Phospholipids the Uredospores of Uromyces phaseoli

Plant Physiol. (1971) 48, 735-739

Phospholipids in the Uredospores of Uromyces phaseoliII. METABOLISM DURING GERMINATION'

Received for publication May 17, 1971

R. J. LANGENBACH' AND H. W. KNOCHEDepartment of Biochemistry and Nutrition, University ofNebraska, Lincoln, Nebraska 68503

ABSTRACT

The levels and types of phospholipids changed in distinctphases during the germination of "P-labeled uredospores ofUromyces phaseoli. During the first 20 minutes of germina-tion, the phospholipid content dropped to 40% of the pre-germination level. Between 2 and 3 hours, phospholipid levelsincreased to approximately 80% of the pregermination levels,and after germination for 5 hours, catabolism had reducedthe "P-lipids to about the same level observed prior to the firstanabolic phase. A second anabolic phase was observed between5 and 10 hours of germination. Phosphatidylcholine and phos-phatidylethanolamine, the major phospholipids, did not un-dergo anabolism and catabolism at the same rates during ger-mination. Only smail quantities of the more polar phospho-lipids were released to the germination media.

Germinating uredospores were capable of utilizing L-methio-nine-methyl-`C, D, L-"C serine, "C-choline, and "C-ethanola-mine for the synthesis of phospholipids. The active one-carbonunits from methionine and serine appear to be involved in themethylation of phosphatidylethanolamine to form phos-phatidylcholine. Preformed ethanolamine and choline may beincorporated into these two phospholipids also. Evidence forthe synthesis of phosphatidylserine, which is subsequentlydecarboxylated to yield phosphatidylethanolamine, was ob-tained.

Detailed studies concerning the metabolism of individualphospholipids in germinating spores of obligately parasiticfungi have not been previously reported. Although the biosyn-thetic pathways for the major phosphatides in animal and bac-terial systems have been extensively studied, relatively littleis known corncerning their biosynthesis in higher plants andfungi. Recently, Kates (7) reviewed the subject of plant phos-pholipids.

While the germination process for uredospores of fungi hasbeen studied extensively, little is known concerning the actualsynthesis of the germ tube and associated membranes. Thiswork was initiated to elucidate the possible pathways for the

'This study was supported by the Nebraska Wheat Division.Published with the approval of the Director as paper No. 3121,Journal Series, Nebraska Agricultural Experiment Station. Researchreported was conducted under project No. 15-18.

2 Present address, McArdle Laboratory for Cancer Research, TheUniversity of Wisconsin, Madison, Wis. 53706. Recipient of a Na-tional Science Foundation Predoctoral Traineeship 1969-1970.

synthesis of phospholipids and to investigate whether possiblerelationships existed between phospholipid metabolism andstructural changes.

MATERIALS AND METHODS

Uredospore Production. The production of 'P-labeled andunlabeled uredospores have been described (1 1, 18).

Germination of Spores. Immediately before germination thespores were washed with distilled water, air dried (18), andthen weighed. Germination was initiated by dispersing thespores evenly on the surface of the medium (1.0 mg spores per1.0 ml medium). Depending upon the quantity of spores re-quired, either Petri dishes (9.4 cm diameter) or Pyrex trays(18.5 X 30 X 4 cm) were used as germination vessels. Themedium was distilled water, except when the incorporation ofspecific substrates was being studied. In those cases the me-dium was a distilled water solution of the appropriate com-pound.

Germination was terminated by filtering the spores andmedium through a glass wool plug. The glass wool plug andentrapped spores were then immersed in a chloroform-metha-nol (2:1 v/v) mixture. The germination period was measuredfrom the time the spores were dispersed on the medium to thetime the spores were immersed in the chloroform-methanolmixture.

Phospholipid Extraction. The methods used in disruptingthe spores and extracting the phospholipids are described inthe previous paper (1 1).

Phospholipid Analysis of Spores. As a measure of total phos-pholipids the radioactivity ("P or '4C) of the chloroform-meth-anol extract was determined. For the determination of radio-activity of specific phospholipids, the chloroform-methanolextract, or an aliquot of the extract, was subjected to thin layerchromatography. The radioactive bands were located by auto-radiography or by spraying a margin of the thin layer platewith one of the reagents given previously (11). The adsorbentfrom the desired area was scraped directly into a counting vial.After adding 1 ml of ethanol and agitating the sample for 10min, 15 ml of the scintillation solution (dioxane-xylene solvent)was added. If a detecting reagent was used, only the unsprayedregion of the plate was used for analysis. For "P-lipids (lo-cated by autoradiography) the counting rates reported wereonly corrected for decay and the proportion of the extractapplied to the plate. By this method more than 90% of theradioactivity (cpm) applied to the chromatogram could beaccounted for by the combined radioactivity (corrected cpm)of the components. For "C-lipids (located by spray reagents)the percentage of each component was calculated on the basisof cpm. The percentage obtained was multiplied by the totalradioactivity of the extract (determined by counting an aliquotand applying a channels ratio quench correction) to obtain the

735

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LANGENBACH AND KNOCHE

14.0

12.0Unw

o 10.0a.U)

coE 8.000

N, 6.0

0

x< 4.0

a.o 20o

0.0

SPORES 0

MEDIUM U

0 2 3 4 5 6

TIME (HOURS)

FIG. 1. The quantity of radioactivity in theextract of "P-labeled uredospores of Uromyc,stages of germination (0) and the chloroform-radioactivity in the aqueous germination metime period, 100 mg of washed spores on 100were germinated in a synchronous manner, w:spores germinating. The specific radioactivityto washing was 4500 cpm/mg.

radioactivity of each component. The areagrams that did not contain compounds weassayed, and the data were included in thetions.

Phospholipid Analysis of the Germinaquantity of phospholipids which were prestion medium was determined by lyophiliziiextracting the dry residue with chlorofcv/v). Care was taken to insure that no ure(ent in the filtered medium prior to the lyopradioactivity in the extract was consideredof the total phospholipids. Assay of specificperformed by the same methods describsection.

Thin Layer Chromatography. Thin laywas performed with Silica Gel G as the abrration of the plates has been described psolvent systems used were system A, chwater (66:30:4, v/v/v) (15), and system Eanol-7.4 N ammonium hydroxide (66:17:3

Chemicals. Reference compounds andthe same sources previously reported (11).(carrier free), L-methionine-methyl-14C (10.serine-3-"C (4.8 mc/mmole), choline-1,2-'mmole), and ethanolamine- 1 , 2-"C hydrommole) were purchased from New EnglandMassachusetts.

was the only physical manifestation of the germination process,and protrusion of the germ tube was not evident until approxi-mately 40 min after the germination was initiated. Between 2and 3 hr the phospholipid levels increased to about 80% of theinitial level. After germination for 5 hr, catabolism had re-duced the 3P-lipids almost to the levels observed prior to theanabolic phase. Synthesis of phospholipids appeared to occuragain between 5 and 10 hr, and in all studies the maximumlevel of 32P in the chloroform-methanol extract was observed atabout 10 hr. Other experiments performed in this laboratoryhave indicated that an additional catabolic phase followed byan anabolic phase occurred between 10 and 24 hr of germina-tion. Also shown in Figure 1 is the amount of phospholipid re-leased to the medium as the germination progressed. Althoughthe phospholipid levels of the medium appeared to change in

,.v an inverse manner with the phospholipid levels in the spores,7 8 9 10 the difference in the levels of the medium was not nearly great

enough to account for the changes in phospholipid level of thegerminating uredospores. Therefore, the disappearance of

chloroform-methanol chloroform-methanol extractable 'P during the first 20 min ofFs phaseoli at various germination represented metabolism of the phospholipids and-methanol extractable not the release of phospholipids to the germination medium.-dium (U). For eachml of distilled water The successive anabolic and catabolic phases of phospho-ith 80 to 90%i of the lipid metabolism were not expected; however, the experimentof the spores prior was repeated several times and in all studies the same basic

metabolic phases were observed. In one experiment, duplicatesamples were germinated for each of the time periods. The

is of the chromato- duplicate samples agreed within -+-10%, and illustrated that there also scraped and early catabolic and anabolic phases of phospholipid metabo-percentage calcula- lism were real and not variations in analyses.

The levels of individual phospholipids at different stages oftion Medium. The germination were investigated by subjecting the chloroform-,ent in the germina- methanol extracts to thin layer chromatography in chloroform-ng the medium and methanol-water (66:30:4, v/v/v), (system A) and determiningirm-methanol (2:1, the radioactivity in the separated components. From the chro-dospores were pres- matographic mobilities of the components and the identifica-)hilization step. The tion of the phospholipids in previous work (11), the identity ofas the radioactivity the components was determined. Figure 2 depicts the changesc phospholipids wasoa ;irn ftha invaltr10PC.eu m tne previous

,er chromatographysorbent. The prepa-reviously (10). Theloroform-methanol-), chloroform-meth-v/v/v) (19).

solvents were fromPhosphoric acid-3P.6 mc/mmole) D,L-"C chloride (5 mc/)chloride (1.5 mc/Nuclear, Waltham,

RESULTS

Metabolism of Endogenous Phospholipids. Uredospores ofUromyces phaseoli, previously labeled with 'P, were germi-nated on distilled water for various time periods, and the totalintracellular phospholipid content was determined. Theamount of phospholipid released to the germination mediumwas also analyzed.The levels of phospholipids appear to change by distinct

phases during the germination process (Fig. 1). The total'P in the chloroform-methanol extract of the spores decreasedby approximately 50% during the first 20 min germinationperiod. In the same time period, a slight swelling of the spores

w

0ILC')

C0

E0

0

a.C.

PE -

0 2 3 4 5 6 7 8 9 10

TIME (HOURS)

FIG. 2. The quantities of radioactivity in individual phospho-lipids at various stages of germination of "P-labeled uredosporesof Uromyces phaseoli. Separation of components was performed bysubjecting aliquots of the chloroform-methanol extracts describedin Figure 1 to thin layer chromatography in the chloroform-meth-anol-water (66:30:4, v/v/v) system. Abbreviations: PC: phosphati-dylcholine; PE: phosphatidylethanolamine; PI: the two phospho-inositides; DPG: diphosphatidylglycerol; 0: the chromatographicorigin.

736 Plant Physiol. Vol. 48, 1971



PHOSPHOLIPIDS OF UROMYCES PHASEOLI. II

in the individual phospholipid levels during the germinationprocess. Diphosphatidylglycerol, phosphatidylethanolamine,and phosphatidylcholine all decreased during the first 20 mingermination period. More dramatic, however, was the decreasein the unidentified 'P-labeled material which remained at theorigin in this chromatography system. Although the identityof these 3P-labeled components was not elucidated, at leastone was believed to contain choline. The levels of the twophosphatidylinositides and of diphosphatidylglycerol did notshow a definite change during the first 20 min period. In thefirst major anabolic phase (between 2 and 3 hr), all of thephospholipid levels increased; however, phosphatidylcholineand the 3P-labeled material at the origin of the chromato-graphic plate showed the most pronounced increases. On apercentage basis, the level of the phosphoinositides increasedsignificantly. The decrease in total phospholipids in the secondcatabolic phase (between 3 and 5 hr) appears to be largely dueto changes in phosphatidylcholine and unidentified 'P materialwhich increased the most during the previous anabolic phase.The second major anabolic phase (between 5 and 10 hr) ap-pears to be the result of synthesis of all phospholipid com-ponents, with phosphatidylethanolamine contributing the mostto the increase in total phospholipid levels. Diphosphatidylglyc-erol, which is a minor component of the phospholipids, did notundergo distinct changes in level. While the level does appearto fluctuate with the total phospholipid level, the differencescould not be considered to be significant when the precision ofthe assay method is considered. In Figures 1 and 2 there is anindication that phospholipid anabolism took place between 20min and 1 hr and that a catabolic phase follows between 1and 2 hr.

Analysis of the phospholipids in the filtered media indicatedthat the more polar phospholipids, phosphatidylcholine, thetwo inositol-containing phosphatides and the unidentified 'P-labeled material which remained at the chromatographic originwere preferentially released. Very little phosphatidylethanola-mine was present in the germination medium regardless of theperiod of incubation. The phospholipids in the spores andthose in the medium at the end of a 2 hr germination periodare shown in Figure 3, which is a photograph of an autoradio-gram obtained by chromatography of the chloroform-methanolextracts of the germinated spores and the lyophylized medium.It appears that those phospholipids that would be expected tohave the greatest solubility in water were present in the highestconcentrations in the germination medium. This does not ruleout the possibility that other phospholipids were transportedfrom the intact spore, but instead of dissolving in the mediumadhered to the spore wall or germ tube.The results of the experiments described thus far indicate

that large changes in phospholipid levels occur during thegermination process and that these changes are primarily dueto the anabolism and catabolism of phosphatidylcholine, phos-phatidylethanolamine, and the 'P-labeled material that re-mains at the origin when the phospholipids are chromato-graphed. Therefore, in addition to changes in the phospholipidlevel, there also appear to be changes in the types of phospho-lipids present during different stages of the germination proc-ess.

Incorporation of Methionine-methyl-14C into Phospholipids.The anabolic phases observed at various stages of the germi-nation process indicated that pathways for the biosynthesis ofphospholipids, particularly phosphatidylcholine and phospha-tidylethanolamine, existed in the germinating spore. For otherbiological systems, two pathways which would achieve a netsynthesis of phosphatidylcholine are known: the successivemethylation of phosphatidylethanolamine to yield phospha-tidylcholine, and the condensation of CDP-choline with an

a,,8-diglyceride (13). In the preceding report (11), it was shownthat labeled phosphatidylcholine, phosphatidyldimethyletha-nolamine, and phosphatidylmonomethylethanolamine weresynthesized when spores were germinated on media containingmethionine-methyl-'4C. In those experiments, methionine wasreadily taken up by spores since up to 30% of the radioactivityoriginally present in the medium could be extracted from ger-minated spores with chloroform-methanol after a 10 hr germi-nation period.

0

w

w

cr0~CLnf

S.Fs

DPG

PE i

PCp I

UN

OR.FIG. 3. An autoradiogram showing the thin layer chromato-

graphic separation of the chloroform-methanol extractable, -P-la-beled components of the germination medium and germinated ure-dospores of Uromyces phaseoli. The germination period was 2 hrand chloroform-methanol-water (66:30:4, v/v/v) was used as thechromatography solvent. Approximately equal quantities of IT-radioactivity were applied to the thin layer chromatography plate.The origin and solvent front are indicated by OR. and SF. re-spectively. DPG: diphosphatidylglycerol; PE: phosphatidylethanol-amine; PC: phosphatidylcholine; PI: phosphatidylinositol; UN: anunidentified inositol-containing phospholipid.

Plant Physiol. Vol. 48, 1971 737

4.11.



LANGENBACH AND KNOCHE

Table 1. Inzcorporationi of Radioactivity i/ito Componients of theChloroform-Methanzol Extract of Uromyces phaseoli Uredospores

Germinated on Media Conitainintg '4C-Cholinieanid 14C-Ethanzolaminie

Radioactivity IncorporatedComponent

14C-choline 14C-ethanolamine

dpmi

Phosphatidylethanolamine 0 40,700Phosphatidylcholine 12,800 8,250Chromatographic origin 3,200 6,050

The incorporation of the "C-labeled methyl group of me-

thionine into individual phospholipids was determined at differ-ent stages of germination. The experiments were carried outby germinating 20 mg of uredospores for various periods oftime. The methionine concentration was 10 [kM, and each Petridish contained 4.4 X 10' dpm of radioactivity. Chromato-graphic separation of the individual phospholipids was accom-

plished with system D. During the first 10 hr, a linear rate ofincorporation of the labeled methyl group into phosphatidyl-choline was observed. Knowing the specific radioactivity of thelabeled methionine, it was calculated that about 33 nmoles ofthe methionine methyl groups were incorporated into phospha-tidylcholine during the 10 hr germination period. Therefore,approximately 17% of the methionine originally present in themedium was utilized in the synthesis of phosphatidylcholine.Phosphatidylmonomethylethanolamine and phosphatidyldi-methylethanolamine appeared to reach a low, steady state levelin the early stages of the germination. These studies indicatethat the successive methylation of phosphatidylethanolamineto synthesize phosphatidylcholine does occur in germinatinguredospores of Uromyces phaseoli. The methylation of phos-phatidylethanolamine to form phosphatidylcholine occurred ata continuous rate over a 10-hr period, yet changes in phos-phatidylcholine levels were observed when 32P-labeled uredo-spores were germinated. In considering several explanations,it seemed likely that in the catabolism of phosphatidylcholine,choline was not degraded and could be reincorporated. There-fore, a pathway for the synthesis of phosphatidylcholine fromcholine and a , /3-diglyceride would be expected to exist.

Incorporation of "C-Choline and "C-Ethanolamine intoPhospholipids. To determine if preformed choline and ethanol-amine could be incorporated into phosphatidylcholine andphosphatidylethanolamine, respectively, uredospores were ger-minated on a medium containing the appropriate labeled sub-strates. The germination period was 10 hr and 20 mg of sporeswere used per sample. Concentrations of the radioactive sub-strates were: choline-1 ,2-"C, 20.0 nmoles/ml (2.32 x 10'dpm/ml) and ethanolamine-1 ,2-"C, 1.59 X 10' nmoles/ml(5.3 x 10" dpm/ml). System A was used in the chromato-graphic separation of the phospholipids. The data obtained are

shown in Table I. Neither choline nor ethanolamine was read-ily taken up by the germinating uredospores. Only 0.35% ofthe radioactivity in the "C-.holine-containing medium was

present in the chloroform-methanol extract of the spores thatgerminated on that medium. However, approximately 80% ofthe "C-choline in the spore was in the form of phosphatidyl-choline. The remainder of the radioactivity did not move fromthe origin of the chromatogram and was believed to be cholineor its derivative.On the basis of radioactivity, approximately 0.52% of the

ethanolamine in the medium was extracted with chloroform-methanol from the germinated spores, and about 74% of that

radioactivity was associated with phosphatidylethanolamine.Apparently, "C-labeled phosphatidylcholine was also synthe-sized by the methylation of phosphatidylethanolamine, or per-haps, ethanolamine.

It appears that choline and ethanolamine can be utilized inthe biosynthesis of phospholipids. Presumably, the anabolicpathway which involves an a, /-diglyceride and the appropriateCDP derivative, CDP-choline or CDP-ethanolamine, doesfunction in the germinating uredospores of Uromnyces phaseoli.

Incorporation of "C-Serine into Phospholipids. Besides thereaction of CDP-ethanolamine with an a, /3-diglyceride, thesynthesis of phosphatidylethanolamine may also occur, insome organisms, by the decarboxylation of phosphatidylserine.To determine if the latter pathway was operative in germinat-ing uredospores, spores were germinated on media containingD ,L-serine-3-`C. For each sample 20 mg of spores were germi-nated 10 hr. The concentration of "C-serine was 43.9 nmoles/ml (4.68 X 10' dpm/ml). In one experiment 2 mm choline wasincluded in the medium and in another 1 mm methionine wasincluded. System A was used in the chromatographic separa-tion of the lipids.

Serine was fairly readily incorporated into lipids, as 3.21%of the "C-activity in the medium was extractable from thegerminated spores with chloroform-methanol. The presence ofcholine in the medium appeared to stimulate the incorpora-tion of serine slightly, whereas when 1 mm methionine was inthe medium, the incorporation of "C-serine into lipids by thegerminating spores was only 0.064%. Table II shows thequantity of radioactivity incorporated into each of the com-ponents of the chloroform-methanol extract of the germinatedspores. With the three types of media, the "4C of serine wasincorporated into phosphatidylethanolamine, phosphatidylcho-line, and a neutral lipid component which consisted of C. ste-roids (unpublished data).The "C of serine could be incorporated into phosphatidyl-

choline by two pathways. The decarboxylation of phospha-tidylserine would yield phosphatidylethanolamine and hencephosphatidylcholine, which would possess the second and thirdcarbon atoms of serine. Methylation of phosphatidylethanola-mine with active one-carbon units from serine would also in-corporate the "C-labeled carbon atom of serine into phospha-tidylcholine. It appears that both routes of incorporation wereoperative when the data of Tables I and II are considered.The ratio of the radioactivity in phosphatidylethanolamine andphosphatidyleholine was 4.9 to 1 when "C-ethanolamine wasused as a substrate. With "C-serine as a substrate the sameratio was 1.6 to 1. The ratios were 2.7 to 1 and 1.3 to 1 when"C-serine was the substrate in media also containing 2 mMcholine and 1 mm methionine, respectively. If the only route ofincorporation of serine-2-"C into phosphatidylcholine was viaphosphatidylethanolamine, one would expect the ratios of theactivity to be the same when either ethanolamine-"C or serine-

Table II. Inicorporationi of Radioactivity inito Componlentts of theChloroform-Methaniol Extract of Uromyces phaseoli Uredospores

Germiinated ont Various Media Conitainintg ' 4C-Seritie

Raoioactivity Incorporated

ComponentiCe 4C-serine 14C-serine"4C-serine plus 2 mme plus 1 mme

choline methionine

dpm

Phosphatidylethanolamine 72,000 115,000 1,200Phosphatidylcholine 45,000 43,000 960Steroids 183,000 202,000 3,840

738 Plant Physiol. Vol. 48, 1971



PHOSPHOLIPIDS OF UROMYCES PHASEOLI. II

"4C was the substrate in the medium. Phosphatidylserine is ap-parently formed in the germinating uredospore, but functionsonly as an intermediate in the synthesis of phosphatidyletha-nolamine and phosphatidylcholine. Serine may also serve as asource of active one-carbon units for the methylation of phos-phatidylethanolamine.

DISCUSSION

Phospholipids in the spore appear to be extensively de-graded in the very early stages of germination and then resyn-thesized at later stages. The various phases indicate there arechanges in the function of phospholipids during the germina-tion period.Whereas the biosynthetic pathways for phospholipids have

been studied in mammalian and bacterial systems, our knowl-edge in plant tissue is incomplete. From this investigation itappears that germinating uredospores of Uromyces phaseoliare capable of catalyzing the successive methylation of phos-phatidylethanolamine to form phosphatidylcholine by the uti-lization of the methyl group of methionine. This pathway hasbeen recently demonstrated in Saccharomyces cervisiae (16,19). On the basis of our incorporation studies, the condensationof choline, ethanolamine, and serine with diglyceride to form,respectively, phosphatidylcholine, phosphatidylethanolamine,and phosphatidylserine does occur, although the intermediatesinvolved have not been identified. In animal tissues, CDP-choline and CDP-ethanolamine react with a diglyceride toform the phosphatides (8, 9) while L-serine condenses withCDP-diglyceride to yield phosphatidylserine in bacterial sys-tems (6). The decarboxylation of phosphatidylserine to givephosphatidylethanolamine appears to take place in germinat-ing uredospores. Serine also furnishes active one-carbon unitsfor the synthesis of phosphatidylcholine as well as the steroidsthat are formed during germination.The pattern of phospholipid levels observed in this study is

similar to that reported for germinating spores of Aspergillusniger (14). In Aspergillus, the rapid catabolism of phospho-lipids was concomitant with an increase in sugar phosphatesand nucleotides, with some of the phosphorus being incorpo-rated into RNA.

Jackson and Frear (5) determined total lipid phosphoruslevels in germinating flax rust uredospores (Melampsora lini)at 2-hr intervals and reported a continuous increase of lipidphosphorus during the first 6 hr of germination. It is possiblethat an initial catabolism of phospholipids also occurs in thisorganism, but the first anabolic phase restores the level ofphospholipids within the first 2-hr period instead of the 2.5- to3-hr period observed in Uromyces phaseoli. However, theother fluctuations of lipid phosphorus levels were not ob-served.The oxygen uptake studies reported by Maheshwari and

Sussman (12) closely parallel the catabolism of phospholipidsobserved in this study with the periods of greatest oxygen con-sumption being concomitant with the periods of greatest phos-pholipid catabolism.The changes in phospholipid levels may reflect a degrada-

tion and resynthesis of membranes. Ultrastructural studies (17)indicate that as early as 0.5 hr after germination has com-menced, morphological changes are visible in Puccinia grami-nis tritici. The electron transparent patches of the cytoplasm

almost occupy the cytoplasm after 1.5 hr of germination. Ona time scale, the appearance of these transparent regions cor-relates well with the initial catabolism of phospholipids byUromyces phaseoli. Williams and Ledingham (20) have re-ported that the diameter of the lipid bodies in ungerminatedspores is 0.8 /_t, while the diameter of lipid bodies in the germtube is about 0.1 ,u. It has been shown in spores of Mucorrouxii that hyphae elongate by tip growth and that the mostactive synthesis of hyphae occurs at the apical segment (2, 3).Assuming a similarity in germ tube growth, the greatest needfor energy and precursors of the cell wall would be at the apicalportion of the elongating germ tube. In spores of rust fungi,the greatest utilization of lipids appears to occur during thetime the germ tubes are rapidly elongating (4). Perhaps afunction of phospholipids is the formation of membraneswhich surround the small lipid droplets for their transportthrough the germ tube.

Acknowledgments-The authors wish to express their appreciation to Dr.J. M. Daly for his advice and review throughout this study. Thanks are dueto Paul Ludden for his assistance in supplying plant and spore material. Thetechnical assistance of Mrs. Kay Rossmiller and Miss Nancy Hemmer is alsoappreciated.

LITERATURE CITED

1. ALLEN, P. J. 1965. Metabolic aspects of spore germination in fungi. Annu.Rev. Phytopathol. 3: 313-342.

2. BART-NICKI-GARCIA, S. 1968. Control of (limorphism in Mucor by hexoses:inhibition of hyphal morphogenesis. J. Bacteriol. 96: 1586-1594.

3. BARTNICKI-GARCIA, S. 1969. Cell wall differentiation in phycomycetes. Phyto-pathology 59: 1065-1071.

4. DALY, J. M., H. W. KNOCHE, AND M. V. WIESE. 1967. Carbohydrate andlipid metabolism during germination of uredospores of Puccinia graministri'tia. Planit Pbysiol. 42: 1633-1642.

5. JACKSON, L. L. AND D. S. FREAR. 1967. Lipids of rust fungi. I. Lipidmetabolism of germinating flax rust uredospores. Can. J. Biochem. 45:1309-1315.

6. KA-ESHIRO, T. AND J. H. LAW. 1964. Phosphatidylcholine synthesis inAgrobacterium tumetaciens. I. Purification and properties of a phos-phatidylethanolamine N-methyltransferase. J. Biol. Chem. 239: 1705-1713.

7. KATES, M. 1970. Plant phospholipids and glycolipids. Advan. Lipid Res. 8:225-265.

8. KENNEDY, E. P. 1961. Biosynthesis of complex lipids. Fed. Proc. 20: 934.9. KENNEDY, E. P. AND S. B. WEISS. 1956. The function of cytidine coenzymes

in the biosynthesis of phospholipids. J. Biol. Chem. 222: 193-214.10. KNOCHE, H. W. 1968. A study on the biosynthesis of cis-9,10-epoxyocta-

decanoic acid. Lipids 3: 163-169.11. LANGENBACH, R. J. AND H. W. KNOCHE. 1971. Phospholipids in the

uredospores of Uromyces phaseoli. I. Identification and localization. PlantPhysiol. 48: 728-734.

12. MAHESHWARI, R. AND A. S. Suss,.%iAN. 1970. Respiratory changes duringgermination of uredospores of Puccinia graminis f. sp. tritici. Phyto-pathology 60:13,57-1364.

13. MAHLER, H. R., AND E. H. CORDES. 1966. Biological Chemistry. Harper andRow, New York. pp. 636-641.

14. NISHI, A. 1961. Role of polyphosphate and phospholipid in germinatingspores of Aspergillus niger. J. Bacteriol. 81: 10-10.

15. SHIVELY, J. M. AND A. A. BENSON. 1967. Phospholipids of Thiobacillusthiooxidans. J. Bacteriol. 94: 1679-1683.

16. STEINER, M. R. A.ND R. L. LESTER. 1970. In vitro study of the methylationpathway of phosphatidylcholine synthesis and the regulation of thispathway in Saccharomyces cerevisiae. Biochemistry 9: 63-69.

17. SissmA.N., A. S., R. J. LOWRY, T. L. DIJRKEE, AN.D R. MAHESHWARI. 1969.Ultrastructural studies of cold-dormant and germinating uredospores ofPuccinia graminis var. tritici. Can. J. Bot. 47: 2073-2078.

18. TROCHA, P. AND J. M. DALY. 1970. Protein and ribonucleic acid synthesisduring germination of uredospores. Plant Physiol. 46: 520-526.

19. WAECHTER, C. J., M. R. STEINER, AND R. L. LESTER. 1969. Regulation ofphosphatidylcholine biosynthesis by the methylation pathway inSaccharomyces cerevisiae. J. Biol. Chem. 244: 3419-3422.

20. WILLIANIS, P. G. AND G. A. LEDINGHAM. 1964. Fine structure of wheat stemrust uredospores. Can. J. Bot. 42:1503-1508.

739Plant Physiol. Vol. 48, 1971



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Phospholipids the Uredospores of Uromyces phaseoli