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The Function of the Pod at Protein Storage in the Seeds of Vicia faba L.
Biochem. Physiol. Pflanzen (BPP), Bd. 166, S. 87 -93 (1974)
Zentralinstitut fiir Genetik und Kulturpflanzenforschung Gatersleben des Forschungszentrums fiir Molekularbiologie und Medizin der Akademie der Wissenschaften der DDR
The Function of the Pod at Protein Storage in the Seeds of Vicia Jaba L. III. Nitrate Reductase in Developing Pods and Seeds of Legu.minosae
By G.
SCHLESIER
and K.
MUNTZ
With 3 figures (Received February 22, 1974) Key Term Index: storage protein, pods, seeds, nitrate reductase, ontogenesis; Vicia faba, Phaseolus vulgaris.
Summary Using the in vivo assay of nitrate reductase it was established that pericarps of the fruits of Vicia faba L. and Phaseolus vulgaris L. are able to reduce nitrate actively. The capacity of nitrate reduction changes in accordance with the developmental stages of pericarp ontogenesis. Nitrate reductase activity was demonstrated in seed coats and cotyledons of ripening fruits of Vicia faba L., too. In contrast to this finding we were unable to demonstrate nitrate reductase activity in ripening seeds of Phoseolus vulgaris L., although in germinating seeds it was possible to induce nitrate reductase at light conditions in the presence of exogenous nitrate. The implications of these results are discussed with respect to the physiological function of pericarps in relation to protein storage in the seeds.
Introduction
The functional homology of pericarps and leaves from leguminoses (MUNTZ 1973a, b) suggests that the pods not only can assimilate carbon dioxide autotrophically (FLINN and PATE 1968, 1970) but might bee able to reduce nitrate, too. Nitrate reduction in fruits would be a source of amino groups in addition to the amino compounds imported by the generative organs from the vegetative parts of the plant (LEWIS and PATE 1973). We investigated pods and seeds of Vicia faba L. and Phaseolus vulgaris L. with respect to their ability to reduce nitrate. The occurrence of nitrate reductase activity in these organs was established, and it was shown that the capacity of nitrate reduction is changing in accordance with different developmental stages of fruit ontogenesis. Material and methods Phaseolus vulgaris L., cv. Declivis Remus, and Ficia faha L., var. minor, cv. Dornburger Ackerbohne, were cultivated in special growth chambers, using a modified HOAGLA~D nutrient solu-
I
i:
2i£i!§.$ ji
88
2EXM¥iL.,
l.i.iii.iJSlIJ"• •
lIfil G.
SCHLESIER
and K.
MUNTZ
tion. The plants were grown under rhythmic changes of illumination and temperature (light: 16 h 24-25 DC, darkness: 8 h 15 DC). In some cases Vicia plants were used from outdoor cultivation. The activity of nitrate reductase was assayed by the in vivo technique of JAWORSKI (1971). This method involved the incubation of tissue discs at 25 DC in the dark during 3 to 5 hours within a medium of 0.1 M phosphate buffer, pH 7.5, containing 0.008 M KN0 3 , 4.15 % n-propanol and 0.001 M chloramphenicol. After separation of the tissue from the medium the nitrite produced in this solution was determined by the sulfanilamide method of SNELL and SNELL (1949). Adhering residues of the incubation mixture were carefully removed from the tissue discs be fore transferring them into a 1.5 . 10-3 M solution of KCN. The tissue was homogenized in this solution, and after centrifugation the nitrite remaining in the tissue was determined in the clear supernatant. The nitrate content of pods and seeds was assayed in centrifuged homogenates using a combination of the methods of MORRIS and RILEY (1963) and ELLIOT and PORTER (1971).
; Results
Nitrate reductase activity in different fruit tissues In vivo and in vitro techniques are available for nitrate reductase assay in plant tissues. Applying both methods to leaf and fruit tissues of our objects, we met some difficulties encountered in the in vitro assay of homogenates from Vicia material. Phenolic compounds (KLEPPER and HAGE~fAN 1969; LEECE, DILLEY and KENWORTHY 1972; DIRR, BARKER and MAYNARD 1972; DIRR, BARKER and MAYNARD 1973) and proteolytic enzymes (MILLERD, SIMON and STERN 1971; MENARY and JONES 1972) may interfer with the in vitro procedures of nitrate reductase assay. Leaves and pericarps of Vicia contain relatively large amounts of phenolic substances, especially in ageing tissues. The proteolytic activities of extracts from leaves and pericarps are low (MUNTZ 1973b). We decided to prefer the in vivo method, which has the additional advantage of working more rapidly than the in vitro assay. To compensate the relatively large standard deviation of mean values from the in vivo technique we always assayed 2-3 parallels in each set of double experiments. Vacuum infiltration (HARPER and HAGEMAN 1972; STREETER and BOSLER 1972) and treatment with n-propanol (JAWORSKI 1971) were compared with respect to their stimulating effect on the velocity of ion influx into leaf and pod tissue discs. We did not find appreciable differences. So we preferred the application of n-propanol, because its use is more simple. Comparing the effects of methanol, ethanol and n-butanol we could reproduce the results of JAWORSKI (1971), who found n-propanol most suitable for in vivo measuring of nitrate reductase activity. Preliminary time course studies have shown that nitrate reductase activity of leaf discs reaches a maximum 7 to 9 hours after start of illumination. Therefore, the plant material was harvested always at this period of light-dark-rhythm. Pericarps of ripening fruits of Phaseolus vulgaris and Vicia faha exerted appreciable nitrate reductase activities without previous experimental enzyme induction. The capacity of pericarps to reduce nitrate decreased from the basis to the apical region of the long pods of Phaseolus (fig. 1). In pods of Vicia faha we never could demonstrate a
i ,
89
The Function of the Pod at Protein Storage in the Seeds etc. 100 % 80
60
40 20
Fig. 1. Zones of different nitrate reductase activity in pods of Phaseolus vulgaris L. ba, mi and ap - basic, middle and apical sections of a pod. Results of experiments with different incubation times: a, 240 min; b, 300 min; c, 360 min.
zonation of nitrate reductase activity. Pericarp tissue of both species contained nitrate at all stages of ontogenetic development (table 1). Seed coat and cotyledons of ripening seeds of Vicia (aba are also able to reduce nitrate. These tissues did not contain detectable amounts of nitrate during the period of maximal nitrate reductase activity (table 1). Only during the last stages of ripening we found nitrate in seed coat and cotyledons. On the contrary, similar tissues from developing Phaseolus seeds never exerted nitrate reductase activity without or with exogenous nitrate, independent of the developmental stage under investigation. We were not able to induce nitrate reductase activity in seed coat and cotyledons of Phaseolus using light and nitrate as inducers. Both tissues contained internal nitrate (table 1) Table 1
Nitrate content (Il-mol NOa/g fresh weight) in different parts of the fruits of Phaseolus vulgaris L. and Vicia faha L.
Species V icia (aha L. Phaseolus vulgaris L.
Developmental stagel ) Pods 6 6 8
0.033 7.750 0.074
Seed coats
1.020 0.127
Cotyledons 0.470 0.241
1) according to the stages used in fig. 2 and 3.
Cotyledons of the germinating seeds of both objects reduce nitrate actively after enzyme induction by a combination of light and nitrate supply. Changes in nitrate reducing acpacity during fruit ripening The characterization of developmental stages of legume fruits by counting the days after anthesis, is a common method applicable to kidney beans, because their flowers
90
G.
SCHLESIER
and K.
MUNTZ
Phaseolus uulgp7'7s 1..
pod
5(}()
r-
100
o
1-
123"~678
Fig. 2. Changes in nitrate reductase activity during ontogenetic development of pods from Phaseolus vulgaris L. 1 = 1-2 days; 2 = 3-5; 3 = 6-8; 4 = 9-11; 5 = 12-20; 6 = 21-30; 7 = 31-40; 8 = > 40 days after anthesis.
are self pollinating and pollination takes place at blossoming. By the same way BOULTER and his group (e. g. 1968) determined the fruit development of field beans. HALL, McLEESTER and BLISS (1972) and MILLERD, SIMON and STERN (1971) found length and fresh weight of the cotyledons as suitable measures to characterize ripening stages of seeds, using Phaseolus vulgaris and V icia faba, respectively. We used the number of days after blossoming to determine the age of fruits, working with kidney beans. The developmental stages of ripening fruits from field beans were characterized on the basis of the fresh weight of seeds. The nitrate reducing capacity of pericarps from developing pods of Phaseolus increased up to a 12 to 20 days period after anthesis (fig. 2). Later on nitrate reductase activity was declining. Fruits of the field beans are growing according to a well established scheme of ontogenetic development (BRIARTY, COULT and BOULTER 1969; PAYNE, BROWNRIGG, YARWOOD and BOULTER 1971; MUNTZ 1973a, b). Pod weight is increasing rapidly during a first developmental stage. At the same time the seeds still remain very small. This is the period of high biosynthetic activity in peri carp tissue. The growth of the seeds starts later on, and in its beginning the testa is growing at a higher rate than the
The Function of the Pod at Protein Storage in the Seeds
91
uteia (aba l.
seedeoaf
pod
cotyledons
3oo+------------r-
100
Fig. 3. Changes in nitrate reductase activity during ontogenetic development of pod, seed coat+ and cotyledons of Vicia faba L. cultivated outdoors. 1 = 50 mg/seed; 2 = 50-100; 3 = 100-150; 4 = 150-250; 5 = 250-350; 6 = 450-550; 7 = 550-650; 8 = 650-850; 9 = 950-1150 mg/seed (fresh weight). Note concerning the column of stage 9 of seed coats: One of the six parallel values was extremely high. Calculating without this value, we receive the short column, indicated by hatching. Taking into account all the six values we receive the large column, without hatching.
embryonic organs. The main increase of dry matter and volume of cytoledons takes place after growth of pericarp and testa had finished. This is the period of protein and carbohydrate storage in cotyledon tissue. In accordance with this scheme, the pericarp tissue of field beans exerted maximal nitrate reductase activity during the first stage of fruit development. At this time the testa showed a much lower nitrate reduction, and it was nearly zero in cotyledons (fig. 3). The maximal nitrate reductase activity of seed coats coincided with a developmental stage where fresh weights of cotyledons reached 50 to 100 mg per seed. Afterwards, nitrate reducing activity of peri carp and testa was decreasing accompanied by an increase of the capacity to reduce nitrate in the cotyledons. Finally, during the latest stage of ripening nitrate reductase activity was decreasing in cotyledon tissue, too.
Diskussion
The results of our experiments demonstrate that nitrate reduction takes place in different tissues of ripening fruits of Leguminosae. This finding suggests that in fruits
92
G. SCHLESIER and K. MUNTZ
a proper shource of amino nitrogen exists in addition to the amino acids imported from vegetative parts of the plant. Consequently, in peri carp tissue autotrophic assimilation of carbon dioxide (FLINN and PATE 1970; MUNTZ 1973a) and nitrate may result in a de novo biosynthesis of amino acids. During the early stages of fruit development, when seeds remain very small and pericarps are growing rapidly, the majority of autotropically generated amino nitrogen may serve for the biosynthesis of amino acids and proteins of the pods. Later on this amino nitrogen will be reactived as part of the free amino compounds and of the proteolytically liberated amino acids which are translocated from the pericarps to the seeds during protein storage in the cotyledons. Moreover, seeds of the field bean are able to reduce nitrate in the testa and in cotyledons, as do seeds of Pisurn (SCHLESIER unpublished). Therefore, V icia seeds posses their own additional autotrophic source of amino nitrogen. Ripening seeds of the kidney bean are lacking in the capacity to reduce nitrate. Endogenous nitrate was present in ripening seeds of Phaseolus vulgaris at any developmental stage investigated in our experiments, but it was impossible to induce nitrate reductase activity by the combination of light and exogenous nitrate supply. The occurrence of nitrate reductase activity in fruit tissues of Vicia faba cultivated outdoors (fig. 3) indicates that this enzyme is not only present in plants grown in nutrient solution containing nitrate but also in plants assimilating parts of their nitrogen by symbiotic nitrogen fixation. Our results are in accordance with findillgs of LLOYD (1972) and MENARY and JoNES (1972), who demonstrated nitrate reductase activity in pericarps and seeds of the papaw fruit (Carica papaya). In different tissues of papaw fruits the activity of nitrate reductase varies with the stages of ontogenetic development. It will be the task of further experiments to elucidate the significance and function of the autotrophic generation of amino nitrogen in different tissues of legume fruits. The parallelism between activity changes and stages of ontogenetic fruit development suggests a functional dependency between the capacity of nitrate reduction and the biosynthetic activity. This is known from other parts of the plant, too. The rhythm of changes in nitrate reductase activity is similar in leaves and pods during organ development (SCHLESIER, unpublished). This result agrees with our hypothesis published previously (MUNTZ 1973a, b) that leaves and peri carps are at least in part functionally homologous organs.
References BOULTER, D., and DAVIS, o. J., Nitrogen metabolism in developing seeds of Vicia {aha. New Phytol. 67,935-946 (1968). BRIARTY, L. G., COULT, D. A., and BOULTER, D., Protein bodies of developing seeds of Vicia {aha. J. expo Bot. 20,358-372 (1969). DIRR, M. A., BARKER, A. V., and MAYNARD, D. N., Nitrate reductase activity in the leaves of the highbush blueberry and other plants. J. Amer. Soc. Hort. Sci. 97, 329 -331 (1972).
The Function of the Pod at Protein Storage in the Seeds
93
-
- - Extraction of nitrate reductase from leaves of Erieaeeae. Phytochemistry 12, 1261-1264 (1973). ELLIOTT, R. J., and PORTER, A. G., A rapid cadmium reduction method for the determination of nitrate in bacon and curing brines. Analyst 96,522-527 (1971). FLINN, A. M., and PATE, .I. S., Biochemical and physiological changes during maturation of fruit of the field pea (Pisum arvense L.). Ann. Bot. (Lond.) N. S. 32, 479-495 (1968). - - A quantitative study of carbon transfer from pod and subtending leaf to the ripening seeds of the field pea (Pisum arvense L.). J. expo Bot. 21, 71-82 (1970). HALL, T. C., McLEESTER, R. C., and BLISS, F. A., Electrophoretic analysis of protein changes during the development of the french bean fruit. Phytochemistry 11, 647-649 (1972). HARPER, J. E., and HAGEMAN, R. H., Canopy and seasonal profiles of nitrate reductase in soybeans (Glycine max. L. Merr.). Plant PhysioI. 49, 146-154 (1972). JAWORSKI, E. C., Nitrate reductase assay in intact plant tissue. Biochem. Biophys. Res. Commun. 43, 1274-1279 (1971). KLEPPER, L., and HAGEMAN, R. H., The occurrence of nitrate reductase in apple leaves. Plant PhysioI. 44,110-114 (1969). LEECE, D. R., DILLEY, D. R., and KENWORTHY, A. L., The occurrence of nitrate reductase in leaves of Prunus species. Plant Physiol. 49, 725 -728 (1972). LEWIS, O. A. M., and PATE, J. S., The significance of transpiration ally derived nitrogen in protein synthesis in fruiting plants of pea (Pisum sativum). J. expo Bot. 24, 596-606 (1973). LLOYD, A. C., Nitrate and nitrate reductase in papaw fruit. J. Agr. Anim. Sci. 29, 85-102 (1972). MENARY, R. C., and JONES, R. H., Nitrate accumulation and reduction in papaw fruits. Austr. J. bioI. Sci. 25, 531-542 (1972). MILLERD, A., SIMON, M., and STERN, H., Legumin synthesis in developing cotyledons of Vicia (aba L. Plant PhysioI. 48,419-425 (1971). MORRIS, A. W., and RILEY, J. P., The determination of nitrate in sea water. Anal. chim. Acta 29, 272-279 (1963). MUNTZ, K., Die Funktion der Hiilsensch1.1e bei der Proteinspeicherung in den Samen von Vicia (aba L. I. Gelelektrophoretischer Vergleich von loslichen Proteinen aus Bliittern und Hiilsen. Biochern. PhysioI. Pflanzen 164, 357 -369 (1973 a). Die Funktion der Hiilsenschale bei der Proteinspeicherung in den Samen von Vicia (aba L. II. Vorkommen einiger Hydrolasen wiihrend der Hiilsenentwicklung bei Vicia (aba L. Biochem. PhysioI. Pflanzen 164,370-382 (1973b). PAYNE, E. S., BROWNRIGG, A., YARWOOD, A., and BOULTER, D., Changing protein synthetic machinery during development of seeds of Vicia (aba. Phytochemistry 10, 2299 - 2303 (1971). SNELL, F. D., and SNELL, T. C., Colorimetric methods of analysis. New York 1949. STREETER, J. G., and BOSLER, M. E., Comparison of in vitro and in vivo assays for nitrate reductase in soybean leaves. Plant PhysioI. 49,448-450 (1972). Authors' address: G. SCHLESIER and K. MUNTZ, 4325 Gatersleben, CorrensstraBe 3.
Verantwortlich fiir die Redaktion: Dr. B. Parthier, 401 HaIle (Saale). Verlag: VEB Gustav Fischer Verlag, 69 Jena, Villengang 2, Telefon 2 4141,24142. Alleinige Anzeigenannahme: DEWAG-Werbung Leipzig, 701 Leipzig, BriihI34-40, Telefon 29740. Fiir Auslandsanzeigen: Interwerbung GmbH, DDR -104 Berlin, TuchoIskystraCe 40, Postfach 230. Satz und Druck: Druckerei "Magnus Poser",69 Jena. - Veriiffentlioht unter der Lizenznummer 1067a des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratisohen Republik. AIle Rechte beim Verlag. Naohdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des VerIa.. er> sowie mit An,.be d3r Qu,11J gll~lttet. Printed in the German Democratic Republic.
Zentralinstitut fiir Genetik und Kulturpflanzenforschung Gatersleben des Forschungszentrums fiir Molekularbiologie und Medizin der Akademie der Wissenschaften der DDR
The Function of the Pod at Protein Storage in the Seeds of Vicia Jaba L. III. Nitrate Reductase in Developing Pods and Seeds of Legu.minosae
By G.
SCHLESIER
and K.
MUNTZ
With 3 figures (Received February 22, 1974) Key Term Index: storage protein, pods, seeds, nitrate reductase, ontogenesis; Vicia faba, Phaseolus vulgaris.
Summary Using the in vivo assay of nitrate reductase it was established that pericarps of the fruits of Vicia faba L. and Phaseolus vulgaris L. are able to reduce nitrate actively. The capacity of nitrate reduction changes in accordance with the developmental stages of pericarp ontogenesis. Nitrate reductase activity was demonstrated in seed coats and cotyledons of ripening fruits of Vicia faba L., too. In contrast to this finding we were unable to demonstrate nitrate reductase activity in ripening seeds of Phoseolus vulgaris L., although in germinating seeds it was possible to induce nitrate reductase at light conditions in the presence of exogenous nitrate. The implications of these results are discussed with respect to the physiological function of pericarps in relation to protein storage in the seeds.
Introduction
The functional homology of pericarps and leaves from leguminoses (MUNTZ 1973a, b) suggests that the pods not only can assimilate carbon dioxide autotrophically (FLINN and PATE 1968, 1970) but might bee able to reduce nitrate, too. Nitrate reduction in fruits would be a source of amino groups in addition to the amino compounds imported by the generative organs from the vegetative parts of the plant (LEWIS and PATE 1973). We investigated pods and seeds of Vicia faba L. and Phaseolus vulgaris L. with respect to their ability to reduce nitrate. The occurrence of nitrate reductase activity in these organs was established, and it was shown that the capacity of nitrate reduction is changing in accordance with different developmental stages of fruit ontogenesis. Material and methods Phaseolus vulgaris L., cv. Declivis Remus, and Ficia faha L., var. minor, cv. Dornburger Ackerbohne, were cultivated in special growth chambers, using a modified HOAGLA~D nutrient solu-
I
i:
2i£i!§.$ ji
88
2EXM¥iL.,
l.i.iii.iJSlIJ"• •
lIfil G.
SCHLESIER
and K.
MUNTZ
tion. The plants were grown under rhythmic changes of illumination and temperature (light: 16 h 24-25 DC, darkness: 8 h 15 DC). In some cases Vicia plants were used from outdoor cultivation. The activity of nitrate reductase was assayed by the in vivo technique of JAWORSKI (1971). This method involved the incubation of tissue discs at 25 DC in the dark during 3 to 5 hours within a medium of 0.1 M phosphate buffer, pH 7.5, containing 0.008 M KN0 3 , 4.15 % n-propanol and 0.001 M chloramphenicol. After separation of the tissue from the medium the nitrite produced in this solution was determined by the sulfanilamide method of SNELL and SNELL (1949). Adhering residues of the incubation mixture were carefully removed from the tissue discs be fore transferring them into a 1.5 . 10-3 M solution of KCN. The tissue was homogenized in this solution, and after centrifugation the nitrite remaining in the tissue was determined in the clear supernatant. The nitrate content of pods and seeds was assayed in centrifuged homogenates using a combination of the methods of MORRIS and RILEY (1963) and ELLIOT and PORTER (1971).
; Results
Nitrate reductase activity in different fruit tissues In vivo and in vitro techniques are available for nitrate reductase assay in plant tissues. Applying both methods to leaf and fruit tissues of our objects, we met some difficulties encountered in the in vitro assay of homogenates from Vicia material. Phenolic compounds (KLEPPER and HAGE~fAN 1969; LEECE, DILLEY and KENWORTHY 1972; DIRR, BARKER and MAYNARD 1972; DIRR, BARKER and MAYNARD 1973) and proteolytic enzymes (MILLERD, SIMON and STERN 1971; MENARY and JONES 1972) may interfer with the in vitro procedures of nitrate reductase assay. Leaves and pericarps of Vicia contain relatively large amounts of phenolic substances, especially in ageing tissues. The proteolytic activities of extracts from leaves and pericarps are low (MUNTZ 1973b). We decided to prefer the in vivo method, which has the additional advantage of working more rapidly than the in vitro assay. To compensate the relatively large standard deviation of mean values from the in vivo technique we always assayed 2-3 parallels in each set of double experiments. Vacuum infiltration (HARPER and HAGEMAN 1972; STREETER and BOSLER 1972) and treatment with n-propanol (JAWORSKI 1971) were compared with respect to their stimulating effect on the velocity of ion influx into leaf and pod tissue discs. We did not find appreciable differences. So we preferred the application of n-propanol, because its use is more simple. Comparing the effects of methanol, ethanol and n-butanol we could reproduce the results of JAWORSKI (1971), who found n-propanol most suitable for in vivo measuring of nitrate reductase activity. Preliminary time course studies have shown that nitrate reductase activity of leaf discs reaches a maximum 7 to 9 hours after start of illumination. Therefore, the plant material was harvested always at this period of light-dark-rhythm. Pericarps of ripening fruits of Phaseolus vulgaris and Vicia faha exerted appreciable nitrate reductase activities without previous experimental enzyme induction. The capacity of pericarps to reduce nitrate decreased from the basis to the apical region of the long pods of Phaseolus (fig. 1). In pods of Vicia faha we never could demonstrate a
i ,
89
The Function of the Pod at Protein Storage in the Seeds etc. 100 % 80
60
40 20
Fig. 1. Zones of different nitrate reductase activity in pods of Phaseolus vulgaris L. ba, mi and ap - basic, middle and apical sections of a pod. Results of experiments with different incubation times: a, 240 min; b, 300 min; c, 360 min.
zonation of nitrate reductase activity. Pericarp tissue of both species contained nitrate at all stages of ontogenetic development (table 1). Seed coat and cotyledons of ripening seeds of Vicia (aba are also able to reduce nitrate. These tissues did not contain detectable amounts of nitrate during the period of maximal nitrate reductase activity (table 1). Only during the last stages of ripening we found nitrate in seed coat and cotyledons. On the contrary, similar tissues from developing Phaseolus seeds never exerted nitrate reductase activity without or with exogenous nitrate, independent of the developmental stage under investigation. We were not able to induce nitrate reductase activity in seed coat and cotyledons of Phaseolus using light and nitrate as inducers. Both tissues contained internal nitrate (table 1) Table 1
Nitrate content (Il-mol NOa/g fresh weight) in different parts of the fruits of Phaseolus vulgaris L. and Vicia faha L.
Species V icia (aha L. Phaseolus vulgaris L.
Developmental stagel ) Pods 6 6 8
0.033 7.750 0.074
Seed coats
1.020 0.127
Cotyledons 0.470 0.241
1) according to the stages used in fig. 2 and 3.
Cotyledons of the germinating seeds of both objects reduce nitrate actively after enzyme induction by a combination of light and nitrate supply. Changes in nitrate reducing acpacity during fruit ripening The characterization of developmental stages of legume fruits by counting the days after anthesis, is a common method applicable to kidney beans, because their flowers
90
G.
SCHLESIER
and K.
MUNTZ
Phaseolus uulgp7'7s 1..
pod
5(}()
r-
100
o
1-
123"~678
Fig. 2. Changes in nitrate reductase activity during ontogenetic development of pods from Phaseolus vulgaris L. 1 = 1-2 days; 2 = 3-5; 3 = 6-8; 4 = 9-11; 5 = 12-20; 6 = 21-30; 7 = 31-40; 8 = > 40 days after anthesis.
are self pollinating and pollination takes place at blossoming. By the same way BOULTER and his group (e. g. 1968) determined the fruit development of field beans. HALL, McLEESTER and BLISS (1972) and MILLERD, SIMON and STERN (1971) found length and fresh weight of the cotyledons as suitable measures to characterize ripening stages of seeds, using Phaseolus vulgaris and V icia faba, respectively. We used the number of days after blossoming to determine the age of fruits, working with kidney beans. The developmental stages of ripening fruits from field beans were characterized on the basis of the fresh weight of seeds. The nitrate reducing capacity of pericarps from developing pods of Phaseolus increased up to a 12 to 20 days period after anthesis (fig. 2). Later on nitrate reductase activity was declining. Fruits of the field beans are growing according to a well established scheme of ontogenetic development (BRIARTY, COULT and BOULTER 1969; PAYNE, BROWNRIGG, YARWOOD and BOULTER 1971; MUNTZ 1973a, b). Pod weight is increasing rapidly during a first developmental stage. At the same time the seeds still remain very small. This is the period of high biosynthetic activity in peri carp tissue. The growth of the seeds starts later on, and in its beginning the testa is growing at a higher rate than the
The Function of the Pod at Protein Storage in the Seeds
91
uteia (aba l.
seedeoaf
pod
cotyledons
3oo+------------r-
100
Fig. 3. Changes in nitrate reductase activity during ontogenetic development of pod, seed coat+ and cotyledons of Vicia faba L. cultivated outdoors. 1 = 50 mg/seed; 2 = 50-100; 3 = 100-150; 4 = 150-250; 5 = 250-350; 6 = 450-550; 7 = 550-650; 8 = 650-850; 9 = 950-1150 mg/seed (fresh weight). Note concerning the column of stage 9 of seed coats: One of the six parallel values was extremely high. Calculating without this value, we receive the short column, indicated by hatching. Taking into account all the six values we receive the large column, without hatching.
embryonic organs. The main increase of dry matter and volume of cytoledons takes place after growth of pericarp and testa had finished. This is the period of protein and carbohydrate storage in cotyledon tissue. In accordance with this scheme, the pericarp tissue of field beans exerted maximal nitrate reductase activity during the first stage of fruit development. At this time the testa showed a much lower nitrate reduction, and it was nearly zero in cotyledons (fig. 3). The maximal nitrate reductase activity of seed coats coincided with a developmental stage where fresh weights of cotyledons reached 50 to 100 mg per seed. Afterwards, nitrate reducing activity of peri carp and testa was decreasing accompanied by an increase of the capacity to reduce nitrate in the cotyledons. Finally, during the latest stage of ripening nitrate reductase activity was decreasing in cotyledon tissue, too.
Diskussion
The results of our experiments demonstrate that nitrate reduction takes place in different tissues of ripening fruits of Leguminosae. This finding suggests that in fruits
92
G. SCHLESIER and K. MUNTZ
a proper shource of amino nitrogen exists in addition to the amino acids imported from vegetative parts of the plant. Consequently, in peri carp tissue autotrophic assimilation of carbon dioxide (FLINN and PATE 1970; MUNTZ 1973a) and nitrate may result in a de novo biosynthesis of amino acids. During the early stages of fruit development, when seeds remain very small and pericarps are growing rapidly, the majority of autotropically generated amino nitrogen may serve for the biosynthesis of amino acids and proteins of the pods. Later on this amino nitrogen will be reactived as part of the free amino compounds and of the proteolytically liberated amino acids which are translocated from the pericarps to the seeds during protein storage in the cotyledons. Moreover, seeds of the field bean are able to reduce nitrate in the testa and in cotyledons, as do seeds of Pisurn (SCHLESIER unpublished). Therefore, V icia seeds posses their own additional autotrophic source of amino nitrogen. Ripening seeds of the kidney bean are lacking in the capacity to reduce nitrate. Endogenous nitrate was present in ripening seeds of Phaseolus vulgaris at any developmental stage investigated in our experiments, but it was impossible to induce nitrate reductase activity by the combination of light and exogenous nitrate supply. The occurrence of nitrate reductase activity in fruit tissues of Vicia faba cultivated outdoors (fig. 3) indicates that this enzyme is not only present in plants grown in nutrient solution containing nitrate but also in plants assimilating parts of their nitrogen by symbiotic nitrogen fixation. Our results are in accordance with findillgs of LLOYD (1972) and MENARY and JoNES (1972), who demonstrated nitrate reductase activity in pericarps and seeds of the papaw fruit (Carica papaya). In different tissues of papaw fruits the activity of nitrate reductase varies with the stages of ontogenetic development. It will be the task of further experiments to elucidate the significance and function of the autotrophic generation of amino nitrogen in different tissues of legume fruits. The parallelism between activity changes and stages of ontogenetic fruit development suggests a functional dependency between the capacity of nitrate reduction and the biosynthetic activity. This is known from other parts of the plant, too. The rhythm of changes in nitrate reductase activity is similar in leaves and pods during organ development (SCHLESIER, unpublished). This result agrees with our hypothesis published previously (MUNTZ 1973a, b) that leaves and peri carps are at least in part functionally homologous organs.
References BOULTER, D., and DAVIS, o. J., Nitrogen metabolism in developing seeds of Vicia {aha. New Phytol. 67,935-946 (1968). BRIARTY, L. G., COULT, D. A., and BOULTER, D., Protein bodies of developing seeds of Vicia {aha. J. expo Bot. 20,358-372 (1969). DIRR, M. A., BARKER, A. V., and MAYNARD, D. N., Nitrate reductase activity in the leaves of the highbush blueberry and other plants. J. Amer. Soc. Hort. Sci. 97, 329 -331 (1972).
The Function of the Pod at Protein Storage in the Seeds
93
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