Radioisotope 45Ca was used to examine changes in levels of bound and soluble 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 45Ca decreased readily in pericarp tissue during ripening of Rutgers tomatoes with only as mall decrease being observed in RIN fruit. No significant increase in soluble 45Ca was observed for either genotype during ripening. Decreasing levels of bound and soluble 45Ca 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 implication of low levels of cell wall bound calcium in relation to catabolic changes associated with ripening are discussed

A.  Uusi-Rauva

J. J. Laine

L. R.  Howard

Agricultural and Food Science

Directory of Open Access Journals

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
(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 8.93 b
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


45Ca mobility and distribution during ripening and maturation of Rutgers and RIN tomatoes

https://journal.fi/afs/article/download/72217/34013

45Ca mobility and distribution during ripening and maturation of Rutgers and RIN tomatoes

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals