- Email: [email protected]
Transport and Utilization of β-alanine by Vicia faba
Biochem. Physiol. Pflanzen (BPP), Bd. 163, S. 225 -228 (1972) University of Kuwait and University of Bonn
Short Communication
Transport and Utilization of ,a-alanine by Vicia faba By MOHAMED HASHEM and WALTER ESCHRICH (Received August 23, 1971)
Introduction
Recent work reported the presence of tJ-alanine (HASHEM 1967) and some other non-proteinogenic amino acids (BVENEPAL 1967) in plants resistant to the infection with fungi or insects, whereas the acids were lacking in the susceptible varieties. Further work demonstrated that the application of tJ-alanine to the leaves of the susceptible variety of Vicia faba prevented its infection with Fusarium oxysporum f. fabae (ABDEL-REHIM et al. 1968). However, the exact mode of action of tJ-alanine and its fate in the plant was only partially explained (HASHEM 1969). Through the use of labelled tJ-alanine it was hoped to find a more satisfactory answer to this question. Material and Methods
! I
l
Seeds of Vicia faba variety "Zwijndrechter Friihe Verbesserte" were surface sterilized and soaked in tap water with continuous aeration for 24 hours. They were then sown in sand and allowed to grow till the third leaf was fully expanded. The plants were then removed from the sand containers, the roots well washed, the testa removed and the seedlings were then transferred to glass jars containing a modified Hoagland's culture solution (ESCHRICH 1966). The roots were aerated through bubbling of air in the culture solution. The plants received a continuous illumination of 6000 Lux and were kept at a temperature of 16°C ± 1. About 20 aphids (Acyrthosiphon pisum Harris) were placed on each third primary leaf and prevented from crawling to the younger parts of the plant through using a loose cylinder of paper whose base was smeared with caterpillar grease. The first primary leaf of each seedling was rubbed gently with carborundum powder, washed, blotted with tissue paper and the radioactive material spotted on the surface. Each seedling received 10,uC of /1-alanine-1-C14 or of /1-alanine3-C14 dissolved in about 40 ,ul of water. The honey dew of the insects was collected on plastic sheets fixed horizontally under the third primary leaf. After 24 hours, the plants were collected, the cotyledons removed, petioles and stems split longitudinally and the plants were then covered with powdered dry ice to freeze. The seedlings were then finally lyophilised. After complete dryness, they were spread on paper sheet, pressed and finally autoradiographed on X-ray film (Agfa-Gevaert) for 35 hours. The contours of the pressed plants were traced on a sheet of paper, the autoradiograms placed over the corresponding drawings and both were then photographed.
226
M. HASHEM and W. ESCHRICH
Insect honey dew was analysed for its amino acid content. For this reason, the honey dew was dissolved in distilled water and run on an ion exchange resin (Amberlite IR 120) in the Hform. The column was washed using carbon dioxide-free distilled water and the amino acids eluted using 2 N ammonia solution. After evaporation in a flash evaporator, the residue was dissolved in 10 % isopropyl alcohol and spotted on thin layer chromatographic plates. The plates were covered with a mixture of cellulose powder MN 300 and silica gel-H in the ratio 5 : 2. The plates were developed in two dimensions using n-butyl alcohol: acetic acid: water (4 : 1 : 1) and n-butyl alcohol: ethyl alcohol: water (4 : 1 : 1). The stems together with the leaf petioles as well as the roots were analysed for their amino acids, organic acids and their sugar content. The lyophilised material was ground with washed calcined sand and were extracted twice using 80 % and 50 % ethyl alcohol under reflux. The combined alcohol fraction was mixed with double its volume of chloroform, shaken and allowed to stand to separate the two layers. The alcohol layer was dried under reduced pressure, dissolved in water, filtered or centrifuged and then slightly acidified. It was finally run on two columns of ion exchange resin connected together (Amberlite IR 120, H-form and Amberlite IR 4- H, OHform). The columns were washed well with carbon dioxidefree distilled water and the washings collected. They were evaporated to dryness in a flash evaporator, the residue dissolved in distilled water and spotted on thin layer "cellulose - silica gel-H" plates. The plates were developed twice in a developer of benzene: n-butanol : pyridine: water (3 : 10 : 5 : 4). The amberlite columns were eluted using 2N ammonia. The eluate from the IR 120 column was chromatographed as before for its amino acid content. The fraction obtained from the IR 4-B column was analysed for its organic acid content. The eluate was dried in vacuum, taken in water and spotted on silica gel-H plates. The plates were developed twice in methyl acetate: isopropyl alcohol: 25 % ammonia (9 : 7 : 4). In all eases, the obtained chromatograms were autoradiographed on X-ray films and the autoradiograms photographed.
Results
The autoradiography of the Vicia faba plants showed that the radioactive material was exported from the first green leaf to the stem and roots of the plant surpassing the second primary leaf. This pattern of distribution indicates that translocation took place via the phloem. The honey dew of the aphids feeding on the third and fourth leaf revealed that p-alanine together with a larger amount of alanine were the major labelled constituents of the a"llino acid fraction. It was also noticed that the honey dew contained some labelled sugars, particularly when t3-alanine-l-C14 was used. Further analysis of the horse bean plants showed that whereas all three examined fractions (amino acids, organic acids and sugars) were more or less equally active on using t3-alanine-l-C14, the activity in case of t3-alanine-3-C14 was concentrated in the organic acid fraction and in the stem in particular. Thus, on using t3-alanine-l-C14, the stem and root showed the presence of almost the same organic acids with more or less equal distribution of activity. However, the use of t3-alanine-3-C14 resulted in a high activity of the organic acids in the stem whereas the organic acids in the root were almost inactive.
T
Transport and Utilisation of fJ-alanine by V icia faba
227
The autoradiograms of the thin layer chromatograms of the free amino acid fraction showed that the only labelled amino acids were both alanine and f3-alanine. Labelled f3-alanine was found to be present in both stem and root. Alanine, on the other hand, was noticed in the amino acid fraction of the stem. It was present in larger concentration on the use of f3-alanine-1-C14 than on the application of f3-alanine3-C14. Labelled sugars were found to be present in both stem and root of V icia faba plants. However, they were more pronounced in the stem than in the root and on using f3-alanine-1-C14 than when f3-alanine-3-C14 was applied.
Discussion
f3-Alanine is known to be a non-proteinogenic amino acid. However, it is also known to enter into a number of peptides, pantothenic acid and in the seed proteins of Vicia faba (HASHEM and ABDEL-REHIM 1967). MEISTER (1965) describes the presence of a f3-alanyl-Co A whose function is not well studied. From the results obtained, it can be noticed that the application of f3-alanine to the leaves of Vicia faba resulted into its transport and utilization by the plant. The transport of f3-alanine took place through the phloem resulting in the distribution of the free amino acid in all parts of the plant. Accordingly, both types of labelled acid were found to be present in the honey dew of the insects as well as in the free amino acid fraction of both the stems and roots. MEISTER (1965) describes two methods for the utilization of f3-alanine. According to PHIL and FRITZSON (1955), f3-alanine undergoes an oxidative deamination with the formation of an unstable semialdehyde which decomposes forming acetic acid and carbon dioxide. The second method of utilization includes a deamination without oxidation resulting in the formation of propionic acid, acetic acid, carbon dioxide and ammonia. This method of utilization has been described by GOLDFINE and STADTMAN (1960) for cell free extracts of Clostridium propionicum. Our results show that the utilization of f3-alanine by Vicia faba must have followed mainly the first type of utilization. In this case, the split carbon dioxide will be fixed into sugars and eventually into several other compounds. Accordingly, the activity of the sugar fraction will be much higher on the use of f3-alanine-1-C 14 than on using f3-alanine-3-C14 which fully agrees with the data obtained for both treatments. The acetic acid residue resulting after decarboxylation will be mainly incorporated into several organic acids. This will result in a higher activity of the organic acid fraction on using f3-alanine-3-C14 than on the application of f3-alanine-1-C14. Again, this assumption agrees fully with our results. The formation of ex-alanine from f3-alanine, however, cannot be explained clearly on the same basis. Nevertheless, it is most probable that some of the semialdehyde
i i
228
M. HASHEM and W. ESCHRICH
formed through the oxidative deamination has undergone rearrangement to pyruvic acid which in turn will produce alanine through amination. This transformation must have taken place very early, since labelled alanine was found in the honey dew of the insects, suggesting its formation prior to transport through the phloem. From the above discussion it can be concluded that ,B-alanine is transported and utilized by Vicia faba. However, its role in the resistance of the plant towards the infection with Fusari1tm oxysporum f. fabae cannot be explained on any other basis than the direct effect of the free amino acid that checks the growth of the fungus. Acknowledgments The authors are deeply indebted to the DFG "Deutsche Forschungsgemeinschaft" for supplying for chemicals and materials necessary for the work. Our gratitude is also due to the DAAD "Deutsche Akademische Austausch Dienst" for the grant given to the first author during his stay in Germany. At last and not the least, we would like to thank Professor Dr. M. STEINER, director of the "Pharmakognostisches Institut der Universitat Bonn" for his interest and encouragement.
Literature ABDEL-REHIM, M. A., MICHAEL, S. M., and MOHAMED HASHEM, On the control of infection of Vicia faba by Fusarium oxysporum f. fabae through the application of tJ-alanine. Flora 159, 135 -140 (1968). BVENEPAL, P. S., and HALL, Charles V., Biochemical studies of plants of Cucurbita pepo varieties as related to feeding responses of squash bug, Anasa Iristis. Proc. Amer. Soc. Hort. Sci. 91, 361-365 (1967). ESCHRICH, W., Translokation uC-markierter Assimilate im Licht und im Dunkeln bei Ficia taba. Plant a (Beri.) 70, 99-124 (1966). HASHEM, MOHAMED, The mechanism of resistance of some varieties of Vicia faba towards the infection with Fusarium oxysporum. Flora 160, 164-167 (1969). - and ABDEL-REHIM, A. M., The possible role of amino acids in resistance of plants to fungal infection. Flora 158, 265-269 (1967). MEISTER, A., Biochemistry of Amino acids. Academic Press N. Y. and London 1965. PHIL, A., and FRITZSON, P., J. BioI. Chern. 215, 345 (1955). Author's address: Prof. Dr. MOHAMED HASHEM, Botany Department, Kuwait University, Kuwait and Prof. Dr. WALTER ESCHRICH, Forstbotanisches Institut Giittingen.
Short Communication
Transport and Utilization of ,a-alanine by Vicia faba By MOHAMED HASHEM and WALTER ESCHRICH (Received August 23, 1971)
Introduction
Recent work reported the presence of tJ-alanine (HASHEM 1967) and some other non-proteinogenic amino acids (BVENEPAL 1967) in plants resistant to the infection with fungi or insects, whereas the acids were lacking in the susceptible varieties. Further work demonstrated that the application of tJ-alanine to the leaves of the susceptible variety of Vicia faba prevented its infection with Fusarium oxysporum f. fabae (ABDEL-REHIM et al. 1968). However, the exact mode of action of tJ-alanine and its fate in the plant was only partially explained (HASHEM 1969). Through the use of labelled tJ-alanine it was hoped to find a more satisfactory answer to this question. Material and Methods
! I
l
Seeds of Vicia faba variety "Zwijndrechter Friihe Verbesserte" were surface sterilized and soaked in tap water with continuous aeration for 24 hours. They were then sown in sand and allowed to grow till the third leaf was fully expanded. The plants were then removed from the sand containers, the roots well washed, the testa removed and the seedlings were then transferred to glass jars containing a modified Hoagland's culture solution (ESCHRICH 1966). The roots were aerated through bubbling of air in the culture solution. The plants received a continuous illumination of 6000 Lux and were kept at a temperature of 16°C ± 1. About 20 aphids (Acyrthosiphon pisum Harris) were placed on each third primary leaf and prevented from crawling to the younger parts of the plant through using a loose cylinder of paper whose base was smeared with caterpillar grease. The first primary leaf of each seedling was rubbed gently with carborundum powder, washed, blotted with tissue paper and the radioactive material spotted on the surface. Each seedling received 10,uC of /1-alanine-1-C14 or of /1-alanine3-C14 dissolved in about 40 ,ul of water. The honey dew of the insects was collected on plastic sheets fixed horizontally under the third primary leaf. After 24 hours, the plants were collected, the cotyledons removed, petioles and stems split longitudinally and the plants were then covered with powdered dry ice to freeze. The seedlings were then finally lyophilised. After complete dryness, they were spread on paper sheet, pressed and finally autoradiographed on X-ray film (Agfa-Gevaert) for 35 hours. The contours of the pressed plants were traced on a sheet of paper, the autoradiograms placed over the corresponding drawings and both were then photographed.
226
M. HASHEM and W. ESCHRICH
Insect honey dew was analysed for its amino acid content. For this reason, the honey dew was dissolved in distilled water and run on an ion exchange resin (Amberlite IR 120) in the Hform. The column was washed using carbon dioxide-free distilled water and the amino acids eluted using 2 N ammonia solution. After evaporation in a flash evaporator, the residue was dissolved in 10 % isopropyl alcohol and spotted on thin layer chromatographic plates. The plates were covered with a mixture of cellulose powder MN 300 and silica gel-H in the ratio 5 : 2. The plates were developed in two dimensions using n-butyl alcohol: acetic acid: water (4 : 1 : 1) and n-butyl alcohol: ethyl alcohol: water (4 : 1 : 1). The stems together with the leaf petioles as well as the roots were analysed for their amino acids, organic acids and their sugar content. The lyophilised material was ground with washed calcined sand and were extracted twice using 80 % and 50 % ethyl alcohol under reflux. The combined alcohol fraction was mixed with double its volume of chloroform, shaken and allowed to stand to separate the two layers. The alcohol layer was dried under reduced pressure, dissolved in water, filtered or centrifuged and then slightly acidified. It was finally run on two columns of ion exchange resin connected together (Amberlite IR 120, H-form and Amberlite IR 4- H, OHform). The columns were washed well with carbon dioxidefree distilled water and the washings collected. They were evaporated to dryness in a flash evaporator, the residue dissolved in distilled water and spotted on thin layer "cellulose - silica gel-H" plates. The plates were developed twice in a developer of benzene: n-butanol : pyridine: water (3 : 10 : 5 : 4). The amberlite columns were eluted using 2N ammonia. The eluate from the IR 120 column was chromatographed as before for its amino acid content. The fraction obtained from the IR 4-B column was analysed for its organic acid content. The eluate was dried in vacuum, taken in water and spotted on silica gel-H plates. The plates were developed twice in methyl acetate: isopropyl alcohol: 25 % ammonia (9 : 7 : 4). In all eases, the obtained chromatograms were autoradiographed on X-ray films and the autoradiograms photographed.
Results
The autoradiography of the Vicia faba plants showed that the radioactive material was exported from the first green leaf to the stem and roots of the plant surpassing the second primary leaf. This pattern of distribution indicates that translocation took place via the phloem. The honey dew of the aphids feeding on the third and fourth leaf revealed that p-alanine together with a larger amount of alanine were the major labelled constituents of the a"llino acid fraction. It was also noticed that the honey dew contained some labelled sugars, particularly when t3-alanine-l-C14 was used. Further analysis of the horse bean plants showed that whereas all three examined fractions (amino acids, organic acids and sugars) were more or less equally active on using t3-alanine-l-C14, the activity in case of t3-alanine-3-C14 was concentrated in the organic acid fraction and in the stem in particular. Thus, on using t3-alanine-l-C14, the stem and root showed the presence of almost the same organic acids with more or less equal distribution of activity. However, the use of t3-alanine-3-C14 resulted in a high activity of the organic acids in the stem whereas the organic acids in the root were almost inactive.
T
Transport and Utilisation of fJ-alanine by V icia faba
227
The autoradiograms of the thin layer chromatograms of the free amino acid fraction showed that the only labelled amino acids were both alanine and f3-alanine. Labelled f3-alanine was found to be present in both stem and root. Alanine, on the other hand, was noticed in the amino acid fraction of the stem. It was present in larger concentration on the use of f3-alanine-1-C14 than on the application of f3-alanine3-C14. Labelled sugars were found to be present in both stem and root of V icia faba plants. However, they were more pronounced in the stem than in the root and on using f3-alanine-1-C14 than when f3-alanine-3-C14 was applied.
Discussion
f3-Alanine is known to be a non-proteinogenic amino acid. However, it is also known to enter into a number of peptides, pantothenic acid and in the seed proteins of Vicia faba (HASHEM and ABDEL-REHIM 1967). MEISTER (1965) describes the presence of a f3-alanyl-Co A whose function is not well studied. From the results obtained, it can be noticed that the application of f3-alanine to the leaves of Vicia faba resulted into its transport and utilization by the plant. The transport of f3-alanine took place through the phloem resulting in the distribution of the free amino acid in all parts of the plant. Accordingly, both types of labelled acid were found to be present in the honey dew of the insects as well as in the free amino acid fraction of both the stems and roots. MEISTER (1965) describes two methods for the utilization of f3-alanine. According to PHIL and FRITZSON (1955), f3-alanine undergoes an oxidative deamination with the formation of an unstable semialdehyde which decomposes forming acetic acid and carbon dioxide. The second method of utilization includes a deamination without oxidation resulting in the formation of propionic acid, acetic acid, carbon dioxide and ammonia. This method of utilization has been described by GOLDFINE and STADTMAN (1960) for cell free extracts of Clostridium propionicum. Our results show that the utilization of f3-alanine by Vicia faba must have followed mainly the first type of utilization. In this case, the split carbon dioxide will be fixed into sugars and eventually into several other compounds. Accordingly, the activity of the sugar fraction will be much higher on the use of f3-alanine-1-C 14 than on using f3-alanine-3-C14 which fully agrees with the data obtained for both treatments. The acetic acid residue resulting after decarboxylation will be mainly incorporated into several organic acids. This will result in a higher activity of the organic acid fraction on using f3-alanine-3-C14 than on the application of f3-alanine-1-C14. Again, this assumption agrees fully with our results. The formation of ex-alanine from f3-alanine, however, cannot be explained clearly on the same basis. Nevertheless, it is most probable that some of the semialdehyde
i i
228
M. HASHEM and W. ESCHRICH
formed through the oxidative deamination has undergone rearrangement to pyruvic acid which in turn will produce alanine through amination. This transformation must have taken place very early, since labelled alanine was found in the honey dew of the insects, suggesting its formation prior to transport through the phloem. From the above discussion it can be concluded that ,B-alanine is transported and utilized by Vicia faba. However, its role in the resistance of the plant towards the infection with Fusari1tm oxysporum f. fabae cannot be explained on any other basis than the direct effect of the free amino acid that checks the growth of the fungus. Acknowledgments The authors are deeply indebted to the DFG "Deutsche Forschungsgemeinschaft" for supplying for chemicals and materials necessary for the work. Our gratitude is also due to the DAAD "Deutsche Akademische Austausch Dienst" for the grant given to the first author during his stay in Germany. At last and not the least, we would like to thank Professor Dr. M. STEINER, director of the "Pharmakognostisches Institut der Universitat Bonn" for his interest and encouragement.
Literature ABDEL-REHIM, M. A., MICHAEL, S. M., and MOHAMED HASHEM, On the control of infection of Vicia faba by Fusarium oxysporum f. fabae through the application of tJ-alanine. Flora 159, 135 -140 (1968). BVENEPAL, P. S., and HALL, Charles V., Biochemical studies of plants of Cucurbita pepo varieties as related to feeding responses of squash bug, Anasa Iristis. Proc. Amer. Soc. Hort. Sci. 91, 361-365 (1967). ESCHRICH, W., Translokation uC-markierter Assimilate im Licht und im Dunkeln bei Ficia taba. Plant a (Beri.) 70, 99-124 (1966). HASHEM, MOHAMED, The mechanism of resistance of some varieties of Vicia faba towards the infection with Fusarium oxysporum. Flora 160, 164-167 (1969). - and ABDEL-REHIM, A. M., The possible role of amino acids in resistance of plants to fungal infection. Flora 158, 265-269 (1967). MEISTER, A., Biochemistry of Amino acids. Academic Press N. Y. and London 1965. PHIL, A., and FRITZSON, P., J. BioI. Chern. 215, 345 (1955). Author's address: Prof. Dr. MOHAMED HASHEM, Botany Department, Kuwait University, Kuwait and Prof. Dr. WALTER ESCHRICH, Forstbotanisches Institut Giittingen.