- Email: [email protected]
Invertase inhibitor in wilting flower petals
Scientia Horticulturae, 40 (1989) 83-90 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
83
I n v e r t a s e Inhibitor in Wilting F l o w e r Petals J. HALABA and R.M. RUDNICKI
Department of Ornamental Plant Preservation, Research Institute of PomoIogy and Floriculture, 96-100 Skierniewice (Poland) (Accepted for publication 9 December 1988)
ABSTRACT
Halaba, J. and Rudnicki, R.M., 1989. Invertase inhibitor in wilting flower petals. Scientia Hortic., 40: 83-90. An invertase inhibitor, which was found in wilting carnation petals, was able to depress the activity of invertase in crude extract made from petals of cut alstroemeria, dahlia, gladiolus, petunia and rose. Extracts made from wilting petals of these flowers were active in depressing the activity of invertase in carnation petals. It is postulated that this invertase inhibitor is possibly formed in the petals of all flowers at the beginning of wilting, and thus controls the translocation of sucrose from wilted petals to other organs of the flower. This hypothesis is supported by the discovery that the inhibitor is also present in the petals of naturally senescing flowers. Keywords: alstroemeria; carnation; cut flowers; dahlia; gladiolus; invertase inhibitor; petal wilting; petunia; rose.
INTRODUCTION
It has been documented that invertase inhibitor is synthesized in wilting petals of Ipomoea (Winkenbach and Matile, 1970) and carnation flowers (Halaba and Rudnicki, 1983, 1988). It was proposed that this inhibitor affects the senescence of petals by blocking sucrose hydrolysis to glucose and fructose in the senescing tissue. Sucrose was found to be the only mobile sugar in petals, and glucose is converted to sucrose before it is translocated (Ho and Nichols, 1975). Hence, inhibition of hydrolysis enables sucrose in wilting petals to be translocated to neighbouring flower organs (Halaba and Rudnicki, 1988 ). Following the earlier discovery by Winkenbach and Matile (1970) of an invertase inhibitor in wilting petals of Ipomoea flowers, and our discovery of a similar protein in carnation petals (Halaba and Rudnicki, 1983 ), it was interesting to know whether invertase inhibitors were present during the wilting process in petals of other flowers. The purpose of the present study was also to find whether the invertase inhibitor extracted from carnation petals affects the activity of 0304-4238/89/$03.50
© 1989 Elsevier Science Publishers B.V.
84 invertase in other flowers, and whether the invertase inhibitors extracted from other flowers affect the activity of invertase in carnation petals. This might indicate a similar mechanism of inhibitory action in various plants and similarity in chemical structure. MATERIALSAND METHODS Plant material. - Cut flowers of alstroemeria (Alstroemeria X hybrida hort. cultivars 'Zebra'), carnation (Dianthus caryophyllus L. cultivars 'White Sire'), dahlia (Dahlia pinnata cultivars 'Snieina Kula'), gladiolus (Gladiolus X tubergenii hort. cultivar 'Dukat' ), petunia (Petunia X hybrida hort. 'Rosa Wolke') and rose (Rosa thea X hybrida hort. 'Charles de Gaulle' (I) and 'Evening Star' (II)) were received from a commercial nursery. Flowers were cut at a fully opened bud stage. Flowers of one lot were placed in vases with distilled water. Petals from these flowers, harvested at the first signs of wilting, were collected and deep frozen. Samples of frozen petals were then used to prepare the invertase inhibitor extracts. From a second lot of freshly cut flowers, petals were harvested directly, frozen, and then used for extraction of invertase. Petals of carnation flowers wilting on mother plants were frozen and used for separate experiments. Preparation of crude extract of invertase inhibitor. - Petals from wilting flowers were macerated in an extraction buffer: 0.05 M NaH2P04, 1 M NaC1 at pH 4.0. Ten ml of extraction buffer was used per g of fresh weight of petal tissue. The slurry was filtered through miracloth and then centrifuged at 35 000 g for 30 min. at 0 ° C. Solid ammonium sulphate was then dissolved in the supernatant at 2°C to produce 80% saturation. After 1 h the precipitate was collected by centrifugation at 10 000 g at 0°C for 10 min. The precipitate was dissolved in a small amount of phosphate buffer: 0.05 M NaH2P04 at pH 4.0. For assays, 2 ml of crude extract of invertase inhibitor were diluted in 25 ml of distilled water. Additionally, purified invertase inhibitor extract from carnation petals was used in one experiment, where it was incubated with invertase extract from the petals of alstroemeria, dahlia, gladiolus, petunia and rose. Invertase inhibitor from wilting carnation petals was purified by column chromatography methods (J. Halaba, K. Manning and R. Nichols, unpublished data, 1983). Ten ml of crude extract of invertase inhibitor were dialysed against 11 of citrate buffer 0.05 M at pH 4.0. After freeze-drying and reconstituting to 1 ml, invertase inhibitor extract was chromatographed on a 1.6 X 85.4 cm column of Sephadex G-75 washed with buffer: 0.01 M acetic acid, 0.05 M NaC1, 0.02% NaN3 at pH 4.0. The flow rate was 21 ml h -1. Thirty fractions, 2.5 ml each, were collected. Preparation of crude extract of invertase. - Fully opened flowers were used for the preparation of crude extracts of invertase. The procedure was similar to
85 the preparation of crude extract of invertase inhibitor, except that buffers with pH 7.0 were used. For assays, 2 ml of crude invertase extract were diluted to 50 ml in distilled water. Determination of invertase and invertase inhibitor activity. - The activity of invertase was determined by incubating 1 ml of crude extract with 2 ml of distilled water, together with 3 ml of 20 mM acetate buffer at a pH 4.8 with 2.92 mM sucrose for I h. The activity of invertase was calculated in Z mole of glucose mg protein- 1 h - 1 as a difference between the reducing sugars concentration after incubation and before incubation. Reducing sugar concentration was determined according to the Nelson (1944) method. Protein was determined by the Coomassie brilliant blue method (Bradford, 1976). To determine the activity of invertase inhibitor, 1 ml of crude extract of invertase was incubated with 2 ml of crude inhibitor extract together with 3 ml of 20 mM acetate buffer at a pH of 4.8 with 2.92 mM sucrose for 1 h. The activity of inhibitor was calculated as the difference between the sum of invertase activity in the samples incubated separately (theoretical activity) and the invertase activity in samples incubated together with invertase extract (experimental activity) as described earlier (Halaba and Rudnicki, 1983). Results of experiments were repeated 3 times. Twenty flowers were used for each treatment. Differences between experimental activity and theoretical activity were statistically evaluated using analysis of variance and Student's t test for determination of LSD at 5%.
RESULTS Investigations of invertase activity in crude extracts prepared from wilting petals showed that invertase inhibitor was present in all of them. Table 1 presents the influence of invertase inhibitor on the activity of invertase from the same species. The invertase activity in a mixture of crude invertase and invertase inhibitor extracts, was lower than the invertase activity theoretically calculated as a sum of both these extracts incubated separately for all tested species. The percent of inhibition was similar for all inhibitors and ranged from 19.9 to 32.0. Results of the investigation of the invertase inhibitor action on the activity of invertase from different species are presented in Table 2. Inhibitors from petals of all species tested decreased the activity of invertase from carnation petals, and their theoretical activity was higher than the experimental activity of all extracts. Invertase inhibitor actions decreased invertase activity nearly one-third except for invertase inhibitor from gladiolus. Additionally purified invertase inhibitor extract from carnation petals was used for investigations of its influence on the inhibition of invertase isolated from other species (Table 3). The invertase inhibitor from wilting carnation
86 TABLE 1 The influence of invertase inhibitor from wilting petals of 5 ornamental species on invertase activity from the same species. Activity of invertase in # mol of glucose mg protein- ~h Extracts of
Invertase activity
Experimental activity of invertase and invertase inhibitor extracts ~ (a)
Theoretical activity~ (b)
% of inhibition b- a - - ~ 100
Invertase, alstroemeria Inhibitor, alstroemeria Invertase, dahlia Inhibitor, dahlia Invertase, gladiolus Inhibitor, gladiolus Invertase, petunia Inhibitor, petunia Invertase, rose (I) Inhibitor, rose (I) Invertase, rose (II) Inhibitor, rose (II)
13.06 2.93 17.12 4.84 23.26 5.87 13.18 6.25 6.42 3.32 6.18 2.17
12.75
15.99
20.3
16.47
22.06
25.3
22.86
29.12
21.5
15.55
19.42
19.9
6.62
9.74
32.0
6.56
5.35
21.4
1(a) and (b) are significantly different according to Student's t test at the 5 % level. TABLE 2 The influence of invertase inhibitors from wilting petals of flowers of 5 ornamental species on invertase from carnation petals. Activity of invertase in # mol of glucose mg protein- 1h Extracts of
Invertase activity
Invertase, carnation Inhibitor, alstroemeria Inhibitor, dahlia Inhibitor, gladiolus Inhibitor, petunia Inhibitor, rose (I) Inhibitor, rose (II)
7.27 2.93 4.84 5.87 6.25 3.32 2.17
Experimental activity of invertase and invertase inhibitor extracts I (a)
Theoretical activity1 (b)
% of inhibition b- a ~ 100
7.14 8.54 11.28 9.56 6.09 6.76
10.20 12.11 13.14 13.52 10.59 9.44
30.0 29.5 14.2 29.3 42.5 28.4
1(a) and (b) are significantly different according to Student's t test at the 5% level.
petals was able to reduce the activity of invertase extracted from all other species. The experimental activity was lower than the theoretical activity calculated for all tested mixtures. The percentages of inhibition varied from 30.4 to 57.1%, depending on the species.
87 TABLE 3 The influence of invertase inhibitor from wilting carnation petals on activity of invertase from flowers of 5 ornamental species. Activity of invertase in/~ mol of glucose mg protein- ~h - 1
Extracts
Invertase
Experimental activity of
Theoretical activity~
% of inhibition b- a
invertaseand invertase inhibitor
(b)
- - ~ 100
13.06 17.21 23.26 13.18 6.42 6.18
57.1 44.2 30.4 39.3 47.4 50.2
extracts ~ (a) Inhibitor, carnation Invertase, alstroemeria Invertase, dahlia Invertase, gladiolus Invertase, petunia Invertase, rose (I) Invertase, rose (II)
0 13.06 17.21 23.26 13.18 6.42 6.18
5.60 9.60 16.18 8.01 3.38 3.07
(a) and (b) are significantly different according to Student's t test at the 5% level. TABLE 4 The influence of inhibitor from wilting carnation petals of non-cut flowers on activity of invertase from carnation. Activity of invertase in #umol glucose mg protein - 1 h Extracts
Invertase activity
Experimental activity invertase and invertase inhibitor extracts
Theoretical activity1 (b)
% of inhibition b- a ~ 100
11.29
41.2
(a) Invertase inhibitor Invertase
4.02 7.27
6.64
1(a) and (b) are significantly different according to Student's t test at the 5% level.
Invertase inhibitor also appeared to be present in wilting petals of non-cut flowers (Table 4). Incubation of both invertase and inhibitor extracts from petals of non-cut carnation flowers gave a lower invertase activity than the sum of invertase activity in both extracts when they were incubated separately. The percentage of inhibition approached 40. DISCUSSION
Results of non-published results (J. Halaba, K. Manning and R.Nichols, 1983) had shown that activityof invertase inhibitor from wilting carnation petals is reduced at 55 °C and higher. It is also reduced in buffers with p H of 5.0 and more. A similar extraction procedure was used in the present study.
88 The sensitivity of invertase inhibitors from wilting petals of alstroemeria, dahlia, gladiolus, petunia and rose were not tested, but a similarity between the invertase inhibitors from different species and tissues exists. Properties of invertase inhibitors extracted by Pressey (1966, 1967, 1968) from potato tubers, sweet potato, red beets and sugar beets are similar to those of the inhibitor from carnation petals (J. Halaba, K. Manning and R. Nichols, unpublished data, 1983). Present results show that the invertase inhibitor is present not only in wilting petals of Ipomoea (Winkenbachand Matile, 1970) and carnation (Halaba and Rudnicki, 1983), but also in many other flowers. Incubation of both invertase and invertase inhibitor extracts of various flowers resulted in lower invertase activity when compared with the sum of invertase activity when these extracts were incubated separately (Table 1 ). This indicates that during the ageing process invertase inhibitors are synthesized in petals of alstroemeria, dahlia, gladiolus, petunia and rose flowers. Thus, it seems that the invertase inhibitors are common during wilting of petals of various species. Petals are the fastest growing organs of the flowers. The requirement for respiratory substances and the carbon source for the growing tissues is satisfied by the hydrolysis of sucrose into glucose and fructose by invertase. Invertase activity is therefore much higher in petals than in other organs (Halaba and Rudnicki, 1981; Hawker et al., 1976; Nichols and Ho, 1975; Winkenbach and Matile, 1970). After pollination, during the natural ageing of petals, the ovary and receptacle grow quickly and senescing petals become one of the main contributors of metabolites. Blockage of invertase action by invertase inhibitor stops sucrose hydrolysis to glucose and fructose and, thereby, enables mobilization of carbohydrate from wilting petals to neighbouring organs. Earlier it was found that treating with cycloheximide blocked the synthesis of the invertase inhibitor and maintained a relatively high level of invertase activity (Halaba and Rudnicki, 1988; Winkenbach and Matile, 1970). Also treating carnation flowers with 14C-sucrose and cycloheximide showed that the receptacles and ovaries not treated with cycloheximide contained from 7 to 10 times more radioactivity compared with treated flowers (Halaba and Rudnicki, 1988). Invertase inhibitors from the storage organs of other cultivated plants play a different physiological role than the inhibitors from petals. Invertase inhibitors in storage tissues are hypothesized to inhibit sucrose hydrolysis during dormancy in stored organs (Pressey, 1966, 1967, 1968). Invertase inhibitor concentration in Pressey's species decreased before the end of dormancy, and the rate of inactivation of inhibitor depended on storage conditions. The breakdown of inhibitor molecules, presumably, is needed to allow invertase action, catalyzing the hydrolysis of sucrose. Although invertase inhibitors from storage organs play a different role than those from flower petals, they have similar properties, being low-molecular proteins with molecular weights 17 800-22 900 (Pressey, 1968), while carnation petal inhibitors have 18 200 (J. Halaba, K. Manning and R. Nichols, un-
89
published data, 1983). They also exhibit similar sensitivities to temperatures and pH. The invertase inhibitor from one plant species can stop the action of invertase from another. This can be explained by the similarity between the composition and the action of both invertase and inhibitor from different species (Pressey, 1967). The results presented in Tables 2 and 3 showed the same relationship between invertase and inhibitors from petals of different species of several floricultural plants, which confirms Pressey's hypothesis. Different invertases and inhibitors acted similarly but not exactly with the same efficiency (Tables 2 and 3). It is very probable that one plant can have more than one isoenzyme of invertase with similar properties, which may explain the different activity of inhibitors from various plants. The presence of invertase inhibitors in petals ageing on the mother plants indicates that after pollination the synthesis of invertase inhibitor enables carbohydrate transfer from the wilting petals to neighbouring organs. This seems to be a general phenomenon for ageing flowers. ACKNOWLEDGEMENTS
This project was partly supported by funds made available from the Maria Sktodowska-Curie Fund, established by contributions from the United States and Polish Governments, Grant No. J-MOA-13 (JB-10), project No. PL-ARS101.
REFERENCES Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 249-254. Halaba, J. and Rudnicki, R.M., 1981. Changes in acid phosphatase and invertase activities in carnation flowers during bud development and senescence. Rogl. Ozdob. Ser. B. 6: 73-81. Halaba, J. and Rudnicki, R.M., 1983. An invertase inhibitor as affecting wilting of carnation petals. Acta Hortic., 138: 261-267. Halaba, J. and Rudnicki, R.M., 1988. Invertase inhibitor-control of sucrose transport from petals to other flower parts. Plant Growth Regulation, 7: 193-199. Hawker, J.S., Walker, R.R. and Ruffner, H.P., 1976. Invertase and sucrose synthase in flowers. Phytochemistry, 15: 1441-1443. Ho, L.C. and Nichols, R., 1975. The role of phloem transport in the translocation of sucrose along the stem of carnation cut flowers. Ann. Bot., 39: 439-436. Nelson, N., 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153: 375-380. Nichols, R. and Ho, L.C., 1975. An effect of ethylene on the distribution of 14C-sucrose from the petals to other flower parts in the senescent cut inflorescence of Dianthus caryophyUus. Ann. Bot., 39: 433-438. Pressey, R., 1966. Separation and properties of potato invertase and invertase inhibitor. Arch. Biochem. Biophys., 113: 667-674.
90 Pressey, R., 1967. Invertase inhibitor from potatoes: purification,characterization, and reactivity with plant invertases. Plant Physiol., 42: 1780-1786. Pressey, R., 1968. Invertase inhibitors from red beet, sugar beet, and sweet potato roots. Plant Physiol., 43: 1430-1434. Winkenbach, F. and Matile, P., 1970. Evidence for de novo synthesis of an invertase inhibitor protein in senescing petals of Ipornoea.Z. Pflanzenphysiol., 63: 292-295.
83
I n v e r t a s e Inhibitor in Wilting F l o w e r Petals J. HALABA and R.M. RUDNICKI
Department of Ornamental Plant Preservation, Research Institute of PomoIogy and Floriculture, 96-100 Skierniewice (Poland) (Accepted for publication 9 December 1988)
ABSTRACT
Halaba, J. and Rudnicki, R.M., 1989. Invertase inhibitor in wilting flower petals. Scientia Hortic., 40: 83-90. An invertase inhibitor, which was found in wilting carnation petals, was able to depress the activity of invertase in crude extract made from petals of cut alstroemeria, dahlia, gladiolus, petunia and rose. Extracts made from wilting petals of these flowers were active in depressing the activity of invertase in carnation petals. It is postulated that this invertase inhibitor is possibly formed in the petals of all flowers at the beginning of wilting, and thus controls the translocation of sucrose from wilted petals to other organs of the flower. This hypothesis is supported by the discovery that the inhibitor is also present in the petals of naturally senescing flowers. Keywords: alstroemeria; carnation; cut flowers; dahlia; gladiolus; invertase inhibitor; petal wilting; petunia; rose.
INTRODUCTION
It has been documented that invertase inhibitor is synthesized in wilting petals of Ipomoea (Winkenbach and Matile, 1970) and carnation flowers (Halaba and Rudnicki, 1983, 1988). It was proposed that this inhibitor affects the senescence of petals by blocking sucrose hydrolysis to glucose and fructose in the senescing tissue. Sucrose was found to be the only mobile sugar in petals, and glucose is converted to sucrose before it is translocated (Ho and Nichols, 1975). Hence, inhibition of hydrolysis enables sucrose in wilting petals to be translocated to neighbouring flower organs (Halaba and Rudnicki, 1988 ). Following the earlier discovery by Winkenbach and Matile (1970) of an invertase inhibitor in wilting petals of Ipomoea flowers, and our discovery of a similar protein in carnation petals (Halaba and Rudnicki, 1983 ), it was interesting to know whether invertase inhibitors were present during the wilting process in petals of other flowers. The purpose of the present study was also to find whether the invertase inhibitor extracted from carnation petals affects the activity of 0304-4238/89/$03.50
© 1989 Elsevier Science Publishers B.V.
84 invertase in other flowers, and whether the invertase inhibitors extracted from other flowers affect the activity of invertase in carnation petals. This might indicate a similar mechanism of inhibitory action in various plants and similarity in chemical structure. MATERIALSAND METHODS Plant material. - Cut flowers of alstroemeria (Alstroemeria X hybrida hort. cultivars 'Zebra'), carnation (Dianthus caryophyllus L. cultivars 'White Sire'), dahlia (Dahlia pinnata cultivars 'Snieina Kula'), gladiolus (Gladiolus X tubergenii hort. cultivar 'Dukat' ), petunia (Petunia X hybrida hort. 'Rosa Wolke') and rose (Rosa thea X hybrida hort. 'Charles de Gaulle' (I) and 'Evening Star' (II)) were received from a commercial nursery. Flowers were cut at a fully opened bud stage. Flowers of one lot were placed in vases with distilled water. Petals from these flowers, harvested at the first signs of wilting, were collected and deep frozen. Samples of frozen petals were then used to prepare the invertase inhibitor extracts. From a second lot of freshly cut flowers, petals were harvested directly, frozen, and then used for extraction of invertase. Petals of carnation flowers wilting on mother plants were frozen and used for separate experiments. Preparation of crude extract of invertase inhibitor. - Petals from wilting flowers were macerated in an extraction buffer: 0.05 M NaH2P04, 1 M NaC1 at pH 4.0. Ten ml of extraction buffer was used per g of fresh weight of petal tissue. The slurry was filtered through miracloth and then centrifuged at 35 000 g for 30 min. at 0 ° C. Solid ammonium sulphate was then dissolved in the supernatant at 2°C to produce 80% saturation. After 1 h the precipitate was collected by centrifugation at 10 000 g at 0°C for 10 min. The precipitate was dissolved in a small amount of phosphate buffer: 0.05 M NaH2P04 at pH 4.0. For assays, 2 ml of crude extract of invertase inhibitor were diluted in 25 ml of distilled water. Additionally, purified invertase inhibitor extract from carnation petals was used in one experiment, where it was incubated with invertase extract from the petals of alstroemeria, dahlia, gladiolus, petunia and rose. Invertase inhibitor from wilting carnation petals was purified by column chromatography methods (J. Halaba, K. Manning and R. Nichols, unpublished data, 1983). Ten ml of crude extract of invertase inhibitor were dialysed against 11 of citrate buffer 0.05 M at pH 4.0. After freeze-drying and reconstituting to 1 ml, invertase inhibitor extract was chromatographed on a 1.6 X 85.4 cm column of Sephadex G-75 washed with buffer: 0.01 M acetic acid, 0.05 M NaC1, 0.02% NaN3 at pH 4.0. The flow rate was 21 ml h -1. Thirty fractions, 2.5 ml each, were collected. Preparation of crude extract of invertase. - Fully opened flowers were used for the preparation of crude extracts of invertase. The procedure was similar to
85 the preparation of crude extract of invertase inhibitor, except that buffers with pH 7.0 were used. For assays, 2 ml of crude invertase extract were diluted to 50 ml in distilled water. Determination of invertase and invertase inhibitor activity. - The activity of invertase was determined by incubating 1 ml of crude extract with 2 ml of distilled water, together with 3 ml of 20 mM acetate buffer at a pH 4.8 with 2.92 mM sucrose for I h. The activity of invertase was calculated in Z mole of glucose mg protein- 1 h - 1 as a difference between the reducing sugars concentration after incubation and before incubation. Reducing sugar concentration was determined according to the Nelson (1944) method. Protein was determined by the Coomassie brilliant blue method (Bradford, 1976). To determine the activity of invertase inhibitor, 1 ml of crude extract of invertase was incubated with 2 ml of crude inhibitor extract together with 3 ml of 20 mM acetate buffer at a pH of 4.8 with 2.92 mM sucrose for 1 h. The activity of inhibitor was calculated as the difference between the sum of invertase activity in the samples incubated separately (theoretical activity) and the invertase activity in samples incubated together with invertase extract (experimental activity) as described earlier (Halaba and Rudnicki, 1983). Results of experiments were repeated 3 times. Twenty flowers were used for each treatment. Differences between experimental activity and theoretical activity were statistically evaluated using analysis of variance and Student's t test for determination of LSD at 5%.
RESULTS Investigations of invertase activity in crude extracts prepared from wilting petals showed that invertase inhibitor was present in all of them. Table 1 presents the influence of invertase inhibitor on the activity of invertase from the same species. The invertase activity in a mixture of crude invertase and invertase inhibitor extracts, was lower than the invertase activity theoretically calculated as a sum of both these extracts incubated separately for all tested species. The percent of inhibition was similar for all inhibitors and ranged from 19.9 to 32.0. Results of the investigation of the invertase inhibitor action on the activity of invertase from different species are presented in Table 2. Inhibitors from petals of all species tested decreased the activity of invertase from carnation petals, and their theoretical activity was higher than the experimental activity of all extracts. Invertase inhibitor actions decreased invertase activity nearly one-third except for invertase inhibitor from gladiolus. Additionally purified invertase inhibitor extract from carnation petals was used for investigations of its influence on the inhibition of invertase isolated from other species (Table 3). The invertase inhibitor from wilting carnation
86 TABLE 1 The influence of invertase inhibitor from wilting petals of 5 ornamental species on invertase activity from the same species. Activity of invertase in # mol of glucose mg protein- ~h Extracts of
Invertase activity
Experimental activity of invertase and invertase inhibitor extracts ~ (a)
Theoretical activity~ (b)
% of inhibition b- a - - ~ 100
Invertase, alstroemeria Inhibitor, alstroemeria Invertase, dahlia Inhibitor, dahlia Invertase, gladiolus Inhibitor, gladiolus Invertase, petunia Inhibitor, petunia Invertase, rose (I) Inhibitor, rose (I) Invertase, rose (II) Inhibitor, rose (II)
13.06 2.93 17.12 4.84 23.26 5.87 13.18 6.25 6.42 3.32 6.18 2.17
12.75
15.99
20.3
16.47
22.06
25.3
22.86
29.12
21.5
15.55
19.42
19.9
6.62
9.74
32.0
6.56
5.35
21.4
1(a) and (b) are significantly different according to Student's t test at the 5 % level. TABLE 2 The influence of invertase inhibitors from wilting petals of flowers of 5 ornamental species on invertase from carnation petals. Activity of invertase in # mol of glucose mg protein- 1h Extracts of
Invertase activity
Invertase, carnation Inhibitor, alstroemeria Inhibitor, dahlia Inhibitor, gladiolus Inhibitor, petunia Inhibitor, rose (I) Inhibitor, rose (II)
7.27 2.93 4.84 5.87 6.25 3.32 2.17
Experimental activity of invertase and invertase inhibitor extracts I (a)
Theoretical activity1 (b)
% of inhibition b- a ~ 100
7.14 8.54 11.28 9.56 6.09 6.76
10.20 12.11 13.14 13.52 10.59 9.44
30.0 29.5 14.2 29.3 42.5 28.4
1(a) and (b) are significantly different according to Student's t test at the 5% level.
petals was able to reduce the activity of invertase extracted from all other species. The experimental activity was lower than the theoretical activity calculated for all tested mixtures. The percentages of inhibition varied from 30.4 to 57.1%, depending on the species.
87 TABLE 3 The influence of invertase inhibitor from wilting carnation petals on activity of invertase from flowers of 5 ornamental species. Activity of invertase in/~ mol of glucose mg protein- ~h - 1
Extracts
Invertase
Experimental activity of
Theoretical activity~
% of inhibition b- a
invertaseand invertase inhibitor
(b)
- - ~ 100
13.06 17.21 23.26 13.18 6.42 6.18
57.1 44.2 30.4 39.3 47.4 50.2
extracts ~ (a) Inhibitor, carnation Invertase, alstroemeria Invertase, dahlia Invertase, gladiolus Invertase, petunia Invertase, rose (I) Invertase, rose (II)
0 13.06 17.21 23.26 13.18 6.42 6.18
5.60 9.60 16.18 8.01 3.38 3.07
(a) and (b) are significantly different according to Student's t test at the 5% level. TABLE 4 The influence of inhibitor from wilting carnation petals of non-cut flowers on activity of invertase from carnation. Activity of invertase in #umol glucose mg protein - 1 h Extracts
Invertase activity
Experimental activity invertase and invertase inhibitor extracts
Theoretical activity1 (b)
% of inhibition b- a ~ 100
11.29
41.2
(a) Invertase inhibitor Invertase
4.02 7.27
6.64
1(a) and (b) are significantly different according to Student's t test at the 5% level.
Invertase inhibitor also appeared to be present in wilting petals of non-cut flowers (Table 4). Incubation of both invertase and inhibitor extracts from petals of non-cut carnation flowers gave a lower invertase activity than the sum of invertase activity in both extracts when they were incubated separately. The percentage of inhibition approached 40. DISCUSSION
Results of non-published results (J. Halaba, K. Manning and R.Nichols, 1983) had shown that activityof invertase inhibitor from wilting carnation petals is reduced at 55 °C and higher. It is also reduced in buffers with p H of 5.0 and more. A similar extraction procedure was used in the present study.
88 The sensitivity of invertase inhibitors from wilting petals of alstroemeria, dahlia, gladiolus, petunia and rose were not tested, but a similarity between the invertase inhibitors from different species and tissues exists. Properties of invertase inhibitors extracted by Pressey (1966, 1967, 1968) from potato tubers, sweet potato, red beets and sugar beets are similar to those of the inhibitor from carnation petals (J. Halaba, K. Manning and R. Nichols, unpublished data, 1983). Present results show that the invertase inhibitor is present not only in wilting petals of Ipomoea (Winkenbachand Matile, 1970) and carnation (Halaba and Rudnicki, 1983), but also in many other flowers. Incubation of both invertase and invertase inhibitor extracts of various flowers resulted in lower invertase activity when compared with the sum of invertase activity when these extracts were incubated separately (Table 1 ). This indicates that during the ageing process invertase inhibitors are synthesized in petals of alstroemeria, dahlia, gladiolus, petunia and rose flowers. Thus, it seems that the invertase inhibitors are common during wilting of petals of various species. Petals are the fastest growing organs of the flowers. The requirement for respiratory substances and the carbon source for the growing tissues is satisfied by the hydrolysis of sucrose into glucose and fructose by invertase. Invertase activity is therefore much higher in petals than in other organs (Halaba and Rudnicki, 1981; Hawker et al., 1976; Nichols and Ho, 1975; Winkenbach and Matile, 1970). After pollination, during the natural ageing of petals, the ovary and receptacle grow quickly and senescing petals become one of the main contributors of metabolites. Blockage of invertase action by invertase inhibitor stops sucrose hydrolysis to glucose and fructose and, thereby, enables mobilization of carbohydrate from wilting petals to neighbouring organs. Earlier it was found that treating with cycloheximide blocked the synthesis of the invertase inhibitor and maintained a relatively high level of invertase activity (Halaba and Rudnicki, 1988; Winkenbach and Matile, 1970). Also treating carnation flowers with 14C-sucrose and cycloheximide showed that the receptacles and ovaries not treated with cycloheximide contained from 7 to 10 times more radioactivity compared with treated flowers (Halaba and Rudnicki, 1988). Invertase inhibitors from the storage organs of other cultivated plants play a different physiological role than the inhibitors from petals. Invertase inhibitors in storage tissues are hypothesized to inhibit sucrose hydrolysis during dormancy in stored organs (Pressey, 1966, 1967, 1968). Invertase inhibitor concentration in Pressey's species decreased before the end of dormancy, and the rate of inactivation of inhibitor depended on storage conditions. The breakdown of inhibitor molecules, presumably, is needed to allow invertase action, catalyzing the hydrolysis of sucrose. Although invertase inhibitors from storage organs play a different role than those from flower petals, they have similar properties, being low-molecular proteins with molecular weights 17 800-22 900 (Pressey, 1968), while carnation petal inhibitors have 18 200 (J. Halaba, K. Manning and R. Nichols, un-
89
published data, 1983). They also exhibit similar sensitivities to temperatures and pH. The invertase inhibitor from one plant species can stop the action of invertase from another. This can be explained by the similarity between the composition and the action of both invertase and inhibitor from different species (Pressey, 1967). The results presented in Tables 2 and 3 showed the same relationship between invertase and inhibitors from petals of different species of several floricultural plants, which confirms Pressey's hypothesis. Different invertases and inhibitors acted similarly but not exactly with the same efficiency (Tables 2 and 3). It is very probable that one plant can have more than one isoenzyme of invertase with similar properties, which may explain the different activity of inhibitors from various plants. The presence of invertase inhibitors in petals ageing on the mother plants indicates that after pollination the synthesis of invertase inhibitor enables carbohydrate transfer from the wilting petals to neighbouring organs. This seems to be a general phenomenon for ageing flowers. ACKNOWLEDGEMENTS
This project was partly supported by funds made available from the Maria Sktodowska-Curie Fund, established by contributions from the United States and Polish Governments, Grant No. J-MOA-13 (JB-10), project No. PL-ARS101.
REFERENCES Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 249-254. Halaba, J. and Rudnicki, R.M., 1981. Changes in acid phosphatase and invertase activities in carnation flowers during bud development and senescence. Rogl. Ozdob. Ser. B. 6: 73-81. Halaba, J. and Rudnicki, R.M., 1983. An invertase inhibitor as affecting wilting of carnation petals. Acta Hortic., 138: 261-267. Halaba, J. and Rudnicki, R.M., 1988. Invertase inhibitor-control of sucrose transport from petals to other flower parts. Plant Growth Regulation, 7: 193-199. Hawker, J.S., Walker, R.R. and Ruffner, H.P., 1976. Invertase and sucrose synthase in flowers. Phytochemistry, 15: 1441-1443. Ho, L.C. and Nichols, R., 1975. The role of phloem transport in the translocation of sucrose along the stem of carnation cut flowers. Ann. Bot., 39: 439-436. Nelson, N., 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153: 375-380. Nichols, R. and Ho, L.C., 1975. An effect of ethylene on the distribution of 14C-sucrose from the petals to other flower parts in the senescent cut inflorescence of Dianthus caryophyUus. Ann. Bot., 39: 433-438. Pressey, R., 1966. Separation and properties of potato invertase and invertase inhibitor. Arch. Biochem. Biophys., 113: 667-674.
90 Pressey, R., 1967. Invertase inhibitor from potatoes: purification,characterization, and reactivity with plant invertases. Plant Physiol., 42: 1780-1786. Pressey, R., 1968. Invertase inhibitors from red beet, sugar beet, and sweet potato roots. Plant Physiol., 43: 1430-1434. Winkenbach, F. and Matile, P., 1970. Evidence for de novo synthesis of an invertase inhibitor protein in senescing petals of Ipornoea.Z. Pflanzenphysiol., 63: 292-295.