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     

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     

Defoliation in White Clover: Regrowth, Photosynthesis and N2 Fixation

Single plants of white clover, grown in a controlled environmentand dependent for nitrogen on fixation in their root nodules,were defoliated once by removing approximately half their shoottissue. Their regrowth was compared with the growth of comparableundefoliated plants. Two similar experiments were carried out:in the first, plants were defoliated at 2.5 g, and in the secondat 1.2 g total plant d. wt. Defoliation reduced rate of N₂ fixation by > 70 per cent,rate of photosynthesis by 83–96 per cent, and rate ofplant respiration by 30–40 per cent. Nodule weights initiallydeclined following defoliation as a result of loss of carbohydratesand other unidentified components. No immediate shedding ofnodules was observed but nodules on the most severely defoliatedplants exhibited accelerated senescence. The original rates of N₂ fixation were re-attained after 5–6or 9 d regrowth, with increase in plant size at defoliation.In general, the rate of recovery of N₂ fixation was relatedto the re-establishment and increase of the plant's photosyntheticcapacity. Throughout the growth of both defoliated and undefoliatedplants nodule respiration (metabolism) accounted for at least23 ± 2 per cent of gross photosynthesis. The unit ‘cost’of fixing N₂ in root nodules, in terms of photosynthate, appearedto be unaffected by defoliation, except perhaps for plants veryrecently defoliated. Similarly, the percentage nitrogen contentsof shoot, root and nodules of defoliated plants became adaptedwithin a few days to those characteristic of undefoliated plants. Trifolium repens, white clover, N₂ fixation, defoliation, photosynthesis, respiration     

Oxygen Supply Limits Nitrogenase Activity of Clover Nodules After Defoliation

The aim of this study was to test the effect of oxygen partialpressure as a possible limiting factor of nitrogen fixationfollowing defoliation. The response of nitrogenase activity(C₂H₂-reduction) of defoliated and undefoliated white and redclover plants (Trifolium repens L. and Trifolium pratense L.)to either 19 kPa oxygen or 55 kPa oxygen was investigated. Priorto defoliation, white clover plants were grown for five weeksin a growth chamber, and red clover plants for 7 or 11 weeksin a glasshouse. The results included measurements of ¹⁶N₂-uptake. Increasing oxygen partial pressure from 19 to 55 kPa severelyrestricted nitrogenase activity of undefoliated white cloverplants; however, 2 h after complete defoliation, the same treatmentcaused a significant increase. A fivefold increase in nitrogenaseactivity upon exposure to the elevated oxygen partial pressurewas found at the end of a 24 h period. This beneficial effectdecreased gradually from 1 to 5 d after defoliation. The responseof recently defoliated red clover plants to 55 kPa oxygen partialpressure was similar to that of white clover, independentlyof plant age. The gradual recovery of nitrogenase activity duringthree weeks of regrowth was associated with a simultaneous changein the response to increased oxygen partial pressure, leadingagain to the response of undefoliated plants. These data suggested that lack of oxygen at the site of nitrogenfixation, resulting from a dramatic increase in oxygen-diffusionresistance, is the main factor limiting nitrogenase activityfollowing defoliation. Trifolium repens L., Trifolium pratense L., white clover, red clover, defoliation, regrowth, nodules, nitrogen fixation, nitrogenase activity, oxygen limitation     

Amylase Activity in Taproots of Medicago sativa L. and Lotus corniculatus L. Following Defoliation

Although the patterns of starch metabolism in taproots of alfalfa(Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatusL.) have been characterized, little is known regarding the activitiesof starch-degrading enzymes in taproots of these species. Ourobjective was to determine how defoliation influences starchdegradation and activities of amylases in taproots of alfalfaand birdsfoot trefoil. In Exp. 1, amylolytic activities andstarch concentrations in taproots of defoliated and undefoliatedplants were compared on days 0, 3, 7, 10, and 14 after defoliation.Taproot starch concentrations declined in defoliated plants,while increasing in taproots of undefoliated plants. Exoamylaseactivities in taproots of defoliated plants did not change withdefoliation, while endoamylase activities increased 2-fold indefoliated alfalfa and 50% in defoliated birdsfoot trefoil plantswhen compared to undefoliated plants. In Exp. 2, activity andisoform complement of amylases were monitored during seedlingdevelopment. High endoamylase activity was found in taprootsof both species at all samplings. In contrast, exoamylase accumulatedin taproots of alfalfa, but not birdsfoot trefoil, in a patternsimilar to starch accumulation. As in Exp. 1, defoliation increasedendoamylase, but not exoamylase activity in taproots of bothspecies. Taproots of both species contained one major and twominor endoamylase isoforms, but the electrophoretic mobilityof these isoforms differed between species. Activities of allisoforms, as indicated on starch-gel blots, increased in responseto defoliation. These results indicate that defoliation increasesactivity of taproot endoamylases, whose activity is associatedwith taproot starch degradation. Key words: Starch degradation, alfalfa, birdsfoot trefoil, enzymes     

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     

Responses of N2 Fixation-linked Respiration to Host-plant Energy Status in White Clover Acclimated to a Controlled Environment

Ryle, G. J. A., Powell, C. E. and Gordon, A. J. 1988. Responsesof N₂ fixation-linked respiration to host-plant energy statusin white clover acclimated to a controlled environment.—J.exp. Bot. 39: 879–887. Single plants of white clover, acclimated to a controlled environmentand dependent for nitrogen on N₂ fixation in their root nodules,were darkened, defoliated or exposed to enhanced CO₂ levelsto establish the quantitative relationships between the photosynthesisof the host plant and the N₂ fixation metabolism of root nodules. The nodule respiration associated with N₂ fixation (FLR) declinedrapidly to 10–15% of its normal rate following plant darkeningearly in the photoperiod. Darkening at progressively later intervalsduring the photoperiod demonstrated a positive, apparently linearrelationship between duration of illumination and total FLRduring the photoperiod and the following night period. Completeor partial defoliation reduced FLR according to the leaf arearemoved: again, there was a strong positive correlation betweencurrent rate of photosynthesis, whether of defoliated or undefoliatedplants and the FLR of root nodules. Doubling the current rateof photosynthesis, by enhancing CO₂ levels around the shoots,promoted FLR within 1–2 h when plants were stressed bylack of light. However, enhanced CO₂ levels increased FLR onlyslowly over a period of several hours in plants entrained tothe normal growing conditions. It is concluded that, in these plants acclimated to a uniformand favourable controlled environment, the supply and utilizationof photosynthetic assimilate in N₂ fixation was finely balancedand quantitatively linked during a single diurnal period andthat nodule functioning was not depressed by lack of energysubstrate. Key words: White clover, N₂ fixation, photosynthesis.     

The Effect of Defoliation on Carbohydrate, Protein and Leghaemoglobin Content of White Clover Nodules

Single white clover plants grown in pots of Perlite in a controlledenvironment and completely dependent on N₂ fixation were defoliatedto various degrees (46–85 per cent of shoot weight removed).The soluble protein content of nodules declined by about 20per cent and leghaemoglobin content by 50 per cent in the first4–7 d after defoliation but increased again to controllevels as new leaf tissue appeared. In the short term (2–3h) carbohydrate content of nodules declined to different extentsdepending on the severity of defoliation. The initial declinein N₂ fixation and the respiration associated with it, appearednot to be related to the instantaneous carbohydrate contentof nodules but rather to the supply of current photosynthatefrom the shoot. After 24–48 h, however, the carbohydratecontent of nodules had declined to low levels, regardless ofthe severity (46 or 71 per cent shoot removed) of defoliation.As new leaf tissue appeared carbohydrate levels in all partsof the plant gradually recovered towards control levels. Microscopic examination of nodule sections indicated that onlyafter very severe defoliation (80–85 per cent shoot removed)was nodule deterioration evident. Even here, as the plant establishednew leaves, the damage to nodules was repaired and no noduleloss was apparent. Trifolium repens, white clover, defoliation, carbohydrate, protein, leghaemoglobin     

Increased Defoliation Frequency Depletes Remobilization of Nitrogen for Leaf Growth in Grasses

Plants ofLolium perenneandFestuca rubrawere grown in sand culturereceiving all nutrients as a complete nutrient solution containing1.5 mMNH₄NO₃, and subjected to one of three defoliation treatments:undefoliated, defoliated on one occasion, or defoliated weekly.¹⁵Nlabelling was used to determine the rate of N uptake, allowingthe amount of N remobilized from storage for the growth of thetwo youngest leaves (subsequently referred to as ‘newleaves’) growing over a 14 d period after defoliationto be calculated. The total plant N uptake by both species wasreduced, compared with undefoliated plants, by both a singleand repeated defoliation, although neither caused complete inhibitionof uptake. Regularly defoliatedL. perennehad a greater reductionin root mass, concomitant with a greater increase in N uptakeper g root than did regularly defoliatedF. rubra. In both species,the amount of N derived from uptake recovered in the new leaveswas unaffected by the frequency of defoliation. BothL. perenneandF.rubramobilized nitrogen to the new leaves after a single defoliation,mobilization being sufficient to supply 50 and 41%, respectively,of the total nitrogen requirement. In regularly defoliated plants,no significant nitrogen was mobilized to the new leaves inL.perenne, and only a small amount was mobilized inF. rubra. Plantsachieved greater leaf regrowth when only defoliated once. Weconclude that increasing the frequency of defoliation of bothL.perenneandF. rubrahad little effect on the uptake of nitrogenby roots which was subsequently supplied to new leaves, butdepleted their capacity for nitrogen remobilization, resultingin a reduction in the rate of growth of new leaves. Lolium perenne; Festuca rubra; defoliation frequency; mobilization; root uptake; nitrogen     

Carbon and Nitrogen Yield, and N2 Fixation in White Clover Plants Receiving Simulated Continuous Defoliation in Controlled Environments

Single plants of white clover grown in controlled environments,and dependent for nitrogen on N, fixation, were defoliated at1 or 2 d intervals to 3, 2 and 1 expanded leaves per stolon(Expt 1), and to 1,0.5 (1 leaf on every alternate stolon) and0 expanded leaves per stolon (Expt 2), for 43–50 days Plants adapted to severe defoliation by developing much smallerleaves with a slightly reduced specific leaf area, more stolons,a smaller proportion of weight in leaf, root and nodules anda greater proportion of weight in stolons. The daily yield (materialremoved by defoliation) of d. wt and nitrogen generally decreasedwith severity of defoliation, as did the residual plant weight.However, the ‘efficiency’ of yield (daily yield/residualweight x 100) of dry matter and nitrogen was greater in themost severely defoliated treatments, attaining a maximum of5–6 % All plants adapted to the imposed defoliation regimes, howeversevere, with the result that even plants maintained withoutany fully expanded leaves invested a similar fraction of theirmetabolic resources in shoot and root as less severely defoliatedplants, and continued to grow and fix N₂, albeit at a very reducedrate of 1–2 mg Nd^–11. The energetic cost of N₂ fixation(acetylene reduction) remained constant in all treatments at31 mole CO₂ mole C₂H₄^–1, but there was some evidence thatrate of N₂ fixation per unit of nodule weight declined in themost harshly defoliated treatment. Trifolium repens, white clover, continous defolation, growth, N₂ fixation     

Nitrogenase Activity in Response to Darkening and Defoliation of Alnus incana

Huss-Danell, K. and Sellstedt, A. 1985. Nitrogenase activityin response to darkening and defoliation of Alnus incana. —J.exp. Bot. 36: 1352–1358 In the Alnus-Frankia symbiosis the nitrogen-fixing root nodulesare one of the sinks for carbon compounds newly formed in photosynthesisand exported from the leaves (source). The source-sink ratioof cloned plants of Alnus incana was reduced by darkening orby total or partial defoliation and the resulting nitrogenaseactivity (C₂H₂-reduction) was measured. Nitrogenase activityhad nearly ceased 5 h after total defoliation but not untilca. 5 d after total darkening. Most of the activity was lostduring the initial hours and days, respectively. When leaf areawas reduced approximately by half nitrogenase activity decreasedslightly less than by half. Removal of upper leaves seemed lessharmful than removal of lower leaves one day after defoliation.On the following 2 d the treatments appeared to be similar.Thus, nitrogenase activity was largely dependent on newly formedassimilates but could also depend on stored reserves that weremobilized. Measurements of in vitro nitrogenase activity inroot nodule homogenates from darkened plants indicated thatnitrogenase gradually became inactivated and/or depleted after1 and 2 d in darkness Key words: Carbon supply, Frankia, nitrogen fixation     

Photorespiratory Enzyme Activities in C3 and CAM Forms of the Facultative CAM Plant, Mesembryanthemum crystallimum L.

The extent to which photorespiration occurs in CrassulaceanAcid Metabolism (CAM) plants has received limited attention.No comparative studies of C₃ and CAM development have been made.To address this problem, activities of several photorespiratoryenzymes were measured in a facultative CAM plant—Mesembryanthemumcrystallinum L.—during induction of CAM by water stress(NaCl-treatment). Salt-treatment over a 22 d period produceda progressive change in metabolism from C₃ to CAM. This wasconfirmed by (I) changes in gas exchange from C₃ fixation tothe characteristic CAM pattern of nocturnal CO2 uptake; (2)increases in did acid fluctuation and (3) a 30-fold increasein phosphoenol pyruvate (PEP) carboxylase activity. In contrast,no significant changes were observed in the activities of glycollateoxidase (GO), NAD-dependent-hydroxypyruvate reductase (HPR),glutamine synthetase (GS) or glutamate dehydrogenase (GDH) whenplants were induced into CAM. Ion exchange chromatography onDEAE Sephacel detected only one GS isoenzyme (GS2, chloroplastic)at all stages of CAM induction, Western blot analysis, however,detected an additional, although minor, band of GS1 (cytosolic),in C₃ plants, which disappeared following CAM induction. Ourresults show that, after development of CAM, these plants stillretain the capacity to photorespire. This may be an essentialrequirement of CAM plants growing in habitats with variablerainfall. When water availability is high, stomata may openin the light allowing ribulose bisphosphate carboxylase oxygenase(Rubisco) activity and photorespiration to occur. The inherentcapacity to photorespire would allow plants to survive variableperiods of rainfall. Key words: Mesembryanthemum, C₃, CAM, photorespiration     

Defoliation in White Clover: Nodule Metabolism, Nodule Growth and Maintenance, and Nitrogenase Functioning During Growth and Regrowth

In two experiments, the functioning and metabolism of nodulesof white clover, following a defoliation which removed abouthalf the shoot tissue, were compared with those of undefoliatedplants. In one experiment, the specific respiration rates of nodulesfrom undefoliated plants varied between 1160 and 1830 µmolCO₂ g^–1h^–1, of which nodule ‘growth and maintenance’accounted for 22 ± 2 per cent, or 27 ± 3.6 percent, according to method of calculation. Defoliation reducedspecific nodule respiration and nodule ‘growth and maintenance’respiration by 60–70 per cent, and rate of N₂ fixationby a similar proportion. The original rate of nodule metabolismwas re-established after about 5 d of regrowth; during regrowthnodule respiration was quantitatively related to rate of N₂,fixation: 9.1 µmol CO₂ µmol^–1N₂. With the possible exception of nodules examined 24 h after defoliation,the efficiency of energy utilization in nitrogenase functioningin both experiments was the same in defoliated and undefoliatedplants: 2.0±0.1 µmol CO₂ µmol^–1 C₂H₄;similarly, there was no change in the efficiency of nitrogenasefunctioning as rate of N₂ fixation increased with plant growthfrom 1 to 22 µmol N₂ per plant h^–1. Exposure of nodulated white clover root systems to a 10 percent acetylene gas mixture resulted in a sharp peak in rateof ethylene production after 1.5–2.5 min; subsequently,rate of ethylene production declined rapidly before stabilisingafter 0.5–1 h at a rate about 50 per cent of that initiallyobserved. Regression of ‘peak’ rate of ethyleneproduction on rate of N₂ fixation indicated a value of 2.9 µmolC₂H₄ µmol^–1 N₂, for rates of N₂ fixation between1 and 22 µmol N₂ per plant h^–1. The relationshipsbetween nitrogenase respiration, acetylene reduction rates andN₂ fixation rates are discussed. Trifolium repens, white clover, defoliation, nodule respiration, N₂, fixation, nitrogenase     

The Effect of Lenient Defoliation on the Nitrogen Economy of White Clover: The Contribution of Mineral Nitrogen to Plant Nitrogen Accumulation During Regrowth

Single plants of white clover (Trifolium repens L.) were grownfrom stolon cuttings rooted in sand. All plants were inoculatedwith Rhizobium trifolii, and for 14 weeks received nutrientsolution containing 0.5 mg N each week, as either ammonium ornitrate. Plants were then leniently defoliated or were leftintact and a ¹⁵N-labelled N source was applied at intervalsof 4 d to replace the unlabelled N. Lement defoliation removedfully expanded leaves only; the remaining immature leaves accountedfor 39–44% of the total. At harvests over the following21 d, leaf numbers were counted and dry matter (DM), N contentsand ¹⁵N enrichments of individual plant organs were determined. Rates of leaf emergence and expansion were accelerated in defoliatedplants; numbers of young leaves were similar in defoliated andintact plants. Total DM and N content were less in defoliatedthan intact plants and were not affected by form of N supplied.DM of young leaves, growing points and stolons and N contentof young leaves were, however, greater when ammonium ratherthan nitrate N was supplied. Rates of increase in the contentof plant total N were 8.2 ± 1.36 mg N d^-1 and 10.2±1.82 mg N d^-1 in defoliated and intact plants respectively.The increases were predominantly due to N₂ fixation, since recoveryof ¹⁵N showed that less than 1% of the increment in plant totalN was assimilated mineral N. Nevertheless, the contributionof mineral N to plant total N was 50% more in defoliated thanin intact plants; higher amounts of mineral N were found particularlyin young leaves and growing points. Partitioning of mineralN to nodulated roots increased over time and was greater whenammonium rather than nitrate N was present. White clover, Trifolium repens L. cv. S184, lenient defoliation, N accumulation, N₂ fixation     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Development of Endopeptidase Activity in Cotyledons of Vigna mungo Seedlings: Effects of Exogenously Applied End-Products and Plant Hormones

The development of endopeptidase activity in cotyledons of Vignamungo seedlings was examined after application of exogenousamino acids, sugars and plant hormones. The endopeptidase activityin the cotyledons fell when germinating seeds were allowed toabsorb a solution of amino acids at high concentrations, andit was postulated that this effect might have been caused inpart by osmotic stress and in part by end-product repression.Protein immunoblotting with an antiserum against SH-EP, themajor cysteine endopeptidase occurring in the cotyledons, showedthat sugars and amino acids at high concentrations also delayedthe post-translational processing of SH-EP intermediates. Endopeptidaseactivity equivalent to nearly twice that in controls was observedwhen GA₃ was applied at 10 to 100 µM to cotyledons thathad been detached from the embryonic axis. In addition, naphthaleneaceticacid at 1 to 100 µM, kinetin at 1 to 10 µM and jasmonicacid at 1 to 10 µM also increased the activity to a limitedextent. Results of pulse-chase experiments suggested that theeffect of GA₁ on the endopeptidase activity in the detachedcotyledons was attributable to suppression of the degradationof the enzyme. Protein immunoblotting revealed the presenceof 34-kOa and 35-kDa intermediates of SH-EP in addition to previouslyreported 36-kDa and 43-kDa intermediates. (Received June 26, 1995; Accepted October 16, 1995)     

Salt Tolerance in the Halophyte, Suaeda maritima (L.) Dum.: Properties of Malic Enzyme and PEP carboxylase

Malic enzyme and phosphenol pyruvate carboxylase activitieshave been isolated and characterized from the shoots of Suaedamaritima plants grown in culture solution (with and withoutNaCl) or in tap water. The enzymes isolated from the lattershowed increases in both specific activity and K_m values incomparison with plants grown in culture solution: however, theaddition of NaCl to the culture solution had no significanteffect on either enzyme. Malate levels were high in plants grownin tap water, reduced by an ordeT of magnitude by the additionof culture solution and then halved by the addition of NaCl. Both enzymes were inhibited in vitro by NaCl, although the additionof high concentrations of betaine and proline to the assay mediumdid not further inhibit enzyme activity. The significance ofthese results is discussed in relation to the proposed roleof betaine and proline as cytoplasmic osmoregulators. Suaeda maritima, halophyte, salt tolerance, malic enzyme, PEP carboxylase     

No Light Activation and High Malate Sensitivity of Phosphoenolpyruvate Carboxylase in Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts (GCP) were prepared from leaves of Commelinacommunis L. and phosphoenolpyruvate carboxylase (PEPc) activityrecorded after injection of the protoplasts directly into theassay medium. The GCP were lysed immediately by the presenceof Triton X-100 and a lowered osmotic concentration in the assaycuvette enabling PEPc activity to be measured with ‘nascent’enzyme. There was no light activation of the enzyme with K_mPEP (about 3.7 mol m^–3) and Vmax being similar in light-ordark-treated protoplasts. Illumination of the GCP in the presenceof CO₂-free air and KCI, a treatment which is known to swellGCP, did not change the kinetics. PEPc activity at saturating PEP was very sensitive to malateinhibition, 20 mmol m^–3 (the I₅₀ value) inhibiting activityby about 50%. Inhibition was similar in light- or dark-treatedprotoplasts. Malate inhibition was, however, much less (I₅₀= 500 mmol m^–3) if the enzyme source was a protoplastextract kept in the absence of glycerol. Inclusion of 20% glycerolin the extraction medium maintained the enzyme in the malate-sensitiveform as occurred in the in vivo assays. The high apparent K_mPEP and the high sensitivity to malate inhibition of GCP PEPcare features unlike those observed with PEPc from leaf tissuesof C₄ and CAM plants and from GCP extracts. PEPc activity increased slightly in the presence of KCI in theassay medium up to about 10 mol m^–3 and thereafter activityslowly declined as KCI concentrations increased further. Key words: Guard cell protoplasts, phosphoenolpyruvate carboxylase     

Studies on the Relation Between Net Photosynthesis and Nitrogenase-linked Respiration in Subterranean Clover

The relation between the rate of nitrogenase-linked respirationand net photosynthesis, and the effect of defoliation on thisrelation, was studied in plants of subterranean clover (Trifoliumsubterraneum L. cv. Seaton Park). Nitrogenase-linked respirationwas estimated as the difference between the rate of nodulatedroot respiration at 21% O₂ and at 3% O₂. The level to which the rate of nitrogenase-linked respirationfell several hours after defoliation was directly proportionalto the decline in the rate of net photosynthesis. Approximately9% of net photosynthesis was always expended in nitrogenaseactivity, irrespective of whether or not the plants were defoliated.This proportion was maintained during the first 3 d of regrowth. To determine whether the decline in nitrogenase-linked respirationafter defoliation was due solely to the decline in the rateof photosynthesis, a further experiment was conducted in whichthe pre-defoliation rate of net photosynthesis was restoredimmediately (with supplementary light) or within 5 min (supplementarylight and CO₂) after defoliation. Restoring the rate of netphotosynthesis did not prevent the post-defoliation declinein nitrogenase-linked respiration. However, when photosynthesiswas reduced to zero by the imposition of darkness, and the rateof nitrogenase-linked respiration allowed to decline to a steadyrate after 3 h, a rapid recovery in the rate of nodulated rootrespiration began within 2 h of returning the plants to thelight. It was hypothesized that a ‘shoot factor’,which was affected by defoliation, could override the apparentrelation between nitrogenase-linked respiration and the rateof current photosynthesis. Key words: Defoliation, N₂ fixation, photosynthesis, nitrogenase-linked respiration, subterranean clover