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Mobilization of carbon during senescence in detached leaves of Lolium temulentum
Plant Science Letters, 25 (1982) 313--319 Elsevier/North-Holland Scientific Publishers Ltd.
313
MOBILIZATION OF CARBON DURING SENESCENCE IN DETACHED LEAVES OF LOLIUM TEMULENTUM
E.J. LLOYD Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, Dyfed. SY23 3EB (U.K.) (Received September 24th, 1981) (Accepted December 3rd, 1981)
SUMMARY
The water-insoluble component of mature leaf extracts of Lolium temulentum contained the majority of radioactivity incorporated from [14C] sucrose fed during leaf expansion. Radioactivity in water-insoluble material was present mainly in the protein fraction. Induced senescence caused a rapid loss of water-insoluble radioactivity mainly attributable to release of label from the protein fraction. Label released from insoluble material by senescence-induced proteolysis accumulated as amino-acids in detached leaves. Changes in the total amino-acid content of hydrolysates and water,soluble extracts paralleled changes in radioactivity. Enhanced 14CO2 respiration by the senescent leaf was associated with radioactivity released by protein degradation.
INTRODUC~ON
Export of metabolites from the senescent leaf to other regions of the plant is one of the fundamental processes occurring during natural sequential senescence [1,2]. During induced senescence of 14C-fed mature, attached, fourth leaves of Lolium ternulenturn, radioactivity released from waterinsoluble material was either translocated or lost through respiration [3]. The experiments in this paper were performed to identify that component of water-insoluble material into which radioactivity was incorporated during leaf expansion and from which label was lost during induced senescence of the mature leaf. Detached leaves were used in this study to prevent export and contain, within the leaf, the products of senescence-induced breakdown. Evidence for determining the source of carbon mobilized during senescence was derived from an examination of the chemical nature of the material in which solubilized radioactivity was recovered. Abbreviations: TFA, trifluoroacetic acid; WIM, water-insoluble material. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
314 MATERIALS AND METHODS Seeds of L. temulentum (Ba 3081 summer annual) were germinated on pads at 20°C with an 8-h p h o t o p e r i o d under a light intensity of 100 Wm -2 . Seedlings were transferred after 7 days and grown under the same conditions in a mineral nutrient solution [4] which was renewed at weekly intervals.
Isotope feeding technique Plants were removed from nutrient solution 5 days after fourth leaf emergence and their roots immersed in water for 24 h before exposure to isotope. Radioactivity was fed to each plant by root absorption of 3.0 ~Ci [14C] sucrose (Amersham) as previously described [3]. In these experiments, ~4C-incorporation was improved (4--5-fold) by adding glucose to the feeding medium at a final concentration of 10 mM [ 5]. After complete uptake of the 14C-labelled sugar medium, plants were transferred to fresh nutrient solution and grown until the fourth leaf was fully expanded.
Leaf treatments At ligule formation, fourth leaves from some of the ~4C-treated plants were excised t o g e t h e r with approx. 2 cm of the leaf basal portion. The leaves were sealed in the dark chambers of a respirometer as previously described [3] and the leaf bases kept submerged in nutrient solution. Attached leaves from other 14C-treated plants were sealed into the light exposed chambers of the respirometer to serve as controls. Leaves were removed from leaf chambers at daily intervals and extractable radioactivity determined.
Analytical procedures Determinations of radioactivity in CO2 and in water-soluble material were obtained as previously described [3]. Water-insoluble material (WIM) was ground in cold water and the suspension brought to a final volume of 10.0 ml. Radioactivity was determined from 1-ml samples after wet digestion to 14CO2 [6]. Treatment with 6 N trifluoroacetic acid (TFA) at l l 0 ° C for 20 h hydrolysed 81.2% of the radioactivity in 2-ml samples of insoluble extracts. Residual particulate material was removed b y low-speed centrifugation. Hydrolysates were then reduced to dryness by rotary-evaporation and made up to a final volume of 2 ml. Samples of acid hydrolysates were separated into protein and non-protein fractions b y ion~exchange chromatography. The protein fraction was defined as the c o m p o n e n t of WIM which yields basic material u p o n hydrolysis and subsequent fractionation by ion~exchange chromatography [ 7 ] . A m i n o acids were assayed in basic fractions by the fluorescamine procedure [8]. Glycine was used as a standard and measurements were made in a Perkin Elmer 3000 Fluorescence Spectrometer. Whole leaf samples were used for the determination of chlorophyll [9] and soluble protein contents [ 10].
315
RESULTS In Table I the contents of total chlorophyll and soluble protein in detached, dark-treated fourth leaves are compared with those of attached light-exposed controls. A marked reduction in the contents of both total chlorophyll and soluble protein occurred when leaves were detached and maintained in darkness, whereas the leaf contents of attached control leaves remained fairly constant over 5 days. Figure 1 shows the changes induced by senescence in the distribution of ~4C incorporated by the fourth leaf during expansion. In leaves extracted at ligule formation, 91% of detectable radioactivity was present in WIM. Similar levels of radioactivity were maintained in control leaves over 5 days, whereas ~4C was progressively lost from the WIM of dark-treated detached leaves. Label lost during senescence from WIM accumulated as soluble radioactivity in detached leaf extracts. Radioactivity did not accumulate in the watersoluble extracts of control leaves. Senescent leaves respired considerably more ~4CO2 than attached control leaves. Samples of WIM were digested with 6 N TFA and the hydrolysate partitioned into basic (protein) acidic and neutral (non-protein) fractions by ionexchange chromatography. The distribution of radioactivity in the major fractions is shown in Fig. 2. Protein fractions contained most of the radioactivity solubilized by acid hydrolysis. In senescent leaves the level of radioactivity in protein declined but the 14C content of protein in control leaves TABLE I CHANGES IN CHLOROPHYLLAND SOLUBLEPROTEIN CONTENTS IN DETACHED, DARK-TREATED AND ATTACHED, LIGHT-EXPOSED MATURE FOURTH LEAVES OF L. T E M U L E N T U M Leaf treatment
Duration
Leaf content (mg)
of senescence
Detached in darkness
Attached in light
(days)
Chlorophyll
Soluble
0 1 2 3 4 5
0.266 0.254 0.185 0.076 0.060 0.055
3.82 3.66 2.80 1.06 1.01 0.84
0 1 2 3 4 5
0.266 0.264 0.294 0.266 0.302 0.231
3.82 3.84 4.36 4.31 4.20 3.12
protein
316
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O I 2 3 4 5 Duration of senescence (Days)
Duration of senescence (Days) Fig. 1. Distribution o f radioactivity during induced senescence of mature, detached 4th leaves of L. temulentum. , detached, dark-treated leaves; . . . . . . . , attached, light-exposed l e a v e s ; . , water-insoluble material; o, water-soluble material; - , 14CO2 respiration. Plants fed 3 pCi ['4C]sucrose 5 days after leaf tip emergence.
Fig. 2. Distribution of radioactivity in basic, acidic and neutral fractions after TFA hydrolysis o f water-insoluble material from mature, detached 4th leaves of L. temulentum. , detached, dark-treated leaves; . . . . . . . . , attached, light-exposed leaves; e, basic fraction (protein); 4, acidic fraction; o, neutral fraction. Plants fed 3 pCi ['4C]sucrose 5 days after leaf tip emergence.
317
was unaffected. The labelling in non-protein fractions was, by comparison, consistently low in hydrolysates from both treatments. Figure 3 shows the distribution of radioactivity in water,soluble extracts of leaves from both treatments after fractionation by ion~exchange chromatography. Radioactivity accumulated in protein components of extracts from senescent detached leaves but there was no comparable increase in the 14C content of this fraction in control leaves. Labelling in the nonprotein fractions of leaf extracts from both treatments remained low and relatively unchanged. Determinations of total amino acid contents in basic fractions of acid hydrolysates and water soluble extracts of leaves from both treatments were performed and the data is shown in Table II. Reduction in the total amino acid content o f hydrolysates from senescent leaves coincided with a marked Detached, dark-treated
Attached,
leaves
dark-treated
leaves
8O
6O
I0 x v
? 40
@
/ 20
0.
.i .
2.
.3 . 4 Duration
0'
5 of
senescence
i'
2'
;
X
5'
(Days)
Fig. 3. Distribution o f radioactivity in basic, acidic and neutral fractions of water-soluble extracts from mature, detached 4th leaves o f L. temulentum. D, basic fraction (protein); A, acidic fraction; o, neutral fraction. Plants fed 3 ~Ci [14C]sucrose 5 days after leaf tip emergence.
318 TABLE II CHANGES IN THE AMINO ACID C O N T E N T OF H Y D R O L Y S A T E S OF WATERINSOLUBLE M A T E R I A L AND OF WATER-SOLUBLE E X T R A C T S FROM DETACHED, D A R K - T R E A T E D AND ATTACHED, LIGHT-EXPOSED M A T U R E F O U R T H LEAVES OF L. TEMULENTUM Data expressed as #mol glycine equivalents.
Duration of senescence (days)
Component Hydrolysate
Water,soluble
Detached in darkness
0 1 2 3 4 5
23.8 27.1 23.0 24.0 12.2 13.2
1.1 3.5 7.5 8.9 10.2 10.8
Attached in light
0 1 2 3 4 5
23.8 27.6 22.4 21.2 21.6 22.6
1.1 1.8 1.0 1.3 1.0 1.9
Leaf treatment
increase in the amino acid pool of water-soluble extracts. By contrast, there were no significant changes in amino acid content in the basic component of either the acid hydrolysates or water soluble extracts of control leaves. DISCUSSION
Labelling the immature leaf with ~4C enabled the destiny of the carbon assimilated during expansion to be traced. At ligule formation, the waterinsoluble component of leaf extracts contained the majority of incorporated radioactivity. Acid hydrolysis of WIM and subsequent fractionation of hydrolysates on ion~exchange resins allowed an assessment of the chemical nature of the material into which radioactivity was incorporated. Recovery, in basic fractions, of most of the radioactivity solubilized by acid hydrolysis provided evidence that carbon assimilated during leaf expansion is directed mainly towards protein synthesis. This evidence is supported by the presence in hydrolysates of considerable amounts of free amino acids. The progressive loss of radioactivity from WIM o f senescent, detached leaves, corresponding with the reduction in both the amino acid and 14C content of the protein fraction of hydrolysates, strongly suggests that carbon is lost from the water-insoluble component of senescent leaves chiefly through the degradation of protein. During senescence of attached leaves [3], radioactivity released from WIM was translocated to other regions of the plant. Leaf detachment allowed a means of containing, within the leaf, the products released during sene-
319
scence. Accumulated water,soluble radioactivity in detached~leaf extracts was composed principally of amino acids. The considerable labelling of amino acids in soluble extracts of detached leaves provides convincing evidence that protein is the major source of carbon mobilized during senescence and emphasises the potential of the senescent leaf as a source of carbon and nitrogen. The 14C content of WIM was not entirely recoverable in water-soluble components. Label was lost through respiration from the soluble pool of accumulated radioactivity with 14CO2 evolution proceeding at a higher rate from detached leaves than from either dark-treated [3] or fight-exposed attached leaves. It has been reported [11] that protein can be a major respiratory substrate during senescence. The presence in basic fractions of water-soluble extracts from detached leaves of radioactivity amounts in great excess of the labelling in acidic and neutral components suggests that the products of protein rather than polysaccharide hydrolysis may provide the major respiratory substrate in the senescent leaf. Energy required to mobilize and export the products of senescence may also be derived from this source. The biochemical changes resulting from protein degradation in the detached leaf are important when considering dry matter and crude protein losses encountered during cutting and conserving of grass crops either as hay or silage. A possible means of improving the nutritive value of conserved herbage may be achieved by chemically treating the conserved crop to restrict protein hydrolysis or by promoting assimilate export and minimising respiratory losses in the standing crop. ACKNOWLEDGEMENTS
The interest shown by Professor J.P. Cooper, FRS, Director of the Welsh Plant Breeding Station and by Drs. J.L. Stoddart, C.J. Pollock and H. Thomas of the Plant Biochemistry Department, is gratefully acknowledged. REFERENCES
1 L. Beevers, in: J.E. Varner and J.D. Bonner (Ed.), Plant Biochemistry, 3rd edn, Academic Press, New York, 1976, p. 771. 2 H. Thomas and J.L. Stoddart, Annu. Rev. Plant Physiol., 31 (1980) 83. 3 E.J. Lloyd, J. Exp. Bot., 31 (1980) 1067. 4 C.J. Pollock, New Phytol., 90 (1982) in press. 5 W. Louwerse, Acta. Bot. Need., 16 (1967) 42. 6 P.S. Dick and T. ap Rees, Phytochemistry, 15 (1976) 255. 7 M.W. Fowler and T. ap Rees, Biochim. Biophys. Acta, 201 (1970) 33. 8 S. Udenfriend, S. Stein, D. Bohlen, W. Dairman, W. Leimgruber and M. Weigele, Science, 178 (1972) 871. 9 G. MacKinney, Jr Biol. Chem., 132 (1940) 91. 10 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 11 T. ap Rees, in: David D. Davies (Ed.), The Biochemistry of Plants, Vol. 2, Academic Press, New York, London, 1980, p.1.
313
MOBILIZATION OF CARBON DURING SENESCENCE IN DETACHED LEAVES OF LOLIUM TEMULENTUM
E.J. LLOYD Welsh Plant Breeding Station, Plas Gogerddan, Aberystwyth, Dyfed. SY23 3EB (U.K.) (Received September 24th, 1981) (Accepted December 3rd, 1981)
SUMMARY
The water-insoluble component of mature leaf extracts of Lolium temulentum contained the majority of radioactivity incorporated from [14C] sucrose fed during leaf expansion. Radioactivity in water-insoluble material was present mainly in the protein fraction. Induced senescence caused a rapid loss of water-insoluble radioactivity mainly attributable to release of label from the protein fraction. Label released from insoluble material by senescence-induced proteolysis accumulated as amino-acids in detached leaves. Changes in the total amino-acid content of hydrolysates and water,soluble extracts paralleled changes in radioactivity. Enhanced 14CO2 respiration by the senescent leaf was associated with radioactivity released by protein degradation.
INTRODUC~ON
Export of metabolites from the senescent leaf to other regions of the plant is one of the fundamental processes occurring during natural sequential senescence [1,2]. During induced senescence of 14C-fed mature, attached, fourth leaves of Lolium ternulenturn, radioactivity released from waterinsoluble material was either translocated or lost through respiration [3]. The experiments in this paper were performed to identify that component of water-insoluble material into which radioactivity was incorporated during leaf expansion and from which label was lost during induced senescence of the mature leaf. Detached leaves were used in this study to prevent export and contain, within the leaf, the products of senescence-induced breakdown. Evidence for determining the source of carbon mobilized during senescence was derived from an examination of the chemical nature of the material in which solubilized radioactivity was recovered. Abbreviations: TFA, trifluoroacetic acid; WIM, water-insoluble material. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
314 MATERIALS AND METHODS Seeds of L. temulentum (Ba 3081 summer annual) were germinated on pads at 20°C with an 8-h p h o t o p e r i o d under a light intensity of 100 Wm -2 . Seedlings were transferred after 7 days and grown under the same conditions in a mineral nutrient solution [4] which was renewed at weekly intervals.
Isotope feeding technique Plants were removed from nutrient solution 5 days after fourth leaf emergence and their roots immersed in water for 24 h before exposure to isotope. Radioactivity was fed to each plant by root absorption of 3.0 ~Ci [14C] sucrose (Amersham) as previously described [3]. In these experiments, ~4C-incorporation was improved (4--5-fold) by adding glucose to the feeding medium at a final concentration of 10 mM [ 5]. After complete uptake of the 14C-labelled sugar medium, plants were transferred to fresh nutrient solution and grown until the fourth leaf was fully expanded.
Leaf treatments At ligule formation, fourth leaves from some of the ~4C-treated plants were excised t o g e t h e r with approx. 2 cm of the leaf basal portion. The leaves were sealed in the dark chambers of a respirometer as previously described [3] and the leaf bases kept submerged in nutrient solution. Attached leaves from other 14C-treated plants were sealed into the light exposed chambers of the respirometer to serve as controls. Leaves were removed from leaf chambers at daily intervals and extractable radioactivity determined.
Analytical procedures Determinations of radioactivity in CO2 and in water-soluble material were obtained as previously described [3]. Water-insoluble material (WIM) was ground in cold water and the suspension brought to a final volume of 10.0 ml. Radioactivity was determined from 1-ml samples after wet digestion to 14CO2 [6]. Treatment with 6 N trifluoroacetic acid (TFA) at l l 0 ° C for 20 h hydrolysed 81.2% of the radioactivity in 2-ml samples of insoluble extracts. Residual particulate material was removed b y low-speed centrifugation. Hydrolysates were then reduced to dryness by rotary-evaporation and made up to a final volume of 2 ml. Samples of acid hydrolysates were separated into protein and non-protein fractions b y ion~exchange chromatography. The protein fraction was defined as the c o m p o n e n t of WIM which yields basic material u p o n hydrolysis and subsequent fractionation by ion~exchange chromatography [ 7 ] . A m i n o acids were assayed in basic fractions by the fluorescamine procedure [8]. Glycine was used as a standard and measurements were made in a Perkin Elmer 3000 Fluorescence Spectrometer. Whole leaf samples were used for the determination of chlorophyll [9] and soluble protein contents [ 10].
315
RESULTS In Table I the contents of total chlorophyll and soluble protein in detached, dark-treated fourth leaves are compared with those of attached light-exposed controls. A marked reduction in the contents of both total chlorophyll and soluble protein occurred when leaves were detached and maintained in darkness, whereas the leaf contents of attached control leaves remained fairly constant over 5 days. Figure 1 shows the changes induced by senescence in the distribution of ~4C incorporated by the fourth leaf during expansion. In leaves extracted at ligule formation, 91% of detectable radioactivity was present in WIM. Similar levels of radioactivity were maintained in control leaves over 5 days, whereas ~4C was progressively lost from the WIM of dark-treated detached leaves. Label lost during senescence from WIM accumulated as soluble radioactivity in detached leaf extracts. Radioactivity did not accumulate in the watersoluble extracts of control leaves. Senescent leaves respired considerably more ~4CO2 than attached control leaves. Samples of WIM were digested with 6 N TFA and the hydrolysate partitioned into basic (protein) acidic and neutral (non-protein) fractions by ionexchange chromatography. The distribution of radioactivity in the major fractions is shown in Fig. 2. Protein fractions contained most of the radioactivity solubilized by acid hydrolysis. In senescent leaves the level of radioactivity in protein declined but the 14C content of protein in control leaves TABLE I CHANGES IN CHLOROPHYLLAND SOLUBLEPROTEIN CONTENTS IN DETACHED, DARK-TREATED AND ATTACHED, LIGHT-EXPOSED MATURE FOURTH LEAVES OF L. T E M U L E N T U M Leaf treatment
Duration
Leaf content (mg)
of senescence
Detached in darkness
Attached in light
(days)
Chlorophyll
Soluble
0 1 2 3 4 5
0.266 0.254 0.185 0.076 0.060 0.055
3.82 3.66 2.80 1.06 1.01 0.84
0 1 2 3 4 5
0.266 0.264 0.294 0.266 0.302 0.231
3.82 3.84 4.36 4.31 4.20 3.12
protein
316
B•.....•.... \.
4 ..... • ...... •
300
® 200
\.
~ " ° "
..."".4
"'•/"1 200
"'O"
150
IO
7
•
j
B
/
==
1013
I00
/"
o
/ O
•/• 50
5O
~'.. . . . .
.&., 4 . . . . . . .
o ..... .. "o
•
0.,.,,0 ..... 0
o
•
•~
• ,A • •"
D
..... •.""
• i
I
!
..' ~ o
f-",o..~.
0
•..... |
. ~ " - k,LT".,.. o
.o.j
8
..: . . . . . .
i
;
O I 2 3 4 5 Duration of senescence (Days)
Duration of senescence (Days) Fig. 1. Distribution o f radioactivity during induced senescence of mature, detached 4th leaves of L. temulentum. , detached, dark-treated leaves; . . . . . . . , attached, light-exposed l e a v e s ; . , water-insoluble material; o, water-soluble material; - , 14CO2 respiration. Plants fed 3 pCi ['4C]sucrose 5 days after leaf tip emergence.
Fig. 2. Distribution of radioactivity in basic, acidic and neutral fractions after TFA hydrolysis o f water-insoluble material from mature, detached 4th leaves of L. temulentum. , detached, dark-treated leaves; . . . . . . . . , attached, light-exposed leaves; e, basic fraction (protein); 4, acidic fraction; o, neutral fraction. Plants fed 3 pCi ['4C]sucrose 5 days after leaf tip emergence.
317
was unaffected. The labelling in non-protein fractions was, by comparison, consistently low in hydrolysates from both treatments. Figure 3 shows the distribution of radioactivity in water,soluble extracts of leaves from both treatments after fractionation by ion~exchange chromatography. Radioactivity accumulated in protein components of extracts from senescent detached leaves but there was no comparable increase in the 14C content of this fraction in control leaves. Labelling in the nonprotein fractions of leaf extracts from both treatments remained low and relatively unchanged. Determinations of total amino acid contents in basic fractions of acid hydrolysates and water soluble extracts of leaves from both treatments were performed and the data is shown in Table II. Reduction in the total amino acid content o f hydrolysates from senescent leaves coincided with a marked Detached, dark-treated
Attached,
leaves
dark-treated
leaves
8O
6O
I0 x v
? 40
@
/ 20
0.
.i .
2.
.3 . 4 Duration
0'
5 of
senescence
i'
2'
;
X
5'
(Days)
Fig. 3. Distribution o f radioactivity in basic, acidic and neutral fractions of water-soluble extracts from mature, detached 4th leaves o f L. temulentum. D, basic fraction (protein); A, acidic fraction; o, neutral fraction. Plants fed 3 ~Ci [14C]sucrose 5 days after leaf tip emergence.
318 TABLE II CHANGES IN THE AMINO ACID C O N T E N T OF H Y D R O L Y S A T E S OF WATERINSOLUBLE M A T E R I A L AND OF WATER-SOLUBLE E X T R A C T S FROM DETACHED, D A R K - T R E A T E D AND ATTACHED, LIGHT-EXPOSED M A T U R E F O U R T H LEAVES OF L. TEMULENTUM Data expressed as #mol glycine equivalents.
Duration of senescence (days)
Component Hydrolysate
Water,soluble
Detached in darkness
0 1 2 3 4 5
23.8 27.1 23.0 24.0 12.2 13.2
1.1 3.5 7.5 8.9 10.2 10.8
Attached in light
0 1 2 3 4 5
23.8 27.6 22.4 21.2 21.6 22.6
1.1 1.8 1.0 1.3 1.0 1.9
Leaf treatment
increase in the amino acid pool of water-soluble extracts. By contrast, there were no significant changes in amino acid content in the basic component of either the acid hydrolysates or water soluble extracts of control leaves. DISCUSSION
Labelling the immature leaf with ~4C enabled the destiny of the carbon assimilated during expansion to be traced. At ligule formation, the waterinsoluble component of leaf extracts contained the majority of incorporated radioactivity. Acid hydrolysis of WIM and subsequent fractionation of hydrolysates on ion~exchange resins allowed an assessment of the chemical nature of the material into which radioactivity was incorporated. Recovery, in basic fractions, of most of the radioactivity solubilized by acid hydrolysis provided evidence that carbon assimilated during leaf expansion is directed mainly towards protein synthesis. This evidence is supported by the presence in hydrolysates of considerable amounts of free amino acids. The progressive loss of radioactivity from WIM o f senescent, detached leaves, corresponding with the reduction in both the amino acid and 14C content of the protein fraction of hydrolysates, strongly suggests that carbon is lost from the water-insoluble component of senescent leaves chiefly through the degradation of protein. During senescence of attached leaves [3], radioactivity released from WIM was translocated to other regions of the plant. Leaf detachment allowed a means of containing, within the leaf, the products released during sene-
319
scence. Accumulated water,soluble radioactivity in detached~leaf extracts was composed principally of amino acids. The considerable labelling of amino acids in soluble extracts of detached leaves provides convincing evidence that protein is the major source of carbon mobilized during senescence and emphasises the potential of the senescent leaf as a source of carbon and nitrogen. The 14C content of WIM was not entirely recoverable in water-soluble components. Label was lost through respiration from the soluble pool of accumulated radioactivity with 14CO2 evolution proceeding at a higher rate from detached leaves than from either dark-treated [3] or fight-exposed attached leaves. It has been reported [11] that protein can be a major respiratory substrate during senescence. The presence in basic fractions of water-soluble extracts from detached leaves of radioactivity amounts in great excess of the labelling in acidic and neutral components suggests that the products of protein rather than polysaccharide hydrolysis may provide the major respiratory substrate in the senescent leaf. Energy required to mobilize and export the products of senescence may also be derived from this source. The biochemical changes resulting from protein degradation in the detached leaf are important when considering dry matter and crude protein losses encountered during cutting and conserving of grass crops either as hay or silage. A possible means of improving the nutritive value of conserved herbage may be achieved by chemically treating the conserved crop to restrict protein hydrolysis or by promoting assimilate export and minimising respiratory losses in the standing crop. ACKNOWLEDGEMENTS
The interest shown by Professor J.P. Cooper, FRS, Director of the Welsh Plant Breeding Station and by Drs. J.L. Stoddart, C.J. Pollock and H. Thomas of the Plant Biochemistry Department, is gratefully acknowledged. REFERENCES
1 L. Beevers, in: J.E. Varner and J.D. Bonner (Ed.), Plant Biochemistry, 3rd edn, Academic Press, New York, 1976, p. 771. 2 H. Thomas and J.L. Stoddart, Annu. Rev. Plant Physiol., 31 (1980) 83. 3 E.J. Lloyd, J. Exp. Bot., 31 (1980) 1067. 4 C.J. Pollock, New Phytol., 90 (1982) in press. 5 W. Louwerse, Acta. Bot. Need., 16 (1967) 42. 6 P.S. Dick and T. ap Rees, Phytochemistry, 15 (1976) 255. 7 M.W. Fowler and T. ap Rees, Biochim. Biophys. Acta, 201 (1970) 33. 8 S. Udenfriend, S. Stein, D. Bohlen, W. Dairman, W. Leimgruber and M. Weigele, Science, 178 (1972) 871. 9 G. MacKinney, Jr Biol. Chem., 132 (1940) 91. 10 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. 11 T. ap Rees, in: David D. Davies (Ed.), The Biochemistry of Plants, Vol. 2, Academic Press, New York, London, 1980, p.1.