Incorporation of lysine-C14 into the developing grain of wheat

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 104, 73-78 (1964)

Incorporation of Lysine-C ~4 into the Developing Grain of Wheat ~ J O H N M. L A W R E N C E AND D. R. G R A N T 2

From the Department of Agricultural Chemistry, Washington State University, Pullman, Washington Received June 5, 1963 Uniformly labeled L-lysine-C~4(5/~c. total, 6.6 X l0 s ~c. per mole) was injected into the top internodes of wheat stems. Injection 20-24 days after flowering (early dough stage) resulted in ripened wheat in which 46-49% of the radioactivity was recovered. About a quarter of the injected tracer was found in the flour fraction. A little over half the total seed C 14was contributed by lysine. About half the total flour activity and about 60% of the activity in the bran fraction was in lysine. Flour lysine and bran lysine had specific activities of about 40,000 and 50,000 uc. per mole, respectively. Injection at a late dough stage of development of the grain resulted in much lower deposition of radioactivity. Quantitative analyses for the free amino acids of total wheat stem juices from the top in~ernode at three times during wheat development showed the presence of considerable amounts of lysine along with other expected amino acids, but no diaminopimelic acid. This finding, together with the relatively high specific activity of wheat lysine following lysine-C 1. injection, suggests that lysine in wheat grain may derive from preformed lysine delivered to the head by way of the stem. INTRODUCTION The nutritional inadequacy of wheat as a source of food protein is known to be due to its relatively low lysine content (1). A low lysine content is characteristic of the seed protein of the Gramineae family (2). Thus, the means by which lysine is produced in the wheat grain and its function in the germinating seed are questions of much interest. McConnell and his associates (3, 7) have developed a technique for studying the incorporation of radioactive-labeled compounds and their metabolic derivatives into wheat seeds by injecting the labeled compound into the stem during maturation of the wheat. They have used the method to study the metabolism in wheat of compounds which serve as precursors of lysine in other organisms (4). Carbon-14 from a-amino1Scientific paper No. 2369, Washington Agricultural Experiment Stations, Pullman, Project No. 1554. 2 Present address: Department of Chemistry, University of Saskatchewan, Saskatoon, Saskatchewan.

adipic acid-6-C 14 was not incorporated into the lysine of the grain proteins to any significant extent (5), but the wheat plant did produce considerable labeled lysine from a,a'-diaminopimelic acid-l,7-C I* (6). The capability apparently resides in the kernel itself. There is also the possibility that lysine m a y be delivered to the wheat spike preformed and incorporated directly into the seed protein. The work reported here shows that when lysine-C 14 is injected into wheat stems, C 14 is incorporated into the wheat protein very efficiently. About half of the protein C I4 is in the form of lysine of much higher specific activity than a n y other amino acid, MATERIALS AND METHODS PRODUCTION OF LABELED W H E A T FRACTIONS

A club wheat (Triticum compactum var. Omar) growing in the field flowered between June 17 and 23, 1960. On July 12, when the wheat was considered to be in an "early dough" stage, and again on July 18, with the wheat in a "late dough" stage, stems were injected in the top internode with 73

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LAWRENCE AND GRANT

uniformly labeled L-lysine-C ~4 (50 ~1. containing 0.11 ms. lysine and 5 uc. of C ~4) by t h e methods of McConnell and co-workers (3, 7). Five of these plants, t a k e n for study, are described in T a b l e I. All were h a r v e s t e d on August 2, h a v i n g a t t a i n e d m a t u r i t y a b o u t a week earlier. The grain was separated from the chaff b y h a n d and was tempered to a b o u t 16% m o i s t u r e b y e q u i l i b r a t i o n in a closed vial. The germ was dissected from each kernel w i i h a small sharpened spatula. After drying in a desiccator t h e combined germs were ground in a mortar. A flour and a b r a n fraction were prepared from t h e degermed grain of each head of wheat by repeated grinding in a mort a r and sifting t h r o u g h No. 20 and No. 54 wire sieves. T h e w h e a t was first ground 2 m i n u t e s and sifted 2 minutes. T h e operation was repeated three times more on t h e material remaining on the sieves, grinding for 3, 4, and 7 m i n u t e s and sifting for 2 m i n u t e s each time. T h e material passing t h r o u g h t h e No. 54 sieve was combined and considered flour. T h e r e m a i n d e r was collected as b r a n a n d a f t e r desiccation was reground in a dental pulverizer. The average yield of the t h r e e fractions was 79% flour, 19% bran, and 2% germ. AMINO ACID DETERMINATIONS ON WHEAT FRACTIONS The amino acid c o n t e n t was determined for flour, bran, germ, and, in some cases, chaff frac-

tions from each of the collected w h e a t heads. Weighed portions were hydrolyzed w i t h 10% redistilled HC1 for 5 hours at 15 psi in a n autoclave. The hydrolysates were e v a p o r a t e d twice to drynesS; the residue was t a k e n u p in water, filtered, and made to a definite volume. F i l t r a t i o n was t h r o u g h specially designed filter planchets, used in t h e m a n n e r of Gooch crucibles, so t h a t t h e insoluble m a t e r i a l could be collected and its radioactivity determined. T h e amino acids of aliquots of t h e filtrates were s e p a r a t e d by ion-exchange column c h r o m a t o g r a p h y using a fraction collector (8). The n i n h y d r i n color of a portion of each fraction was determined (9, 10) and a n o t h e r portion was used for radioactivity counting. CARBON-14 DETERMINATIONS T h e samples, e i t h e r of t o t a l soluble hydrolysates or of amino acid fractions, were spread en planchets w i t h the aid of lens p a p e r inserts. T h e insoluble residues from t h e hydrolyses were collected on special planchets, as m e n t i o n e d above, consisting of p e r f o r a t e d a l u m i n m n discs on which circles of filter p a p e r were clamped b e n e a t h a l u m i n u m rings. The samples, after being plated, were dried first at room t e m p e r a t u r e , t h e n for 20 minutes at ll0~ in a n air oven, weighed, and counted in a Geiger-Mflller counter with an efficiency of 23%. Self-absorption corrections to zero thickness were made. S t a n d a r d deviations for t h e

TABLE I DESCRIPTION OF SAMPLES Sample No.

Type of tillera

Date of flowering

Date of injection

1 2 3

Secondary Secondary Primary

17 J u n e -18 J u n e

12 July 12 July 12 July

4

Secondary

22 J u n e

12-18 July

5

Secondary

22 J u n e

18 J u l y

Mode of injection 50 ~1. 50 ~1. + 50 ~1. w a t e r 50 ~l. + 50 ul. amino acid mixture b 10 ~l. each on 12, 13, 14, 15, and 18 July 50 ~l.

a A t h a r v e s t , the p l a n t s were uprooted a n d the roots were examined to d e t e r m i n e w h e t h e r t h e injected tiller represented t h e p r i m a r y stem from t h e seed, one of t h e secondary tillers arising from t h e m a i n axis, or one of the t e r t i a r y tillers arising from secondary axes. b For a rough approximation of t h e amino acid milieu in t h e stem of early dough stage wheat, rye was analyzed since it reached t h e corresponding stage sufficiently sooner to allow time for t h e analyses. A b o u t 50 top internodes of rye stems were collected on J u l y 3 (early dough stage) a n d pressed in a C a r v e r press. To the pressed juice an equal volume of 10% trichloroacetic acid was added to p r e c i p i t a t e t h e proteins. The proteins were centrifuged out a n d the free amino acids of t h e solution were determined (8). Amino acids were t h e n injected in a b o u t t h e same ratio to t h e injected lysine as the corresponding ratio in the protein-free rye stem juice. T h e a m o u n t s injected were as follows: L-aspartic acid, 100 ~g.; Lserine, 80 uS. ; nL-threonine, 85 uS. ; L-asparagine, 20 #g. ; L-glutamine, 30 ~g. ; L-glutamic acid, 90 ~g. ; Lproline, 260 uS. ; g!ycine, 45 uS. ; DL-alanine, 190 ~g. ; DL-valine, 90 uS. ; L-methionine, 20 ~g. ; DL-isoleucine, 20 ~g. ; L-leucine, 40 ~g. ; nL-phenylalanine, 25 ~g. ; L-tyrosine, 20 ~g. ; L-histidine, 25 ~g. ; L-arginine, 85 uS. ; and DL-tryptophan, 10 ~g.

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INCORPORATION OF LYSINE-C14 INTO WHEAT TABLE II INCORPORATION OF C 14 FROM L y S I N E - C 14 INJECTED INTO WI-IEA_T G R A I N % Injected radioactivity recovered in:

Plant No.

2 3 4 5

Flour

Bran

Germ

26 27 28 5

20 19 16 7

3.6 2.8 2.3 1.0

(~c./g.)

Total Grain

counting varied from less than 0.5% for lysine, to about 2% for moderate activity materials such as the proline peak, to nearly 10% for very low activity peaks. FREE AMINO ACIDS IN WHEAT JUICES Top internodes of stems of Omar wheat were collected in the morning on June 21 (4 days before flowering), July 10 (late milk stage), and July 24, 1962 (late dough stage, 7 days before harvest). As much juice as could be obtained was pressed out of the combined sections for each date in a Carver press. Absolute ethanol was added to a final concentration of 70% (v/v). The precipitate was centrifuged out, the supernatant solution was evaporated to dryness under a stream of warm air, and the residue was redissolved in 0.25 N citrate buffer pH 2.91 for amino acid analysis in the Technicon amino acid analyzer (11). RESULTS The wheat stems that were treated with lysine-C 14 at the early dough stage incorporated almost half this radioactivity into the adjacent spike. Treatment 6 days later (plant 5) resulted in much less recovery in the grain. The results are in Table II. For this purpose, the radioactivity of both the soluble and insoluble fractions from the hydrolyses were added together to give the total radioactivity of the grain fractions. In plant 2 and plant 5, respectively, 0.6 % and 0.7 % of the injected radioactivity was found in the chaff. Plant 1 was used to study the fate of the approximately 52 % unaccounted for. Only 1.36 % was found in the rest of the injected tiller after the spike was removed, and only 0.04 % was found in all the rest of the plant, i.e., the roots and 5 other tillers with their spikes. I t can be assumed that the unaccounted for C 14 has been released as respiratory COs. About half the radioactivity deposited in

50 49 46 13

Flour

Bran

Germ

1.1 1.1 1.6 0.3

3.3 3.2 3.8 1.9

5.5 4.0 4.9 2.4

TABLE III RADIOACTIVITY IN AMINO ACIDS FROM FLOUR HYDROLYSATES

Amino acid

% Total activity in flour hydrolysate due to individual amino acids Plant 2

Lysine Glutamic acid Proline Aspartic acid Arginine Nonamino acid

47 17

Plant 5

44 21

Specific activity (~c./molc) Plant 2

Plant 5

37,900 1450

15,800 420

8.2 1.9

5.5 1.1

760 780

310 210

1.6 16

0.9 16

1410

300

the seed was in the form of lysine. In fact, 47, 55, 44, and 49 % of the total seed radioactivity was found in the lysine of the seed hydrolysate for plants 2, 3, 4, and 5, respectively. About 10 % of the seed radioactivity was in the insoluble material remaining after hydrolysis and some of this probably derived from lysine. Without replicates the statistical significance of the differences cannot be determined, but the data suggest that the lysine m a y be deposited as such somewhat more efficiently and with less conversion to other amino acids when it is supplied to the plant in the presence of a balanced mixture of other amino acids as in plant 3. The predominance of lysine as a repository of C 14 occurs in spite of the relatively small amount of lysine in seeds as compared with m a n y of the other amino acids. This is shown in Table I I I , in which are compared the more strongly labeled amino acids in the flour fractions from the grain of early and late treated plants. While there was 2-3 times

76

LAWRENCE AND GRANT TABLE IV L Y S I N E RADIOACTIVITY IN FLOUR~ BRAN~ AND GERM

Plant No.

2 3 4 5

% of Total activity in hydrolysate of each fraction due to lysine-C14

Specific activity (# c./mole)

Flour

Bran

Germ

Flour

Bran

Germ

47 53 42 44

60 68 58 62

56 69 71 51

37,900 40,500 41,300 15,800

54,200 44,800 49,400 45,200

31,400 21,400 26,900 11,900

TABLE VI

TABLE V DISTRIBUTION OF L Y S I N E - C FRACTIONS Plant No.

TM

IN G R A I N

F R E E AMINO ACIDS IN W H E A T STEM JUICE a

% Total in: Flour

Bran

Collection date Germ

Amino acid

21 June

10 July

24 July

(~moles/lOO internodes)

2 3 4 5

46 48 52 31

46 45 40 61

8 7 8 7

as much radioactivity in lysine as in glutamic acid, the specific activity was 25-30 times as high. I n the bran and germ the situation was similar, with a somewhat greater proportion of the activity being found in the lysine, as shown in Table IV. I t is reasonable that since a larger proportion of the bran and germ animo acids is represented by lysine, a larger proportion of the bran and germ radioactivity would be found in lysine. Actually, specific activities of the lysine in bran were somewhat higher than in flour. The way in which the lysine-C I* was distributed among the different parts of the seed is summarized in Table V. Late injection (plant 5) resulted in proportionately more labeled lysine in the bran. Bilinski and McConnell (12) found that when labeled acetate was injected, bran proteins showed increased activity relative to the flour with later injection time. One m a y ask to what extent lysine and possible precursors of lysine are present in the stem enroute to the developing seed. Being unable to devise a method of separating the xylem constituents from those of the other plant juices, we resorted to analyzing for the free a m i n o acids in the total juices of the top intern0de of wheat stems. The

Lysine Aspartic acid Threonine + asparagine + glutamine Serine Glutamie acid Alanine Proline Glycine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine 7-Aminobutyric acid fl-Alanine Ornithine Tryptophan

18 15 ca. 90

7 17 ca. 50

1 0.1 ca. 6

50 6 90 92 13 24 8 15 19 5 5 6 8 190 8 12 0

25 8 73 480 5 17 4 7 9 3 5 5 10 71 trace 4 0

2 0.5 7 47 1 5 3 2 2 1 2 2 1 8 trace 1 1

a The total volumes which could be pressed out were 28 ml. on June 21, 14 ml. on July 10, and 1.0 ml. on July 24. results appear in Table VI. Identification was by position on the chromatogram only, but the only identification for which we feel any doubt is that for ornithine. I n no case was there any trace of a peak for diaminopimelic acid. DISCUSSION The results of treating wheat with lysineC I~ can be compared w i t h those of M c -

INCORPORATION OF LYSINE-C14 INTO WHEAT Connell and co-workers, who have studied the utilization by wheat of labeled acetate (7), pyruvate (13), glutamic acid (14), serine (15), a-aminoadipic acid (5), and a,a'-diaminopimelic acid (6). Dosages were similar, and the shorter time in the present work between treatment and harvest probably corresponds to faster maturation in a warmer climate. In agreement with the Canadian workers, we found that much less of C~4goes into the wheat when the treatment time is later than the early dough stage. With injection at the early dough stage about 46-49 % of the activity could be recovered in the grain. With most of the compounds he studied, McConnell found about the same recovery. Differences occurred between the results with lysine and with other compounds. For example, after treatment with pyruvate, serine, a-aminoadipic acid, or glutamic acid, about half the C 14 in the wheat appeared in the starch, while in the case of lysine treatment, the material representing primarily starch (nonamino acid portion of the hydrolysate of whole flour) contained only 16 % of the flour activity. In addition, some of the approximately 10% of the flour activity which remained in the insoluble residue after hydrolysis probably derived from starch. The difference is probably real in spite of the wide difference in fractionation methods. It probably derives from the less diverse metabolic activity of lysine and its lesser contribution to the metabolic pool. Carbon14 fl'om ]ysine was much more prone to go into bran (also differently defined) than was the radioactivity from other compounds. This might reasonably be related to the fact that a higher proportion of the total wheat lysine is found in the bran, than is the case for most other amino acids or other constituents. Specific activities of individual amino acids cannot be compared directly either. McConnell and his collaborators reported on those in the gluten fraction. The most comparable results in the present work are those based on the amino acids in a hydrolysate of whole flour. However, after due allowance for differences in amounts of activity injected, the present data shows a much higher specific activity in the wheat flour

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lysine than was found in any of the amino acids in McConnell's work. The specific activity of lysine after lysine treatment was more than 20 times as great as after treatment with a,a'-diaminopimelic acid (6). Again this is probably due to less dissipation of the injected material in the plant's metabolism. The ready incorporation of radioactive lysine in the wheat seed plus the absence of direct precursors of lysine among the free amino acids of the stem juice suggests that wheat grain lysine is synthesized elsewhere in the plant and is delivered to the grain l~reformed. However, the synthesis of lysine from diaminopimelic acid as a normal pathway in the wheat seed cannot be ruled out. Some precursor of diaminopimelic acid, rather than the acid itself, may be transported by way of the stem. Aspartate and pyruvate have been found to supply the carbon chain of diaminopimelic acid in Escherichia coli (16). Bilinski and McConnell (13) found that lysine was one of the least radioactive of the amino acids present after treatment of wheat with pyruvate-2-C 14. Much of the pyruvate, though, was apparently used for other syntheses by way of acetate. Another possibility is that lysine in the grain is synthesized from precursors that arose from photosynthetic activity in the spike itself. The evidence indicates considerable such activity during maturation of the grain (17). Such precursors might be converted to lysine by way of diaminopimelic acid. It may be noted that the total amount of lysine injected was 0.11 rag. or 0.75 t~moles, which is over 10 times as much as was present in all the juice of the internode at the time. McConnell used this much or more of the compounds he injected. What distortions, if any, of the normal metabolic mechanisms of the plant may result from this are unknown. There were certainly no obvious visible signs of maladjustment. ACKNOWLEDGMENTS The authors are indebted to Mr. D. B. Milne for assistance with the radioactive counting, and to Dr. C. F. Konzak for advice on the field aspects of this work and for the use of wheat in his experimental plots for the experiments.

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LAWRENCE AND GRANT 8. MOORE, S., SPACEMAN, D. H., AND STEIN, W. H., Anal. Chem. 30, 1185 (1958). JANSEN, G. R., J. Nutr. 76, Suppl. 1, Part II, 9. GRANT,D. R., Anal. Biochem. 6, 109 (1963). No. 2, 1-17 (1962). 10. ROSEN, H., Arch. Biochem. Biophys. 67, 10 LAWRENCE,J. M., CONE, VERA.M., XND DAY, (1957). KATHERINE M., Plant Physiol. 32, lvi (1957). 11. PIEz, K. A., AND MORRIS, LOUISE, Anal. Biochem. 1, 187 (1960). McCONNELL, W. S., AND RAMACHANDRAN, L. K., Can. J. Biochem. Physiol. 34, 180 12. BILINSKI, E., AND McCoNNELL, W. B., Cereal Chem. 35, 66 (1958). (1956). 13. BILINSKI, E., ANDMcCoNNELL, W. B., Can. J. VOGEL, H. J., Proc. Natl. Acad. Sci. U. S. 45, Biochem. Physiol. 36, 381 (1958). 1717 (1959). 14. McCONNELL, W. B., Can. J. Biochem. Physiol. ~ NATH, R., AND McCONNELL, W. B., Can. J. 37, 933 (1959). Biochem. Physiol. 38, 904 (1960). 15. NATR, R., AND McCoNNELL, W. B., Can. J. FINLAYSON, A. J., AND McCONNELL, W. B., Biochem. Physiol. 38,533 (1960). Biochim. Biophys. Acta 45, 622 (1960). 16. RHULAND,L. E., Nature 185, 224 (1960). BILINSKI, E., AND McCONNELL, W. B., Can. J. 17. BUTTROSE, M. S., Australian J. Biol. Sci. 15, Biochem. Physiol. 35, 357 (1957). 611 (1962). REFERENCES

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