Planta (Berl.) 124, 89--91 (1975) 9 by Springer-Verlag 1975
Quantitative Analysis of Seasonal Variation in the Amino Acids in Phloem Sap of Salix alba L. 1). ~ . L e c k s t e i n a n d M. L l e w e l l y n Biology Department, Queen Elizabeth College, Campden Hill, London W8 7AH, U.K. Received 29 January; accepted 6 March 1975
Summary. Phloem sap of Salix alba L. was collected at monthly intervals between May and October. Amino acid analysis was carried out by ion exchange chromatography. The concentrations of individual amino acids are reported. Introduction A l t h o u g h t h e q u a n t i t a t i v e a m i n o acid composition of p h l o e m sap has been r e p o r t e d for several p l a n t s (Hall a n d B a k e r , 1972 ; Ziegler, 1974) seasonal v a r i a t i o n has been s t u d i e d o n l y b y q u a l i t a t i v e m e t h o d s (Ziegler, 1956; Mittler, 1953, 1958; 1)eel a n d W e a t h e r l y , 1959). M i t t l e r (1953, 1958) r e p o r t e d on t h e a m i n o a c i d comp o s i t i o n of p h l o e m sap in Salix acuti/olia. He f o u n d t h a t a s p a r t i c acid, g l u t a m i c acid, serine, threonine, alanine, valine, leucine a n d / o r iso-leucine, p h e n y l a l a n i n e , a s p a r a g i n e , g l u t a m i n e a n d p o s s i b l y y - a m i n o b u t y r i c acid were p r e s e n t d u r i n g b u d b u r s t , leaf d e v e l o p m e n t a n d senescence. D u r i n g leaf m a t u r i t y , however, o n l y a s p a r t i e acid, g l u t a m i c acid, g l u t a m i n e a n d p o s s i b l y a s p a r a g i n e were present. This p a p e r r e p o r t s t h e q u a n t i t a t i v e analysis of free a m i n o acids in t h e p h l o e m sap of S. alba var. vitellina from M a y t o October, using ion e x c h a n g e c h r o m a t o g r a p h y .
Materials and Methods Sap was collected from the stems of saplings (0.9 to 1.5 cm dia.) of S. alba var. vitellina by severing the stylets of the willow aphid, Tuberolachuus salignus (Gmelin) which were feeding from the phloem. This technique was used originally by Mittler (1958) and was modified by using microscissors rather than razor blades to cut the stylets. The sap exuding from cut stylets was collected in 10 mm a pipettes for up to 12 h and stored at --20 ~ C. The saplings were grown in the open in 17.5 cm diameter pots filled with John Innes :No. 2 compost. Sampling was carried out in a laboratory at 20 ~:]: 2 ~ C and at a light intensity of 3000 lux. Sap samples from May to October were collected from several different saplings over a 10-day period during the middle of each month, phloem sap samples for each month were pooled. During May and June the sapling leaves were in active growth, in July and August mature, in September senescence commenced, and for most of October no leaves remained on the plant. Amino acid analysis of samples was carried out using a Technieon Auto-analyser with an ion exchange resin column 130 cm long packed with Chromobeads A. Each sample was made up to a volume of 1 cma using distilled water and acidified with 0.1 cm8 0.1 N hydrochloric acid. Amino acids in the phloem sap were identified by comparing the chromatograms with those of standard amino acid mixtures. Concentrations were obtained by the incorporation of a nor-leucine standard into each sample of phloem sap.
90
P.M. Leckstein and M. Llewellyn
Table 1. The volume o~ phloem sap collected and the concentration of individual amino acids and amides. + = presence of small amount; -- = absence Concentration in
Aspartic acid Threonine } Asparagine Glutamine Serine Glutamic acid Glycine Alanine Valine Cysteine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Vol. of sap collected in mm 3
~g mm -3
May
June
July
August
Sept.
Oct.
2.60344
0.06788
-]-
0.31145
0.27685
0.43358
0.34969
-[-
2.92074
0.15498
0.24770
0.13643
-1.91857 0.12423 0.09711 -~ ---~ -~ -{-~ -}--
0.04099 1.55811 0.06776 0.04187 ~ -{~ -~-~ 0.05286 0.00292 ~ A-4-
0.88906 2.05541 0.16752 0.11225 0.14995 ~-0.12200 0.10757 -~ 0.21310 0.15350 0.16757 A0.14091
1.33780 0.18686 0.03953 -4-0.10813 ~0.09576 0.13512 -~ 0.05286 0.17104 0.15671 --}-
0.56328 0.67974 0.27670 0.10869 0.15815 -~ 0.14036 0.10101 -[0.10737 0.08479 0.14585 -~
1.88847 0.08813 0.22399 0.09087 ~-
17.3
17.3
38.2
40.9
24.9
-[-~ -0.09941 -~ -t-21.8
Results and Discussion The volumes of phloem sap collected during each m o n t h are shown in Table 1, together with the concentration of individual amino acids and amides present in the sap, expressed in 9g m m -3. The amino acids eluted from the ion exchange column in the order shown in the Table. Sufficient phloem sap was available for only one determination in each m o n t h and the Auto-analyser was unable to give a complete separation of all amino acids and amides. Consequently the peaks for asparagine, glutamine and threonine were unresolved. I n addition it was n o t always possible to calculate concentrations of certain amino acids which were present in small amounts. Between 16 and 18 amino acids and amides were f o u n d in the sap of S. alba whereas 14 were reported in S. penduli/olia (Ziegler, 1974), 13 in S. x rubens (Ziegler, 1974), 12 in S. acuti/olia (Mittler, 1953, 1958) and 10 in S. viminalis (Peel and Weatherly, 1959). The identification of arginine, cysteine, glycine, histidine, lysine, methionine and t r y p t o p h a n in S. alba in addition to those amino acids and amides in S. acuti/olia m a y be due to the greater sensitivity of ion exchange chromatography. Mittler (1953, 1958) reported t h a t y-amino butyric acid m a y be present in the sap of S. acuti]olia b u t it was not found in S. alba. Our results confirm t h a t aspartie acid, glutamic acid, serine asparagine, glutamine and threonine are the m a j o r amino acid components of the sap. Aspartie acid, glutamic acid, asparagine, glutamine and threonine account for
Seasonal Variation in the Amino Acids in Phloem
91
95.7 % of the total amino acid concentration of the sap in May, 88.7 % in J u n e and 69.1% in July, whereas in August, September and October they account for only 23.8 %, 41.7 % and 22.2 %. The proportion of serine in the amino acid composition of the sap however is higher in October (63.8%), August (48.6%) and September (19.5 % ) than in J u l y (12.3 % ), June (2.2 % ) and May (0 % ). I t is apparent t h a t aspartic acid, glutamic acid, asparagine, glutamine and threonine are translocated principally during the periods of leaf development (May and June), whereas in the later stages of senescence (October) the major amino acid component of the sap is serine. We wish to thank Dr. H. Laidlaw and the Director of the Forest Products Research Laboratory, Princes Risborough, Bucks., for allowing us the use of facilities. Dr. N. J. King, Mr. G. Smith and Mrs. A. Stringer-Calvert gave valuable assistance with the analysis of phloem sap.
References Hall, Shelagh M., Baker, D. A. : The chemical composition of Ricinus phloem exudate. Planta (Berl.) 106, 131-140 (1972) Mittler, T. E. : Amino acids in the phloem sap and their excretion by aphids. Nature (Lond.) 172, 207 (i953) Mittler, T.E.: Studies on the feeding and nutrition of Tuberolachnus salignus (Gmelin) (Homoptera, Aphididae). II. The nitrogen and sugar composition of ingested phloem sap and excreted honeydew. J. exp. Biol. 35, 74-84 (1958) Peel, A. J., Weatherly, P. E. : Composition of sieve-tube sap. Nature (Lond.) 184, 1955-1956 (1959) Ziegler, H. : Untersuchungen fiber die Leitung und Sekretion der Assimilate. Planta (Berl.) 47, 447-500 (1956) Ziegler, H.: Biochemical aspects of phloem transport. In: Transport at the cellular level. Symp. Soc. Exp. Biol. XXVIII, p. 43-62, Sleigh, M. A., and Jennings, D. H., eds. Cambridge: Cambridge University Press 1974
Quantitative Analysis of Seasonal Variation in the Amino Acids in Phloem Sap of Salix alba L. 1). ~ . L e c k s t e i n a n d M. L l e w e l l y n Biology Department, Queen Elizabeth College, Campden Hill, London W8 7AH, U.K. Received 29 January; accepted 6 March 1975
Summary. Phloem sap of Salix alba L. was collected at monthly intervals between May and October. Amino acid analysis was carried out by ion exchange chromatography. The concentrations of individual amino acids are reported. Introduction A l t h o u g h t h e q u a n t i t a t i v e a m i n o acid composition of p h l o e m sap has been r e p o r t e d for several p l a n t s (Hall a n d B a k e r , 1972 ; Ziegler, 1974) seasonal v a r i a t i o n has been s t u d i e d o n l y b y q u a l i t a t i v e m e t h o d s (Ziegler, 1956; Mittler, 1953, 1958; 1)eel a n d W e a t h e r l y , 1959). M i t t l e r (1953, 1958) r e p o r t e d on t h e a m i n o a c i d comp o s i t i o n of p h l o e m sap in Salix acuti/olia. He f o u n d t h a t a s p a r t i c acid, g l u t a m i c acid, serine, threonine, alanine, valine, leucine a n d / o r iso-leucine, p h e n y l a l a n i n e , a s p a r a g i n e , g l u t a m i n e a n d p o s s i b l y y - a m i n o b u t y r i c acid were p r e s e n t d u r i n g b u d b u r s t , leaf d e v e l o p m e n t a n d senescence. D u r i n g leaf m a t u r i t y , however, o n l y a s p a r t i e acid, g l u t a m i c acid, g l u t a m i n e a n d p o s s i b l y a s p a r a g i n e were present. This p a p e r r e p o r t s t h e q u a n t i t a t i v e analysis of free a m i n o acids in t h e p h l o e m sap of S. alba var. vitellina from M a y t o October, using ion e x c h a n g e c h r o m a t o g r a p h y .
Materials and Methods Sap was collected from the stems of saplings (0.9 to 1.5 cm dia.) of S. alba var. vitellina by severing the stylets of the willow aphid, Tuberolachuus salignus (Gmelin) which were feeding from the phloem. This technique was used originally by Mittler (1958) and was modified by using microscissors rather than razor blades to cut the stylets. The sap exuding from cut stylets was collected in 10 mm a pipettes for up to 12 h and stored at --20 ~ C. The saplings were grown in the open in 17.5 cm diameter pots filled with John Innes :No. 2 compost. Sampling was carried out in a laboratory at 20 ~:]: 2 ~ C and at a light intensity of 3000 lux. Sap samples from May to October were collected from several different saplings over a 10-day period during the middle of each month, phloem sap samples for each month were pooled. During May and June the sapling leaves were in active growth, in July and August mature, in September senescence commenced, and for most of October no leaves remained on the plant. Amino acid analysis of samples was carried out using a Technieon Auto-analyser with an ion exchange resin column 130 cm long packed with Chromobeads A. Each sample was made up to a volume of 1 cma using distilled water and acidified with 0.1 cm8 0.1 N hydrochloric acid. Amino acids in the phloem sap were identified by comparing the chromatograms with those of standard amino acid mixtures. Concentrations were obtained by the incorporation of a nor-leucine standard into each sample of phloem sap.
90
P.M. Leckstein and M. Llewellyn
Table 1. The volume o~ phloem sap collected and the concentration of individual amino acids and amides. + = presence of small amount; -- = absence Concentration in
Aspartic acid Threonine } Asparagine Glutamine Serine Glutamic acid Glycine Alanine Valine Cysteine Methionine Iso-leucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan Vol. of sap collected in mm 3
~g mm -3
May
June
July
August
Sept.
Oct.
2.60344
0.06788
-]-
0.31145
0.27685
0.43358
0.34969
-[-
2.92074
0.15498
0.24770
0.13643
-1.91857 0.12423 0.09711 -~ ---~ -~ -{-~ -}--
0.04099 1.55811 0.06776 0.04187 ~ -{~ -~-~ 0.05286 0.00292 ~ A-4-
0.88906 2.05541 0.16752 0.11225 0.14995 ~-0.12200 0.10757 -~ 0.21310 0.15350 0.16757 A0.14091
1.33780 0.18686 0.03953 -4-0.10813 ~0.09576 0.13512 -~ 0.05286 0.17104 0.15671 --}-
0.56328 0.67974 0.27670 0.10869 0.15815 -~ 0.14036 0.10101 -[0.10737 0.08479 0.14585 -~
1.88847 0.08813 0.22399 0.09087 ~-
17.3
17.3
38.2
40.9
24.9
-[-~ -0.09941 -~ -t-21.8
Results and Discussion The volumes of phloem sap collected during each m o n t h are shown in Table 1, together with the concentration of individual amino acids and amides present in the sap, expressed in 9g m m -3. The amino acids eluted from the ion exchange column in the order shown in the Table. Sufficient phloem sap was available for only one determination in each m o n t h and the Auto-analyser was unable to give a complete separation of all amino acids and amides. Consequently the peaks for asparagine, glutamine and threonine were unresolved. I n addition it was n o t always possible to calculate concentrations of certain amino acids which were present in small amounts. Between 16 and 18 amino acids and amides were f o u n d in the sap of S. alba whereas 14 were reported in S. penduli/olia (Ziegler, 1974), 13 in S. x rubens (Ziegler, 1974), 12 in S. acuti/olia (Mittler, 1953, 1958) and 10 in S. viminalis (Peel and Weatherly, 1959). The identification of arginine, cysteine, glycine, histidine, lysine, methionine and t r y p t o p h a n in S. alba in addition to those amino acids and amides in S. acuti/olia m a y be due to the greater sensitivity of ion exchange chromatography. Mittler (1953, 1958) reported t h a t y-amino butyric acid m a y be present in the sap of S. acuti]olia b u t it was not found in S. alba. Our results confirm t h a t aspartie acid, glutamic acid, serine asparagine, glutamine and threonine are the m a j o r amino acid components of the sap. Aspartie acid, glutamic acid, asparagine, glutamine and threonine account for
Seasonal Variation in the Amino Acids in Phloem
91
95.7 % of the total amino acid concentration of the sap in May, 88.7 % in J u n e and 69.1% in July, whereas in August, September and October they account for only 23.8 %, 41.7 % and 22.2 %. The proportion of serine in the amino acid composition of the sap however is higher in October (63.8%), August (48.6%) and September (19.5 % ) than in J u l y (12.3 % ), June (2.2 % ) and May (0 % ). I t is apparent t h a t aspartic acid, glutamic acid, asparagine, glutamine and threonine are translocated principally during the periods of leaf development (May and June), whereas in the later stages of senescence (October) the major amino acid component of the sap is serine. We wish to thank Dr. H. Laidlaw and the Director of the Forest Products Research Laboratory, Princes Risborough, Bucks., for allowing us the use of facilities. Dr. N. J. King, Mr. G. Smith and Mrs. A. Stringer-Calvert gave valuable assistance with the analysis of phloem sap.
References Hall, Shelagh M., Baker, D. A. : The chemical composition of Ricinus phloem exudate. Planta (Berl.) 106, 131-140 (1972) Mittler, T. E. : Amino acids in the phloem sap and their excretion by aphids. Nature (Lond.) 172, 207 (i953) Mittler, T.E.: Studies on the feeding and nutrition of Tuberolachnus salignus (Gmelin) (Homoptera, Aphididae). II. The nitrogen and sugar composition of ingested phloem sap and excreted honeydew. J. exp. Biol. 35, 74-84 (1958) Peel, A. J., Weatherly, P. E. : Composition of sieve-tube sap. Nature (Lond.) 184, 1955-1956 (1959) Ziegler, H. : Untersuchungen fiber die Leitung und Sekretion der Assimilate. Planta (Berl.) 47, 447-500 (1956) Ziegler, H.: Biochemical aspects of phloem transport. In: Transport at the cellular level. Symp. Soc. Exp. Biol. XXVIII, p. 43-62, Sleigh, M. A., and Jennings, D. H., eds. Cambridge: Cambridge University Press 1974
