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Effect of Puccinia graminis tritici on OrganicAcid Content of Wheat Leaves'

J. M. Daly & L. R. Krupka 3

University of Nebraska, Lincoln

IntroductionAlthough greatly increased rates of oxidative me-

tabolisnm result from infection of higher plants byrust fungi (1,6), the significance of the high ratesfor the development of the rust pathogen is not under-stood. Previous work has indicated that the increasein respiratory rates may be correlated at least partial-ly with the development of the parasite (5, 6). Atthe flecking stage, when visible symptoms of infectionare first noticed and only vegetative myceliunm ispresent, there is a moderate increase in rates withoutany detectable change in the nature of the respiratorypathways. The subsequent larger increases in rattesassociated with the process of sporulation are char-acterized by a decline in C61/C1 ratios. This latterfeature of the metabolic alterations induced by allrust infections studied suggests that an oxidativepentose, or similar, pathway may be of importanice inobligate parasitism. Shu and Ledingham (23) haveshown that the germinating spore of the wheat rustfungus has all the enzymes of the pentose pathwavand the respiration is characterized by lowv C6/C1ratios. Consequently, if the mycelium of the parasiteis largely responsible for the increased rates of res-piration via a pathway requiring a C, decarboxylation(6), major changes should be apparent in the amountsor activity of metabolic components either directly in,or closely related to, the pathways of glucose catabo-lism. In contrast to other tissues in which lowC /C, ratios have been induced (2, 17), data to bepublished on rust-affected plants do show increases incarbohydrate compounds which arise after decar-boxylation of the aldehydic group of glucose.

The effects of sodium fluoride on the respirationof safflower hypocotyls (8) suggested the cyclic (3)operation of the pentose pathway and suggest alsothat the tricarboxylic acid cycle (TCA) would notnecessarily function at rates appreciably differentfrom those in uninfected tissue. Similar inhibitor ex-periments with wheat (7, 21) and bean (7) rust-affected plants were not consistent. However, results

I Received for publication Sept. 5, 1961.* Published with the approval of the Director as Paper

No. 1148, Journal Series, Nebraska Agricultural Experi-ment Station. Supported by funds from Grant E-2000.U.S.P.H. and Research Council, University of Nebraska.

3Pathologist and Postdoctoral Fellow.

obtained by Farkas and Kiraly (10) and Bauermeister(4) with malonic and fluoroacetic acids have beeninterpreted as indicating a change in respiratorypathways, presumably TCA cycle reactions, duringdisease development. In both instances rust-affectedwlheat plants were less sensitive to these inhibitorsthan normal tissue, but the competitive nature of mal-onate inhibition presents kinetic difficulties (7).

A quantitative study of organic acid concentra-tions in wheat infected with the leaf rust fungus wasmade by Staples (24) while Bauermeister (4) notedchanges in size of spots of organic acids separated bypaper chromotography. In each instance moderateincreases in malic and citric acids were observedafter infections were well established and the maiorrespiratory changes had occurred. The significantunexplained exception noted by both workers wasaconitic acid which actually declined 3 to 6 daysafter infection.

The anomalies of the changes anmong TCA cycleacids and the detection of moderate increases onlywhen infections were well advanced might be ex-

plained by a reduction in TCA activity during diseasedevelopment followed by an active translocation ofacids from other leaves as respiration rates increased.Previous work (20) has indicated a capacity of in-fected tissue to accumulate exogenous metabolites.

The data to be presented are part of a comparativestudy of organic acid changes in wheat and beantissue infected by rust fungi in wlhiclh a stage char-acterized by vegetative growth of the parasites (fleckstage) was compared to the sporulating stage of de-velopment. Particular attention was paid to vari-ables such as infection intensity, ontogenic host driftsin metabolism, diurnal changes in metabolism, andenvironmental control, aspects whiclh often are notassessed adequately. Since wheat plants under our

conditions showed characteristics which normal beantissue did not possess, more extensive data on beanrust infections will be discussed elsewlhere.

Materials & Methods

Growth and inoculation of wheat was as describedpreviously (6, 7, 8). The first leaf of Little Clubwheat (Triticumiit aestizvum L.) was inoculated ap-proximately one week after planting with Pucciniiagramiiinis tritici (Eriks & Henn.) race 56. A tem-

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perature of 21 + 1 C and a photoperiod of 1,200 ft-cfrom cool white fluorescent lights for 14 hours was

maintained during subsequent development of rust.There are reports of diurnal fluctuations in or-

ganic acid content of wheat (24). Since diurnalchanges in R.Q. have been demonstrated for bothhealthy and rust-affected wheat leaves under thesegrowth conditions (7), preliminary experiments were

carried out to establish the magnitude of the possibkchanges in organic acids. It was found that malicacid content particularly was altered, in some in-stances increasing by approximately 50 % after 7hours of exposure of plants to light. For this reason

samples were collected shortly before or at the onsetof the light period.

The flecking stage in these experiments occurredon the 4th and 5th days after inoculation. Samplesobtained at sporulation were collected 4 days later.

The procedures for extraction and analysis were

essentially the same as those of Staples (24) andPalmer (14). Usually 200 leaves, representing 12to 15 g of tissue, constituted a sample for analysis.After weighing, the sample was blended for 1 to 2minutes in 100 ml 80 % ethanol. The extract was

filtered through cheesecloth and centrifuged. Theresidues were blended twice again in 100 ml of 80 %ethanol and the alcohol extracts combined for passage(1-2 ml per min under pressure) through a 10 X0.8 cm column of Dowex-1X (200-400 mesh) in theformate form.

Gradient elution of the acids (14) with 6 N formicaci(I with an automatic fraction collector was stand-ar(lized so that reproducible recovery patterns of theacids were obtained. Fractions, 2 ml, were dried at46 C by blowing a stream of air into the collectiontubes for 2 to 3 hours. Distilled water, 2 ml, was

a(lded and the fractions titrated with 0.01 NaOH de-livered from a microburette. Phenol Red \-as theindicator. The order and fractioin number for re-

covery of acids was: Di-carboxylic amino 12-20; suc-

cinic 28-35; malic 38-45; citric 66-80; fumaric 110-120; phosphoric 130-160; aconitic 160-190.

The indlividual titration values for each fractionwere summle(l and the concentrations in AtI of acidfor each peak were calculated. The peaks wvere dis-tinct except between phosphate and aconitate, andoccasionally between malic and succinic acids wlheneither of the latter pair was in high concentrationi.Even in such cases the peaks were sufficiently sepa-

rate so that a reasonably accurate estimate of amountsof acid was obtained from the slhape of the elutioncurves. If care is exercised in the drying process,recovery of acids is 90 % or greater.

In experiments involving incorporation of isotopiccarbon from glucose, tissue collected at the start ofthe light period was cut into sections approximately0.5 cm in length and floated on 0.1 Ai phosphatebuffer (pH 4.6) containing 1 mg/liter ml of uni-formly labeled glucose-C'4 (activity equalled 106cpmimg). The glucose U-C'4 was purchased fromVolk Radiochemical Co. Tissue, 2 g, was placed on

20 ml of isotope solution in standard petri plates for5 hours in the dark. After collection of each 2 mlfraction, an aliquot was transferred to a stainlesssteel planchet and dried. Activity then was measuredin a Tracerlab automatic flow-counter. In all cases,radioactivity in the elution curve correspondedl totitration values, indicating that the radioactivity waspresent only in recoverable organic acids andl not inresidues formed possibly from labile acidls such asoxalacetic or glyoxylic (14).

The identity and purity of the individual aci(d peaksafter elution was determined by paper chromatographyin two solvents: Ethyl ether, formic acid, water(5: 2: 1); N-butanol, benzl alcohol, formic acid, water(43:43: 5: 9).

The location of acid spots was determined withbrom phenol blue indicator spray while ninhydrin wasemployed for amino acids. When radioactive samiipleswere chromatographed, activity was located with aTracerlab chromatogram scanner. Except in sporu-lating tissue, the amino acid peak consisted of glutamicand aspartic acids. At sporulation two additionalbarely detectable spots with higher Rf values wereevident, but no radioactivity was noted. The succinlic,malic, and aconitic peaks were pure.

It has been shown (14) that fractions correspondl-ing to citric acid (66-80 in our experiments) may con-tain citric, isocitric, pyruvic, malonic (23), andl aphosphate compound (25), perhaps a derivative ofcitric. Pyruvic and malonic acids were not (letecte(lin wheat although they may have existed in smallamounts.

Since citrate and isocitrate have identical Rfvalues in all solvents employed, it was necessary tocheck for the presence of isocitric acid by formationof isocitric lactone. The lactone has an Rf signifi-cantly higher than either citric or isocitric acids.Formation of the lactone was accomplished by evapo-rating to dryness a solution of combinedl fractions60 to 80 in 1 N HCL and heating for 2 hours. Theresulting material was suspended in water and chro-matographed. Good conversion of known isocitratewas obtained, but healthy or diseased tissue extractsdid not form isocitric lactone.

Radioactivity data indicated a second componelntof the citric acid peak which remained at the originwhen chromatographed. It was thought first thatsince citric acid is a known chelating agent, it waspossible for citric acid to complex with ions presentin the stainless steel planchets; but pure citric 5-C'4carried through the same procedures did not resultin a spot at the origin. Neither did pyruvic U-C'4.

Vickery and Palmer have reported phosphoricacid in the citric acid fraction of tobacco (24). How-ever, the presence of C14 and the low Rf in the solventsemployed is indicative of an organic phosplhate.Mr. MIarlin Bolar has shown that hexose monophos-phates are eluted in the same fractions. The presenceof such esters would not alter the data obtained onaci(l concentrations in leaves since the (lissociationconstants of the secondl and third hydrogens are low.

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DALY & KRUPKA-RUST EFFECT ON ORGANIC ACID CONTENT

In the data for radioactivity, the unknown is treatedseparately unitil its identity is known. The natureof the results will not be altered appreciably. Theevidence from chromatograms indicates that the onlytitrable acid was citric.

The concentrations of organic acids in leaves ispresented in tables 1 to 4 as /AM acids per 10 g leaftissue. The use of /LM, rather than milliequivalents(24) or mg, of acid appears more suitable for ex-pressing the relationships among the acids which dif-fer in numbers of carboxyl groups and molecularweights. The radioactivity data are expressed incpm. In the case of the amino and citric acid peaks,the relative percentage of radioactivity in each of thematerials present in the peaks was determined eitherby a chromatogram scanner or by cutting out the acidspots from the chromatogram and counting the activi-ty of each spot with the flow counter. From this data,the absolute level of radioactivity in each componentthen was calculated from the previously determinedtotal activity of the peak.

Results

- Concentration of Acids in Healthy & InfectedLeaves. Tables I and II record the amounts oforganic acids obtained when strong flecks were ap-parent and 4 days later when 100 % of the infectionsites were sporulating. Levels of acids in healthywheat were much higher in the experiment of table Ithan in the experiment of table II, yet the develop-ment of rust and general appearance of the plantswere the same. Although no critical experimentswere performed, it is possible this difference resiclesin the nutritional status of the plants. Plants usedfor experiments of tables I and III were grown onsoil which had been sterilized and re-used severaltimes, while the data for tables II and IV were ob-tained on soil recently composted. This situationwas desirable since it permits an evaluation of possi-bly significant trends in disease development in hosttissue of different acid status.

From the data of table I, with an infection level of125 pustules per leaf at flecking, when respiratory

Table I

gm Acids per 10 g of Healthy (H) & Rust-Affected (D)1st Leaves of Wheat at Flecking &

Sporulating Stages

Acid Flecking Sporulating*H D** H D

Amino 32 40 38 102Succinic 7.0 11 6.0 27Malic 28 65 39 160Citric 22 30 22 118Fumaric .. 20Aconitic 90 36 92 46

* 4 Days after flecking sample.** 125 Pustules per leaf.

rates are only slightly elevated (6), it can be seenthat the levels of all components, except aconitic, werehigher in rusted than in healthy leaves. Note, how-ever, that the increases are not uniform. Malic acidincreased most markedly on a molecular basis (ap-proximately 40 ,M/10 g). On a percentage basis, thedifference was 232 % while the other acids were:amino 125; succinic 157; citric 136. Aconitate levelswere only 40 % of the control tissue.

At sporulation the differences in all acids weremore pronounced and in diseased tissue fumaric acidwas detected. Although aconitic acid concentrationswere below the control plants, there was a significant(approximately 30 %) increase in the diseased plantswhen compared to tissue collected 4 days previously.

Table IIInfluence of Infection Intensity on Changes in Acid

Content* of 1st Leaf of Wheat

Acid Flecking Sporulating**H 75 p/l*** 200 p/! H 75 p/l 200 p/1

Amino 12.1 8.6 5.9 24 49 49Succinic 1.4 1.5 4.3 2.3 8.0 14Malic 4.3 5.8 18 7.4 49 89Citric 2.8 3.0 6.1 5.2 34 61Aconitic 3.5 3.6 6.3 1.8 15 17

* /.i per 10 g tissue.** 4 Days after flecking sample.* Pustules per leaf.

The increases in citric and malic acids, as well asthe amino acids, were approximately the same at thisstage of disease development.

The data of table II show that when the acidlevels are low in host tissue, different effects are ob-served in the amino and aconitic fractions. Thesedata also show that the level of infection has a pro-nounced bearing on the magnitude of the effects ob-served. Although 75 pustules per leaf appears vis-ually to be a heavy infection and is appreciably higherthan has been reported in some experiments, it isobvious that levels of infection may not be sufficientto permit detection of changes early in the develop-ment of rust.

In contrast to the data of table I, at the fleckingstage the dicarboxylic amino acids are lower in con-centration and the difference is a function of the levelof infection. Of equal importance is the fact thataconitic acid levels are the same with moderate in-fections but are increased with heavy infections.Despite the differences in these components, at fleck-ing there was a marked increase in malic acid atthis stage of the heavy infection.

At sporulation, both intensities of infection pro-duced significant increases in levels of all componentsbut the effect with high intensities is more marked.In agreement with the data of table I, the differencesbetween healthy and diseased tissue in citric acid at

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PLANT PHYSIOLOGY

sporulation are of the same order of magnitude (ap-proximately tenfold) as the differences in malic acid,while at flecking the differences are twofold andfourfold, respectively.0 Synthesis of Acid Components. Although thedifferences observed in tables I and II could not beattributedl readily to translocation and accumulation oforganic acids per se, twvo additional lines of evidencefor synthesis of organic acids at infection sites were

obtained. Table III shoxvs increases in organic acidsin excised healthy and diseased leaves whiclh were

placed in 10 nml of 0.1 AM KH..PO4 containing 1 mg per

ml glucose. The cut einds xvere placed in the solu-

Table IVIncorporation* of Activity From Glucose U-C14 Into

Acids in Healthy & Rust-Affected1st Leaves of Wheat. cpm X 10-3

Acid Flecking Sporulating

H 75p/l 200p/l H 75 p/l 200p/lAmino**AsparticGlutamicSuccinicMalicCitricOriginCitric

Aconitic

63.916.047.94.9

12.150.629.621.0-0.75

38.938.9N.D.4.7

13.940.420.220.20.70

12.212.2N.D.6.6

19.638.217.221.01.0

113.245.367.96.8

21.738.521.217.30.5

115.940.575.48.1

26.156.012.343.71.8

108.330.378.010.636.075.219.655.62.4

Table IIIChanges in Acids (iM per 10 g) of Healthy &Rust-Affected 1st Leaves After Excision*

Acid Healthy 70 % Sporulation115 p/l

hr hr0 5 10 0 5 10

Amino 22 32 33 49 48 49Succinic 5.1 4.5 3.4 11 12 9.6Malic 38 65 59 89 130 114Citric 11 13 10 33 36 39Aconitic 146 195 179 84 100 71

* 200 Leaves placed with cut ends immersed in 10 ml of0.1, KH.,PO4 containing 1 mg glucose per ml.

tion to a depth of about one-half inch undler the same

light and temperature conditions as those und(ler xvhichthe plants were growvn. The disease(l tissue sup-

ported 115 pustules per leaf and approximately 70 %of the pustules were sporulating. At this stage. themetabolisml of the acid components should possess

characteristics associated with both the fleckiiig stageand complete sporulation. In healthy tissue tlhel-e xvas

an increase in all componenits. except succiniic, (luringthe first 5 hours. In the next 5 hours there was a

decline in all acids except the aamino acids. Indiseased tissue the amino acids did not clhange ap-

preciably witlh time. The other acids increased dur-ing the first 5 hours, but subsequently, with the ex-

ception of citric acid, decline(l.The above data indicate that syntheses of acids

may occur in diseased leaves, but do not indicaterelative rates of synthesis. This may be due, in part,to a more rapid utilization of individual acidls by

diseased tissue in addition to a general decrease in

acid content because of excision of tlle leaves. \Vehave observed that both wvheat and beanl tissue, wlvenfloated on glucose solution in the dark, show a de-crease in acid content.

The data of table IV wvere obtained on aliquotsobtained from the tissue usedl for the experiment oftable II. Tw o granms of tissue sections were sup-

plied 20 mg glucose uniformly labelled vitfl C' for

* 2 g of tissue comparable to that of table II werefloated for 4 hours on 20 ml of 0.1 M KH.,PO4 con-tainiing 1 mg/ml of glucose U-CI4. cpm equals 106per ml.

** At sporulation four ninhydrin-reacting compoundswere observed, but only aspartic and glutamic haddetectable radioactivity.

5 hours, in the dark. The incorporation of C14 intoinclividual acids is similar to the chiainges ohserved inthe concentrations found in intact tissue. It shouldhe noticed that, at flecking, the greatest percentageincrease of incorporation into Kreb's cycle intermle(li-ates was in the four carbon acids, malic and succinic,vhein comlpared to the control tissue. At sporulation.however, imialic was not as markedly dliffereint as atflecking.

The total amount of radioactivity in the citric aci(dfraction was actually lowNer in disease(d tissue at fleck-ing, but the decrease apparently was due largely to adifferenice in the unknowin material present in thepeak. The amount of radioactive label in citric acidappears to be the samlie. The other 6-carbon aci(l.aconiitic, also lhad approximately the samiie rate of in-corporatioin. At sporulation, however, both citric aindaconitic acids had Imuclh higher lexvels of activity indisease(l tissue wlheni comiipared to healthy.

The pattern of labelling in aspartic and glutaiinicaci(ls at the two stages of disease (levelopinieint re-flected, in general, the (listributionl of label in orgallicacids. At flecking, Ino radioactivity was (letected inglutamliic acid separated from aspartic aci(d by- clhro-matography. Visual estimation of the size of theglutamiiic acid spot of the chronmatogramii indicatedthat the concentration of glutanmic aci(d as only one-third the concentrationi of aspartic aci(l. In the laterstages of inifection the reverse situation xvas fotlnd(l.Approximately tvx-o-thirds of tlle total incorporationinlto dicarboxvlic aminiiio acids wvas associated wx ithglutaiiiic aci(l.

Discussion

The data ind(licate that despite clhaniges in the patlh-Nvavs oftglucose catabolism in diseased tissue ( 1, 0.

280



DALY & KRUPKA-RUST EFFECT ON ORGANIC ACID CONTENT

8, 21), acids associated with the TCA cycle are syn-

thesized and accumulate in rust-affected wheat leaves.The observed changes in wheat are much greaterthan suggested by previous results (4, 24) and havebeen found consistently in experiments not describedhere. It is apparent that levels of infection are ofimportance in determining the magnitudes of inducedalterations in acid concentrations. The relativelyslight changes observed by other workers may be a

consequence of low infection intensities. The declinein aconitic acid (4,24) is not a unique feature ofinfection by rust fungi, but is associated with theinitial levels of acids present during establishment ofthe fungus.

The large increases resulting from infection may

help to interpret several aspects of the literature on

obligate parasitism. The failure to obtain (4, 10) as

much inhibition of respiration of diseased tissue withmalonate or fluoroacetate is explained most readily bythe size of substrate pools. The influence of environ-ment, particularly light intensity, on the degree ofinhibition by malonate during growth (4, 10) may

be a reflection of light-controlled diurnal fluctuationsof acid content.

The possibility that differences in enzyme activityin extracts prepared from healthy and rust-affectedtissue (12,15) may be a function of different pH or

redox values during preparation or assay must also beconsidered. Kaul and Shaw (11) have reportedmarked changes in redox potentials in wheat sap as

a result of infection. Attempts to correlate (11 )such phenonomena with enzyme functions in vivoseeml premature until the contributions of acids(probably located in the vacuole) to the measuredpotentials can be determined.

Although greater amounts of acids in diseasedtissue can account for the insensitivity to competitiveinhibitors such as malonate, the absence of soundkinetic data or labelling patterns preclude a decisionas to whether the Kreb's cycle is operating at higheror even at the same levels in diseased tissue when com-

pared to healthy tissues. A solution for this basicquestion has been attempted for various tissues in-cluding filamentous fungi (13, 22) and wheat (5)but kinetic uncertainties make any estimate of TCAcycling difficult. The conspicuous changes in malate,measured either on a molecular basis or by incorpora-

tion data, at the flecking stage of development may beof considerable significance. The recent recognitionof auxiliary mechanisms [malate synthetase (9),carbon dioxide fixation (18)] in formation of malateniight suggest that the initial changes in acids, whenrates of respiration are beginning to rise, occur byreactions other than the formation of citrate via con-

densing enzyme. It might be argued, however, thatfailure to detect increases in citric or aconitic acidsat this stage is only a reflection of greater utilizationof these acids when compared to malate at this stage.

This concept is supported by the pronounced de-clinie in aconitic in tissues initially high in organicaci(ls aind by the absence of an appreciable pool of

glutamic acid in diseased, but not healthy, wheat atflecking. Utilization of glutamic acid for synthesisof components not detected in this study would ac-count for such results. Glutamine, for example, hasbeen demonstrated to be involved in reactions leadingto the formation of glucosamine (16), an integral unitof fungal cell w-alls. In a kinetic situation, wherea-ketoglutaric acid was continually siphoned fromTCA cycling, additional non-cycle reactions leadingto malate formation would be of considerable impor-tance in maintaining steady-state conditions for citrateformation. In this respect the observation of SenGupta and Sen (19) that indole acetic acid increasesCOo, fixation into malate may be pertinent, since in-fection by rust fungi increases indole acetic acidlevels (7).

The marked accumiulatioin of citric and glutamicacids at sporulation also has been found consistently.At present the functional significance of this ac-cumulation is not uncderstood nor is the site (hostversus parasite) of the accumulation known. Sincespores of the fungus contain relatively large amountsof lipoidal materials, it is possible that the transitionfrom active vegetative growth to sporulation resultsin a relative decrease in metabolic demandls for aminoacids furnished by Kreb's cycle intermediates. Sincerespiration continues to increase during sporulation.the accumulation of citric and glutamic acidls mightbe expected.

A separate more detailed comparison of infectedbean tissue and germinatinig bean rust spores hasshown the same general trendls with infectedl tissueas those described above. It would appear that theinitial increase in malate at flecking followed bycitrate and glutamate accumulation at sporulation ischaracteristic of infection by rust fungi. Until re-action pathways are determinie(l. the correlationi ofsuch changes w-ith metabolic alterations previouslyfound cannot be evaluated.

Summary

Healthy and rust-affected first leaves of wheathave been examined for changes in organic acids as-sociated witlh the TCA cycle during the vegetative(flecking) and sporulating stages of parasite dlevelop-ment. Although large increases in total acid arefound, the acid status of the host plant and the amountof infection by rust fungi determine the degree ofdifferences for individual acids. In all instances,how%vever, an increase was found in the malic andsuccinic acids during vegetative development. Citricacid accumulates to a greater extent at sporulation.Increases in other acids are not so pronounced.Aconitic acid actually- decreases if present originallyin large amounts in normal host tissue.

The significance of the results for obligate para-sitisImi is discussed, particularly the possibility ofancillary formation of malate by reactions normallynot considered as part of the Kreb's cycle.

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PLANT PHYSIOLOGY

Literature Cited

1. ALLEN, P. J. 1959. Metabolic considerations ofobligate parasitism. Ch. 12. Plant PathologyProblems & Progress 1908-1958. C. S. Holtonet al., eds. The University of \Visconsin Press.Madison.

2. AP REES, T. & H. BEEVERS. 1960. Pentose Path-way as a major component of induced respirationof carrot and potato slices. Plant Physiol. 35:839-848.

3. AXELROD, B. & H. BEEVERS. 1956. MIechanisms ofcarbohydrate breakdown in plants. Ann. Rev. ofPlant Physiol. 7: 267-299.

4. BAUERMEISTER, R. S. 1961. Bioclhemische 1Uniter-suchungeni Zum WVirt-Parasit-Verhaltnis am Bei-speil von Puiccinia graminis tritici. Phvtopath. Z.40: 221-244.

5. BILINSKi, E. & W. B. MCCONNELL. 1958. StudiesoIn wheat plants using C14 compounds. VII. Utili-zation of pyruvate-2-C14. Can. J. Biochem.Physiol. 36: 381-388.

6. DALY, J. M., A. A. BELL, L. R. KRUPKA. 1961.Respiratory changes during development of rustdiseases. Phytopathology 51: 461-471.

7. DALY, J. M. & R. E. INMAN. 1958. Changes inauxin levels in safflower hypocotyls infected withPuccinia carthamiii. Phytopathology 48: 91-97.

8. DALY, J. M., R. MI. SAYRE, & J. H. PAZUR. 1957.The hexose monophosphate shunt as the majorrespiratory pathway during sporulation of rust ofsafflower. Plant Physiol. 32: 44-48.

9. DOYLE, W. P., R. HUFF, & C. H. WANG. 1960.Role of glyoxylate in biosynthesis of acids in to-mato fruit. Plant Physiol. 35: 745-751.

10. FARKAS, S. L. & Z. KIRALY. 1955. Studies on therespiration of wheat infected xx ith stem rust &powdery mildew. Physiol. Plantarum. 8: 877-888.

11. KAUL, R. & M. SHAWV. 1960. TIhe p)hlysiology ofhost-parasite relations. V-I. Oxidation-re(luctionchanges in xvheat leaf sap caused by rust infectionis.Can. J. Botany 38: 399-407.

12. KIRALY, Z. & (G. L. FARKAS. 1957. Decrease inglycolic acid oxidlase activity in wheat leaves in-fected with Puccinia gramimnis var. tritici. Phyto-pathology 47: 277-278.

13. MOSES, V. 1957. The metabolic significance of thecitric acid cycle in the growth of the fungus Zygor-hynchts moelleri. J. Gen. Microbiol. 161: 534-549.

14. PALMER, J. K. 1955. Chemical investigations of thetobacco plant. X. Determination of organic acidsby ion exchange chromatography. Conn. Agric.Exp. Sta. Bull. 589: 1-31.

15. PILET, P. E. 1960. Auxin contenit & auxini catabo-lism of the stems of Eutphorbia cyparrissias L. in-fected by Uroinyces pisi (Pers.). Phytopath. Z.40: 75-90.

16. POGWVELL, B. M. & R. M. GRYDER. 1957. Enzymaticsyntheses of glucosamine 6-phosphate in rat liver.J. Biol. Chem. 228: 701-712.

17. ROMBERGER, J. A. & G. NORTON. 1961. Chang-ing respiratory pathways in potato tuber slices.Plant Physiol. 36: 20-30.

18. SALTMAN, P., G. KUNITAKE, H. SPOTTER, & C.STITTE. 1956. The dark fixation of CO2 by suc-culent leaves: The first products. Plant Physiol.31: 464-465.

19. SEN GUPTA, A. & S. P. SEN. 1961. Carboni dioxidefixation by auxin treated tissues. Plant Physiol.36: 374-380.

20. SHAWV, M. & D. J. SA-MBORSKI. 1956. The physi-ology of host-parasite relations. I. The accumula-tion of radioactive substances at infections offacultative & obligate parasites including tobaccomosaic virus. Can. J. Botany 34: 389-405.

21. SHAW, M. & D. J. SAMBORSKI. 1957. The physi-ology of host-parasite relations. III. The patternof respiration in rusted & mildewed cereal leaves.Can. J. Botany 35: 389-407.

22. SHU, P., A. FUNK, & A. C. NEISH. 1954. MIechan-ism of citric acid formation from glucose by Asper-gill/us niger. Can. J. Biochem. Physiol. 32: 68-80.

23. SHU, P. & G A. LEDINGHAAI. 1956. EInzymiies re-lated to carbohydrate metabolism in Urediosporesof wheat stem rust. Can. J. Microbiol. 2: 489-495.

24. STAPLES, R. C. 1957. Changes in the organic acidcomposition of wheat leaves infected xvith the leafrust fungus. Contrib. Boyce Thompson Inst. 19:1-18.

25. VICKERY, H. B. & J. K. PALAIEIR. 1957. The nme-tabolism of the organic acids of tobacco lea-ves.XIII. Effect of culture of excised leaves in solutionof potassium carbonate. J. Biol. Chem. 227: 69-81.

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