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The Effect of Embryo Axis, 6-benzylaminopurine and Kinetin on Protein Metabolism in Protein Bodies during Germination of Lentil Seeds
The Effect of Embryo Axis, 6-benzylaminopurine and Kinetin on Protein Metabolism in Protein Bodies during Germination of Lentil Seeds JESUS ALVAREZ, HILARIO GUERRA, LUISA MARTIN
CALVARRO'~)
and NIEVES VILLALOBOS'~)
Departamento de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Salamanca, 37007. Salamanca, Espana
*) Departamento de Fisiologia Vegetal, Facultad de Biologia, Universidad de Salamanca, 37008. Salamanca, Espana Received March 19, 1986· Accepted June 9,1986
Summary Excision of the embryonic axis reduces globulin degradation of lentil (Lens culinaris, Medik. cv Castellana) by 36 %. Of the protease activities detected in the protein bodies of these seeds only carboxypeptidase activity is significantly affected (negatively) by excision of the embryonic axis. Application of 6-benzylaminopurine or kinetin has opposite effects on globulin degradation according to whether it is performed during the germination of intact seeds (reduced degradation) or during the incubation of seeds whose embryonic axis has been excised (increased degradation). However, these cytokinins - increase the proteolytic levels of the protein bodies, both in the presence and the absence of the embryonic axis. Analysis of the endogenous cytokinins in cotyledons reveals their presence in ungerminated seeds together with a decrease in them after 168 hours of germination. Key words: Lens culinaris, Leguminosae, Cytokinins, Protease Activities, Protein Bodies.
Introduction The increase in protease activity, concomitant with the degradation of storage proteins, is a widely accepted fact for the majority of seeds (Ashton, 1976). However, in many cases the specific function of individual proteolytic enzymes and their cooperative actions at successive stages of germination remain to be elucidated. The possible compartmentalization of part of the protease activity of the cotyledons might act as a modulating mechanism (Wilden et aI., 1980), and in this sense the protein bodies would play an important role in the development of protease activity during germination. In many works, attempts have been made to clarify the mechanism by which the embryonic axis controls the mobilization of reserves (Davies and Chapman, 1979; Metivier and Paulilo, 1980). However, few results have been obtained regarding the effect of the embryonic axis on the composition and variation of protein bodies. In other plant species, the results seem to show that the embryonic axis is able to exert Abbreviation list: BA, 6-benzylaminopurine; KIN, Kinetin; MBP, Membrane Bound Proteins; NCBZ, N-carbobenzoxiphenylalanilalanine; TCA, Trichloroacetic Acid.
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its effect on the degradation of storage compounds by acting as a sink for the hydrolysis products (Varner et aI., 1963) or perhaps through the release to the cotyledons of some factor which acts on the normal development of protease activities (Penner and Ashton, 1967; Metivier and Paulilo, 1980. In some legumes, this factor is probably a cytokinin since following exogenous application of these substances it is able to replace the effect of excision of the embryonic axis (Penner and Ashton, 1967; Sze and Ashton, 1971). The aim of the present work was to study the physiology and control of the protease activities present in the protein bodies of lentil cotyledons, studying the control exerted by the embryonic axis on the degradation of storage proteins and the application both to intact seeds and seeds without axis of the phytohormones which most commonly affect proteolysis.
Materials and Methods Plant material Lentil (Lens culinaris Medik., var. Castellana) seeds were bought from growers at Espino de la Orbada (Province of Salamanca, Spain). The seeds were soaked for 20 min in a disinfectant solution (0.1 % Captan) at room temperature and were germinated in the dark at 80% RH on glass plates wrapped in a layer of moist filter paper and introduced into glass-covered seed trays for different periods of time. In some cases the embryonic axis was separated in ungerminated seeds or in seeds germinated for different periods of time, after which the cotyledons were placed in the conditions indicated; in other cases aqueous extracts were made of the embryonic axes excised at different germination times and these extracts were employed as germination media. In order to determine the effect of KIN and BA on the mobilization of proteins in protein bodies, both intact seeds and seeds without axes were germinated or incubated in 1O- 4 M concentrations of these substances under the same conditions as reponed above. To avoid possible bacterial contamination, 750 units of acucilin were added to the germination medium.
Isolation
0/protein bodies
Protein bodies were extracted from cotyledons according to the method, slightly modified, of Huang and Beevers (1971) and the proteins were fractionated according to the criterion of Osborn (1924) into albumins and globulins; following this, the MBP fraction of these organelles were extracted according to the method described in a previous work (Alvarez and Guerra, 1985).
Assays Protein concentrations were measured by the Coomassie blue technique of Bradford (1976). Caseinolytic activity was determined by measuring the TCA-soluble material either by the amounts of amino acids - hydrolysis of substrate into aminoacids - (Yomo and Varner, 1973), or of peptides - hydrolysis of substrate into peptides - (Kunitz, 1947), released at 37°C in a reaction mixture containing 1 % W IV casein as the substrate. Enzyme activity was expressed as units of activity (/Lg of aminoacids released per hour or the amount of enzyme that would increase absorption at 280nm by 0.001 per minute) per seed. Carboxypeptidase activity was measured using N-carbobenzoxiphenylalanilalanine (NCBZ) according to the method of Mikola and Kolhemainen (1972). One of enzyme activity was considered to be the amount of enzyme that would induce an increase in absorption of 0.001 per min at 37°C. Cytokinins were extracted from the cotyledons separating the free and bound fractions, according to the method of Smith and Van Staden (1978) and were evaluated according to the bio-
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Control of protein metabolism in protein bodies of lentil seeds
321
22 A
18
14
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.,
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0
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. - - 0 _____ - - - - - _____ 0 _ _ _ ___________ 0 0 0 0
o
24
48
72
96
120
144
Germination
168
(Hours)
Fig. 1: Variations of carboxypeptidase activity in protein bodies (closed symbols) and effect of cycloheximide (open symbols) during germination of lentil seeds. A: albumin fraction, B: globulin fraction and C: MBP fraction. Values are the means of four separate experiments with four determinations in each. The standard deviation of the indicated values does not exceed 10%. assay based on induction of chlorophyll formation in etiolated cotyledons of Cucumis sativa cv Calahorra (Fletcher et aI., 1982). Results were expressed in ng of Zeatin per cotyledon by a straight Zeatin standard taken under the same conditions.
Results and Discussion
Carboxypeptidase activity In earlier studies it was observed that the three protein fractions (albumins, globulins, and MBP) of protein bodies isolated as described elsewhere did not show any
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A
B
14
10
6
2
24
72
120 Germination
(hours)
168
24
72
120
168
Germination (hours)
Fig. 2: Changes in the globulin content of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 10- 4 M BA (triangles). Values are the means of five separate experiments with four determinations in each. The standard deviation of the indicated values does not exceed 12 %.
cross contamination, and that globulins constitute the principal fraction of storage proteins in the protein bodies (Alvarez and Guerra, 1985). In protein bodies from lentil seeds there is a certain carboxypeptidase activity which increases during germination and reaches its maximum level on the 5th day (Fig. 1). This protease is mainly present in the albumin fraction since approximately 70 % of the carboxypeptidase activity detected in the protein bodies is present in this protein fraction. In the globulin fraction, carboxypeptidase activity is largely absent and hardly undergoes any variations during the period studied. However, in the MBP fraction ca. 24 % of the carboxypeptidase activity is present and between day 3 and 5 undergoes a similar, though less pronounced rise than in the albumin fraction. Incubation of the seeds in the presence of cycloheximide abolished the rise, suggesting that under normal conditions de novo synthesis of enzyme takes place as from the first day of germination. Carboxypeptidase activity, and caseinolytic activity (Alvarez and Guerra, 1985) are the only activities so far discovered in lentil seeds (Guerra and NicoLis, 1983) which are located within the protein bodies and which appear to be responsible for the degradation of storage proteins compartmentalized in these organelles. Several proteolytic enzymes have been found in protein bodies of other species (Wilden et aI., 1980; Harris and Chrispeels, 1975; and Morris et aI., 1970). In the cotyledons of germinating mung beans an increase was detected in carboxypeptidase activity from the first day of germination (Chrispeels and Boulter, 1975), in agreement with our findings. In other cases, however, high levels of exoprotease activities, which decrease during germination, have been reported in ungerminated seeds (Preston and Kruger, 1976 and Tully and Beevers, 1978).
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323
B
168
120
72
24
168
120
72
24
Germination
Germination
(hours)
(hours)
Fig.3: Changes in carboxypeptidase activity of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 10- 4 BA (triangles). Values are the means of five separate experiments with four determinations in each. The standard deviation of the indicated values does not exceed 12%. Table 1: Effect of excision of the embryonic axis on carboxypeptidase activity in protein bodies of lentil seeds. Values are the means ± standard deviation of five separate experiments with four determinations in each. Time of excision of embryonic axis (hours) no excIsIon 120 96 72 48 24 0
Enzyme activity (units/seed) Incubation (hours) 72 96
120
168
31.5± 1.3
38.5± 1.3
27.0±2.1 23.5±2.1 24.5±2.5 19.3±2.0 19.2±2.0 19.3±2.0 16.4± 1.5 15.5± 1.4 20.3±2.1
38.0±3.9 28.1±2.9 25.0±2.1 22.2±2.3 21.0±2.1
34.4±2.2 37.8±2.1 37.9±2.5 27.9±2.4 25.4±2.1 22.3 ±2.1 20.9±2.0
0
24
48
16.4± 1.0
19.2± 1.2
23.2± 1.3
16.9± 1.5
23.9± 1.3
Effict 0/ the embryonic axis
In intact seeds about 50 % of the globulins from protein bodies is degraded by the 7th day of germination (Fig. 2 A). Excision of the embryonic axis reduces this degradation - by 36 % (Fig. 2 B). Variations in the degradation of storage proteins caused by excision of the embryonic axis has been described for many other kinds of seeds such as the mung bean (Kern and Chrispeeles, 1978), or Cucumis sativa (Davies and Chapman, 1979). The mechanism by which the embryonic axis acts on proteolysis remains unclear, or is a matter for contention, owing to the great variability in the results obtained. This effect of the embryonic axis may be due to the fact that in its absence the levels of activity of the proteases responsible for globulin degrada-
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JESUS ALVAREZ
18
et al.
A
B
A_A-A A/._.-·
14
10
6
2
-.-.--
/./
/~ .........
~~
.-.
;I'" . "
•
I
24
72
120
24
168
72
120
Germination
Germination
(hours)
(hours)
168
Fig. 4: Changes in caseinolytic activity (amino acids released from the casein molecule) of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 1O- 4 M BA (triangles). Values are the means of five separate experiments with five determinations in each. The standard deviations of the indicated values does not exceed 12 %.
B
A 175
125 75
25 24
72
120
168
24
72
120
Germination
Germination
(hours)
(hours)
168
Fig. 5: Changes in caseinolytic activity (peptides released from the casein molecule) of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 1O- 4 M BA (triangles). Values are the means of five separate experiments with five determinations in each. The standard deviation of the indicated values does not exceed 12 %.
tion are altered, as has been described for the pea (Guardiola and Suttcliffe, 1971; Yomo and Varner, 1973) and Vigna species (Kern and Chrispeels, 1978; Morohashi, 1982). Of the two activities studied by us, only carboxypeptidase levels were clearly reduced by excision of the embryonic axis (Fig. 3). The axis is thus seen to be consistently essential for enzyme activity to develop normally, since its removal at different
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Control of protein metabolism in protein bodies of lentil seeds
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Table 2: Effect of the embryonic extracts on the protease activities of protein bodies from lentil cotyledons. Values are the means ± standard deviation of three separate experiments with five determinations in each. Germination Intact seeds (hours)
Seeds without Incubation aXIs axis 5h
axis 24h
Carboxypeptidase activity 96 120 (units!cotyledon)
15.75± 0.67 10.15±0.25 19.25± 0.73 10.50±0.34
13.40 ± 0.75 14.60±0.74
14.10±0.49 IS.70±0.63
Caseinolytic activity (Amino acids released) (units! cotyledon)
96 120
15.60± 0.99 13.80±0.83 14.80± 1.13 13.60±0.S2
13.90±0.89 13.40 ± 0.50
14.60±0.81 13.50±0.51
Caseinolytic activity (Amino acids released) (units! cotyledon)
96 120
175 ±8 200 ±9
220 ±11 225 ± 5
189 ±8 206 ±6
180 ±9 201 ±9
1400
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Fig.6: Changes in endogenous levels of cytokinins from cotyledons during germination of lentil seeds. A: total cytokinins. B: (.) bound cytokinins and (0) free cytokinins. Values are the means of two separate experiments with ten determinations in each. The standard deviation of the indicated values does not exceed 15 %.
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germination times immediately leads to constant levels of carboxypeptidase activity (Table 1). Moreover, caseinolytic activity as evaluated by its two forms (hydrolysis of the casein molecule into amino acids or into peptides) shows a slight reduction (Figs. 4 and 5) which varies between 11 % and 12 %, according to the form employed for evaluation. It is possible that the embryonic axis releases some substance to the cotyledons since extracts of 24 h-old embryonic axes partially invert the effect induced on carboxypeptidase activity by removal of the embryonic axis (Table 2), similar to what has been proposed for the pumpkin by Penner and Ashton (1967).
Effect of BA and KIN Fig. 2 shows the variations in globulins of the protein bodies, both during germination of intact seeds (Fig. 2 A) and during incubation of seeds without their embryonic axes (Fig. 2 B), following the application of BA and after KIN. The effect of cytokin ins is dependent on the presence of the embryonic axis; hence, if application is performed with intact seeds the rate of globulin mobilization is reduced on the 7th day by 83 % for BA and by 80 % for KIN. Contrariwise, if cytokinins are applied during the germination of seeds without their embryonic axis, the rates of degradation are even greater than those reached in intact seedlings germinated in water. However, both cytokinins caused increases in the levels of protease activity of the protein bodies, regardless of the presence of the embryonic axis. This effect was more pronounced for carboxypeptidase activity (Fig. 2 A) and for caseinolytic activity as evaluated by the hydrolysis of substrate into amino acids (Fig. 3). In many seeds growth substances have been proposed as regulatory factors for the degradation of storage proteins. In cereals, these substances usually belong to the gibberellin group (Bewley and Black, 1984), whereas in legumes this regulatory function seems to be carried out by cytokinins, since both BA (Penner and Ashton, 1967; Sze and Ashton, 1971) and KIN (Gepstain and Ilan, 1970) have been described as the promoting factors of a number of enzyme systems. As suggested by Metivier and Paulilo (1980) for seeds of Phaseolus vulgaris, it is possible that the effect of cytokinins takes place through the stimulation of protease biosynthesis. However it is difficult to account for the reduction in globulin degradation during the germination of intact seeds in the presence of BA or KIN, in spite of the strong increase in protease activities. It is possible that in some unknown way the action of the enzymes on the substrate is blocked. A similar finding has been described for the a-amylases of lentil cotyledons (Revilla and Fernandez-Tarrago, 1986). The levels of this enzymatic activity reach much higher values when isolated cotyledons are incubated than with intact seeds and yet no starch degradation takes place.
Endogenous levels of cytokinins The variation in the endogenous levels of total cytokinins is shown in Fig. 5 A. Cotyledons of ungerminated seeds, have considerable amounts of cytokinins which increase between 24 - 48 h and later decrease until 168 h. The bound and free cyto-
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Control of protein metabolism in protein bodies of lentil seeds
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kinins (Fig. 5 B) vary differently during germination, the former maintaining their levels practically constant during the first 48 h, after wich they decrease until 168 h, whereas the latter reach peak values on the 2nd day, thereafter decreasing until the 4th day, after which they increase again until 168 h. These results point to a possible transformation of bound into free cytokinins; such a change has been reported for maize seeds Gulin-Tegelman, 1979). The fact that the total cytokinins reach peak values on the 2nd day (this being immediately prior to the moment of maximum protease .activity) points to the importance of these substances in the mobilization of storage proteins. Acknowledgements The authors are grateful to Prof. J. Fernandez Tarrago for his useful suggestions. This work was supported by a grant from the «Comisi6n Asesora de Investigaci6n Cientffica y Tecnica».
References ALVAREZ, J. and H. GUERRA: Biochemical and morphological changes in protein bodies during germination of lentil seeds. J. Exp. Bot. 36, 1296-1303 (1985). ASHTON, F. M.: Mobilization of storage protein of seeds. Ann. Rev. Plant Physiology 27, 95117 (1976). BEWLEY, J. D. and M. BLACK: Seeds: physiology of development and germination. Plenum Press. New York (1984). BRADFORD, M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Ana!. Biochem. 72, 248-254 (1976). CHRISPEELS, M. J. and D. BOULTER: Control of storage protein metabolism in the cotyledons of germinating mung beans: A role of endopeptidase. Plant Physio!. 55, 1031-1037 (1975). DAVIES, H. V. andJ. M. CHAPMAN: The control of food mobilization in seeds of Cucumis sativus L. I. The influence of embryonic axis and testa on protein and lipid degradation. Planta 146, 579-584 (1979). FLETCHER, R. A., W. KALLIDUMBILL, and P. STEELE: An improved bioassay for cytokinins using cucumber cotyledons. Plant Physio!. 69, 675-677 (1982). GEPSTAIN, S. and I. ILAN: A promotive action of kinetin on amylase activity in cotyledons of Phaseolus vulgaris. Plant and Cell Physio!. 11, 819-822 (1970). GUARDIOLA, J. L. and J. F. SUTTCLIFFE: Control of protein hydrolysis in the cotyledons of germinating pea (Pisum sativum L.) seeds. Ann. Bot. 35,791-807 (1971). GUERRA, H. and G. NICOLAS: Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils. Rev. Esp. Fisio!. 39,277 -282 (1983). HARRIS, N. and M. J. CHRISPEELS: Histochemical and biochemical observations of storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. Plant Physio!. 56, 292-299 (1975). HUANG, A. H. C. and H. BEEVERS: Isolation of microbodies from plant tissues. Plant Physio!. 48,637-641 (1971). JULIN-TEGELMAN, A.: The changes in endogenous cytokinin-like substances in Zea mays seeds during germination. Plant Sci. Lett. 14, 259-262 (1979). KERN, R. and M. J. CHRISPEELS: Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings. Plant Physio!. 62, 815-819 (1978). KUNITZ, M.: Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physio!. 30, 291-310 (1947). METIVIER, J. M. T. PAULILO: The utilization of cotyledonary reserves in Phaseolus vulgaris L. cv. Carioca. J. Exp. Bot. 31, 1271-1282 (1980).
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MIKOLA, ]. and L. KOLHEMAINER: Localization and activity of various peptidases in germinating barley. Planta 104, 167 -177 (1972). MOROHASHI, Y.: Control of development of amylolytic and proteolytic activities in cotyledons of germinating black gram seeds. Plant Physiol. 56, 189-193 (1982). MORRIs, G. F. 1., D. A. THURMAN, and D. BOULTER: The extraction and chemical composition of aleurone gains (protein bodies) isolated from seeds of Vicia faba. Phytochemistry 9, 1707 -1714 (1970). OSBORN, T. B.: The vegetable proteins. Longmans, Green. New York. (New York) (1924). PENNER, D. and F. M. ASHTON: Hormonal control of proteinase activity in squash cotyledons. Plant Physiol. 42, 791-796 (1967). PRESTON, K. P.and J. E. KRUGER: Purification and properties of two proteolytic enzymes with carboxypeptidase activity in germinating wheat. Plant Physiol. 58, 516-520 (1976). REVILLA, M. A. and]. FERNANDEZ-TARRAGO: The effect of the seed coat, embryonic axis and aeration conditions on starch degradation in cotyledons of Lens culinaris.Plant Physiol. In press. (1986). SMITH, A. R. and J . VAN STADEN: Changes in endogenous cytokinin levels in kernels of Zea mays L. during imbibition and germination. ]. Exp. Bot. 29, 1067 -1075 (1978). SZE, H. and F. M. ASHTON: Dipeptidase development in cotyledons in Cucurbita maxima during germination. Phytochemistry 10, 2935-2942 (1971). TULLY, R. E. and H. BEEVERS: Proteases and peptidases of castor bean endosperm enzyme characterization and changes during germination. Plant Physiol. 62, 746-750 (1978). VARNER, ]. E., L. V. BALLE, and R. C. HUANG: Senescence of cotyledons of germinating peans. Influence of axis tissue. Plant Physiol. 38, 89-92 (1963). WILDEN, W. VAN DER, E. M. HERMAN, and M. J. CHRISPEELS: Protein bodies of mung bean cotyledons as autophagic organelles. Proc. Nat!. Acad. Sci. 77, 428-432 (1980). YOMO, H. and J. E. VARNER: Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol. 51, 708-713 (1973).
J Plant Physiol.
Vol. 126. pp. 319-328 (1987)
CALVARRO'~)
and NIEVES VILLALOBOS'~)
Departamento de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Salamanca, 37007. Salamanca, Espana
*) Departamento de Fisiologia Vegetal, Facultad de Biologia, Universidad de Salamanca, 37008. Salamanca, Espana Received March 19, 1986· Accepted June 9,1986
Summary Excision of the embryonic axis reduces globulin degradation of lentil (Lens culinaris, Medik. cv Castellana) by 36 %. Of the protease activities detected in the protein bodies of these seeds only carboxypeptidase activity is significantly affected (negatively) by excision of the embryonic axis. Application of 6-benzylaminopurine or kinetin has opposite effects on globulin degradation according to whether it is performed during the germination of intact seeds (reduced degradation) or during the incubation of seeds whose embryonic axis has been excised (increased degradation). However, these cytokinins - increase the proteolytic levels of the protein bodies, both in the presence and the absence of the embryonic axis. Analysis of the endogenous cytokinins in cotyledons reveals their presence in ungerminated seeds together with a decrease in them after 168 hours of germination. Key words: Lens culinaris, Leguminosae, Cytokinins, Protease Activities, Protein Bodies.
Introduction The increase in protease activity, concomitant with the degradation of storage proteins, is a widely accepted fact for the majority of seeds (Ashton, 1976). However, in many cases the specific function of individual proteolytic enzymes and their cooperative actions at successive stages of germination remain to be elucidated. The possible compartmentalization of part of the protease activity of the cotyledons might act as a modulating mechanism (Wilden et aI., 1980), and in this sense the protein bodies would play an important role in the development of protease activity during germination. In many works, attempts have been made to clarify the mechanism by which the embryonic axis controls the mobilization of reserves (Davies and Chapman, 1979; Metivier and Paulilo, 1980). However, few results have been obtained regarding the effect of the embryonic axis on the composition and variation of protein bodies. In other plant species, the results seem to show that the embryonic axis is able to exert Abbreviation list: BA, 6-benzylaminopurine; KIN, Kinetin; MBP, Membrane Bound Proteins; NCBZ, N-carbobenzoxiphenylalanilalanine; TCA, Trichloroacetic Acid.
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Vol. 126. pp. 319-328 (1987)
320
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its effect on the degradation of storage compounds by acting as a sink for the hydrolysis products (Varner et aI., 1963) or perhaps through the release to the cotyledons of some factor which acts on the normal development of protease activities (Penner and Ashton, 1967; Metivier and Paulilo, 1980. In some legumes, this factor is probably a cytokinin since following exogenous application of these substances it is able to replace the effect of excision of the embryonic axis (Penner and Ashton, 1967; Sze and Ashton, 1971). The aim of the present work was to study the physiology and control of the protease activities present in the protein bodies of lentil cotyledons, studying the control exerted by the embryonic axis on the degradation of storage proteins and the application both to intact seeds and seeds without axis of the phytohormones which most commonly affect proteolysis.
Materials and Methods Plant material Lentil (Lens culinaris Medik., var. Castellana) seeds were bought from growers at Espino de la Orbada (Province of Salamanca, Spain). The seeds were soaked for 20 min in a disinfectant solution (0.1 % Captan) at room temperature and were germinated in the dark at 80% RH on glass plates wrapped in a layer of moist filter paper and introduced into glass-covered seed trays for different periods of time. In some cases the embryonic axis was separated in ungerminated seeds or in seeds germinated for different periods of time, after which the cotyledons were placed in the conditions indicated; in other cases aqueous extracts were made of the embryonic axes excised at different germination times and these extracts were employed as germination media. In order to determine the effect of KIN and BA on the mobilization of proteins in protein bodies, both intact seeds and seeds without axes were germinated or incubated in 1O- 4 M concentrations of these substances under the same conditions as reponed above. To avoid possible bacterial contamination, 750 units of acucilin were added to the germination medium.
Isolation
0/protein bodies
Protein bodies were extracted from cotyledons according to the method, slightly modified, of Huang and Beevers (1971) and the proteins were fractionated according to the criterion of Osborn (1924) into albumins and globulins; following this, the MBP fraction of these organelles were extracted according to the method described in a previous work (Alvarez and Guerra, 1985).
Assays Protein concentrations were measured by the Coomassie blue technique of Bradford (1976). Caseinolytic activity was determined by measuring the TCA-soluble material either by the amounts of amino acids - hydrolysis of substrate into aminoacids - (Yomo and Varner, 1973), or of peptides - hydrolysis of substrate into peptides - (Kunitz, 1947), released at 37°C in a reaction mixture containing 1 % W IV casein as the substrate. Enzyme activity was expressed as units of activity (/Lg of aminoacids released per hour or the amount of enzyme that would increase absorption at 280nm by 0.001 per minute) per seed. Carboxypeptidase activity was measured using N-carbobenzoxiphenylalanilalanine (NCBZ) according to the method of Mikola and Kolhemainen (1972). One of enzyme activity was considered to be the amount of enzyme that would induce an increase in absorption of 0.001 per min at 37°C. Cytokinins were extracted from the cotyledons separating the free and bound fractions, according to the method of Smith and Van Staden (1978) and were evaluated according to the bio-
J. Plant Physiol.
Vol. 126. pp. 319-328 (1987)
Control of protein metabolism in protein bodies of lentil seeds
321
22 A
18
14
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. - - 0 _____ - - - - - _____ 0 _ _ _ ___________ 0 0 0 0
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24
48
72
96
120
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Germination
168
(Hours)
Fig. 1: Variations of carboxypeptidase activity in protein bodies (closed symbols) and effect of cycloheximide (open symbols) during germination of lentil seeds. A: albumin fraction, B: globulin fraction and C: MBP fraction. Values are the means of four separate experiments with four determinations in each. The standard deviation of the indicated values does not exceed 10%. assay based on induction of chlorophyll formation in etiolated cotyledons of Cucumis sativa cv Calahorra (Fletcher et aI., 1982). Results were expressed in ng of Zeatin per cotyledon by a straight Zeatin standard taken under the same conditions.
Results and Discussion
Carboxypeptidase activity In earlier studies it was observed that the three protein fractions (albumins, globulins, and MBP) of protein bodies isolated as described elsewhere did not show any
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A
B
14
10
6
2
24
72
120 Germination
(hours)
168
24
72
120
168
Germination (hours)
Fig. 2: Changes in the globulin content of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 10- 4 M BA (triangles). Values are the means of five separate experiments with four determinations in each. The standard deviation of the indicated values does not exceed 12 %.
cross contamination, and that globulins constitute the principal fraction of storage proteins in the protein bodies (Alvarez and Guerra, 1985). In protein bodies from lentil seeds there is a certain carboxypeptidase activity which increases during germination and reaches its maximum level on the 5th day (Fig. 1). This protease is mainly present in the albumin fraction since approximately 70 % of the carboxypeptidase activity detected in the protein bodies is present in this protein fraction. In the globulin fraction, carboxypeptidase activity is largely absent and hardly undergoes any variations during the period studied. However, in the MBP fraction ca. 24 % of the carboxypeptidase activity is present and between day 3 and 5 undergoes a similar, though less pronounced rise than in the albumin fraction. Incubation of the seeds in the presence of cycloheximide abolished the rise, suggesting that under normal conditions de novo synthesis of enzyme takes place as from the first day of germination. Carboxypeptidase activity, and caseinolytic activity (Alvarez and Guerra, 1985) are the only activities so far discovered in lentil seeds (Guerra and NicoLis, 1983) which are located within the protein bodies and which appear to be responsible for the degradation of storage proteins compartmentalized in these organelles. Several proteolytic enzymes have been found in protein bodies of other species (Wilden et aI., 1980; Harris and Chrispeels, 1975; and Morris et aI., 1970). In the cotyledons of germinating mung beans an increase was detected in carboxypeptidase activity from the first day of germination (Chrispeels and Boulter, 1975), in agreement with our findings. In other cases, however, high levels of exoprotease activities, which decrease during germination, have been reported in ungerminated seeds (Preston and Kruger, 1976 and Tully and Beevers, 1978).
J. Plant Physiol. Vol.
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Control of protein metabolism in protein bodies of lentil seeds
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B
168
120
72
24
168
120
72
24
Germination
Germination
(hours)
(hours)
Fig.3: Changes in carboxypeptidase activity of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 10- 4 BA (triangles). Values are the means of five separate experiments with four determinations in each. The standard deviation of the indicated values does not exceed 12%. Table 1: Effect of excision of the embryonic axis on carboxypeptidase activity in protein bodies of lentil seeds. Values are the means ± standard deviation of five separate experiments with four determinations in each. Time of excision of embryonic axis (hours) no excIsIon 120 96 72 48 24 0
Enzyme activity (units/seed) Incubation (hours) 72 96
120
168
31.5± 1.3
38.5± 1.3
27.0±2.1 23.5±2.1 24.5±2.5 19.3±2.0 19.2±2.0 19.3±2.0 16.4± 1.5 15.5± 1.4 20.3±2.1
38.0±3.9 28.1±2.9 25.0±2.1 22.2±2.3 21.0±2.1
34.4±2.2 37.8±2.1 37.9±2.5 27.9±2.4 25.4±2.1 22.3 ±2.1 20.9±2.0
0
24
48
16.4± 1.0
19.2± 1.2
23.2± 1.3
16.9± 1.5
23.9± 1.3
Effict 0/ the embryonic axis
In intact seeds about 50 % of the globulins from protein bodies is degraded by the 7th day of germination (Fig. 2 A). Excision of the embryonic axis reduces this degradation - by 36 % (Fig. 2 B). Variations in the degradation of storage proteins caused by excision of the embryonic axis has been described for many other kinds of seeds such as the mung bean (Kern and Chrispeeles, 1978), or Cucumis sativa (Davies and Chapman, 1979). The mechanism by which the embryonic axis acts on proteolysis remains unclear, or is a matter for contention, owing to the great variability in the results obtained. This effect of the embryonic axis may be due to the fact that in its absence the levels of activity of the proteases responsible for globulin degrada-
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324
JESUS ALVAREZ
18
et al.
A
B
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72
120
Germination
Germination
(hours)
(hours)
168
Fig. 4: Changes in caseinolytic activity (amino acids released from the casein molecule) of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 1O- 4 M BA (triangles). Values are the means of five separate experiments with five determinations in each. The standard deviations of the indicated values does not exceed 12 %.
B
A 175
125 75
25 24
72
120
168
24
72
120
Germination
Germination
(hours)
(hours)
168
Fig. 5: Changes in caseinolytic activity (peptides released from the casein molecule) of protein bodies during germination of intact seeds (A) or during incubation of seeds without axis (B), in water (circles), in 1O- 4 M KIN (squares) and in 1O- 4 M BA (triangles). Values are the means of five separate experiments with five determinations in each. The standard deviation of the indicated values does not exceed 12 %.
tion are altered, as has been described for the pea (Guardiola and Suttcliffe, 1971; Yomo and Varner, 1973) and Vigna species (Kern and Chrispeels, 1978; Morohashi, 1982). Of the two activities studied by us, only carboxypeptidase levels were clearly reduced by excision of the embryonic axis (Fig. 3). The axis is thus seen to be consistently essential for enzyme activity to develop normally, since its removal at different
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Table 2: Effect of the embryonic extracts on the protease activities of protein bodies from lentil cotyledons. Values are the means ± standard deviation of three separate experiments with five determinations in each. Germination Intact seeds (hours)
Seeds without Incubation aXIs axis 5h
axis 24h
Carboxypeptidase activity 96 120 (units!cotyledon)
15.75± 0.67 10.15±0.25 19.25± 0.73 10.50±0.34
13.40 ± 0.75 14.60±0.74
14.10±0.49 IS.70±0.63
Caseinolytic activity (Amino acids released) (units! cotyledon)
96 120
15.60± 0.99 13.80±0.83 14.80± 1.13 13.60±0.S2
13.90±0.89 13.40 ± 0.50
14.60±0.81 13.50±0.51
Caseinolytic activity (Amino acids released) (units! cotyledon)
96 120
175 ±8 200 ±9
220 ±11 225 ± 5
189 ±8 206 ±6
180 ±9 201 ±9
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Fig.6: Changes in endogenous levels of cytokinins from cotyledons during germination of lentil seeds. A: total cytokinins. B: (.) bound cytokinins and (0) free cytokinins. Values are the means of two separate experiments with ten determinations in each. The standard deviation of the indicated values does not exceed 15 %.
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germination times immediately leads to constant levels of carboxypeptidase activity (Table 1). Moreover, caseinolytic activity as evaluated by its two forms (hydrolysis of the casein molecule into amino acids or into peptides) shows a slight reduction (Figs. 4 and 5) which varies between 11 % and 12 %, according to the form employed for evaluation. It is possible that the embryonic axis releases some substance to the cotyledons since extracts of 24 h-old embryonic axes partially invert the effect induced on carboxypeptidase activity by removal of the embryonic axis (Table 2), similar to what has been proposed for the pumpkin by Penner and Ashton (1967).
Effect of BA and KIN Fig. 2 shows the variations in globulins of the protein bodies, both during germination of intact seeds (Fig. 2 A) and during incubation of seeds without their embryonic axes (Fig. 2 B), following the application of BA and after KIN. The effect of cytokin ins is dependent on the presence of the embryonic axis; hence, if application is performed with intact seeds the rate of globulin mobilization is reduced on the 7th day by 83 % for BA and by 80 % for KIN. Contrariwise, if cytokinins are applied during the germination of seeds without their embryonic axis, the rates of degradation are even greater than those reached in intact seedlings germinated in water. However, both cytokinins caused increases in the levels of protease activity of the protein bodies, regardless of the presence of the embryonic axis. This effect was more pronounced for carboxypeptidase activity (Fig. 2 A) and for caseinolytic activity as evaluated by the hydrolysis of substrate into amino acids (Fig. 3). In many seeds growth substances have been proposed as regulatory factors for the degradation of storage proteins. In cereals, these substances usually belong to the gibberellin group (Bewley and Black, 1984), whereas in legumes this regulatory function seems to be carried out by cytokinins, since both BA (Penner and Ashton, 1967; Sze and Ashton, 1971) and KIN (Gepstain and Ilan, 1970) have been described as the promoting factors of a number of enzyme systems. As suggested by Metivier and Paulilo (1980) for seeds of Phaseolus vulgaris, it is possible that the effect of cytokinins takes place through the stimulation of protease biosynthesis. However it is difficult to account for the reduction in globulin degradation during the germination of intact seeds in the presence of BA or KIN, in spite of the strong increase in protease activities. It is possible that in some unknown way the action of the enzymes on the substrate is blocked. A similar finding has been described for the a-amylases of lentil cotyledons (Revilla and Fernandez-Tarrago, 1986). The levels of this enzymatic activity reach much higher values when isolated cotyledons are incubated than with intact seeds and yet no starch degradation takes place.
Endogenous levels of cytokinins The variation in the endogenous levels of total cytokinins is shown in Fig. 5 A. Cotyledons of ungerminated seeds, have considerable amounts of cytokinins which increase between 24 - 48 h and later decrease until 168 h. The bound and free cyto-
J. Plant Physiol. Vol.
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Control of protein metabolism in protein bodies of lentil seeds
327
kinins (Fig. 5 B) vary differently during germination, the former maintaining their levels practically constant during the first 48 h, after wich they decrease until 168 h, whereas the latter reach peak values on the 2nd day, thereafter decreasing until the 4th day, after which they increase again until 168 h. These results point to a possible transformation of bound into free cytokinins; such a change has been reported for maize seeds Gulin-Tegelman, 1979). The fact that the total cytokinins reach peak values on the 2nd day (this being immediately prior to the moment of maximum protease .activity) points to the importance of these substances in the mobilization of storage proteins. Acknowledgements The authors are grateful to Prof. J. Fernandez Tarrago for his useful suggestions. This work was supported by a grant from the «Comisi6n Asesora de Investigaci6n Cientffica y Tecnica».
References ALVAREZ, J. and H. GUERRA: Biochemical and morphological changes in protein bodies during germination of lentil seeds. J. Exp. Bot. 36, 1296-1303 (1985). ASHTON, F. M.: Mobilization of storage protein of seeds. Ann. Rev. Plant Physiology 27, 95117 (1976). BEWLEY, J. D. and M. BLACK: Seeds: physiology of development and germination. Plenum Press. New York (1984). BRADFORD, M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Ana!. Biochem. 72, 248-254 (1976). CHRISPEELS, M. J. and D. BOULTER: Control of storage protein metabolism in the cotyledons of germinating mung beans: A role of endopeptidase. Plant Physio!. 55, 1031-1037 (1975). DAVIES, H. V. andJ. M. CHAPMAN: The control of food mobilization in seeds of Cucumis sativus L. I. The influence of embryonic axis and testa on protein and lipid degradation. Planta 146, 579-584 (1979). FLETCHER, R. A., W. KALLIDUMBILL, and P. STEELE: An improved bioassay for cytokinins using cucumber cotyledons. Plant Physio!. 69, 675-677 (1982). GEPSTAIN, S. and I. ILAN: A promotive action of kinetin on amylase activity in cotyledons of Phaseolus vulgaris. Plant and Cell Physio!. 11, 819-822 (1970). GUARDIOLA, J. L. and J. F. SUTTCLIFFE: Control of protein hydrolysis in the cotyledons of germinating pea (Pisum sativum L.) seeds. Ann. Bot. 35,791-807 (1971). GUERRA, H. and G. NICOLAS: Changes in nitrogen fractions and proteolytic activities in the cotyledons of germinating lentils. Rev. Esp. Fisio!. 39,277 -282 (1983). HARRIS, N. and M. J. CHRISPEELS: Histochemical and biochemical observations of storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. Plant Physio!. 56, 292-299 (1975). HUANG, A. H. C. and H. BEEVERS: Isolation of microbodies from plant tissues. Plant Physio!. 48,637-641 (1971). JULIN-TEGELMAN, A.: The changes in endogenous cytokinin-like substances in Zea mays seeds during germination. Plant Sci. Lett. 14, 259-262 (1979). KERN, R. and M. J. CHRISPEELS: Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings. Plant Physio!. 62, 815-819 (1978). KUNITZ, M.: Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physio!. 30, 291-310 (1947). METIVIER, J. M. T. PAULILO: The utilization of cotyledonary reserves in Phaseolus vulgaris L. cv. Carioca. J. Exp. Bot. 31, 1271-1282 (1980).
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MIKOLA, ]. and L. KOLHEMAINER: Localization and activity of various peptidases in germinating barley. Planta 104, 167 -177 (1972). MOROHASHI, Y.: Control of development of amylolytic and proteolytic activities in cotyledons of germinating black gram seeds. Plant Physiol. 56, 189-193 (1982). MORRIs, G. F. 1., D. A. THURMAN, and D. BOULTER: The extraction and chemical composition of aleurone gains (protein bodies) isolated from seeds of Vicia faba. Phytochemistry 9, 1707 -1714 (1970). OSBORN, T. B.: The vegetable proteins. Longmans, Green. New York. (New York) (1924). PENNER, D. and F. M. ASHTON: Hormonal control of proteinase activity in squash cotyledons. Plant Physiol. 42, 791-796 (1967). PRESTON, K. P.and J. E. KRUGER: Purification and properties of two proteolytic enzymes with carboxypeptidase activity in germinating wheat. Plant Physiol. 58, 516-520 (1976). REVILLA, M. A. and]. FERNANDEZ-TARRAGO: The effect of the seed coat, embryonic axis and aeration conditions on starch degradation in cotyledons of Lens culinaris.Plant Physiol. In press. (1986). SMITH, A. R. and J . VAN STADEN: Changes in endogenous cytokinin levels in kernels of Zea mays L. during imbibition and germination. ]. Exp. Bot. 29, 1067 -1075 (1978). SZE, H. and F. M. ASHTON: Dipeptidase development in cotyledons in Cucurbita maxima during germination. Phytochemistry 10, 2935-2942 (1971). TULLY, R. E. and H. BEEVERS: Proteases and peptidases of castor bean endosperm enzyme characterization and changes during germination. Plant Physiol. 62, 746-750 (1978). VARNER, ]. E., L. V. BALLE, and R. C. HUANG: Senescence of cotyledons of germinating peans. Influence of axis tissue. Plant Physiol. 38, 89-92 (1963). WILDEN, W. VAN DER, E. M. HERMAN, and M. J. CHRISPEELS: Protein bodies of mung bean cotyledons as autophagic organelles. Proc. Nat!. Acad. Sci. 77, 428-432 (1980). YOMO, H. and J. E. VARNER: Control of the formation of amylases and proteases in the cotyledons of germinating peas. Plant Physiol. 51, 708-713 (1973).
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Vol. 126. pp. 319-328 (1987)