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The Effect of Calcium on Respiration of Apples1
Miklos Faust and C. B. Shear2
Agricultural Research Service,
U. S. Department o f Agriculture,
Beltsville, Maryland
Abstract. Respiration of apples was inversely related to Ca content of flesh. Respiration increased markedly
if Ca concn was below 110 ppm. High N levels also increased respiration. High Ca successfully counteracted
the N effect and kept the respiration at a low level. When N was supplied as ammonium, respiration was
higher than when a similar quantity of N was supplied as nitrate. Calcium counteracted the increased
respiration induced by ammonium-N. Low Ca fruit lost 30-70% of its capacity to synthetize proteins and
nucleic acids as measured by the incorporation of labelled valine and uracil. Low-Ca fruit was also high in
ethanol insoluble solids, the major portion of which was probably cellulose. The effect of Ca on respiration
can explain the negative relation between certain disorders (internal breakdown and watercore)
characterized by overmaturity and Ca content of fruit.
Calcium appears to have an im portant regulating role in
metabolism of apple fruit. Metabolic disorders such as bitter pit
(6), cork spot (13), internal breakdown (6), lenticel breakdown
(12), watercore (6), and Jonathon spot (3) are all decreased in
severity if Ca is present in the fruit in sufficiently high
quantities. Several of the above disorders are associated with a
high rate of respiration or overmaturity of the fruit. This
suggests that Ca may regulate respiration and perhaps other
matabolic processes in the maturing fruit (4). We are presenting
evidence that Ca is indeed involved in regulating the respiration
of apples.
Materials and Methods
‘York Imperial’ apples were harvested from trees receiving
various nutrition in controlled-nutrient culture. Experimental
trea tments consisted of 2 sources of N (all NO3"and 3/4 NH$ +
1/4 NO3), 2 levels of N (28 and 112 ppm), and 2 levels of Ca
(20 and 320 ppm). All other essential nutrients were supplied at
nutritionally adequate levels. Details of the composition of
solutions are published elsewhere (14).
Apples were harvested about 2 weeks prior to commercial
harvest of this cultivar. Fruit respiration (CO2 production) was
determined for 3 years, 1969, 1970, and 1971. In 1971,
additional information about respiratory pathways was
collected. Since total respiration was similar for all 3 years and
the pattern of respiration was identical for each year, data for 1
year only are presented with the biochemical data collected in
1971.
Total respiration of fruit was determined with an infrared
CO2 analyzer on lots o f 10 apples weighing approx 700 g. The
co n t inu o u s ai r flow through the system was 200
cc/min/container. Apples were placed into the respirometer
immediately after picking, and CO2 evolution was determined
every 50 min for each container. Data obtained 24 hr after
picking are presented here.
For metabolic studies 1 g of flesh and 0.5 g of peel was used.
Tissue was placed in each flask o f an 8-unit radiometric
respirometer (5). To each flask we added 10 ml o f 0.2M
potassium phosphate buffer at pH 5.7 containing 1 pc of
labelled substrate (specific radioactivities were glucose-1 -14c
(G-1-14C), 5.0 mc/mmole; glucose-6-14c (G-6-14C, 4.5
mc/m mole; malate-U-14c, 28 mc/mmole; pyruvate-3-14c, 1.5
mc/mmole; valine-1-14c, 34.2 mc/mmole; and uracil-2-14c, 7.2
mc/mmole). The flasks were stoppered, attached to the res-
pirometer and shaken gently for 2 hr in a mechanical
1 Received for publication December 6, 1971.
^Plant Physiologists, Plant Science Research Division.
water-bath shaker at 25°C. C02-free air was passed through the
flasks at a flow rate of 50 cc/min. Respiratory CO2, trapped in
0.5N NaOH, was precipitated from the alkali with saturated
BaCl2- The BaC03 was collected on glass-fiber filter disks and
counted in a gas-flow scaler.
Following incubation, tissues were rinsed in tap water,
ground in a mortar and made to 10 ml with 80% (v/v) ethanol.
The ethanol extract was filtered and both the ethanol extract
and the ethanol insoluble residue were counted. The total
uptake by the tissue was calculated by adding the activities
obtained from counting BaC03, the ethanol extract, and the
ethanol insoluble residue. The respiratory CO2 and the amount
of label incorporated into ethanol insoluble residue were
calculated as percent of total the ethanol uptake. The C6/C1
ratio was calculated by dividing the percent of activity
recovered as CO2 for G-6-14c by that for G-l-14 c. CO2 was
not collected in the uracil and valine experiment; consequently,
the total uptake was obtained by adding only the 2 fractions,
ethanol and ethanol insoluble residue.
Mineral analysis was done by an emission spectrophotometer.
Details of this analysis are described elsewhere (2). Nitrogen was
determined by the Kjeldahl method.
Results and Discussion
Total respiration of fruit was inversely related to the Ca
concn of the flesh of the fruit. At Ca concn above 110 ppm,
fruit respiration was relatively stable, b ut as concn dropped
below 110 ppm, respiration increased markedly (Fig. 1).
Respiration increased with increased levels of N as either N O^
or NHj bu t only when Ca supply was low. High Ca
counteracted the effect of high N. When N was supplied as NHj,
respiration was higher than with the same level of N supplied as
NOJ. However, Ca concn of fruit from NHjf-treated trees was
lower than that of fruit from trees receiving the same level of N
as NO3 (Table 1).
Peel and flesh tissues of apples metabolized the various
labelled compounds to different degrees. Peel tissue metabolized
more G -6 -l4c, G-1-14C and pyruvate- 3 -14c, but no more
malate-U-14c than did the flesh tissue. The C6/C1 ratio of peel
was considerably lower than that of flesh except for high Ca and
high N (Table 2).
Fle s h tissue from low-Ca fruit metabolized more
malate-U-14c and less G-1-14C and G-6-14C at both N levels
than did tissue from high-Ca fruit. Decarboxylation of pyruvate
was not affected by Ca levels (Table 2).
With the exception of G -l-14c more of all the labelled
compounds tested were incorporated into the ethanol insoluble
residue of the flesh of the high-Ca fruit than of the low Ca fruit
PPM CALCIUM
Fig. 1. Relation of respiration of ‘York Imperial’ apple to the Ca content
of the flesh.
(Table 3). The difference was im portant with uracil-2-14c and
with valine-l-14c. The least difference was observed with
Table 1. E ffect of Ca and N on respiration of fruits of ‘York Imperial’
apples.
Level o f nutrition CO2 evolution Compositio n of flesh
Ca NO3-nh |by w hole fruit NCa
mg/kg/hr % dry wt ppm.
Low low 36.0 .167± .016 93 ± 3
High low 27.4 . 17 8± .018 14 6± 4
Low high 56.5 ,3 24 ± .040 8 6 ± 2
High high 26.6 .340 ± .024 166 ± 5
Low Low 65.2 .23 2± .027 71 ± 1
High low 20.4 .343 ± .029 11 6± 1
Low high 82.5 .5 12 ± .073 60 +9
High high 35.2 .348 ± .024 126± 3
G-1-14C and with pyruvate-3-14c. Level of Ca supply affected
only the incorporation of G-l-l^C into the peel tissue.
The to tal ethanol insoluble residue was higher in both flesh
and peel tissue from those fruit that received low Ca, regardless
of N level (Table 4).
Although the effect of Ca on respiration is only slight at
levels above 110 ppm, the effect is dramatic at lower concn.
Fruit with 90 ppm Ca or less is subject to breakdown soon after
harvest.
Calcium is able to preserve cellular organization (8).
Electronmicroscope studies revealed extensive disintegration of
m ito c o n dr i a , endoplasmic reticulum, and cytoplasmic
membranes in various Ca-deficient plants (8). Increased
respiration in ripening apples is accompanied by cellular
disorganization (1). It also is likely that the reverse may occur;
cellular disorganization induced by Ca insufficiency may be
accompanied by increased respiration.
Letham (9) noted that, P, had an effect similar to that
observed by us for Ca. Phosphorus could effectively counteract
the increased respiration caused by N. He used a similar
explanation to that which we are using for the Ca effect. He
reasoned that P is an important component o f phospholipids
which are insufficient in cell membranes if P is limiting. The
similarity of effects o f Ca and P on respiration is easily
u n d er s tan d ab l e . Calcium interacts with phospholipid
membranes whereas P is an integral part of these membranes (8,
9).
Another notable effect of high Ca is the higher level of
protein and nucleic acid synthesis in high-Ca cells as shown by
incorporation of labelled substrates into the ethanol insoluble
fraction of the tissue. Martin and Lewis (10) suggested that
breakdown in cells may be due to their greater difficulty in
maintaining protein. In the present investigation, one of the
functions of Ca may be to maintain protein synthesis. Thus Ca
may preserve the cellular organization n ot only by preserving
the cell membranes, but also by maintaining the nucleic acid
and protein synthesis.
Calcium n utrition had little effect on the metabolism of
various labelled compounds in the peel, whereas it affected the
metabolism of these compounds in the flesh. Calcium is 3 to 4
times higher in the peel of the apple than in the flesh (7). It is
likely th at Ca concn in the peel is high enough to maintain
normal metabolism even under low-Ca conditions. In fact,
lenticel breakdown, the only Ca-associated disorder th at affects
the skin, develops only under conditions of extremely low Ca.
Cell structure o f low-Ca apples appears to be more rigid.
Such apples crack deeply when exposed to high internal turgor
pressures (12). High cellulose content of these apples may play a
role in the rigidity of cell walls.
It is important for the horticulturist to know whether low-Ca
apples respire more because their overall respiration is higher or
because they mature at a faster rate. We did not have enough
apples to determine respiration on sequential pickings nor could
we carry apples in the respirometer long enough to see the
Table 2. CO2 evolution from various substrates metabolized by flesh and peel tissues o f ‘York Imperial’ apples.2
Glucose
6-l^C
Glucose
1-14C
C6/C1
ratio
Pyruvate
3-14c
Malate
U-14C
Flesh
Low Ca low N .90 ± .1 3.5 ± .7 .25 ±. 07 1.0 3± .34 29.5 ± 7.2
High Ca low N 1.7 5+ .3 6.8± 2.6 .28 ± .02 1.47± .65 2 3.9± 5.8
Low Ca high N 2. 15 ± .2 6.7 ± .9 ,3 2± .01 1.92± .62 30.2± 6.4
High Ca high N 2.90 ± .7 7.5 ±3 .6 .1 4± .01 1.38± .39 26 .7± 5.0
Peel
Low Ca low N 6. 3± 2.1 37.1± 15.7 .17 + 02 5. 92 ± 2.0 0 3 4.2± 9.1
High Ca low N 5.3 + 1.8 38.4 ± 17.9 .15 ±.01 2 .5 0± .62 20 .7± 6.1
Low Ca high N 5.1 ± 2.6 34.9 ± 16.1 . 14 +0 2 3 .94 ± .74 22.8± 3.6
High Ca high N 5 .5 ± 2.2 32.9 ± 12.0 .1 6+0 1 3.44 ± 1.35 29.1 ± 3.4
zFruit in these experiments was taken from trees receiving N O3 nutrition.
Glucose
6-14C
Glucose
1-14C
Pyruvate
3-14C Malate
U-14C
Uracyl
2-l4C Valine
l-l4c
Flesh
Low Ca low N .6 3 ± . 06 .3 6 ± .15 1.5 0 ± .14 ,5 2 ± . 09 ,7 0 ± .17 1.6 8 1 . 1 7
High Ca low N 1.0 6 + .28 .3 3 + . 2 0 1.7 9 + .24 . 67 ± .0 7 1.0 2 ± .0 9 3.3 6 ± .5 4
Low Ca high N .4 0 ± .1 0 .45 ± .2 5 1 .4 8 ± .20 .55 ± .0 1 .9 5 ± .2 2 1.0 0 ± .3 0
High Ca high N .7 3 ± .15 .6 6 ± .1 5 1.9 0 + .25 .6 3 ± . 01 1.0 8 ± .15 1.5 7 ± .5 2
P e e l
Low Ca low N .93 ± .15 .6 6 ^ . 1 1 1 .2 8 ± .4 0 .5 8 ± .02 2.05 ± .47 1 .41 ± ; 0 7
High Ca low N .9 0 ± .1 0 .9 0 ± .2 0 1. 27 ± .4 4 .6 5 ± . 2 0 2. 03 ± .50 3.6 7 ± . 21
Low Ca high N . 86 ± .20 .5 3 ± .1 5 .9 4 ± .2 1 .51 ± . 0 2 1 .6 2 + .51 3.0 5 ± .18
High C ahig hN .9 3 ± .32 .8 0 ± .10 1 .1 7 ± .1 6 .5 3 ± .18 1 .7 7 ± .18 3.2 1 ± .1 8
zFruit in these experiments was taken from trees receiving N O-3 nutrition.
clim a cteric because o f breakdown of low-Ca apples.
Determination of maturity is inaccurate on low-Ca apples
whether it is measured by color change or by pressure test.
Low-Ca apples lose their chlorophyll much before maturity (12)
Table 4. Ethanol insoluble fraction of tissues from ‘York Imperial’ apple
fruit.
Flesh
mg/g
Peel
mg/g
Low Ca low N 53 .9 ± 8.75 149 .2±2 .46
High Ca low N 34.7± 2.68 126.4 ±1 .15
Low Ca high N 77 .5± 4.78 1 60 .4 ±5 .50
High Ca high N 4 6.5 ± 3.25 1 08 .4± 5. 06
and the pressure test is probably influenced by high cellulose
levels of such fruit. Therefore, we do not have positive evidence
on which to base an answer to the above question. However, the
higher rate of decarboxylation of malate-U-14c in low-Ca apples
indicates that these apples were more mature. Malic enzyme
activity usually increased with maturity (11). The increase in
decarboxylation o f malate, however, does not account for the
increase in total respiration of the low-Ca fruit. Lethan (9)
reported that fruit from high-N trees was higher in respiration
throughout the growing season and matured several days earlier
than did fruit o f other treatments. We suspect that low-Ca
apples also have a higher in respiration and mature faster.
However, more data are needed on this point.
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1 4 .
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