Adenosine Diphosphate-Glucose Pyrophosphorylase Control ... · Theprimary leaves ofhost plants wereinoculated in a settling tower 10 days after planting with lyophilized uredospores

Plant Physiol. (1970) 46, 126-135

Adenosine Diphosphate-Glucose Pyrophosphorylase Controlof Starch Accumulation in Rust-infected Wheat Leaves'

Received for publication February 25, 1970

PAUL W. MACDONALD2 AND GARY A. STROBEL3Department ofBotany and Microbiology, Montana State University, Bozeman. Montana 59715

ABSTRACT

The variation in starch content in healthy and Pucciniastriiformsi-infected wheat leaves was measured from 5 to15 days after inoculation. The starch content of diseasedleaves relative to healthy leaves decreased from 5 to 9 days,increased from 9 to 12 days to twice that of healthy leaves,and decreased from 12 to 15 days after inoculation. Electronmicrographs of plant tissues indicated that the starch ac-cumulated in the chloroplasts of host cells adjacent tofungal hyphae. Variations in sugar phosphates, ATP, andinorganic phosphate were measured during the infectionprocess. ADP-glucose pyrophosphorylase was extracted andpartially purified from healthy and diseased leaves. Whenproportionate concentrations of sugar phosphates and in-organic phosphate found in healthy and diseased leavesduring the infection process were placed in the assay mix-ture, ADP-glucose pyrophosphorylase activity was similarto the pattern of starch accumulation and was almost theinverse of the variation observed in inorganic phosphate indiseased leaves during the infection process. A mechanismto explain the accumulation of starch is presented and dis-cussed. This mechanism is based on the regulation of ADP-glucose pyrophosphorylase by changes in effector moleculeconcentrations during the infection process. Reasons forthese changes are presented.

Starch commonly accumulates in plants infected by obligateparasites. Generally, it accumulates in granules in chloroplasts ofphotosynthetic tissues (1) and in host cytoplasm in nonphoto-synthetic tissue (29). Little detailed information is available onthe regulation of starch accumulation in diseased plants. Usually,the starch content decreases around parasitic colonies soon afterinfection, followed by a subsequent increase before and duringsporulation and a decrease thereafter. Mirocha and Zaki (15)reported that starch decreased at infection sites of rusted bean

1 From a thesis presented by P. W. MacDonald in partial fulfillmentof the requirements for the Ph.D. degree in Botany at Montana StateUniversity. Supported in part by National Science Foundation GrantGB-12956, and specific grant for research P.L. 85-943, CooperativeStates Research Service, United States Department of Agriculture.Montana Agricultural Experiment Station Publication 147, JournalSeries.

2Present address: Department of Plant Pathology, University of Cal-ifornia, Riverside, California 92502.

3 United States Public Health Service Research Career DevelopmentAwardee 1-K4-GM-42, 475-01 from the National Institute of GeneralMedical Sciences.

leaves soon after infection, increased sharply just before sporula-tion, and then decreased sharply after spcrulation. Keen andWilliams (10) observed that starch accumulated in the cyto-plasm of host cells infected with Plasmod&ophora brassicae Wor.during vegetative growth and decreased during sporulation of thefungus.

Several physiological mechanisms have been proposed toexplain starch accumulation in diseased plants. Tanaka and Akai(27) have hypothesized that increased starch content in riceleaves infected with Cochbiobolus miyabeanus (Ito and Kuribay)Dickson was due to a decrease in ,s-amylase activity. Keen andWilliams (10) found increased specific activities of the starchsynthetic enzymes, UDP-glucose pyrophosphorylase and starchsynthetase, during starch accumulation in cabbage hypocotylsinfected with Plasmodiophora brassicae, but they offered noexplanation for these changes in enzyme activities.Through the work of Preiss' group it appears that regulation

of starch biosynthesis (reactions 1 and 2)

ATP + a-D-glucose 1-phosphate ADP-glucose + PPi (1)ADP-glucose + a-1, 4-glucan = -1,4-glucosyl-glucan + ADP (2)

in green algae (19, 22) and higher plants (6-8) occurs at the levelof ADP-glucose pyrophosphorylase (reaction 1). The regulationof this enzyme involves activation by glycolytic intermediates andinhibition by inorganic phosphate. Glycerate-3-P is the mostpotent activator. No activation of plant a-1 , 4-glucan synthetases(reaction 2) by glycolytic intermediates has been observed (5, 7).It seemed logical that regulation ofADP-glucose pyrophosphoryl-ase might account for starch accumulation in diseased plants.The purpose of this report is to investigate the validity of the

hypothesis that changes in effector molecule concentrations asthe result of host-parasite interaction are at least partially respon-sible for the regulation of starch accumulation in wheat leavesinfected with Puccinia striiformis West.

METHODS

Triticum vulgare L. Rego was chosen for this investigation.The host plants were grown in a walk-in environment chamberrigidly controlled and programmed for temperature, relativehumidity, and light. The diurnal temperature profile was 15C/24 C (dark/light). The relative humidity was about 95%during the dark period and 65%, during the light. The photo-period was 12 hr. Light intensities were increased stepwise from300 to 1800 to a maximum of 3500 ft-c at the middle of thephotoperiod and then decreased through a similar range. Underthese conditions Rego produces a three-infection type (moder-ately susceptible: uredia abundant, chlorosis). The host plantswere grown in 4-inch clay pots in sandy loam-peat moss-sand(1:1:1).The primary leaves of host plants were inoculated in a settling

tower 10 days after planting with lyophilized uredospores of126

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STARCH ACCUMULATION IN RUSTED WHEAT LEAVES

Puccinia striiformis A.T.C.C. PR No. 35 according to the methodof Sharp (25) to obtain uniform spore distribution. A CO2 gunwas used where plants were horizontally oriented to a sporeshower. A faster method of inoculation was also used in whichthe spores were applied to the leaves with a camel hair brush. Afterinoculation, the plants were placed in a dark dew chamber for 48hr for spore germination to occur, and then they were returned tothe controlled-environment chamber. Control (noninoculated)plants were also placed in the dew chamber to keep treatmentssimilar.

Sugars, sugar phosphates, nucleotide phosphates, pyridinenucleotides, and enzymes for sugar phosphate analyses were pur-chased from Sigma Chemical Company. Sodium pyrophosphate-32p was purchased from Amersham/Searle, Des Plaines, Illinois.

Radioactivity was quantitatively measured with a Nuclear-Chicago liquid scintillation counter. The scintillation fluid con-sisted of 4% Cab-O-Sil gel (Beckman Instruments) in toluenecontaining 40 ml of Spectrafluor and 50 mg of p-bis-2-(5-phen-oxazolyl)benzene per liter. Radioactivity on planchets wascounted on a Nuclear-Chicago gas flow counter. 32p cpm wereconverted to dpm by dividing by the percentage efficiency whichwas determined by counting samples of known radioactivity. Theloss in radioactivity of 32p each day was corrected for on the basisthat 32p has a half-life of 14.3 days.

Starch Determinations. One gram fresh weight of primary leafblades from both healthy and diseased plants was harvested at10 AM, cut into 15-mm sections, and boiled in 80% (v/v) ethanol(three changes) to remove chlorophyll and soluble sugars. Theextracted leaf sections were placed in large test tubes. The starchremaining behind in the leaves was then hydrolyzed with 15 mlof 52% perchloric acid for 24 hr at 24 C in a Brunswick Psychro-therm shaker at 10 rpm. Two-milliliter samples were taken andneutralized with an equal volume of 9 N NaOH.Glucose concentrations were determined by the arsenomolyb-

date method of Nelson (17). A standard curve was establishedwith a-D-glucose. The glucose concentrations obtained weremultiplied by 0.9 to calculate starch concentrations. Rates ofhydrolysis of known amounts of amylose, amylopectin, andcellulose were measured. It was determined that 80% amylose,70% amylopectin, and 20% cellulose were hydrolyzed after 24hr. Hydrolysis of cellulose was not corrected in the starch deter-minations.

Electron Microscopy. Electron microscopy was used to deter-mine the location of the starch in diseased leaf tissue. The follow-ing procedure was used. Leaf sections, 2 mm2, were cut with arazor blade from healthy and diseased leaves and fixed for 1 hr in2.5%/o glutaraldehyde in 0.05 M potassium phosphate buffer, pH7.3, followed by three 15-min washings in phosphate buffer. Thesections were then fixed for 4 to 12 hr in 2% osmium tetroxidefollowed by three 15-min washings in phosphate buffer.The sections were dehydrated at room temperature in 20, 50,

70, and 100% acetone for 5, 10, 10, and 15 min, respectively, andin propylene oxide liquid for 15 min.The sections were then infiltrated with embedding plastic and

propylene oxide (1:1, v/v) for 1 hr at 40 C. The embeddingplastic consisted of CIBA Araldite epoxy resin (No. 6005) andDDSA hardener (1.43 volumes of resin/1.0 volume of hardener),and BDMA accelerator (0.034 volume of accelerator/2.43volumes of resin-hardener mixture). The specimens were em-bedded at 60 C for 24 hr to allow polymerization of plastic.Specimen blocks were trimmed with a razor blade, thin-

sectioned with glass knives, and mounted on copper grids. Sec-tions were then stained with 2%/o aqueous uranyl acetate for M hrand with Reynold's lead citrate (20) for 3 to 5 min. Sections ofseveral hundred cells from four specimen blocks at each samplingdate were examined with a Zeiss EM-9A electron microscopeoperating at 60 kv.

Photosynthesis and Respiration. A Gilson differential respirom-eter equipped with a light bar was used to measure photosynthe-sis and respiration rates. Measurements were made at 25 C.Readings were taken at 10-min intervals for 50 min following a30-min equilibrium period. The change in 02 occurring between20 and 40 min was used to calculate rates. This change was con-verted to ul of dry gas at standard conditions. Gross photo-synthesis was calculated by adding the respiration rate to thephotosynthetic rate.For photosynthetic measurements the C02 concentration was

maintained at 0.03% in the flasks with a 0.2 M NaCO3 buffer, pH9.9 (28). Four or five freshly harvested primary leaf blades wereplaced in 3.0 ml of the buffer; buffer only was used as a blank.Respiration rates were measured in the dark. Center wells con-tained 0.2 ml of 20% KOH to absorb CO2 in the system, andstrips of filter paper were placed therein to increase the surfacearea of the KOH. Four or five leaf blades were placed in eachflask with 3.0 ml of distilled water. Following measurements,leaves were oven-dried and weighed.

Determination of Sugar Phosphates, ATP, and Inorganic Phos-phate. Leaves from healthy and diseased plants were har-vested, weighed, and frozen at - 15 C until all samples wereobtained. Then they were lyophilized overnight, weighed, andboiled twice in 80% (v/v) ethanol. The ethanol extracts werecombined, extracted twice with petroleum ether (ligroin 30-60C) to remove chlorophyll, vacuum-evaporated to approximately5 ml, taken to dryness with an air blower at room temperature,and stored desiccated at -15 C. The dried material was dis-solved in 1 ml of distilled water and passed through 1.0-cm x2.0-cm columns of Dowex 50W-X8 (H+ form), 200 to 400 mesh,then Dowex 1-X8 (formate form), 200 to 400 mesh. The anionfraction which contained the sugar phosphates, ATP, and in-organic phosphate was eluted off Dowex 1 with 6 N formic acid,taken to dryness with the air blower, and stored at -15 C.The sugar phosphate and ATP concentrations were determined

according to the method of Latzko and Gibbs (11) whereby thesemetabolites were enzymatically coupled to a pyridine nucleotide-requiring reaction and the change in absorbance of reducedpyridine nucleotide was measured. The compounds were as-sayed in the following sequence: glucose 6-phosphate, fructose6-phosphate, glucose 1-phosphate, ATP, dihydroxyacetone-phosphate, glyceraldehyde 3-phosphate, fructose 1, 6-diphosphateand 3-phosphoglyceric acid.

Concentrations of compounds were determined in a finalvolume of 3.0 ml with a Beckman DU spectrophotometer at340 nm; distilled water was used as a blank.

Glucose-6-P, Fructose-6-P, Glucose-i-P, and ATP. The follow-ing solutions were added per ml: initially 100 ,umoles of tri-ethanolamine-HCl buffer, pH 7.6; 0.2 pmole of TPN; 5 ,umolesof MgCl2 ; 0.5 unit of glucose-6-P dehydrogenase (AA = glucose-6-P), then the addition of 0.5 unit of phosphohexoseisomerase(AA = fructose-6-P), then the addition of 0.5 unit of phospho-glucomutase (AA = glucose-1-P), and finally the addition of 3Amoles of glucose and 0.5 unit of hexokinase (AA = ATP).

Dihydroxyacetone-P, Glyceraldehyde-3-P, Fructose-i, 6-diP.Initially, 100 ,umoles of triethanolamine-HCl buffer, pH 7.6;50 ,umoles of EDTA, pH 7.6; 0.1 Amole of DPNH; 0.5 unit ofa-glycerophosphate dehydrogenase (AA = dihydroxyacetone-P),then the addition of 0.27 unit of triose-P isomerase (AA = glyc-eraldehyde-3-P), and finally the addition of 0.55 unit aldolase(AA = dihydroxyacetone-P + glyceraldehyde-3-P from fructose-1,6-diP).

Glycerate-3-P. First, 100 MAmoles of triethanolamine-HCI buffer,pH 7.6; 5 jzmoles of MgCl2 ; 0.1 Mmole of DPNH; 2.8 units ofglyceraldehyde-3-P dehydrogenase (AA = glycerate-1, 3-P); andthen the addition of 0.2 unit of glycerate-3-P kinase (AA =

Plant Physiol. Vol. 46, 1970 127

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MACDONALD AND STROBEL

Inorganic phosphate was determined by the method of Fiskeand SubbaRow (12). A standard curve was established with pureK2HPO4 in distilled water.

Preparation of ADP-Glucose Pyrophosphorylase. The enzymewas prepared by a method modified from Ghosh and Preiss (8).Protein concentration was measured by the method of Lowryet al. (13).

Step 1. Ten-gram quantities of healthy leaves were cut into1-cm sections, placed in an OmniMixer (Sorvall) with 70 ml ofchilled acetone, and homogenized at full speed for 1 min. Thehomogenate was passed under vacuum through a Buchner funnelcontaining Whatman No. 42 filter paper and followed byseveral volumes of chilled acetone. The acetone powder wasdried on the filter paper and stored desiccated at -15 C. Thefilter paper was cut up into small pieces and taken up in 0.05 Mtris-HCl buffer, pH 7.5, containing 1 mm EDTA and 2.5 mMreduced glutathione, passed through two layers of cheeseclothto remove cellulose fibers and filter paper, and centrifuged at40,000g for 15 min. The supernatant fluid was used as thesource of pyrophosphorylase.

Step 2. ADP-glucose pyrophosphorylase was further purifiedby heat denaturation at 65 C for 5 min in a water bath, thenquickly cooled in cold water. Denatured protein was removed bycentrifugation at 20,000g for 15 min.

Step 3. The supernatant, which contained all enzymaticactivity, was fractionated with solid ammonium sulfate into 0to 40% and 40 to 60% saturation fractions, centrifuged at 20,000gfor 15 min, taken up in 0.05 M tris-HCl buffer containing 1 mmEDTA and 2.5 mM GSH, and dialyzed overnight against thesame buffer at 4 C. The 40 to 60% ammonium sulfate fractionwas used for enzyme assays. No further purification of theenzyme was made. The ammonium sulfate fraction was storedat 4 C.Assay of Pyrophosphorylase Activity. Pyrophosphorylase

activity was assayed according to the method of Shen and Preiss(26) whereby pyrophosphorolysis of ADP-glucose was followedby the formation of ATP-32P in the presence of P32Pi . Sodiumpyrophosphate-32P (specific radioactivity 189.5 mc/mmole) wastaken up in 53 ml of 0.01 M sodium pyrophosphate to yield 5 x104 dpm/,ll at the commencement of experiments. The reactionmixture, which contained 30 ,Amoles of tris-HCl buffer (pH 7.5),3 pmoles of MgCl2, 0.2 ,umole of ADP-glucose, 0.5 j,mole ofP32Pi (specific radioactivity 0.4-2.2 Ac/Amole), 5 Amoles of KF,and the enzyme preparation in a final volume of 0.5 ml, wasincubated in a conical centrifuge tube at 37 C for 10 min in awater bath. The reaction was stopped by the addition of 3 ml of5% cold trichloroacetic acid and 0.1 ml of 0.1 M unlabeled sodiumpyrophosphate was added to dilute the P32Pi. Then, 0.1 ml ofNorit A suspension (150 mg/ml) was added to adsorb the ATP-32p formed. The Norit A suspension was centrifuged in a clinicalcentrifuge, and the supernatant was discarded, washed twice morewith 3 ml of cold 5% trichloroacetic acid, and once with 3 ml ofcold distilled water. After washing, the Norit A was suspended in2 ml of aqueous solution of 50% ethanol containing 0.1% NH3 .One milliliter of this suspension was dried in a planchet andcounted in the gas flow counter; or alternatively dried Norit Awas counted in the liquid scintillation counter.

RESULTS

Preliminary results with IKI staining indicated the presence ofstarch in diseased wheat leaves. The positive reaction for starchwas most pronounced 12 days after inoculation. Under the micro-scope the starch appeared to be located in chloroplasts of hostcells adjacent to intercellular fungal hyphae, and in immatureuredospores at the periphery of pustules. The chloroplastsappeared more "granular" with starch and smaller than those inhealthy leaves.

Pattern of Starch Accumulation. The starch content of wheat

leaves inoculated with P. striiformis was followed from 5 through15 days after inoculation (Fig. 1). Relative to the starch contentin healthy leaves the starch content of diseased leaves decreasedfrom 5 to 9 days during the flecking stage, increased from 9 to 12days with the sharpest increase occurring on 12 days, and thendecreased sharply from 12 to 15 days. At 12 days after inoculation,the starch content of diseased leaves was 1.8 times greater thanthat in healthy leaves.

Labeling experiments with 14CO2 and sucrose-U-"4C demon-strated the accumulation of carbohydrate at infection sites (14).Photosynthetic fixation ofCO2 and accumulation of carbohydratefrom other plant parts may account for the source of carbohy-drate for starch. Furthermore, leaves which were administeredlabeled '4CO2 and then extracted of chlorophyll and solublesugars were hydrolyzed with perchloric acid, paper-chromato-graphed, and autoradiographed. The results showed that thelabel was predominantly in glucose with some in xylose suggestingthat the accumulated label was probably in starch.

Electron Microscopy. Thin sections of healthy and diseasedleaves were examined under the electron microscope at 7, 10, 12,and 15 days after inoculation. Figures 2 and 3 show typical chlor-oplasts at 12 days after inoculation in healthy and diseased leaves,respectively. Chloroplasts in healthy leaves at all sampling dateswere normal in appearance. The grana lamellae were well devel-oped and interconnected at regular intervals by intergrana lamel-lae. The internal lamellar system was embedded in a finely granu-lar stroma and bounded by the chloroplast envelope (an innerand outer membrane). No starch granules were observed in anyof the chloroplasts examined. Small osmiophilic globules wereseen in some chloroplasts.

Chloroplasts in diseased leaves were similar in appearance tothose observed in comparable healthy leaves at 7 days afterinoculation, and also in host cells distant from the fungus at latersampling dates. At 10 days after inoculation chloroplasts incells adjacent to intercellular fungal hyphae departed from normalplastid appearance. Chloroplasts with osmiophilic globules werecommonly observed. These bodies were larger in size and lesselectron opaque than those observed in plastids of healthy leaves

+8.0-

+6.0-

I

0: 0

'-+2.0-4.0

5 7

Flecking Sporylotion

p 9 lDAYS

1 13 15

FIG. 1. Variation in starch content in wheat leaves infected withPuccinia striiformis from 5 to 15 days after inoculation expressed as thedifference in starch content between diseased and healthy leaves. Thevertical lines indicate the division of stages of host-parasite interaction.Each point on this graph represents the average oftwo separate determi-nations, each replicated four times, for both averages of two determina-tions. The value obtained for healthy leaves was subtracted from thevalue obtained from diseased leaves to give each point on the graph.

128 Plant Physiol. Vol. 46, 1970

llllll.

I



Plant Physiol. Vol. 46, 1970 STARCH ACCUMULATION IN RUSTED WHEAT LEAVES

No starch granules were observed in plastids of diseased tissuesat 7 or 10 days after inoculation.At 12 days after inoculation (Fig. 3) starch granules were

observed in many chloroplasts of cells adjacent to fungal hyphae.

Usually one or two small starch granules were seen within achloroplast section. Each granule appeared to be surrounded byan electron transparent region. The evident displacement ofchloroplast contents near the starch granules seemed to indicate

t. W a-TA#

*-

FIG. 2. A chloroplast in a cell of a noninfected wheat leaf (control) 12 days after inoculation showing no evidence of disorganization or starchgranules. X 24,000. CE: Chloroplast envelope; G: grana lamellae, I: intergrana lamellae; 0: osmiophilic globules; ST: stroma; and T: tonoplast

.- .~~~jv. '.A

_ 1..;/

t'1

at

FIG. 3. Chloroplasts in a cell of wheat leaf infected with P. striiformis each containing one starch granule. Osmiophilic globules are present. X22,600. CE: Chloroplast envelope; G: grana lamellae; I: intergrana lamellae; 0: osmiophilic globules; ST: stroma; and S: starch granule.

129

Alp,



MAcDONALD AND STROBEL Plant Physiol. Vol. 46, 1970

that the granules had increased in volume within the plastids.Some plastids became distended, and some produced invagina-tions of their envelopes. Rupturing of the plastidial envelopes,however, resulting from increased size of starch granules wasrare. Osmiophilic globules were commonly observed in thestroma and were more numerous and often less electron-opaquethan those observed in plastids of healthy leaves.

+2.0

L.

n + I1.001

E

0

-jo0

Flecking SporulationAK4

AiI a_ \~A

*0 *A-A Gross photosynthesis.-. Respiration

3 5 7 9 11 13 15DAYS

FIG. 4. Respiration and gross photosynthetic rates of leaves infectedwith P. striiformis from 3 to 16 days after inoculation expressed as thedifference in rates between diseased and healthy leaves. These data arethe means of five replications.

L.

00

+500-1 __Glucose-I-Phosphate

+400 - a+300 -

E +200-

E + 100

Cl)U)F-

blJILz

- 100--200--300 -

-400 -

5

ij +500 -

I +400I7 4300-

3 +200-w + 100

Fructose-6-PhosphateC

Unz - IC7, -2CLuLL -3C5 -4C

DO--

DO -I I_

8 9 10 11 12 13 14DAYS

Fructose -- 4 6-Diphosphate-d

8 9 10 11 12 13 14DAYS

FIG. 5. Variation in the quantities of metabolites in leaves infectedwith P. striiformis from 8 to 14 days after inoculation expressed as thedifferences in concentrations between diseased and healthy leaves. Eachpoint on these graphs represents the average of two separate determina-tions, each replicated twice, for both healthy and diseased leaves. Thevalue obtained for healthy leaves was subtracted from that of diseasedeaves to give each point on the graph.

4500Dihydroayacetone

\ +400 - PhosphateE +300 -eE:t +200 -E +100 -

D 0U)() - 100 -

0 -200 -wzI -300--

C,)wLL -400-zf

Z+500-_ i+o 3-Phosphogyeic

3 +400 - AcidZ-4300- gww +200-w + 100

o -100-zw -200-w.L -300U_.0) ..400

I_3-Phosphog0ycera/dehyde- f _

_ "S _

A TPh

A11--

8 9 10 11 12 13 1InAYS

FIG. 5. e-h

At 15 days after inoculation very few plastids were observed tocontain starch granules. The most striking feature was the pres-ence of many large osmiophilic bodies in some chloroplasts nearfungal tissue. These appeared larger in size compared to thoseobserved earlier in the infection process. These bodies appearedto have displaced some of the chloroplast contents, which indi-cated that they may have increased in size. Degeneration andgeneral disarray of internal structure of some chloroplasts wasobserved in some cases, probably the result of approaching celldeath.

Respiration and Gross Photosynthesis. The differences betweenthe respiration and gross photosynthetic rates of healthy anddiseased leaves appear in Figure 4. Both photosynthesis andrespiration rates in diseased leaves gradually increased during thedisease cycle through the flecking stage. Photosynthesis peakedat 8 days and then gradually decreased, and respiration ratepeaked at 10 days and then gradually decreased during sporula-tion. The respiration rate of diseased leaves was 3 times greaterthan healthy leaves at 10 days; and photosynthesis was 1.7 and1.6 times greater in diseased leaves than in healthy leaves at 8and 10 days, respectively, after inoculation. The peak of respira-tion and gross photosynthetic rates in diseased leaves coincideswith the beginning of sporulation but precedes maximum starchaccumulation by several days.Changes in Levels of Phosphorylated Intermediates. Differences

between diseased and healthy leaves in concentrations of meta-bolic intermediates involved in starch biosynthesis during theinfection process are shown in Figures 5 and 6. At 8 days afterinoculation, all measured sugar phosphates, ATP, and Pi indiseased leaves were at about the same levels as in healthy leaves.Then, as starch increased from 9 to 12 days (Fig. 1), some markedchanges had taken place. Glucose-i-P (Fig. 5a), a substrate ofADP-glucose pyrophosphorylase, decreased sharply from 8 to 10days in relation to healthy leaves, incre-sed sharply from 10 to

130

III8 9 10 11 12 13 14

DAYS

--r.,.

t-. It




11 days, and then decreased slightly at 12 days. Glucose-6-P(Fig. 5b) increased sharply from 8 to 10 days and then decreasedsharply from 10 through 12 days. Fructose-6-P (Fig. Sc) andfructose-1, 6-diP (Fig. Sd) showed little change during this period.Dihydroxyacetone-P (Fig. Se) increased in concentration from8 to 10 days and then decreased from 10 to 11 days. Glyceralde-hyde-3-P (Fig. Sf) showed an increase on 12 days. Glycerate-3-P(Fig. 5g) showed a slight change in concentration during thisperiod; and ATP increased slightly at day 11. Inorganic phos-phate (Fig. 6), an inhibitor of ADP-glucose pyrophosphorylase,increased sharply from 8 to 10 days, decreased sharply from 10to 11 days, and then increased from 11 to 12 days.When the starch content decreased from 12 through 15 days

after inoculation, glucose-l-P increased slightly to that ofhealthy leaves; glucose-6-P increased sharply, and fructose-6-Pshowed a slight increase in concentration. Fructose-1 , 6-P anddihydroxyacetone-P showed decreases in concentration. Glyceral-dehyde-3-P showed a slight decrease and glycerate-3-P and ATPshowed no change. Inorganic phosphate increased slightly from12 to 14 days.ADP-Glucose Pyrophosphorylase Experiments. The purifica-

tion procedure used for the preparation of ADP-glucose pyr-phosphorylase from healthy wheat leaves is summarized in TableI. A purification of 8.5-fold was achieved under the specified

F/eckinqS/g lion

+60 -

>, +40-t0 -

a, +20-

0-

E -

t -20 -

I- -

: -40-Cl)O -60-

-80-

7 9 11DAYS

13 15

FIG. 6. Variation in inorganic phosphate concentration in leavesinfected with P. striiformis from 8 to 14 days after inoculation expressedas the difference in concentration between diseased and healthy leaves.The vertical line indicates the division of stages of host-parasite inter-action. Each point on this graph represents the average of two separatedeterminations, each replicated twice for both healthy and diseasedleaves. The value obtained for healthy leaves was subtracted from thatof diseased leaves to give each point on the graph.

Table I. Purification of ADP-Glucose Pyrophosphorylase fromHealtlhy Wheat Leaves

1. Acetone powder2. Heat treatment3. Ammonium sulfate

0-40%40-60%-o

ml

6053

mg/ml

2.731.37

SpecificActivity

munits'/mg

4.79.6

3.2 5.13 6.85.1 3.72 39.8

Total Yield Purifi-lActivityl cation

mlnits'

760745

112750

10098

1599

Table II. Substrate Specificity of Enzyme Preparations fromHealthy and Diseased Leaves

The conditions of the experiment are described in the assayprocedure. The 40 to 60% ammonium sulfate fraction was used.The concentration of substrate was 0.38 mm. Diseased leaves wereharvested 11 days after inoculation.

Substrate Source of Enzyme ATP Formed

my& moles/mg per10 min

ADP-glucose Healthy leaves 22.8Diseased leaves 8.9

UDP-glucose Healthy leaves 10.2Diseased leaves 5.9

7 50z0

400'-

#

E0 30

cnw-Jo 20

E

6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

pH

FIG. 7. Effect of pH on ADP-glucose pyrophosphorolysis with en-zyme preparations from healthy and P. striiformis-infected wheatleaves. Carbonate buffer was used at pH 6.0, a tris-HCl buffer was usedfrom pH 7.0 to 8.5, and a glycine-NaOH buffer from pH 9.0 to 10.0.The conditions are described in the assay procedure except that thebuffers varied.

conditions, and 99% of the total activity of the enzyme was in the40 to 60% ammonium sulfate fraction. The same purificationprocedure was followed for the preparation of the enzyme fromdiseased plants harvested 11 days after inoculation.Some of the other properties of the enzyme were previously

examined and reported (14). ADP-glucose, inorganic pyro-phosphate, and enzyme were simultaneously required for thereaction to occur, and MgCl2 was also required for optimalactivity. The rate of ATP formation versus protein concentra-tion was linear, the Km for ADP-glucose as the substrate asdetermined from a Lineweaver-Burk (12) plot was 1.08 X 10-3M, and the Vma. was 1.18 X 10-2 m,mole/mg. 10 min in theabsence of activator.

Table II shows the substrate specificity of enzyme preparationsfrom healthy and diseased leaves for ADP-glucose and UDP-glucose. ADP-glucose as the substrate resulted in greater enzymeactivity with both preparations than did UDP-glucose. It ispossible that there are two glucose-nucleotide phosphorylasereactions in these ammonium sulfate fractions or that one ADP-

I One munit is defined as 1 mnumole of ATP formed under theconditions described in the assay procedure.

131



MAcDONALD AND STROBEL

glucose pyrophosphorylase had greater activity for ADP-glucose than for UDP-glucose.

Figure 7 shows the pH optima for pyrophosphorolysis ofADP-glucose in the presence of the enzyme preparations from healthyand diseased leaves in various buffers. The pH optima for theenzyme preparations from healthy and diseased leaves were 70and 7.5, respectively.

Tables III and IV show activation or inhibition of ADP-glucose pyrophosphorylase in the presence of the preparationfrom healthy and diseased leaves respectively, with variousmetabolites at pH 7.5. The best activator was glycerate-3-P,giving 14.5- and 42-fold increases in activity in healthy and dis-eased tissue preparations, respectively, compared to preparationswith no activator. Fructose-1 ,6-diP was one-eighth and one-seventh as effective as glycerate-3-P in the preparations fromhealthy and diseased leaves, respectively. Dihydroxyacetone-Pgave a 1.6-fold stimulation to the enzyme preparation fromdiseased leaves but was inactive with the enzyme preparationfrom healthy leaves. Glucose-6-P and fructose-6-P were inactivewith the preparation from diseased leaves but were inhibitory withthe preparation from healthy leaves. Enzyme inhibition wasobserved in both preparations with glyceraldehyde-3-P. Ribose-5-P was tested only with the enzyme preparation from diseasedleaves and was effective as fructose-1 , 6-P as an activator. Itshould be mentioned here that enzyme activity was measuredthrough pyrophosphoryolysis and not via synthesis of ADP-

Table III. Effect of Metabolic Initermediates oni ATP Synithesiswith the Enzzyme Preparationi from Healthy Leaves

The conditions of the experiment are described in the assay pro-cedure. The concentrations of compounds present in the reactionsmixture are listed in the table.

RelativeCompound Concn ATP Formed Increase or

Decrease

mAmoles/mngper 10 mnn

None ... 34.4Glucose-6-P 0.91 21.8 0.6Fructose-6-P 0.91 21.0 0.6Fructose-1,6-diP 0.91 57.0 1.7Dihydroxyacetone-P 0.88 34.0 1.0Glyceraldehyde-3-P 0.91 8.9 0.3Glycerate-3-P 0.88 420.0 14.5

Table IV. Effect of Metabolic Intermediates oni ATP Synithesiswith the Enizyme Preparationi from Diseased Wheat Leaves

The conditions of the experiment are described in the assay pro-

cedure. The concentrations of compounds present in the reactionmixtures are listed in the table. The enzyme was prepared from dis-eased wheat leaves 11 days after inoculations.

] RelativeCompound Concn ATP formed Increase or

Decrease

m.u m,Amoles/ing per10min

None ... 8.9 ...

Glucose-6-P 0.91 7.7 0.9Fructose-6-P 0.91 10.0 1.1Fructose-1, 6-P 0.91 49.4 5.5Dihydroxyacetone-P 0.88 14.2 1.6Glyceraldehyde-3-P 0.91 2.3 0.3Glycerate-3-P 0.88 376.0 42.0Ribose-5-P 0.91 50.0 5.6

glucose, which is the true physiological function of the enzyme.

The effects of various metabolites on the enzyme may therefore beaffected by the direction in which the enzyme is assayed; i.e., theextent of inhibition or activation may be different in the directionof synthesis as compared to pyrophosphorolysis.

Figure 8 shows the effect of activator concentration on thestimulation of ATP synthesis by varying the concentration ofglycerate-3-P. The saturation curve for glycerate-3-P did notfollow first order (Michaelis-Menton) kinetics, but was sigmoidalin nature, suggesting that more than one molecule was bound tothe enzyme and that these activator sites were interacting. The Hillequation (3) yielded a K value of 7.4 x 10-s mM. When the logv/(Vmax - v) was plotted against the log of the glycerate-3-Pconcentration (insert Fig. 8), a straight line was obtained with a

slope of 2.16, suggesting that there were more than two, perhapsthree or four binding sites on the enzyme for this activator.

Inorganic phosphate was a potent inhibitor of ADP-glucosepyrophosphorylase. Figure 9 shows the effect of phosphate on therate of ATP synthesis. Plotting the data as log V/max - v) versus

log of inorganic phosphate concentration (insert Fig. 9) gave an

interaction coefficient of 1.47 and the Ki was 1.41 X 10-2 mM

x

z 60z-

0

° 50

40(1)

-J

0 30

:1tE 20

0

E 10

ILO

0 5 10 15 20 25 30 35 40v 100 500

3-PHOSPHOGLYCERATE,mM x 10-2

FIG. 8. The dependence of the rate of ADP-glucose pyrophospho-rolysis on the concentration of the activator, 3-phosphoglycerate(3-PGA). The reaction conditions are described in the assay procedureexcept that the concentration of 3-phosphoglycerate was varied. Theinsert shows a plot of log v/V.n,x-v versus log of 3-phosphoglycerateconcentration.

100 1.0V

Z -0-0.0

ta>~ ~ ~ LO20__)<_ LG[

60LO- 2< v~~~max- K,'=.4/x/O- mMA

n/1.47

<c 40-

Z -3.0 -2.0 1.0 0.0

20 LOG [P]

O0 0.5 1.0 2.0 5.00.0

INORGANIC PHOSPHATE, mM

FIG. 9. Inhibition of ATP synthesis by inorganic phosphate (Pi).The reaction conditions are described in the assay procedure except that

the concentrations of Pi varied. The insert shows a plot of log v/ Vmax-vversus log of inhibitor concentration. Vlar5 is defined as the velocity in

the absence of the inhibitor.

|.0

0.0

0 -1.0E K'=7.4x /O3mM'~* n r2.16

.0 -2.0fp,_I-J -2.0 -1.0 0.0

LOG [3-PGA]1.- --L-- - .--1


I

II

I




Table V. Co,icenitrationis of Metabolites Used in the Assay of ADP-Glucose PyrophosphorylaseThese values were calculated proportionately from concentrations of metabolites found in healthy and diseased leaves assuming 250

munits' of enzyme per g dry weight of healthy leaves and 2.7 units of enzyme per assay mixture.2

Metabolite

Glucose-6-P

Fructose-6-P

Fructose-I,6-diP

Dihydroxyacetone-P

Glyceraldehyde-3-P

Glycerate-3-P

Inorganic phosphate

HealthyDiseasedHealthyDiseasedHealthyDiseasedHealthyDiseasedHealthyDiseasedHealthyDiseasedHealthyDiseased

Days after Inoculation

8 10 11 12 14

1.500.680.750.160.181.670.130.000.000.000.620.84

246267

0.316.340.000.000.231.350.612.710.310.140.460.55

162686

mumoles/assay

2.152.480.820.490.000.090.830.830.661.001.331.00

1040181

I One munit is defined as 1 mMmole of ATP formed under the conditions described in the assay procedure.mMmoles mpmoles assay

2 The absolute values of metabolites may be calculated as follows: = Xg dry wt assay 2700 munits

9.892.244.532.990.760.751.511.490.001.490.510.60

365130

0.943.010.590.192.960.154.430.200.000.301.482.00

17065

250 munitsg dry wt

z

+1.0

00C,D-J -1.0a0

E -2.0

8 9 10 11 12 13 14

DAYS

FIG. 10. Variation in the rate of ADP-glucose pyrophosphorylaseactivity in the presence of proportionate concentrations of metabolitesfound in healthy and P. striiformis-infected wheat leaves from 8 to 14days after inoculation. The data are expressed as the differences inenzyme activity between simulated metabolite preparations from dis-eased and healthy leaves. The assays were made with an enzyme prep-aration from healthy leaves. The vertical line indicates the division ofstages of host-parasite interaction.

Table V shows the concentrations of metabolites placed in theassay mixture with the enzyme simulating those concentrationsfound in healthy and diseased leaves from 8 to 14 days afterinoculation.These values were calculated proportionately fromconcentrations of metabolites found in healthy and diseasedleaves assuming 250 munits of ADP-glucose pyrophosphorylaseper g dry weight of healthy leaves and 2.7 munits of enzyme usedper assay. An enzyme preparation from healthy leaves was used

for all assays. The results of these simulations are plottedin Figure 10 as the differences in enzyme activity between simu-lated metabolite preparations from diseased and healthy leaves.ATP and glucose-i-P were not used since they are products of thereaction in the enzyme assay. Figure 10 shows that pyrophos-phorylase activity decreased from 8 to 10 days, increased from 10to 12 days, and then decreased from 12 to 14 days after inocula-tion. This pattern closely resembles that of starch accumulation(Fig. 1). These results are evidence for the regulation of starchaccumulation by changes in metabolite concentrations during theinfection process.

DISCUSSION

The evidence presented indicates that starch accumulates inwheat leaves infected with Puccinia striiformis. Starch determina-tions from 5 to 15 days after inoculation (Fig. 1) show that starchdecreases during the flecking stage, increases in concentrationthrough the beginning of sporulation, and then decreases. Theseresults are supported by electron micrographs of healthy and dis-eased host cells which show starch granules in some chloroplastsof leaf cells of diseased plants only at 12 days after inoculation(Fig. 3). Only one or two small starch granules were seen inchloroplast sections of cells adjacent to fungal hyphae at the timeof maximal starch accumulation. Presumably this quantity ofstarch contained in several hundred chloroplasts could accountfor most of the accumulated starch found in diseased leaves. Nostarch accumulated in the host cytoplasm, as occurs in nonphoto-synthetic tissue (10), but a positive reaction for starch with I-KIwas observed in immature spores at pustule peripheries. Thisobservation warrants further investigation.

Other evidence supporting starch accumulation was the posi-tive reaction to I-KI for starch in chloroplasts of host cells ad-jacent to fungal tissue giving chloroplasts of diseased leaves a"granular" appearance at 12 days after inoculation (preliminaryresults). These may be the starch granules observed in electronmicrographs (Fig. 8). Further evidence was the demonstration ofthe accumulation of carbohydrates at diseased leaf tips following14C02 and sucrose-'4C labeling experiments (14). Accumulation

133



MACDONALD AND STROBEL

of metabolites at infection sites has been shown to be an activeprocess in diseased tissue by Shaw and Samborski (25), and itprobably accounts for the carbohydrate accumulated as starch andfor other metabolites required for pathogen growth.These results generally agree with reports for other obligate

plant parasites. The actual timing of starch accumulation intissues during the infection process seems to vary with the para-site. In fungal diseases starch has been found to accumulateduring the vegetative growth of the fungus and then decreaseduring sporulation (1, 10, 15). In viral diseases, starch seems toaccumulate in deteriorating plastids at cell death (2). Discrep-ancies in results of various authors may be largely due to thelack of uniformity in the conditions of their experiments. Starchcontent will vary depending on environment, plant age, and stageof infection (9).The reaction producing ADP-glucose from ATP and glucose-i-

P seems to be a main control point for polysaccharide synthesisin bacteria and green plants (8, 18, 21). Preiss proposed a mecha-nism to explain diurnal starch variations in spinach leaf based onthe regulation of ADP-glucose pyrophosphorylase (18). A similarmechanism might explain variations in starch content in diseasedwheat leaves.Some basic assumptions had to be made to propose a mecha-

nism to explain starch variations in rust-diseased leaves; (a) themechanism proposed by Preiss is assumed to be correct; (b) har-vesting leaves at the same time each day eliminates most variationin metabolite levels due to diurnal fluctuations; (c) nondiseasedwheat leaves have a relatively low rate of starch biosynthesis sinceno starch granules were observed in chloroplasts from nondis-eased leaves at any sampling date; and (d) the host and pathogencould not be separated; therefore metabolite changes observed indiseased leaves are a reflection of what is occurring in both hostand pathogen. Another factor which is recognized but not dealtwith is compartmentation of metabolites. No data are availableon the concentrations of metabolites at enzyme sites.The data presented establish the presence of ADP-glucose pyro-

phosphorylase in healthy wheat leaves (Table I) and also in dis-eased wheat leaves (14). It is activated and inhibited by variousmetabolites, suggesting that it is an allosteric or regulatory en-zyme (Tables II and III). The saturation curves for glycerate-3-P(Fig. 8) and Pi (Fig. 9) indicate the presence of allosteric sites onthe enzyme (8). The properties of ADP-glucose pyrophospho-rylase for wheat leaves generally agree with those found forother plant tissues (18, 21).ADP-glucose is the sole glucosyl precursor for starch synthesis

(18); therefore, it appears that starch synthesis is controlled bythe regulation of ADP-glucose synthesis. The levels of activatorssuch as glycerate-3-P and fructose-1,6-diP (and possibly otherglycolytic intermediates) and inhibitors such as Pi would con-trol the rate of starch synthesis.

In nondiseased plants the balance between activators and inhi-bitors (glycerate-3-P and Pi in particular) in light and dark regu-late the rate of ADP-glucose synthesis (8, 18, 21). The data sug-gest that in diseased leaves the levels of Pi and, to a lesser extent,the levels of effector molecules regulate the rate of starch synthesisthrough ADP-glucose pyrophosphorylase.The decrease in starch content in diseased leaves from 7 to 8

days after inoculation (Fig. 1) can be explained on the basis thatthe levels of Pi in nondiseased leaves are somewhat inhibitory toADP-glucose pyrophosphorylase. At 8 days after inoculation thelevel ofPi is about the same in diseased leaves as in healthy leaves,and thus inhibitory. Thus, since starch is probably not being syn-thesized at a very rapid rate and the respiration rate of the host-parasite interaction is near a peak (Fig. 4) as the result of fungalrespiration and enhancement of host respiration by the fungus(23), carbohydrate reserves are probably being depleted.The increase in starch from 9 to 10 days after inoculation is

difficult t explain in relation to ADP-glucose pryophosphorylasesince Pi increases in diseased leaves and thus should inhibit starchbiosynthesis. The increase in Pifrom 8 to 10 days after inoculationmay be contributed in part by the use of ATP by the fungus forbiosynthetic processes involved in reproduction, and in part byaccumulation from other plant parts, as observed by Mukherjeeand Shaw (16). The carbohydrate for starch synthesis may be theresult of accumulation of carbohydrate from other plant parts,and from photosynthesis (Fig. 4).The starch content increases sharply from 10 to 12 days after

inoculation. At the same time the Pi level decreases sharply from10 to 11 days, followed by an increase from 11 to 12 days. Thisdecrease seems to be a most significant factor in the accumulationof starch on those days. Inhibition of ADP-glucose pyrophos-phorylase activity would be released by the decrease in Pi, andstarch biosynthesis could occur from 10 to 12 days after inocula-tion. A contributing factor to starch biosynthesis might be activa-tion of pyrophosphorylase by glycerate-3-P (Fig. Sg), the mostpotent activator of ADP-glucose pyrophosphorylase. Althoughthe concentration of this compound does not greatly changeduring the infection process, the combination of a decrease in Piconcentration and the concentration of glycerate-3-P presentcould conceivably cause activation of the enzyme (Fig. 10).Other contributing factors to the increase in starch content are

the changes in other sugar phosphate concentrations. Glucose-6-P, which is slightly inhibitory to ADP-glucose pyrophosphorylase(Table III), decreased in concentration from 10 to 12 days afterinoculation (Fig. Sb). This may have resulted in a release ofinhibition of the enzyme, allowing starch synthesis to occur. Thesame might be true for fructose-6-P (Fig. Sc), which showed a

slight decrease in concentration from 10 to 12 days after inocula-tion. The possibility of other or unknown activators stimulatingstarch synthesis also exists, for example, phosphoenolpyruvate,an activator which was not tested, or pentose pathway interme-diates.The decrease in starch content in diseased leaves from 12 to 15

days after inoculation can be attributed to the increase of Pi con-centration to levels resulting in inhibition of starch biosynthesis.The starch content then, would decrease as the result of the ac-

tivity of starch-degradative enzymes.Experimental evidence to support this mechanism of starch

accumulation is the activity of ADP-glucose pyrophosphorylasefrom healthy plant leaves in the presence of proportionate con-centrations of sugar phosphates and Pi found in healthy and dis-eased plants during the infection process (Fig. 10). The activity ofthe enzyme closely parallels the starch curve (Fig. 1) and is al-most the reciprocal of the phosphate curve (Fig. 6). When thephosphate content of diseased leaves was high, the enzyme activ-ity was low, and, when phosphate levels were low, enzyme activitywas high. The increase in starch from 9 to 10 days is not explainedby these data since enzyme activity decreased between 8 and 10days. As stated before, the presence of an unknown activator indiseased leaves could conceivably overcome inhibition by Pi .

The concentration of ADP-glucose pyrophosphorylase was ob-served to increase in diseased leaves (1.5-fold) from 10 to 12 daysand to decrease (to one-third) from 12 to 14 days after inoculation(14). The specific activity was also observed to decrease from 12to 14 days after inoculation. Increased enzyme concentrationwould increase the capacity of the system for starch synthesis atthe time when starch was increasing during the infection process.This factor along with the regulatory control of pyrophosphory-lase itself would help explain the changes in starch content in dis-eased leaves.The experimental evidence does not support an enzyme contri-

bution by the fungus to account for increased enzyme levels in dis-eased leaves (14). An enzyme preparation from fungal spores wasnot activated by 3-phosphoglycerate, whereas the enzyme prep-





aration from diseased wheat leaves was stimulated more by gly-cerate-3-P than the enzyme preparation from healthy leaves(Tables III and IV). Also, the pyrophosphorylase fungal uredo-spores had a greater specificity for UDP-glucose than enzymepreparations from diseased leaves (14).An interesting observation which deserves further study is the

greater stimulation of enzyme activity by activators with the prep-aration from diseased leaves (Tables III and IV). The activatorsglycerate-3-P and fructose-1,6-diP stimulated the enzyme prep-aration from diseased leaves about three times more effectivelythan enzyme preparation from healthy leaves. At least two possi-bilities exist for increased sensitivity to activators of the enzymefrom diseased leaves. One is that the fungus is secreting an enzymeor enzymes which cleave or alter portions of the host pyrophos-phorylase tertiary and quaternary structure, conceivably causinga change in the binding properities of the enzyme for activatorsor substrate, or both. Another possibility is that metal ions ac-cumulating at infection sites could bind with the pyrophosphory-lase, altering its structure.Some limitations in the interpretation of these results are: (a)

The concentrations of metabolites at the actual site of ADP-glucose pyrophosphorylase are not known. (b) The host andpathogen could not be separated so that the contributions of eachto the metabolite and enzyme concentrations in the determina-tions are not known. (c) Different results may have been obtainedusing metabolite simulations with the enzyme from diseasedleaves.The importance to the fungus of starch accumulation is not

known. It seemed to accumulate too late in the infection processto be of greatest value to the fungus during sporulation. It couldbe used by the fungus for late sporulation during the host tissuesenescence. In Nicotiana infected with tobacco mosaic virus (2)starch accumulates during chloroplast deterioration with no ap-parent advantage to the virus. It is quite possible that starch ac-cumulates in diseased plants because the altered balance ofmetab-olites which affect ADP-glucose pyrophosphorylase is conducivefor starch biosynthesis. In other words, the host-parasite interac-tion alters the metabolism in the host in such a way that starch issynthesized as a result of increased ADP-glucose pyrophosphory-lase activity.

Acknowledgments-The authors wish to acknowledge Dr. E. L. Sharp of this depart-ment for the use of his environmental chambers, spore collections, and other equipmentand materials; and Dr. G. Julian of the Department of Chemistry, Montana StateUniversity, for the use of his gas flow counter.

LITERATURE CITED1. AKAI, S., M. FUKUTOMI, N. ISHIDA, AND H. KUNOH. 1967. An anatomical approach

to mechanism of fungal infection in plants. In: C. J. Mirocha and 1. Uritani, eds.,The Dynamic Role of Molecular Constituents in Plant-Parasite Interaction.American Phytopathology Society, St. Paul. pp. 1-21.

2. CARROLL, T. W. AND T. KOSUGE. 1969. Changes in structure of chloroplasts ac-companying necrosis of tobacco leaves systemically infected with tobacco mosaicvirus. Phytopathology 59: 953-963.

3. CHANGEUX, J. P. 1963. Allosteric interactions on biosynthetic L-threonine deami-nase from E. coli K12. Cold Spring Harbor Symp. Quant. Biol. 28: 497.

4. FiSKE, C. H. AND Y. SUBBARow. 1925. The colorimetric determination of phos-phorus. J. Biol. Chem. 66: 375-403.

5. FRYDMAN, R. 1963. Starch synthetase of potatoes and waxy maize. Arch. Biochem.Biophys. 102: 242-248.

6. GHOSH, H. P. AND J. PREISS. 1965. Biosynthesis of starch in spinach chloroplasts.Biochemistry 4: 1354-1361.

7. GHOSH, H. P. AND J. PREISS. 1965. The biosynthesis of starch in spinach chloro-plasts. J. Biol. Chem. 240: 960-962.

8. GHOSH, H. P. AND J. PREISS. 1966. Adenosine diphosphate glucose pyrophosphory-lase. A regulatory enzyme in the biosynthesis of starch in spinach leaf chloro-plasts. J. Biol. Chem. 241: 4491-4504.

9. INMAN, R. E. 1962. Disease development, disease intensity, and carbohydrate levelsin rusted bean leaves. Phytopathology 52: 1207-1211.

10. KEEN, N. T. AND P. H. WILLIAMS. 1969. Synthesis and degradation of starch andlipids following infection of cabbage by Plasmodiophora brassicae. Phytopathol-ogy 59: 778-785.

11. LATZKO, E. AND M. GiBBs. 1969. Level of photosynthetic intermediates in isolatedspinach chloroplasts. Plant Physiol. 44: 396-402.

12. LiNEWEAVER, H. AND D. BURK. 1934. The determination of enzyme dissociationconstants. J. Amer. Chem. Soc. 56: 658-666.

13. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Proteinmeasurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

14. MAcDONALD, P. W. 1970. Relationship of ADP-glucose pyrophosphorylase tostarch accumulation in wheat leaves infected with Puccinia striiformis West.Ph.D. thesis. Montana State University, Bozeman.

15. MIROCHA, C. J. AND A. I. ZAKI. 1966. Fluctuations in amount of starch in hostplants invaded by rust and mildew fungi. Phytopathology 56: 1220-1224.

16. MUKHERJEE, K. L. AND M. SHAW. 1962. The physiology of host-parasite relations.XI. The effect of stem rust on the phosphate fractions in wheat leaves. Can. J.Bot. 40: 975-985.

17. NELSON, N. 1944. A photometric adaption of the Somogyi method for the deter-mination of glucose. J. Biol. Chem. 153: 375-380.

18. PREIss, J., H. P. GHOSH, AND J. WITrKoP. 1967. Regulation of the biosynthesis ofstarch in spinach leaf chloroplasts. In: T. W. Goodwin, ed., Biochemistry ofChloroplasts, Vol. It. Academic Press, New York. pp. 131-153.

19. PREISS, J. AND E. GREENBERG. 1967. Biosynthesis of starch in Chlorella pyrenoidosa.1. Purification and properties of the adenosine diphosphoglucose: a-1 ,4-glucan,a-4-glucosyl transferase from Chlorella. Arch. Biochem. Biophys. 118: 702-708.

20. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaquestain in electron microscopy. J. Cell Biol. 17: 208-212.

21. SANWAL, G. G., E. GREENBERG, J. HARDIE, E. C. CAMERON, AND J. PREISS. 1968.Regulation of starch biosynthesis in plant leaves: Activation and inhibition ofADP glucose pyrophosphorylase. Plant Physiol. 43: 417-427.

22. SANWAL, G. G. AND J. PREISS. 1967. Biosynthesis of starch in Chlorella pyrenoidosa.II. Regulation of ATP: a-glucose-l-phosphate adenyl transferase (ADP glucosepyrophosphorylase) by inorganic phosphate and 3-phosphoglycerate. Arch.Biochem. Biophys. 119: 454-469.

23. Scorr, K. J. 1965. Respiratory and enzymatic activities in the host and pathogen ofbarley leaves infected with Erysiphe graminis. Phytopathology 55: 438-441.

24. SHARP, E. L. 1965. Prepenetration and post-penetration environment and develop-ment of Puccinia striiformis on wheat. Phytopathology 55: 198-203.

25. SHAW, M. AND D. J. SAMBORSKI. 1956. The physiology of host-parasite relations. 1.The accumulation of radioaztive substances at infections of facultative and obligate parasites including tobacco mosaic virus. Can. J. Bot. 34: 389-405.

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27. TANAKA, H. AND S. AKAT. 1960. On the mechanism of starch accumulation in tissuesurrounding lesions on rice leaves due to the attack of Cochliobolus miyabeanus.II. On the activities of beta-amylase and invertase in tissues surrounding spots.(In Japanese, English summary) Phytopathol. Soc. Japan Ann. 25: 80-84.

28. UMBRErT, W. W., R. H. BURRIS, AND J. E. STAUFFER. 1957. Manometric Techniques.Burgess Publishing Co., Minneapolis.

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Documents

Adenosine Diphosphate-Glucose Pyrophosphorylase Control ... · Theprimary leaves ofhost plants wereinoculated in a settling tower 10 days after planting with lyophilized uredospores