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Galactosyl transferase activity of chloroplast envelopes from senescent soybean cotyledons
Plant Science Letters, 14 (1979) 1--6 © Elsevier/North-Holland Scientific Publishers Ltd.
GALACTOSYL TRANSFERASE ACTIVITY OF CHLOROPLAST ENVELOPES FROM SENESCENT SOYBEAN COTYLEDONS DAVID DALGARN, PAUL MILLER, TERRY BRICKER, NANCY SPEER, JAN G. JAWORSKI* and DAVID W. NEWMAN** Departments of Botany and *Chemistry, Miami University, Oxford, 0H45056 (U.<~.A.)
(Received June 28th, 1978) (Revision received August 29th, 1978) (Accepted August 29th, 1978)
SUMMARY
During the senescence of photosynthetic tissues, the level of chloroplast galactolipids markedly declines. The final assembly of these lipids occurs in the plastid envelope. An investigation was made to determine the activity of plastid envelope UDP.galactose-diglyceride galactosyl transferase (galactosyl transferMe), which makes this final assembly, in envelopes from greening and senescing soybean cotyledons. When the tissue greened there was an increase in the galactosyl transferase activity. As the tissue senesced there was a decline of the activity of this enzyme as well as a decrease in cotyledon chlorophyll, nucleic acid, protein, mono- and di-galactosyl diacylglycerols, phosphatidyl glycerol, phosphatidyl ethanolamine and phosphatidyl choline levels. It is suggested that the decreased plastid envelope galactosyl transferase activity in senescent tissue may explain, at least in part, the decreased levels of mono- and digalactosyl diacylglycerols in these tissues.
INTRODUCTION
Senescence involves changes in many compounds in previously green, photosynthetic tissues [1]. For example, in soybean cotyledons there is a reduction in the protein, R N A , and chlorophyll levels.As well, there are changes in the chloroplast lipidswhich seem to be correlated with ultrastructuralalterations of the chloroplast [2]. In particularthere seem to be marked reductions in the galactolipids of the chloroplast and, as would be expected, a decline in the relativeamount of linolenic acid, much of which seems to be ~sterifiedto l
**To whom reprint requests should be sent. Abbreviations: DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; PE, phosphatidyl ethanc~amine; PC, phosphatidyl choline; PG, phosphatidyl glycerol.
the galactolipid, monogalactosyl diacylglycerol (MGDG) [3]. The question arises as to why there seems to be a more rapid decline in the chloroplast galactolipids during senescence than in the phospholipids most commonly associated with extrachloroplastic membranes [4]. The final assembly of the galactolipids of the chloroplast seems to occur in the enveI6pe fraction by way of galactosyl ~mlsferase [ 5--8]. It follows that possibly there is a decreased capacity for this final assembly in the chloroplasts of senescent soybean cotyledons. We, therefore, made an attempt to determine if, indeed, this might be the case. MATERIALS AND METHODS
Growth conditions Soybean (Glycine max cv. Wayne) were grown under continuous light of 32 W/m2 at 28°C. The plants were irrigated on alternate days with water or with a complete nutrient solution. Analy ticM procedures Chlorophyll analyses were made spectrophotometrically [9]. Protein was assayed by a m~ification of the Lowry method [10]. Cherry's [11] method was used to determine the total nucleic acid content of the tissue. This method is based on reeJing the absorbance difference at 260 and 290 nm. The free amino acids o~ the tissue were extracted by gently refluxing leaves in 80% ethanol for 30 rain. An aliquot of the ethanol extract was used to determine the free amino content by the Moore and Stein [ 12] method. A standard curve was made using a variety of amino acids. The lipids were extracted with chloroform/methanol (2 : 1, v/v). The homogenate was filtered, the filtrate transferred to a separatory funnel, and washed 3 times with 70 ml of water acidified with 2 drops of 5N H2SO4. The chloroform phase containing the lipophilic compounds was dried with anhydrous Na2SO4 and stored under N2 at -20°C. The lipids were separated directly by thin-layer chromatography. The two dimensional system which was routinely used consisted of chloroform/methanol/water (65 : 25 : 4, v/v) for the first dimension and acetone/ acetic acid/water (100 : 2 : 1, v/v) for the second dimension" [13]. Three methods were used to visualize the separated lipids on the thin layer plates. Iodine vapors will detect all of the lipids. A molybdenum spray was used to identify the phospholipids [ 14]. The third and non
lation medium [17] and ground at low speed in a blender for two 2-s bursts. The resulting suspension was filtered through two layers of cheesecloth on top of two layers of Miracloth (Calbiochemical Co.) and centrifuged at 1000 g for 1--2 min. The chloroplast pellets (containing mostly intact chloroplasts) from an initial 450 g of cotyledons were combined; the rupture and fractionation procedures of Douce et ai. [ 18] were followed. The major envelope fraction located in the second band of the sucrose gradient was collected and pooled from six tubes. This band was light yellow and found on the gradient exactly as reported previously [ 18]. Galactosyl transferase activity was assayed following the method of Douce [6] with minor modifications -- the incubation mixture contained 2.0 ml of envelope band, 0.45 ml of buffer (tricine-NaOH (pH 7.5); 0.8 ~M MgCI-~, and 0.05 ml of label (750 000 d.p~n. ['4C] UDP-galactose; specific activity 300 mCi/mmol). The various lipid products were separated by one dimensi: ,~al thin-layer chromatography using chloroform/methanol/acetic acid/;vater (85 : 15 : 10 : 4, v/v). The radioactivity was counted by liquid scintillation spectrophotometry. Envelope protein was measured by the method of Kalb et al. [19].
RESULTS AND DISCUSSION As was expected from the work of Krul [ 1], as the cotyledons aged, the chlorophyll, nucleic acid, and protein levels declined (Table I). In addition, the level of amino acids declined. A possible reason for this concomitant decline in the amino nitrogen level, assuming increased proteolytic activity, was that these nitrogenous compounds, as soon as formed by the digestion of the protein, were transported out of the cotyledon under our experimental conditions. It will be noticed that there was initially a much more rapid decline in the protein level than in the chlorophyll level, suggesting a rapid digestion of the storage proteins outside of the chloroplast. The chloroplast galactolipids decreased faster than did the phospholipids more commonly associated with the nonchloroplast membranes (PE and PC) (Table II; 4). There was initially a more rapid decline in PG, as compared to that of MGDG, but this decline slowed by the 12th and 15th days. Table III illustrates the changes'in envelope galactosyl transferase activity during senescence. As the cotyledons were greening and maturing the activity increased. During ~nescence the activity dropped off to a low value. The observed changes in enzyme activity may be due to decreased level of the galactosyl transferase or because of the limiting diglyceride substrate. This decrease in activity would limit the final assembly of MGDG and DGDG. Much less decrease was observed in the ability of the envelope to assemble DGDG. However, at no time was this activity great. Perhaps the assay conditions were less favorable for DGDG than for MGDG assembly, e.g. differing pH optima [20] or the enzyme synthesizing the digalactolipid is less tightly bound to the membranes [21 ] and thus was lost during envelope isolation. Van Besouw and
TABLE I CHLOROPHYLL, NUCLEIC ACID, PROTEIN AND AMINO ACID CONTENT OF 8OYBEAN COTYLEDONS GROWN UNDER CONTINUOUS LIGHT Six.day tissue = 100%; Amounts per two cotyledons; Three to 8 replications were used to determine each mean and minimu_m size for each was 50 cotyledons/replicate.
(d s) m
Chlorophyll mg 8D % Nucleic acids ~g SD % Protein ~g SD % Amino acids
~M SD %
,,,,
,
6
9
12"
15
18
0,47 0.05 I00
0.40 0.03 85
0.35 0,08 74
0.16 0.01 34
0.04 0.01 8
1036 108 100
582 130 56
514 124 50
268 14 26
153 42 15
23237 3749 100
7935 2417 34
6601 1089 28
2768 1189 12
1849 200 8
0.098 0.010 100
0.068 0.032 69
0.050 0.016 51
0.018 0,007 18
0.011 0.001 11
TABLE II LWID CONTENT OF SOYBEAN COTYLEDONS GROWN UNDER CONTINUOUS LIGHT Six,lay tissue = 100%; Amounts/three cotyledons; Four replications were used to determine each mean. Age (days)
MGDG ,M SD % DGDG ~M SD % ~M SD % PE ~M SD % PC ,M SD %
6
9
12
15
18
1.41 0.I0 100
1.20 0.25 85
0.62 0.26 44
0.24 0.02 17
0.06 0.00 4
0.96 0.08 I00
0.72 0.17 75
0.4,~ 0.12 50
0.19 0.07 20
0 04 0.01 4
0.14 0.0,3 100
0.10 0.01 71
0.0(0.00 43
0.04 0.01 29
0.02 0.01 14
0.15 0.02 100
0.13 0.01 87
0.10 0.04 67
0.08 0.02 53
0.06 0.00 40
025 0.06 !00
0.21 0.02 84
0.15 0.04 60
0.14 0.02 56
0.12 0.01 48
TABLE HI GALACTOSYL TRANSFERASE ACTIVITY Amount of radioactivity incorporated into galactolipids by soybean chloroplast envelopes. The numbers are averages of two values. Age
6 days 11 days 16 days
C.pjn. per mg envelope fraction protein MGDG
DGDG
660 24100 3800
29 1080 500
Wintermans [5] suggest t h a t t h e digalactolipid is synthesized as follows: 2 monogalactolipid -~ digalactolipid + diglyceride, which might suggest the low D G D G assembly observed in vitro due to insufficient precursor. The observed loss o f galactosyl transferase activity in senescing tissue probably c o n t r i b u t e s t o the loss o f MGDG and DGDG in late stages of senescence. The influence o f the transferase activity on the galactolipid levels at early stages o f s e n e s c e n c e is less clear. We have observed t h a t the time of initiation of senescence varies considerably from one e x p e r i m e n t to another, and thus clear correlations between e n z y m e activities and c o n c e n t r a t i o n s of cellular comp o n e n t s are difficult to m a k e during this part o f t h e growth cycle. Nevertheless, we have d e m o n s t r a t e d that at later stages of senescence, both the galactosyl transferase activity and the levels o f MGDG and DGDG decreased. REFERENCES
1 W.R. Krui, Plant Physiol., 54 (1974) 36. 2 D.J. Huber and D.W. Newman, J. Expti. Bot., 27 (1976) 490. 3 C. Hitchcock and B.W. Nichols, Plant Lipid Biochemistry, Academic Press, London, 1971, p. 91. 4 D. Dalgarn, D. Huber and D.W. Newman, Physiol. Plant., 40 (1977) 153. 5 A. Van Besouw and J.F.G.M. Wintermans, Biochim. Biophys. Acta, 529 (1978) 44. 6 R. Douce, Science, 183 (1974) 852. 7 H. van Hummel, T. Huisebos and J.F.G.M. Wintermans, Biochim. Biophys. Acta, 380 (1975)219. 8 B. Liedvogel and H. Kleinig, Planta, 129 (1976) 19. 9 D.I. Arnon, Plant Physiol., 24 (1949) 1. 10 G.R. Schacterle and R.L. Pollack, Anal. Biochem., 51 (1973) 654. 11 J.H. Cherry, Molecular Biology of Plants, Columbia University Press, New York, 1973, p. 18. 12 S. Moore and W.W. Stein, J. Biol. Chem., 176 (1948) 376. 13 H.W. Gardner, J. Lipid Res., 9 (1968) 139. 14 J.C. Dittmer and R.L. Lester, J. Lipid Res., 5 (1964) 126. 15 P.G. Roughan and R.D. Batt, Anal. Biochem., 22 (1968) .74. 16 G.R. Bartlett, J. Biol. Chem., 234 (1959) 466. 17 C.G. Kannangara, B.S. Jacobson and P.K. Stumpf, Plant Physiol., 52 (1973) 156. 18 R. Douce, R.B. Holt and A.A. Bermon, J. Biol. Chem., 248 (1973) 7215. 19 V.F. Kalb and R.W. Bemlohr, Anal. Biochem., 82 (1977) 362. 20 J. Joyard and R. Douce, Physiol. Veg., 14 (1976) 31. 21 A. Ongun and J. Mudd, J. Biol. Chem., 243 (1968) 1558.
GALACTOSYL TRANSFERASE ACTIVITY OF CHLOROPLAST ENVELOPES FROM SENESCENT SOYBEAN COTYLEDONS DAVID DALGARN, PAUL MILLER, TERRY BRICKER, NANCY SPEER, JAN G. JAWORSKI* and DAVID W. NEWMAN** Departments of Botany and *Chemistry, Miami University, Oxford, 0H45056 (U.<~.A.)
(Received June 28th, 1978) (Revision received August 29th, 1978) (Accepted August 29th, 1978)
SUMMARY
During the senescence of photosynthetic tissues, the level of chloroplast galactolipids markedly declines. The final assembly of these lipids occurs in the plastid envelope. An investigation was made to determine the activity of plastid envelope UDP.galactose-diglyceride galactosyl transferase (galactosyl transferMe), which makes this final assembly, in envelopes from greening and senescing soybean cotyledons. When the tissue greened there was an increase in the galactosyl transferase activity. As the tissue senesced there was a decline of the activity of this enzyme as well as a decrease in cotyledon chlorophyll, nucleic acid, protein, mono- and di-galactosyl diacylglycerols, phosphatidyl glycerol, phosphatidyl ethanolamine and phosphatidyl choline levels. It is suggested that the decreased plastid envelope galactosyl transferase activity in senescent tissue may explain, at least in part, the decreased levels of mono- and digalactosyl diacylglycerols in these tissues.
INTRODUCTION
Senescence involves changes in many compounds in previously green, photosynthetic tissues [1]. For example, in soybean cotyledons there is a reduction in the protein, R N A , and chlorophyll levels.As well, there are changes in the chloroplast lipidswhich seem to be correlated with ultrastructuralalterations of the chloroplast [2]. In particularthere seem to be marked reductions in the galactolipids of the chloroplast and, as would be expected, a decline in the relativeamount of linolenic acid, much of which seems to be ~sterifiedto l
**To whom reprint requests should be sent. Abbreviations: DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; PE, phosphatidyl ethanc~amine; PC, phosphatidyl choline; PG, phosphatidyl glycerol.
the galactolipid, monogalactosyl diacylglycerol (MGDG) [3]. The question arises as to why there seems to be a more rapid decline in the chloroplast galactolipids during senescence than in the phospholipids most commonly associated with extrachloroplastic membranes [4]. The final assembly of the galactolipids of the chloroplast seems to occur in the enveI6pe fraction by way of galactosyl ~mlsferase [ 5--8]. It follows that possibly there is a decreased capacity for this final assembly in the chloroplasts of senescent soybean cotyledons. We, therefore, made an attempt to determine if, indeed, this might be the case. MATERIALS AND METHODS
Growth conditions Soybean (Glycine max cv. Wayne) were grown under continuous light of 32 W/m2 at 28°C. The plants were irrigated on alternate days with water or with a complete nutrient solution. Analy ticM procedures Chlorophyll analyses were made spectrophotometrically [9]. Protein was assayed by a m~ification of the Lowry method [10]. Cherry's [11] method was used to determine the total nucleic acid content of the tissue. This method is based on reeJing the absorbance difference at 260 and 290 nm. The free amino acids o~ the tissue were extracted by gently refluxing leaves in 80% ethanol for 30 rain. An aliquot of the ethanol extract was used to determine the free amino content by the Moore and Stein [ 12] method. A standard curve was made using a variety of amino acids. The lipids were extracted with chloroform/methanol (2 : 1, v/v). The homogenate was filtered, the filtrate transferred to a separatory funnel, and washed 3 times with 70 ml of water acidified with 2 drops of 5N H2SO4. The chloroform phase containing the lipophilic compounds was dried with anhydrous Na2SO4 and stored under N2 at -20°C. The lipids were separated directly by thin-layer chromatography. The two dimensional system which was routinely used consisted of chloroform/methanol/water (65 : 25 : 4, v/v) for the first dimension and acetone/ acetic acid/water (100 : 2 : 1, v/v) for the second dimension" [13]. Three methods were used to visualize the separated lipids on the thin layer plates. Iodine vapors will detect all of the lipids. A molybdenum spray was used to identify the phospholipids [ 14]. The third and non
lation medium [17] and ground at low speed in a blender for two 2-s bursts. The resulting suspension was filtered through two layers of cheesecloth on top of two layers of Miracloth (Calbiochemical Co.) and centrifuged at 1000 g for 1--2 min. The chloroplast pellets (containing mostly intact chloroplasts) from an initial 450 g of cotyledons were combined; the rupture and fractionation procedures of Douce et ai. [ 18] were followed. The major envelope fraction located in the second band of the sucrose gradient was collected and pooled from six tubes. This band was light yellow and found on the gradient exactly as reported previously [ 18]. Galactosyl transferase activity was assayed following the method of Douce [6] with minor modifications -- the incubation mixture contained 2.0 ml of envelope band, 0.45 ml of buffer (tricine-NaOH (pH 7.5); 0.8 ~M MgCI-~, and 0.05 ml of label (750 000 d.p~n. ['4C] UDP-galactose; specific activity 300 mCi/mmol). The various lipid products were separated by one dimensi: ,~al thin-layer chromatography using chloroform/methanol/acetic acid/;vater (85 : 15 : 10 : 4, v/v). The radioactivity was counted by liquid scintillation spectrophotometry. Envelope protein was measured by the method of Kalb et al. [19].
RESULTS AND DISCUSSION As was expected from the work of Krul [ 1], as the cotyledons aged, the chlorophyll, nucleic acid, and protein levels declined (Table I). In addition, the level of amino acids declined. A possible reason for this concomitant decline in the amino nitrogen level, assuming increased proteolytic activity, was that these nitrogenous compounds, as soon as formed by the digestion of the protein, were transported out of the cotyledon under our experimental conditions. It will be noticed that there was initially a much more rapid decline in the protein level than in the chlorophyll level, suggesting a rapid digestion of the storage proteins outside of the chloroplast. The chloroplast galactolipids decreased faster than did the phospholipids more commonly associated with the nonchloroplast membranes (PE and PC) (Table II; 4). There was initially a more rapid decline in PG, as compared to that of MGDG, but this decline slowed by the 12th and 15th days. Table III illustrates the changes'in envelope galactosyl transferase activity during senescence. As the cotyledons were greening and maturing the activity increased. During ~nescence the activity dropped off to a low value. The observed changes in enzyme activity may be due to decreased level of the galactosyl transferase or because of the limiting diglyceride substrate. This decrease in activity would limit the final assembly of MGDG and DGDG. Much less decrease was observed in the ability of the envelope to assemble DGDG. However, at no time was this activity great. Perhaps the assay conditions were less favorable for DGDG than for MGDG assembly, e.g. differing pH optima [20] or the enzyme synthesizing the digalactolipid is less tightly bound to the membranes [21 ] and thus was lost during envelope isolation. Van Besouw and
TABLE I CHLOROPHYLL, NUCLEIC ACID, PROTEIN AND AMINO ACID CONTENT OF 8OYBEAN COTYLEDONS GROWN UNDER CONTINUOUS LIGHT Six.day tissue = 100%; Amounts per two cotyledons; Three to 8 replications were used to determine each mean and minimu_m size for each was 50 cotyledons/replicate.
(d s) m
Chlorophyll mg 8D % Nucleic acids ~g SD % Protein ~g SD % Amino acids
~M SD %
,,,,
,
6
9
12"
15
18
0,47 0.05 I00
0.40 0.03 85
0.35 0,08 74
0.16 0.01 34
0.04 0.01 8
1036 108 100
582 130 56
514 124 50
268 14 26
153 42 15
23237 3749 100
7935 2417 34
6601 1089 28
2768 1189 12
1849 200 8
0.098 0.010 100
0.068 0.032 69
0.050 0.016 51
0.018 0,007 18
0.011 0.001 11
TABLE II LWID CONTENT OF SOYBEAN COTYLEDONS GROWN UNDER CONTINUOUS LIGHT Six,lay tissue = 100%; Amounts/three cotyledons; Four replications were used to determine each mean. Age (days)
MGDG ,M SD % DGDG ~M SD % ~M SD % PE ~M SD % PC ,M SD %
6
9
12
15
18
1.41 0.I0 100
1.20 0.25 85
0.62 0.26 44
0.24 0.02 17
0.06 0.00 4
0.96 0.08 I00
0.72 0.17 75
0.4,~ 0.12 50
0.19 0.07 20
0 04 0.01 4
0.14 0.0,3 100
0.10 0.01 71
0.0(0.00 43
0.04 0.01 29
0.02 0.01 14
0.15 0.02 100
0.13 0.01 87
0.10 0.04 67
0.08 0.02 53
0.06 0.00 40
025 0.06 !00
0.21 0.02 84
0.15 0.04 60
0.14 0.02 56
0.12 0.01 48
TABLE HI GALACTOSYL TRANSFERASE ACTIVITY Amount of radioactivity incorporated into galactolipids by soybean chloroplast envelopes. The numbers are averages of two values. Age
6 days 11 days 16 days
C.pjn. per mg envelope fraction protein MGDG
DGDG
660 24100 3800
29 1080 500
Wintermans [5] suggest t h a t t h e digalactolipid is synthesized as follows: 2 monogalactolipid -~ digalactolipid + diglyceride, which might suggest the low D G D G assembly observed in vitro due to insufficient precursor. The observed loss o f galactosyl transferase activity in senescing tissue probably c o n t r i b u t e s t o the loss o f MGDG and DGDG in late stages of senescence. The influence o f the transferase activity on the galactolipid levels at early stages o f s e n e s c e n c e is less clear. We have observed t h a t the time of initiation of senescence varies considerably from one e x p e r i m e n t to another, and thus clear correlations between e n z y m e activities and c o n c e n t r a t i o n s of cellular comp o n e n t s are difficult to m a k e during this part o f t h e growth cycle. Nevertheless, we have d e m o n s t r a t e d that at later stages of senescence, both the galactosyl transferase activity and the levels o f MGDG and DGDG decreased. REFERENCES
1 W.R. Krui, Plant Physiol., 54 (1974) 36. 2 D.J. Huber and D.W. Newman, J. Expti. Bot., 27 (1976) 490. 3 C. Hitchcock and B.W. Nichols, Plant Lipid Biochemistry, Academic Press, London, 1971, p. 91. 4 D. Dalgarn, D. Huber and D.W. Newman, Physiol. Plant., 40 (1977) 153. 5 A. Van Besouw and J.F.G.M. Wintermans, Biochim. Biophys. Acta, 529 (1978) 44. 6 R. Douce, Science, 183 (1974) 852. 7 H. van Hummel, T. Huisebos and J.F.G.M. Wintermans, Biochim. Biophys. Acta, 380 (1975)219. 8 B. Liedvogel and H. Kleinig, Planta, 129 (1976) 19. 9 D.I. Arnon, Plant Physiol., 24 (1949) 1. 10 G.R. Schacterle and R.L. Pollack, Anal. Biochem., 51 (1973) 654. 11 J.H. Cherry, Molecular Biology of Plants, Columbia University Press, New York, 1973, p. 18. 12 S. Moore and W.W. Stein, J. Biol. Chem., 176 (1948) 376. 13 H.W. Gardner, J. Lipid Res., 9 (1968) 139. 14 J.C. Dittmer and R.L. Lester, J. Lipid Res., 5 (1964) 126. 15 P.G. Roughan and R.D. Batt, Anal. Biochem., 22 (1968) .74. 16 G.R. Bartlett, J. Biol. Chem., 234 (1959) 466. 17 C.G. Kannangara, B.S. Jacobson and P.K. Stumpf, Plant Physiol., 52 (1973) 156. 18 R. Douce, R.B. Holt and A.A. Bermon, J. Biol. Chem., 248 (1973) 7215. 19 V.F. Kalb and R.W. Bemlohr, Anal. Biochem., 82 (1977) 362. 20 J. Joyard and R. Douce, Physiol. Veg., 14 (1976) 31. 21 A. Ongun and J. Mudd, J. Biol. Chem., 243 (1968) 1558.