Carbohydrate Metabolism in Taproots of Medicago sativa L. during Winter Adaptation and Spring Regrowth

Our objective was to identify amylases that may participate in starch degradation in alfalfa (Medicago sativa L.) taproots during winter hardening and subsequent spring regrowth. Taproots from field-grown plants were sampled at intervals throughout fall, winter, and early spring. In experiment 1, taproots were separated into bark and wood tissues. Concentrations of soluble sugars, starch, and buffer-soluble proteins and activities of endo- and exoamylase were determined. Starch concentrations declined in late fall, whereas concentrations of sucrose increased. Total amylolytic activity (primarily exoamylase) was not consistently associated with starch degradation but followed trends in soluble protein concentration of taproots. This was especially evident in spring when both declined as starch degradation increased and shoot growth resumed. Activity of endoamylase increased during periods of starch degradation, especially in bark tissues. In experiment 2, a low starch line had higher specific activity of taproot amylases. This line depleted its taproot starch by late winter, after which taproot sugar concentrations declined. As in experiment 1, total amylolytic activity declined in spring in both lines, whereas that of endoamylase increased in both lines even though little starch remained in taproots of the low starch line. Several isoforms of both amylases were distinguished using native polyacrylamide electrophoresis, with isoforms being similar in bark and wood tissues. The slowest migrating isoform of endoamylase was most prominent at each sampling. Activity of all endoamylase isoforms increased during winter adaptation and in spring when shoot growth resumed. Endoamylase activity consistently increased at times of starch utilization in alfalfa taproots (hardening, spring regrowth, after defoliation), indicating that it may serve an important role in starch degradation.     

Amylolytic Activity in Taproots of Diploid and Tetraploid Medicago sativa L.

Our objective was to determine whether starch degradation intaproots of alfalfa (Medicago sativa L.) after defoliation wasassociated with activity and isoform complement of endo- andexo-amylases. Taproots of genetically comparable diploid (2x)and tetraploid (4x) populations and the tetraploid cultivarHi-Phy were recovered immediately after defoliation and at approx.4-d intervals thereafter. Taproot tissues were analysed forstarch concentrations and activities of endo- and exo-amylases.An electrophoretic blotting technique was used to examine amylaseisoforms. Starch degradation was most rapid in taproots of Hi-Phy,slowest in taproots of the 2x population, with the 4x populationbeing intermediate. The 4x population had a greater initialincrease in endo-amylase activity compared to the 2x population;however, Hi-Phy averaged eightfold greater endo-amylase activitythan either 2x or 4x populations. Although exo-amylase specificactivity was at least 500-fold greater than endo-amylase specificactivity in all populations, changes in endo-amylase activitywere more closely associated with trends in starch degradation.Multiple isoforms of endo- and exo-amylase were observed intaproots of all populations. Taproots of Hi-Phy contained anendo-amylase isoform that was not apparent in the 2x or 4x populationsthat may contribute to the high activity of this amylase intaproots of this cultivar. These results, although correlative,suggest an important role for endo-amylase in taproot starchhydrolysis after defoliation. Medicago sativa (L.), alfalfa, taproots, herbage, diploid, tetraploid, starch, endo-amylase, exo-amylase, isoforms, electrophoresis     

Starch Grain Distribution in Taproots of Defoliated Medicago sativa L

Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.     

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     

Ethanol synthesis and loss from flooded roots of Medicago sativa L. and Lotus corniculatus L.

Abstract Field flooding of established alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) for up to 12 d resulted in a significant increase in alcohol dehydrogenase activity (ADH) and an increase in the K_m of ADH in both species. Root concentration of ethanol increased throughout the flooding regime in alfalfa roots. No ethanol was detected in any trefoil root samples. Alfalfa plants which had shoots removed 5 d prior to flooding accumulated significantly higher levels of root ethanol and showed flooding injury sooner, indicating a significant effect of shoots on development of flooding injury. Alfalfa and trefoil plants grown in the greenhouse were flooded and ethanol in the transpiration effluent was trapped and measured. Alfalfa transpired measurable quantities of ethanol which peaked just prior to development of shoot injury symptoms. No ethanol was detected in the transpiration effluent from trefoil shoots. Flooded roots of both alfalfa and trefoil excreted ethanol but alfalfa roots synthesized more total ethanol and retained a larger proportion in the roots than did trefoil. While the ethanol accumulation response in alfalfa and trefoil are consistent with the ethanol ‘self-poisoning’ hypothesis of flooding injury, the very small quantities of ethanol found in these roots still raises questions as to its absolute effect in the plant.     

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     

Defoliation-Induced Stress in Nodules of White Clover: II. IMMUNOLOGICAL AND ENZYMIC MEASUREMENTS OF KEY PROTEINS

Changes in key nodule proteins following defoliation of whiteclover plants were assessed by measurements of enzyme activitiesand by use of antibodies to specific nodule proteins. Defoliation caused major declines in protein and leghaemoglobinlevels and in the activities of invertase, sucrose synthase,UDP glucose pyrophosphorylase, aspartate amino transferase,glutamine synthetase, phosphoenolpyruvate carboxylase, and malatedehydrogenase. In continuously defoliated plants the activities of these noduleenzymes continued to decline throughout the course of the 17d experiment. In plants defoliated once and then allowed toregrow new leaves, nodule enzyme activities declined for 7 to10 d before increasing again to control levels by the end ofthe experiment. In this single defoliation/recovery treatmentonly the activities of PEP carboxylase and glutamine synthetasedeclined to a greater extent than the general decline in proteincontent. Alcohol dehydrogenase, increased in specific activityfollowing a single defoliation and only declined in nodulesof continuously defoliated plants. Amino peptidase activity declined in concert with other enzymeactivities described above. Endopeptidase activity, in contrast,increased significantly after 4 d following either a singleor continuous defoliation. In the plants allowed to regrow newleaves endopeptidase activity declined to control levels again,whereas in plants continuously defoliated the activity rose5-fold. However, endopeptidase increased only after significantdeclines in protein and other enzyme activities had alreadyoccurred. Western immunoblotting confirmed that the declines in glutaminesynthetase activity and leghaemoglobin levels were due to thedisappearance of antigen. Declines in nitrogenase componentsI and II indicated that bacteroid proteins were affected bydefoliation over the same time-scale as host plant encoded proteins.As new leaves grew and nodule N₂ fixation was reestablished,these specific nodule proteins were again detectable by immunoblotting. Key words: Trifolium repens, white clover, defoliation, nodules, enzymes, antibodies     

Photosynthate Partitioning and Enzymes of Sucrose Metabolism in Sugarbeet Roots

Populations of sugarbeet (Beta vulgaris L.) plants that differed in taproot/leaf weight ratio and in photosynthate partitioning between taproots and fibrous roots did not differ in root/shoot ratio as indicated by relative dry weight distribution. Based on the hypothesis that dry weight distribution is influenced by the metabolism of imported sucrose, we examined the relationships between the activity of the enzymes of sucrose metabolism and dry weight distribution as a function of genotype and ontogeny. A decreased specific activity of acid invertase in taproots was associated with increased taproot/fibrous root weight ratio at 21 and at 28 days post-emergence. Alkaline invertase activity was negatively correlated with taproot/fibrous root weight ratio at 28 days. Sucrose synthetase specific activities of taproots were not correlated with dry matter distribution. Acid invertase may influence photosynthate partitioning between the taproot and fibrous roots via regulation of sucrose levels in the region of fibrous root initiation.     

Etiolated Growth as Measure of Non-structural Biomass in Lucerne Taproots

Enzymatic hydrolysis of starch in lucerne (Medicago sativa L.)taproots is the conventional method used to determine the quantityof carbohydrates allocated to regrowth. Etiolated growth froma taproot could be used to quantify total root biomass allocatedto regrowth. This study compared concentrations of non-structuralcarbohydrates, as measured by -amylase hydrolysis of starchto glucose, to concentrations of non-structural biomass, asmeasured by etiolated growth from lucerne taproots placed inan incubator and plants in situ. The concentration of starchfrom enzymatically assayed taproots was 325 g kg^-1 expressedas glucose equivalents. Etiolated growth and weight loss byrespiration from plants grown in the incubator accounted for524 g of actual biomass per kg of root. There was 46·2g kg^-1 of N, 3·1 of P, and 33·1 of K in the etiolatedgrowth. An 88% increase in etiolated growth dry weight was observedfrom plants in situ compared to taproots placed in the incubator.Accurate quantification on non-structural biomass should notbe limited to sampling just the taproot, but must included theentire root system. Compared to determining non-structural carbohydratesby enzymatic hydrolysis of starch, the procedure used in determiningnon-structural biomass by etiolate growth gave results in unitsrelative to the plant. The use of etiolate growth also providedinformation on mineral nutrient partitioning from root to shoots,was less technically demanding, and could be applied to theentire root system.Copyright 1993, 1999 Academic Press Medicago sativa, root carbohydrates, etiolated growth, taproot     

Long-term Partitioning, Storage and Re-mobilization of 14C Assimilated by Lolium perenne (cv. Melle)

The youngest fully expanded leaves of single tillers of vegetativeperennial ryegrass plants were exposed to ¹⁴CO₂. Thereafter,quantitative and fractional analysis of the partitioning, storageand re-mobilization after defoliation of the ¹⁴C-labelled assimilatewas sequentially conducted over a 22 d period. In undefoliated plants, most ¹⁴C reached its final destinationwithin 5–6 of feeding. Forty per cent of assimilated ¹⁴Cwas subsequently lost through respiration, while 13.5, 8.5 and34 per cent remained in roots, stem bases and tops respectively.At least some ¹⁴C was distributed to tillers throughout theplant, but secondary tillers subtended by the fed tiller madethe greatest demand on ¹⁴C translocated from the fed tiller. A small, but significant portion of ¹⁴C was invested into longterm storage in undefoliated plants, four per cent of the totalassimilated still being present in a labile chemical form inroots and stem bases 22 d after feeding. In plants that wereseverely defoliated 4 d after feeding, depletion of reserve¹⁴C was observed relative to undefoliated plants. The depletiontook place from stem bases, not roots, and both low and highmolecular weight storage compounds were involved. A portionof the depleted ¹⁴C was incorporated into new growth after defoliation. Lolium perenne, perennial ryegrass, assimilate partitioning, storage, re-mobilization, defoliation     

Metabolic response of Medicago sativa L. and Lotus corniculatus L roots to anoxia

Abstract Differential rates of fermentation and energy production have been implicated in the response of plant species to extended root anoxia. This study describes the metabolic response to anaerobiosis of waterlogging-tolerant birdsfoot trefoil (Lotus corniculatus L.) and waterlogging-sensitive alfalfa (Medicago sativa L.). Studies were carried out on glasshouse-grown plants subjected to root anaerobiosis in nutrient solution. Rate of fermentation, as estimated by CO₂ evolution, declined significantly upon anaerobiosis in both species but was proportionally less, relative to the aerobic control, in trefoil. Another indicator of carbon flux through glycolysis, the concentration of glucose-6-phosphate, was also significantly lower in trefoil roots relative to aerobic controls. Both species showed significantly increased root exudation of K⁺, sugars and andno-N, especially during the first 2 d of root anaerobiosis, indicating changes in membrane selective permeability. The energy status of roots subjected to anaerobiosis declined sharply in both species but trefoil roots maintained higher ATP/ADP ratios for up to 4 d of anaerobiosis. The results are consistent with the hypothesis that increased fermentation activity maintains a more favourable root energy status. This higher energy status may facilitate survival by maintaining crucial root activities, such as maintenance of membrane stability.     

Supply and partitioning of assimilates to roots of Medicago sativa L. and Lotus corniculatus L. under anoxia

Abstract A current explanation of the mechanism of flooding injury to roots suggests that oxygen deficiency depresses the supply of respirable carbohydrates sufficiently to inhibit fermentation. However, even though it has been shown that phloem transport of assimilate is sharply reduced to anaerobic roots, inhibition of assimilate metabolism has also been suggested to be an important factor. This study examines these hypotheses by relating assimilate supply and metabolic activity in anoxic roots of alfalfa (Medicago sativa L.), a flood-intolerant species, and birdsfoot trefoil (Lotus corniculatus L.), a flood-tolerant plant. Roots were made anoxic (severe O₂ deficiency) for 2, 4 or 6 d and shoots were labelled with ¹⁴CO₂. Assimilate transport to the roots and metabolism to structural components were significantly decreased in both species in response to anoxia. Trefoil exhibited significantly greater ¹⁴C incorporation into the residue fraction at 4 d anoxia than did alfalfa, and this was consistent with the greater flooding tolerance of trefoil. When assimilate supply to O₂-deficient roots was decreased by shoot shading, shoot fresh weight was reduced by both anoxia and light treatments. Root-soluble sugars were significantly decreased by shading but were greatly increased in response to anoxia. Root starch concentration also increased under anoxia. Root K⁺ concentration was reduced by anoxia only. The energy status (ATP/ADP) of roots was significantly decreased by shading; however, anoxia reduced the energy status only in unshaded plants. The data indicate that carbohydrate supply to anaerobic roots does not appear to be a limiting factor in the metabolic response of alfalfa roots. Alternatively, metabolism of assimilate in anoxic roots may be an important determinant of survival.     

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     

Composition and Structure of Starch from Taproots of Contrasting Genotypes of Medicago sativa L

The objective of this study was to examine the composition and branch chain lengths of alfalfa (Medicago sativa L.) taproot starch during starch utilization and reaccumulation in response to defoliation. Genotypes were propagated vegetatively and well-established plants were sampled at defoliation and at weekly intervals thereafter. Starch granules from root tissues were dispersed in dimethyl sulfoxide and starch components separated using gel permeation chromatography. Root starches also were debranched enzymically, and branch chain lengths were examined. Results indicate that, irrespective of starch concentration, starch from taproots of the high starch genotype was composed of approximately 80% high molecular weight starch with I₂-Kl absorbance characteristics similar to amylopectin. The remaining 20% of the starch was low molecular weight with I₂-Kl absorbance characteristics similar to amylose. Starches of the low starch genotype contained approximately 85% high molecular weight polysaccharide at high root starch concentrations (>50 grams per kilogram). At low root starch concentrations (<10 grams per kilogram), starch from the low starch genotype had nearly equal proportions of low and high molecular weight polysaccharide. The I₂-Kl absorbance properties of the low molecular weight starches from roots of the low starch genotype indicated that some branching may be present. The distribution of chain lengths from amylopectin did not change during starch degradation and reaccumulation for the high starch genotype. In the low starch genotype, the proportion of low molecular weight branches having a degree of polymerization between 1 and 30 was decreased at the very low starch concentrations observed on the 14th day of regrowth. Higher concentrations and/or quantities of starch in roots of the high starch genotype were not associated with greater rate of herbage regrowth, when compared to the low starch genotype.     

Purification and properties of starch hydrolyzing enzymes in mature roots of sugar beets

Mature roots of sugar beets, which accumulate large amounts of sucrose but not starch, nevertheless contained acid and neutral amylases, judging from their pH optima, as well as pullulanase. Acid and neutral amylases were partially purified by procedures including fractionation with ammonium sulfate, ion exchange column chromatography, and gel filtration. Acid amylase was classified as an exoamylase, since it produced only glucose from soluble starch, amylopectin. β-limit dextrin, and rabbit liver glycogen. Neutral amylase was classified as an endoamylase, since it liberated maltose as the main product plus a small amount of glucose and oligosaccharides, and was capable of hydrolyzing β-limit dextrin. Pullulanase was purified to apparent homogeneity by procedures including fractionation with ammonium sulfate, Diethylaminoethyl-cellulose column chromatography and affinity chromatography. Pullulanase was capable of hydrolyzing soluble starch, amylopectin, β-limit-dextrin, and pullulan. Debranching of amylopectin was further evident by an increase in extinction coefficient, and by a shift of λ_max from 530 to 560 nm when the debranched amylopectin formed a complex with I₂-KI.     

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     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

The Influence of Defoliation on the Distribution of Assimilates in Lolium multiflorum Lam

Autoradiographs were made of plants of Lolium multiflorum Lam.after ¹⁴CO₂ had been fixed by selected leaves. The results showedthat labelled compounds were not translocated to other tillersbut were moved to the whole root system. This pattern of distributionwas changed when all or some of the tillers on the plant weredefoliated. Where a single undefoliated tiller remained, itinitially supplied the cut tillers with ¹⁴C-containing products,thus reintegrating a system of apparently independent tillers.When all the tillers were partially defoliated, labelled compoundswere no longer translocated to the root system. A further experimentsuggested that root reserves were not mobilized for regrowthfollowing defoliation. These results are discussed in termsof the integration of a grass plant in the vegetative state.     

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.