- Email: [email protected]
Changes in amylase activity during rose bud opening
Scientia Horticulturae, 16 (1982) 283--289
283
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CHANGES IN AMYLASE ACTIVITY DURING ROSE BUD OPENING
JOHN B.W. HAMMOND
Glasshouse Crops Research Institute, Littlehampton, Sussex BN16 3PU (Gt. Britain) (Accepted for publication 12 June 1981)
ABSTRACT Hammond, J.B.W., 1982. Changes in amylase activity during rose bud opening. Scientia Hortic., 16: 283--289. Following homogenisation and centrifugation of rose petals (cultivar 'Sonia'), amylolytic activity was found in the supernatant fraction and associated with the particulate matter, especially the starch granules. The latter activity was released into solution by treatment with non-ionic detergent. The activity in the supernatant fraction showed no significant change during the ageing of cut flowers, but the particulate activity increased. The release of activity from the starch-containing fraction was achieved more quickly with material isolated from stored roses than from fresh flowers. Properties of the enzyme derived from the starch fraction suggest that it is different to the soluble enzyme. The possible role of amylase in rose petal senescence is discussed.
INTRODUCTION
Starch contents of up to 11% dry wt. have been reported in petals of the cultivated glasshouse rose (Sacalis and Chin 1976; Ho and Nichols, 1977). Starch breakdown is accompanied by a rise in fructose and glucose levels in the petals even when the cut flowers are stood in water alone (Kaltaler and Steponkus, 1974). It has been suggested that sugar accumulation is a mechanism to reduce petal water potential, promoting water influx for cell enlargement and flower opening (Ho and Nichols, 1977). Enzymes responsible for starch breakdown ,may therefore be important in the control of opening of harvested roses. It has been proposed that a-amylase is the only enzyme capable of attacking intact starch granules (Dunn, 1974). Thus the possibility that amylase activity increases in rose petals after harvest was investigated. MATERIALS AND METHODS
Roses (cultivar 'Sonia') were harvested at Stage 1 or 2 of development {petals tightly furled to petals just opening), the stems trimmed to 30 cm and all leaves except the upper 2 mature compound leaves removed (Ho and 0304-4238/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
284
Nichols, 1977). The flowers were stood in tap water and stored at 18 ° C, 60% r.h., under warm white fluorescent lights (15 W m -2) illuminated from 09.00 a.m. for 8 h. After 2 days they had developed to Stage 3--4 (Ho and Nichols, 1977). When roses were allowed to develop on the plant, t h e y were harvested at the appropriate age and treated immediately. Petals from 5 flowers were pooled for each enzyme analysis. All material was harvested between 10.00 and 11.00 a.m. and the petals frozen in liquid N2 within 30 min of harvest. e x t r a c t i o n . - - The frozen petals were ground in liquid N2 and homogenised for 1 min in cold 0.1 M acetate buffer (pH 5.3) containing 5 mM calcium chloride, sampled for dry wt. determination and centrifuged for 15 min at 3000 × g. The supernatant was dialysed against 0.001 M acetate buffer (pH 5.3) for 3 h prior to amylase assay (initial supernatant). The pellet was re-suspended in buffer and then centrifuged for 15 min at 3000 × g. The resulting pellet was then filtered using Miracloth, with the addition of further cold buffer to wash the suspension through until no more suspended matter was seen in the filtrate. The residue was suspended in buffer for amylase assay. The Miracloth filtrate was centrifuged for 10 min at 6000 × g to give a pellet (crude starch fraction). Amylase was solubilised from the crude starch fraction by incubation in acetate buffer (pH 5.3 with 5 mM calcium chloride containing Tween 20) for 1 h at 40 ° C. The suspension was centrifuged at 6000 × g for 10 min after incubation.
Amylase
a s s a y . - - The enzyme preparation was added to an equal volume of 5% boiled soluble starch in water, mixed and incubated for 2 h at 40 ° C. Samples were taken at the start and finish of incubation and assayed for reducing sugar b y the Nelson--Somogyi m e t h o d using glucose standards (Herbert et al., 1971). No evidence was found for activity changes caused by inhibitory or other materials in the extracts after checking b y assay of mixtures of extracts from fresh and stored roses. Units of amylase activity were expressed as pg glucose equivalents produced h -1. /~-limit dextrin was prepared by incubating 10 ml 1% boiled soluble starch with 1000 units ~-amylase (Sigma, T y p e 1B) in acetate buffer (pH 4.8) for 4 h at 25 ° C. The digest was dialysed against water overnight at 5 ° C. Assays with Amylopectin Azure (Calbiochem) used 0.05 mg substrate in 2 ml enzyme preparation. After 2 h at 40°C the suspension was filtered and the absorbance measured at 500 nm using a buffer blank. Determination of the extent of enzyme inactivation by trypsin and EDTA was carried o u t after pre-incubation for 1 h at 20°C with 20 000 units trypsin (Sigma, T y p e III) with/without 2 pmoles EDTA ml-'. The action pattern was determined as described b y Tung and Nordin (1969). Solubilised enzyme and washed amylose were incubated at 40°C using a shaking water-bath. Samples were taken at zero time and at intervals up to 7 h. Samples were inactivated by addition of 1 drop 0.02 M silver ni-
Amylase
285 trate and centrifuged at 30 000 X g for 10 min. The supernatants were filtered through a membrane filter (Millipore 0.22 # m pore size} and assayed for reducing power and total carbohydrate by Nelson--Somogyi and anthrone assays, respectively (Herbert et al., 1971). Glucose standards were used. RESULTS A m y l o l y t i c activity in petal homogenates. -- Amylolytic activity was consistently present in both the initial supernatant and the crude starch fraction; about 80% of the activity in the latter sedimented after 2 min at 1500 × g. The residue of cell debris after Miracloth filtration showed a small but variable a m o u n t of amylolytic activity, possibly because of incomplete separation from the crude starch fraction. TABLE I Response of supernatant and solubilised starch fraction amylolytic activity from petals of 2-day-stored roses to treatments used to characterise amylase Treatment
% of control activity remaining after treatment Supernatant
Solubilised
(crude fraction) Heat at 70°C for I0 min Pre-incubation with trypsin Pre-incubation with trypsin + E D T A p-limit dextrin substrate (% of activity against soluble starch)
52 98 95 0
58 99 39 100
Amylopectin azure substrate (Relative to activity of solubilised enzyme)
57
100
Amylolytic activities in both supernatant and crude starch fraction showed similar resistance to heating (Table I). Incubation with trypsin did not affect the activity from either source, but when EDTA was added together with the trypsin there was a reduction in the amylolytic activity of the crude starch fraction. The enzyme from the starch fraction showed no significant loss in activity with ~-limit dextrin as substrate compared with starch, and greater activity against Amylopectin azure than the initial supernatant. The products of starch digestion by the solubilised starch fraction enzyme were identified by thin-layer chromatography as glucose, maltose, isomaltose and oligosaccharides. The rate of release of amylase activity from the crude starch fraction by Tween 20 varied with the development of the flower. Solubilisation of 80% of the total solubilised activity took over 20 min with material from fresh roses and less than 2 min when material from roses stored for 2 days was treated (Fig. 1).
286
l
3.0
to.8
(min)
'~ ~ 2-5 o ~
10--
o
5--
~, 2.0 u 0
1
I
I
I
0
1 Time from harvest (days)
2
#
0
I
I
I
L
I
0,2 0.3 0.4 0.5 0.6 glucose equivalents produced
l
0.7 0.8 pmol.
Fig. 1. Time taken for Tween 20 mediated release of amylase from starch grains from roses stored for different periods, t0.8 is the time taken for 80% of the total activity solubilised to be released. Mean Of 2 experiments, bars show range of mean. Fig. 2. Action pattern produced by solubilised starch fraction amylase using amylose as substrate. The graph shows the relationship between the ratio of total carbohydrate released (after removal of substrate) to reducing power and reducing groups liberated during the enzyme reaction.
The action pattern of the solubilised enzyme on amylose was that of an endo-attack (Tung and Nordin, 1969). The ratio of total carbohydrate, after removal of amylose substrate, to reducing power decreased during the incuba tion (Fig. 2). Endo-attack results in early release of large oligosaccharides followed by their b r e a k d o w n to smaller components. Thus, more reducing power is observed per product molecule as the reaction proceeds and the oligosaccharides get smaller. This gives a decreasing plot, as seen in Fig. 2. With exo-attack only single, final product molecules are released, thus there is no change in the ratio of total carbohydrate released to reducing power released and a straight horizontal line is given. Changes in amylase activity during development. -- Amylase activity in the crude starch fraction increased after harvest (Table IIa). There was a large variation between experiments in amylase levels at harvest, although an increase in activity was seen in all experiments. The mean increase was about 65%, most of this occurring in the first 24 h; in some cases the activity decreased between 24 and 48 h after harvest. The mean increase in activity between harvest and the highest observed post-harvest levels was 286 units. This increase was significant (P > 0.002) when tested by the paired sample method. The variations in activity at harvest appeared to be related to the periodic increases in rose flower production, known as flushes, which are caused b y pruning the plants followed by parallel development and then harvest of the new flower buds. Higher amylase levels at harvest were general-
287
TABLE
II
Changes in amylolytic activity in rose petals during storage. Flowers harvested at tight-bud stage (5 flowers/sample) (units g dry wt.-i of petals)
(a) Changes in crude starch fraction amylase activity (values given for solubilised enzyme) Days stored
Date of harvest
13.7.78
20.7.78
0 1
234.2 161.1
91.2 299.7
2
474.1
341.3
6.9.78
11.9.78
14.9.78
2.10.78
12.10.78 Means
73.7 104.8
201.5 246.1
623.5 839.5
365.3 845.4
333.9 799.7
274.8 490.9
272.5
351.1
660.5
464.7
600.1
452.0
(b) Changes in initial supernatant activity Daysstored
0 1 2
Date o f h a r v e ~ 6.9.78
11.9.78
2.10.78
12.10.78
Means
147.7 251.3 1577.1
1432.6 124.1 808.4
1369.8 1649.3 1503.5
866.5 799.7 668.9
954.2 706.1 1139.5
ly present in flowers taken at the height of a flush, when the plant was supporting the greatest number of flowers. The mean amylolytic activity in the supernatant increased after harvest, but the increase was not statistically significant {Table IIb). The weight of the crude starch fraction decreased during storage of the roses; an initial value of 8% dry weight decreased to 4.9% dry weight after 2 days storage. These figures are similar to those for enzymically analysed starch given by Ho and Nichols {1977). DISCUSSION
The identification of amylase activity in rose petals provides a possible mechanism for the breakdown of starch reported in harvested flowers (Ho and Nichols, 1977). Although there was amylolytic activity in the soluble fraction of the petal homogenate, the starch fraction amylase was of greater interest because it increased after harvest. The properties of the starch fraction amylase (Table I) suggest that it may be a-amylase (E.C. 3.2.1.1) (Fischer and Stein, 1960); final identification must await further purification. The supernatant activity exhibited different properties and showed no consistent increase after harvest. Binding of amylase to the starch fraction could be caused by precipitation with tannins during extraction (Goldstein and Swain, 1965; Hawker, 1969).
288
Extraction in the presence of Polyclar AT, 0.25% bovine serum albumin or ovalbumin as protectants against protein precipitation resulted in no significant transfer of amylase activity into the supernatant (J.B.W. Hammond, 1978, unpublished data). This suggests that protein precipitation was not the primary cause of amylase binding to the starch fraction. Amylase binding to starch granules has been proposed as a pre-condition to starch hydrolysis (McLaren, 1963; Sargeant and Walker, 1978). The variation in ease of removal of amylase from the starch fraction could be a function of such binding and hydrolysis during flower storage. The granule surface could then form a less stable binding medium. The lower percentage solubilisation of total activity from stored flowers could also result from granule digestion. Internal erosion of starch granules has been observed (Chandorkar and Badenhuizen, 1967; Nayyar and Ram, 1977), and this would presumably make the digesting enzyme inside the granule more difficult to extract. Starch-bound amylase activity showed up to 3-fold increase after harvest and appeared to peak at 800--850 units g dry wty'. Buds harvested during a flush had higher starch-bound amylase activity at harvest and activity fell during the second day of storage. The increase in starch-bound amylase activity after harvest suggests that it is involved in the rapid post-harvest breakdown of starch. The effect of changes in bound amylase activity on the rate of starch hydrolysis, and of flower opening, remains to be tested, as does the importance of the supernatant amylolytic activity in these processes. However if, as seems likely, amylase attack is the first step in starch breakdown, a rise in amylase activity would increase the rate of breakdown. Thus, suppression of amylase activity by artificial means could offer a way of retarding the opening of harvested rose flowers.
REFERENCES Chandorkar, K.R. and Badenhuizen, N.P., 1967. The fate of ADPG-alpha glucan glucosyltransferase during amylolytic corrosion of starch granules, and its relation to starch granule structure. Cereal Chem., 44: 27--38. Dunn, G., 1974. A model for starch breakdown in higher plants. Phytochemistry, 13: 1341--1346. Fischer, E.H. and Stein, E.A., 1960. In: P.D. Boyer, H. Lardy and K. Myrback (Editors), The Enzymes,Vo]. 4. AcademicPress, London, pp. 313--343. Go]dstein, J.L. and Swain, T., 1965. The inhibition of enzymes by tannins. Phytochemistry, 4: 185--192. Hawker, J.S., 1969. Insoluble invertasefrom grapes: an artefact of extraction. Phytochemistry, 8: 337--344. Herbert, D., Phipps, P.J. and Strange, R.E., 1971. In: J.R. Norris and D.W. Ribbons (Editors), Methods in Microbiology,Vol. 5B. AcademicPress, London, pp. 266--301. Ho, L.C. and Nichols, R., 1977. Transloeationof ,4 C-sucrose in relation to changesin carbohydrate content in rose corollas cut at different stages of development.Ann. Bot., 41 : 227--242.
289 Kaltaler, R.E.L. and Steponkus, P.L., 1974. Uptake and metabolism of sucrose in cut roses. J. Am. Soc. Hortic. Sci., 99: 490--493. McLaren, A.D., 1963. Enzyme reactions in structurally restricted systems. IV. The digestion of insoluble substrates by hydrolytic enzymes. Enzymologia, 26: 237--246. Nayyar, V.L. and Ram, H.Y.M., 1977. Ultrastructure of starch grain breakdown in cotyledons of Ca]anis ca]an during germination. Phytomorphology, 27: 187--190. Sacalis, J.N. and Chin, C.K., 1976. Metabolism of sucrose in cut roses. I. Comparison of sucrose pulse and continuous sucrose uptake. J. Am. Soc. Hortic. Sci., 101: 254--257. Sargeant, J.G. and Walker, T.S., 1978. Adsorption of wheat s-amylase isoenzymes to wheat starch. Starke, 30: 160--163. Tung, K.K. and Nordin, J.H., 1969. Determination of the action patterns of glycanases. Anal. Biochem., 29: 84--90.
283
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
CHANGES IN AMYLASE ACTIVITY DURING ROSE BUD OPENING
JOHN B.W. HAMMOND
Glasshouse Crops Research Institute, Littlehampton, Sussex BN16 3PU (Gt. Britain) (Accepted for publication 12 June 1981)
ABSTRACT Hammond, J.B.W., 1982. Changes in amylase activity during rose bud opening. Scientia Hortic., 16: 283--289. Following homogenisation and centrifugation of rose petals (cultivar 'Sonia'), amylolytic activity was found in the supernatant fraction and associated with the particulate matter, especially the starch granules. The latter activity was released into solution by treatment with non-ionic detergent. The activity in the supernatant fraction showed no significant change during the ageing of cut flowers, but the particulate activity increased. The release of activity from the starch-containing fraction was achieved more quickly with material isolated from stored roses than from fresh flowers. Properties of the enzyme derived from the starch fraction suggest that it is different to the soluble enzyme. The possible role of amylase in rose petal senescence is discussed.
INTRODUCTION
Starch contents of up to 11% dry wt. have been reported in petals of the cultivated glasshouse rose (Sacalis and Chin 1976; Ho and Nichols, 1977). Starch breakdown is accompanied by a rise in fructose and glucose levels in the petals even when the cut flowers are stood in water alone (Kaltaler and Steponkus, 1974). It has been suggested that sugar accumulation is a mechanism to reduce petal water potential, promoting water influx for cell enlargement and flower opening (Ho and Nichols, 1977). Enzymes responsible for starch breakdown ,may therefore be important in the control of opening of harvested roses. It has been proposed that a-amylase is the only enzyme capable of attacking intact starch granules (Dunn, 1974). Thus the possibility that amylase activity increases in rose petals after harvest was investigated. MATERIALS AND METHODS
Roses (cultivar 'Sonia') were harvested at Stage 1 or 2 of development {petals tightly furled to petals just opening), the stems trimmed to 30 cm and all leaves except the upper 2 mature compound leaves removed (Ho and 0304-4238/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
284
Nichols, 1977). The flowers were stood in tap water and stored at 18 ° C, 60% r.h., under warm white fluorescent lights (15 W m -2) illuminated from 09.00 a.m. for 8 h. After 2 days they had developed to Stage 3--4 (Ho and Nichols, 1977). When roses were allowed to develop on the plant, t h e y were harvested at the appropriate age and treated immediately. Petals from 5 flowers were pooled for each enzyme analysis. All material was harvested between 10.00 and 11.00 a.m. and the petals frozen in liquid N2 within 30 min of harvest. e x t r a c t i o n . - - The frozen petals were ground in liquid N2 and homogenised for 1 min in cold 0.1 M acetate buffer (pH 5.3) containing 5 mM calcium chloride, sampled for dry wt. determination and centrifuged for 15 min at 3000 × g. The supernatant was dialysed against 0.001 M acetate buffer (pH 5.3) for 3 h prior to amylase assay (initial supernatant). The pellet was re-suspended in buffer and then centrifuged for 15 min at 3000 × g. The resulting pellet was then filtered using Miracloth, with the addition of further cold buffer to wash the suspension through until no more suspended matter was seen in the filtrate. The residue was suspended in buffer for amylase assay. The Miracloth filtrate was centrifuged for 10 min at 6000 × g to give a pellet (crude starch fraction). Amylase was solubilised from the crude starch fraction by incubation in acetate buffer (pH 5.3 with 5 mM calcium chloride containing Tween 20) for 1 h at 40 ° C. The suspension was centrifuged at 6000 × g for 10 min after incubation.
Amylase
a s s a y . - - The enzyme preparation was added to an equal volume of 5% boiled soluble starch in water, mixed and incubated for 2 h at 40 ° C. Samples were taken at the start and finish of incubation and assayed for reducing sugar b y the Nelson--Somogyi m e t h o d using glucose standards (Herbert et al., 1971). No evidence was found for activity changes caused by inhibitory or other materials in the extracts after checking b y assay of mixtures of extracts from fresh and stored roses. Units of amylase activity were expressed as pg glucose equivalents produced h -1. /~-limit dextrin was prepared by incubating 10 ml 1% boiled soluble starch with 1000 units ~-amylase (Sigma, T y p e 1B) in acetate buffer (pH 4.8) for 4 h at 25 ° C. The digest was dialysed against water overnight at 5 ° C. Assays with Amylopectin Azure (Calbiochem) used 0.05 mg substrate in 2 ml enzyme preparation. After 2 h at 40°C the suspension was filtered and the absorbance measured at 500 nm using a buffer blank. Determination of the extent of enzyme inactivation by trypsin and EDTA was carried o u t after pre-incubation for 1 h at 20°C with 20 000 units trypsin (Sigma, T y p e III) with/without 2 pmoles EDTA ml-'. The action pattern was determined as described b y Tung and Nordin (1969). Solubilised enzyme and washed amylose were incubated at 40°C using a shaking water-bath. Samples were taken at zero time and at intervals up to 7 h. Samples were inactivated by addition of 1 drop 0.02 M silver ni-
Amylase
285 trate and centrifuged at 30 000 X g for 10 min. The supernatants were filtered through a membrane filter (Millipore 0.22 # m pore size} and assayed for reducing power and total carbohydrate by Nelson--Somogyi and anthrone assays, respectively (Herbert et al., 1971). Glucose standards were used. RESULTS A m y l o l y t i c activity in petal homogenates. -- Amylolytic activity was consistently present in both the initial supernatant and the crude starch fraction; about 80% of the activity in the latter sedimented after 2 min at 1500 × g. The residue of cell debris after Miracloth filtration showed a small but variable a m o u n t of amylolytic activity, possibly because of incomplete separation from the crude starch fraction. TABLE I Response of supernatant and solubilised starch fraction amylolytic activity from petals of 2-day-stored roses to treatments used to characterise amylase Treatment
% of control activity remaining after treatment Supernatant
Solubilised
(crude fraction) Heat at 70°C for I0 min Pre-incubation with trypsin Pre-incubation with trypsin + E D T A p-limit dextrin substrate (% of activity against soluble starch)
52 98 95 0
58 99 39 100
Amylopectin azure substrate (Relative to activity of solubilised enzyme)
57
100
Amylolytic activities in both supernatant and crude starch fraction showed similar resistance to heating (Table I). Incubation with trypsin did not affect the activity from either source, but when EDTA was added together with the trypsin there was a reduction in the amylolytic activity of the crude starch fraction. The enzyme from the starch fraction showed no significant loss in activity with ~-limit dextrin as substrate compared with starch, and greater activity against Amylopectin azure than the initial supernatant. The products of starch digestion by the solubilised starch fraction enzyme were identified by thin-layer chromatography as glucose, maltose, isomaltose and oligosaccharides. The rate of release of amylase activity from the crude starch fraction by Tween 20 varied with the development of the flower. Solubilisation of 80% of the total solubilised activity took over 20 min with material from fresh roses and less than 2 min when material from roses stored for 2 days was treated (Fig. 1).
286
l
3.0
to.8
(min)
'~ ~ 2-5 o ~
10--
o
5--
~, 2.0 u 0
1
I
I
I
0
1 Time from harvest (days)
2
#
0
I
I
I
L
I
0,2 0.3 0.4 0.5 0.6 glucose equivalents produced
l
0.7 0.8 pmol.
Fig. 1. Time taken for Tween 20 mediated release of amylase from starch grains from roses stored for different periods, t0.8 is the time taken for 80% of the total activity solubilised to be released. Mean Of 2 experiments, bars show range of mean. Fig. 2. Action pattern produced by solubilised starch fraction amylase using amylose as substrate. The graph shows the relationship between the ratio of total carbohydrate released (after removal of substrate) to reducing power and reducing groups liberated during the enzyme reaction.
The action pattern of the solubilised enzyme on amylose was that of an endo-attack (Tung and Nordin, 1969). The ratio of total carbohydrate, after removal of amylose substrate, to reducing power decreased during the incuba tion (Fig. 2). Endo-attack results in early release of large oligosaccharides followed by their b r e a k d o w n to smaller components. Thus, more reducing power is observed per product molecule as the reaction proceeds and the oligosaccharides get smaller. This gives a decreasing plot, as seen in Fig. 2. With exo-attack only single, final product molecules are released, thus there is no change in the ratio of total carbohydrate released to reducing power released and a straight horizontal line is given. Changes in amylase activity during development. -- Amylase activity in the crude starch fraction increased after harvest (Table IIa). There was a large variation between experiments in amylase levels at harvest, although an increase in activity was seen in all experiments. The mean increase was about 65%, most of this occurring in the first 24 h; in some cases the activity decreased between 24 and 48 h after harvest. The mean increase in activity between harvest and the highest observed post-harvest levels was 286 units. This increase was significant (P > 0.002) when tested by the paired sample method. The variations in activity at harvest appeared to be related to the periodic increases in rose flower production, known as flushes, which are caused b y pruning the plants followed by parallel development and then harvest of the new flower buds. Higher amylase levels at harvest were general-
287
TABLE
II
Changes in amylolytic activity in rose petals during storage. Flowers harvested at tight-bud stage (5 flowers/sample) (units g dry wt.-i of petals)
(a) Changes in crude starch fraction amylase activity (values given for solubilised enzyme) Days stored
Date of harvest
13.7.78
20.7.78
0 1
234.2 161.1
91.2 299.7
2
474.1
341.3
6.9.78
11.9.78
14.9.78
2.10.78
12.10.78 Means
73.7 104.8
201.5 246.1
623.5 839.5
365.3 845.4
333.9 799.7
274.8 490.9
272.5
351.1
660.5
464.7
600.1
452.0
(b) Changes in initial supernatant activity Daysstored
0 1 2
Date o f h a r v e ~ 6.9.78
11.9.78
2.10.78
12.10.78
Means
147.7 251.3 1577.1
1432.6 124.1 808.4
1369.8 1649.3 1503.5
866.5 799.7 668.9
954.2 706.1 1139.5
ly present in flowers taken at the height of a flush, when the plant was supporting the greatest number of flowers. The mean amylolytic activity in the supernatant increased after harvest, but the increase was not statistically significant {Table IIb). The weight of the crude starch fraction decreased during storage of the roses; an initial value of 8% dry weight decreased to 4.9% dry weight after 2 days storage. These figures are similar to those for enzymically analysed starch given by Ho and Nichols {1977). DISCUSSION
The identification of amylase activity in rose petals provides a possible mechanism for the breakdown of starch reported in harvested flowers (Ho and Nichols, 1977). Although there was amylolytic activity in the soluble fraction of the petal homogenate, the starch fraction amylase was of greater interest because it increased after harvest. The properties of the starch fraction amylase (Table I) suggest that it may be a-amylase (E.C. 3.2.1.1) (Fischer and Stein, 1960); final identification must await further purification. The supernatant activity exhibited different properties and showed no consistent increase after harvest. Binding of amylase to the starch fraction could be caused by precipitation with tannins during extraction (Goldstein and Swain, 1965; Hawker, 1969).
288
Extraction in the presence of Polyclar AT, 0.25% bovine serum albumin or ovalbumin as protectants against protein precipitation resulted in no significant transfer of amylase activity into the supernatant (J.B.W. Hammond, 1978, unpublished data). This suggests that protein precipitation was not the primary cause of amylase binding to the starch fraction. Amylase binding to starch granules has been proposed as a pre-condition to starch hydrolysis (McLaren, 1963; Sargeant and Walker, 1978). The variation in ease of removal of amylase from the starch fraction could be a function of such binding and hydrolysis during flower storage. The granule surface could then form a less stable binding medium. The lower percentage solubilisation of total activity from stored flowers could also result from granule digestion. Internal erosion of starch granules has been observed (Chandorkar and Badenhuizen, 1967; Nayyar and Ram, 1977), and this would presumably make the digesting enzyme inside the granule more difficult to extract. Starch-bound amylase activity showed up to 3-fold increase after harvest and appeared to peak at 800--850 units g dry wty'. Buds harvested during a flush had higher starch-bound amylase activity at harvest and activity fell during the second day of storage. The increase in starch-bound amylase activity after harvest suggests that it is involved in the rapid post-harvest breakdown of starch. The effect of changes in bound amylase activity on the rate of starch hydrolysis, and of flower opening, remains to be tested, as does the importance of the supernatant amylolytic activity in these processes. However if, as seems likely, amylase attack is the first step in starch breakdown, a rise in amylase activity would increase the rate of breakdown. Thus, suppression of amylase activity by artificial means could offer a way of retarding the opening of harvested rose flowers.
REFERENCES Chandorkar, K.R. and Badenhuizen, N.P., 1967. The fate of ADPG-alpha glucan glucosyltransferase during amylolytic corrosion of starch granules, and its relation to starch granule structure. Cereal Chem., 44: 27--38. Dunn, G., 1974. A model for starch breakdown in higher plants. Phytochemistry, 13: 1341--1346. Fischer, E.H. and Stein, E.A., 1960. In: P.D. Boyer, H. Lardy and K. Myrback (Editors), The Enzymes,Vo]. 4. AcademicPress, London, pp. 313--343. Go]dstein, J.L. and Swain, T., 1965. The inhibition of enzymes by tannins. Phytochemistry, 4: 185--192. Hawker, J.S., 1969. Insoluble invertasefrom grapes: an artefact of extraction. Phytochemistry, 8: 337--344. Herbert, D., Phipps, P.J. and Strange, R.E., 1971. In: J.R. Norris and D.W. Ribbons (Editors), Methods in Microbiology,Vol. 5B. AcademicPress, London, pp. 266--301. Ho, L.C. and Nichols, R., 1977. Transloeationof ,4 C-sucrose in relation to changesin carbohydrate content in rose corollas cut at different stages of development.Ann. Bot., 41 : 227--242.
289 Kaltaler, R.E.L. and Steponkus, P.L., 1974. Uptake and metabolism of sucrose in cut roses. J. Am. Soc. Hortic. Sci., 99: 490--493. McLaren, A.D., 1963. Enzyme reactions in structurally restricted systems. IV. The digestion of insoluble substrates by hydrolytic enzymes. Enzymologia, 26: 237--246. Nayyar, V.L. and Ram, H.Y.M., 1977. Ultrastructure of starch grain breakdown in cotyledons of Ca]anis ca]an during germination. Phytomorphology, 27: 187--190. Sacalis, J.N. and Chin, C.K., 1976. Metabolism of sucrose in cut roses. I. Comparison of sucrose pulse and continuous sucrose uptake. J. Am. Soc. Hortic. Sci., 101: 254--257. Sargeant, J.G. and Walker, T.S., 1978. Adsorption of wheat s-amylase isoenzymes to wheat starch. Starke, 30: 160--163. Tung, K.K. and Nordin, J.H., 1969. Determination of the action patterns of glycanases. Anal. Biochem., 29: 84--90.