DAVI S - Plant physiology · PHENOLASEACTIVITY IN RELATIONTO SEEDVIABILITY W. C. DAVIS (WITH THREE FIGURES) Introduction Oxidase and catalase activity of plant tissues are so frequently

PHENOLASE ACTIVITY IN RELATION TO SEED VIABILITY

W. C. DAVI S

(WITH THREE FIGURES)

Introduction

Oxidase and catalase activity of plant tissues are so frequently used ascriteria of metabolism and viability that methods for their quantitativemeasurement are becoming increasingly important in physiological tech-nique. -Quantitative data for catalase, however, are more numerousthan those of other oxidases, probably because of the difficulty en-countered in making accurate determinations of oxygenases and peroxidases.There are, nevertheless, several important reasons for more frequent estima-tions of oxidase activity. The physiological function of catalase is stillobscure. Oxidases, on the other hand, have been closely associated withthe ability of living organisms to bring about transformations of materialsotherwise stable at ordinary temperatures. Though our knowledge of oxida-tion-reduction reactions in vivo is as yet fragmentary, the abundant anddiverse experimental evidence which has accumulated makes inescapable theconclusion that oxidases are essential to co-ordinated respiratory and otheroxidative activities of plants. The correlation between oxidases and physi-ological processes has been so definite that these enzymes have been placedin a causal relationship to them. The desirability of a convenient quantita-tive method for oxidase determination is consequently self-evident.

Colorimetric methods for estimation of oxidases have been widely used.These methods have involved the use of such reagents as alpha-naphthol,hydroquinon, pyrogallol, paracresol, ortho- and para-aminophenol andothers. Though solutions of these reagents are to varying degrees autoxidiz-able in air, the amount of spontaneous oxidation can be minimized by keep-ing the hydrion concentration within definite limits for each reagent (6).All of them, however, are appreciably autoxidized in nearly neutral media.This is especially true of two common reagents, phenylenediamine hydro-chloride and demethyl-phenylenediamine, which have as a consequence beenlittle used as such in colorimetric estimations of oxidases.

In an attempt to procure an understanding of enzymatic processes inplants, objection arises to any appreciable modification of the hydrion con-centration of the reaction medium from that approximately normal to theplant. Realization of this fact has resulted in a search for reagents whichare not greatly autoxidized in neutral media (6). A few oxidase reagentsof this type, such as alpha-naphthol and paracresol are available but they donot produce the steep color gradients of more sensitive chromogens. Other

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things being equal, sensitive reagents reveal more minute differences andproduce sharper contrasts. These characteristics and the fact that the pro-cedure with certain precautions can be made quantitative readily justifythe use of these reagents despite the apparent complexity of the process. Anexperiment was undertaken with the Nadi reagent to determine its utility inthe estimation of indophenol oxidase activity in certain plant tissues. Thisreagent has found general application in animal physiology but only infre-quent use thus far in plant problems (7, 9, 10, 12). This report describesthe use of the Nadi reagent in a study of seed viability.

Experimental procedure

The calorimetric procedure for determination of phenolase activity in-volved essentially the following steps, namely, the preparation of a com-pletely oxidized color standard in terms of which the phenolase activity andspontaneous autoxidation are expressed, the determination of the phenolaseactivity of a unit quantity of seed powder, and the spontaneous autoxida-tion of the reagent containing a unit quantity of inactivated seed powder.The calorimetric reading of the latter was then subtracted from the readingobtained for the active enzymatic material and the result recorded as theactual phenolase activity.

The color standard was prepared by taking 125 cc. of M/100 a-phenyl-enediamine, an equal amount of M/100 a-naphthol, 62 cc. of 0.25 per cent.sodium carbonate and placing them in a 500-cc. bottle through which washedair was vigorously bubbled for five days to insure maximum coloration. Thesolution was then made up by volume to 70 per cent. alcohol. It was ob-served that the percentage of alcohol, acidity, and alkalinity of the solutiongreatly influenced the color. The resulting oxidized solution had a pH of7.0. Since the accuracy of the colorimeter, other things remaining constant,depends upon having nearly the same color intensity in the standard andunknown, the two were always balanced as nearly as possible. Dilutions ofthe standard made were primarily for the purpose of increasing the ac-curacy of the colorimeter determinations.

A series of convenient dilutions was made by pipetting 1 cc. of the com-pletely oxidized Nadi reagent into 40 cc. of 70 per cent. alcohol, 1 in 60,1 in 80, 1 in 100, 1 in 150, 1 in 500, and 1 in 1000. These color standardswere designated in the order given as 40, 60, 80, etc. The enzyme powderwas in all cases maintained at a concentration of 1 per cent. while the in-tensity of the color standard employed was chosen with respect to thephenolase activity.A small number of assorted seeds was ground in a Nixtamal mill, only

seeds apparently free from mechanical injury, and infection being se-lected. Each group of seeds was labelled to identify it in relation to variety

128



DAVIS: PHENOLASE ACTIVITY IN RELATION TO SEED VIABILITY

and age. Lots of seeds were then analyzed at random to avoid possible errordue to preconceived notions. The powder was then screened through an 80mesh sieve. In the case of wheat a few determinations were made withcoarser material which passed through the 40 mesh but not the 80 meshscreen. In all other determinations only the material which passed the80 mesh screen was used, the object being to secure material as nearly uni-form in physical texture and composition as possible. The powder, thoughno definite attempt was made to differentiate the various parts of the seeds,was predominantly endosperm and embryo tissue.

In determination of phenolase activity the sieved powder was weighedand made up to 1 per cent. by volume with boiled, distilled water at pH6.8 (2, 3, 14). This solution was then allowed to stand for 90 minutes. Inthe meantime small quantities of dimethyl-para-phenylenediamine hydro-chloride (hereafter referred to as phenylenediamine), a-naphthol, andsodium carbonate were weighed. The sodium carbonate was used to neu-tralize the acidity of the phenylenediamine. When the aqueous enzymesolution has stood for 90 minutes at room temperature (230 C. to 260 C.)the weighed quantities of a-naphthol and phenylenediamine were made upwith distilled water to a concentration of M/100. This is approximately theconcentration employed by other workers in similar investigatons (5, 7, 10),and it was found to give satisfactory results with the seeds used in thisexperiment.

The phenylenediamine and a-naphthol solutions were not made up andmixed until used as they are relatively unstable and autoxidizable in the pres-ence of air. The substances, however, are quite stable in the solid form.a-naphthol as used in this experiment was made up by adding equal parts ofdistilled water and 95 per cent. alchohol. An aqueous solution of a-naphtholwas also prepared by boiling 8 gm. of the solid a-naphthol in 1 liter of dis-tilled water for about 150 minutes. The solution was then made up to 1 literby volume, cooled, and filtered. A few determinations were then made to seeif the 9 per cent. alcohol retarded the reaction. No retardation was observedand consequently the alcoholic solution of a-naphthol was used in all of theexperimental work. It was found that 1 cc. of 0.25 per cent. sodium car-bonate was sufficient to neutralize the phenylenediamine, giving the final pHas 7.5 for the total substrate. A 2-cc. portion of a-naphthol, an equalamount of phenylenediamine, 1 cc. of sodium carbonate and 5 cc. of aninfusion made from 1 part of seed powder in 100 parts of distilled waterwere then pipetted into a 9.5 cm. petri dish. The shallow petri dish wasused to increase the surface area in contact with air (5). The reaction wasallowed to proceed for a given period of time with occasional shaking andthe solution was then made up by volume to a 70 per cent. alcohol concen-

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PLANT PHYSIOLOGY

tration. The frequency and duration of shaking were constant for eachgroup of seeds.

It was observed that the increase in the alcoholic concentration in-hibited the enzyme action but did not stop the autoxidation of the reagents.A time period of ten minutes was adequate for the resulting di-amino-com-pound to become uniformly dispersed in the alcohol. DYE (5) workingwith animal tissues found it necessary to allow 15 minutes for completedispersion of indophenol in alcohol. A difference in the physico-chemicalproperties in plant and animal extracts may account for the difference inthe time period required for the reaction.A volume of the colored solution was then decanted and taken out of the

petri dish and its color intensity determined in a Duboscq colorimeteragainst the color standard. For a control, 2 cc. of phenylenediamine andequal volume of a-naphthol, 1 cc. of sodium carbonate, and 5 cc. of enzymesolution were pipetted into a petri dish and made up by volume to 70 percent. alcohol. This made the concentration of the control similar to that ofthe unknown, thus affording a fairly accurate measure of the spontaneousoxidation of the Nadi reagents in the presence of atmospheric oxygen.Temperature variations in all of the experimental work remained in therange of 22.9 to 26.3° C. The per cent. oxidation of indophenol whichmight be formed was divided into the actual amount formed as determinedin the calorimeter, and this was expressed as percentage.

In the determination of catalase activity an apparatus similar to that em-ployed by APPLEMAN (1) was used. The determinations were made byplacing 1 gm. of the seed powder in a 250-cc. Erlenmeyer flask, with 20 cc.of hydrogen peroxide, with reaction period (ten minutes) and the shakingconstant throughout. Corrections for variations in atmospheric pressurewere made and the corrected gas volume was recorded.

The per cent. germination of seeds was taken by selecting multiple lotsof 25 apparently perfect seeds. These were then treated for 5 minutes in0.5 per cent. mercuric chloride solution, washed with distilled water, andthen placed in sterile petri dishes containing a small amount of uniformlymoistened absorbent cotton. The seeds were at all times subjected to asaturated atmosphere. A germination count was made at the end of eachday for 5 days. Temperature variation during the germination period wasfrom 25 to 340 C. All seeds remained free, from infection during thegermination period.

The seeds used in the experiments had been stored under ordinarylaboratory conditions and although no attempt had been made to provideabsolutely uniform conditions throughout the period of storage, they werenot exposed to abrupt temperature or moisture fluctuations.

The experimental data are presented in the accompanying tables I-V.

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DAVIS: PHENOLASE ACTIVITY IN RELATION TO SEED VIABILITY 131

TABLE IAGE, PHENOLASE ACTIVITY, AND GERMINATION

AGE | POWDER REACTION OXIDATION GERMINATIONI ~~~TEMPERATURE

MARQUIS WHEAT

years mesh °C. per cent. per cent.3 80 23.8 14 245 80 23.1 23 606 80 23.0 24 767 80 23.0 16 288 80 23.1 13 123 40 23.5 9 245 40 23.0 25 606 40 22.9 24 767 40 23.4 14 288 40 23.3 13 12

MICHIGAN AMBER WHEAT

1 80 25.7 21.8 326 80 25.6 21.4 0

13 80 25.6 13.0 01 40 25.8 16.0 326 40 25.8 12.0 0

13 40 25.8 10.0 0

TABLE IIAGE, PHENOLASE ACTIVITY, AND GERMINATION

AGE | TEMPERATURE OXIDATION GERMINATION

PINK OF WINTER WHEAT

years 0C. per cent. per cent.1 25.1 50 643 25.0 29 84 24.3 39 326 24.4 18 01 25.1 37 643 25.0 33 84 24.2 29 326 24.8 26 0

SUCCESS BEARDLESS BARLEY

1 25.9 23 644 25.7 22 12

11 26.1 18 8



PLANT PHYSIOLOGY

TABLE IIIAGE, PHENOLASE ACTIVITY, AND GERMINATION

AGE TEMPERATURE REACTIONTOTAL OXIDATION GERMINATION

SWEDISH SELECT SPRING OATS

OG.

25.525.525.925.525.525.325.325.225.125.325.325.325.3

min.10101010101010101045454545

min.202020202020201202060606060

per cent.18.3318.1916.8616.2114.0015.1012.7612.0010.4575.9075.6075.8075.96

per cent.16165252

12160

0

0

16120

0

TABLE IVAGE, PHENOLASE ACTIVITY, AND GERMINATION

TEMPERATURES TIME GERMIONNA-INCUBATIONJ REACTION REACTION TOTAL IION

ARLINGTON WHITE SPINE CUCUMBER

years 0C. CG mi. min. per cev t. per cent.2 25.4 25.4 20 40 27.0 563 25.4 25.4 20 40 26.8 524 25.4 25.4 20 40 27.2 282 26.7 26.1 10 20 15.0 563 26.1 26.1 10 20 14.0 524 26.1 26.1 10 20 10.0 282 52 26.3 10 20 13.0 563 52 26.3 10 20 12.0 524 52 26.3 10 20 8.0 28

MANCHURIA SPRING BARLEY

1 25.9 25.9 10 20 34 764 25.9 25.9 10 20 27 46

11 25.9 25.9 10 20 24 121 52 [ 26.4 10 20 30 764 52 26.4 10 20 26 46

11 52 26.4 10 20 24 12

132

years1 ...............

2...............3...............4...............5...............6...............7...............8...............9...............1...............2...............5...............7...............



DAVIS: PHENOLASE ACTIVITY IN RELATION TO SEED VIABILITY

TABLE VRELATION OF CATALASE ACTIVITY AND AGE IN SEEDS

AGE AMOUNT POWDER AMOUNT OF GAS GERMINATION

ARLINGTON WHITE SPINE CUCUMBER

years gin. mesh cc. per cent.4 0.2 40 14.55 564 0.4 40 26.4 563 0.2 40 14.8 523 0.4 40 32.0 522 0.2 40 15.1 282 0.4 40 33.1 28

MICHIGAN AMBER WHEAT

1 0.4 60 34.0 326 0.4 60 27.5 0

13 0.4 60 17.0 0

DiscussionIn view of differences in interpretation of the indophenol reaction (7)

and because of the exploratory character of the present investigation noattempt has been made in this report to adopt a critical definition of theterms oxidase and phenolase. The terms are employed simply to designatethe oxidizing components of certain seeds. Although there is a certainamount of doubt regarding the exact course of the Nadi reaction there isconsensus of opinion about the principle involved and the nature of theend products. It seems entirely justifiable, therefore, to employ the Nadimethod in testing the phenolase activity of seeds. The Nadi method has al-ready been proved experimentally satisfactory, it is quantitative, and thereis agreement concerning the fundamental character of the reaction.

Analysis of the data (tables I-V) reveals that there is no simple quantita-tive correlation between phenolase activity, age and germination as de-termined by the Nadi reaction. If, however, both the age and percentagegermination are taken into consideration then a definite though not propor-tional relationship becomes apparent. Those samples which were taken fromrelatively young seeds and had a high degree of germination gave corre-spondingly a large oxidation percentage. The fact revealed in table I, thatthe three year old seeds, in the case of Marquis wheat, have a lower per-centage oxidation than 5 year old seeds is explained, in part at least, by thefact that the percentage of germination of the 5 year old grain is more thantwice as great as that of the 3 year old. Oxidase activity consequently wasin this instance more closely correlated with- viability than with age.

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PLANT PHYSIOLOGY

Similar results have been reported by CROCKER and HARRINGTON (4) forAmaranthus retroflexus. Examination of the experimental results obtainedfrom the 7 year old wheat with a germination of 28 per cent. as constrastedto 8 year old grain with 12 per cent. germination shows a decrease inoxidation as the concomitant of age and low vitality.A difference in the amount of oxidation with variation in size of the

mesh through which the seed powder has passed was also noted (table I).The powder which passed through the 40 but not the 80 mesh sieve gavemore irregular results for both Marquis and Michigan amber wheat. Whilethe percentage oxidation was more variable in the 40 mesh than in the 80mesh powder it will be noted that the relationship to age and percentagegermination of the seeds had the same general trend. The percentage ofoxidation for the two varieties of wheat at the same age is not identicalnor is there a definite correlation of Marquis and Michigan wheat in relationto germination and oxidation. It will also be noted (table I) that the 6and 15 year old Michigan amber wheat did not germinate but that the lat-ter gave a lower oxidation percentage. This indicates that there is a definiterelation between age and phenolase activity.

The parallelism in catalase and phenolase activity in seeds of given ageis of interest. The latter may prove to be the more reliable index to viabilityif the procedure for its determination can be standardized and simplified.

There appears to be a positive relationship between catalase and pheno-lase activity in Arlington white spine cucumber and Michigan amber wheat(fig. 1). The phenolase and catalase activity approximately parallel eachother in relation to the age of seeds. The decrease in activity of the twoenzymes accompanying senescence indicates that the intensity of the catalase

t35,_

C RC AT A L AS E'C 3

G *,25-A aS S 20-LIB 1 15-E oPHENOLASER IQ.,/T .10- "

D N5

-MICHIGAN AMBER WHEAT

... ARLINGTON CUCUMBBER1 9 4 5 _ 7 R Ft

AGE OF SEEDS IN YEARSFIG. 1. Comparison of phenolase and catalase activity in seeds of wheat and cucumbers-

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DAVIS: PHENOLASE ACTIVITY IN RELATION TO SEED VIABILITY 135

activity had no appreciable effect upon the amount of indophenol pro-duced (7).

The same generalizations in relation to age and per cent. of germinationapply to the Pink of Winter wheat (table II). The three year old Pink ofWinter wheat with 8 per cent. germination gave a lower percentage oxida-tion than the four year old wheat of the same variety but with 32 per cent.germination. This situation is similar to that occurring in Marquis wheat(table I). These two cases indicate that there is a greater amount of cor-relation between oxidation and germination than between age and oxidationin the case of wheat. Success beardless barley, on the other hand (tableII), disclosed a slow decrease in the rate of oxidation in relation to both ageand germination for the first 4 years and then a relatively large decreasein relation to viability and senescence.

The drop in percentage oxidation is shown graphically (fig. 2). Barley,oats, and cucumber show a definite decrease in the phenolase activity in rela-tion to age of the seeds. In wheat, on the other hand, there is an initial in-crease in the percentage oxidation which is subsequently followed by adecrease. It is, however, to be noted that the increase in the phenolase ac-tivity of wheat is also accompanied by an increase in the percentage germina-tion. In the other determinations (fig. 2) the germination percentage de-

SAft156SALE OATS

35. §~~~~~.Acm on""SWHEAT§35AAAT_CrUMSKi30.

25.

20

5

E A4 14 i6

FIG. 2. Phenolase activity curves for various seeds of different ages. The numbers inthe circles give the per cent. germination.

creases as the age increases in all species except oats. In this case, despitethe increase in the per cent. germination, there is a decrease in degree ofoxidation with age.

HARRISON (7) and other workers (8, 11) have shown that the indophenolformed in the Nadi reaction may in turn react with reducing substances



PLANT PHYSIOLOGY

such as glutathione and hypoxanthine, being thereby reduced to its color-less leuco base. The final quantity of indophenol thus may be no reliablecriterion of peroxidase activity if the concentration of reducing substancespresent is high. In a study of the type here reported, the utility of theNadi reaction is not lost on this account, because reduction reactions indormant seeds are inappreciable in comparison with those in actively grow-ing tissues. The dilutions of the seed extracts and the brief time allowedfor the reaction still further minimized reduction of indophenol.

Error of greater magnitude may possibly be introduced into the Nadireaction by the presence of an appreciable amount of catalase, due to itsdecomposition of the peroxide upon which the formation of indophenol de-pends. Several investigations (7, 10) have shown that the inactivation ofthe more thermosensitive catalase at moderate temperatures actually resultsin an increase of indophenol by peroxidases. To test the effect of catalaseon the formation of indophenol by phenolase, several samples of enzymaticseed extracts were subjected to a mild heat treatment.Heat treatment at 520 C. for 90 minutes in a water bath without shak-

ing (7, 10) decreased rather than increased phenolase activity in Manchuriaspring barley and Arlington white spine cucumber (fig. 3). In the case of

OUNTREATED 15 COTREALED

E 31R 6CUCUNMBE

1 33 4 5

A~~~ ~ EA30

1NOT25NAGE2(* U~~~~BRLEY

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 3. Effect of heat treatment on phenolase activity.

barley of considerable age, the temperature treatment had very little effectupon the rate of oxidation. The effects of catalase in the dilutions of seedextracts employed thus appear to have been negligible.

The results of catalase determinations (table V) show a decrease in ac-tivity with age and low percentages of germination. Many experimentshave been made on catalase in search of a method to determine viability,especially, of seeds. SAMPIETRO (13) and CROCKER and HARRINGTON (4)show a relationship between catalase activity, age and germination. Thecorrelation, however, is not definite enough to make reliable forecasts of the

136



DAVIS: PIIENOLASE ACTIVITY IN RELATION TO SEED VIABILITY

germination percentage. The greater part of the work so far done withcatalase in relation to germination apparently has been unable to evaluateprecisely the effect of growth and storage conditions on enzymatic activity.

In conclusion, the Nadi reaction, involving short reaction periods anddilutions of seed extracts as above described, was found to yield concordantresults in the study of phenolase activity of dry seeds of varying age.When the limitations of the Nadi reaction are circumvented, its sensivitymakes it a simple and valuable indicator of oxidase activity. Phenolaseactivity as herein determined showed a high degree of correlation with seedviability.

SummaryA modification of the Nadi reaction for phenolase determination is de-

scribed. Phenolase activity was high in young seeds with a high percentageof germination and low in old seeds of low percentage of germination.Enzymatic material heat treated at 25° C. for 90 minutes did not show in-crease in phenolase activity. Phenolase and catalase activity in general wereparallel in cucumber and wheat.

UNIVERSITY OF HAWAII,HONOLULU, OAHU.

LITERATURE CITED

1. APPLEMAN, C. 0. Some observation on catalase. Bot. Gaz. 50: 182-192. 1910.

2. BEIJERINCK, M. W., and VAN HEST, J. J. Lebedev 's "Hefemazera-tionssaft." Folio microbiol. Delft. 4: 107-118. 1916.

3. BUNZELL, H. H. The relationship existing between the oxidase activityof plant juices and their hydrogen ion concentrations, with a noteon the cause of oxidase activity in plant tissues. Jour. Biol. Chem.28: 315-333. 1916-4917.

4. CROCKER, WILLIAM, and HARRINGTON, G. T. Catalase and oxidasecontent of seeds in relation to their dormancy, age, vitality, andrespiration. Jour. Agr. Res. 15: 137-174. 1918.

5. DYE, J. A. Improved colorimetric method for determining quantita-tively the indophenol oxidase content of animal tissues. Proc. Soe.Exp. Biol. and Med. 24: 640-642. 1927.

6. FONG, W. Y., and CRUESS, W. V. Comparison of several indicators forfruit oxidase. Amer. Jour. Bot. 16: 799-802. 1929.

7. HARRISON, D. C. The indophenol reaction in biological oxidations.Biochem. Jour. 23: 982-999. 1929.

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8. , and THURLOW, S. The secondary oxidation of some sub-stances of physiological interest. Biochem. Jour. 20: 217-231.1926.

9. KASTLE, J. H. The oxidases. Hygienic Laboratory. Bull. no. 59.1909.

10. KEILIN, D. Cytochrome and respiratory enzymes. Proc. Roy. Soc.London. B 104: 206-252. 1929.

11. KODAMA, K. Studies on xanthine oxidase. VIII: The oxidation reduc-tion potential of the oxidase system. Biochem. Jour. 20: 1095-1103. 1926.

12. REY-PAILHADE, J. Sur 1'existence simultanee de deux ferments d'oxy-dation dans certains vegetaux. Compt. Rend. Soc. Biol. 48: 479-480. 1896.

13. SAMPIETRO, G. Investigations on the relationship between catalase ofrices and their germinative capacity. Giorn. risicoltura. 18: 133-141. 1923. Chem. Abs. 18: 1317. 1924.

14. SMALL, J. Hydrogen-ion concentration in plant cells and tissues.GebrUder Borntraeger. Berlin. 1929.

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Documents

DAVI S - Plant physiology · PHENOLASEACTIVITY IN RELATIONTO SEEDVIABILITY W. C. DAVIS (WITH THREE FIGURES) Introduction Oxidase and catalase activity of plant tissues are so frequently