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Biosynthesis of pipecolic acid in calcium-deficient wheat plants
Kurze Mitteilungen
Department of Plant Physiology, Charles University, Praha, Czechoslovakia
Biosynthesis of Pipecolic Acid in Calcium-deficient Wheat Plants ALENA CINCEROVA and EVA CERNA With 5 figures Received May 27, 1974
Summary The rate of uptake of 14C-L-Iysine by roots of intact plants, and its subsequent translocation and degradation were studied in wheat plants cultivated in both a Ca-deficient and a complete nutrient solution. The Ca-deficient plants showed a more rapid uptake of L-Iysine as well as its more rapid translocation into the shoots and degradation to the pipecolic acid and a-aminoadipic acids. The plants with a complete nutrition accumulated lysine in their shoots; the transformation of lysine was slow and less conspicuous in them. The incorporation of radioactivity into proline in the plants given complete nutrition indicates possible differences between the metabolic pathways involved in them and in the Ca-deficient plants.
Our previous paper (CINCERovA and CERNA, 1973) has demonstrated an increased production of pipecolic acid, which is a non-proteinogenous aminoacid, in young wheat plants cultivated in a nutrient solution lacking calcium. Lysine has been proved to be the initial compound for the biosynthesis of pipecolic acid in these plants. The same observation has also been made in plants which contain relatively large amounts of pipecolic acid in their aminoacid pool, even when cultivated under favourable trophic conditions, e.g. in some species of Fabaceae (GROBBELAAR and STEWARD, 1953; Lowy, 1953; HYLIN, 1964). Our own experiments with aseptic plants cultures excluded the possiblity of microorganisms taking part in the biosynthesis of pipecolic acid. Its formation thus appears to be a result of an altered plant metabolism under the trophic conditions of nutrient deficiency. The present paper attempts a quantitative evaluation of the changes with the time in the metabolic transformation of 14C-Iabelled L-Lysine taken up by roots of intact plants, both Ca-deficient and control ones. Plant cultivation: Plants of wheat (Triticum aestivum L., cv. FANAL) were cultivated under constant conditions of temperature (19 ± 2 0q, illumination (fluorescent light tubes Z. P/lanzenphysiol. Bd. 74, S. 366-370. 1974.
Pipecolic Acid in Wheat Plants
367
combined with bulbs, light intensity 2500 Lx, 16 hours'photoperiod) and relative air humidity (60 to 70 %). Two experimental variants were compared: 1. Knop nutrient solution (LASTUVKA and MINAR, 1967): Ca/N0 3/ 2 - 3,487 mmol/l H 2 0, KN0 3 -1.414 mmol, KCI - 0.953 mmol, KH 2 P0 4 - 0.821 mmol, MgS0 4 -1.187 mmol, EDTA Fe - 5 p.p.m. 2. Knop solution without Ca, which was substituted by an equivalent amount of Na. The wheat caryopses were allowed to germinate in Petri dishes for 3 days. The germinating caryopses were then transplanted into darkened beakers, whose tops were covered with para fine-impregnated gauze. Each beaker contained 250 ml of nutrient solution and supported 12 plants. The plants were cultivated in the respective solutions for 7 days. Afterwards, one hour after the beginning of the photoperiod, the plants were transferred into the same fresh nutrient solutions (Knop, -Cal, both containing in addition 8 mg of 14Clabelled L-Iysine (Chechoslovakia, nonspecifically labelled, total activity 25 ,uc per 250 ml). Identification of aminoacids: The shoots of experimental plants were frozen, powdered, lyophilized and extracted with 80 % ethanol (for 24 hours, 2 Qq. The extract was purified on the Dowex column in H+ from, washed with 1.5 N ammonia and transferred into 10 0 /0 isopropanol. For one-dimensional chromatography, the mixture of butanol, acetic acid and water (4 : 1 : 5) was used. Prior to the aminoacid detection, the chromatograms were measured by means of the measuring set N. Z. Q. 719 and radiochromatograph N. O. Q. 611 (Tesla-Premyslenl, Czechoslovakia). In order to obtain radioautograms, the chromatograms were exposed on an X-ray film Medix Rapid (Fotochema, Czechoslovakia) for 30 days. The final detection was accomplished with a ninhydrin reagent containing cadmium acetate (HEILIN et aI., 1957) and with an isatin-containing detection reagent for a safe identification of aminoacids (JIRACEK et aI., 1967).
180 135
a
90
E
~180
IJ
135
b
90
Fig. 1: Extracts from plants 1 hour after commencing lysine feeding. Fig. 1-5: Radiochromatographic reccords of 14C-Iabelled substances of 14C-L-Iysine conversion. Ethanolic extracts of wheat plants were separated chromatographicly in n-butanolacid-water (4:1 :5). RF values of amino acids are expressed in R Leu (RF of leucines = 1). 1 - lysine, 2 - a-amino adipic acid, 3 - proline, 4 - pipecolic acid. a - variant Knop, b - variant -Ca.
Z. Pflanzenphysiol. Bd. 74, S. 366-370. 1974.
368
A.
CINCEROVA
and E.
CERNA
a
135
90
o
1·0
0·23 RLeu
Fig. 2: Extracts from plants 3 hours after commencing lysine feeding.
The figures 1-5 show the radiochromatographic record of the transformation of 14C-L-lysine taken up by the roots of the wheat plants. The lysine was translocated more rapidly into the shoots of the Ca-deficient plants (-Ca), which showed a higher activity than the control plants after as early as 3 hours (Figs. 2 a, 2 b). After 6 hours, the control plants (Knop) also showed a high activity of lysine; less radiocativity had been incorporated into pipecolic acid (Fig. 3 a). The Cadeficient plants had accumulated markedly more lysine by that time; more radioactivity had also been incorporated into their pipecolic and a-aminoadipic acids (Fig. 3 b). 1801r-----------------------------------~
135
a
135
b
o
0·65
0·84
R leu
Fig. 3: Extracts from plants 6 hours after commencing lysine feeding.
Z. Pflanzenphysiol. Bd. 74, S. 366-370. 1974.
1·0
Pipecolic Acid in Wheat Plants
369
12 hours after the commencement of lysine feeding, its transformation to the pipecolic acid and a-aminoadipic acids continued in the Ca-deficient plants (Fig. 4 b). In the control plants, the pipecolic acid showed only a weak activity (Fig. 4 a), while the accumulation of lysine was quite pronounced. 180r-------------------------------------,
o
0·84
0'23
1·0
RLeu
Fig. 4: Extracts from plants 12 hours after commencing lysine feeding.
After 24 hours, the Ca-deficient plants showed high aCtiVIty of pipecolic acid and a poorer activity of a-amino adipic acid (Fig. 5 b). In the control plants, only poor activity was recorded in the pipecolic acid, proline and a-aminoadipic acid (5 a). Proline activity was not found at all in the Ca-deficient plants.
o
0·65
0·23
0·76 0'84
1·0
RLeu
Fig. 5: Extracts from plants 24 hours after commencing lysine feeding. Z. PJlanzenphysiol. Bd. 74, S. 366-370. 1974.
370
A. CINCEROVA and E. CERNA
Lysine thus appears to have been transported more rapidly into the shoots of the Ca-deficient plants, where it was readily degraded to pipecolic acid. The rapid incorporation of radioactivity into a-aminoadipic acid justifies the assumption that lysine is degraded in these plants roughly in the way described by ROTHSTEIN and MILLER (1954): lysine --+ a-keto-E-aminocaproic acid --+ jl-piperideine-2-carboxylic acid --+ pipecolic acid --+ jl-piperideine-6-carboxylic acid --+ a-aminoadipic-o-semialdehyde --+ a-amino adipic acid --+ a-keto adipic acid -7 --+ glutaric acid. As compared with Ca-deficient plants, those cultivated in a complete nutrient solution absorbed, translocated and degraded lysine less rapidly. As proline was recorded during the lysine transformations only in the control plants, one cannot exclude the possibility of other additional mechanisms taking part in the degradation pathway of lysine. References CINCEROVA, A., and E. CERNA: Acta Univ. Caroliae - BioI. 1971, 149 (1973). GROBBELAAR, N., and F. C. STEWARD: J. Am. Chern. Soc. 75,4341 (1953). Lowy, P. H.: Arch. Biochem. Biophys. 47, 228 (1953). HYLIN, J. W.: Phytochmistry 3,161 (1964). LASTUVKA, Z., and J. MINAR: Folia Fac. Sci. Natur. Univ. Purkynianae brunnensis, Biologica 8, 1 (1967). HEILMANN, J., J. BARROLLIER, and E. WATZKE, E.: Z. physioI. Chemie 309, 219 (1957). JIRACEK, V., J. KOSTIR, and B. JIRSIKovA: Rostlinna vyroba 13, 165 (1967). ROTHSTEIN, M., and L. L. MILLER: J. BioI. Chern. 211, 851 (1954).
ALENA CINCEROV A and EVA CERNA, Department of Plant Physiology, Charles University, Vinicna 5, Praha 2, Czechoslovakia.
z.
PJlanzenphysiol. Bd. 74, S. 366-370. 1974.
Department of Plant Physiology, Charles University, Praha, Czechoslovakia
Biosynthesis of Pipecolic Acid in Calcium-deficient Wheat Plants ALENA CINCEROVA and EVA CERNA With 5 figures Received May 27, 1974
Summary The rate of uptake of 14C-L-Iysine by roots of intact plants, and its subsequent translocation and degradation were studied in wheat plants cultivated in both a Ca-deficient and a complete nutrient solution. The Ca-deficient plants showed a more rapid uptake of L-Iysine as well as its more rapid translocation into the shoots and degradation to the pipecolic acid and a-aminoadipic acids. The plants with a complete nutrition accumulated lysine in their shoots; the transformation of lysine was slow and less conspicuous in them. The incorporation of radioactivity into proline in the plants given complete nutrition indicates possible differences between the metabolic pathways involved in them and in the Ca-deficient plants.
Our previous paper (CINCERovA and CERNA, 1973) has demonstrated an increased production of pipecolic acid, which is a non-proteinogenous aminoacid, in young wheat plants cultivated in a nutrient solution lacking calcium. Lysine has been proved to be the initial compound for the biosynthesis of pipecolic acid in these plants. The same observation has also been made in plants which contain relatively large amounts of pipecolic acid in their aminoacid pool, even when cultivated under favourable trophic conditions, e.g. in some species of Fabaceae (GROBBELAAR and STEWARD, 1953; Lowy, 1953; HYLIN, 1964). Our own experiments with aseptic plants cultures excluded the possiblity of microorganisms taking part in the biosynthesis of pipecolic acid. Its formation thus appears to be a result of an altered plant metabolism under the trophic conditions of nutrient deficiency. The present paper attempts a quantitative evaluation of the changes with the time in the metabolic transformation of 14C-Iabelled L-Lysine taken up by roots of intact plants, both Ca-deficient and control ones. Plant cultivation: Plants of wheat (Triticum aestivum L., cv. FANAL) were cultivated under constant conditions of temperature (19 ± 2 0q, illumination (fluorescent light tubes Z. P/lanzenphysiol. Bd. 74, S. 366-370. 1974.
Pipecolic Acid in Wheat Plants
367
combined with bulbs, light intensity 2500 Lx, 16 hours'photoperiod) and relative air humidity (60 to 70 %). Two experimental variants were compared: 1. Knop nutrient solution (LASTUVKA and MINAR, 1967): Ca/N0 3/ 2 - 3,487 mmol/l H 2 0, KN0 3 -1.414 mmol, KCI - 0.953 mmol, KH 2 P0 4 - 0.821 mmol, MgS0 4 -1.187 mmol, EDTA Fe - 5 p.p.m. 2. Knop solution without Ca, which was substituted by an equivalent amount of Na. The wheat caryopses were allowed to germinate in Petri dishes for 3 days. The germinating caryopses were then transplanted into darkened beakers, whose tops were covered with para fine-impregnated gauze. Each beaker contained 250 ml of nutrient solution and supported 12 plants. The plants were cultivated in the respective solutions for 7 days. Afterwards, one hour after the beginning of the photoperiod, the plants were transferred into the same fresh nutrient solutions (Knop, -Cal, both containing in addition 8 mg of 14Clabelled L-Iysine (Chechoslovakia, nonspecifically labelled, total activity 25 ,uc per 250 ml). Identification of aminoacids: The shoots of experimental plants were frozen, powdered, lyophilized and extracted with 80 % ethanol (for 24 hours, 2 Qq. The extract was purified on the Dowex column in H+ from, washed with 1.5 N ammonia and transferred into 10 0 /0 isopropanol. For one-dimensional chromatography, the mixture of butanol, acetic acid and water (4 : 1 : 5) was used. Prior to the aminoacid detection, the chromatograms were measured by means of the measuring set N. Z. Q. 719 and radiochromatograph N. O. Q. 611 (Tesla-Premyslenl, Czechoslovakia). In order to obtain radioautograms, the chromatograms were exposed on an X-ray film Medix Rapid (Fotochema, Czechoslovakia) for 30 days. The final detection was accomplished with a ninhydrin reagent containing cadmium acetate (HEILIN et aI., 1957) and with an isatin-containing detection reagent for a safe identification of aminoacids (JIRACEK et aI., 1967).
180 135
a
90
E
~180
IJ
135
b
90
Fig. 1: Extracts from plants 1 hour after commencing lysine feeding. Fig. 1-5: Radiochromatographic reccords of 14C-Iabelled substances of 14C-L-Iysine conversion. Ethanolic extracts of wheat plants were separated chromatographicly in n-butanolacid-water (4:1 :5). RF values of amino acids are expressed in R Leu (RF of leucines = 1). 1 - lysine, 2 - a-amino adipic acid, 3 - proline, 4 - pipecolic acid. a - variant Knop, b - variant -Ca.
Z. Pflanzenphysiol. Bd. 74, S. 366-370. 1974.
368
A.
CINCEROVA
and E.
CERNA
a
135
90
o
1·0
0·23 RLeu
Fig. 2: Extracts from plants 3 hours after commencing lysine feeding.
The figures 1-5 show the radiochromatographic record of the transformation of 14C-L-lysine taken up by the roots of the wheat plants. The lysine was translocated more rapidly into the shoots of the Ca-deficient plants (-Ca), which showed a higher activity than the control plants after as early as 3 hours (Figs. 2 a, 2 b). After 6 hours, the control plants (Knop) also showed a high activity of lysine; less radiocativity had been incorporated into pipecolic acid (Fig. 3 a). The Cadeficient plants had accumulated markedly more lysine by that time; more radioactivity had also been incorporated into their pipecolic and a-aminoadipic acids (Fig. 3 b). 1801r-----------------------------------~
135
a
135
b
o
0·65
0·84
R leu
Fig. 3: Extracts from plants 6 hours after commencing lysine feeding.
Z. Pflanzenphysiol. Bd. 74, S. 366-370. 1974.
1·0
Pipecolic Acid in Wheat Plants
369
12 hours after the commencement of lysine feeding, its transformation to the pipecolic acid and a-aminoadipic acids continued in the Ca-deficient plants (Fig. 4 b). In the control plants, the pipecolic acid showed only a weak activity (Fig. 4 a), while the accumulation of lysine was quite pronounced. 180r-------------------------------------,
o
0·84
0'23
1·0
RLeu
Fig. 4: Extracts from plants 12 hours after commencing lysine feeding.
After 24 hours, the Ca-deficient plants showed high aCtiVIty of pipecolic acid and a poorer activity of a-amino adipic acid (Fig. 5 b). In the control plants, only poor activity was recorded in the pipecolic acid, proline and a-aminoadipic acid (5 a). Proline activity was not found at all in the Ca-deficient plants.
o
0·65
0·23
0·76 0'84
1·0
RLeu
Fig. 5: Extracts from plants 24 hours after commencing lysine feeding. Z. PJlanzenphysiol. Bd. 74, S. 366-370. 1974.
370
A. CINCEROVA and E. CERNA
Lysine thus appears to have been transported more rapidly into the shoots of the Ca-deficient plants, where it was readily degraded to pipecolic acid. The rapid incorporation of radioactivity into a-aminoadipic acid justifies the assumption that lysine is degraded in these plants roughly in the way described by ROTHSTEIN and MILLER (1954): lysine --+ a-keto-E-aminocaproic acid --+ jl-piperideine-2-carboxylic acid --+ pipecolic acid --+ jl-piperideine-6-carboxylic acid --+ a-aminoadipic-o-semialdehyde --+ a-amino adipic acid --+ a-keto adipic acid -7 --+ glutaric acid. As compared with Ca-deficient plants, those cultivated in a complete nutrient solution absorbed, translocated and degraded lysine less rapidly. As proline was recorded during the lysine transformations only in the control plants, one cannot exclude the possibility of other additional mechanisms taking part in the degradation pathway of lysine. References CINCEROVA, A., and E. CERNA: Acta Univ. Caroliae - BioI. 1971, 149 (1973). GROBBELAAR, N., and F. C. STEWARD: J. Am. Chern. Soc. 75,4341 (1953). Lowy, P. H.: Arch. Biochem. Biophys. 47, 228 (1953). HYLIN, J. W.: Phytochmistry 3,161 (1964). LASTUVKA, Z., and J. MINAR: Folia Fac. Sci. Natur. Univ. Purkynianae brunnensis, Biologica 8, 1 (1967). HEILMANN, J., J. BARROLLIER, and E. WATZKE, E.: Z. physioI. Chemie 309, 219 (1957). JIRACEK, V., J. KOSTIR, and B. JIRSIKovA: Rostlinna vyroba 13, 165 (1967). ROTHSTEIN, M., and L. L. MILLER: J. BioI. Chern. 211, 851 (1954).
ALENA CINCEROV A and EVA CERNA, Department of Plant Physiology, Charles University, Vinicna 5, Praha 2, Czechoslovakia.
z.
PJlanzenphysiol. Bd. 74, S. 366-370. 1974.