL PARAMETERS AND PROTEIN SYNTHESIS

Nodule function and protein synthesis were studied in defoliationstressed white clover plants. Uncut control plants (C) werecompared with plants from two defoliation treatments: (1) continuousdefoliation (CD) where all leaves and petioles were removedeach day; and (2) defoliated/recovered (DR) where, after removalof all leaves and petioles, new leaves were then allowed toregrow. After a single defoliation N₂ fixation (acetylene reductionactivity) and nitrogenase-linked respiration declined by morethan 80% within 3 h and by nearly 100% by 24 h. DR plants beganto fix nitrogen again at a very low level 3 d later and thereafterrose to control levels by 15 d. Continuously defoliated plantsnever recovered N₂ fixation capacity. Nodule protein complementwas assessed by polyacrylamide gel electrophoresis. Major changesoccurred in buffer soluble protein band patterns by 6 d in CDplants, but few changes were evident in SDS soluble proteins.By 9 and 14 d significant disruption of all proteins was evident.The prominent host plant protein, leghaemoglobin (Lb) had disappearedby 14 d. In DR plants the intensity of staining was reducedbut no major changes in band patterns were evident and by 21d nodules were rejuvenated. [³⁵S]-labelled methionine was incorporated into nodule proteinsfrom all treatments throughout the experiment. However, continuousdefoliation caused increasing variability between replicatesin the labelled band patterns. By 21 d CD, much of the labelledprotein was present as amorphous low M_r material which suggestseither disruption of the protein synthesizing machinery or rapidhydrolysis by proteolytic enzymes. Surprisingly [³⁵S]-methionine was never found in Lb from nodulesof any treatment. It is possible that white clover Lb does notcontain any methionine residues or that no synthesis of Lb occurred. Key words: Trifolium repens, white clover, defoliation, protein synthesis, nodules     

Nitrate Effects on Nodule Oxygen Permeability and Leghemoglobin (Nodule Oximetry and Computer Modeling)

Two current hypotheses to explain nitrate inhibition of nodule function both involve decreased O2 supply for respiration in support of N2 fixation. This decrease could result from either (a) decreased O2 permeability (PO) of the nodule cortex, or (b) conversion of leghemoglobin (Lb) to an inactive, nitrosyl form. These hypotheses were tested using alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus) plants grown in growth pouches under controlled conditions. Nodulated roots were exposed to 10 mM KNO3 or KCI. Fractional oxygenation of Lb under air (FOLair), relative concentration of functional Lb, apparent PO, and O2-saturated central zone respiration rate were all monitored by nodule oximetry. Apparent PO and FOLair in nitrate-treated nodules decreased to <50% of values for KCI controls within 24 h, but there was no decrease in functional Lb concentration during the first 72 h. In nitrate-treated alfalfa, but not in birdsfoot trefoil, FOLair, apparent PO, and O2-saturated central zone respiration rate decreased during each light period and recovered somewhat during the subsequent dark period. This species difference could be explained by greater reliance on photoreduction of nitrate in alfalfa than in birdsfoot trefoil. Computer simulations extended the experimental results, showing that previously reported decreases in apparent PO of Glycine max nodules with nitrate exposure cannot be explained by hypothetical decreases in the concentration or O2 affinity of Lb.     

Extreme Phosphate Deficiency Decreases the In Vivo CO2/O2 Specificity Factor of Ribulose 1,5-Bisphosphate Carboxylase-Oxygenase in Intact Leaves of Sunflower

Sunflower plants were grown under controlled environmental conditionswith either 0 or 10 mol m^–3 phosphate (Pi). From steady-statemeasurements of gas exchange and chlorophyll fluorescence madeon intact leaves, the in vivo CO₂/O₂ specificity factor (invivo K_sp) of ribulose 1,5-Aisphosphate carboxylase-oxygenase(Rubisco) was determined following two methods based on modelsof C₃ photosynthesis by Brooks and Farquhar (1985) and Peterson(1989). The two methods gave in vivo K_sp values for controlsunflower leaves which were similar to published values forhigher plants. Extreme Pi deficiency decreased in vivo K_sp,in sunflower leaves compared to adequate Pi. This suggests thatPi deficiency affected photorespiration less than photosynthesis.The decrease in in vivo K_sp may be due to a real change in theenzyme kinetics favouring oxygenation more than carboxylationor due to an increase in the number of CO₂ molecules releasedper oxygenation; in which case the observed decrease in thein vivo K_sp determined on intact leaves will not agree numericallywith the true K_sp of Rubisco determined in vitro using purifiedenzyme from the same leaf. We discuss the implications of therelatively large photorespiration in Pi-deficient sunflowerleaves with respect to the increased dissipation of photosyntheticelectrons and photorespiratory recycling of Pi in thechloroplaststroma. Although our results on in vivo K_sp suggested a relativelylarger photorespiratory potential in Pi-deficient than controlsunflower leaves, photosynthesis was insensitive to O₂ in Pi-deficientleaves; the possible reasons for this phenomenon are discussed.Under extreme Pi deficiency, O₂ sensitivity of photosynthesisis not a reflection of the in vivo photorespiratory rates. Determinationof in vivo K_sp of Rubisco is a useful approach to study thephotorespiratory potential of intact leaves. Key words: Chlorophyll fluorescence, phosphate deficiency, photorespiration, photosynthesis, PSII quantum yield, Rubisco specificity factor     

The Effect of Reduction in the Number of Nodules on Nodule Activity of Faba Bean (Vicia faba cv. Fiord)

Faba bean (Vicia faba L. cv. Fiord) plants were raised in agrowth room for 5 weeks and then transplanted to a hydroponicsystem. After 48 h for acclimation, nine plants were removed(day 0) for the measurement of nitrogenase activity by acetylenereduction (AR), for determination of nodule number and noduleweight, volume of the active N₂ fixing region (VAR), and volumeof the senescent N₂, fixing region (VSR). Half the nodule populationon a further 18 plants was excised, and nine of these plantswere assayed for AR. The nine plants from which the noduleshad been removed (treated plants) and nine control plants witha full complement of nodules, were left to grow for 5 d, afterwhich they were all harvested and assayed. The average weight of nodules and VAR remained constant in thecontrol plants between day 0 and day 5, whereas the nodulesleft on the treated plants increased in weight by 1.2 timesand VAR by 2.2 times. By day 5, VAR per plant was the same inthe control plants as in the treated, whereas VSR of controland treated plants increased by 4.6 and 2.2 times, respectively.Removal of half of the nodules at day 0 halved the AR activityper plant, but specific activity remained the same. After 5d, however, the nodules of the treated plants showed the sametotal activity as those of the control. Thus the specific activityof the nodules left on the treated plants doubled after 5 din response to excision. The indeterminate nodule of faba bean appears to be able toincrease its specific activity substantially in response toincrease in the demand for fixed N. N₂ fixation per nodule wasresponsive to substrate supply, in that halving the number ofnodules on a plant induced the remaining nodules to increaseactivity, presumably because they could use the assimilate previouslydistributed over a larger number of nodules. Victa faba, faba bean, nodule number, nodule activity, acetylene reduction, volumes of active and senescent N₂ fixing regions of nodules, hydroponic system