Plant and Soil 7 5 , 4 3 5 - 4 4 2 (1983). Ms. 5560 9 1983 Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in the Netherlands.

Changes during development in the free amino acid constituents of fababean ( Vicia faba L.) plants D. G. HILL-COTTINGHAM and J. V. PURVES

Long Ashton Research Station, University of Bristol, Long Ashton, Bristol, BS18 9AF, U.K. Received 1 July 1983. Revised September 1983

Key words Amino acids Asparagine Dopa Fababean Plant organs Viciafaba L.

Summary Nodulated fababeans growing in unfertilized soil were sampled at intervals during development and the plants divided into their constituent organs before extraction and determination of the separate free amino acid contents. At each developmental stage asparagine and 3,4-dihydroxyphenylalanine (Dopa) were the most abundant free amino acids in all organs except the seeds. Of the second-order constituents, there were respectively, modest amounts of serine and glutamine in the leaves, tyrosine in the pods and alanine in the young seeds. During plant development there were marked differences in both the amount and composition of the free amino acid fraction in some organs. The concentration of asparagine increased sharply during pod-flU in both stems and pods and it is suggested that this is the form in which much N is moved into the seeds for protein synthesis. Young seeds contained much free asparagine, glutamine and alanine but with time the two latter constituents were replaced by arginine. There was a rapid accumulation of Dopa in the fruits and leaves during early pod-fill but the amount decreased as the plants aged and the Dopa content of the mature seeds were extremely low.

Introduction There have been many reports of the free, or uncombined, amino acid content of the seeds of Vicia faba, but much less attention has been given to these constituents in other organs of the plant and to the chances that take place during development. Only about one-tenth of the total N in the mature seed is not combined into protein, much of this soluble fraction being present as arginine. This amino acid was found to be degraded rapidly in the two weeks after germination and glutamine was claimed to be the major soluble constituent in the young seedling2. Some of the corresponding changes taking place during the growth and development of the pods and seeds have also been reported 3. Dihydroxyphenylalanine (Dopa) is a non-protein amino acid known to occur in certain organs of Vicia faba 7. Later workers examined the free amino acid content of whole fababean plants and reported that Dopa was the most abundant single constituent 9. 435

436

HILL-COTTINGHAM AND PURVES

The experiment described below was designed to determine, qualitatively and quantitatively, the changes in the uncombined amino acids of all parts of fababean plants during their development. Exp~iment~ Seeds of cv. Dacre, inoculated with Rhizobium 1001 ( R o t h a m s t e d Experimental Station), were sown in c o m p o s t w i t h o u t added N fertilizer and the plants grown in pots in an unheated glasshouse. On six occasions from before flowering until seed maturity two representative plants were sampled for analysis. Initially the plants were divided into roots, stem and leaves only, b u t at later stages t h e stem was separated into old and y o u n g portions by cutting at node 10 or 11, t h e lowest flowering node. T h e leaves from the two stem portions were also analysed separately. When present, flowers and y o u n g (< 4 cm) pods were combined, b u t older fruits were divided into pods and seeds, and the seeds were separated into cotyledons and testa. At t h e fifth sampling t h e fruit from the lower four podded nodes, which had started to ripen, and t h e upper, green fruits were analysed separately, as were the roots and nodules. For the final sample only the ripe fruits were collected. After sub-division all material was weighed fresh and t h e n portions were either oven- or freeze-dried. The total N c o n t e n t was determined colorimetrically after a Kjeldahl digestion. Freeze-dried samples were extracted with ethanol-chloroform-water ( 1 2 - 5 - 3 v/v) at - - 1 5 ~ for 1 8 h ~. After filtration, further addition o f chloroform and water enabled t h e pigments to be removed b y phase separation. T h e aqueous ethanolic phase containing the amino acids was evaporated to dryness below 35~ with a Buchler Evapomix. The amino acids were separated using a Rank-Hilger Chromaspek with a lithium buffer gradient and were detected fluorimetrically after reaction with o-phthalaldehyde. Nor-leucine was added as internal standard. Peak areas were measured using a Hewlett-Packard 3354 data processor. When required, Dopa was also determined colorimetrically 1~

Results

Total dry weight and N content The accumulated total dry weights and N content of the principal organs o f the plant are shown in Figs. 1a and 1b, respectively. Initially there was a steady rise in both the dry weight and N content in the roots, stem and leaves, but from the c o m m e n c e m e n t of pod-fill (day 50) the rates increased approximately three-fold, most of the additional material accumulating in the seeds. Amino acids At all sampling times asparagine and Dopa were by far the most abundant free amino acids in all organs except the seeds. There was also a high concentration of asparagine in the seeds at all times and, in addition, their arginine content increased as they matured until it became the largest single constituent. Table 1 gives the mean values obtained for the concentration o f total soluble N in each plant part sampled on day 108, when the most

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comprehensive survey was made, together with the percentage distribution of this N between the different amino acids. These results show that the composition varied b o t h between the different organs and also between the older and younger parts of the same organ. Thus the soluble N in the roots comprised about 50% Dopa and 30% asparagine, b u t in the nodules these proportions were reversed. The percentage composition of the two stem sections was very similar, asparagine being b y far the largest constituent, b u t the concentration of soluble N in the upper stem was almost ten times that o f the lower section. In contrast, the leaves contained less free asparagine than all other parts and there was little difference between the old and y o u n g leaves in b o t h the amount of soluble N and the distribution of amino acids. All leaves contained marked amounts of uncombined serine, glutamine and alanine. The concentration of total soluble N in the pods was much greater than that in their seeds, while there was also more in the testa than in the seed cotyledon. The y o u n g seeds contained high concentrations of asparagine, glutamine and alanine, b u t the amounts of the two latter compounds declined, whilst that of arginine increased, as the seeds matured. The testa contained less arginine than the cotyledon. Most of the other protein amino acids, together with ~/-aminobutyric acid, were present as minor constituents of all parts of these plants. Although not listed in Table 1, traces of cystine and tryptophan were also found in many extracts, and in some there were small amounts of ~-alanine and ornithine, together with an unidentified c o m p o u n d running between valine and methionine/Dopa. The buffer system used with the amino acid analyzer did not permit

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separation of Dopa from methionine. However, colorimetric analysis showed that in all the root, stem, leaf and pod extracts most, if not all, the material in this joint peak could reasonably be attributed to Dopa. The concentration of Dopa in both cotyledons and testa were extremely low.

Changes during development Detailed analyses similar to those given in Table 1 were made for all plant material from each sampling. However, because asparagine and Dopa together accounted for 70-90% of the total uncombined amino acids in all plant parts except the seeds at all times, only the results of these two compounds are illustrated below. Fig. 2 shows the changes with time in the concentrations of asparagine, Dopa and the total of the other amino acids as calculated for the whole roots, stems and leaves. The corresponding changes in the flowers and pods are presented in Fig. 3, together with those for asparagine, alanine and arginine in the seeds. The weight and total N concentration of each organ have been included in these Figures for ease of reference. The free amino acid composition varied both qualitatively and quantitatively during development in the different organs of the plant (Figs. 2 and 3). The concentration of soluble N in the roots and stem increased as their growth rate decreased from day 66 onwards. This

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t o o k the form of a small and short-lived change in all constituents in the roots, whereas in the stem there was a four-fold increase persisting for the next 40 days, entirely attributable to a boost in the asparagine content. The concentration of soluble N in the leaves increased from day 50, when they were ~till growing rapidly, b u t declined later in advance of senescence. Young pods contained more free amino acids than any other organ; the concentration reached a peak at day 78, after which it fell steadily as the seeds grew and matured. Asparagine was always the dominant soluble constituent o f the pods b u t there was a build-up of arginine in the seeds as they ripened. Discussion

Analysis showed that the plants used in these studies had a normal major nutrient status and that their main amino acid constituents were similar to those in recent reports 8. However these results differ from some data of earlier workers who, for example, had claimed that the ungerminated seed contained mostly histidine b u t little asparagine 2, or who had failed to find Dopa in developing pods 3.

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Nodules contained considerably more asparagine than the roots (Table 1). This is consistent with asparagine being the major product of N fixation in Vicia faba, since it was present in a far greater concentration in xylem sap from plants inoculated with Rhizobium than from those grown on nitrate only s. It is also of interest that, after asparagine and Dopa, serine was the most abundant amino acid in the leaves (Table 1), for this compound was the first to be labelled in petioles o f broad bean leaves assimilating 14CO28 . Later in that experiment pods were found to contain radioactive serine, alanine, glutamic acid, glutamine and tyrosine, whereas asparagine, Dopa and some other compounds present in considerable quantity remained unlabelled. The proportion of total N present as uncombined amino acids also varied widely both between the different plant organs and during development. Roots and leaves never had more than 6 - 7 % soluble N, but there was a greater proportion in the stems, particularly from pod-fill onwards, until by days 78 and 108 over one-third of the total N was of a soluble form, most of it being free asparagine (Fig. 2b). There were also marked differences within the fruit, in that almost half the total N in the young pods was soluble compared with only 4 - 5 % in the seeds. There was an increase in free asparagine in stems and pods coinciding with the period of most rapid growth of the seeds (Figs. 2 and

442

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3), which suggests that asparagine is the form in which much of the N is moved into the seeds of Vicia faba for protein synthesis. This may well be a c o m m o n situation in other legumes since it has been shown that labelled asparagine fed to shoots o f Lupinus albus results in lSN transfer to a wide range of amino acids in the seed protein ~. Since Dopa is not a constituent of proteins, the total amounts in the plants can be determined by analysis of the soluble extracts. There was a rapid accumulation of Dopa in the pods and leaves during the early pod-fill stage but the total content started to decrease in advance o f senescence (Fig. 4). The relative distribution between the different organs as in general agreement with that given previously 9. Our present studies, however, have indicated a greater concentration ( 6 0 0 m g plant -1 ) than that in the earlier report 9, but this might be attributable to intercultivar variation. It is evident from Table 1 that pods which contain a large concentration of Dopa also have much more tyrosine than other organs but similar phenylaline contents. This has metabolic significance because it is known that tyrosine is a precursor of Dopa but phenylaline is not 4. Acknowledgements The authors thank Miss M. E. Holgate for her help with the statistical analysis. Long Ashton Research Station is financed through the Agricultural Research Council, London.

References

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6 7 8 9 10

Atkins C A, Pate J S and Sharkey P J 1975 Asparagine metabolism - Key to the nitrogen nutrition of developing legume seeds. Plant Physiol. 5 6 , 8 0 7 - 8 1 2 . Boulter D and Barber J T 1963 Amino acid metabolism in germinating seeds of Vicia faba L. in relation to their biology. New Phytol. 62, 301-316. Boulter D and Davis O J 1968 Nitrogen metabolism in developing seeds of Vicia faba. New Phytol. 6 7 , 9 3 5 - 9 4 6 . Griffith T and Conn E E 1973 Biosynthesis of 3,4-dihydroxyphenylaline in Vicia faba. Phytochemistry 12, 1651-1656. HiU-Cottingham D G 1983 Chemical constituents and biochemistry. In The Faba Bean (Vicia faba L.) A Basis for Improvement. Ed. P D Hebblethwaite, pp 159-180, Butterworths, London. HiU-Cottingham D G and Cooper D R 1969 Extraction and analysis of amino acids from apple tree material. J. Sci. Food Agric. 2 0 , 6 6 2 - 6 6 5 . Kenten R H 1957 Latent phenolase in extracts of broad bean (Vicia faba L.) leaves. Biochem. J. 6 7 , 3 0 0 - 3 0 7 . Kipps A E and Boulter D 1974 Origins of the amino acids in pods and seeds of Vicia faba L. New Phytol. 73,675-684. Longo R, Castellani A, Sberze P and TiboUa M 1974 Distribution of L-Dopa and related amino acids in Vicia. Phytochemistry 13,167-171. Shiman R, Akino A and Kaufman S 1971 Solubilization and partial purification of tyrosine hydroxylase. J. Biol. Chem. 246, 1330-1340.