Electrophoretic studies of the relationship of peroxidases, polyphenol oxidase, and indoleacetic acid oxidase to cold tolerance of alfalfa

CRYOBIOLOGY

12, 62-80

( 1975)

Electrophoretic Studies of the Relationship of Peroxidases, Polyphenol Oxidase, and lndoleacetic Acid Oxidase to Cold Tolerance of Alfalfa I32 M. KRASNUK,

G. A. JUNG,

AND

F. H. WITHAM

Department of Biology, The Pennsyluania State University; a& U.S. Regional Pasture Reseawh Laboratory, Agricultural Research Service, U.S. Department of

Agriculture, University Park, Pennsylvania The relationship of soluble proteins to cold tolerance in alfalfa (Medicago sativa L.) has been well documented (2, 6, 10, 14, 17-19, 31). Electrophoresis of root extracts of six genotypes of alfalfa by Coleman et al. (6) resulted in the separation of 17-20 soluble protein bands with several highly charged protein bands prevalent in cold-hardened material. GerIoff et al. (14) reported the electrophoretic separation of approximately 18-20 soluble protein components from root extracts of several alfalfa cultivars; however, no significant differences in component bands were detected between cultivars that differed in inherent hardening capacities. Although the concentrations of various soluble proteins varied according to environment, no specific protein was found to appear during hardening and disappear during dehardening. Electrophoretic investigations of the possible enzymatic nature of the soluble proteins associated with cold tolerance have been reported, but studies of different enzymes in alfalfa are limited to peroxidases. Gerloff et al. (14) observed a significant increase Received May 31, 1974. *Supported in part by Grant GB-25187 from the National Science Foundation, 2 Authorized for publication on May 1, 1974 as journal series paper No. 4686 of the Pennsylvania Agricultural Experiment Station. 62 Copyright All rights

1975 by Academic Press, o9 reproduction in any form

Inc.

reserved.

16802

in total peroxidase in extracts of alfalfa roots during hardening and qualitative changes (two new bands) in electrophoretie patterns of the isoperoxidases, The work of McCown et al. (24, 25) indicated qualitative and quantitative differences in peroxidases of several other species of coldhardened plants, whereas Roberts (30) noted mainly quantitative changes in isoperoxidases of wheat during hardening. Results of electrophoretic analyses of peroxidases from Salix during rapid deacclimation led Hall ef al. (15) to conclude that peroxidases did not confer hardiness directly, but they could be important as a hydroperoxide degradation system during hardening. Electrophoretic studies of polyphenol oxidase, IAA oxidase, and cold tolerance are lacking: however, the assay data of Korovin and Barskaya (20) indicated a decreased polyphenol oxidase activity in potato at low temperatures. Sysoev and Krasnaya (35) reported a sign&ant increase in poIypheno1 oxidase of spring wheat during hardening, and BoIduc et al. (1) detected a tenfold increase in IAA oxidase activity in winter wheat seedlings during hardening. Information concerning these enzymes in alfalfa is not available. One purpose of this study, therefore, was to compare, by electrophoresis, isoenzymes of polyphenol oxidase, IAA oxidase, and

PEROXIDASES,

OXIDASES,

peroxidases of cold-tolerant and coId-sensitive alfalfa during hardening, dehardening, and in the uuhardened state. This study further differs from other investigations, in that three protein extractants were employed. Since pH and ionic nature of extractants influence protein solubility, a second purpose of this study was to compare effects of the extractants on the enzymes in relation to different levels of cold tolerance.

AND

COLD

TOLERANCE

63

implemented to reduce carbohydrate reserves and protein levels to a minimum for comparison with other samples. These sampies will be referred to as Vz (for Vernal Summer 2) and SZ (for Sonora Summer 2). Cold-hardened pIants were obtained directly from the fieId during the winter. Autumn temperatures were above normal and killing frosts were not observed until late November. Also, soil temperature was lower in February than in December. Approximately 1000 plants of each cuItivar were removed from the field the second MATERIALS AND METHODS week of December, but only plants of the Growth of Plant Material cold-tolerant Vernal were removed in Iate February. Reference to these samples will Seeds of two cultivars of alfalfa, coldbe Vs (for Vernal December), S, (for tolerant ‘Vernal’ and cold-sensitive ‘Sonora,’ Sonora December, and Va (for Vernal were sown in the field during the spring. February), After growing for five months in the field, Partially dehardened plant materia1 was 500 plants of each cultivar were potted (10 produced by potting 150-200 plants of each plants/pot) and moved to the greenhouse. cultivar (10 plants/pot), removed from the After five weeks, 24 pots of each cultivar field the second week of December, and were placed in a growth chamber and growing them for 77 days in the greenmaintained for 67 days under a 16-hr photoperiod (light intensity of 19,332 lux) at house. Photoperiod, light quality, and light 27°C and an 8-hr dark period at 16°C intensity in the greenhouse were those of the natura1 environment, but the higher in( summer conditions). PIants were watered daiIy and rotated within the chamber fre- door day and night t.emperatures were suffiquently. Malathion was sprayed on the cient to allow topgrowth to develop. Conplants when necessary. To maintain some ditions, however, were not conducive to morphologicat uniformity among all sam- flowering. These samples will be referred ples, pIants were clipped to a height of 10 to as V, for partially dehardened Vernal cm. Plants were clipped at full bloom, or and S, for partially dehardened Sonora. 47 days from the time they were placed in the chamber. PIants were clipped again Sampling eight and 14 days after initial clipping, to Plants obtained from each environment maintain IO-cm height until they were sam- were sampled in the same manner. After pled. These samples will be referred to as all debris was removed, the pIants were V1 (for Vernal Summer 1) and Si (for thoroughly washed and cut to a 2.5cm Sonora Summer 1). crown length above and a 2.5”cm root A second group of plants was grown Iater length beIow the cotyledonary node. Part under similar conditions, but for a %-day of the pIant tissue was reserved for assessperiod. These were clipped at full bIoom, ment of cold tolerance, and the remainder or 47 days after they were placed in the was immediately frozen in liquid nitrogen, growth chamber; however, they were Iyophilized, ground in a WiIey Mill to pass clipped again three and six days after the through a 40-mesh screen, and stored at initia1 cIipping. This clipping regime was -20°C in airtight containers.

KRASNUK,

64 TABLE

JUNG AND WITHAM

Extrnction

1

EVALUATION OF COLD TOLITRANCYop ALFALFA CROWN AND ROOT T~ssuss -4s RILLATED TO PERCENTAGEOF ELECTROLYTESEFFLUXED BWTlpleS 1. 2. 3. 4. 5. 6. 7. 8. 9.

Vernal summer (V,) Vernal summer (V,) Sonora summer (8,) Sonora summer (82) Vernal December (V,) Vernal February (V,) Sonora December (Sa) Vernal partially dehardened (V,) Sonora partially dehardened (56)

*/oEflh' 91.9 > I

83.4 67.2 63.9 84.9 81.2 84.4

QValues above 70% indicate relatively high cold injury, whereas, percentages between 40 and 70 indicate moderate cold injury. Each value is an average of the percentage ion efflux from four 10-g samples. Any two values compared are significantly different if their difference is greater than 4.9 percentage units.

Cold Tolerance Tests CoId tolerance of all samples was evaluated using an electrical conductivity method described by Dexter et al. (8). Four 10-g replicates of each sample, consisting of 2.5-cm crown and 2.5cm root tissue, were placed in deep petri dishes and exposed to -8°C for 4 hr. Samples were removed from the freezer, immersed in a volume of distilled water equivalent to 5~ their fresh weight, and held at a temperature of 2-5°C for 20 hr. The conductance of the decanted liquid containing effluxed electrolytes was measured at 25°C with a conductivity bridge. After homogenization of the sample tissues and addition of the decanted liquid, the conductivity at 25°C was measured again to determine the total electroIyte content. A numerical value (percentage electrolytes effluxed) was obtained by dividing the former conductance value by the latter and multiplying by 100. A high percentage (above 70) indicates little or no cold tolerance, whiIe mid (40-70) and Iower (below 40) percentages indicate moderate and high levels, respectively (Table 1).

of Soluble Protein

Soluble protein was extracted by mixing 500 mg of lyophilized powder of each sample with 25 ml of the following precooIed (4°C) solvents: double-distilled water, pH 7.0, 0.05 M Tris-HCl buffer, pH 7.0; and 0.05 M sodium borate buffer, pH 9.0. The extracts were stirred vigorously for approximately 1 min and placed in the cold (4°C ) , where extraction was continued for 16 hr. After 16 hr, the extracts were stirred again for 1 min, filtered through four layers of cheesecloth, and clarified by centrifugation at 14,000 g for 10 min at 04°C ( 10). Soluble Protein Estimation Concentrations of soluble protein in the extracts were determined according to Lowry ef al. (22), as modified by Shih et aE. (31). For each sample extract lOO-mg dry-weight equivalents (5.0 ml) were mixed with .5.0 mI of cold 20% trichloroacetic acid and placed in the cold (4°C) for 12 hr. The treated extracts were centrifuged at 17,500 g for 10 min at 0-4”C and the resuhing precipitate was washed with 5.0 ml of 95% ethanol containing 0.1 N potassium acetate. After recentrifugation at 17,500 g for 10 min at 04°C the precipitate was dissolved with 0.5 ml of 1 N NaOH and dihrted to a 5.0”ml volume with double-distilled water. Soluble protein estimations, determined according to the standard L,owry technique (22), were compared with standard curve values of known concentrations of bovine serum albumin. EZectrophoresis SolubIe proteins present in extracts were separated by polyacrylamide disc gel electrophoresis according to the methods of Ornstein (28) and Davis (7). Modifications suggested by Steward and Barber (32), and Faw and Jung (10) also were used.

PEROXIDASES,

OXIDASES,

Long gel tubes (12 cm) were used in this study so that the length of the small pore running gel was 5.5 cm. Spacer gels were increased in volume, as advised by Davis (7), and were aIso photopolymerized [ 32). Sample extracts equivalent to either 4 mg dry weight of tissue (Cl,2 ml) or 100 rg of protein were applied onto the spacer gels, and the remaining voIume of the gel tubes was Wed with Iarge pore gel solution. After photopoIymerization of sample gels, gel tubes were placed in the cold (4°C) for 15 min before electrophoresis. Electrophoretic separations were performed with coId (4°C) 0.005 M Trisglycine buffer, pH 8.3, and an ice bath was maintained around the Iower reservoir to dissipate heat build-up. A current of 2.0 mA/geI column was used initially until the bromphenol blue tracer band had migrated into the small pore gel. The current was then increased to 3.0 mA/gel column and was maintained at this level until the tracer band was 1.0 cm from the bottom of the small pore running ge1. Total running time was 2 hr. Amide Gels

Black Staining

and Destaining

of

A visible pattern of the separated soluble proteins on the geIs was obtained by immersing one set of gels, after electrophoresis, in a solution of 1.0 g of amido black dye in 100 ml of 7% acetic acid, After staining for 1 hr, the gels were immersed in 7% acetic acid for approximately 12 hr and then destained electrically (7). The gels were subsequent’ly stored in 7% acetic acid and photographed with a Polaroid Land camera. Enzyme Localization

on Gels

The enzyme systems, which were all anionic, were localized on the gels after electrophoresis by immersing the gels in a reaction mixture specific for a particuIar enzyme rather than the staining procedure.

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85

Controls consisted of incubating geIs in reaction mixtures, with or without enzyme, and in the presence or absence of substrate. (a) Peroxidase. The peroxidases were localized on gels, according to Gerloff et al. (14), with some modifications. The first method consisted of incubating gels in 10 ml of aqueous 0.10 M guaiacol for 30 min at 35°C with subsequent transfer to 0.3% H,O, for 10 min. Gels were immediately photographed after a lo-min incubation in hydrogen peroxide. A second similar method utihzed aqueous 0.10 M catechol as the hydrogen donor. For the third method of localizing peroxidases on gels, 10 m1 of aqueous 0.20 M pyrogallo1 were used as the hydrogen donor, and gels were photographed after a 5-min incubation in hydrogen peroxide. Controls in these systems consisted of gel incubation in reaction mixtures plus or minus appropriate hydrogen donors ( guaiacol, catechol, pyrogalIo1) followed by immersion in or omission of Hz02. (b) Polyphenol oxidase. The procedure was adapted from methods described by Bwstone (4). GeIs were incubated for 1 hr at 35°C in a reaction mixture consisting of 10 ml 0.10 M potassium phosphate buffer, pH 7.3-7.4 containing 0.1% L-dihydroxyphenyIaIanine ( L-DOPA), and photographed. Controls consisted of geIs incubated in reaction mixtures in the presence and absence of L-DOPA. (c) IAA oxidose. Indoleacetic acid oxidase (IAA) was localized on the gels according to Frenkel (11) with modifications of Meudt and Gaines (27). Gels were incubated for 4 hr at 32°C in a reaction mixture consisting of 10 ml 0.10 M potassium phosphate buffer, pH 6.0; 2 mM IAA, 0.1, mM 2,4 dichloropheno1, 0.1 mM MnC&. The gels were then immersed for 1 hr in a solution of 0.5% p-N, N-dimethylaminocinnamaldehyde (DMACA) in 1 N HCl. Since band patterns were too light to be photographed, diagrams were made of the patterns. Control gels were incubated in reac-

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tion mixtures plus or minus IAA, followed by immersion in or omission of DMACA. Tissue A.rsays (a) Peroxidase assay. Peroxidase activity present in 5.0 mg dry weight equivalents of doubled-distilled water extracts of the samples was assayed according to the methods of Chance and Maehly (5), as described by Gerloff et al. (14). The reaction was performed at room temperature. At zero time, 1.0 ml of 0.3% H202 was added to a spectrophotometer tube, with a 1%cm internal diameter containing, 1.0 ml 0.10 M potassium phosphate buffer, pH 7.0, I.0 ml 0.02 M guaiacol, 3.0 ml double-distilled water, and 1.0 ml aqueous extract corresponding to 5.0 mg dry weight of tissue. The tube was shaken and optical density readings of tetraguaiacol formation were taken at 470 nm at IO-set intervals, for a 2-min period. Controls consisted of reaction mixtures with or without enzyme extract, guaiacol, or H202. Boiled enzyme also was used as a control. Results were plot.ted on the basis of optical density and enzyme units. The enzyme unit is defined as the quantity of enzyme needed to produce a 0.1 change in optical density (OD 470 nm) per minute at room temperature in a reaction mixture with a volume of 7.0 ml and light path of 1.2 cm under initial velocity conditions. (d) IAA oxidase assay. IAA oxidase activity present in 10 mg dry weight equivalents of doubIe-distilled water extracts was measured in 50-ml Erlenmeyer flasks at room temperature (3, 21,37). At zero time 1.0 ml of IAA solution (350 pg/ml double-distilled water) was added to a reaction mixture consisting of 1.0 ml 1O-3 M 2,4 dichlorophenol, 1.0 ml lo-$ M MnCL, 6.0 ml 0.05 M potassium ph0sphat.e pH 4.5, and 1.0 ml aqueous extract equivalent to IO mg dry weight of sample tissue. After 15, 30, and 60 min, l.O-ml aliquots of the reaction mixture were removed and

added to 4.0 ml of Salkowski reagent (500 ml double-distilled water, 300 ml concentrated HzS04, 15 ml 0.50 M FeCh) prepared according t.o Tang and Banner (36). A quantitative estimate of residual IAA was made with a Klett-Summerson photoelectric calorimeter, equipped with a No. 54 filter. Comparison with standard curve values of IAA plus Salkowski reagent indicated the amount of residual IAA. ControIs consisted of reaction mixtures containing IAA with and without boiled enzyme. RESULTS AND DISCUSSION

Cold Tolerance Vernal winter (V,) and (V,) samples were considered as moderately hardened since percentage efflux values of 40% or less in the cold-tolerance assay would be representative of fully hardened plant material (Table 1). The high value for the Sonora (S,) sample reflects little or no hardening although the plants removed from the field in December were not damaged as evidenced by the growth of these plants in the greenhouse. The percentage effiux for the Sonora samples was approximately the same at all times and was, in fact, lower for the Sonora summer sample (ST) than for the corresponding Vernal summer sample (V,). The differences in summer samples were probably a result of endogenous differences developed in response to frequent clipping of topgrowth and reduction in reserve materials; however, it might also be possible that Sonora was inherently more hardened to higher summer temperatures than Vernal. The high efflux values for field plants, transferred to the greenhouse in December ( Vg, S,) indicated a reduced level of hardiness. Although photoperiod and light intensity were essentially those of the natural environment., the higher indoor temperatures resulted in the production of topgrowth and a subsequent loss of hardiness. This

PEROXIDASES,

OXIDASES,

AND

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TOLERANCE

67

60 !

I23456789

-

DISTILLED WTER, pH 7.0

123456769

I23456769

OD5M TRIS-ICI. pH 7.0

0.05M BORATE, fl

90

FIG. 1. Comparison of the levels of trichIoroacetic acid-precipitable soluble proteins extracted with three solvents from unhardened, hardened, and partially dehardened Vernal and Sonora alfalfa crown and root tissues. For each extraction system, Nos. l-9 represent the following samples: 1,2-Vernal summer samples; 3,GSonora summer samples; 5,6-Vernal. December and February field samples, respectively; 7-Sonora December field sample; 8,9Vernal and Sonora dehardening samples, respectively. SampIes 2 and 4 were subjected to heavier clipping treatment. The pattern is representative of the total protein in ten separate extracts from each cultivar and experimental condition indicated.

was assumed to be a partial since flowering did not occur. Extraction

dehardening

Systems

Some differences in the 1eveIs of trichloracetic acid-precipitable soluble proteins were evident with respect to extractant, cultivar, and environment (Fig. 1). Water extracts of Vernal and Sonora samples exhibited the greatest differences in soluble protein content between cultivars for all environments. The soluble protein content of both cultivars increased dramatically in the winter ( Samples 5,6,7), and decreased during dehardening (Samples 8, 9). Also, lower soIubIe protein Ievels were detected in Samples 2 and 4 as compared with Sampies 1, and 3, presumably an effect of frequent clipping. With Tris-HCl buffer as the extractant, dif!erences observed in soluble protein levels between cultivars were generally less. Fluctuations with respect to environment were still evident; however, the de-

gree, in most cases, was less than with the aqueous system. The 0.05 M borate buffer at pH 9.0 was the most eflkient of the three extractants. Although this buffer yielded larger amounts of soluble protein than the other extractants, it did not reflect any greater cultivar differences in the levels or environmental of soluble proteins extracted. Since the distilled-water extraction in these studies and in previous studies (9) resuhed in the greatest soluble protein differences between cultivars with respect to environment, this system appears appropriate for studying protein changes in relation to coId tolerance. However, the use of water as an extractant was criticized by Gerloff & al. (14) on the assumption that endogenous differences in concentrations of organic acids, etc., between cultivars, could influence the quantity and quality of proteins extracted. This criticism may be valid since changes in tissue pH during hardening have been detected (23, 37) and

68

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results presented in Fig. 1 indicate that the use of a miId buffer reduced cultivar differences. Conversely, any genuine in uiwo cultivar differences associated with coId tolerance may be minimized or negated by the use of buffered extractants (9). Therefore, the best extraction medium for studying the reIationship of soluble proteins to cold tolerance is difficult to determine. Consequently, all experiments were performed with three extractants to compare any quantitative or qualitative soluble protein differences between cultivars with the gain or loss of cold tolerance. Amide Black Staining of Soluble Proteins The results of amido black staining (data not shown) indicated the presence of approximately 18-20 soluble protein bands on most of the gels, which were similar to results obtained by others (6, 9, 10, 14). Gels containing soluble proteins extracted from winter samples of both cultivars ( Vs, S$, V,) contained higher Ievels of soluble protein for the three extractants used whiIe lower amounts were detected on gels of summer and partially dehardened samples. The results paralleled the soluble protein levels (Fig. 1) and coId-tolerance changes (Table 1) associated with the different environments, Cultivar differences were present; however, differences in intensities of the soluble protein components, as a function of the extractant used, made evaluation difficult. Several differences in bands were visible, especially the increased intensity of slowly migrating bands near the gel origins of winter samples (Va, Sa, V,), which appeared to be the result of increases in specific proteins as well as a higher resolution of these components. Similar pattern changes were noted by Coleman et al. (6). Intensity increases among the faster migrating proteins of water and Tris-HCl-extracted winter samples of both cultivars were observed; however, the intensity in-

creases were greater in gels containing cold-tolerant Vernal extracts compared to the Sonora extracts. Aside from cultivar and environmental effects, any quantitative, as well as qualitative differences, in soluble proteins extracted from tissues depended on the type and pH of the extractant used. This fact is of utmost importance, especially in studies of enzyme changes related to cold tolerance since results could be greatly distorted by the extraction system used (9,10). Thus, the presence or absence of specific proteins (enzyme forms) in hardened and unh.ardened samples, could be more of a function of their soIubility in a specific extractant than of their relationship to hardening. Localization Guaiacol

of Peroxidases on Gels with

Isoperoxidase band patterns on gels incubated in 0.10 M guaiacol were basically similar and related to environmental, and to a lesser extent, cuhivar differences (Fig. 2). Sample gels containing distilled-water or Tris-HCl-extracted proteins exhibited an active midband region consisting of three closely migrating bands, two Iower or faster migr.ating isoenzymes, and two or three light bands in the upper portions of the gels. The lowest band, present near the bottoms of these gels and a11others, is not enzymatic in nature, but is the tracer dye band. A similar pattern was observed in geIs containing borate extracts, however, migration of the two faster moving bands was greater. In addition, more prominent upper bands were present in some borate sample gels, but were diffuse in others. The results illustrated mainly quantitative changes in individual isoperoxidases of both cultivars, so that intensity increases or decreases of constitutive isoenzymes were observed with all samples obtained from the different environments. These results follow, to some extent, the data of Roberts (30) in studies of peroxidases in relation

PEROXIDASES,

OXIDASES,

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69

abcdef FIG, 2. Peroxidase zymograms after electrophoresis of soluble proteins in extracts of unhardened, hardened, and partially dehardened Vernal (V) and Sonora (S ) alfalfa crown and root tissues. Gels were incubated in guaiacol for 30 min before transfer to hydrogen peroxide, Environments from which samples were obtained (Nos. 1-S) were as follows: l-Summer conditions; 2-Summer conditions with frequent clipping; 3-Winter field conditions (December); &-Winter field conditions (February); 5-Dehardening conditions in greenhouse. Peroxidases present in 4.0 mg dry weight of tissue (a, b, c) and 100 pg equivalents of protein (d, e, f) were separated by electrophoresis of double-distilled water extracts (a, d), 0.05 M Tris-HCl extracts (b, e ), and 0.05 M sodium borate extracts (c, f).

of wheat, However, an to cold tolerance additional band was also detected in the Vernal winter sample Va extracts and was Iocated on the gels as the lowest band (fastest migrating band). The additional band was lighter than the two other lower bands and was not easily discernible due to the other intensely staining isoenzymes. A somewhat similar result was obtained by Gerloff et aZ. (14) who reported the presence of two new peroxidases in cold-hardened Vernal aIfaIfa roots. The formation of new isoperoxidases in other plants during hardening has also been observed (24, 25). For the extractants used, sodium borate buffer extraction yieIded a higher quantity and possibly a different quality of enzyme forms in upper portions of sample gels. Differences in these regions were not ap-

parent between gels containing distilledwater or Tris-HCl extracts. Isoperoxidase activity, based on equal dry weights of tissue or equal qu.antities of protein, increased during the winter and decreased during dehardening for both cultivars, regardless of the extractant used. The isoperoxidases of Vernal exhibited higher intensities than those of Sonora, however, quantitative differences between cuhivars were less in the December ( Vlr Ss) sampies. Although both cultivars exhibited increased isoperoxidase activities in December, they displayed differences in this capacity during the summer and dehardening. These differences reflected either greater amounts of isoenzymes in Vernal (VI, V2 ), although soluble protein levels of Vernal samples were almost equal to or slightly

70

KRASNUK,

JUNG

AND

abcdef

abcdef

abcdef

abcdef

abcdef

abcdef

WITHAM

abcdef

abcdef

abcdef

FIG. 3. Peroxidase zymograms after electrophoresis of soluble proteins in extracts of unhardened, hardened, and partially dehardened Vernal (V) and Sonora ( S) alfalfa crown and root tissues. Gels were incubated in catechol for 30 min before transfer to hydrogen peroxide. Environments from which samples were obtained (Nos. l-5) were as follows: I-Summer conditions; Z-Summer conditions with frequent clipping; 3-Winter field conditions (December) ; Q-Winter field conditions ( February) ; SIDehardening conditions in greenhouse. Peroxidases present in 4.0 mg dry weight of tissue (a, b, c) and 100 &g equivalents of protein

(d, e, f) were separated by electrophoresis of double-distilled water extracts (a, d), 0.05 M Tris-HC1

(b, e), and 0.05 M sodium borate extracts

less than those of corresponding Sonora samples ( SI, &), or a greater substrate specificity of Sonora isoperoxidases, since lighter intensities were detected with the monophenol guaiacol. Localization

of Peroxidases

on Gels with

Catechol Isoperoxidase band patterns obtained from gels incubated in 0.10 M catechol before transfer to H&z were similar to those detected with guaiacol. There were two or three upper bands, three closely migrating midbands, and two fast moving lower bands present in sample gels of distilled water, Tris-HCI, and sodium borate extracts (Fig. 3). For both cultivars, band patterns were qualitativeIy similar for all environments

(c, f).

and showed evidence of increases or decreases in activity of existing isoenzymes, Exceptions concerning qualitative similarity of isoenzyme patterns seemed apparent, since the two lower bands on gel samples S1, SZ, Va, Vg, S, appeared to be absent. However, in these cases, the lower bands were present but were very light. The presence of these bands on gels assayed with guaiacol (Fig. 2) indicated these isoenzymes were either in very low concentration or were very substrate specific and did not influence the oxidation of catechol. For both cultivars and for all environments, slight differences existed between extractants. Comparisons of the band patterns with respect to the three extractants used, indicated that the borate buffer extracted greater quantities of peroxidase,

PEROXIDASES,

OXIDASES,

especiaIIy the slower migrating forms. Distilled water and Tris-HCI extracted basically the same kinds of isoperoxidases but the Tris buffer extracted higher quantities of upper forms. On the basis of equal dry weights, the degree of activity exhibited by the isoperoxid,ases with catechol was related to the extractant used, and the environment from which the samples were obtained. Greater differences in bands than those observed with guaiacol (Fig. 2) were evident in this system. The isoenzymes exhibked increased intensities during hardening, and lower intensities in summer and during dehardening, However, the VernaI (V,) sample seemed to be an exception on the basis of equal dry weight and equal amounts of protein, although borate sample gels appeared almost equivalent to the ( Va and Ss) December samples. This result was also noted in the guaiacol study (Fig. 2). Environment.aI differences in isoperoxidase activities were compIex when gels were compared on the basis of equal amounts of protein. For all extractants, Sonora isoenzymes ( S:{) generally exhibited higher intensities in December, as compared to summer samples, whereas peroxidases of Vernal winter samples ( Vl, V,) exhibited only slight increases in intensity in some bands and decreases in others. In fact, the Vernal (V,) samples apparently exhibited the lowest activity on the basis of equal protein, which could reflect either reductions in peroxidase components or a nonprefe.rentiaI increase in peroxidase with respect to other proteins, since total soluble protein content was highest in the V, sample. Isoperoridase activity of Vernal with catechol seemed to be different from that of Sonora on the basis of equal amounts of protein, While peroxidase components exhibited increased activity in Sonora (S,) sample gels, the highest intensity IeveIs in Vernal sample gels were present in the summer samples.

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71

Slight oultivar differences in individual isoenzymes were evident in the Va, S3 sample gels, but the greatest cultivar dif7erences were noted in summer sample gels, on the basis of equal dry weight and of equal protein. Intensity differences were minimized in the December samples (Va, Sa) although the two lower bands were more intense in Va and a midband change in Sa was also evident. Differences on gels of partially dehardened sampIes were also observed. Therefore, both cultivars apparently produced simiIar types of isoperoxidases in December as during the summer, however the activities of these individual components varied. Since both cultivars apparently produced qualitatively equivalent forms of isoperoxidase during summer, differences in peroxidase activity between cultivars probably resulted from quantitative differences in individual isoenzymes, or from differences in substrate specificity, or both. When results of Figs. 2 and 3 are compared, basically, the same isoperoxidase band patterns are present in summer samples of both cultivars, but intensities are greater for Vernal enzymes, reflecting a higher quantity of the same isoperoxidases. However, the higher isoenzyme intensities of VernaI samples could reflect greater substrate specificity of the Sonora isoperoxidases. Possibly, the peroxidase complement of VernaI can utilize either guaiacol or catechol more efficiently than the peroxidases of Sonora, Therefore, differences in intensity could result from a combination of quantitative and specificity differences. In relating these results to cold-tolerance several possible inferences development, can be made. First, the higher quantity of isoenzymes or reduced substrate specificity of the isoperoxidase complement of Vernal summer samples indicates a greater capacity of the cold-toIerant cultivar to utlhze various types of endogenous substrates in peroxidatic reactions. This capacity could

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be important to metabolism during periods of stress and especially during environmental changes which precede or are associated with cold-hardening. Since very little is known concerning the function of the individual isoenzymes it is d&cult to determine a functional relationship of each form to cold tolerance. Although each isoenzyme functions as a peroxidase it is not known how the isoperoxidases function in relation to IAA-oxidation, lignin biosynthesis, hydroperoxide degradation, etc. Thus, as environment changes, demands for certain components or elimination of other products may be critical and the cultivar having a greater abundance of those isoenzymes capable of utilizing more diverse substrates, or those isoenzymes necessary for the critical reactions, may have the metabolic advantage. Second, the intensity differences exhibited on gels may reflect a higher overall activity in Vernal at all times. Since differences in individual isoperoxidase activities between Vernal and Sonora samples appeared minimal in December, possibly an increased total peroxidase activity in the winter samples may not be as important as the rate at which the increase occurs. Gerloff et al. (14) reported that a

AND

WITHAM

cold-tolerant cultivar developed a maximum peroxidase activity sooner in autumn than a cold-sensitive cultivar. Thus, the differential rate of activity increase between cultivars during the critical hardening period could be more important metabolically as far as cold toIerance is concerned. But again, it is possible that an overall increase may not be as critical as the increased quantity and function of specific isoenzymes, and it is the functions of the individual isoperoxidases which must be determined to understand the roles of these enzymes in cold tolerance. Although the results of this study only include samples from summer and winter environments, the higher isoperoxidase activities already present in Vernal, during summer, could provide this cultivar with a better “start” when cold-hardening is initiated. Therefore, any additional increase in peroxidases, produced as more soluble protein is synthesized during hardening, may further increase the functional activities of specific isoenzymes which may be necessary for survival. Although isoperoxidase activities were equivalent in the December samples of both cultivars, Sonora had not developed any degree of tolerance greater

TIME (seconds) FIG. 4. Tissue assay of peroxidase activity present in extracts of unhardened, hardened, and partially dehardened Vernal and Sonora alfalfa crown and root tissues. Double-distilled water extracts equivalent to 5.0 mg dry weight of tissue were assayed with guaiacol as the hydrogen donor. Numbers 1-9 represent the following samples: 1,2-Vernal summer samples; 3,4Sonora summer samples; 5,6-Vernal December and February field samples, respectively; ‘?-Sonora December field samples; &g-Vernal and Sonora dehardening samples, respectively. Samples 2 and 4 were subjected to heavier clipping treatment. The pattern of peroxidase activity is representative of three separate experiments.

PEROXIDASES,

OXIDASES,

than that of summer samples, while VernaI had moderat+ hardened.

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73

comparing gels was difficult. Band patterns on gels of distilled-water or Tris-HC1 extracts were simiIar for both cultivars and Tissue Assay of Peroxidase with Guaiacol all environments, namely two to three upper bands, three closely positioned midAn assay of distilled-waler extracts of the bands, and two lower bands, but with the samples was performed with guaiacol as addition of another very fast migrating hydrogen donor. Protein levels can be exdiffuse band, which was apparent directly trapolated from reference to results of behind the dye band. The new band was Fig. 1. also apparent in gels of borate-buffer exThe results (Fig. 4) indicated differences tracts, which contained the highest quantiin total peroxidase activity associated with ties of the isoenzymes. cultivar and environment. Peroxidase acBand patterns obtained with distilledtivity was greater in winter samples 5, 6, water extracts and Tris-HCl extracts were and 7, than in respective summer samples similar, but in some cases the Tris-HCI. 1, 2, 3, 4, or partiaIIy dehardened samples buffer extracted slightly higher amounts of 8, and 9. Also, activity was highest in hardithe slowly migrating isoenzymes. Quantiest sampIes 5, and 6, and higher for Vernal tative changes were apparent with respect extracts 1, 2, 5, 6, and 8 than for correto environment or extractant and the addisponding Sonora extracts 3, 4, 7, and 9. tional band previously detected in Vernal Vernal extracts contained a higher quantity winter samples [Fig. 2, 5’4) was not deof total peroxidase than Sonora extracts tected with pyrogallol. and the greater reaction rates of Vernal On the basis of equal dry weight isoperoxidases support this. The rate differperoxidase activities increased in the winter ence was especially noticeable between samples, for all extracts of both cultivars, December samples 5 and 7, as well as beand decreased during dehardening. On the tween summer samples 1,2,3, and 4. When basis of equal protein, slightly higher band samples 5 and 7 were compared, soluble intensities were also observed in winter prot.ein levels were higher in Vernal than samples (Va, S,). However, the exception, in Sonora, so that the Sonora activity would again, was the Vernal sample (V,), since be less. However, levels of soluble protein gels containing equal amounts of protein (Fig. 1) were nearly similar or sIightly were lighter than corresponding gels of higher in the summer Sonora samples 3, December samples, and 4 compared to Vernal samples 1, 2, Cultivar differences in isoperoxidase acsuggesting a preferential increase in pertivities detected previously with guaiacol oxidases in Vernal during summer. Speand catechol, were not as great with pyrocificity differences also may have been gall01 since all isoenzymes were present in involved in the reactions so that isoperoxisummer and winter samples. Band intensidases of Sonora did not utilize guaiacol as ties of Vernal winter (V,) gels seemed efficiently as Vernal isoenzymes. equivalent to those of Sonora (&) gels on an equal dry weight and equal protein Localization of Peroxidases on Gels with basis, although some differences in lower Pyrogallol bands were noted between cultivars. However, the most interesting results were eviIsoperoxidases detected with pyrogallol (Fig. 5) resembled those of previous ex- dent in summer sample gels. If band patterns of summer sample gels incubated in periments involving guaiacol and catechol. pyrogaIlo1 (Fig. 5) are compared with Because the reaction product pulpurogallin those of Figs. 2 and 3, all isoperoxidases diffused into the tubes during photography,

KRASNUK,

74

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JUNG

AND

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WITHAM

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FIG. 5. Peroxidase zymograms after electrophoresis of soluble proteins in extracts of unhardened, hardened, and partially dehardened Vernal ( V) and Sonora (S ) alfalfa crown and root tissues. Gels were incubated in pyrogallol for 30 min before transfer to hydrogen peroxide. Environments from which samples were obtained (Nos. 1-5) were as follows: I-Summer conditions; &Summer conditions with frequent clipping; 3-Winter field conditions (December) ; P-Winter field conditions ( February) ; 5-Dehardening conditions in greenhouse. Peroxidases present in 4.0 mg dry weight of tissue (a, b, c) and 100 pg equivalents of protein (d, e, f) were separated by electrophoresis of double-distilled water extracts (a, d), 0.05 M Tris-HCl extracts (b, e), and 0.05 M sodium borate extracts (c, f).

(upper, mid, and lower bands) are evident in summer gels of both cultivars. Cultivar differences in reactions of isoenzymes detected with guaiacol or catecho were minimal with pyrogallol, although, with pyrogallol, a slightly higher intensity was also exhibited by Vernal summer gel samples. Several conclusions can be inferred from these results. First, both Vernal and Sonora apparently have a similar qualitative complement of isoperoxidases, as indicated by reaction with the triphenol pyrogallol. Second, the Vernal sample gels exhibit slightly higher band intensities, which may reflect higher quantities of these isoenzymes. Speculation as to substrate specificity differences of the isoperoxidases may be valid, since minimal specificity of the

isoenzymes of both cultivars towards pyrogall01 was exhibited in contrast to results observed with guaiacol and catechol. Therefore, apparently with higher quantities of isoperoxidases, as well as a minimal degree of substrate specificity of these isoperoxidases, Vernal is capable of a greater overall peroxidatic reaction rate than Sonora. This differential capacity between cultivars in the unhardened state could be important, as cold hardening begins in late summer or early autumn. The possible involvement of peroxidases in controlling endogenous hydroperoxide buildup during lipoxidolysis that may adversely affect membrane permeability [ 15, 24), in the oxidation of the growth regulator TAA (12, 13, 21, 26, 29)) and in the biosynthesis of

PEROXIDASES,

OXIDASES,

AND

hgnin matrix for cell walk (16, 34) suggests that the functions of all, or of specific isoperoxidases could be important during the period when hardening occurs. Thus, cultivar differences observed in isoperoxidase activities during summer could result in significant differences in metabolism or growth at the onset of hardening, as the plant adapts to impending low temperatures and reduced photoperiods. Localization of Polgphenol with L-DUPA

COLD

TOLERANCE

75

With L-DOPA as substrate isoenzyme patterns on gels of distiIled water and TrisHCI extracts were similar with three to four bands on upper areas of the gels, three midbands, and two lower (fast migrating) bands (Fig. 6). Separation patterns obtained with borate extracts resembled those of water and Tris-HCl samples, but were very diffuse. No qualitative differences in band patterns between cultivars or related to environment were evident, but slight quantitative differences were observed. Distilled water and Tris-HC! seemed to extract similar amounts of isoenzymes, but in some cases Tris-HC1 extracted a slightly higher amount of the upper forms. The b0rat.e buffer also extracted higher quantities of

Oxidase on Gels

Polyphenol oxidases have been implicated as regulators of phenolic substances that indirectly control activity of peroxidases (33,34).

1

2

3

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5

4

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FIG. 6. Polyphenol oxidase zymograms after electrophoresis of soluble proteins in extracts of unhardened, hardened, and partially dehardened Vernal (V) and Sonora (S) alfalfa crown and root tissues. Gels were incubated in L-DOPA for 1 hr. Environments from which samples I-Summer conditions; Z-Summer conditions were obtained (Nos. I-5) were as folIows: with frequent clipping; &Winter field conditions (December); 4-Winter field conditions (February) ; %Dehardening conditions in greenhouse. Polyphenol oxidases present in 4.0 mg dry weight of tissue (a, b, C) and 100 pg equivalents of protein (d, e, f) were separated by electrophoresis of double-distilled water extracts (a, d), 0.05 M Tris-HC1 extracts (b, e), and 0.05 M sodium borate extracts ( c, f ) .

76

KRASNLJK,

JUNG

abcdef

AND

WITHAM

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a

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FIG. 7. Indoleacetic acid (IAA) oxidase zymograms after electrophoresis of unhardened, hardened, and partialIy dehardened Vernal (V) and Sonora ( S ) alfalfa crown and root tissues. Gels were incubated in a reaction mixture containing JAA for 4 hr and were developed in DMACA for 1 hr. Environments from which samples were obtained (Nos. l-5) were as follows: l--Summer conditions; 2-Summer conditions with frequent clipping; 3-Winter field conditions ( December); 4-Winter field conditions (February) ; 5-Dehardening conditions in greenhouse. Indoleacetic acid oxidases present in 4.0 mg dry weight of tissue (a, b, c), and 100 pg equivalents of protein (d, e, f) were separated by electrophoresis of doubledistilled water extracts (a, d), 0.05 M Tris-HCl extracts (b, e), and 0.05 M sodium borate extracts (c, f).

the slower migrating enzymes present in upper areas of the gels. On the basis of eqnivalent dry weights, some individual band increases were visible in winter gel samples of Vernal (V,, V,) and Sonora (Sa), as compared to gels of summer and partially dehardened samples, On the basis of equal amounts of protein, evaluations were more complex. Lower bands on gels of VernaI winter samples generally were darker and more resolved than those of the summer samples, whereas less change was noted for upper and midband forms. Isoenzyme activities on gels of Sonora sampies folIowed a similar trend, but intensities of some bands on summer sample gels seemed to be either equivalent or darker

than those of the winter samples. Isoenzymes of Sonora generally showed higher activities than corresponding Vernal components, which were especiahy noticeable in summer on an equal dry weight and equal protein basis. Both varieties exhibited some changes in polyphenol oxidase isoenzymes which were influenced by environment. However, since there are changes in individual components, it is difficult to speculate whether or not an overall increase occurred during hardening. The slight changes that were detected in the winter sampIes could be important since the differences in activity of the various isoenzymes could reflect the demand for various metabolic products, such as lignins, flavi-

PEHOXIDASES,

OXIDASES,

nols, and alkaloids, since polyphenol oxidase has been linked to biosynthesis of these compounds ( 23 ) . Localization on Gels

of Indoleacetic

Acid Oxidase

After electrophoresis of various alfalfa sampIes, IAA oxidase activity was assayed on the gels. Since bands were too Iight to be photographed, diagrams were made. The results (Fig. 7) show the separation patterns of IAA oxidases extracted. Isoenzyme patterns detected on gels of distilled water and Tris-HC1 extracts were similar for both cultivars from all environments. The patterns on gels of borate extracts resembled those of other extractants except for the presence of an additional, fast moving band. Also, another faint fast migrating isoenzyme was detected on gels containing borate extracts of partialIy dehardened Sonora (S,). Other samples (Sa, S5, Vq, V5) with quaIitative differences in band patterns on upper areas of gels of the

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TOLERANCE

77

borate samples could have been due to greater resolution. Cultivar or environmental differences were minimal except possibly in the band patterns of partially dehardened samples of V, and Ss. No quantitative estimates could be performed accurately because of fading, but bands on upper areas of all gels were generally darkest. Tissue Assay uf Indoleacetic

Acid Oxidase

Since no quantitative estimate of IAA oxidase activity could be determined from the previous study, an assay was performed with IO-mg dry weight equivalents of distilIed-water extracts. Differences in IAA oxidase activity between cultivars were apparent (Fig. 8), however, Vernal samples I, 2, 5, 6, and 8 were more effective in destroying IAA than corresponding Sonora sampIes 3, 4, 7, and 9. Indoleacetic acid oxidase activity in both cultivars increased in winter (samples 5, 6, and 7) and decreased during dehardening (sampIes 8 and 9). A slightly higher

3

TIME

[minutes)

FIG. 8. Tissue assay of indoleacetic acid (IAA) ox&se activity present in extracts of unhardened, hardened, -and partially dehardened Vernal and Sonora alfalfa crown and root tissues. Double-distilIed water extracts equivalent to 10 mg dry weight of tissue were added to reaction mixtures containing IAA and the residual IAA remaining at different times was determined. Numbers 1-9 represent the following samples: &e-Vernal summer samples; 3,P Sonora summer samples; S,B-Vernal December and February winter field samples, respecand Sonora dehardening samples, retively; ‘i-Sonora December field sampIe; &S-Vernal spectively. Samples 2 and 4 were subjected to heavier clipping treatment. The pattern of the IAA oxidase activity is representative of three sparate experiments.

7s

KRASNUK,

JUNG AND WITHAM

activity was also noted in summer sampIes as a result of frequent clipping treatments. The increased IAA oxidase activity in alfalfa during hardening agrees somewhat with the results of Bolduc et al. (l), who found a tenfold increase in IAA oxidase activity in wheat after a 40-day cold treatment. Although reactions of all extracts eventuaIly went to completion, the rate at which completion was finally attained was greater for Vernal. Indoleacetic acid oxidase activity inversely followed growth rate to a great extent. Enzyme activity was lower in samples 1, 2, 3, 4, 8, and 9 during periods of high growth rate and higher in samples 5, 6, and 7 were growth rate was lower. The exceptions to this observation were the Vernal summer samples 1 and 2. The functional interreIationship between IAA oxidase and peroxidase activities may explain to some extent the inverse relationship between growth rate and cold tolerance. Peroxidase activity (Fig. 4) with respect to cultivar and environment paralIeled IAA oxidase activity (Fig. 8). Since IAA oxidation is thought to be catalyzed by peroxidases (2, 15, 16, 27, 32, 35) the IAA oxidase reactions (Figs. 7, 8) were probabIy due to peroxidases. Thus, the hardy cultivar Vernal, which contained greater quantities of peroxidase (Figs. 2, 3, 4) consequently exhibited greater IAA oxidase activity than Sonora. The differential capacity to control growth rate may be the result of inherent differences between cultivars in producing quantities of specific peroxidases which control endogenous levels of IAA. The initialIy higher peroxidase and IAA oxidase activities in Vernal summer samples, as compared to Sonora, indicates that any additional increases in activity, during hardening, which are related to increased enzyme quantity could give the coId toIerant cultivar a metabolic advantage over the cold sensitive cultivar. Therefore, increases in enzymatic

components associated with increases in soluble proteins during hardening would accentuate further the activity differences between cultivars. SUMMARY

Two cultivars of alfalfa (Me&ago sativa L. ), coId-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to investigate the relationship of soluble proteins and enzyme activity and solubility characteristics to cold tolerance. Evaluations of cold-tolerance levels developed in crown and root samples were compared with results of soluble protein analyses and were in agreement with previously reported observations. Soluble protein content was associated with increases in cold tolerance and related to the environment from which samples were obtained; however, the degree of protein differences within samples of the same cultivar as well as between the two cultivars seemed to be influenced by the type of extractant used. Polyacrylamide disc gel electrophoresis of the extracted soluble proteins was performed on the basis of equal dry weights and equal quantities of protein. Amido black straining of gels indicated mainly quantitative changes and slight qualitative differences in component bands influenced by environment and extractant. Gels assayed for peroxidase, polyphenol oxidase, and indoleacetic acid oxidase enzymes exhibited mainly quantitative differences in constitutive isoenzyme components of both cultivars which were associated with environmenta changes. Enzyme activities generally increased in winter, as cold tolerance and soluble protein content increased, and decreased during dehardening. The few qualitative differences in isoenzyme bands that were detected, appeared to be influenced by cuhivar, environment, extractant, or substrate specificity difFerences.

PEROXIDASES,

OXIDASES,

Variation in isoenzyme components between cultivars was maximum in summer samples, and minimum in winter samples, suggesting that overall reaction rates or activities of individual isoenzymes, preceding or during hardening, could be a limiting factor in cold-tolerance development. REFERENCES 1. Bolduc, R. J,, Cherry, J. H., and Blair, B. 0. Increase in indoleacetic acid oxidase activity of winter wheat by cold treatment and gibberelIic acid. Plant Physiol. 45, 481464 (1970). 2. Bula, R. J., Smith, D., and Hodgson, H. J, Cold resistance in alfalfa at two diverse latitudes. Agron. J. 48, 153-156 (1956). 3. Burke, R. Nature of indoleacetic acid oxidase preparations from Parthenocissus tricmpidata crown-ga!l tissue cultures. Pennsylvania State Univ. M.S. thesis ( 1972). 4. Burstone, ?vl. S. “Enzyme Histochemistry,” pp. 465-466. Academic Press, New York, 1962. 5. Chance, B., and Maehly, A. C. Assay of catalases and peroxidases. In “Methods of Enzymology” (S. P. Colowick and N. 0. KapIan, Eds.), Vol. II, pp. 764-775. Academic Press, New York, 1955. 6. Coleman, E. A., Bula, R. J., and Davis, R. L. EIectrophoretic and immunological comparisons of soluble root proteins of Medicago sat& L. genotypes in the coId hardened and nonhardened conditions. Plant Phgsiol. 41, 1681-1685 (1966). 7. Davis, B. J. Disc electrophoresis. II. Method and application of human serum proteins.

Ann. N.Y. Acad. SC& 121, 404-427 (1964). 8. Dexter, S. T., Tottingham, W. E., and Graber, L. F. Investigations of the hardiness of plants by measurement of electrical conductivity. Plant Physiol. 7, 63-78 ( 1932). 9. Faw, W. F. Electrophoretic studies of soluble proteins in alfalfa (Me&ago s&vu L.) in relation to cold hardiness. West Virginia Univ. Ph.D. thesis ( 1969). IO. Faw, W. F., and Jung, G. A. Electrophoretic protein patterns in relation to low temperature tolerance and growth regulation of a!falfa. CryobioZogy 9, 548-555 (1972). 11. Frenkel, C. Involvement of peroxidase and indole3-acetic acid ox&se isozymes from pear, tomato and blueberry fruit in ripening. Pkmt Physiol. 49, 757-763 ( 1972). 12. Galston, A. W., Bonner, J., and Baker, B. S.

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COLD

TOLERANCE

Flavoprotein and peroxidase as components of the indoleacetic acid oxidase system of peas. Arch. Biochem. Biophs. 42, 456-470 (1953). Galston, A. W., Lavee, S., and Siegel, B. Z. The induction and repression of peroxidase isozymes by 3-indole acetic acid. In “Biochemistry and Physiology of Plant Growth Substances” (G. Setterfield and F. Wightmann, Ed.), pp. 455-472. Runge Press, . Ottawa, 1968. Gerloff, E. D., Stahmann, M. A., and Smith, D. Soluble proteins in alfalfa roots as related to cold hardiness. Plant Physiol. 42, 895-899 ( 1967). Hall, T. C., McLeester, R. C., McCown, B. H., and Beck, G. E. Enzyme changes during deacclimation of wilIow stem. Cryobiology 7, 130-135 (1970). Higuchi, T. Biochemical studies o$ lignin formation. III. Physiol. Plant. 10, 633-648

(1957). 17. Jung, G. A., Shih, S. C., and Shelton, D. C. Influence of purines and pyrimidines on cold hardiness of plants. III. Associated changes in soluble protein and nucleic acid content and tissue pH. Pkmt Physiol. 42, 1653-1657 ( 1967) _ 18. Jung, G. A., Shih, S. C., and Shelton, D. C. Seasonal changes in soluble protein, nucleic acids, and tissue pH related to cold hardiness of alfalfa. Cryobiolofiy 4, II-16 (1967). 19. Jung, G. A., and Smith, D. Trends of cold resistance and chemical changes over winter in roots and crowns of alfalfa and medium red clover. I. Changes in certain nitrogen and carbohydrate fractions. Agron. J. 53, 359-364 ( 1961). 20. Korovin, A. I., and Barskaya, T. A. Effect of soil temperature on respiration and activity of oxidative enzymes of roots in cold resistant and thermophilic plants. Sooiet Plant P&&l. (Trans. from Fiziol F&t.) 9, 331333 (1962). 21. Lipetz, J., and Galston, A. W. Indole acetic acid oxidase and peroxidase in normal and crown-gall tissue cultures of Putihenocissu~ tricuspiduta. Amer. J. Bat. 46, 193-196 (1959). 22. Lowry, 0. H., Rosebrough, N. J., Farr, L. J., and Randall, R. J. Protein measurement with the Folin phenol reagent. I. Biol. C&m. 193,265-275 (1951). 23. Mason, H. S. Comparative biochemistry of the phenolase complex. Adv. Enzymol. 16, 106 184 ( 1955).

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24. McCown, B. H., Hall, T. C., and Beck, G. E. Plant leaf and stem proteins. II. Isozymes Phgsiol. and environmental change. I%& 44,210-216 (1969). 25. McCown, B. H., McLeester, R. C., Beck, G. E., and Hall, T. C. Environment-induced changes in peroxidase zymograms in the stems of deciduous and evergreen plants. Cq/ohioZogy 5, 41&412 ( 1969). 26. McCune, D. C. Multiple peroxidases in corn. Ann. N.Y. Aced. Sci. 94, 723-730 ( 1961). 27. Meudt, W. J., and Gaines, T. P. Studies on the oxidation of indole-3-acetic acid by peroxidase enzymes. I. Calorimetric determination of indole-3-acetic acid oxidation products. Plant PhysioE. 42, 1395-1399 (1967). 28. Ornstein, L. Disc electrophoresis. I. Background and theory. Ann. NY. Aced. Sd. 121, 321349 ( 1964). 29. Ray, P. M. The destruction of indoleacetic acid. III. Relationships between peroxidase action and indoleacetic acid oxidation. Ad. Biochem. Biophys. 87, 19-30 ( 1960). 30, Roberts, W. D. A. A comparison of the peroxidase isozymes of wheat plants grown at 6°C and 20°C. Canad. J. Bat. 47, 26% 265 ( 1969). 31. Shih, S. C., Jung, G. A., and Shelton, D. C. Effects of temperature and photoperiod on

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metabolic changes in alfalfa in relation to cold hardiness. Crop Sci. 7, 385-389 ( 1967). Steward, F. C., and Barber, J. R. The use of acrylamide gel electrophoresis in the investigation of the soluble proteins of plants. Ann. N.Y. Acud. Sci. 121, 525-531 (1964). Stonier, T., Singer, R. W., and Yang, H. M. Studies on auxin protectors. IX. Inactivation of certain protectors by polyphenol oxidase. Pht Phr~sicd.46,454457 ( 1970). Stonier, T., and Yang, H. M. Studies on auxin protectors. XI. Inhibition of peroxidase-catalyzed oxidation of glutathione by auxin protectors and o-dihydroxyphenols. Plant Physid. 51,391-395 ( 1973). Sysoev, A. F., and Krasnaya, T. S. Changes in activity of certain oxidative enzymes in wheat seedlings during low temperature hardening. Dokl. Akad. Nauk. USSR 173, 472-474 ( 1967 ) . Tang, Y. W., and Bonner, J. The enzymatic inactivation of indole acetic acid. I. Some characteristics of the enzyme contained in pea seedlings. Arch. Biochem. 13, 11-25 (1947). Witham, F. II., and Gentile, A. C. Some characteristics and inhibitors of indoleacetic acid oxidase from tissue cultures of crowngall. J. Exp. Bat. 12, 188-198 (1961).