Role of Sucrose Synthase and Other Related Enzymes. in Sucrose Accumulation in Peach Fruit

J. Japan. Soc. Hort. Sci. 60 (3) : 531 538. 1991. Role of Sucrose Synthase and Other Related Enzymes in Sucrose Accumulation in Peach Fruit Takaya Moriguchi1, Yuri Ishizawa1, Tetsuro Sanada1, Sayuri Teramoto2 and Shohei Yamaki2 1 Division of Breeding, Fruit Tree Research Station, MAFF, Ibaraki 305 2 Laboratory of Horticulture, School of Agriculture, Nagoya University, Chikusa, Nagoya 464 Summary Four peach cultivars differing in their maturation time and sugar composition were analyzed for the detection of seasonal changes in the content of sugars in the flesh and peduncle, and in the activity of sucrose-metabolizing enzymes. Besides the accumulation of sucrose as a major sugar at the mature stage in all the cultivars, the fructose content was similar to glucose content in 'Akatsuki' and 'Yuzora'. While in 'Naganoyaseito-early' and 'Hokimomo' the fructose content was low. Of the enzymes assayed, there were no clear differences in the activity among the peach cultivars. In all the cultivars, the sucrose synthase (SS) activity increased with sucrose accumulation, suggesting the importance of its role in sucrose accumulation in peach fruit. On the contrary, the sucrose-phosphate synthase (SPS) activity did not increase with sucrose accumulation. The acid invertase activity decreased with fruit development. The decrease in the acid invertase activity appeared to reflect fruit maturation, during which sucrose accumulated. The UDPglucose pyrophosphorylase activity was also assayed in these peaches throughout the development of fruit. However, typical seasonal fluctuations of the activity were not observed among the four cultivars. Kinetic parameters of SS from immature and mature peach fruit suggested that SS tended to be involved in sucrose synthesis in the mature peach fruit compared with the immature one. The differences in sucrose synthesis between peach and other fruits were also discussed, based on the activities and properties of these enzymes. Introduction The sucrose content is low in young peach fruit, but rapidly increases to account for more than 70% of total soluble sugars at maturation (10). This change reflects the change in the sucrose metabolism (16). In the previous paper using only one peach cultivar ('Hakuto'), the sucrose synthase (SS) (E.C.2.4.1.13) activity was found to increase with maturation during which the sucrose content increased rapidly, while the sucrose-phosphate synthase (SPS) (E.C.2.4.1.14) activity which is assumed to play an important role in sucrose synthesis, remained low during maturation. On the contrary, the acid invertase (E.C.3.2.1.26) activity was relatively high at the early developing stage and decreased along with sucrose accumulation Received for publication 13 May 1991. Contribution No. A-274 of the Fruit Tree Research Station. (10). On the other hand, we reported that large quantities of sucrose accumulated during maturation in all the varieties including the early- and late-maturing ones, and in some varieties fructose also accumulated along with sucrose (11). Moreover, in many plants (3, 6, 14, 19), SS is known to be involved in sucrose cleavage rather than synthesis, while SPS is actively involved in sucrose synthesis (3, 19). Thus, studies were carried out to determine whether the enzymes involved in sucrose accumulation in 'Hakuto' cultivar played the same role in other peach cultivars that differed in the maturation time and sugar composition. 1. Materials Materials and Methods Approximately 10~50 fruits of the Prunus per- 531

532 T. MORIGUCHI, Y. ISHIZAWA, T. SANADA. S. TERAMOTO AND S. YAMAKI sica cvs. 'Yuzora', 'Naganoyaseito-early' and 'Hokimomo' were harvested throughout the developing season. 'Akatsuki' (early- to middlematuring type) and 'Yuzora' (late-maturing type) are known to accumulate large amounts of fructose, while `Naganoyaseito-early' (middle-maturing type) and 'Hokimomo' (late-maturing type) accumulate small amounts. A portion of each fruit and peduncle was subsampled and diced into small pieces. Fruit samples ranging from 1.5 to 10 g in fresh weight were used for enzyme assays and sugar determinations. Peduncle samples of 0.4 g in fresh weight were used for sugar determinations. For all the enzyme assays and sugar determinations there were 2 replications. For partial purification of SS, 100 to 200 g of fruit cv. 'Hakuto' was harvested at the immature stage. 2. Determination of sugar content and composition Sugars were analyzed according to the methods described in the previous report (11). 3. Enzyme extraction Unless indicated otherwise, all the steps were carried out at 4 Ž. The SS, SPS and UDPglucose pyrophosphorylase (E.C.2.7.7.9) were extracted by homogenizing peach fruit in 0.2 M K-phosphate buffer (ph 7.8) containing 10 mm K-ascorbate, 5 mm MgCl2, 1 mm dithiothreitol (DTT) and 10% polyvinylpolypyrrolidone. After centrifugation at 12,000 ~ g for 20 min, a portion of the supernatant was dialyzed for 5~8 hr at 4 Ž against 10 mm Tris-HCl buffer (ph 7.2) containing 1 mm DTT. Acid invertase was extracted by homogenizing peach fruit in 0.1 M K-phosphate buffer (ph 7.0) containing 10 mm K-ascorbate and 1 mm DTT. After centrifugation at 12,000 ~ g for 20 min, the precipitate was washed again with 0.1 M K- phosphate buffer (ph 7.0) containing 10 mm K- ascorbate and 1 mm DTT, then recentrifuged at 12,000 ~ g for 20 min. The combined supernatant and the residue remaining after centrifugation were dialyzed against 10 mm Tris-HCl buffer (ph 7.2) containing 1 mm DTT. For further purificaiton of SS from immature peach fruit cv. Hakuto, the supernatant was adjusted to 80% saturation with ammonium sulfate, then centrifuged at 12,000 ~ g for 10 min. The precipitate was dissolved in a small volume of 5 mm Tris-HCl buffer (ph 7.5) containing 0.2 mm EDTA and 2 mm 2-mercaptoethanol (buffer A), dialyzed against the same buffer overnight, and applied to a column (3 cm2 ~ 15 cm) of DEAEcellulose (DE-52) which had been previously equilibrated with buffer A. After the column was washed with buffer A, SS was eluted with 0.5M KCl in buffer A. After the eluate was concentrated in a collodion bag, the solution was applied to a column (2 cm2 ~ 78 cm) of Sepharose CL-6B which had been equilibrated with 25 mm Tris-HC1 buffer (ph 7.5) containing 1 mm EDTA and 5 mm 2-mercaptoethanol, then eluted with the same buffer. Fractions exhibiting the SS activity were collected and used for the kinetics studies. 4. Enzyme assay The reaction mixture (1.2 ml) used to determined the SS activity contained 15 mm Hepes-KOH buffer (ph 8.5), 2 mm UDPglucose, 15 mm fructose, 5 mm MgCl2 and an adequate volume of sample. The mixtures were incubated at 30 Ž and the reaction terminated at 0 and 20 min with 0.25 ml of 0.4N NaOH. The mixture was boiled in a water bath for 20 mm to destroy unreacted fructose molecule. The sucrose formed was then hydrolyzed by 30% HCl and the resultant ketose was measured colorimetrically with resorcinol (15). The method for the analysis of the SPS activity was identical with that for SS except that fructose-6-phosphate and Hepes-KOH buffer (ph 7.5) were substituted for fructose and Hepes-KOH buffer (ph 8.5), respectively. Furthermore, 1.3 mm NaF was added to the reaction mixture. Data were expressed as micromoles of sucrose or sucrose-p produced per hour. Acid invertase was assayed in 0.5 ml of a reaction mixture containing 75 mm acetate buffer (ph 5.0), 95 mm sucrose and an adequate volume of sample. The mixture was incubated at 30 Ž for 20 min. The amount of reducing sugar produced was determined by the method of Somogyi-Nelson (13). Data were expressed as micromoles of reducing sugar produced per hour. UDPglucose pyrophosphorylase activity was determined by the spectrophotometric measurement of NADPH formation accompanying the enzymecoupled conversion to 6-phosphogluconate through glucose-6-phosphate. Reaction mixture (3 ml) contained 42 mm Tris-HC1 buffer (ph 7.8), 1.5 mm

ROLE OF ENZYMES RELATING TO SUCROSE ACCUMULATION IN PEACH FRUIT 533 PPi, 1 mm UDPglucose, 6.6 mm MgCl2, 0.38 mm NADP, 0.02 mm glucose-1,6-bisphosphate, 6.6 U ml-1 phosphoglucomutase, 930 mu ml-1 glucose-6-phosphate dehydrogenase and the sample. The reaction was started by the addition of NADP, and the NADPH formation at 30 Ž was continuously monitored at 340 nm. Data were expressed as micromoles of glucose-l-phosphate per hour. For the kinetics of SS, the activity for the synthesis of sucrose was assayed by an enzymecoupling method for the production of UDP, according to the procedure reported previously (9). The activity for the cleavage of sucrose was assayed by using excess UDPglucose dehydrogenase for the production of UDPglucose, according to the procedure reported by More11 and Copeland (8). Results 1. Sugar accumulation in fruit flesh In all the cultivars examined, the sucrose content per g fresh weight which was low at the immature stage increased rapidly with fruit maturation, to finally account for 62-88% of total sugars (Fig. 1). In 'Akatsuki' and 'Yuzora', the fructose content was similar to that of glucose, and showed the same pattern of change throughout the fruit development. On the contrary, the fructose content of 'Naganoyaseito-early' and 'Hokimomo' was low throughout the fruit development. These results coincided with those reported previously (11). 2. Changes of sugar content in peduncle In all the cultivars, sorbitol was the main translocating sugar and the content per g fresh weight increased gradually with the fruit development (Fig. 2). Glucose and fructose were also present in various amounts, and large amounts of sucrose was also detected. 3. Changes in SS and SPS activity SS activity which was high in immature fruits, decreased rapidly with fruit development (Figs. 3 and 4). Its activity rose again along with sucrose accumulation, but to a lesser extent in Naganoyaseito-early' compared with `Akatsuki', ' 'Yuzora' and 'Hokimomo'. The SPS activity remained low throughout the development (Figs. 3 and 4). Although the SPS activity in 'Akatsuki', Yuzora' and 'Hokimomo' increased slightly ' at the mature stage, the increase was not significant compared with that of the SS activity. In Naganoyaseito-early', the SPS activity remained ' constant throughout the fruit development. Fig. 1. Seasonal changes in sugar content of peach fruit flesh. œ; sucrose, Ÿ; fructose, ; glucose, ; sorbitol.

534 T. MORIGUCHI. Y. ISHIZAWA, T. SANADA, S. TERAMOTO AND S. YAMAKI 4. Changes in acid invertase activity Acid invertase consisted of two types, bound and soluble. In all the cultivars, the activity of the bound enzyme was roughly equal to that of the soluble one during the immature stage, but the bound enzyme tended to be predominant at the mature stage (Fig. 5). When the data were expressed as the sum of the activity of the two types, the invertase activity was highest at the immature stage, then it rapidly decreased throughout the fruit development. In 'Yuzora' and 'Hokimomo', the acid invertase activity slightly increased at the mature stage and the activity in 'Hokimomo' Fig. 2. Seasonal changes in sugar content of peach fruit peduncle. œ ; sucrose, Ÿ; fructose, ; glucose, ; sorbitol. Fig. 3. Seasonal changes in SS and SPS activity in peach fruit flesh. - œ- ; SS of 'Akatsuki', -- œ--; SPS of 'Akatsuki', - - ; SS of 'Yuzora', -- --; SPS of 'Yuzora'. Fig. 4. Seasonal changes in SS and SPS activity in peach fruit flesh. - - ; SS of 'Naganoyaseitorearly', -- --; of SPS Vaganoyaseito-early', SS of -- --;SPS - -; 'Hokimomo', of 'Hokimomo'.

ROLE OF ENZYMES RELATING TO SUCROSE ACCUMULATION IN PEACH FRUIT 535 decreased again with further development. 5. Changes in UDPglucose pyrophosphorylase activity UDPglocose pyrophosphorylase activity was highest among the enzymes examined (Fig. 6). In all the cultivars, the UDPglucose pyrophosphorylase activity was highest at the immature stage, then it rapidly decreased, and slightly rose again along with fruit maturation except in the case of Naganoyaseito-early'. There were no differences ' in the pattern of activity among the four cultivars at all the developmental stages. 6. Kinetic parameters of partially purified SS from immature peach fruit Kinetic parameters of SS from immature peach fruit 'Hakuto' are investgated. For sucrose synthesis, the Km values were 4.3 and 0.029 mm for fructose and UDPglucose, respectively. For sucrose cleavage, the Km value was 5.3 or 0.03 mm each for sucrose or UDP (Table 1). along with fruit maturaiton. Sucrose accumulation was associated with the increase of the SS activity in all the cultivars. High SS activity at the early developmental stage may be considered as follows: 1) SS may act sucrose cleavage direction rather than sucrose synthesis direction, and the resultant UDPglucose may be used as the precursor of cell wall polysaccharides; 2) Sucrose synthesized by SS reaction may be easy to cleave through high acid invertase activity at this stage. At the mature stage, SS appeared to play an important role in sucrose accumulation in peach fruit, unlike SPS which displayed a weak activity with fluctuations frequently unrelated to the sucrose content. These observations were in agreement with previous results (10). As SPS is a labile enzyme, careful extraction is essential (3). However, when the extraction procedures were compared in particular in the case of desalting methods, including ultrafiltration, Discussion Sucrose was the major sugar that accumulated in all the cultivars, and its content rapidly increased Fig. 6. Seasonal changes in UDP glucose pyrophospherylase activity in peach fruit flesh. œ; 'Akatsuki', ; 'Yuzora' ; 'Naganoyaseito -early', ; 'Hokimomo'. Fig. 5. Seasonal changes in acid invertase activity in peach fruit flesh. - œ-; total acid invertase activity 'Akatsuki', in -- œ--; acid invertase activity in 'Akatsuki' soluble, - - ; Table 1. Kinetic parameters of SS from immature peach fruit cv. 'Hakuto'. total acid invertase activity in 'Yuzora', -- --; soluble acid invertase activity 'Yuzora', acid invertase activity in - -; total 'Naganoyaseito-early', soluble acid invertase in -- --; activity in 'Naganoyaseito-early', - -; total acid invertase activity in 'Hokimomo', -- --; soluble acid invertase activity in 'Hokimomo'.

536 T. MORIGUCHI, Y. ISHIZAWA, T. SANADA, S. TERAMOTO AND S. YAMAKI gel filtration and dialysis, no significant differences were detected in terms of SPS activity among these methods (data not shown). Therefore, the values of SPS activity recorded in these experiments may not result from a loss of activity during the extraction method. It appears that sucrose was synthesized at the mature stage by the reaction of SS in all the peach cultivars studied regardless of the maturation time of fruit and the content of other sugars than sucrose. In the peduncle tissues, the sorbitol content increased gradually with fruit development, and sorbitol was a major sugar at the mature stage. However, sucrose could also be detected to some extent, suggesting that sucrose is also an important translocating sugar in peach. It was assumed that sucrose itself translocated from the phloem may accumulate into the fruit flesh without affecting the fruit metabolism. In peach fruit, the acid invertase was major type and the alkaline invertase was minor one (12). The acid invertase activity which decreased with fruit development appeared to reflect fruit maturation, and facilitate sucrose accumulation. In Asian pear, the acid invertase activity also decreased with fruit maturation in inverse proportion to the sucrose accumulation (18). Similar results were reported in sucrose-accumulating tissues (5, 7, 16, 17). UDPglucose pyrophosphorylase contributes to the synthesis of UDPglucose which is an important glucose donor in the synthesis of many carbohydrates such as sucrose, starch, cell wall polysaccharides and the precursors of the sugar nucleotides (2). The higher activity in immature fruit may reflect the active synthesis of polysaccharides or sugar nucleotides. The resumption of the increase at the mature stage appeared to coincide with the requirement of UDPglucose as the substrate of sucrose synthesis by SS. When the sucrose accumulation pattern was compared between peach and pear fruits, sucrose in peach fruit was mainly synthesized by SS. On the contrary, SPS in peach fruit did not play an important role in sucrose accumulation, although it is assumed to be the major sucrose-synthesizing enzyme (1). On the other hand, in pear fruit which accumulated a large amount of sucrose, both SS and SPS activities increased along with sucrose accumulation. The SS and SPS enzymes appeared to play a major role in sucrose accumulation in pear fruit (unpublished data). Hubbard et al. (3) reported that SPS was also a major determinant of the sucrose content in muskmelon. In citrus fruit, the SS activity was high in the tissues involved in transport (vascular bundles and segment epidermis), while the SPS activity was high in the phloem-free sink tissue (juice sacs) (6). These results suggest that SPS plays an important role in sucrose accumulation in juice sacs. Moreover, Yelle et al. (19) reported that SS was involved in sucrose cleavage in two tomato species which differed in the soluble solids level. Thus, the activity of SS and SPS for sucrose accumulation appears to vary among fruit species. The kinetic parameters of SS purified from mature peach fruit 'Hakuto', indicated a high affinity of the fructose (Km; 4.8 mm) and UDPglucose (Km; 0.033 mm), substrates for sucrose synthesis (9) compared with a low affinity of the sucrose (Km; 62.5 mm). These results show that SS plays an important role in sucrose synthesis and accumulation in peach fruit. In immature peach fruit, the Km value for sucrose (Km; 5.3 mm) was smaller than that in mature peach, whereas the Km value for fructose or UDPglucose in immature peach was similar to that in mature one. These findings suggest that the accumulation of sucrose in immature peach fruit is lower than that in mature peach fruit. Consequently, immature peach fruit accumulated a large amount of hexose. When the kinetic parameters of SS were compared between mature peach and pear fruits, the Km value for sucrose (Km =11.2~16.3 mm) in mature pear fruit was smaller (about 1/4 1/5 of mature peach fruit) than that of mature peach fruit. Sucrose cleavage in pear fruit appeared to occur more readily than in peach fruit, judging from the Km values for sucrose. The differences in the Km values for sucrose observed among mature peach and pear fruits may reflect the differences in the ratio of sucrose and hexose contained in both mature fruits. It was shown that pear fruit contained a large amount of hexose except for sucrose (4, 18), while in peach fruit sucrose was the predominant sugar at the mature stage. To conclude, more detailed studies at the molecular level should be carried out to clarify the differences in the role played by SS and SPS in various fruits.

ROLE OF ENZYMES RELATING TO SUCROSE ACCUMULATION IN PEACH FRUIT 537 Acknowledgments We are grateful to Mr. H. Kyotani and K. Nishimura for their suggestions and to Mrs. S. Hayashi for her technical assistance. Literature Cited 1. Hawker, J.S. 1985. Sucrose. p. 1-51. In: Dey and Dixon (eds.). Biochemistry of storage carbohydrates in green plants. Academic Press, New York. 2. Hondo, T., A. Hara and T. Funaguma. 1983. The purificaiton and some properties of the UDPglucose pyrophosphorylase from pollen of Typha latifolia L. Plant Cell Physiol. 24: 61-69. 3. Hubbard, N.L., S.C. Huber and D.M. Pharr. 1989. Sucrose phosphate synthase and acid invertase as determinants of sucrose concentration in developing muskmelon (Cucumis melo L.) fruits. Plant Physiol. 91 : 1527-1534. 4. Kajiura, I., S. Yamaki, M. Omura, T. Akihama and Y. Machida. 1979. Improvement of sugar content and composition in fruits, and classifications of East Asian pears by the principal component analysis of sugar compositions in fruits. Japan. J. Breed. 29 : 1-12. (In Japanese with English summary). 5. Lingle, S. E. and J. R. Dunlap. 1987. Sucrose metabolism in netted muskmelon fruit during development. Plant Physiol. 84 : 386-389. 6. Lowell, C.A., P.T. Tomlinson and K.E. Koch. 1989. Sucrose-metabolizing enzymes in transport tissues and adjacent sink structures in developing citrus fruit. Plant Physiol. 90: 1394-1402. 7. McCollum, T.G., D.J. Huber and D.J. Cantliffe. 1988. Soluble sugar accumulation and activity of related enzymes during muskmelon fruit development. J. Amer. Soc. Hort. Sci. 113 : 399-403. 8. Morell, M. and L. Copeland. 1985. Sucrose synthase of soybean nodules. Plant Physiol. 78 : 149-154, 9. Moriguchi, T. and S. Yamaki. 1988. Purification and characterization of sucrose synthase from peach (Prunus persica) fruit. Plant Cell Physiol. 29 : 1361-1366. 10. Moriguchi, T., T. Sanada and S. Yamaki. 1990. Seasonal fluctuations of some enzymes relating to sucrose and sorbitol metabolism in peach fruit. J. Amer. Soc. Hort. Sci. 115: 278-281. 11. Moriguchi, T., Y. Ishizawa and T. Sanada. 1990. Differences in sugar composition in Prunus persica fruit and the classificaiton by the principal component analysis. J. Japan. Soc. Hort. Sci. 59 : 307-312. 12. Moriguchi, T., T. Sanada and S. Yamaki. 1991. Properties of acid invertase purified from peach fruits. Phytochemistry 30 : 95-97. 13. Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153 : 375-380. 14. Robinson, N.L., J.D. Hewitt and A.B. Bennett. 1988. Sink metabolism in tomato fruit. I. Developmental changes in carbohydrate metabolizing enzymes. Plant Physiol. 87 : 727-730. 15. Roe, J.H. 1934. A colorimetric method for the determination of fructose in blood and urine. J. Biol. Chem. 107 : 15-22. 16. Schaffer, A.A., B. Aloni and E. Fogelman. 1987. Sucrose metabolism and accumulation in developing fruit of Cucumis. Phytochemistry 26 : 1883-1887. 17. Yamaki, S. and K. Ishikawa. 1986. Roles of four sorbitol related enzymes and invertase in the seasonal alteration of sugar metabolism in apple tissue. J. Amer. Soc. Hort. Sci. 111 : 134-137. 18. Yamaki, S. and T. Moriguchi. 1989. Seasonal fluctuation of sorbitol-related enzymes and inver- Japanese pear (Pyrus serotina Rehder var. cotta Rehder) fruit. J. Japan. Soc. Hort. Sci. 57 : 602-607. 19. Yelle, S., J.D. Hewitt, N.L. Robinson, S. Damon and A.B. Bennett. 1988. Sink metabolism in tomato fruit. III. Analysis of carbohydrate assimilation in a wild species. Plant Physiol. 87 : 737-740.

538 T. MORIGUCHI, Y. ISHIZAWA, T. SANADA, S. TERAMOTO AND S. YAMAKI