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Metabolism of lysine and leucine derived from storage protein during the germination of maize
Biochimica et Biophysica Acta, 304 (1973) 353-362
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 27087
METABOLISM OF LYSINE A N D L E U C I N E DERIVED F R O M STORAGE P R O T E I N D U R I N G T H E G E R M I N A T I O N OF MAIZE
LADASLAV SODEK* and CURTIS M. WILSON** Department of Agronomy, University of Illinois and Agricultural Research Service, U.S. Department of Agriculture, Urbana, Ill. 61801 (U.S.A.)
(Received January 2nd, 1973)
SUMMARY The metabolism of [U-X4C]leucine or [U-14C]lysine during the germination of maize was studied using seed in which the storage protein was labelled. The labelled amino acids were injected below the ear of maize plants during seed development. The storage protein contained a large excess of leucine but insufficient lysine for synthesis of new protein in the developing axis. The axis preferentially utilized leucine and lysine (and possibly proline) derived from storage protein for synthesis of new protein. Excess leucine was respired as CO2 with little conversion to other amino acids. The lysine deficit was met by synthesis of new unlabelled carbon skeletons.
INTRODUCTION During the germination of cereals, the endosperm proteins are hydrolyzed to amino acids, most of which appear to be transported to the growing axis and incorporated into protein ~. Endosperm proteins contain different proportions of amino acids than do the embryo proteins. Dupuy et al. 2 reported that aspartic acid, glycine, and lysine increased in absolute amount during the germination of maize, while glutamic acid, proline, leucine, and alanine decreased. Folkes and Yemm 3 concluded that the endosperm of barley could provide almost three-fourths of the needed amino acids without interconversion of carbon chains, and that glutamic acid, the amides, and proline were the most important sources of nitrogen for the synthesis of additional amino acids. Oaks 4 supplied exogenous leueine or acetate to maize root tips to demonstrate that the tip is supplied with sufficient leucine from the endosperm to repress leucine synthesis. Joy and Folkes 5 found that amino acids close to the pathways of carbohydrate metabolism were interconverted to other compounds and to CO2, while leucine and lysine, far removed from carbohydrates, were utilized without change by excised barley embryos. * Present address: L. Sodek, C.E.N.A., ESALQ, Caixa Postal, 96, Piracicaba, Est. S~.oPaulo, Brazil. ** Author to whom requests for reprints should be sent.
354
L. SODEK, C. M. WILSON
[U-t4C]Lysine and [U-14C]leucine injected into the shank below the ear of maize plants during the period of endosperm development are incorporated into endosperm storage protein 6. Seeds in which the amino acids of the storage protein are labelled should be a useful innow.tion in studies of the metabolism of amino acids during germination. We report here a preliminary experiment on the metabolism of leucine and lysine during germination which may serve as a model for improved experiments with endogenously labelled amino acids. MATERIALS AND METHODS
Seed was obtained from self-pollinated maize plants (Zea mays L.). The hybrid R804 • R802 was used with [U -14C]leucine and the inbred M l4 with [U -~ 4C]lysine. Twenty-six days after pollination, 100 #1 of [U-~4C]leucine (6.25 ~tCi) or [U-14C]lysine (2/~Ci) were injected into the shank below the ear and the wounds were sealed with lanolin. The mature seed was stored at about 12 ?(, moisture at 4 ~'C. Seeds were surface sterilized in 0.2 ~o sodium hypochlorite solution and germinated for 24 h at 28 °C in the dark on wet paper towels in glass trays covered with a plastic film. Selected seeds were transferred to an air-tight plastic box in which a pla~tform of filter paper was supported by a coil of filter paper standing in 10 -4 M CaCI 2. The chamber was kept at 28 °C in the dark and was flushed continuously with a stream of CO2-free air. The respired CO 2 was trapped by passage through a fritted glass tube in 20 ~o KOH. At 24-h intervals the K O H solution was transferred to centrifuge tubes, an excess of 10 ~ BaCI2~-I ~o NH4C1 was added, the tubes were stoppered, and shaken. The B a C O 3 w a s immediately centrifuged down and washed twice with hot wa.ter. Thc final precipitate, in less than 1-g lots, was suspended in 10 ml Bray's scintillator 7 with about 2.5 ~,, Cab-O-Sil and counted at near 60 o~ efficiency. Twenty seeds were divided into endosperm and embryo (scutellum plus axis), while twenty 6-day-old seedlings were divided into endosperm, scutellum, and axis. The endosperm was frozen with solid CO2, lyophilized, and ground in a ball mill. Nitrogen fractions designated as free amino acid, albumin, globulin, zein-1, zein-2, glutelin, and residue were extracted and analyzed for ~4C and nitrogen content as previously described 6'8. The scutellum (with or without the axis) was frozen in solid CO2, lyophilized, and homogenized in 80 ~o ethanol. The homogenate was filtered through a glass-fiber disc e.nd the residue was washed with 80 ~, ethanol. The axis was dropped into boiling 80 ~o ethanol immediately after dissection and was homogenized in a blender, filtered, collected, and washed with 80 % ethanol. The alcohol-soluble fraction from the axis (containing the free amino acids) was taken to dryness in a rotary evaporator. The residue was taken up in 1 M HCI and heated in a screwcap vial at 110 °C for 4 h to hydrolize amides prior to amino acid analysis. The amino acid contents of the alcohol-insoluble fractions were determined on an automatic a~mino acid an~.lyzer9 after HCI hydrolysis. The 14C contents of the total hydrolysa.tes, the separated amino acids, and the non-amino acid compounds (not adsorbed by the resin column) were determined as previously described 6. RESULTS
Amino acid composition The amino acid compositions of the endosperm and the insoluble fraction of
M E T A B O L I S M O F L Y S I N E A N D L E U C I N E IN M A I Z E
355
the scutellum before germination and of the axis 6 days after germination are given in Table I. The amino acid composition of endosperm reserve protein differs from the protein newly synthesized in the axis. For example, endosperm protein is low in lysine but high in leucine. Axis protein, on the other hand, contains 7.4 % lysine and 9 % leucine, and is generally more balanced in the other amino acids. The actual supply and demand of the amino acids can be more readily appreciated when the data are expressed as #moles per part. Six days after germination the axis contained 4.1 #moles of lysine and 5.4 #moles of leucine, while the endosperm originally contained 3.1 #moles of lysine and 28 #moles of leucine. Since one-fourth of the total nitrogen remained in the endosperm at 6 days (Table II), it is apparent that there is a large surplus of leucine, but a deficit of lysine, in the seed. The scutellum contains only about 15 % of the seed amino acids, but there is almost as much lysine in the scutellum as in the endosperm, and its amino acid composition is similar to the axis. Unfortunately, we did not include the 6-day scutellum in the balaoce sheet, but during germination the 14C content (Tables I1 and IV) and the nitrogen content 1o of the scutellum increase, and we are assuming that the endosperm was the ultimate source of the axis amino acids. TABLE I AMINO ACID COMPOSITION OF THE ENDOSPERM AND SCUTELLUM BEFORE GERM I N A T I O N A N D IN THE 6-DAY-OLD AXIS*
0 days Endosperm
Scutellum
Axis
Whole
Insoluble
Soluble
mole %
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine** (half) Valine Methionine** Isoleucine Leucine Tyro~,ine Phenylalanine Total
6 days
#moles/ part
mole %
#moles/ part
Insoluble
mole %
1.7 1.9 1.7 5.7 3.5 5.9 19.1 11.0 4.9 12.3 -5.5 1.7 3.9 15.2 2.0 4.0
3.1 3.5 3.1 10.4 6.4 10.9 35.2 20.3 9.0 22.6 -10.1 3.1 7.2 28.0 3.7 7.4
6.3 2.7 7.6 8.2 4.3 5.8 13.8 4.6 11.5 10.0 1.1 7.2 1.2 3.5 7.0 1.8 3.4
2.06 0.88 2.48 2.67 1.42 1.90 4.52 1.50 3.76 3.28 0.36 2.36 0.39 1.15 2.28 0.60 1.10
0.8 3.4 0.3 40.2 4.6 7.0 12.1 3.4 6.1 7.3 . 6.3 -2.3 2.7 1.7 1.8
100.0
184.0
100.0
32.66
100.0
#moles/ part
mole %
#moles/ part 3.90 1.05 2.62 5.47 2.78 3.30 5.55 2.92 4.95 5.47
1.65 -0.60 0.70 0.43 0.46
7.4 2.0 5.0 10.4 5.3 6.3 10.6 5.6 9.4 10.4 . 7.7 0.9 4.4 9.0 2.0 3.6
26.11
100.0
52.48
0.20 0.90 0.08 10.50 1.20 1.84 3.15 0.90 1.59 1.91 .
.
4.05 0.45 2.32 4.73 1.05 1.87
* This data is from the R804 × R802 ear, but the mole % data from the M14 ear was similar. ** Precautions against air oxidation during hydrolysis were not taken.
356
L. S O D E K , C. M. W I L S O N
T A B L E II D I S T R I B U T I O N O F 14C A N D N I T R O G E N IN E N D O S P E R M PROTEIN FRACTIONS BEFORE AND AFTER GERMINATION O F S E E D C O N T A I N I N G [~4C]LEUC1NE [ ' * C ] L e u c i n e was injected into the s h a n k o f an R 8 0 4 × R802 hybrid 26 days after pollination, and the ear was harvested at 58 days. T h e average d p m (per seed) was 8450 before g e r m i n a t i o n and 9530 at 6 days.
% Total in endosperm 0 days
Free a m i n o acids Albumin Globulin Zein- I Zein-2 Glutelin Residue
6 days
~4C
N
~C
N
1.1 0.5 I.I 36.5 32.4 20.3 8.1
3.0 3.5 32.7 31.1 27.3
15.3 3.2 3.0 31.2 18.3 21.5 7.5
5.5 6.0 27.5 19.0 27.0
34 200 7 150 18.33 3.83 207 I 300
d p m / g dry wt dpm/endosperm m g N/g dry wt mg N/endosperm m g dry w t / e n d o s p e r m dpm/scutellum
17 200 I 720 10.00 1.00 100 2 010
T A B L E III L A B E L L I N G O F A M I N O A C I D S A N D O T H E R C O M P O U N D S IN P A R T S O F T H E C O R N S E E D L I N G B E F O R E A N D A F T E R G E R M I N A T I O N O F S E E D S C O N T A I N I N G [14C]LEUCINE*
6 days
0 days Endosperm %**
Leucine G l u t a m i c acid Proline Aspartic acid O t h e r a m i n o acids N o n - a m i n o acids ??t
97.5
I*?
dpm/ Itrnole
248
Scutellum
Axis
Insoluble
Soluble***
Insoluble t
",~
dpm/ ftmole
o/~,
dpm/ #mole
",~
dpm/ Itmole
90.5
405
19.0 5.6 0.8t* 18.0
190 12.5 6.2 12.0
70.0 5.6 1.1 ?+ 3.7 5.7 13.6
193.0 13.1 4.9 8.8
55
* See Table I!. * * ~4C recovered in each fraction as percentage of t~C applied to the column. * * * 700 d p m / p a r t . 1300 d p m / p a r t . ?? C o u n t s were less t h a n twice the b a c k g r o u n d count. ?t? Probably sugars a n d organic acids.
METABOLISM OF LYSINE A N D LEUCINE IN MAIZE
357
Metabolism of [U-14 C]leucine during germination The distribution in the seed, the storage proteins and the seedling parts of nitrogen and of 14C derived from injected [U -14C]leucine are shown in Tables II and III. Before germination, 85 ~ of the total 14C was found within the endosperm. The remainder was located in the scutellum, except for a very small fraction in the axis. Virtually all the label in the seed was present as leucine, and the 14C of the endosperm was located almost entirely in the storage proteins. The major storage proteins, zein and glutelin, contained most of the label. When the seed was left to geIminate, 74 ~o of the endosperm nitrogen and 76 of the ~4C was transported out of the endosperm during a 6-day period. Depletion of the major storage proteins, especially zein-2, accounted for most of the nitrogen loss. The albumin and globulin fractions were depleted to a lesser extent, and for this reason contained a higher proportional of the total ~4C and nitrogen at 6 days. Enzymes synthesized for the mobilization of storage material might contribute to the albumin and globulin fractions. 14C accumulated in the free amino acid fraction during germination, probably from the release of [14C]leucine from the reserve protein by proteolytic action. However, only small amounts of amino acids accumulate in the endosFerm during germination1°, and the 15 ~ ~4C in the soluble fraction represents only three times as much total activity as before germination. Analysis of the seedling after the 6-day germination period revealed the fate of the [~4C]leucine transported out of the endosperm. The axis contained 21 ~o of the total ~4C, only 18 ~ remained within the endospelm, while 40 ~ was respired as CO 2. 14CO2 was produced at an even rate after a lag period (Fig. 1). The scutellum contained a higher proportion of the label (21 ~o) than it did before germination, but alcohol-soluble compounds did not accumulate. Although the label in the seed before germination was virtually all in leucine, only about half of the ~4C in the 6-day seedling axis was in leucine (Table III). Most of the axis leucine was found in the insoluble (protein) fraction, with a specific activity similar to that of endosperm leucine before germination. The non-leucine labelled
c~ 3c o
"6 2¢
o~ lc
o
o
1
2
3
4
5
6
Days a f t e r germination
Fig. 1. Evolution of ~4CO2 during germination of maize seeds. [U-14C]leucine or lU-14C]lysine was injected below the ear during development, then the seeds were germinated. The ~4CO2 evolved is expressed as percent of the total 14C in the lot of seeds. 0 - - 0 , ~4CO2 from [U-14C]leucine; O - - O , ~4CO2 derived directly or indirectly from [U-~4C]lysine.
358
L. S O D E K , C. M. W I L S O N
compounds in the axis included glutamic acid, proline, and aspartic acid, as well as unidentified compounds which would include starch in the insoluble fraction and sugars and organic acids in the soluble fraction (where 55 ~ of the label was in nonamino acid compounds).
Metabolism of [U-14C]lysine during germination The distributions in the seed, seedling parts, and storage proteins of nitrogen and of 14C derived from injected [U -14C]lysine are shown in Tables IV and V. More than 80 ~o of the 14C in the seed before germination was located in the endosperm, but very little was present in the axis. The major storage proteins, zein and glutelin, were the most highly labelled endosperm fractions. Although zein was relatively less labelled than glutelin, in contrast to the leucine experiment, this can be attributed to the different leucine/lysine contents of the different proteins 6,8. As in the leucine experiment, a high proportion of the endosperm 14C and nitrogen, principally derived from the major storage proteins, was removed from the endosperm during germination. Again, there were increases in percentage (though not absolute amounts) of albumins and globulins, together with an accumulation of 14C in the free amino acid fraction. In contrast to the endosperm, the 14C content of the scutellum changed little after germination. During the 6-day germination period somewhat less I4C derived from lysine was mobilized from the endosperm than was the case for [14C]leucine; only 20 ~/o was respired as CO2 while 29 ~o was recovered in the axis. The pattern of 14CO2 T A B L E IV D I S T R I B U T I O N O F 14C A N D N I T R O G E N IN E N D O S P E R M P R O T E I N F R A C T I O N S B E F O R E A N D A F T E R G E R M I N A T I O N O F SEED C O N T A I N I N G [14C]LYSINE A N D ITS METABOLITES* [~4C]Lysine was injected into the shank of an M I 4 inbred 26 days after pollination, and the ear was harvested at 58 clays. The average dpm (per seed) was 27 840 before germination and 26 410 at six days.
%o of total ~4C and N in endosperm 0 days
Free amino acids Albumin Globulin Zein-I Zein-2 Glutelin Residue d p m / g dry wt dpm/endosperm mg N / g dry wt mg N / e n d o s p e r m mg dry wt/endosperm dpm/scutellum
6 days
14 C
N
~4C
N
4.7 4.8 6.2 21.6 14.0 30.8 18.0
3.9 4.9 42.2 20.6 24.0
30.0 6.8 10.0 12.9 6.0 15.0 19.3
7.5 5.8 34.2 15.0 18.3
130 885 22 800 20.4 3.55 174 5 040
74 480 6 700 12.0 1.08 90 6 300
METABOLISM OF LYSINE AND LEUCINE IN MAIZE
359
TABLE V LABELLING OF AMINO ACIDS AND OTHER COMPOUNDS IN PARTS OF THE CORN SEEDLING BEFORE AND AFTER GERMINATION OF SEED CONTAINING [14C]LYSINE AND ITS METABOLITES* 6 days
0 days
Lysine Glutamic acid Proline Aspartic acid Other amino acids Non-amino acidstt
Endosperm
Scutellum
Axis
%**
Insoluble
Soluble***
lnsolublet
%
dpm/ l~mole
%
dpm/ ttmole
%
dpm/ I~mole
89.5 9.3 9.3 9.3 9.3 9.3
3660
30 42 42 42 42 22
--
77 4 8 2 6 2
1280 43 155 26
dpm/ I~mole
47.5 4320 21.5 177 11.5 165 2.5 77 9.0 4.5
* See Table IV. ** ~4C recovered in fraction as ~ of ~4C applied to column. *** 2160 dpm/part. 5600 dpm/part. tt Probably sugars and organic acids. production (Fig. 1) was similar to that in the leucine experiment. For the purposes of this study, lysine is less suitable than leucine, in that much of the 14C in the endosperm was present in glutamic acid and proline (Table V). Only 47.5 % was in lysine, in line with our previous report 6. In contrast, 89.5 % of the label in the scutellum was in lysine, to further complicate the picture. The proportion of ~4C recovered in lysine in the insoluble axis fraction (77 %) was greater than that originally present in the endosperm, suggesting that glutamate and proline were catabolized to a much greater extent than lysine. In the 6-day axis protein the specific activities of lysine and glutamic acid, but not proline, was considerably less than in the original endosperm. DISCUSSION Amino acid metabolism The amino acid composition of the two fractions of the axis (Table I) are similar to those reported by Oaks t for maize root tips, except that the relative concentrations of glutamate and aspartate were reversed in the soluble fraction. The changes upon germination are similar to those found by Dupuy et al. 2, in that there are large decreases in glutamic acid, proline, leucine, and alanine, and large increases in lysine and aspartic acid. Aspartic acid accumulated mainly in the soluble fraction of the axis, presumably as newly synthesized asparagine. In contrast, proline and leucine are present in very low amounts in the soluble fraction considering the quantity available from the endosperm. Thus the growing seedling must possess an active metabolic system for catabolism of the carbon skeletons of these two amino acids. Glutamate, of
360
L. SODEK, C. M. WILSON
course, is centrally located in well-known metabolic systems. The low level of free lysine is to be expected, and lysine must be synthesized to provide enough for new protein. In the 6-day axis (Table IIl) the specific activity of leucine was not much less than in the endosperm, evidence that leucine from the endosperm was directly incorporated into axis protein, and that the surplus leucine had inhibited leucine synthesis in the axis, as suggested by Oaks 4. Extensive degradation of leucine occurred, for only one-sixth of the 14C transported out of the endosperm was recovered as leucine. Oaks 11 concluded that leucine was not extensively metabolized in excised maize roots, but this was after a 2-h incubation with exogenous [U -14C]leucine. Splittstoesser 12 reported that only 6 % of the metabolized [U -14C]leucine supplied to an excised maize axis was lost as CO2, while 74 o/ /o was recovered from the residue after 24 h. Joy and Folkes 5 also found much more 14C from leucine in protein than in CO 2 after 18 h incubation of excised barley embryos. Beevers and Splittstoesser 13 found somewhat more metabolism of [U -14C]-leucine which was injected into the cotyledons of germinating peas. It seems that experiments with excised maize tissues greatly underestimated the proportion of leucine metabolized to CO2 and other compounds within the normal seedling. A possible explanation is that the isolated tissue, removed from the endosperm, no longer has surplus leucine available for catabolism. The reduced specific activity of [14C]lysine in the axis protein fraction (Table V) shows that the labelled lysine was diluted by newly synthesized unlabelled lysine. However, rough calculations indicate that the 6-day seedlings contained almost as much [14C]lysine as the original seed, suggesting that there was little degradation. The labelled CO2 probably was derived from glutamic acid, proline, and other nonlysine compounds. We may note that the axis protein contained twice as much [14C] proline as [14C]glutamic acid, the reverse of the situation in the endosperm. The specific activities in the seed and in the axis suggest that new, unlabelled glutamic acid was extensively synthesized, while there was little synthesis ofproline. It has previously been reported that proline is rapidly metabolized within the maize root tip, but is not synthesized there 14.15. The metabolic relationships of lysine and leucine to protein synthesis are reversed in the developing maize endosperm and the growing seedling. In the developing endosperm lysine is provided in excess, and [14C]lysine is converted to other amino acids, while [14C]leucine is incorporated into protein without change 6. Assays for free amino acids in the developing endosperms of wheat 16 and corn (Wilson, C. M., unpublished) show low levels of leucine relative to the amount needed for protein synthesis, while [ysine levels are relatively high. In the growing axis, leucine is supplied in excess from storage proteins, whereas the lysine supply is insufficient to meet the needs of protein synthesis (Table I). Here leucine synthesis is restricted 4 and excess ieucine is catabolized, while lysine may be conserved and certainly must be synthesized. The present results are consistent with the amino acid hypothesis of Folkes and Yem 3, that leucine, lysine, and proline liberated in the endosperm are translocated to the embryo and incorporated into new protein in the embryo, and that the amino acids present in excess of requirements are metabolized to other compounds or respired to C02.
In vivo labelling Of storage proteins To our knowledge, the use of seed in which the storage protein amino acids are
METABOLISM OF LYSINE A N D LEUCINE IN MAIZE
361
labelled with t 4C is a new approach to the study of the metabolism of amino acids during germination. Usually labelled amino acids are injected into the storage organ immediately prior to germination 13 or are present in the medium in which the seedlings s or parts thereof 4' 11,12 are incubated. In such experiments there are the dangers of upsetting the steady state or that the exogenous tracer will follow pathways not representative of the normal metabolism of the endogenous compound under investigation 17. The most distinct advantage of the new method is that one can be certain that the labelled amino acid will go through the natural transport and metabolic processes. There are, however, several problems with the new technique which our preliminary experiment revealed: (1) The amino acid administered may undergo metabolic changes before incorporation into the seed reserve protein, i.e. [14C]lysine was extensively converted into [14C]glutamate and [14C]proline in the developing endosperm 6. Less conversion of [14C]lysine may occur in seeds possessing proteins with higher lysine contents, such as opaque-2 maize 6 or most of the non-cereal plants. (2) The scutellum proteins are also labelled, and, in the case of the [14C]lysine experiment, labelling was different from the endosperm. It is not known if turnover of scutellum proteins occurred, with utilization by the growing axis of the amino acids thus released. (3) The injected labelled amino acids were diluted before incorporation into storage protein. The resulting low level of r~.dioactivity made it especially difficult to measure accurately the 14C-content of the non-leucine and non-lysine compounds. Also, the labelled amino acids were supplied to the endosperm over a short period of time by a single injection at 26 days after pollination, about the mid-point of N accumulation. Thus most of the protein of the mature endosperm was formed before and after the ~4C was available. We have assumed that the amino acids were released at a constant specific activity during germination, but some of our interpretations would be in error if there was a change. However, ~4CO2 was released evenly throughout this period, and the specific activities of the endosperm and of the protein fractions were similar before and after germination in the leucine experiment (Table II). Continuous or repeated administration of labelled amino acids through the shank would give higher and more evenly distributed activity. ACKNOWLEDGEMENTS
This study was supported by the Agricultural Research Service, U. S. Department of Agriculture, Cooperative Agreement No. 12-14-100-9359(34), administered by the Plant Science Research Division, Beltsville, Md., and by a grant from the University of Illinois Research Board.
REFERENCES 1 2 3 4
Oaks, A. (1966)Plant Physiol. 41, 173-180 Dupuy, .l'., Boutin, J. and Dupaigne, G. (1971) C. R. Acad. Sci., Ser. D (Paris) 272, 2548-2551 Folkes, B. F. and Yemm, E. W. (1958) New Phytol. 57, 106-131 Oaks, A. (1965) Plant Physiol. 40, 149-155
362 5 6 7 8 9 lO II 12 13 14 15 16 17
L. SODEK, C. M. WILSON
Joy, K. W. and Folkes, B. F. (1965) J. Exp. Bot. 16, 646-666 Sodek, L. and Wilson, C. M. (1970) Arch. Biochem. Biophys. 140, 29-38 Bray, G. A. (1960) Anal. Biochem. l, 279-285 Sodek, L. and Wilson, C. M. (1971) J. Agric. Food Chem. 19, 1144-1150 Moore, S., Spackman, D. H. and Stein, W. H. (1958) Anal. Chem. 30, 1185-1190 Ingle, J., Beevers, L. and Hageman, R. H. (1964) Plant Physiol. 39, 735-740 Oaks, A. (1965)PlantPhysiol. 40, 142-149 Splittstoesser, W. E. (1967) Phytochemistry 6, 933-939 Beevers, L. and Splittstoesser, W. E. (1968) J. Exp. Bot. 19, 698-711 Barnard, R. A. and Oaks, A. (1970) Can. J. Bot. 48, 1155-1158 Oaks, A., Mitchell, D. J., Barnard, R. A. and Johnson, F. J. (1970) Can. J. Bot. 48, 2249-2258 Jennings, A. C. and Morton, R. K. (1963) Austr. J. Biol. Sci. 16, 384-394 Sodek, L. and Wilson, C. M. (1971) Plant CelIPhysiol. 12, 889-893
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METABOLISM OF LYSINE A N D L E U C I N E DERIVED F R O M STORAGE P R O T E I N D U R I N G T H E G E R M I N A T I O N OF MAIZE
LADASLAV SODEK* and CURTIS M. WILSON** Department of Agronomy, University of Illinois and Agricultural Research Service, U.S. Department of Agriculture, Urbana, Ill. 61801 (U.S.A.)
(Received January 2nd, 1973)
SUMMARY The metabolism of [U-X4C]leucine or [U-14C]lysine during the germination of maize was studied using seed in which the storage protein was labelled. The labelled amino acids were injected below the ear of maize plants during seed development. The storage protein contained a large excess of leucine but insufficient lysine for synthesis of new protein in the developing axis. The axis preferentially utilized leucine and lysine (and possibly proline) derived from storage protein for synthesis of new protein. Excess leucine was respired as CO2 with little conversion to other amino acids. The lysine deficit was met by synthesis of new unlabelled carbon skeletons.
INTRODUCTION During the germination of cereals, the endosperm proteins are hydrolyzed to amino acids, most of which appear to be transported to the growing axis and incorporated into protein ~. Endosperm proteins contain different proportions of amino acids than do the embryo proteins. Dupuy et al. 2 reported that aspartic acid, glycine, and lysine increased in absolute amount during the germination of maize, while glutamic acid, proline, leucine, and alanine decreased. Folkes and Yemm 3 concluded that the endosperm of barley could provide almost three-fourths of the needed amino acids without interconversion of carbon chains, and that glutamic acid, the amides, and proline were the most important sources of nitrogen for the synthesis of additional amino acids. Oaks 4 supplied exogenous leueine or acetate to maize root tips to demonstrate that the tip is supplied with sufficient leucine from the endosperm to repress leucine synthesis. Joy and Folkes 5 found that amino acids close to the pathways of carbohydrate metabolism were interconverted to other compounds and to CO2, while leucine and lysine, far removed from carbohydrates, were utilized without change by excised barley embryos. * Present address: L. Sodek, C.E.N.A., ESALQ, Caixa Postal, 96, Piracicaba, Est. S~.oPaulo, Brazil. ** Author to whom requests for reprints should be sent.
354
L. SODEK, C. M. WILSON
[U-t4C]Lysine and [U-14C]leucine injected into the shank below the ear of maize plants during the period of endosperm development are incorporated into endosperm storage protein 6. Seeds in which the amino acids of the storage protein are labelled should be a useful innow.tion in studies of the metabolism of amino acids during germination. We report here a preliminary experiment on the metabolism of leucine and lysine during germination which may serve as a model for improved experiments with endogenously labelled amino acids. MATERIALS AND METHODS
Seed was obtained from self-pollinated maize plants (Zea mays L.). The hybrid R804 • R802 was used with [U -14C]leucine and the inbred M l4 with [U -~ 4C]lysine. Twenty-six days after pollination, 100 #1 of [U-~4C]leucine (6.25 ~tCi) or [U-14C]lysine (2/~Ci) were injected into the shank below the ear and the wounds were sealed with lanolin. The mature seed was stored at about 12 ?(, moisture at 4 ~'C. Seeds were surface sterilized in 0.2 ~o sodium hypochlorite solution and germinated for 24 h at 28 °C in the dark on wet paper towels in glass trays covered with a plastic film. Selected seeds were transferred to an air-tight plastic box in which a pla~tform of filter paper was supported by a coil of filter paper standing in 10 -4 M CaCI 2. The chamber was kept at 28 °C in the dark and was flushed continuously with a stream of CO2-free air. The respired CO 2 was trapped by passage through a fritted glass tube in 20 ~o KOH. At 24-h intervals the K O H solution was transferred to centrifuge tubes, an excess of 10 ~ BaCI2~-I ~o NH4C1 was added, the tubes were stoppered, and shaken. The B a C O 3 w a s immediately centrifuged down and washed twice with hot wa.ter. Thc final precipitate, in less than 1-g lots, was suspended in 10 ml Bray's scintillator 7 with about 2.5 ~,, Cab-O-Sil and counted at near 60 o~ efficiency. Twenty seeds were divided into endosperm and embryo (scutellum plus axis), while twenty 6-day-old seedlings were divided into endosperm, scutellum, and axis. The endosperm was frozen with solid CO2, lyophilized, and ground in a ball mill. Nitrogen fractions designated as free amino acid, albumin, globulin, zein-1, zein-2, glutelin, and residue were extracted and analyzed for ~4C and nitrogen content as previously described 6'8. The scutellum (with or without the axis) was frozen in solid CO2, lyophilized, and homogenized in 80 ~o ethanol. The homogenate was filtered through a glass-fiber disc e.nd the residue was washed with 80 ~, ethanol. The axis was dropped into boiling 80 ~o ethanol immediately after dissection and was homogenized in a blender, filtered, collected, and washed with 80 % ethanol. The alcohol-soluble fraction from the axis (containing the free amino acids) was taken to dryness in a rotary evaporator. The residue was taken up in 1 M HCI and heated in a screwcap vial at 110 °C for 4 h to hydrolize amides prior to amino acid analysis. The amino acid contents of the alcohol-insoluble fractions were determined on an automatic a~mino acid an~.lyzer9 after HCI hydrolysis. The 14C contents of the total hydrolysa.tes, the separated amino acids, and the non-amino acid compounds (not adsorbed by the resin column) were determined as previously described 6. RESULTS
Amino acid composition The amino acid compositions of the endosperm and the insoluble fraction of
M E T A B O L I S M O F L Y S I N E A N D L E U C I N E IN M A I Z E
355
the scutellum before germination and of the axis 6 days after germination are given in Table I. The amino acid composition of endosperm reserve protein differs from the protein newly synthesized in the axis. For example, endosperm protein is low in lysine but high in leucine. Axis protein, on the other hand, contains 7.4 % lysine and 9 % leucine, and is generally more balanced in the other amino acids. The actual supply and demand of the amino acids can be more readily appreciated when the data are expressed as #moles per part. Six days after germination the axis contained 4.1 #moles of lysine and 5.4 #moles of leucine, while the endosperm originally contained 3.1 #moles of lysine and 28 #moles of leucine. Since one-fourth of the total nitrogen remained in the endosperm at 6 days (Table II), it is apparent that there is a large surplus of leucine, but a deficit of lysine, in the seed. The scutellum contains only about 15 % of the seed amino acids, but there is almost as much lysine in the scutellum as in the endosperm, and its amino acid composition is similar to the axis. Unfortunately, we did not include the 6-day scutellum in the balaoce sheet, but during germination the 14C content (Tables I1 and IV) and the nitrogen content 1o of the scutellum increase, and we are assuming that the endosperm was the ultimate source of the axis amino acids. TABLE I AMINO ACID COMPOSITION OF THE ENDOSPERM AND SCUTELLUM BEFORE GERM I N A T I O N A N D IN THE 6-DAY-OLD AXIS*
0 days Endosperm
Scutellum
Axis
Whole
Insoluble
Soluble
mole %
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine** (half) Valine Methionine** Isoleucine Leucine Tyro~,ine Phenylalanine Total
6 days
#moles/ part
mole %
#moles/ part
Insoluble
mole %
1.7 1.9 1.7 5.7 3.5 5.9 19.1 11.0 4.9 12.3 -5.5 1.7 3.9 15.2 2.0 4.0
3.1 3.5 3.1 10.4 6.4 10.9 35.2 20.3 9.0 22.6 -10.1 3.1 7.2 28.0 3.7 7.4
6.3 2.7 7.6 8.2 4.3 5.8 13.8 4.6 11.5 10.0 1.1 7.2 1.2 3.5 7.0 1.8 3.4
2.06 0.88 2.48 2.67 1.42 1.90 4.52 1.50 3.76 3.28 0.36 2.36 0.39 1.15 2.28 0.60 1.10
0.8 3.4 0.3 40.2 4.6 7.0 12.1 3.4 6.1 7.3 . 6.3 -2.3 2.7 1.7 1.8
100.0
184.0
100.0
32.66
100.0
#moles/ part
mole %
#moles/ part 3.90 1.05 2.62 5.47 2.78 3.30 5.55 2.92 4.95 5.47
1.65 -0.60 0.70 0.43 0.46
7.4 2.0 5.0 10.4 5.3 6.3 10.6 5.6 9.4 10.4 . 7.7 0.9 4.4 9.0 2.0 3.6
26.11
100.0
52.48
0.20 0.90 0.08 10.50 1.20 1.84 3.15 0.90 1.59 1.91 .
.
4.05 0.45 2.32 4.73 1.05 1.87
* This data is from the R804 × R802 ear, but the mole % data from the M14 ear was similar. ** Precautions against air oxidation during hydrolysis were not taken.
356
L. S O D E K , C. M. W I L S O N
T A B L E II D I S T R I B U T I O N O F 14C A N D N I T R O G E N IN E N D O S P E R M PROTEIN FRACTIONS BEFORE AND AFTER GERMINATION O F S E E D C O N T A I N I N G [~4C]LEUC1NE [ ' * C ] L e u c i n e was injected into the s h a n k o f an R 8 0 4 × R802 hybrid 26 days after pollination, and the ear was harvested at 58 days. T h e average d p m (per seed) was 8450 before g e r m i n a t i o n and 9530 at 6 days.
% Total in endosperm 0 days
Free a m i n o acids Albumin Globulin Zein- I Zein-2 Glutelin Residue
6 days
~4C
N
~C
N
1.1 0.5 I.I 36.5 32.4 20.3 8.1
3.0 3.5 32.7 31.1 27.3
15.3 3.2 3.0 31.2 18.3 21.5 7.5
5.5 6.0 27.5 19.0 27.0
34 200 7 150 18.33 3.83 207 I 300
d p m / g dry wt dpm/endosperm m g N/g dry wt mg N/endosperm m g dry w t / e n d o s p e r m dpm/scutellum
17 200 I 720 10.00 1.00 100 2 010
T A B L E III L A B E L L I N G O F A M I N O A C I D S A N D O T H E R C O M P O U N D S IN P A R T S O F T H E C O R N S E E D L I N G B E F O R E A N D A F T E R G E R M I N A T I O N O F S E E D S C O N T A I N I N G [14C]LEUCINE*
6 days
0 days Endosperm %**
Leucine G l u t a m i c acid Proline Aspartic acid O t h e r a m i n o acids N o n - a m i n o acids ??t
97.5
I*?
dpm/ Itrnole
248
Scutellum
Axis
Insoluble
Soluble***
Insoluble t
",~
dpm/ ftmole
o/~,
dpm/ #mole
",~
dpm/ Itmole
90.5
405
19.0 5.6 0.8t* 18.0
190 12.5 6.2 12.0
70.0 5.6 1.1 ?+ 3.7 5.7 13.6
193.0 13.1 4.9 8.8
55
* See Table I!. * * ~4C recovered in each fraction as percentage of t~C applied to the column. * * * 700 d p m / p a r t . 1300 d p m / p a r t . ?? C o u n t s were less t h a n twice the b a c k g r o u n d count. ?t? Probably sugars a n d organic acids.
METABOLISM OF LYSINE A N D LEUCINE IN MAIZE
357
Metabolism of [U-14 C]leucine during germination The distribution in the seed, the storage proteins and the seedling parts of nitrogen and of 14C derived from injected [U -14C]leucine are shown in Tables II and III. Before germination, 85 ~ of the total 14C was found within the endosperm. The remainder was located in the scutellum, except for a very small fraction in the axis. Virtually all the label in the seed was present as leucine, and the 14C of the endosperm was located almost entirely in the storage proteins. The major storage proteins, zein and glutelin, contained most of the label. When the seed was left to geIminate, 74 ~o of the endosperm nitrogen and 76 of the ~4C was transported out of the endosperm during a 6-day period. Depletion of the major storage proteins, especially zein-2, accounted for most of the nitrogen loss. The albumin and globulin fractions were depleted to a lesser extent, and for this reason contained a higher proportional of the total ~4C and nitrogen at 6 days. Enzymes synthesized for the mobilization of storage material might contribute to the albumin and globulin fractions. 14C accumulated in the free amino acid fraction during germination, probably from the release of [14C]leucine from the reserve protein by proteolytic action. However, only small amounts of amino acids accumulate in the endosFerm during germination1°, and the 15 ~ ~4C in the soluble fraction represents only three times as much total activity as before germination. Analysis of the seedling after the 6-day germination period revealed the fate of the [~4C]leucine transported out of the endosperm. The axis contained 21 ~o of the total ~4C, only 18 ~ remained within the endospelm, while 40 ~ was respired as CO 2. 14CO2 was produced at an even rate after a lag period (Fig. 1). The scutellum contained a higher proportion of the label (21 ~o) than it did before germination, but alcohol-soluble compounds did not accumulate. Although the label in the seed before germination was virtually all in leucine, only about half of the ~4C in the 6-day seedling axis was in leucine (Table III). Most of the axis leucine was found in the insoluble (protein) fraction, with a specific activity similar to that of endosperm leucine before germination. The non-leucine labelled
c~ 3c o
"6 2¢
o~ lc
o
o
1
2
3
4
5
6
Days a f t e r germination
Fig. 1. Evolution of ~4CO2 during germination of maize seeds. [U-14C]leucine or lU-14C]lysine was injected below the ear during development, then the seeds were germinated. The ~4CO2 evolved is expressed as percent of the total 14C in the lot of seeds. 0 - - 0 , ~4CO2 from [U-14C]leucine; O - - O , ~4CO2 derived directly or indirectly from [U-~4C]lysine.
358
L. S O D E K , C. M. W I L S O N
compounds in the axis included glutamic acid, proline, and aspartic acid, as well as unidentified compounds which would include starch in the insoluble fraction and sugars and organic acids in the soluble fraction (where 55 ~ of the label was in nonamino acid compounds).
Metabolism of [U-14C]lysine during germination The distributions in the seed, seedling parts, and storage proteins of nitrogen and of 14C derived from injected [U -14C]lysine are shown in Tables IV and V. More than 80 ~o of the 14C in the seed before germination was located in the endosperm, but very little was present in the axis. The major storage proteins, zein and glutelin, were the most highly labelled endosperm fractions. Although zein was relatively less labelled than glutelin, in contrast to the leucine experiment, this can be attributed to the different leucine/lysine contents of the different proteins 6,8. As in the leucine experiment, a high proportion of the endosperm 14C and nitrogen, principally derived from the major storage proteins, was removed from the endosperm during germination. Again, there were increases in percentage (though not absolute amounts) of albumins and globulins, together with an accumulation of 14C in the free amino acid fraction. In contrast to the endosperm, the 14C content of the scutellum changed little after germination. During the 6-day germination period somewhat less I4C derived from lysine was mobilized from the endosperm than was the case for [14C]leucine; only 20 ~/o was respired as CO2 while 29 ~o was recovered in the axis. The pattern of 14CO2 T A B L E IV D I S T R I B U T I O N O F 14C A N D N I T R O G E N IN E N D O S P E R M P R O T E I N F R A C T I O N S B E F O R E A N D A F T E R G E R M I N A T I O N O F SEED C O N T A I N I N G [14C]LYSINE A N D ITS METABOLITES* [~4C]Lysine was injected into the shank of an M I 4 inbred 26 days after pollination, and the ear was harvested at 58 clays. The average dpm (per seed) was 27 840 before germination and 26 410 at six days.
%o of total ~4C and N in endosperm 0 days
Free amino acids Albumin Globulin Zein-I Zein-2 Glutelin Residue d p m / g dry wt dpm/endosperm mg N / g dry wt mg N / e n d o s p e r m mg dry wt/endosperm dpm/scutellum
6 days
14 C
N
~4C
N
4.7 4.8 6.2 21.6 14.0 30.8 18.0
3.9 4.9 42.2 20.6 24.0
30.0 6.8 10.0 12.9 6.0 15.0 19.3
7.5 5.8 34.2 15.0 18.3
130 885 22 800 20.4 3.55 174 5 040
74 480 6 700 12.0 1.08 90 6 300
METABOLISM OF LYSINE AND LEUCINE IN MAIZE
359
TABLE V LABELLING OF AMINO ACIDS AND OTHER COMPOUNDS IN PARTS OF THE CORN SEEDLING BEFORE AND AFTER GERMINATION OF SEED CONTAINING [14C]LYSINE AND ITS METABOLITES* 6 days
0 days
Lysine Glutamic acid Proline Aspartic acid Other amino acids Non-amino acidstt
Endosperm
Scutellum
Axis
%**
Insoluble
Soluble***
lnsolublet
%
dpm/ l~mole
%
dpm/ ttmole
%
dpm/ I~mole
89.5 9.3 9.3 9.3 9.3 9.3
3660
30 42 42 42 42 22
--
77 4 8 2 6 2
1280 43 155 26
dpm/ I~mole
47.5 4320 21.5 177 11.5 165 2.5 77 9.0 4.5
* See Table IV. ** ~4C recovered in fraction as ~ of ~4C applied to column. *** 2160 dpm/part. 5600 dpm/part. tt Probably sugars and organic acids. production (Fig. 1) was similar to that in the leucine experiment. For the purposes of this study, lysine is less suitable than leucine, in that much of the 14C in the endosperm was present in glutamic acid and proline (Table V). Only 47.5 % was in lysine, in line with our previous report 6. In contrast, 89.5 % of the label in the scutellum was in lysine, to further complicate the picture. The proportion of ~4C recovered in lysine in the insoluble axis fraction (77 %) was greater than that originally present in the endosperm, suggesting that glutamate and proline were catabolized to a much greater extent than lysine. In the 6-day axis protein the specific activities of lysine and glutamic acid, but not proline, was considerably less than in the original endosperm. DISCUSSION Amino acid metabolism The amino acid composition of the two fractions of the axis (Table I) are similar to those reported by Oaks t for maize root tips, except that the relative concentrations of glutamate and aspartate were reversed in the soluble fraction. The changes upon germination are similar to those found by Dupuy et al. 2, in that there are large decreases in glutamic acid, proline, leucine, and alanine, and large increases in lysine and aspartic acid. Aspartic acid accumulated mainly in the soluble fraction of the axis, presumably as newly synthesized asparagine. In contrast, proline and leucine are present in very low amounts in the soluble fraction considering the quantity available from the endosperm. Thus the growing seedling must possess an active metabolic system for catabolism of the carbon skeletons of these two amino acids. Glutamate, of
360
L. SODEK, C. M. WILSON
course, is centrally located in well-known metabolic systems. The low level of free lysine is to be expected, and lysine must be synthesized to provide enough for new protein. In the 6-day axis (Table IIl) the specific activity of leucine was not much less than in the endosperm, evidence that leucine from the endosperm was directly incorporated into axis protein, and that the surplus leucine had inhibited leucine synthesis in the axis, as suggested by Oaks 4. Extensive degradation of leucine occurred, for only one-sixth of the 14C transported out of the endosperm was recovered as leucine. Oaks 11 concluded that leucine was not extensively metabolized in excised maize roots, but this was after a 2-h incubation with exogenous [U -14C]leucine. Splittstoesser 12 reported that only 6 % of the metabolized [U -14C]leucine supplied to an excised maize axis was lost as CO2, while 74 o/ /o was recovered from the residue after 24 h. Joy and Folkes 5 also found much more 14C from leucine in protein than in CO 2 after 18 h incubation of excised barley embryos. Beevers and Splittstoesser 13 found somewhat more metabolism of [U -14C]-leucine which was injected into the cotyledons of germinating peas. It seems that experiments with excised maize tissues greatly underestimated the proportion of leucine metabolized to CO2 and other compounds within the normal seedling. A possible explanation is that the isolated tissue, removed from the endosperm, no longer has surplus leucine available for catabolism. The reduced specific activity of [14C]lysine in the axis protein fraction (Table V) shows that the labelled lysine was diluted by newly synthesized unlabelled lysine. However, rough calculations indicate that the 6-day seedlings contained almost as much [14C]lysine as the original seed, suggesting that there was little degradation. The labelled CO2 probably was derived from glutamic acid, proline, and other nonlysine compounds. We may note that the axis protein contained twice as much [14C] proline as [14C]glutamic acid, the reverse of the situation in the endosperm. The specific activities in the seed and in the axis suggest that new, unlabelled glutamic acid was extensively synthesized, while there was little synthesis ofproline. It has previously been reported that proline is rapidly metabolized within the maize root tip, but is not synthesized there 14.15. The metabolic relationships of lysine and leucine to protein synthesis are reversed in the developing maize endosperm and the growing seedling. In the developing endosperm lysine is provided in excess, and [14C]lysine is converted to other amino acids, while [14C]leucine is incorporated into protein without change 6. Assays for free amino acids in the developing endosperms of wheat 16 and corn (Wilson, C. M., unpublished) show low levels of leucine relative to the amount needed for protein synthesis, while [ysine levels are relatively high. In the growing axis, leucine is supplied in excess from storage proteins, whereas the lysine supply is insufficient to meet the needs of protein synthesis (Table I). Here leucine synthesis is restricted 4 and excess ieucine is catabolized, while lysine may be conserved and certainly must be synthesized. The present results are consistent with the amino acid hypothesis of Folkes and Yem 3, that leucine, lysine, and proline liberated in the endosperm are translocated to the embryo and incorporated into new protein in the embryo, and that the amino acids present in excess of requirements are metabolized to other compounds or respired to C02.
In vivo labelling Of storage proteins To our knowledge, the use of seed in which the storage protein amino acids are
METABOLISM OF LYSINE A N D LEUCINE IN MAIZE
361
labelled with t 4C is a new approach to the study of the metabolism of amino acids during germination. Usually labelled amino acids are injected into the storage organ immediately prior to germination 13 or are present in the medium in which the seedlings s or parts thereof 4' 11,12 are incubated. In such experiments there are the dangers of upsetting the steady state or that the exogenous tracer will follow pathways not representative of the normal metabolism of the endogenous compound under investigation 17. The most distinct advantage of the new method is that one can be certain that the labelled amino acid will go through the natural transport and metabolic processes. There are, however, several problems with the new technique which our preliminary experiment revealed: (1) The amino acid administered may undergo metabolic changes before incorporation into the seed reserve protein, i.e. [14C]lysine was extensively converted into [14C]glutamate and [14C]proline in the developing endosperm 6. Less conversion of [14C]lysine may occur in seeds possessing proteins with higher lysine contents, such as opaque-2 maize 6 or most of the non-cereal plants. (2) The scutellum proteins are also labelled, and, in the case of the [14C]lysine experiment, labelling was different from the endosperm. It is not known if turnover of scutellum proteins occurred, with utilization by the growing axis of the amino acids thus released. (3) The injected labelled amino acids were diluted before incorporation into storage protein. The resulting low level of r~.dioactivity made it especially difficult to measure accurately the 14C-content of the non-leucine and non-lysine compounds. Also, the labelled amino acids were supplied to the endosperm over a short period of time by a single injection at 26 days after pollination, about the mid-point of N accumulation. Thus most of the protein of the mature endosperm was formed before and after the ~4C was available. We have assumed that the amino acids were released at a constant specific activity during germination, but some of our interpretations would be in error if there was a change. However, ~4CO2 was released evenly throughout this period, and the specific activities of the endosperm and of the protein fractions were similar before and after germination in the leucine experiment (Table II). Continuous or repeated administration of labelled amino acids through the shank would give higher and more evenly distributed activity. ACKNOWLEDGEMENTS
This study was supported by the Agricultural Research Service, U. S. Department of Agriculture, Cooperative Agreement No. 12-14-100-9359(34), administered by the Plant Science Research Division, Beltsville, Md., and by a grant from the University of Illinois Research Board.
REFERENCES 1 2 3 4
Oaks, A. (1966)Plant Physiol. 41, 173-180 Dupuy, .l'., Boutin, J. and Dupaigne, G. (1971) C. R. Acad. Sci., Ser. D (Paris) 272, 2548-2551 Folkes, B. F. and Yemm, E. W. (1958) New Phytol. 57, 106-131 Oaks, A. (1965) Plant Physiol. 40, 149-155
362 5 6 7 8 9 lO II 12 13 14 15 16 17
L. SODEK, C. M. WILSON
Joy, K. W. and Folkes, B. F. (1965) J. Exp. Bot. 16, 646-666 Sodek, L. and Wilson, C. M. (1970) Arch. Biochem. Biophys. 140, 29-38 Bray, G. A. (1960) Anal. Biochem. l, 279-285 Sodek, L. and Wilson, C. M. (1971) J. Agric. Food Chem. 19, 1144-1150 Moore, S., Spackman, D. H. and Stein, W. H. (1958) Anal. Chem. 30, 1185-1190 Ingle, J., Beevers, L. and Hageman, R. H. (1964) Plant Physiol. 39, 735-740 Oaks, A. (1965)PlantPhysiol. 40, 142-149 Splittstoesser, W. E. (1967) Phytochemistry 6, 933-939 Beevers, L. and Splittstoesser, W. E. (1968) J. Exp. Bot. 19, 698-711 Barnard, R. A. and Oaks, A. (1970) Can. J. Bot. 48, 1155-1158 Oaks, A., Mitchell, D. J., Barnard, R. A. and Johnson, F. J. (1970) Can. J. Bot. 48, 2249-2258 Jennings, A. C. and Morton, R. K. (1963) Austr. J. Biol. Sci. 16, 384-394 Sodek, L. and Wilson, C. M. (1971) Plant CelIPhysiol. 12, 889-893