PLANT Vol. 39 No. 6

PHYSIOlOGY November 1964

Influence of Environment on Metabolism of Organic Acids andCarbohydrates in Vitis Vinifera. I. Temperature 1 2

W. Mark KliewerDepartment of Viticulture and Enology, University of California, Davis, California

Temperature has a considerable effect on theamount of organic acids and sugars in grapes. Ger-ber (6) in 1898 investigated the influence of organicacids in grapes and showed that the respiratory quo-tient changed with temperature, indicating a changein respiratory substrate. These changes were cor-related with the levels of several sugars and organicacids present. Gerber reported that when the tem-perature of grapes exceeded 350 tartrates were re-spired, while malic acid was respired at 30° andsugars were respired at much lower temperatures.

Bremond (4) noted that low temperatures at nightstimulated the formation of acids, and that tempera-tures higher than 300 caused the acid levels to declinein grapes grown in Algeria. Peynaud and Maurie(18) also observed that the acids in grapes are me-tabolized by respiration at high temperatures, whereasat low temperatures they are formed. Hennig andBurkhardt (11) found that malic acid in grapes wasless in warm than in cool seasons. Observationssuch as those mentioned above have led to the gen-eralization that a variety will be higher in acid atthe same degree Balling in a cool season than in awarm or hot season. However, there are numerousexceptions to this rule. Peynaud (16) did not finda constant relationship between the amount of malicacid retained by the same varieties in the warm yearof 1937 and that retained in the cool year of 1938.Amerine's (2) data also failed to indicate a uniformdifference between cool and warm years. More re-cent data of Peynaud and Maurie (18) has led themto withdraw their earlier published statement thatthe respiratory oxidation of acids in grapes is instrict relationship to the temperature during ripeningin the fall. The synthesis and metabolism of organicacids and sugars in plants is a complex process thatrequires numerous enzyme systems. These un-doubtedly have different temperature optima, andconsequently can be expected to function differentlyat various temperatures. It should be emphasizedalso that temperature is only one aspect of the en-

'Received Feb. 24, 1964.2 This work was supported in part by a Grant (GB-

1746) from the National Science Foundation.869

vironment and not until all environmental factors aredefined can one expect to predict with accuracy whatthe influence of one factor will have on a metabolicproduct.

Reuther and Reichardt (23) recently studied theeffect of temperature on bleeding and metabolism inVitis vinifera. They found that the starch to sugarequilibrium in the stem responds quickly to changesin temperature. Also, they noted a positive correla-tion between sugar content in the xylem fluid of themain stem and that of the shoot. This relationshipwas affected by soil and air temperatures.

Langridge (12) has recently thoroughly reviewedthe biochemical aspects of temperature response inbacteria and plants. A considerable amount of workhas been done on the effect of temperature and lighton the metabolism of Crassulacean plants and iscovered in a review by Ranson and Thomas (21).

The present study was undertaken to gain directinformation on the effect of temperature on synthesisand metabolism of organic acids and sugars in grapes.(The term grape as used in this paper will mean inall cases the berry rather than the plant as a whole.)The experiments were conducted under controlledenvironmental conditions; C1402 was used to followthe incorporation of carbon into organic acids andsugars of berries from shoots that were cultured atone of 6 different temperatures.

Materials and Methods

Plant Material. Shoots were used from 8-year-old, cane-pruned vines of Vitis vinifera L., varietySultanina (syn. Thompson Seedless), growing in anirrigated vineyard at Davis. Two temperature expe-riments were conducted at widely different stages ofberry maturity. Experiment I was initiated on July16, 1963 when the immature berries had a degreeBalling reading of 6.5. Experiment II was startedon September 9, 1963 when the degree Balling read-ing of the nearly ripe berries was 16.0.

Application of C1402. Six shoots from a singlevine, each shoot bearing a medium-sized cluster,were cut back to the sixth or seventh node from thelateral arm. Approximately 3 mg of BaC1403,

PLANT PHYSIOLOGY

having a specific activity of 1.99 mc/mg carbon, wereused as the radioactive source. The radioactive car-bon was presented to the shoots by the method ofHale and Weaver (9) with the modification thatfoam rubber padding was used to seal the tygon tub-ing and polyethylene bags to the shoots. After theaddition of lactic acid to the vials containing theBaC1403 the system was aspirated 4 times for periodsof 1 minute each to ensure complete mixing of theC140 with the atmosphere around the leaves. Thebags were removed from around the shoots after an

exposure of 1 hour. The C1402 was administeredto the shoots between 9: 30 and 10: 30 AM and thelight intensity ranged between 10,000 to 11,000 ft-c.

Incubation and Sampling of Shoots. The shootswere excised from the vine 11 hours (experiment I)and 8 hours (experiment II) after they had beenexposed to the C1402; the stems immediately were

placed in individual 1-liter Erlenmyer flasks contain-ing 1/10 Hoagland nutrient solution. A treatedshoot was placed in each of the 6 temperature controlrooms and kept in the dark at temperatures rangingfrom 10° to 370 (tables I and II). Temperaturein the rooms were maintained constant within ±0.5°.A seventh shoot, located on a vine adjacent to the onefrom which the shoots were excised, was treated inthe same manner as described above except that it wasnot removed from the vine. It was used to comparethe labeling pattern in the intact vine with that inthe excised shoots. A thermograph was placed underthis vine to record the temperature during the 96-hoursampling period. Twenty to 40 berries were pickedat random from the cluster on each of the 7 shoots attime intervals of 8 or 11, 24, 48, 72 and 96 hours.These were put into plastic bags, which were thensealed and the berries immediately frozen with pow-

Table I. Summary of Radioactivity of Organic Acid and Sugar Fractions from Immature GrapesThese grapes were kept at 6 different temperatures and sampled at 5 time intervals. The experiment was initiated

July 16, 1963 when the green berries had a degree Balling reading at 6.5.

Temperature Time sampled Fr wt of No. of Radioactivity of fractions*treatment of after Fr berriesexcised application berries berries Organic Original Percent

shoots of C1402 analyzed in samples acid Sugar Extract recovery

°C Hr g cpm/gfrwt X 10-3 %F.T.**(28) 11 *** 20 42 32 98 141 93.5

10 24 20 41 34 160 202 97.015 24 20 52 84 91 185 96.220 24 20 53 399 460 896 97.325 24 20 54 393 442 875 96.430 24 20 52 363 336 757 94.437 24 20 55 969 866 2051 90.7

F.T.**(23) 24 20 40 114 405 565 92.510 48 20 43 68 188 291 89.315 48 20 51 196 183 402 96.220 48 20 55 662 336 1070 93.325 48 20 54 567 305 901 98.030 48 20 52 638 520 1289 91.337 48 20 54 876 1128 2077 101.0

F.T.**(23) 48 20 53 342 364 787 91.510 72 20 45 96 193 331 89.415 72 20 46 288 190 507 95.520 72 18.7 53 734 382 1205 93.125 72 20 55 501 334 911 92.630 72 20 54 557 466 1096 94.837 72 12 30 926 808 1962 91.8

F.T.*(24) 72 10.6 26 262 480 793 94.210 96 20 35 120 413 578 93.815 96 20 45 234 246 512 95.320 96 20 55 718 288 1106 92.025 96 20 52 637 271 963 95.330 96 20 57 671 308 1097 91.537 96 9.6 27 922 800 1905 95.0

F.T.*(23) 96 6.4 22 253 123 397 96.1* The organic acid fraction consists of all material eluted from Dowex 1 with 1.5 N (NH4)2 C03, and the sugar

fractions consist of neutral substances in the effluent from the Dowex 1 column.** F.T. stands for field temperature; the figure in parenthesis indicates the mean temperature between sampling

times. The berry sample was taken from a treated shoots still attached to the vine.* The data obtained 11 hours after application of the C1402 represent the average value obtained from 7 berry sam-

ples taken from the treated shoots just before they were removed from the vine.

870

KLIEWER-TEMPERATURE AND METABOLISM OF ORGANIC ACIDS AND SUGARS

dered dry ice. The berries were stored at -20° untilthey were analyzed. The stems of the excised shootswere kept submerged under dilute nutrient solutionat all times, the rooms were kept dark and the rela-tive humidity of the temperature control rooms was

maintained between 70 and 90 %. Under theseconditions the shoots remained turgid for 5 to 6 days.

Extraction Procedure. Twenty g of berries were

sliced into fourths with a razor blade and droppedinto 80 ml of boiling 95 % ethanol in a 400-ml beaker.The beaker was covered with a watch glass, and themixture then simmered 1 hour on a steam bath. Afterthe alcohol extract was decanted, the tissue was trans-ferred to a 50-ml stainless steel container and mace-

rated in 40 ml of 80 % ethanol with a omnimixeroperating at full speed for 2 minutes. The homogen-ate was returned to the beaker with several washingsand then simmered on a steam bath for 30 to 40minutes. Following cooling, the supernatant fluidwas decanted and the residue extracted with 50 ml of50 % ethanol for 30 minutes. The supernatant fluid

and the residue were filtered with suction throughWhatman No. 2 filter paper. The residue waswashed several times with distilled water and thefiltrate was transferred to a 1-liter boiling flask.The ethanol was evaporated at 450, under vacuum,using a rotary evaporator. The resulting mixturewas filtered through Whatman No. 2 filter paper andmade up to 200 ml with distilled water. This solu-tion, hereafter, will be referred to as the original ex-tract. A 0.1 ml sample was withdrawn for deter-mination of radioactivity.

Preliminary Separation of Amino Acid, OrganicAcid and Sugar Fractions. A sample of the originalextract (usually 100 ml) equivalent to 10 g freshweight of the berries was passed through a 1.5 X 20cm column of Dowex 50-X8, 50- to 100-mesh cationexchange resin in the H+ form, at a rate of about2 ml per minute. The column was washed with 3resin-bed volumes of distilled water and the washingswere combined with the effluent.A fraction containing positively charged com-

Table II. Summary of Radioactivity of Organic Acid and Sugar Fractions from Nearly Ripe GrapesThese grapes were kept at 6 different temperatures and sampled at 5 time intervals. The experiment was initiated

September 9, 1963 when the ripening berries had a degree Balling reading of 16.0.

Temperature Time sampled Fr wt of No. of Radioactivity of fractions*treatment of after . ,

excised application berriesd brinapes Organic Sgr Original Percentshoots of C1402 analyed insmles acid Sugar extract recovery

°C Hr g cpm/gfrwtX 10-3F.T.**(29) 8*** 20 15.0 3 94 102 97.5

10 24 20 14.0 15 73 97 96.715 24 20 13.5 18 96 129 92.520 24 20 12.0 14 255 287 95.425 24 20 16.0 26 831 890 97.730 24 20 14.5 22 842 887 98.637 24 20 18.0 11 630 670 96.5

F.T.(22) 24 20 15.0 12 707 731 100.210 48 20 14.5 22 92 123 98.315 48 20 13.5 14 98 117 100.820 48 20 13.5 46 395 475 98.125 48 20 15.0 87 712 869 98.930 48 20 14.5 88 700 875 98.037 48 20 19.0 42 603 719 97.9

F.T.(23) 48 20 15.0 56 713 812 98.510 72 20 15.0 18 138 168 96.415 72 20 14.0 8 107 126 95.220 72 20 15.0 60 979 1088 99.425 72 20 16.0 62 1001 1125 98.430 72 20 15.5 69 1007 1157 97.437 72 20 20.5 49 763 898 97.8

F.T.**(21) 72 20 14.5 66 838 951 98.910 96 20 15.0 16 174 209 95.015 96 20 14.0 10 105 122 99.120 96 20 14.0 142 843 1096 98.925 96 20 16.0 155 999 1298 97.830 96 20 15.0 100 824 1015 98.937 96 20 21.0 57 646 760 97.5

F.T.**(21) 96 20 14.5 88 527 703 98.4* Refer to footnotes in table I for explanation.

* The data obtained 8 hours after the application of C1402 represent the average value obtained from 7 berry sam-ples taken from the treated shoots just before they were removed from the vine.

871

PLANT PHYSIOLOGY

pounds, such as amino acids, was eluted from thecation exchange column with 150 ml of 10 % NH40H.The column was washed with 3 bed volumes of distilledwater, and the eluate and washings were combinedand evaporated to dryness over a steam bath. Theresidue was dissolved in 5 ml of distilled water. Thisis designated as the amino acid fraction. Since theamount of radioactivity in the amino acid fraction wassmall and very little affected by temperature it willnot be dealt with any further in this paper.

The sugars in the effluent from the Dowex 50column were separated from the organic acids by pas-sage through a Dowex 1-X8, 100-mesh anion ex-change column, 2 cm in diameter and 100 cm long,filled with about 30 ml of resin. The resin was pre-viously converted to the CO3- form to avoid decom-position of the sugars (22). The column was washedwith 3 bed-volumes of distilled water and the wash-ings were combined with the effluent. The sugarfraction was evaporated to dryness at 400 under thevacuum with the aid of a rotary evaporator, and theresidue was dissolved in 10 ml of distilled water.

The organic acids were eluted from the Dowex 1column with 300 ml of 1.5 N(NH4)2CO3; the columnwas washed with 3 bed-volumes of distilled water.The washings and elute were combined and evapo-rated to dryness under vacuum at 500. The organicacids also were dissolved in 10 ml of distilled water.

Identification of Sugars and Organic Acids.Paper chromatography was used to separate andidentify the individual sugars and organic acids.Samples of the sugar and organic acid fractions(0.01 and 0.02 ml, respectively) were spotted onWhatman No. 1 chromatographic paper with the aidof micro pipettes. Each treatment was spotted induplicate. One was used to locate the respectivesugars or organic acids and the other in determiningthe radioactivity of the compounds.

The sugars were separated by descending, 1-di-mensional chromatography, using a fresh solution ofn-propyl alcohol, benzyl alcohol, 85 % formic acid,and water (50: 72: 20: 20; v/v) (7) or butyl al-cohol-acetic acid-water (4: 1:5, v/v). Sucrose, glu-cose, and fructose were identified by comparison withthe RF values of known samples of these sugars. Forconfirmation of the identity of glucose, the glucosearea was eluted and treated with glucose oxidase andthe reaction mixture rechromatographed to identifygluconic acid. Sucrose was identified by hydrolysiswith invertase and chromatographic identification ofthe resulting monosaccharides components. Rechro-matography of the reaction products resulting fromtreatments of the eluted fructose area with calciumhyroxide showed a normal distribution of activity be-tween the epimers, fructose, mannose, and glucose.

The chromatograms spotted with the organic acidsolutions were developed with butyl alcohol, water,formic acid (10: 10:1, v/v) and also with a solutionof pentyl aloohol and SM formic acid (1: 1, v/v) (5).The formic acid was removed by suspending thechromatogram in a steam cabinet for 30 minutes.

Tartaric, citric, and malic acids were identified by Rpvalues and by elution from chromatograms and co-chromaitography with known samples of these acids.

The sugars were detected by spraying the chro-matograms with a modified p-anisidine solution (13)and heating in an oven at 95 to 1000 for 3 minutes;the organic acids were detected by spraying with0.1 % brom-cresol green in 95 % ethanol brought topH 4.0.

Measurement of Radioactivity. To determineradioactivity 0.1 ml of each fraction was pipetted ontoa 1 X 3 cm strip of Whatman No. 1 chromatographicpaper in a 30 ml pyrex counting vial. The paperwas dried overnight at room temperature and thebottles then were filled with scintillator solution con-sisting of 5 g of 2,5-diphenyloxazole (PPO) and 0.3of 2,2-p-phenylenebis-( 5-phenyloxazole) (POPOP)per liter of toluene (10). Total activity in the orig-inal extract was determined in the same manner.

The chromatograms with the located sugars andorganic acids were superimposed over their corre-sponding chromatograms and the areas cut out.These were placed in counting bottles to which scin-tillator fluid was added. Each sample was countedtwice with a Packard liquid scintillator counter for10 minutes. The sum of the radioactivity of the indi-vidual sugars and organic acids generally was within5 % of the total radioactivity applied to the chromatog-raphic paper. The data on radioactivity are expressedas percentages of the total radioactivity or as countsper minute per g fresh weight of berries. The counterhad a counting efficiency of approximately 60 % forC14.

The total dissolved acids in the juice of berrieswas measured by a temperature corrected hand re-fractometer. For convenience the values are ex-pressed as g of sugar solids per 100 g of juice ordegree Balling.

Results

Activities of the fraction in each experiment aresummarized in tables I and II. The activities arecorrected for background only. The percent of radio-activity recovered in the amino acid, organic acid,and sugar fractions ranged from 89 to 101 % of thetotal activity in the original extract. Most of theactivity lost during the fractionation occurred duringthe passage of the extract through the Dowex 50 ionexchange column.

In nearly every instance there was an increase inradioactivity in each fraction between the time theshoots were removed from the vine until the 24-hoursample of berries was taken, indicating that organicconstituents were being translocated from the leavesto the berries faster than they were being respired.Activity in the organic acid and sugar fractions gen-erally continued to increase with time, especially atlower temperatures; however, there were several ex-ceptions. At temperatures of 200 and above therewere considerably larger amounts of C14 incorporated

872

KLIEWER-TEMPERATURE AND METABOLISM OF ORGANIC ACIDS AND SUGARS

into the various fractions than at temperatures below200, indicating that translocation and/or biosyn-thesis of the various metabolites were increased atthe higher temperatures. Percent radioactivity inthe various fractions from grapes taken from theshoot that was left on the vine in the field generallyfell within the range of values for excised shootscultured between 15 and 250. The mean field tem-perature was also within this range.

The relative proportions of radioactivity in theorganic acid and sugar fractions of immature berrieskept at 10 to 370 and sampled at 4 different timesare shown in figure 1. In the green berries (6.50Balling) organic acids were synthesized in greatestamounts between 20 and 250. At 100 there was a

greater percentage of radioactivity in the sugar frac-tion than in the organic acid fraction while at 15 and370 the organic acid and sugar fractions had approxi-mately equal radioactivity. These data indicate thattemperature has a large influence on the relative

90 I 1

80K24 HOUR

70

60 -

50 -

40 - SUGAR

'30 -

O20lo _

z 0_

80.2 72 HOUR

O 70 -

I

U 0

aL50

amounts of organic acids and sugars produced inberries from grape shoots.

Total radioactivity in the organic acid and sugar

fractions from berries treated with C1402 when theywere nearly ripe (160 Balling), was much differentthan in fractions from berries treated while they were

still green (fig 2). The proportion of radioactivityin the sugar fraction generally was increased slightlyby an increase in temperature. This fraction usuallycontained 80 % or more of the total radioactivity atall temperatures. In contrast to the effect on green

berries, lower temperatures (10 and 150) favoredincreased incorporation of label into organic acids inthe nearly ripe fruit (fig 2).

The distribution of radioactivity among the or-

ganic acids obtained both from green berries and fromnearly ripe berries, as influenced by the temperatureat which the berries were kept, is shown in figures3 and 4. The activity of the individual organic acidsis expressed as a percentage of total radioactivity of

"10 15 20 25 30 37 10 15 20 25 30 37TEMPERATURE -°C

FIG. 1. Influence of temperature on the relative amount of C14 incorporated into amino acid (A.A.), organic acid(O.A.), and sugar fractions from immature berries sampled at 4 time intervals. The figures along the ordinate axisrefer to the percent of the total radioactivity in the original extract.

873

87490

PLANT PHYSIOLOGY

>30

0 20

090 A19 A. A. 0-J: ,__n_HW=_- A. A.

0

I-

110 11

wi 90QuJCL 80-

0 T0. A. I I A. A. I

10 15 20 25 30 37 10 15 20 25 30 37TEMPERATURE_ °C

FIG. 2. Influence of temperature on the relative amount of C14 incorporated into the amino acid, organic acid,and sugar fractions from nearly ripe berries sampled at 4 time intervals. The figures along the ordinate axis referto the percent of the total radioactivity in the original extract.

the organic acid fraction. Malic acid was consider-ably more radioactive than any of the other acids;it contained 50 to 85 % of the activity in the greenand nearly ripe berries. Tartaric acid containedfrom 4 to 21 % of the total radioactivity and theamount found in green berries was nearly twice thatobtained from the ripe berries, indicating that theenzyme system catalyzing the formation of tartaricacid was considerably more active during earlygrowth of the berry.

The temperature at which the grape shoots werecultured affected quite differently the proportion oflabel in malic acid from green berries as comparedto that in ripe berries (fig 3,4). The percentage ofthe total radioactivity in malic acid from green ber-ries was lowest at 100, somewhat higher at tempera-tures between 15 and 300, and greatest at 37°. Incontrast, malic acid extracted from ripe berries cul-tured at 100 contained 80 % or more of the label,while at higher temperatures there was a considerabledecrease in the amount of C14 incorporated into the

acid. Several other workers (19, 29, 30) have ob-served that low temperature stimulated malic acidformation in plants. However, at 30 and 37° therewas generally an increase in the relative amount oflabel in malic acid from nearly ripe berries as com-pared to that in the other organic acids.

The amount of label in tartaric acid was not in-fluenced greatly by temperature; however, both thegreen and ripe berries cultured at temperatures be-tween 15 and 300 consistently contained more labelthan did fruit kept at lower or higher temperatures.The influence of temperature on the proportion ofradioactivity in citric and several unidentified organicacids in the nearly mature fruit generally followed thepattern of tartaric acid.

The temperature at which the grape shoots werekept had considerable effect on the proportion of ac-tivity in the various grape sugars extracted from theimmature grapes; however, the effect was much lesswith mature grapes (fig 5, 6). Green berries heldat 100 had approximately the same amount of C14 in

KLIEWER-TEMPERATURE AND METABOLISM OF ORGANIC ACIDS AND SUGARS

glucose as in fructose; temperatures above 100 re-sulted in an increased amount of labeled glucose anda correspondingly decreased amount of C14 in fruc-tose. The influence of temperature on the proportionof activity in the sugars was most pronounced afterthe first 24 hours. The percent of C14 in glucosetended also to increase with the length of time fromapplication of C1402, while generally there was a de-crease in the amount of label in fructose with time.The proportion of activity in sucrose extracted fromimmature berries was less than 5 % of the totalactivity in the sugar fraction and was not affectedgreatly by temperature.

Unlike with green berries, the proportion of ac-tivity in glucose and fructose extracted from nearlyripe berries was not affected strongly by temperature.Glucose contained from 37 to 52 % of the total activityin the sugar fraction, and fructose had 35 to 48 %.However, after 96 hours of culture there were nearly

identical amounts of C14 in glucose and fructose at alltemperatures. The proportion of activity in sucrosefrom ripe berries was generally 2 to 3 times greaterthan that in green berries. Temperature at whichthe shoots were cultured had a striking effect on theproportion of label in the sucrose extracted from themature berries. After the initial 24-hour culturedperiod, sucrose extracted from berries held at 10°had more than twice as much activity as the berriescultured at higher temperatures.

Discussion

A comparison of figures 1 and 2 shows a greatdifference between the proportions of label in thesugar and in the organic acid fractions from berriestreated with C1402 at widely different stages ofgrowth development. The greatest proportion ofthe carbon photosynthesized in shoots with immature

70 1

I I I I24 HOUR

- -

MA.LIC

.o~~- 0--~ -M. 0t3m~-0 _ _1sop_-o TAROT"A"RIC"

72 HOUR

30 -

20 [

O- _ -_ TARTARICI It

I I48 HOUR

_ f IC~~~~~~~AI

3r TARTARIC

96 HOUR

MALIC

00 -TARTARIC -

I I10 15 20 25 30 37 10 15 20 25 30 37

TEMPERATURE -'DCFIG. 3. Influence of temperature on the relative amount of C14 found in tartaric and malic acids from immature

berries sampled at 4 time intervals. The figures along the ordinate axis refer to the percent of the total radioactivityin the acid fraction.

90

80

60

50

I-4

>30

o 200OCLz 10-J

oU

= 80U-0

z

tY

'U'U0.

60

50

40

10

a

I 9 I

I i I

875

70

AnLr

I

F

I

PLANT PHYSIOLOGY

90

80

70

60

50

An

30 -

201T^RTARIC,__ UNKNOWN r iTAR~~UKNOWNTARTARIC

o20 UNKNOWN A ITARICI0~~ ~

10 15 20C25I30T37R10I-20 2530T37

72HOURE96 HOURz80U-o70MAI

UU 60-uJCL 50-AI

40

30-

20TARTARIC ~ UNNOWI~~.UUNKNOWN1TARTARIC

0I

'' ITRIC1"¶ T~ ICITRIC t10 15 20 25 30 37 10 IC 20 25 30 37

TEMPERATURE - "

FIG. 4. Influence of temperature on the relative amount of C14 found in tartaric and malic acids from nearly ripeberries sampled at 4 time intervals. The figures along the ordinate axis refer to the percent of the total radioactivityin the acid fraction.

berries was converted to production of organic acids,while the nearly ripe berries were incorporating mostof the label into sugars. It is well known that as theberry ripens the acid content, particularly that ofmalic, decreases. This generally is attributed to in-creased respiration rates and a reduction in theamount of acid translocated from the leaves (2).The reason for the increased acid respiration in theberry at the time when the sugar content is at amaximum is not known. The reduction and also thesynthesis of malic acid in the berry as it ripens couldbe due to a loss in activity of the CO2-fixing me-chanism or to the loss of enzymic activity necessaryfor reactions intermediate to malate synthesis. Itmigh-t also be possible that malic and other organicacids are used for carbohydrate synthesis to a greaterextent as the berry ripens.

Sissakian and his co-workers (27) have shownthat at the beginning of ripening and until full phy-

siological maturity of grapes the ability of acid forma-tion of grape vines starts decreasing. Hale (8) haspostulated that tartaric acid synthesis in the grapemay be related to some aspect of growth metabolism.The data in figures 1 and 2 show a much greaterincorporation of label into the organic acids of im-mature, rapidly growing berries compared to theamount found in the nearly ripe fruit and are in ac-cord with this postulation.

The amount of C1402 incorporated into tartaricacid indicates that its rate of synthesis is much slowerthan that of malic acid. It is well known that bothmalic and tartaric acids accumulate in the berry.However, as the fruit approaches maturity the amountof tartaric acid generally exceeds that of malic acid(2). It therefore follows that once tartaric acid issynthesized it is metabolized very slowly. Payesand Laties (14,15) have suggested that reactionsleading to tartaric acid may serve as regulators of

876

KLIEWER-TEMPERATURE AND METABOLISM OF ORGANIC ACIDS AND SUGARS

metabolism. Once tartaric acid is formed in theberry, it probably cannot be actively metabolizedbecause of the lack of either an active enzyme systemcapable of metabolizing this acid, or because of thepresence of a substance that inhibits its breakdown.

Malic acid generally contained from 50 to 90 %of the total radioactivity found in the organic acidfraction; in the green berries it accounted for nearlya quarter of the total radioactivity in the originalextracts. This indicates that probably enzyme sys-tems other than those in the tricarboxylic acid cycleare also responsibile for malic acid synthesis andaccumulation in grapes. The dark fixation of CO2into malic acid can be carried out by any one of 3enzyme systems. Malic enzyme, which catalyzes thesynthesis of malic acid by the ,B-carboxylation of py-ruvic acid, has been shown to be present in grapeberries (25). Phosphoenylpyruvic carboxylase andphosphoenol-pyruvate carboxykinase also are able toform malic acid via oxalacetic acid. More recently,malate synthetase, which catalyzes the condensation

of acetyl CoA and glyoxalate to form malate, hasbeen found in plants (31). Payes and Laties (15)have recently shown another way that malic acid canbe synthesized in plants from y-OH a-ketoglutarate.The enzymic formation of this compound from gly-oxylate and pyruvate has recently been demonstratedin plant extracts (15).

The proportion of radioactivity found in thevarious organic acids of grapes are in agreement withthe results of other workers. Hale (8) found thattartaric and malic acid from immature ThompsonSeedless berries harvested after 11 and 47 hours fromthe time they were treated with C1402 contained 13 to15 % and 62 to 73 % respectively of the total radio-activity. Stafford and Loewus (26) exposed de-tached mature grape leaves to C1402 and found 76to 90 % of the label in the organic acid fraction wasin malic acid and only 0.01 to 0.08 % in tartaric acid.Ribereau-Gayon (24) found 8 to 9 % of the radio-activity in the organic acid fraction from immatureberries was in tartaric acid, 52 to 75 % in malic

I I I24 HOUR

GLUCOSE

1%"I,%

FRUCTOSE

SUCROSEw--o- .-o, , _ _

I I

N,h

- A"._

FRUCTOSE

48 HOUR

GLUCOSE

- -on _< FRUCTOSE

SUCROSE

*. - .--- - -- L96 HOUR

GLUCOSE

,,~FRUCTOSE_^.f '9e~4-M-_.mm "

IU SUCROSE ,SUCROSE

Tb- .. 01b- I*_ 9----- 0@4 -0- - - O.-

v10 15 20 25 30 37 10 15 20 25 30 37

TEMPERATURE °CFIG. 5. Influence of temperature on the relative amount of C14 incorporated into glucose, fructose and sucrose from

immature berries sampled at 4 time intervals. The figures along the ordinate axis refer to the percent of the totalradioactivity in the sugar fraction.

877

90f I I I I Iw I I I I l

80

70

60 1

I.FIF

50

40I.-

' 30

o 200

m 10-J

0I--

= 80ILo 70zUJ 60UX5a- 50

40

30 1-

20 _-

i I a 1 F I

H . I I I I w- I -t a I

72 HOUR

GLUCOSE.

1%. q%ft"fta"4,

1nII

878 PLANT PHYSIOLOGY

- -A - _A -~ FRUCTOSE \- FRUCTOSE

30-

o 20- SUCROSE

10 - * *-0- L_UCOS

0 0

w 72 HOUR 96 HOUR80_

U.o70

z60-

a.-A040 ~ - \GLUCOSE

30

20 SUCROSE~ ~ ~ ~ p ~ .~~SCRS

10 15 20 25 30 37 10 15 20 25 30 37TEMPERATURE -°C

FIG. 6. Influence of temperature on the relative amount of C14 incorporated into glucose, fructose, and sucrose fromnearly ripe berries sampled at 4 time intervals. The figures along the ordinate axis refer to the percent of the totalradioactivity in the sugar fraction.

acid, 3 to 7 % in citric acid and 5 to 9 % in glycolicacid after exposure of shoots to C1402 for 2 and 15hours.

The extracts from green berries showed increasesin the proportion of label in glucose as compared tothat in fructose, with increases in temperature. Thismay have been due to more active enzyme synthesisof glucose, compared to fructose, at this stage ofberry development. However, in the nearly ripe ber-ries, the proportions of activity in glucose and fruc-tose were approximately equal, and remained rela-tively constant over the entire range of culture tem-peratures, indicating that the enzymes synthesizingglucose and fructose were approximately equally ac-tive. These observations are in general agreementwith the established finding that glucose is generallypresent in immature berries in greatest amount, whilein ripe berries glucose and fructose usually are pres-ent in approximately equal amounts (13).

Low temperatures were found to increase theamount of label in sucrose from ripe berries. Rabson

(20) has also reported a greater incorporation ofC14 into sucrose from potato plants held at low tem-peratures (40 to 500 F) than at higher temperatures.Other workers (19, 28, 29) have shown an increasedproduction of organic acids at low temperature andthis agrees with the findings reported above withripe berries. Increased production of organic acidsat low temperatures has generally been ascribed tothe fact that the oxidation of carbohydrates to or-ganic acids is a reaction that liberates energy andshould accordingly be promoted by a decrease intemperature (19). The reason for the differencebetween immature and mature berries in regard tothe relative amount of label found in the organicacid fraction at low temperatures is not known.

In addition to the presence of tartaric, citric andmalic acids in the organic acid fraction, 2 and often3 well-defined spots showed up when the chromato-grams were sprayed with brom-cresol green. TheRF values of these ranged between 0.06 and 0.18.Determination of the radioactivity and the amount

KLIEWER-TEMPERATURE AND METABOLISM OF ORGANIC ACIDS AND SUGARS

of acid present of these unidentified organic acidsshowed that they often had specific activities higherthan any of the above mentioned organic acids.Work is presently in progress to identify these com-pounds.

Summary

Shoots of mature grape vines (Vitis vinifera L.,variety Sultanina), each with an attached fruitcluster, were presented C1402 at an early stage ofberry development and again when the berries werenearly ripe. After a period of 8 to 11 hours from thetime the label initially was administered, the shootswere removed from the vines and cultured in the darkat 6 temperatures ranging from 10 to 37°. The rela-tive amounts of C14 incorporated into the organicacid and sugar fractions of berries cultured at thevarious temperatures were determined over a 96-hourperiod. Likewise, the relative amounts of C14 inmalic, tartaric and citric acids, and in glucose, fruc-tose, and sucrose were measured.

Maturity of the berries greatly influenced the rela-tive amounts of label in the sugar and organic acidfractions. Green berries (6.50 Balling) incorporatedC14 predominantly into organic acid; the optimumtemperature for organic acid synthesis was between20 and 250. The nearly ripe fruit (160 Balling), incontrast, had approximately 80 % or more of the totallabel in the sugar fraction at all temperatures. Theamount of C14 incorporated into the organic acids ofthe nearly ripe berries was 2 to 3 times greater at10 to 150 than at the higher temperatures.

Malic acid was the principal labeled organic acid,containing 40 to 90 % of the activity in the organicacid fraction; tartaric acid had 4 to 21 %, and citricacid had 2 to 10 %. The proportion of activity intartaric acid from green berries was several timesgreater than that from ripe berries, indicating moreactive synthesis of tartaric acid during early berrygrowth. Increasing the culture temperature from 10to 250 generally increased the amount of label intartaric and citric acid; however, temperatures higherthan this resulted in a reduction of activity in theseacids. The relative amount of activity in malic acidfrom green berries generally showed small increaseswith increasing temperature; in nearly ripe berriesthere was a decrease in activity when the culturetemperatures were increased from 10 to 250.

The relative amounts of C14 in glucose fromgreen berries was greatly increased at the higherculture temperatures, while fructose had correspond-ing decreases in activity. Sucrose from green ber-ries contained less than 5 % of the total activity ofthe sugar fraction. Glucose and fructose from nearlyripe berries, on the other hand, had approximatelyan equal amount of activity, and were little affectedby temperature. The relative activity of sucrose fromnearly ripe berries kept at 100 was 2 to 3 timesgreater than that from berries held at 200 or higher.Sucrose from ripe berries was 2 to 12 times more

active (on a percentage basis) than sucrose fromgreen berries.

Acknowledgment

The technical assistance of Mr. R. E. Combs is grate-fully acknowledged.

Literature Cited

1. AMERINE, M. A. 1951. The acids of Californiagrapes and wines. II. Malic acid. Food Technol.5: 13-16.

2. AMERINE, M. A. 1956. The maturation of winegrapes. Wines and Vines 37(10): 27-32, (11):53-55.

3. AMERINE, M. A. AND G. THOUKIS. 1958. The glu-cose-fructose ratio of California grapes. Vitis 1:224-29.

4. BREMOND, E. 1937. Contribution a l'etude anaty-tique et physico-chemique de l'acidite des vins.Algiers: Imprimeries La Typo-Litho et Jules Car-bonel Reunies.

5. BUSH, M. L., R. MONTGOMERY, AND W. L. PORTER.1952. Identification of organic acids on paperchromatograms. Anal. Chem. 24: 489-91.

6. GEBER, C. 1897. Recherches sur la maturation desfruits charnus. Ann. Sci. Nat. Botan. (8) 4: 1-6.

7. GIOVANNOZZI-SERMANNI, G. 1956. Solvent forquantitative paper chromatography of sugars. Na-ture 177: 586-87.

8. HALE, C. R. 1962. Synthesis of organic acids inthe fruits of the grape. Nature 195: 917-18.

9. HALE, C. R. AND R. J. WEAVER. 1962. The effectof developmental stage on direction of translocationof photosynthate in Vitis vinifera. Hilgardia 33:89-131.

10. HAYES, F. N. 1962. Solutes and solvents for liquidscintillation counting. Packard Technical Bull. No.1.

11. HENNIG, K. AND R. BURKHARDr. 1951. Die polar-graphische Bestimmung Apfel-siiure in Wein. Z.Lebensm. Untersuch.-Forsch. 92: 245-52.

12. LANGRIDGE, J. 1963. Biochemical aspects of tem-perature response. Ann. Rev. Plant Physiol. 14:441-62.

13. MUKHERJEE, S. AND H. C. SRIVASTAVA. 1952. Im-proved spray reagents for detection of sugars.Nature 169: 330.

14. PAYES, B. AND G. G. LATIES. 1964. The enzymaticcondensation of glyoxylate to yield a-keto-o-hy-droxysuccinate. Biochem. Biophys. Res. Commun.In Press.

15. PAYES, B. AND G. G. LATIES. 1963. The enzymaticconversion of y-OH a-ketoglutarate to malate:A postulated step in the cyclic oxidation of glyoxyl-ate. Biochem. Biophys. Res. Commun. 13: 179-85.

16. PEYNAUD, E. 1947. Contribution a 1'Etude bio-chemique de la maturation du raisin et de la com-position des vins. Lille: Imp. G. Santi et Fils.

17. PEYNAUD, E. AND A. MAURIE. 1953. Evolution desacides organiques dans le grain de raisin au coursde la maturation en 1951. Ann. Technol. Agr. 2:83-94.

18. PEYNAUD, E. AND A. MAURIE. 1958. Synthesis oftartaric and malic acids by grape vines. Am. J.Enol. 9: 32-6.

879

PLANT PHYSIOLOGY

19. PUCHER, G. W., C. S. LEAVENWORTH, W. D. GINm,AND H. B. VICKERY. 1948. Studies in the metabo-lism of crassulacean plants: The effect of tempera-ture upon the culture of excised leaves of Bryophyl-lum culycinuwm. Plant Physiol. 23: 123-32.

20. RABSON, R. 1956. Interactions of the environmentand plant metabolism with special reference to therole of keto acids. Ph.D. thesis, Cornell Univ.,Ithaca, N. Y.

21. RANSON, S. L. AND M. THOMAS. 1960. Crassula-cean acid metabolism. Ann. Rev. Plant Physiol.11: 81-110.

22. RESNIK, F. E., A. L. LEE, AND W. A. POWELL. 1955.Chromatography of organic acids in cured tobacco.Anal. Chem. 27: 928-31.

23. REUTHER, G. AND A. REICHARDT. 1963. The effectsof temperature upon the bleeding and metabolismin Vitis vinifera. Planta 59: 391-410.

24. RIBEREAU-GAYON, G. AND P. RIBEREAu-GAYON.1963. Utilisation de C1402 et de glucose U14Cpour l'etude du metabolism des organiques deVitis vinifera L. Comptes Rendus: 257: 778480.

25. RODOPULO, A. K. 1960. Oxidation-reduction trans-formations of the organic acids in the process ofgrape ripening. Biokhim. Vinodeliya 6:132-70.

26. STAFFORD, H. A. AND F. A. LOEWUS. 1958. Thefixation of C1402 into tartaric and malic acids ofexcised grape leaves. Plant Physiol. 33: 194-99.

27. SISSAKIAN, N. M., I. A. EGOROV, AND B. A. AFRI-KlAN. 1948. Biolimicheskie osobennosti vinogradai ikh sviaz's tekhnologiei vina. Biokhim. Vino-deliya 2.

28. THOMAS, M. AND RANSON, S. L. 1954. Physio-logical studies on acid metabolism in green plants.III. Further evidence of C02-fixation during darkacidification of plants showing crassulacean acidmetabolism. New Phytologist 53: 1-30.

29. VICKERY, H. B. 1954. The effect of temperatureon the behavior of malic acid and starch in leaves ofBryophyllum calycinum cultured in darkness. PlantPhysiol. 29: 385-92.

30. WOLF, J. 1938. Beitrage zur Kenntnis des Saure-stoffwechsels sukkulenter Crassulaceen. III.Stoffliche Zusammenhange zwischen garfahigenKohlehydraten und organischen Sauren. Planta 28:60-86.

31. YAMAMOTO, Y. AND H. BEEVERS. 1960. Malatesynthetase in higher plants. Plant Physiol. 35:1024.

Lipolysis and the Free Fatty Acid Pool in Seedlings'Allen J. St. Angelo and Aaron M. Altschul

Seed Protein Pioneering Research Laboratory,2 New Orleans, Louisiana

Introduction

Seeds of high lipid content metabolize most of theirstored lipids within a week after the start of germina-tion. The sequence of events from oxidation of thefatty acids from the acyl CoA derivatives through thenet synthesis of carbohydrates has been well described(2, 3, 10, 18). The first step of lipid metabolism, theremoval of acyl groups from the triglycerides, dependson a lipase which either preexists in the resting seedas active enzyme or precursor, or is synthesized upongermination. The free fatty acids thus produced areactivated in the presence of ATP and transferred tothe CoA derivatives.

As part of a study of the mobilization of lipids inoilseeds, we were interested in the nature of the lipasesin several different oilseeds. There is confusion inthe literature on the types of lipases which exist inthe resting state and which develop on germination.

1 Revised manuscript received Mar. 2, 1964.2 One of the laboratories of the Southern Utilization

Research and Development Division, Agricultural Re-search Service, United States Department of Agriculture.

Castor bean endosperm contains an acid lipase witha pH optimum at 4.3 (13, 14). It has been statedthat a second lipase develops in castor bean on germi-nation which has a pH optimum at neutrality (20).A lipase was reported in germinating cottonseed witha pH optimum range of 7 to 8 (12). There has beena -report of an acid lipase in the germinating peanut(15). We therefore selected castor bean, Ricinuscommunis, cottonseed, Gossypium hirsutum, and pea-nut, Arachis hypogaea, as the 3 seeds to be germinatedfor study of the lipases. To avoid erroneous conclu-sions arising from use of artificial substrates, westudied these enzymes on their endogenous substrates.A second point about the mobilization of seeds is

the control of the lipolysis reaction. Is this uncon-trolled and therefore can there be an accumulation offree fatty acids (FFA) in the seed, or is the forma-tion of free fatty acid tied to other metabolic steps sothat there is no accumulation of this intermediate?Here again there are contradictory reports in theliterature (8, 11, 18, 20). It could be that lipaseactivity released on grinding the seed for analysis ofthe lipids is responsible for the FFA observed. We,

880