Fruit Photosynthesis and Phosphoenolpyruvate Carboxylase Activity As Affected by Lightproof Fruit Bagging in Satsuma Mandarin
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J. AMER.SOC.HORT.SCI. 137(4):215–220. 2012. Fruit Photosynthesis and Phosphoenolpyruvate Carboxylase Activity as Affected by Lightproof Fruit Bagging in Satsuma Mandarin Shin Hiratsuka1, Yuka Yokoyama, Hiroshi Nishimura, Takayuki Miyazaki, and Kazuyoshi Nada Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan ADDITIONAL INDEX WORDS. chlorophyll, rind, sugar content ABSTRACT. To clarify why fruit bagging reduces sugar content at harvest, we investigated its effect on carbon dioxide assimilation by Satsuma mandarin (Citrus unshiu) fruit through photosynthesis and phosphoenolpyruvate carboxylase –2 –1 (PEPC; enzyme code 4.1.1.31). Seasonal changes in gross photosynthesis ranged from 70 to 400 mmolÁd Áh O2 with a peak at 99 days after full bloom (DAFB) when the assimilation rate of fruit was comparable to that of leaves. However, a peak showing net photosynthesis appeared at 112 DAFB because of high fruit respiration. When fruit were bagged at 85 DAFB, the net photosynthetic peak disappeared, perhaps as a result of the decline in chlorophyll content in the rind. Sugar and organic acid content in the bagged fruit were 0.3% and 0.16% less, respectively, than controls at the mature stage (204 DAFB). PEPC activity in the rind was much higher than in leaves on a protein basis; it increased between 92 and 112 DAFB and showed a peak of 72 units. The PEPC activity peak was also 90% of control after fruit bagging. Thus, just before their color development, mandarin fruit assimilate CO2 actively through photosynthesis and PEPC. However, these activities are inhibited by bagging, likely resulting in lower sugar content at harvest. The concomitant activation of PEPC and photosynthesis between 99 and 126 DAFB indicates that CO2 fixed by PEPC might be used for photosynthesis in mandarin fruit, because photosynthesis in several fruit such as apple (Malus pumila) and pea (Pisum sativum) is considered to have an intermediate status among C3, non-autotrophic tissue, and C4/CAM photosynthesis. 14 In Japanese commercial fruit tree orchards, young fruit often Jones, 1981), and in citrus (Citrus sp.) fruit, CO2 assimilation are covered with paper bags to prevent fungal, insect, and by the fruit and distribution among the fruit tissues have been physical damage and to promote color development on the fruit well studied. In grapefruit (Citrus paradisi), the 14C in the juice skin. Fruit bagging also seems to be useful for reducing the use of tissue was significantly higher when fruit were incubated in the pesticides. However, bagging usually lowers sugar content at dark than in the light between 2 and 6.5 months after anthesis, harvest (Arakawa et al., 1994; Huang et al., 2009; Proctor and and little 14C-photosynthate was detectable (Yen and Koch, 14 Lougheed, 1976; Watanabe et al., 2011). This phenomenon 1990). Bean and Todd (1960) proved CO2 uptake by young generally is considered to be caused by inhibiting fruit photosyn- ‘Valencia’ orange (Citrus sinensis) fruit; incorporated 14C was thesis by shading, weakening fruit sink strength resulting from distributed into sugars, amino acids, and organic acids in rind lower light and increased temperature and moisture in the bag, or tissues under light conditions, but not into sugars in the juice both. However, little supportive experimental evidence has been sacs. In experiments determining the role of fruit CO2 assimi- provided. lation in citric acid accumulation of Citrus natsudaidai juice Photosynthesis occurs in fruit of many crop plants. Photo- sacs, the level of juice 14C was also greater in fruit placed into the synthetic activity of coffee fruit (Coffea arabica)seemsto dark than the light (Akao and Tsukahara, 1979). Thus, CO2 fixed contribute 20% to 30% of the total photosynthesis of the tree by photosynthesis in fruit does not seem to be accumulated in the (Lopez et al., 2000). Pods of pea and soybean (Glycine max) juice, although carbon assimilated by PEPC would be stored as photosynthesize in two distinct layers; the outer layer, comprising organic acids and/or amino acids in citrus. chlorenchyma of the mesocarp, captures CO2 from the outside CO2 assimilation by fruit PEPC has been well studied atmosphere and the inner layer, a chloroplast-containing epider- (Diakou et al., 2000, Moing et al., 2000, Munoz et al., 2001), mis lining the pod gas cavity, reassimilates the CO2 released and a possible role for PEPC in organic acid accumulation from respiring seeds in the pod cavity (Atkins et al., 1977; in the juice was reported in peach (Prunus sp.) (Moing et al., Quebedeaux and Chollet, 1975). However, in fleshy fruit, the 2000), although other metabolic factors such as vacuolar storage subepidermal layers contain chlorophyll during the early de- ability also were linked to organic acid accumulation (Moing velopmental stages, and photosynthesis is confirmed in some et al., 1999). The pods of pea and soybean possess higher PEPC plant species. CO2 exchange through the epidermis has been activity than the leaves, and pod PEPC seems to participate in best documented in young apple fruit (Blanke and Lenz, 1989; CO2 assimilation (Atkins et al., 1977; Quebedeaux and Chollet, 1975). However, there is little information on the physiological role of fruit PEPC in sugar accumulation of mature fruit. Received for publication 31 Jan. 2012. Accepted for publication 24 May 2012. According to Blanke and Lenz (1989), fruit have an intermediate This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 19658013 status between C3 and C4/CAM photosynthesis, and thus fixation and 23658029). of CO2 by PEPC might contribute to sugar accumulation in fruit. 1Corresponding author. E-mail: [email protected]. Recently, Walker et al. (2011) suggested that organic acids J. AMER.SOC.HORT.SCI. 137(4):215–220. 2012. 215 dissimilated through phosphoenolpyruvate carboxykinase using a glass homogenizer. The homogenate was centrifuged at (PEPCK) are used for gluconeogenesis in cherry (Prunus avium) 20,000 gn for 10 min and the supernatant was passed through a fruit. Sephadex G-25 (Pharmacia, Uppsala, Sweden) column to remove The present study was conducted to verify the lightproof fruit polyphenols. The protein fraction gathered was saturated with bagging effect on sugar concentration at harvest in Satsuma 80% ammonium sulfate and the protein was collected by mandarin. Then, the effect of fruit bagging on fruit photosynthesis centrifugingat 20,000 gn for 10 min. The sediment was redis- and PEPC activity was characterized during fruit development. solved in a small amount of the buffer and dialyzed against the same buffer overnight. All procedures were carried out below Materials and Methods 4 °C. The protein concentration of the sample was determined according to the method of Bradford (1976) and the samples PLANT MATERIALS. Three adult trees of Satsuma mandarin (cv. were stored at –80 °C until use. Okitsuwase) were used at an experimental farm of Mie PEPC activity was determined as NADH oxidation using University, Tsu, Mie, Japan. The 90 fruit (30 fruit per tree) were a coupling reaction with malate dehydrogenase (MDH) (Kumar covered with paper bags (18 cm long · 14 cm wide; Kobayashi et al., 1989). The reaction mixture consisted of 100 mg protein, Bag Mfg. Co., Iida, Japan) coated black inside, which cut the 20 units MDH (Wako Chemical, Osaka, Japan), 1.5 mM NADH, transmitted light more than 99%, at 85 d after full bloom. This bag 1mM dithiothreitol, 10 mM NaHCO3, and 5 mM MgCl2 in 0.05 has been used for improving fruit coloration of peach in practical M Tris-HCl buffer (pH 7.8). The total volume of the mixture culture of Japan. Nine fruit from three trees (three fruit per tree) was 1.4 mL. The reaction was started by adding phosphoenol- were sampled at 92, 99, 112, 126, 140, and 161 DAFB and used pyruvate (PEP) at a final concentration of 2.3 mM; the reaction for determination of chlorophyll content, photosynthesis and was allowed to stand for 2 h at 25 °C and the decrease in OD respiration rate, and PEPC activity. Nine unbagged fruit from 340 nm was recorded. As a control, buffer without any protein three trees (three fruit per tree) were used as controls for each date. was used. Before use, the buffer had been bubbled with N2 gas To determine the effect of bagging on sugar and acid content for at least 1 h and stored in a desiccator containing 2 M NaOH. in mature fruit, 15 bagged fruit and 15 unbagged fruit were The experiment was repeated three times. Enzyme activity was sampled randomly from the canopy surface of the same trees expressed in units (1 unit = 1 mmol NADH oxidation per milligram (five fruit per tree) at 204 DAFB. protein per hour). DETERMINATION OF CHLOROPHYLL CONTENT IN THE RIND. For DETERMINATION OF SUGAR AND ACID CONTENT. At fruit chlorophyll determination, 15 rind discs (4 mm diameter) were maturation (204 DAFB), 15 fruit each were sampled from prepared from three orientations of five fruit using a cork borer, bagged and unbagged treatments as described previously, and and albedo tissue was removed by a razor blade. The five some juice was squeezed from each fruit. Then, the sugar content randomly selected discs were homogenized in a glass homoge- was determined by a refractometer (Atago, Tokyo, Japan) and nizer containing 80% acetone at 0 °C. After centrifugation at the organic acid content by titration with 0.1 M NaOH. Based on 15,000 gn for 5 min, the supernatant volume was adjusted to 3 mL the titration value, the citric acid content was calculated (1 mL with 80% acetone. Based on the method of Arnon (1949), of 0.1 M NaOH = 6.4 mg citric acid).