Immunochemical Studies on Arachis hvpogaea ProteinsWith Particular Reference to the Reserve Proteins.

II. Protein Modification During GerminationJ. Daussant¾, N. J. Neucere2, and E. J. Conkerton'

Southern Utilization Research and Development Division, Agricultural Research Service,United States Department of Agriculture, New Orleans, Louisiana 70119

Received September 16, 1968.

Abstract. a-Conarachin and a-arachin are metabolized differently during germination.The amount of a-conarachin decreases without the appearance of antigenic intermediary com-pounds. a-Arachin retains its original antigenic structure and beoomes more anodic duringgermination, but a number of intermediary proteins, differing widely in electrophoretic mobility,form up to 14 days germination. The electrophoretic shift of a-arachin, detectable after 1 dayof germination, might be caused by progressive deamidation of glutamine and asparagine.Decreases in amide content of protein extracted after germination support the hypothesis andsuggest that a-arachin provides the first source of ammonia to the new seedling.

New antigenic proteins are synthesized in both roots and cotyledons during germination.Some appear organ specific, whereas others are identical in both the cotyledons and the roots.Appearance and disappearance of each new protein is unique with respect to period of germi-nation and hence might correspond to specific events of germination.

It is generally assumied that the reserve proteinisof the seed are hydrolyzed during germiiniationi pro-viding amlino acids for the synthesis of niew proteins(2). a-Arachin and a-colnarachin, the major reserveproteins of classic arachinl and conarachin in A4rachishypoga,ea, progressively decrease during germination(4). They are located in different subcellular frac-tions and have different characteristics (3). Thissuggests different metabolic functions and modifica-tions during germination. The aim of this study wasto follow the fate of these proteins during germina-tion. The results suggest a particular metabolic rolefor a-arachin.

Materials and Methods

Germtinatioz. Seeds of Arachis hypogaea weregerminated in vermiculite at 220 in lots of 20, eaclhseed weighing 1 ± 0.1 g. Sample lots were with-drawn at 2, 5, 8, 9, and 14 days of germlinationl.Germination was performed in the light.

Extraction. Twenty cotvledons from each germli-nated sample were extracted with 60 ml of buffer;2 g of roots were extracted with 10 ml of buffer asdescribed previously, (3).

Immntunochemnical Methods. Double diffusion ac-cording to Ouchterlony (12), immunoelectrophoretic

1 Present address: Service de Chimie Microbienne,Institut Pasteur, Paris 15e, France.

2 Present address: Oilseed Crops Laboratory, South-ern Utilization Research and Development Division.

anialysis (IEA) according to Grabar and \NVilliams(7), and iminmunie serumii absor,ption technique as de-scribed previously wxere employed (3). Immunesera used were: aniti-arachin, anti-a-conarachlin,anti-cotyledon of ungerminated seeds, anti-cotyledonand anti-root of 5-day old seedlings. Immun,e seraagainst the seedlings Aere prepared by the methodused for immune serum anti-cotyvledon of ungermi-nated (quiescent) seeds (3).

An ide Nitrogen, Determinationi. Extracts iso-lated from peanuts before and during germinationwvere adjusted, on the basis of protein contents !(11),to contain equivalent amounts of protein. Portionscontainiing fromii 3 to 10 mlg of protein were placedin test tubes (150 X 25 mm). The required volumeof 2 N HCl was added, (1 ml of acid per mg protein),and a micromagnetic stirring bar placed in each tube.The tubes were stoppered. heated at 1000 for 3 hr,then cooled immediately to room temperature (10).For determinatioln of amiiide nitrogein, a 2 ml portionof the acid solution was analyzed by the micro-Kj eldahl technique, using appropriate reductions inquantity of reagents. Total nitrogen content wasdetermined on an unhydrolyzed aliquot by the Kjel-dahli method (14). Results are expressed as thepercent of total nitrogen that is amide nitrogen.

Results

Estimations of Protein Concentrationzs. The re-sults reported in table I show that the content ofextractable protein per cotyledon decreases as germi-nation progresses. This decrease in protein includes

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DAUSSANT ET AL.-IMMUNOCIIEMICAL STUDIES OF THE PEANUT PROTEINS

Table I. Relativ)e Amiiounts of Total Proteils, a-Arachint, al- and a2,-Conarachin Extracted From thc ArachisCotyledons at Various Stages of Germiination

Limit dilutions where precipitin band is still visible

Germination Protein concn. a-arachin a1-conarachin a2-conarachindays ilng/ml

0 57 1280 80 402 46 1280(-) 80 405 30 320 40 or 20 40

(or 640)9 20 160 20 or 10 20 or 4014 12 160 10 20

Table lI. Percentage of Proteint Amiiide Nitrogen ittCotyledonary Extracts

Days Amide nitrogengerminated % of total N

0 12.52 ...5 11.48 8.9

14 7.0

both a-arachliii and a,-conarachin. In contrast,a2-conarachin is not notably affected during germi-nation.

A nide Nitrogeni. Reproducible determinationswere not obtained with different samples germinatedfor 2 days-1 sample slightly decreased, whereasanother slightly increased. Until additional materialis available,, these discrepancies must be consideredwithin experimental error. However, there is aprogressive decrease in amide nitrogen content ofthe extracted proteins after 2 days of germination(table II).

a-Arachin. Particular features concerning themodification of a-arachin upon germination wereobserved. IEA of cotyledonary extracts of seedsbefore and during germination using anti-arachinimmune serum shows 2 characteristic changes in theelectrophoretic behavior of a-arachin (Fig. 1).First, the molecules become more anodic upon germi-nation, with a notable shift in electrophoretic mobilityafter 1 day of germination. Second, a-arachin ap-pears composed of molecules with the same anti-genicity, but with varying electrophoretic mobilitiesduring germination. This is shown by the elongatedprecipitin band which occurs in IEA of the samplesgerminated 2, 5, and 9 days. After 14 days a-ara-chin becomes more homogeneous by electrophoreticanalysis. It is noteworthy that the electrophoreticmobility of the a-arachin contaminant previouslydescribed (3) also shows a small shift, but ratherhomogeneous, as germination proceeded. Appar-ently, this elongation of the precipitin band is uniqueto a-arachin and reaches a maximum before the fifthday of germination (Fig. 1-3).

a-Arachin retains its original antigenicity upongermination. A mixture of 2 cotyledonary extractsfrom quiescent seeds and from 14-day old seedlings,was analyzed by IEA. The 2 antigenic populationsof a-arachin form 2 precipitin bands in differentareas of the migration field. The coalescence ofthese precipitin bands at the point of junction reflectthe antigenic identity of a-arachin in both quiescentand germinating seeds (Fig. 1-6).

a-Conarachlin. IEA of cotyledonary extracts ofuingernminated and germinated seeds showed that theprecipitin band corresponding to a,-conarachin de-creases in intensity as germination progresses. Incontrast to a-arachin, no change in the electro-phoretic mobility of a1 and a.,-conarachin was de-tected.

New Antigenic Proteinis in Roots. Ainalyses ofproteins extracted from roots after 5 days are shownin Fig. 2. Some of these proteins apparently havean antigenic structure similar to that of proteins inmature cotyledons since extracts from ungerminatedcotyledons react with the anti-root immune serum(Fig. 2-1). Fig, 2-2 corresponds to the 5-day rootextract. Analyses of proteins having an antigenicstructure different from those of cotyledonary pro-teins of mature seeds are shown in Fig. 2-3. Anti-root immune serum below 3 was previously absorbedwith a cotyledonary extract of mature seeds. Thevalidity of the absorption technique is verified by thefact that different quantities of cotyledonary extractdo not show any precipitin bands when reacted withthe absorbed immune serum (Fig. 2-4).

The appearance and disappearance of new rootproteins during growth is shown by IEA of samplesextracted 2, 5, 9, and 14 days of germination (Fig.2-5,6, /, 8). The new proteins do not appear anddisappear at the same time; for example, the pre-cipitin 'band consisting of 3 arcs forming a continuousline occurs first and subsides last during growth ofthe root. These 3 joined arcs indicate the existenceof 3 populations of molecules that have the sameantigenic structure but different electrophoretic mo-bilities. The most anodic constituent, which seemsto be t'he major one, persists the longest in the grow-ing root.

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PLANT PHYSIOLOGY

I

2

3I+

4

5;

6

FIG. 1. IEA of cotyledonary extracts of quiescent seeds (1), of seeds germlinated 2 days (2), 5 days (3), 9 days(4), 14 days (5), and a mixture of cotyledonary extracts of nongerminated seeds and of seeds germinated 14 days(6), with anti-arachin as the immule serumiii.

New Antigenic Proteins in Cotyledons. Identicalexperiments designed to study the eventual appear-ance of new antigenic proteins in cotyledons duringgermination and growth of the seedlings are reportedin Fig. 3. Again, as in the roots, it is found thatthe cotyledons, too, are the site of synthesis of pro-teins whose antigenic structures do not occur incotyledonary proteins of mature seeds. This isdemonstrated by the appearance of precipitin linesof samples taken 5 days after germination usinganti-cotyledonary immune serum previously absorbedwith a cotyledonary extract of mature seeds (Fig.

3-3). Again, it appears that occurrence and dis-appearance of cer-tain proteins is not simultaneousfor all components (Fig. 3-5, 6, 7, 8).

Direct comparisons between the new antigenicproteins in cotyledons and in roots were not made.Nevertheless, the continuous band of 3 arcs thatform in both extracts suggests the presence of thesame structural proteins in both organs. The shapeand the position of the strong precipitin band remain-ing at the origin after IEA in both extracts furthersuggests that identical proteins *exist in both rootsand cotyledons at a certain stage of growth.

4832

. 0 0 0 .

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DAUSSANT ET AL.-IMMUNOCHEMICAL STUDIES OF THE PEANUT PROTEINS

maintain their initial antigenic specificity. Thissuggests that the modification might be caused byarachain, a proteolytic "trypsin like" enzyme foundin Arachis by Irving and Fontaine (8).

Electrophoretic shifts toward either the anode orthe cathode have been reported for other proteinsduring germination (5, 6, 9). These shifts could beattributed to modifications of antigenically unimpor-tant sites on the molecule. A loss of basic residues

3 that are not involved in the antigenic specificitv ofa-arachin may cause this shift. For example, pro-gressive deamidation of glutamine or asparagine

4 would make the protein more acidic. .a-Arachin isknown to have a high content of glutamic acid(21/100) and of aspartic acid (16/100) part of whichmay be in the amide form (1). Deamidation wouldresult in an anodic shift when electrophoresis isconducted at pH 8.2. Hence, this phenomenon cotuld

6

7

8

FIG. 2. IEA of cotyledonary extracts of mature seeds(1), of extracts of roots removed 5 days after the be-ginning of germination (2 and 3) is shown using thecorresponding antiroot immune serum (trough below 1)and using this serum previously absorbed with the coty-ledonary extract of mature seeds (trough below 3).Wells in (4) were filled after electrophoresis with dif-ferent quantities of the cotyledonary extract of matureseeds to check the absorption technique. IEA usingthe preceding absorbed immune sera of extracts of rootsremoved 2 days (5), 5 days (6), 9 days (7), and 14days (8) after the beginning of germination.

0..... ... ............... ............ .. ......I. .......

.

Discussion

Semiquantitative evxaluations showed that a1-con-arachin and a-arachin disappear during germination,confirming results of Dechary et al. (4). The dis-appearance of a2-conarachin was not as obvious,hence, a2-conarachin cannot be considered a reserveprotein in the category of a1-conarachin and at-ara-chin.

at-Arachin is metabolized differently from theother cotyledonary proteins during germination. Theanodic shift in electrophoretic mobility and the elon-gation of its precipitin band is evident from the firstto nearly the tenth day of germination, A similaranodic shift for ce-arachin was induced by trvpsin(3). In both cases the final antigenic constituents

- -.1

7

8

FIG. 3. IEA of cotyledonary extracts of mature seeds(1), of seeds 5 days after the beginning of the germination(2 and 3), is shown using the anti-cotyledon of ger-minated seeds as the immune serum (trough below 1),and with immune serum previously absorbed with thecotyledonary extracts of mature seeds (trough below3) Wells in (4) were filled after electrophoresis withdifferent quantities of cotyledonary extracts of matureseeds to check the absorption technique. IEA usingthis absorbed immune serum is shown of cotyledonaryextracts from seeds germinated 2 days (5), 5 days (6),9 days (7), and 14 days (8).

0_

..

_ng__s ..

O

0.. ... ........

2

3

4

5

6

MINWA.W.=i......m---m ,.Mll .......1-1770 .. IVIRPM

483

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PLANT PHYSIOLOGY

be ascribed to varying degrees of deamidation duringgermination. This hypothesis is supported in firstapproximation by the decrease in amide nitrogencontent during germination. These results suggestthat the reserve proteins not only provide amino acidsfor -the synthesis of new protein, but also thata-arachin, in particular, may be the first source ofammonia for the new seedling.

The existence of organ specific proteins in rootsis shown by the presence of antigens with uniquestructures as reflected by their antigenic specificities.The proportions of these new proteins in roots varvduring germination. This constitutes further supportfor the idea of Steward et al. (13) that proteins arecharacteristic for different stages of developmentwithin an organ.

Some of the root proteins are antigenicallv relatedto those of the ungerminated seed. This suggeststhat certain proteins synthesized during maturationof the seed are again synthesized in new organs ofthe seedling during germination and growthl. Theexistence of antigenically identical proteins in dif-ferent organis has also been reported for Vicia faba(5).

The occurrence of new antigens in the cotyledonduring germination and growth provides anotherexample of protein synthesis in the storage organs ofthe seed (15 ). Some of these new proteins seemidentical to those synthesized in the roots.

Acknowledgments

The authors thank Dr. Robert L. Ory for valuablediscussions, W. K. Bailey for furnishing the seed, JackBergquist for the photography, and W. B. Carney fortechnical assistance.

Literature Cited

1. BLOCK, R. AND D. BOLLING. 1951. SummaryTables. In: The Amino Acid Composition ofProteins and Foods. 2nd ed., C. C. Thomas, pub-lisher. Springfield, Illinois. p 491.

2. DANIELSON, C. E. 1956. Plant Proteins Ann. Rev.Plant Physiol. 7: 213-36.

3. DAUSSANT, J., N. J. NEUCERE, AND L. YATSU. 1969.Immunochemical studies on Arachis hypogaea pro-teins with particular reference to the reserve pro-teins. I. Characterization, distribution, and prop-erties of a-arachin and a-conarachin. Plant Phy-siol. 44: 471-79.

4. DECHARY, J. M., K. F. TALLUTO, W. J. EVANS, W.B. CARNEY, AND A. M. ALTSCHUL. 1961. a-Conarachin. Nature 190: 1125-26.

5. GHETIE, V. AND L. BUZILA. 1962. Immunologicalinvestigations of seed germination of horse beans(Vicia faba). St. Cerc. Biochem. 5: 541-50.

6. GRABAR, P. AND J. DAUSSANT. 1964. Study ofbarley and malt amylases by immunochemicalmethods. Cereal Chem. 41: 523-32.

7. GRABAR, P. AND C. A. WILLIAMS. 1953. Methodepermettant l'etude conjugee des proprietes electro-phoretiques et immunochimiques d'un melange deproteines. Application au serum sanguain Bio-chim. Biophys. Acta 10: 193-94.

8. IRVING, G. W. AND T. D. FONTAINE. 1945. Puri-fication and properties or arachain, a newly discov-ered proteolytic enzyme of the peanut, Arch. Bio-chem. 6: 351-64.

9. Ki.oz, J., A. TURKOVA, AND E. KLOZOVA. 1966.Proteins found during maturation and germinationof seeds of Phaseolus vulgaris L. Biologia Plan-tarum (Praha) 8: 16473.

10. LEACH, S. J. AND E. M. J. PARKHILL. 1955. In-ternational WTool Textile Research Conference Pro-ceedings (Australia). C92-C1O1.

1 1. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, ANDR. G. RANDALL. 1951. Protein measurement withthe Folin-phenol reagent. J. Biol. Chem. 193:265-75.

12. OUCHTERLONY, 0. 1949. Antigen-antibody reac-tions in gel. Acta Pathol. Microbiol. Scand. 26:507-15.

13. STEWARD, F. C., R. F. LYNDON, AND J. T. BARBER.1965. Acrylamide gel electrophoresis of solubleplant proteins: A study on pea seedlings in rela-tion to development. Am. J. Botany 52: 155-64.

14. STEYERMARK, A. 1951. Quantitative organic mi-croanalysis. The Blakiston Company, New York,New York. p 134.

15. VARNER, J. E. 1965. Seed development and ger-mination. In: Plant Biochemistry. J. Bonner andJ. Varner, eds. Academic Press, New York. p763-92.

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