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Studies on nitrogen metabolism during somatic embryogenesis in carrot. I. Utilization of α-alanine as a nitrogen source
Plant Science Letters, 33 (1984) 7--13 Elsevier Scientific Publi~ers Ireland Ltd.
STUDIES ON N I T R O G E N METABOLISM D U R I N G SOMATIC EMBRYOGENESIS IN C A R R O T . I. U T I L I Z A T I O N O F a-ALANINE AS A N I T R O G E N SOURCE
HIROSHI KAMADA and HIROSHI HARADA
Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-hen, 305 (Japan) (Received May 16th, 1983) (Revision received July 28th, 1983) (Accepted July 28th, 1983)
SUMMARY
a-Alanine added to a culture medium was incorporated into cells and/or e m b r y o s during rapid cell proliferation and globular e m b r y o formation, in which an active protein synthesis was occurring. After the incorporation into cells, a-alanine seemed to be quickly transformed to glutamic acid by alanine aminotransferase and utilized as a nitrogen source. Alanine aminotransferase activity was observed in cells and/or embryos, although the activity decreased during culture period. Utilization of a-alanine by cultured cells as a nitrogen source was discussed in relation to its stimulative effect on somatic embryogenesis. Key words: a-Alanine -- Alanine aminotransferase -- Nitrogen source -Somatic e m b r y o g e n e s i s - Daucus carota L. INTRODUCTION
Since the first observation in 1958 of somatic e m b r y o formation in carrot tissue culture [ 1,2], a n u m b e r of research workers have investigated somatic embryogenesis from histological, physiological and biochemical points of view. These investigations clearly demonstrated that the kind of nitrogen source in culture medium was a very important factor in controlling somatic embryogenesis. In many cases, the presence of reduced nitrogenous c o m p o u n d s together with nitrate was necessary for somatic embryogenesis [3--7 ]. Among various reduced nitrogenous compounds, a-alanine, Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; M~A, embryo-forming medium; MSAD, non-embryo-forming medium. 0304-4211/84/503.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
7
glutamine and glutamic acid were found to be very stimulatory in inducing embryo differentiation [5,6]. W e found, however, accumulation neither of a-alanine-rich proteins nor a large amount of a-alanine in a free form in the cellswhich were cultured in an a-alanine-richm e d i u m [8]. These observations prompted us to study further the role of a-alanine in somatic embryogenesis. Accordingly, we investigated first the consumption of a-alanine added to a culture medium, and secondly the changes in alanine aminotransferase (glutamic pyruvic transaminase (EC 2.6.1.2) activity during carrot somatic embryogenesis. MATERIALS A N D M E T H O D S
Cell culture and induction o f somatic embryos Methods for cell culture and induction of somatic embryos of D. carota L. cv. US-Harumakigosun were described earlier [6]. Cell suspension culture derived from hypocotyls was subcultured at 2-week intervals in the Murashige and Skoog's liquid medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (1 mg/1). Small cell clusters (37--63/~m in diameter) collected from 14
Enzyme extraction and assay of alanine aminotransferase (glutamic pyruvic transaminase E C 2.6.1.2) Cell clusters and/or embryos (1 g) were homogenized with 1 rnl of 0.1 M potassium phosphate buffer (pH 7.0) containing E D T A (1 raM). The homogenate was centrifuged at 10 000 X g for 10 rain at 4°C. The supernatant was then re-centrifuged at 150 000 X g for 30 rain at 4°C. After the supernatant was passed through a Sephadex G-25 column (1 X 5 cm) which was
equilibrated with the same buffer as the extraction buffer, alanine aminotransferase activity in the void volume fraction was measured by a spectrophotometric method involving the coupled reaction of lactic dehydrogenase and NADH [9]. The reaction mixture was composed of 1 M phosphate buffer (pH 7.3; 0.3 ml), 41 mM a-ketoglutarate (0.3 ml), 170 mM L-alanine (0.3 nil), lactic dehydrogenase (Miles Lab. Ltd; 50 units/ml, 0.1 ml), NADH (Boehringer Manheim GmbH; 4 rag/l, 0.1 ml) and distilled water (1.9 ml). Enzyme reaction was started by the addition of enzyme solution (0.1 ml) at 35°C and the change in absorbance at 340 nm was recorded. The enzyme activity was expressed by the following unit: 1 unit = oxidation of 1 ~mole NADH per minute. Protein contents in the enzyme solutions were determined with Coomasie brilliant blue reagent (Bio-Rad Lab.). RESULTS
The time course indicating the changes in the number of embryos formed during culture was described earlier [6,8]. When cells were cultured in MSA medium, globular embryos were observed on the 4th day of culture and their number increased rapidly to reach a maximum value on the 10th day of culture. Heart- and torpedo-shaped embryos appeared on the 7th day of culture and their numbers also increased rapidly. On the other hand, in MSAD medium, compact cell clusters appeared on the 4th day of culture, but clearly differentiated embryos could not be observed throughout the culture period. With MSA medium, the fresh weight (rag fresh wt. per ml medium) of cells and/or embryos increased rapidly after the 4th day of culture (Fig. 1A). As regards MSAD medium, a similar tendency was observed, but the rate of increase was slow (e.g. about 10% of the maximal) E E 20
A
10
o & ~
B 20
Ib
L
0
I
L
A
4 7 10 Days of c u l t u r e
/
13
Fig. 1. Changes in fresh weight (A) and protein content (B) in cells and/or embryos cultured in MSA (s s) or MSAD (A ~) medium during 13 days of culture.
10 r
g~
s
o
o
o
4 Days of
7
,0
73
cullure
Fig. 2. Decrease in the quantity of a-alanine detected in MSA (o (A A) medium during 13 days of culture.
I) or M S A D
during the late stage of culture, especially from the 10th to the 13th days. With MSA medium, protein contents (mg protein per g fresh wt.) in cells and/or embryos decreased about 20% between the 1st and 4th days of culture, then slowly increased during globular embryo formation (Fig. 1B). With MSAD medium, protein content decreased b y 75% up to the 4th day of culture, then increased sharply and more rapidly than with MSA medium toward the 10th day of culture. Figure 2 shows the decrease in a-alanine content in M S A and M S A D media during the culture period. The quantities of a-alanine in both media decreased to less than a half of the initiallevel by the 7th day of culture, and became nearly zero by the 10th day of culture. Table 1 shows the effects of various components in the reaction mixture on the activity of alanine aminotransferase. E n z y m e extracts used were obTABLE I E F F E C T S O F D I F F E R E N T C O M P O N E N T S IN A R E A C T I O N M I X T U R E A L A N I N E A M I N O T R A N S F E R A S E ACTIVITY
ON
Enzyme extractsused were prepared from initialcells(day 0 in culture)as describedin Materialsand Methods. Reaction mixture
Activity (units/reaction mixture)
Relative activity
~
0.1130 0.0085 0.0110 0.0085
I00.0 7.5 9.7 7.5
0.0000
0.0
0.0090
8.0
Complete Complete minus a-ketoglutarate Complete minus L-alanine Complete minus L-alanine minus a-ketoglutarate (~ Complete minus a-ketoglutarate plus pyruvate (~) Complete minus lactic dehydrogenase True enzyme activity = (~) -- (~) = 0.1020.
11 1.0
0.05
0JB
c 0.04
B
O
"i 0s
o.
0.03
& ~ 0.4
~' 0.02
.-- 0.2
'E 0.01
i-
0£
t
i
o
4
•
;o
Days of culture
,3
000
|
I
o
,
m
";
,o
,'3
Days of c u l t u r e
Fig. 3. Changes in alanine aminotransferase activity in cells a n d / o r e m b r y o s c u l t u r e d in MSA (o 8 ) o r M S A D (A ~) m e d i u m . T h e activity was expressed in units per g fresh wt. (A) or units per m g protein (B).
tained from initialcells (day 0 in culture) as described in Materials and Methods. The value of its activity with a complete reaction mixture was 0.1130, but if one of the components was eliminated, each respective value was reduced to less than one tenth of the figure obtained with a complete mixture. Thus, in subsequent experiments, real enzyme activity was calculated by subtracting the value obtained with a complete reaction mixture minus L-alanine ((~) from the value obtained with a complete reaction mixture ((~)). Changes in alanine aminotransferase activity during the culture period are shown in Fig. 3. With M S A medium, the activity diminished by 7 5 % toward the 7th day of culture, then remained more or less at a constant level. With M S A D medium, on the other hand, the activity (on both fresh wt. and protein basis) became low on the 4th day of culture, then transiently recovered to the initiallevel on the 7th day of culture before decreasing again. This tendency in alanine aminotransferase activity, especially the transient increase on the 7th day of culture with M S A D medium, was observed in three separate experiments. DISCUSSION
Our results presented above showed that a-alanine added to the culture m e d i u m disappeared rapidly from the culture m e d i u m during active cell proliferation and globular embryo formation. It was reported that L-alanine added to a culture m e d i u m was readily absorbed by rice cellscultured in vitro [10,11]. The analysis of amino acids remaining in the culture medium, indicated that no amino acid was found in a large amount in the culture medium. Thus, it is quite conceivable that a-alanine added to the culture niedium was incorporated into cells and/or embryos. In a previous paper [8], w e showed that neither an appreciable amount of a-alanine nor the accumulation of a-alanine-rich proteins was found in carrot cells and/or embryos cultured in the a-alanine-rich medium, and that the quantities
12
of glutamic acid and glutamine in the cells were transiently increased on the 7th day of culture, while a-alanine was rapidly disappeared from the culture medium. Working with the same experimental system, we also found that the amount of nitrate added together with a-alanine to a culture medium was utilized only after the disappearance of a-alanine from the culture medium (data not shown here). These results indicate that a-alanine incorporated into cells and/or embryos might be transformed into glutamic acid and utilized as a nitrogen source. In fact, we detected alanine aminotransferase activity in our material, though its activity decreased during culture period except for a transient increase mentioned above. The reason for this transient increase in the activity is not clear at present but seems to be consistent, and so of interest. It is not known if alanine aminotransferase is a limiting enzyme in nitrogen metabolism. Our results showed that a-alanine rapidly disappeared even when the activity of alanine aminotransferase was very low. It may be said that even a very low activity of alanine aminotransferase is sufficient to catabolize aminotransfer from a-alanine to 2-oxoglutamte. The persistence of nitrate added with a.alanine suggests additionally suppression of nitrate reductase activity under these conditions. As reported earlier, rapid cell proliferation and active protein synthesis were observed during somatic embryogenesis in carrot [12,13]. The addition to culture medium of a-alanine or glutamine as a sole nitrogen source strongly stimulated carrot embryogenesis [5,6]. These facts and the results presented above indicate that these amino acids were readily incorporated into cells and utilized as a nitrogen source during rapid cell proliferation required for globular embryo formation. We are presently studying the effects of a-alanine on nitrogen metabolism, including nitrate utilization and changes in nitrate reductase activity during somatic embryogenesis. ACKNOWLEDGEMENT
Present work was supported in part by a grantAn-aid from the Science Research Fund of the Ministry of Education, Culture, and Science to one of the authors (H.H.). REFERENCES 1 2 3 4 5 6 7 8
J. Reinert, Ber. Dt,ch. Bot. Ge,., 71 (1958) 15. F.C. Steward, M.O. Map~ and K. Meats, Am. J. Bot., 45 (1958) 705. W. Halperin and D.IC Dougall, Nature, 205 (1965) 519. J. Reinert, M. Tazawa and S. Semenoff, Nature, 216 (1967) 1215. D.F. Wetherell and D.K. Dougall, Phy,iol. Plant., 37 (1976) 97. H. Kamada and H. Harada, Z. Pfianzenphysiol., 91 (1979) 453. K.A. Walker and S.J. Sato, Plant Cell, Ti~mue and Organ Culture, 1 (1981) 109. H. Kamada and H. Harada, Plant Cell Physiol., 25 (1984) in press.
13 9 H.L. Segal and T. Matauzawa, L-alanine aminotrnn-fera~, Methods in Enzymology Vol. XVII A, p. 153. 10 H. Manabe and K. Ohira, Soil Sci. Plant Nutr., 26 (1980) 517. 11 H. Manabe and L. Ohira, Soil Sci. Plant Nutr., 27 (1981) 455. 12 D. Verma and D.K. Dougall, In vitro, 14 (1978) 183. 13 T. Fujimura, A. Komamine and H. Matsumoto, Physiol. Plant., 49 (1980) 255.
STUDIES ON N I T R O G E N METABOLISM D U R I N G SOMATIC EMBRYOGENESIS IN C A R R O T . I. U T I L I Z A T I O N O F a-ALANINE AS A N I T R O G E N SOURCE
HIROSHI KAMADA and HIROSHI HARADA
Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-hen, 305 (Japan) (Received May 16th, 1983) (Revision received July 28th, 1983) (Accepted July 28th, 1983)
SUMMARY
a-Alanine added to a culture medium was incorporated into cells and/or e m b r y o s during rapid cell proliferation and globular e m b r y o formation, in which an active protein synthesis was occurring. After the incorporation into cells, a-alanine seemed to be quickly transformed to glutamic acid by alanine aminotransferase and utilized as a nitrogen source. Alanine aminotransferase activity was observed in cells and/or embryos, although the activity decreased during culture period. Utilization of a-alanine by cultured cells as a nitrogen source was discussed in relation to its stimulative effect on somatic embryogenesis. Key words: a-Alanine -- Alanine aminotransferase -- Nitrogen source -Somatic e m b r y o g e n e s i s - Daucus carota L. INTRODUCTION
Since the first observation in 1958 of somatic e m b r y o formation in carrot tissue culture [ 1,2], a n u m b e r of research workers have investigated somatic embryogenesis from histological, physiological and biochemical points of view. These investigations clearly demonstrated that the kind of nitrogen source in culture medium was a very important factor in controlling somatic embryogenesis. In many cases, the presence of reduced nitrogenous c o m p o u n d s together with nitrate was necessary for somatic embryogenesis [3--7 ]. Among various reduced nitrogenous compounds, a-alanine, Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; M~A, embryo-forming medium; MSAD, non-embryo-forming medium. 0304-4211/84/503.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
7
glutamine and glutamic acid were found to be very stimulatory in inducing embryo differentiation [5,6]. W e found, however, accumulation neither of a-alanine-rich proteins nor a large amount of a-alanine in a free form in the cellswhich were cultured in an a-alanine-richm e d i u m [8]. These observations prompted us to study further the role of a-alanine in somatic embryogenesis. Accordingly, we investigated first the consumption of a-alanine added to a culture medium, and secondly the changes in alanine aminotransferase (glutamic pyruvic transaminase (EC 2.6.1.2) activity during carrot somatic embryogenesis. MATERIALS A N D M E T H O D S
Cell culture and induction o f somatic embryos Methods for cell culture and induction of somatic embryos of D. carota L. cv. US-Harumakigosun were described earlier [6]. Cell suspension culture derived from hypocotyls was subcultured at 2-week intervals in the Murashige and Skoog's liquid medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (1 mg/1). Small cell clusters (37--63/~m in diameter) collected from 14
Enzyme extraction and assay of alanine aminotransferase (glutamic pyruvic transaminase E C 2.6.1.2) Cell clusters and/or embryos (1 g) were homogenized with 1 rnl of 0.1 M potassium phosphate buffer (pH 7.0) containing E D T A (1 raM). The homogenate was centrifuged at 10 000 X g for 10 rain at 4°C. The supernatant was then re-centrifuged at 150 000 X g for 30 rain at 4°C. After the supernatant was passed through a Sephadex G-25 column (1 X 5 cm) which was
equilibrated with the same buffer as the extraction buffer, alanine aminotransferase activity in the void volume fraction was measured by a spectrophotometric method involving the coupled reaction of lactic dehydrogenase and NADH [9]. The reaction mixture was composed of 1 M phosphate buffer (pH 7.3; 0.3 ml), 41 mM a-ketoglutarate (0.3 ml), 170 mM L-alanine (0.3 nil), lactic dehydrogenase (Miles Lab. Ltd; 50 units/ml, 0.1 ml), NADH (Boehringer Manheim GmbH; 4 rag/l, 0.1 ml) and distilled water (1.9 ml). Enzyme reaction was started by the addition of enzyme solution (0.1 ml) at 35°C and the change in absorbance at 340 nm was recorded. The enzyme activity was expressed by the following unit: 1 unit = oxidation of 1 ~mole NADH per minute. Protein contents in the enzyme solutions were determined with Coomasie brilliant blue reagent (Bio-Rad Lab.). RESULTS
The time course indicating the changes in the number of embryos formed during culture was described earlier [6,8]. When cells were cultured in MSA medium, globular embryos were observed on the 4th day of culture and their number increased rapidly to reach a maximum value on the 10th day of culture. Heart- and torpedo-shaped embryos appeared on the 7th day of culture and their numbers also increased rapidly. On the other hand, in MSAD medium, compact cell clusters appeared on the 4th day of culture, but clearly differentiated embryos could not be observed throughout the culture period. With MSA medium, the fresh weight (rag fresh wt. per ml medium) of cells and/or embryos increased rapidly after the 4th day of culture (Fig. 1A). As regards MSAD medium, a similar tendency was observed, but the rate of increase was slow (e.g. about 10% of the maximal) E E 20
A
10
o & ~
B 20
Ib
L
0
I
L
A
4 7 10 Days of c u l t u r e
/
13
Fig. 1. Changes in fresh weight (A) and protein content (B) in cells and/or embryos cultured in MSA (s s) or MSAD (A ~) medium during 13 days of culture.
10 r
g~
s
o
o
o
4 Days of
7
,0
73
cullure
Fig. 2. Decrease in the quantity of a-alanine detected in MSA (o (A A) medium during 13 days of culture.
I) or M S A D
during the late stage of culture, especially from the 10th to the 13th days. With MSA medium, protein contents (mg protein per g fresh wt.) in cells and/or embryos decreased about 20% between the 1st and 4th days of culture, then slowly increased during globular embryo formation (Fig. 1B). With MSAD medium, protein content decreased b y 75% up to the 4th day of culture, then increased sharply and more rapidly than with MSA medium toward the 10th day of culture. Figure 2 shows the decrease in a-alanine content in M S A and M S A D media during the culture period. The quantities of a-alanine in both media decreased to less than a half of the initiallevel by the 7th day of culture, and became nearly zero by the 10th day of culture. Table 1 shows the effects of various components in the reaction mixture on the activity of alanine aminotransferase. E n z y m e extracts used were obTABLE I E F F E C T S O F D I F F E R E N T C O M P O N E N T S IN A R E A C T I O N M I X T U R E A L A N I N E A M I N O T R A N S F E R A S E ACTIVITY
ON
Enzyme extractsused were prepared from initialcells(day 0 in culture)as describedin Materialsand Methods. Reaction mixture
Activity (units/reaction mixture)
Relative activity
~
0.1130 0.0085 0.0110 0.0085
I00.0 7.5 9.7 7.5
0.0000
0.0
0.0090
8.0
Complete Complete minus a-ketoglutarate Complete minus L-alanine Complete minus L-alanine minus a-ketoglutarate (~ Complete minus a-ketoglutarate plus pyruvate (~) Complete minus lactic dehydrogenase True enzyme activity = (~) -- (~) = 0.1020.
11 1.0
0.05
0JB
c 0.04
B
O
"i 0s
o.
0.03
& ~ 0.4
~' 0.02
.-- 0.2
'E 0.01
i-
0£
t
i
o
4
•
;o
Days of culture
,3
000
|
I
o
,
m
";
,o
,'3
Days of c u l t u r e
Fig. 3. Changes in alanine aminotransferase activity in cells a n d / o r e m b r y o s c u l t u r e d in MSA (o 8 ) o r M S A D (A ~) m e d i u m . T h e activity was expressed in units per g fresh wt. (A) or units per m g protein (B).
tained from initialcells (day 0 in culture) as described in Materials and Methods. The value of its activity with a complete reaction mixture was 0.1130, but if one of the components was eliminated, each respective value was reduced to less than one tenth of the figure obtained with a complete mixture. Thus, in subsequent experiments, real enzyme activity was calculated by subtracting the value obtained with a complete reaction mixture minus L-alanine ((~) from the value obtained with a complete reaction mixture ((~)). Changes in alanine aminotransferase activity during the culture period are shown in Fig. 3. With M S A medium, the activity diminished by 7 5 % toward the 7th day of culture, then remained more or less at a constant level. With M S A D medium, on the other hand, the activity (on both fresh wt. and protein basis) became low on the 4th day of culture, then transiently recovered to the initiallevel on the 7th day of culture before decreasing again. This tendency in alanine aminotransferase activity, especially the transient increase on the 7th day of culture with M S A D medium, was observed in three separate experiments. DISCUSSION
Our results presented above showed that a-alanine added to the culture m e d i u m disappeared rapidly from the culture m e d i u m during active cell proliferation and globular embryo formation. It was reported that L-alanine added to a culture m e d i u m was readily absorbed by rice cellscultured in vitro [10,11]. The analysis of amino acids remaining in the culture medium, indicated that no amino acid was found in a large amount in the culture medium. Thus, it is quite conceivable that a-alanine added to the culture niedium was incorporated into cells and/or embryos. In a previous paper [8], w e showed that neither an appreciable amount of a-alanine nor the accumulation of a-alanine-rich proteins was found in carrot cells and/or embryos cultured in the a-alanine-rich medium, and that the quantities
12
of glutamic acid and glutamine in the cells were transiently increased on the 7th day of culture, while a-alanine was rapidly disappeared from the culture medium. Working with the same experimental system, we also found that the amount of nitrate added together with a-alanine to a culture medium was utilized only after the disappearance of a-alanine from the culture medium (data not shown here). These results indicate that a-alanine incorporated into cells and/or embryos might be transformed into glutamic acid and utilized as a nitrogen source. In fact, we detected alanine aminotransferase activity in our material, though its activity decreased during culture period except for a transient increase mentioned above. The reason for this transient increase in the activity is not clear at present but seems to be consistent, and so of interest. It is not known if alanine aminotransferase is a limiting enzyme in nitrogen metabolism. Our results showed that a-alanine rapidly disappeared even when the activity of alanine aminotransferase was very low. It may be said that even a very low activity of alanine aminotransferase is sufficient to catabolize aminotransfer from a-alanine to 2-oxoglutamte. The persistence of nitrate added with a.alanine suggests additionally suppression of nitrate reductase activity under these conditions. As reported earlier, rapid cell proliferation and active protein synthesis were observed during somatic embryogenesis in carrot [12,13]. The addition to culture medium of a-alanine or glutamine as a sole nitrogen source strongly stimulated carrot embryogenesis [5,6]. These facts and the results presented above indicate that these amino acids were readily incorporated into cells and utilized as a nitrogen source during rapid cell proliferation required for globular embryo formation. We are presently studying the effects of a-alanine on nitrogen metabolism, including nitrate utilization and changes in nitrate reductase activity during somatic embryogenesis. ACKNOWLEDGEMENT
Present work was supported in part by a grantAn-aid from the Science Research Fund of the Ministry of Education, Culture, and Science to one of the authors (H.H.). REFERENCES 1 2 3 4 5 6 7 8
J. Reinert, Ber. Dt,ch. Bot. Ge,., 71 (1958) 15. F.C. Steward, M.O. Map~ and K. Meats, Am. J. Bot., 45 (1958) 705. W. Halperin and D.IC Dougall, Nature, 205 (1965) 519. J. Reinert, M. Tazawa and S. Semenoff, Nature, 216 (1967) 1215. D.F. Wetherell and D.K. Dougall, Phy,iol. Plant., 37 (1976) 97. H. Kamada and H. Harada, Z. Pfianzenphysiol., 91 (1979) 453. K.A. Walker and S.J. Sato, Plant Cell, Ti~mue and Organ Culture, 1 (1981) 109. H. Kamada and H. Harada, Plant Cell Physiol., 25 (1984) in press.
13 9 H.L. Segal and T. Matauzawa, L-alanine aminotrnn-fera~, Methods in Enzymology Vol. XVII A, p. 153. 10 H. Manabe and K. Ohira, Soil Sci. Plant Nutr., 26 (1980) 517. 11 H. Manabe and L. Ohira, Soil Sci. Plant Nutr., 27 (1981) 455. 12 D. Verma and D.K. Dougall, In vitro, 14 (1978) 183. 13 T. Fujimura, A. Komamine and H. Matsumoto, Physiol. Plant., 49 (1980) 255.