carbohydrates in soybean nodules · with roots for carbohydrates, especially the disaccharides...

Plant Physiol. (1980) 66,4714760032-0889/80/66/047 1/06/$00.50/0

Carbohydrates in Soybean NodulesII. DISTRIBUTION OF COMPOUNDS IN SEEDLINGS DURING THE ONSET OF NITROGEN FIXATION1

Received for publication March 6, 1980 and in revised form May 19, 1980

JOHN G. STREETERDepartment ofAgronomy, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691

ABSTRACT

During the first few days of nitrogen fixation activity by soybean (Glycinemax (L.) Meff) root nodules, D-chiro-inositoL myo-inositol, sucrose, a,a-trehalose, and maltose accumulate rapidly and reach concentrations severalfold greater than concentrations in other plant organs. Concentrations ofD-pinitol in nodules (-1.0 milligrams per gram fresh weight) were similarto concentrations in leaf blades. The concentration of fructose in noduleswas lower than concentrations in other plant organs.

Comparison of nonnodulated roots, nodulated roots (after removal ofnodules), and nodules indicated that nodules may compete successfullywith roots for carbohydrates, especially the disaccharides sucrose, a,a-trehalose, and maltose. Based on the isolation of protoplasts and bacte-roids, it was tentatively concluded that the highest concentrations ofcyclitols in soybean nodules are located in the infected region and that,inside infected cells, the highest concentrations of D-pinitol and myo-inositol are outside of bacteroids.

Evidence for the identification of D-chiro-inositol and maltose in soybeannodules is presented.

In the summary of a paper written 45 years ago, Allison (1)concluded that "the quantity of nitrogen fixed by legumes growingunder conditions favorable for fixation usually, but not always,varies closely with the carbohydrate supply." More recent effortshave confirmed the idea that major portions of legume photosyn-thate are translocated to nodules and consumed there in supportofN fixation, nodule maintenance, and assimilation of ammonia(12, 16, 18). Recent estimates of carbohydrate utilization by leg-ume nodules or nodulated roots suggest the oxidation of 3 to 7 gof C/g N fixed (12, 18).

In spite of the obvious importance of carbohydrates in nodulefunction, there have been few attempts to understand the utiliza-tion of carbohydrates at the level of individual compounds. Bachet al. (5) exposed soybean plants to '4CO2 for several h andmeasured the distribution of radioactivity in carbohydrates, or-ganic acids, and amino acids in nodules. The labeling of sucrose,glucose, fructose, and several unknown neutral compounds wasreported and the inclusion of a dark period after 14CO2 exposurewas found to influence the distribution of label (5).

Recently, most of the H20-soluble carbohydrates in soybeannodules were identified (23). A significant finding was that threecyclitols, myo-inositol, D-pinitol, and D-chiro-inositol, are majorconstituents of nodules and that D-pinitol concentration appearedto be correlated with nitrogenase activity (23). Here, a detaileddescription of the distribution of cyclitols and sugars in soybean

'Approved for publication as Journal Article 37-80 of the Ohio Agri-cultural Research and Development Center.

plants during the onset of N fixation in nodules is reported.Evidence to support the identification of D-chiro-inositol andmaltose is also presented.

MATERIALS AND METHODS

Plant Culture. Soybean seeds (Glycine max (L.) Merr.), cv."Beeson", were planted in silica sand and inoculated with acommercial preparation of Rhizobium japonicum (21). N-free nu-trient solution was supplied four times/day, as described previ-ously (21), except that no NO3 was supplied at any stage ofgrowth. In the comparison of nodulated and nonnodulated plants,all pots were planted with Beeson soybeans but half of the potswere not inoculated. Because the sand used contains no R. japon-icum, uninoculated plants were completely devoid of nodules.Plants were grown in a greenhouse with supplemental fluorescentand incandescent light and a photoperiod of 15 h.

Analysis of Seedlings. Nitrogenase activity was determinedusing nodulated roots and 10% (v/v) C2H2 as described previously(21). After removal of nodules for determination of fresh weight,these roots and nodules were discarded.

For analysis of carbohydrate, duplicate samples each consistingof six to nine plants were washed free of sand, divided intocotyledon, leaf blade, stem plus petiole, root, and nodule portions,weighed, and immediately ground in 95% (v/v) ethanol. Aftercentrifugation, the residue was re-extracted three or four timeswith 80%1o (v/v) ethanol. Combined extracts were evaporated todryness in vacuo, dissolved in H20, and stored over a few mlchloroform at 2 C. Carbohydrate composition was determined byreacting dry portions of crude extracts with hydroxylamine toform the oximes of glucose, fructose, and maltose, followed byformation of TMS2 derivatives using hexamethyldisilazane andtrifluoroacetic acid (23). GLC was the same as described previ-ously (23) using f3-phenylglucose as an internal standard.

Isolation of Bacteroids and Protoplasts. For bacteroid isolation,nodules were ground in a mortar in 2 volumes 20 mm KH2PO4+ 0.20 M ascorbate (pH 7.4), and 2 g insoluble PVP (PolyclarAT)/g nodule was added to the slurry (9). After straining themixture through four layers of cheesecloth and centrifugation at50OOg for 15 min, the pellet (bacteroid fraction) was resuspendedin 15 volumes phosphate-ascorbate buffer and recentrifuged at5,000g for 10 min to give bacteroid and bacteroid-wash fractions.The original supernatant was centrifuged at 40,000g for 15 min toyield 40,000g pellet and "cytosol" fractions. Bacteroid and 40,00Ogpellet fractions were extracted several times with 75% (v/v) ethanolcontaining 0.33 pmol HCl/ml. After evaporation of most of theethanol under a stream of air, extracts were passed throughcolumns of Dowex 50-H+ and Dowex 1 formate at 2 C to removecations and anions. Supernatant fractions were passed throughion-exchange columns directly (2 C). Effluent from the ion-ex-

2 Abbreviations: TMS: trimethylsilyl; DAP: days after planting.

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Plant Physiol. Vol. 66, 1980

change columns was adjusted to pH = 7.0 ± 0.1, dried under astream of air (with chloroform), and analyzed for carbohydratecomposition.

Protoplasts were isolated by incubation of nodule slices (10 to15 g fresh weight) with commercial preparations of hydrolyticenzymes (8). A medium containing salts (14) instead of mannitolas the osmoticum was used to allow removal of the osmoticum byion exchange before GLC analysis of carbohydrates. Incubationmedium and all glassware were sterilized by autoclaving beforethe experiment, but sterile conditions could not be maintainedduring all operations.Use of the medium (14) and enzymes (8) described here results

in complete solution of cell walls in the infected region of soybeannodules in 2 to 6 h but leaves most of the cortex intact, even after24 h. Three to five cell layers of the inner cortex may be freed,however, so that the separation of infected protoplasts and cortexis not perfect. Infected protoplasts were separated from corticalrings with 100-mesh screen and, after settling, the medium wasdrawn off and protoplasts were very gently washed twice withmedium lacking enzymes. Medium and washes were discardedand protoplasts and cortical rings were macerated in 95% (v/v)ethanol using TenBroeck homogenizers. After centrifugation andtwo re-extractions with 75% (v/v) ethanol, combined extracts weredried, re-dissolved in water, passed through columns of Dowex50-H+ and Dowex 1 formate at 2 C to remove salts, and analyzedfor carbohydrate concentration.

RESULTS AND DISCUSSION

CARBOHYDRATES IN NODULES

A typical chromatogram of the neutral compounds in soybeannodules is shown in Figure 1. Peak 4 has recently been identifiedas sequoyitol. This cyclitol was not quantified in the experimentsdescribed here. Work on the identification of the unknown shownas peak 7 (Fig. 1) remains incomplete and, except for somepreliminary data (Fig. 5), discussion of this compound will bedeferred until its identity is known.The chromatogram shown in Figure 1 was obtained with a

nodule extract passed through ion-exchange resins. Crude extracts,which are generally analyzed, yield more complex chromatogramsdue to the presence of ionic compounds (e.g. quinic acid, gluconicacid) which are silylated under the conditions employed. Thepractice of routinely analyzing crude extracts (23) is still recom-mended because of the danger of hydrolyzing disaccharides on

ion exchange resins and because there seems to be little or nointerference by ionic compounds with the quantitation of neutralcompounds. A chromatogram of neutral compounds was chosenfor Figure 1 to illustrate that most of the major mono- anddisaccharides in soybean nodules have been accounted for.

D-Chiro-inositol. The identity of chiro-inositol was previouslyclaimed (23), but with only meager documentation. Electronimpact mass spectra of the TMS-ether of this compound weresubsequently obtained. The fragmentation pattem of peak 2 (Fig.1) was in good agreement with the results of Sherman et al. (20)for authentic chiro-inositol. The fragmentation patterns for unsub-stituted cyclitols tend to be unique among carbohydrates withvery prominent fragments having mle 305 and 318, and prominentfragments with mle 432, 433, and 507 (20). The mass spectrum ofpeak 2 indicated that the compound was one of the nine possibleunsubstituted cyclitols (4, 20).

Identity of the compound can be narrowed down by comparingthe proportions ofvarious fragments among isomers. For example,the ratio of mle 318 to mle 305 (found 1.7) eliminates myo-, epi-,and cis-inositol (20). The ratio of mle 432 to mle 507 (found 1.4)eliminates scyllo-, allo-, and muco-inositol (as well as epi- andcis-)(20). The elimination of allo- and scyllo-inositols is leastcertain based on the mass spectra alone. However, allo-inositol is

4-SED3-FAt 5-GW 7-UNK I-TRE2-OIN 9-SUCR

FIG. 1. Gas-liquid chromatogram of TMS derivatives of the neutralcompounds in an ethanol extract of whole soybean nodules. PIN: D-pinitol; CIN: D-chiro-inositol; FRU: fructose; SEQ: sequoyitol; GLU:glucose; MIN: myo-inositol; UNK: unknown; IS: internal standard (,B-phenylglucose); SUCR: sucrose; TRE: a,a-trehalose; and MALT: maltose.The retention time of maltose is between 17 and 18 min.

not known to occur in nature (4, 10) and scyllo-inositol can bedistinguished easily from chiro-inositol by its migration in variouspaper chromatography solvents (I 1). The compound correspond-ing to peak 2 in Figure 1 has been purified from soybean nodulesand has a RF identical to authentic chiro-inositol in solvents A, E,and H described by Kindl (1 1). Based on all ofthese considerationsplus the fact that the purified compound is strongly dextrorotatory,peak 2 can unequivocally be identified as D-chiro-inositol.

Maltose. A mass spectrum of the TMS-ether of the (presumed)oxime of peak 11 (Fig. 1) was also obtained. Mass spectra ofTMS-disaccharides are not especially diagnostic and have notbeen extensively studied (7, 13). However, the mass spectrum ofpeak 11 served to at least rule out compounds like galactopinitol,which may elute near maltose in GLC analysis and has recentlybeen purified from soybean seeds (19).The compound represented by peak I1 (Fig. 1) was degraded

by a-glucosidase (Sigma, Type III, boiled enzyme control) whichmeans that it is almost definitely an a-glucosyl disaccharide. Thea-glucosyl disaccharides sucrose, trehalose, turanose, and isomal-tose were ruled out by virtue of distinctly different retention timeson a column of 3.0%o OV-17 (23). The a-glucosyl disaccharideskojibiose and nigerose were ruled out based on the elution ofoxime TMS ethers of these compounds relative to maltose from1.5% OV-17 and 1.5% SE-52 columns (24).There are a few a-glucosyl disaccharides reported in plants (see

ref. 15) which could not be ruled out as peak 11. However, the co-chromatography of peak 11 and maltose has been observed underseveral different GLC conditions. For example, the shift in relativeretention time of maltose TMS ether relative to the oxime TMSether is large compared to other disaccharides (24). The shift inrelative retention time of the TMS ether of peak 11 was exactlypredicted by the results of Toba and Adachi for maltose (24). Thecombined GLC results plus the hydrolysis of the compound bya-glucosidase strongly suggest that peak 11 is maltose.

472 STREETER

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CARBOHYDRATES IN SOYBEAN NODULES

Root Nodules of Other Legumes. Carbohydrate composition ofroot nodules from two other legumes-birdsfoot trefoil (Lotuscorniculatus L.) and red clover (Trifolium pratense L.)-was as-sessed to establish that compounds shown (Fig. 1) are not uniqueto soybean nodules. With the exception of sequoyitol, the presenceof which was not clearly established, all of the compounds shown(Fig. 1) were positively identified in nodules of both plant species.

DISTRIBUTION OF COMPOUNDS IN SEEDLINGS

Experiment I. In this experiment, plants were sampled at 3- to7-day intervals from 4 to 43 DAP, and distribution of carbohydratecompounds in nodules, roots, stems + petioles, and leaf bladeswas determined. Two samples were obtained on each harvest date,but differences between concentrations of compounds in duplicatesamples were so small (almost always <10o) that ranges were notplotted for Figures 2 and 3.The increase in fresh weight (Fig. 2) illustrates the growth of

these plants. Fresh weight of stems + petioles (not shown) rangedfrom 0.16 g at 7 DAP to 7.13 g/plant at 43 DAP. Microscopicexamination of roots and nodules indicated many radial divisionsof root cortex 7 DAP, differentiation of an infected "region" 10DAP, and vascular bundles near the infected region 13 DAP.Very rapid growth and differentiation of nodules was notedbetween 13 and 17 DAP, the sampling date when nodules werefirst collected for carbohydrate analysis and when C2H2 reductionactivity was first observed.

D-PinitOl was a major component of cotyledons and very youngroots but concentrations declined to less than 0.1 mg/g freshweight by 20 DAP. Pinitol in cotyledons and young roots may bederived from galactopinitol which is a component of soybeanseeds (19). Pinitol has previously been reported as a major con-stituent of soybean leaf blades (17) and concentrations in noduleswere found to be similar to concentrations in leaf blades (Fig. 2).D-Chiro-inositol in young cotyledons (Fig. 2) may be derived bydemethylation of D-pinitol (3, 10). From 17 to 43 DAP, there wasa linear increase in the concentration of chiro-inositol in contrastto the essentially complete absence of this compound in adjacentroots. The pattern of myo-inositol distribution was very similar tothe pattern of chiro-inositol distribution with the exception ofrelatively low concentrations of myo-inositol in young cotyledons.Both chiro- and myo-inositol were detectable in leaf blades, but atconcentrations much lower than those in nodules.

Sucrose was readily detected in all tissues (Fig. 3). At 4 DAP,sucrose concentration was higher in cotyledons than in othertissues but, after 17 DAP, the highest sucrose concentration wasfound in nodules. In contrast, glucose and fructose concentrationsin nodules were not high, relative to other tissues. In fact, fructoseconcentration in nodules is so low that it is often difficult toquantify, as reported earlier (23). Trehalose concentration innodules was 10-fold or more higher than the concentration inother organs. However, it should be noted that trehalose concen-tration was generally only 10%o as large as sucrose concentrationin nodules.Data for stems plus petioles were not plotted in order to avoid

complicating Figures 2 and 3. Glucose, fructose, and sucrose werethe major carbohydrates in stems + petioles from 17 DAP on,when concentrations of each sugar ranged from 1.0 to 3.7 mg/gfresh weight. In samples 7, 10, and 13 DAP, D-pinitol was themajor carbohydrate in stems + petioles. During this period, smallquantities (-0. 15 mg/g fresh weight) of D-chiro- and myo-inositolwere also detected but, after this period, these cycitols were notdetected. Traces of trehalose (c0.1 mg/g fresh weight were alsofound in stems + petioles.Data for maltose were recorded for only some of the analyses

in this experiment, but some idea of the distribution of maltosewas obtained. Maltose was detected in cotyledons, stems + pet-ioles, and leaf blades, although concentrations were almost always

at l~~~~~~~~~~~1.2 D:W)DoP 6 AF|1.0

I44

U.3 COT

~ 42 - ROOT

OO20 30 40 O 10 20 30 4

0~~~~~~

1.6Dchiro-INOSITOL myE(NOTOL) l.

1.4 CO

wum 1.2-

N FIXATION N FIXATION

z

4i .8 -

ofn NOD

z0

.4 LEAFtions in nodules ragedfrom0.1to0.35mg/LEAF Dea .2 BLD

a_ COTnrtoess A atr iiart h atr o

I0ir0ROOTRT

Th mos impnn obevain in exeimn IinldeI a

o 10 20 30 40 0 10 20 30 40SEEDLING AGE (DAYS AFTER PLANTING)

FIG. 2. Fresh weight of, and distribution of cyclitols in, soybean seed-lings from 4 to 43 DAP. Infection of Rhizobia was detected microscopically7 DAP but C2H2 reduction (N fixation) activity was nil until 17 DAP whenthe first nodule samples (0.10 g/plant) were obtained for analysis ofcarbohydrates. C212 reduction activity averaged 7.2aimol/g fresh weightofnoduleshfor samples at 17 and 20 DAP. COT: cotyledon; NOD:nodule. The mean of two observations was plotted.less than 0.05 mg/g fresh weight. In contrast, maltose concentra-tions in nodules ranged from 0.15 to 0.35 mg/g fresh weight witha concentration versus DAP pattemr similar to the patte fortrehalose (Fig. 3). Maltose has previously been observed as aconstituent of soybean plants (2) but concentrations were notreported.The most important observations in experiment I included (a)

the rapid accumttlation of cyclitols in nodules during the periodof rapid nodule growth and just after the onset of N fixationactivity (Fig. 2) and (b) the very high concentrations Of D-chiro-inositol, myo-inositol, a,a-trehalose, sucrose, and maltose in nod-ules relative to any other plant organ (Figs. 2 and 3).

Experiment 1I. Roots and nodules were sampled at 1-day inter-vals from 12 to 19 DAP. Shoots were not extracted and analyzed.Carbohydrate composition of nodules and roots, from whichnodules had been removed, was compared to carbohydrate com-

Plant Physiol. Vol. 66, 1980 473




'-.2

x~~~~~~~2 EAF

I) BLADE

Z O

w

IL.

E ROOT

: ROOT COT

~ ~ ~ 0z

GLUCOSE FRUCTOSE0

N FIXATIO

d 4l. N FIXATION 4

0

3.- 3.-

- -~~~~~~ROOT coT

COT

~~~~~~~~2.

-ROOT

ANODLEAF

ED

0 10 20 30 40 0 10 20 30 40

SEEDLING AGE (DAYS AFTER PLANTING)

FIG. 3. Distribution of a,a-trehalose, sucrose, glucose, and fructose in

soybean seedlings. See Figure 2 legend for details.

position of roots (of the same soybean variety) which had no

nodules. Non-nodulated (uninoculated) plants received no N in

the nutrient solution but did not become N-deficient because ofthe short span of the experiment.Nodules were first sampled 13 DAP when total nodule weight

averaged 5.8 mg fresh weight/plant. The increase in nodule weightwas approximately linear up to 19 DAP when nodule weightaveraged 116 mg fresh weight/plant, a 20-fold increase in 6 days.Nitrogenase activity was nil 15 DAP but was positive 16 DAPand in all harvests thereafter (Fig. 4). Between 14 and 16 DAPthere was a >3-fold increase in the total carbohydrate concentra-tion ofnodules (computed as the sum of all compounds measured).

Several patterns were evident in the distribution of individualcompounds: (a) the concentration of the compound in noduleswas many fold greater than concentration in either nodulated ornon-nodulated roots (pinitol, chiro-inositol, myo-inositol, un-known); (b) the concentration in nodules > non-nodulated roots> nodulated roots (the disaccharides sucrose, trehalose, maltose);and (c) the concentration in non-nodulated roots > nodulatedroots > nodules (fructose) (Figs. 4 and 5). The pattern for sucrose,

10

I--z

60 0

OW

2

0'

WEIGHT

12 14

TOTAL

I I A a I I 1 I I I I I I Vol12 14 16 18 12 14 16 ls

SEEDLING AGE (DAYS AFTER PLANTING)

FIG. 4. Fresh weight, nitrogenase, and carbohydrate concentrations innodules (NOD), nodule-free infected roots (N-ROOT), and noninfectedroots (NN-ROOT) of soybean seedlings at daily intervals from 12 to 19DAP. Total carbohydrate is the sum of all compounds measured by GLC.The mean and range of two observations was plotted.

an important translocation form of carbohydrate in soybeans (6),and, especially, the pattern for total carbohydrate suggests thatnodules are a very strong sink for carbohydrate. The superior"competitiveness" of nodules for carbohydrate, relative to roots,may have been the cause for the low weight of nodulated roots,relative to non-nodulated roots, after day 16 (Fig. 4). The patternfor fructose suggests that it may be more rapidly metabolized innodules, relative to glucose for example (Fig. 4). This suggestionis consistent with the finding of Bach et al. (5) that fructoseincreases the incorporation of `5N2 by nodule slices more thansucrose or glucose.The overall patterns of carbohydrate distribution in experiment

II were consistent with those observed in experiment I. The use ofdaily observations in experiment II revealed a very close temporalrelationship between the onset ofN fixation and the accumulationof all compounds except fructose in nodules. The comparison withnon-nodulated roots indicated a preferential accumulation ofdisaccharides in nodules relative to adjacent roots. Three com-pounds (D-chiro-inositol, myo-inositol, unknown) which accumu-lated in large quantities in nodules were essentially absent in roots,whether or not the roots bore nodules.

DISTRIBUTION OF CYCLITOLS WITHIN NODULES

The accumulation of large quantities of cyclitols in nodulesduring the onset of N fixation (Figs. 2 and 5) leads to questions

474 STREETER



Plant Physiol. Vol. 66, 1980 CARBOHYDRATES IN SOYBEAN NODULES

1.6

1.2 F

0.8-

0.4!-_

¢ 0 N-ROOT

UJ 12 14 16 I8U. Q8C

chiro- INOSITOL

0.6EQ6_.z

0

!i

or 0.4 - O/1--z

w

0

°0. NOD0

o

x 12 14 16 18

*1myo- INOSITOL

I

U)

xI.a

z

0

mi

I.-

0

0

0

0.6

0.4

Q2

o ~- N-ROOT

1.0

Table 1. Distribution of Cyclitols in Infected Protoplasts versus Cortex ofSoybean Nodules after Incubation ofNodule Slices with Hydrolytic

Enzymesfor Various Time PeriodsFresh weight of protoplasts and cortex was estimated from volume

measurements and the assumption of density = 1 g/cc. Data are averagesof two GLC analyses of each sample.

Incuba- Cyclitol Concentration

Sample tion DTime D-Pinitol -chiro- myo-Ino-

Inositol sitolh mg/gfresh wt

Whole nodules 0 1.11 2.38 1.80Protoplast cortex 1.5 0.23 1.59 1.09

0.16 0.61 0.44Protoplast cortex 3.0 0.25 1.79 1.21

0.13 0.34 0.25

12 14 16 I8

UNKNOWN ,

0.8 F

0.6-

NOD

0.4

0.2 F

oI I,I I Il

12 14 16 18 12 14 16 18SEEDLING AGE (DAYS AFTER PLANTING )

FIG. 5. Concentration of carbohydrates in nodules (NOD), nodule-freeinfected roots (N-ROOT), and noninfected roots (NN-ROOT) of soybeanseedlings at daily intervals from 12 to 19 DAP. The calculation ofunknownconcentration is based on glucose. D-Chiro-inositol was not detected in anyroot sample. The mean and range of two observations was plotted.

about their distribution and possible function in nodules. Use ofhydrolytic enzymes to prepare protoplasts for analysis of carbo-hydrate composition has potentially serious pitfalls. For example,I suspected that the commercial enzyme preparations might haveseveral disaccharidase activities and, upon testing, found very highhydrolytic activity for sucrose, trehalose, and maltose in "Rho-zyme" and high maltase activity in "Cellulase" and "Macero-zyme" (8). These problems were confirmed when analysis ofsugars in protoplasts showed little resemblance to the patterns inthe original nodules.

Cyclitol metabolizing ability of enzymes used for protoplastpreparation was not measured. But the recoveries of D-chiro-inositol and myo-inositol were about 90% relative to whole nodulesafter a 1.5-h incubation (the shortest incubation time when pro-toplasts could be obtained) (Table I). Recovery did not decreasesignificantly after a 3-h incubation period. (With a 6.75-h incu-bation, not shown in Table I, cyclitol concentrations declined toabout 50%o of the concentrations found at 3 h.) The concentrationof cyclitols in protoplasts was two or more times the concentrationof cyclitols found in nodule cortex (Table I). Since the protoplastfraction is derived almost exclusively from the infected region (seeunder "Materials and Methods"), it is tentatively concluded thatcyclitols in nodules are localized in infected cells.The use of bacteroid isolation to study the distribution of

compounds in nodules is complicated by problems similar to those

Table II. Distribution of Cyclitols in Fractions of Soybean NodulesDerived by Centrifugation of Macerated Nodules

Quantity ofcarbohydrate in nodule fractions was calculated by dividingthe total amount of each cyclitol in each fraction by the total weight of theinitial sample (19.0 g). Results for whole nodules are from duplicatesubsamples (0.5 g) taken from the main sample (20.0 g) and extractedimmediately.

Cyclitol Concentration

Sample D-Pini- D-chiro- myo-

tol Inositol Inositol

ug/glfresh wt

Whole nodules 739 823 1378Nodule fractions

Bacteroid 81 130 147Cytosol (40,000g supernatant) 438 207 41240,000g pellet 9 6 12Bacteroid wash 21 24 30

Sum of fractions 549 367 601

associated with protoplast isolation. For example, soybean noduleshave significant enzyme activity for the hydrolysis of sucrose (22),trehalose, and maltose (unpublished). Recovery of compoinqds,relative to the initial sample, can be used as a rough indicator ofmetabolism during the isolation process. The recovery of pinitolwas about 75% and the ratio of concentrations in cytosol andbacteroid fractions was 5:1 (Table II). The recovery of chiro-inositol was only 45% and the ratio of concentrations in cytosoland bacteroids was only 1.6: 1. For myo-inositol, recovery was also45% and the cytosol to bacteroid ratio was 2.8:1. It is possible thatthe concentration of cyclitols in the cytosol is due to diffusionfrom bacteroids during isolation. I can only report that a secondexperiment yielded a similar pattern of cyclitol distribution; thecytosol: bacteroid ratios for D-pinitol, D-chiro-inositol, and myo-inositol were 2.3, 0.8, and 2.3, respectively.

It can be tentatively concluded that cyclitols in soybean nodulesare localized in the infected region and that pinitol and myo-inositol are concentrated outside of bacteroids, but it should beemphasized that this conclusion needs to be confirmed by othermeans. A larger proportion of the D-chiro-inositol is present inbacteroids than for D-pinitol or myo-inositol. This distributionplus the relative absence of D-chiro-inositol from roots (Figs. 2 and5) leads to the suggestion that D-chiro-inositol may be synthesizedby bacteroids.

It is difficult to speculate about the function of cyclitols innodules because so little is known about their function in plants,with the exception of myo-inositol (3). Myo-inositol and pinitolhave been reported to enhance the activity of auxin and cytokinin

475




in the initiation of secondary roots (25). Myo-inositol may beinvolved not only in the formation of cell wall polysaccharide butalso in the formation ofmembranes (26). The positive relationshipbetween pinitol concentration and C2H2 reduction activity (23)suggests that the cyclitols may play some role in N fixation.

Acknowledgments-I thank Dr. Edward Fairchild and the Department of Phar-macy, Ohio State University, for mass spectra; Professor Otto Hoffmann-Ostenhofand Dr. Wolfgang Loeffelhardt, Institute for General Biochemistry, University ofVienna, for assistance with the purification of D-chiro-inositol and for authentic D-chiro-inositol; Mary Kilpatrick and Sheila Bylica for general technical assistance.

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carbohydrates in soybean nodules · with roots for carbohydrates, especially the disaccharides...

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