JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen A ikakauskirja Vol. 58: 27—32, 1986 45Ca mobility and distribution during ripening and maturation of Rutgers and RIN tomatoes. L.R. HOWARD, 1 A. UUSI-RAUVA and J.J. LAINE 2 University of Arkansas, Department of Food Science, Route 11, Fayetteville, AR, 72703 University of Helsinki, Faculty of Agriculture and Forestry, Instrument Centre, Viikki, SF-00710 Helsinki, Finland University of Helsinki, Department of Food Chemistry and Technology SF-00710 Helsinki, Finland Abstract. Radioisotope 45 Ca was used to examine changes in levels of bound and solu- ble calcium during tomato fruit ripening, and the distribution of calcium in different regions of Rutgers and Rin (non-ripening) tomato tissue. Levels of cell wallmiddle lamella bound 45 Ca decreased readily in pericarp tissue during ripening of Rutgers tomatoes with only a small de- crease being observed in RIN fruit. No significant increase in soluble 45Ca was observed for either genotype during ripening. Decreasing levels of bound and soluble 45 Ca were observed from calyx to blossom end of pericarp tissue in Rutgers and RIN fruits. Low levels of bound 45Ca were found in the inner locular walls at an early stage of tomato ripening. The implica- tion of low levels of cell wall bound calcium in relation to catabolic changes associated with ripening are discussed. Index words: Ca mobility in tomato tissue. Rutgers and RIN tomatoes. Introduction Calcium has been shown to affect many as- pects of fruit ripening. In tomatoes, posthar- vest application of calcium retarded the ripen- ing process of mature green fruit and fruit that had commenced ripening (Wills and Tirma- zi, 1979). The retardation of the ripening process was attributed to calcium inhibition of mitochondrial and pectic enzymes (Wills and Rigney, 1979). Many studies have impli- 1 To whom correspondence should be sent. 2 Present Address; Finnish Meat Research Centre, Box 56, 13101 Hämeenlinna, Finland cated the pectic enzyme polygalacturonase (PG) in softening of tomato tissue (Pressey, 1979; Hobson, 1981; Huber, 1984). Calcium plays a major role in structural integrity of cell walls due to its ability to crosslink pectic sub- stances (Grant et al., 1973; Rees, 1975). In- corporation of calcium into the cell wall re- sults in enhanced tissue resistance to PG, al- though the ecaxt chemical nature of resistance is unresolved (Wills and Tirmazi, 1979; Buescher and Hobson, 1982). Several studies have reported that cell wall bound calcium declines prior to, or simulta- neously with the initiation of ripening 27 https://www.c-info.fi/en/info/?token=PiL_i-hn-IpF5e7U.X4cQrnsJhS3yYbN-Xw9Wkg.wjZ4DLpSoFVw5yHuHBLDs6k2lExQsK1Y0E7Vz_OJwu4tJOOdFTGJYFZSyZDAaykD4QNH0nq5ZS_AAe7y3fWXqFodYj_0usVjzaqYBkNEXY5Vy3NGBRfSxmS4f5ZU9K5fKHonzzNXlSVz4240pQeSYHY_cAhRWPc34XorThrR-O8YPmv1K5b9GbZmOGO6-PZBt12U8WGe5w (Suwaan and Poovaiah, 1978; Poovaiah, 1979; Rigney and Wills, 1981). Rigney and Wills, (1981) have suggested that mobility of cell wall bound calcium might initiate ripening and precede PG activity which has also been postulated to initiate tomato ripening (No and Tigchelaar, 1977; Tigchelaar et al., 1978; Poovaiah and Nukaya, 1979). Studies performed on the nonripening, RIN, tomato mutant lend further support to the scheme, since bound calcium levels in RIN increase throughout maturationand little or no PG ac- tivity develops (Suwann and Poovaiah, 1978; Buescher and Tigchelaar, 1975). Recent studies have indicated possible mech- anisms of calcium removal from cell walls. Buescher and Hobson (1982) demonstrated that calcium removal by chelators such as EDTA or citrate allowed extensive degrada- tion of tomato cell walls by PG. Brady et al. (1985) reported that pectic polymers of a firm and soft tomato cultivar were equally suscep- tible to PG hydrolysis in the presence of cit- rate which also demonstrates a strong role for calcium in limiting degradation by PG. Brady et al. (1985) failed to observe a de- crease in levels of cell wall bound calcium dur- ing ripening, results that conflict with those reported by others (Suwaan and Poovaiah, 1978; Poovaiah, 1979; Rigney and Wills, 1981). Furthermore, Rushing and Huber (1985) were unable to induce ripening in ma- ture green and RIN tomatoes by vacuum in- filtration of various chelating agents. Thus, the exact role of calcium in tomato ripening is still unresolved. In this study, we utilized the radioistope 45Ca as a trace in or- der to follow calcium solubilization and dis- tribution in RIN and Rutgers tomato fruit. Use of radioisotopes in plant nutrition studies is widespread, however, 45 Ca has not been previously used to observe changes in cell wall bound Ca during tomato ripening. Materials and methods Plant Material Tomatoes (Lycopersicon esculentum Mill) cultivars Rutgers and RIN (isogenic to Rut- gers) were grown in sand in a temperature controlled greenhouse (19—23°C), with sup- plemental lighting. The supplimentary light- ning was 250 W/m 2 by Hg-LX-400 mercury lamps and the period was 16 hours/day. The humidity of theroom ranged between 50—70 °7o during the cultivation period. Plants re- ceived a standard nutrient solution daily containing as follows: macronutrients (mEq/1) N 16.2, P 1.8,K 9.9, Ca 8.0, Mg 3.7, 53.8, and micronutrients (ppm): Fe 1.38, Mn 1.38, B 0.28, Zn 0.14, Co 0.14, Mo 0.03. Plants were trained to one stem and fruits were tagged at the time of anthesis. Blossoms were thinned to allow two to three fruits per flower cluster with a maximum of ten fruits per plant. Rutgers fruits were harvested and sorted into the following ripeness classes: ma- ture green (MG), breaker (B), turning (T), pink (p), and red (R) using a U.S.D.A. Visual Aid TM-L-1 (ANON., 1975). Days from an- thesis that corresponded to the color stages of Rutgers were used to obtain fruit of the non- ripening (RIN) mutant at known stages of de- velopment. Stage one fruit corresponding to mature green were harvested 60—63 days af- ter anthesis while stage two fruit correspond- ing to red ripe were harvested 79 —81 days af- ter anthesis. Sample Preparation and Analysis Tomato pericarp tissue including the peel was dissected into three equal portions from the distal to proximal end of the fruit. A fourth sample was taken from the locular walls at the interior of the fruit. Dissected samples were immediately freeze dried and stored at —2O°C. Representative samples were finely ground to pass through a 60 mesh screen. The samples were analyzed for solu- ble and bound 45 Ca by a modified method of 28 Suwaan and Poovaiah (1978). The tissue was leached with distilled water for 1 hr in a cold room (4°C). Samples were centrifuged at 15,000 g for 30 min and the supernatant filter- ed through Whatman No. 541 ashless filter paper. Two additional aliquots of distilled wa- ter were added to each sample followed by centrifugation and filtration. Filtrate was col- lected and brought to final 100 ml volume and used to analyze soluble 45 Ca. The pellet was ashed at 600°C for 6—7 hours, dissolved in 5 % FICI and used to determine bound 45Ca. Isotope Application and Procedure Radioisotope 45Ca, as 45 CaCl2 (2.1. mCi/ ml original activity) was applied at 100 /tCi per plant upon flowering of the first clusters of approximately 80 % of the plants. Samples (1.0 ml) from soluble and bound 45 Ca fractions were placed into vials which contained 10 ml of PCS liquid scintillation cocktail (Amersham). Radioactivity was mea- sured with a liquid scintillation counter (LKB Wallac Rackbeta 1215). Activity was ex- pressed as disentigrations per minute (dpm). All samples were corrected for background and radioactive decay. Statistical Analysis Duplicate determinations of three represent- ative fruit from each maturity stage were ex- amined. Five maturity stages were examined from Rutgers and two stages from RIN. Va- riance analysis with LSD (0.05 level) was used to compare the means. Results and discussion Rutgers and RIN tomatoes both contained a large proportion of 45 Ca bound to cell wall material (Fig. 1). Assumably the majority of bound 45Ca serves as a structural component of the CW-ML complex. Low levels of soluble 45 Ca were observed in both Rutges and RIN tomatoes. In Rutgers, a significant decrease in bound 45Ca of percarp tissue was observed as fruits ripened from mature green to pink. After the pink stage, bound 45 Ca continued to decline. No increase in soluble 45Ca was observed during ripening of Rutgers fruit. Bound 45 Ca in pericarp tissue of RIN fruit decreased from stage one to stage two. These two maturity classifications, based on days from anthesis, correspond to mature green and red Rutgers fruit, respectively. Decreased levels of bound 45 Ca were not accompanied by an increase in soluble 45 Ca. RIN fruits contained greater levels of bound and soluble 45Ca than Rutgers throughout ripening. These results support previous observations that bound calcium in Rutgers tomatoes de- clines during ripening (Suwaan and Poo- Fig. I. Levels of bound and soluble 45 Ca during ripen- ing of Rutgers and aging of RIN tomato fruits. Broken lines indicate RIN, continuous lines, Rut- gers. Open points (o) = bound 45Ca, closed points (•) = soluble 45Ca. Points on each curve followed by the same letter are not significantly different at the 0.05 level (LSD). 29 vaiah, 1978; Poovaiah, 1979; Rigney and Wills, 1979). In contrast to the results of these authors no large increase in soluble cal- cium was observed. Brady et al. (1985) also failed to observe an increase in soluble cal- cium. They attributed lack of calcium solu- bilization to the interaction of soluble calcium with intracellular acids, which resulted in for- mation of insoluble complexes. Possibly a si- milar mechanism occurred in this study or cal- cium was translocated out of the fruit. Stu- dies performed on apples have demonstrated calcium translocation from the fruit during moisture stress (Wilkinson, 1968; Martin, 1969). Martin (1967) demonstrated that 45 Ca injected into apple peel and carpel tissue was retranslocated to the nearby leaves and shoots. A similar study in tomato fruit would be of value in determining the role of calcium trans- location during ripening. Calcium distribution of soluble and bound forms according to tissue location in Rutgers and RIN tomatoes is presented in Table 1. Bound calcium in Rutgers fruits was highest at the calyx and lowest at the blossom end for both mature green and red tomatoes. A simi- lar trend for bound calcium was also observ- ed for RIN tomatoes at corresponding matur- ities. It is apparent that bound calcium is not distributed uniformily within the fruit, pre- sumably due to immobility of the ion in phloem and nonvascular tissue (Bangerth, 1973, Ferguson, 1979). Decreasing levels of soluble 45 Ca from the calyx to blossom end of the fruit were also ob- served in Rutgers and RIN tomatoes. Low lev- els of calcium at the blossom end of the fruit could play a role in promotion of blossom end rot, either through reduction of cellular co- hesion or increased membrane permeability (Bangerth, 1973, Shear, 1975; Simon, 1978). Levels of bound 45 Ca tended to be very low at the interior of both RIN and Rutgers fruit during ripening. Tomato locular and sur- rounding tissues are a rich source of organic acids, particularly citric and malic acids (Ste- vens et al., 1977). These acids could have a two fold effect on calcium removal from cell walls through: 1)displacement of calcium by ionized protons, 2) chelation of calcium by free carboxyl groups. This is of intrest since incipient ripeness of Rutgers fruits is initiated in the interior tissues. Removal of calcium from cell walls in the interior of matur green Rutgers fruit could possibly trigger the chain of events as envisioned by Rigney and Wills (1981). At the very least this could lead to re- duced resistance of the polygalacturonan chain to polygalacturonase. Decreasing levels of bound calcium in the interiorand lower re- gions of Rutgers fruits during ripening corre- sponds with visual observance of color devel- opment during fruint ripening, since ripening commences in the inner locular walls and pro- ceeds upward from blossom to calyx end of the fruit. This trendwas not observed in RIN fruit which fail to ripen. These results demonstrate that a substan- tial reduction in bound calcium of Rutgers to- matoes occurs during ripening. We have also Table 1. 45Calcium Distribution and Incorporation Into Soluble and Bound Forms In Rin and Rutgers Tomatoes. Rutger (MG) (R) RIN (1)* (2) Position x Bound Soluble Bound Soluble Bound Soluble Bound Soluble 1 y49.15a z 4.30 b 25.82 a 4.15 a 63.43 a 9.68 a 53.73 a 10.52 a 2 38.48 b 3.98 b 22.37 a 3.50ab 46.03b 6.97 b38.65 b 8.93ab 3 30.06 c 2.76 b 18.38 b 2,10 b 32.80 c 5.85 b 30.07 c 7.35 b 4 30.52 c 6.47 a 16.48 b 4.36 a 28.09 c 4.70 b 22.28 d 9.52 a w Stage 1 and 2 Rin fruit correspond to mature green (MG) and red (R) Rutgers fruit according to days after anthe- sis. x Positions I—3 refer to apical, middle, and distal regions of pericarp tissue. Position 4 refers to inner locular walls, y Values are expressed as dpm/mg, z Means labeled by the same letter within each column are not significantly different from each other at the 0.05 level (LSD). 30 demonstrated that a decreasing gradient of bound calcium exists from the calyx and pro- ceeds to the blossom end of both RIN and Rutgers tomatoes. It is suggested that during the early ripening stages, a reduction in bound calcium in the interior and lower portions of Rutgers fruits could severely limit the fruit’s resistance to deteriorative changes. References Anon. 1975. Color classification requirements in toma- toes. U.S.D.A. Visual Aid TM-L-1. U.S.D.A., Agric. Marketing Service, The John Henry Company, Lan- sing, ML Bangerth, F. 1973. Investigations upon Ca related phys- iological disorders. Phytopath. Z. 77: 20—37. Brady, C.J., McGlasson, W.8., Pearson, J.A., Meld- rum, S.K. and Kopeliovitch, E. 1985. Interactions between the amount and molecular forms of polyga- lacturonase, calcium, and firmness in tomato fruit. J. Amer. Soc. Hort. Sci. 110: 254—258. Buescher, R.W. and Hobson, G.E. 1982. Role of cal- cium and chelating agents in regulating the degrada- tion of tomato fruit tissue by polygalacturonase. J. Food Biochem. 6: 147—160. Buescher, R.W. and Tigchelaar, E.C. 1975. Pectines- terase, polygalacturonase, Cx-cellulase activities and softening of the rin tomato mutant. Hort Sci. 10: 624—625. Ferguson, LB. 1979. The movement of calcium in non- vascular tissue of plants. Comm, in Soil Sci. and Plant Anal. 10: 217—224. Ferguson, LB. 1984. Calcium in plant senescence and fruit ripening. Plant, Cell and Environ. 7: 477—489. Grant, G.T., Morris, E.R., Rees, P.A., Smith, P.J.C., and Thom, D. 1973. Biological interactions between polysaccharides and divalent cations: the eggbox mod- el. F.E.B.S. Letters. 32: 195—198. Hobson, G.E. 1981. Enzymes and texture changes during ripening. In Recent Advances in the Biochemistry of Fruit and Vegetables, (J. Friend and M.J.C. Rhodes, Eds.), Academic Press, London, pp. 121 130. Huber, P.J. 1984. The role of cell wall hydrolases in fruit softening. Hortic. Rev. 5: 169—219. Martin, D. 1967. 4! Ca movement in apple trees. Exper- iments in Tasmania 1960—1965. Fed. Sta. Rec. C.5.1.R.0. 6: 49—54. Ne, T.J. and Tigchelaar, E.C. 1977. Action of the non- ripening (nor) mutant on fruit ripening of tomato. J. Amer. Soc. Hort. Sci. 102: 504—509, Poovaiah, B.W. 1979. Role of calcium in ripening and Acknowledgement. The senior author would like to thank the Department Head of Food Chemistry and Tech- nology, Dr. P. Koivistoinen for providing funding and facilities to carry out this work while on leave from the University of Arkansas. This investigation was also sup- ported in part by a research grant from Oy Gustav Pau- lig Ab, SF-00810 Helsinki, Finland. Sincere appreciation is also extended to Dr. R. W. Buescher for disucssion and revision of the manuscript. senescence. Commun. Soil Sci. Plant Anal. 10: 83—88. Poovaiah, B.W. and Nukaya, A. 1979. Polygalacturon- ase and cellulase enzymes in the normal Rutgers and mutant rin tomato fruit and their relationship to the respiratory climacteric. Plant Physiol. 64: 534—537. Pressey, R. 1977. Enzymes involved in fruit softening. In Enzymes in Food and Beverage Processing, (Ory R.L. and St. Angelo, A. eds.). A.C.S. Symp. Series No. 47, pp. 172—191. Rigney, C.J. and Wills, R.B.H. 1981. Calcium move- ment, a regulating factor in the initiation of tomato fruit ripening. Hort. Sci. 16: 532—551. Rushing, J.W. and Huber, D.J. 1985. Initiation of to- mato fruit ripening with copper. J. Amer. Soc. Sci. 110: 316—318. Shear, C.B. 1975. Calcium-related disorders of fruits and vegetables. Hort. Sci. 10: 361—365. Simon, E.W. 1978. The symptoms of calcium deficiency in plants. New Phytol. 80: I—ls. Stephens, M.A., Kader, A.A., and Albright-holton, M. 1977. Intercultivar variation in composition of lo- cular and pericarp portions of fresh market tomatoes. J. Amer. Soc. Hort. Sci. 102: 689—692. Suwaan, M.A. and Poovaiah, B.W. 1978. Association between elemental content and fruit ripening in rin and normal tomatoes. Plant Physiol. 61: 883—885. Tigchelaar, E.C., McGlasson, W.B. and Buescher, R.W. 1978. Genetic regulation of tomato fruit ripening. Hort. Sci. 13: 508—573. Wilkinson, B.G. 1968. Mineral composition of apples. IX. Uptake of calcium by the fruit. J. Sci. Ed. Agr. 19: 646—647. Wills, R.B.H. and Rigney, C.J. 1979.Effect of calcium on activity of mitochondria and pectic enzymes isolated from tomato fruits. J. Food Biochem. 3: 103—110. Wills, R.B.H. and Tirmazi, S.I.H. 1979. Effect of cal- cium and other minerals on ripening of tomatoes. Aus- tral. J. Plant Physiol. 6: 221—227. Ms. received November 19, 1985 31 SELOSTUS 45 Ca-isotoopin liikkuvuus ja jakautuminen Rutgers ja RIN-tomaattilajikkeissa kypsymisen aikana L.R. Howard 1 , A. Uusi-Rauva, J.J. Laine 2 University of Arkansas, Department of Food Science, Route 11, Fayetteville, AR, 72703 University of Helsinki, Faculty ofAgriculture and Forestry, Instrument Centre, Viikki, SF-00710 Helsinki, Finland University of Helsinki, Department of Food Chemistry and Technology SF-00710 Helsinki, Finland Radioaktiivista 45Ca-isotooppia käytettiin tutkittaessa sitoutuneen ja liukoisen kalsiumin määrän muutoksia to- maatissa sen kypsyessä sekä kalsiumin jakautumista kyp- syraättöraän tomaatin eri osissa. Tomaattilajikkeeet oli- vat Rutgers ja RIN. Soluseinien keskilevyjen sitoma 45Ca-isotoopin määrä pienentyi voimakkaasti perikarp- pisolukossa Rutgers lajikkeen kypsyessä, kun taas RIN 1 To whom correspondence should be sent. 2 Present Address: Finnish Meat Research Centre, Box, 56, 13101 Hämeenlinna, Finland lajikkeella pienentyminen oli vähäistä. Liukoisen 45Ca- isotoopin määrässä ei havaittu merkittävää lisäystä kum- mankaan lajikkeen kypsymisvaiheessa. Tutkimuksessa ha- vaittiin molemmilla lajikkeilla liukoisen ja sitoutuneen 45Ca-isotoopin määrissä pienentymistä perikarppisolu- kossa verhiöstä kukkapohjaan. Tomaatin kypsymisen var- haisessa vaiheessa todettiin sitoutuneen 45Ca-isotoopin alhainen määrä sisemmissä vaskulaariseinämissä. Tutki- muksessa selvitettiin lisäksi soluseinään sitoutuneen kal- siumin alhaisen määrän vaikutus katabolisiin muutoksiin kypsymisvaiheessa. 32