Shin Hiratsuka

Sucrose accumulation and its related enzyme activities in the juice sacs of satsuma mandarin fruit from trees with different crop loads

Abstract Sucrose accumulation and its related enzyme activities in the juice sacs were compared between the fruit from conventional crop load (CCL, leaf–fruit ratio is about 25) and low crop load (LCL, leaf–fruit ratio is more than 50) trees in satsuma mandarin (Citrus unshiu Mark). Sucrose in the juice began to increase in September and the increase continued to harvest (late November) in fruit on trees with both crop loads, but the rate of increase was significantly higher in CCL fruit. Synthetic sucrose synthase (SS, EC 2.4.1.13) increased greatly as sucrose accumulated in the juice sacs, whereas its cleavage activity decreased. In spite of lower accumulation of sucrose in LCL fruit, synthetic SS activity was higher than in CCL fruit. Acid invertase (EC 3.2.1.26) activity, which decreased with fruit development, was significantly higher in LCL fruit than in CCL ones until late October. Thus, sucrose synthesized by SS may be broken through this higher activity of acid invertase in LCL fruit, resulting in repression of sucrose accumulation. When diurnal changes in SS activity in juice sacs were measured under orchard conditions in mid-November, sucrose increase was estimated to be 0.4% per fruit per day. This activity is enough to accumulate sucrose to harvest level (7%) within 20 days. In satsuma mandarin fruit, therefore, sucrose concentration in the juice may be regulated by both synthetic SS and acid invertase activities in the juice sacs, and crop load of the tree may greatly affect sucrose accumulation by controlling these enzyme activities.

Enzyme activity changes during anthocyanin synthesis in 'Olympia' grape berries

Abstract Anthocyanin synthesis began in the skin of ‘Olympia’ ( Vitis labruscana Bailey) grape when the berry growth reached a late stage in the second sigmoid growth-curve. During berry development, shikimate NADP oxidoreductase (SORase, EC.1.1.1.25), phenylalanine ammonia-lyase (PAL, EC.4.3.1.5), and chalcone flavanone isomerase (CHFI, EC.5.5.1.6) were investigated in the skin. These enzyme activities were high during early development of the young berry, but rapidly decreased thereafter, and were activated again when rapid anthocyanin synthesis subsequently occurred. When PAL from the young berry was analyzed by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE), it showed a single activity band with a p I value of approximately 5.4, whereas PAL from the ripe berry showed two activity bands with p I s of 5.4 and 6.5. Thus, SORase, PAL, and CHFI seem to be involved in anthocyanin accumulation simultaneously, and newly synthesized isozyme of PAL may play a key role in pigment formation in ‘Olympia’ skin. The function of these enzyme activities in young berry is also discussed in this text.

Comparison of an S-protein expression between self-compatible and -Incompatible Japanese pear cultivars

Abstract An S4-allele-associated protein (S4-protein) was identified by both isoelectricfocusing (IEF) polyacrylamide-gel electrophoresis and N-terminal amino-acid sequences in self-incompatible Nijisseiki (self-in- compatibility genotype=S2S4) and self-compatible Osa-Nijisseiki (S2S4SM, SM=stylar-part mutant) styles of the Japanese pear, Pyrus serotina Rehd. var. culta Rehd. Expression of the protein in the cultivars was compared during flower bud development and at post-transcriptional levels.In mature styles, S4-protein could be detected on IEF gel in both cultivars and the N-terminal amino acid sequences were identical, although Osa-Nijisseiki contained only one-third the amount contained in Nijisseiki. No difference was observed in S2-protien amounts. In Nijisseiki styles, S4-protein was already detectable 8 days before anthesis (DBA) and it was synthesized consistently until 2 days after anthesis (DAA); the amount increased 4.7-fold during these 10 days. In contrast, S4-protein in Osa-Nijisseiki was not detected earlier than 6 DBA; a small amount was found at 4 DBA, and it increased gradually as flowers developed. Thus, expression of Osa-Nijisseiki S4-protein is developmentally controlled in the same way as that of Nijisseiki S4-protein, but with a time lag of several days; the protein level at 2 DAA corresponded to that of Nijisseiki earlier than 4 DBA. S4-proteins from both Nijisseiki and Osa- Nijisseiki showed RNase activity and the activity was also developmentally controlled; it increased about fourfold during the interval from 8 DBA to 2 DAA in Nijisseiki, and 3.3-fold during the interval from 4 DBA to 2 DAA in Osa-Nijisseiki. Activity at 2 DAA, however, was twice as high in Nijisseiki. In vitro protein synthesis showed that poly(A)+ from mature Osa-Nijisseiki styles could form S4-like protein in a manner similar to that of Nijisseiki. These results suggest that the self-compatibility of Osa-Nijisseiki is due to a low level of S4-protein expression, a mechanism very similar to the low level of S-protein and weak incompatibility in immature styles of self-incompatible Nijisseiki. Part of this protein repression may be regulated during post-transcriptional events.

Characterization of an S-allele associated protein in Japanese pear

SummaryThis paper describes some characteristics of a stylar protein associated with the S2 self-incompatibility allele (S2-protein) in Japanese pear reported earlier (Hiratsuka, 1992). The term ‘style’ refers to style plus stigma in this paper unless indicated otherwise.The S2-protein, which is a relatively major component of styles with a pI of 6.5, was present only in the style, and the stigmatic zone involved the protein much higher in quantity than upper half of the style, followed by lower half. Molecular weight was assumed to be 24,000 judged from migration distance in sodium dodecyl sulfate (SDS) polyacrylamide gel. Immature styles from 8 to 4 days before anthesis also contained the protein though the amount was relatively small. Neither the heat treatment of prepollinated styles nor the pollination (compatible or incompatible) altered the pI value and staining concentration of S2-protein in the gel. The protein did not have so strong ribonuclease (RNase) activity as reported in S-proteins of Nicotiana alata (McClure et al., 1989) and the RNase activities of extractable stylar proteins from self-incompatible strains were almost the same as those from self-compatible strains.

Incompatible pollen tube growth and protein composition in styles of Japanese pear following high temperature treatments

SummaryImmersing the excised-prepollinated styles of self-incompatible Japanese pear, Pyrus serotina Rehd., into distilled water at 45°C for 1.5–2.5 min promoted the growth of self-pollen tubes; both the number of styles penetrating the pollen tubes through styles to cut ends and of pollen tubes protruding from cut ends were increased. The subsequent growth of the protruded pollen tubes on agar media was also enhanced by the treatments. Treatments at 40°C did not promote the growth of self-pollen tubes and those at 50°C led the styles to necrosis. In the stylar proteins analyzed by isoelectricfocusing polyacrylamide gel electrophoresis, several changes in banding pattern were found following the heat treatment; one of the bands was a glycoprotein that increases in quantity in developing styles as self-incompatibility of the style becomes stronger (Hiratsuka et al., 1986). This protein band was stained more weakly by Coomassie Brilliant Blue-G250 dye. Antigenic substances in the styles examined by immunodiffusion seemed not to be denatured by high temperature treatments.

Detection and inheritance of a stylar protein associated with a self-incompatibility genotype of Japanese pear

SummaryStylar proteins in 6 cultivars of Japanese pear (Pyrus serotina Rehd.) with different self-incompatibility alleles were examined by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE). Cultivars used involved 1 self-compatible mutant ‘Osa-Nijisseiki’ obtained from ‘Nijisseiki’ (S2S4). Banding patterns of proteins were almost the same among the 6 cultivars except for 3 bands; one of which associated clearly with self-incompatibility S2-allele (hereafter the protein referred to as S2-protein) and was easily detectable because of its large amount and its pI value differed from many other proteins. Two other bands were seemingly associated with S3- and S4-alleles, respectively. The S2-protein was apparently present in the styles of ‘Osa-Nijisseiki’.The inheritance of S2-protein was studied by using 15 selfed plants of ‘Osa-Nijisseiki’. Eight out of 15 plants possessed the S2-protein as a major component of the stylar proteins and 3 plants as a minor component. The remainder 4 plants did not have the protein. This result may support that the S2-protein is the actual S2-gene product.

Fruit photosynthesis in Satsuma mandarin

To clarify detailed characteristics of fruit photosynthesis, possible gas exchange pathway and photosynthetic response to different environments were investigated in Satsuma mandarin (Citrus unshiu). About 300 mm(-2) stomata were present on fruit surface during young stages (∼10-30 mm diameter fruit) and each stoma increased in size until approximately 88 days after full bloom (DAFB), while the stomata collapsed steadily thereafter; more than 50% stomata deformed at 153 DAFB. The transpiration rate of the fruit appeared to match with stoma development and its intactness rather than the density. Gross photosynthetic rate of the rind increased gradually with increasing CO2 up to 500 ppm but decreased at higher concentrations, which may resemble C4 photosynthesis. In contrast, leaf photosynthesis increased constantly with CO2 increment. Although both fruit and leaf photosynthesis were accelerated by rising photosynthetic photon flux density (PPFD), fruit photosynthesis was greater under considerably lower PPFD from 13.5 to 68 μmolm(-2)s(-1). Thus, Satsuma mandarin fruit appears to incorporate CO2 through fully developed and non-collapsed stomata, and subject it to fruit photosynthesis, which may be characterized as intermediate status among C3, C4 and shade plant photosynthesis. The device of fruit photosynthesis may develop differently from its leaf to capture CO2 efficiently.