Article

pubs.acs.org/JAFC

Potential Use of a Weak Ethylene Receptor Mutant, Sletr12, as

Breeding Material To Extend Fruit Shelf Life of Tomato
Syariful Mubarok,, Yoshihiro Okabe, Naoya Fukuda, Tohru Ariizumi, and Hiroshi Ezura*,

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 305-8572 Japan
Department of Agronomy, Faculty of Agriculture, Padjadjaran University, Bandung, 45363 Indonesia

ABSTRACT: Mutations in the ethylene receptor gene (SlETR1), Sletr1-1 and Sletr1-2, are eective in reducing ethylene
sensitivity and improving fruit shelf life. In this study the eect of Sletr1-1 and Sletr1-2 mutations was investigated in F1 hybrid
lines. These two mutants and control were crossed with four commercial pure-line tomatoes. The Sletr1-1 mutation showed
undesirable pleiotropic eects in the F1 hybrid lines. The Sletr1-2 mutation was eective in improving fruit shelf life of F1 hybrid
lines for 45 days longer. It was also eective in improving fruit rmness without change in fruit size, ethylene production,
respiration rate, and total soluble solids or a great reduction in fruit color, lycopene, and -carotene, although the titratable acidity
was increased by Sletr1-2 mutation. These results indicate that the Sletr1-2 mutant allele has the potential to improve fruit shelf
life via incorporation in tomato breeding programs.
KEYWORDS: ethylene, fruit shelf life, Sletr1-1, Sletr1-2, tomato

INTRODUCTION
Tomato (Solanum lycopersicum L.) is a popular eshy fruit that
is grown worldwide on an estimated total of 4.98 million
hectares of land.1 The qualities of the tomato are important
whether the tomato fruit is sold on the market or to industry.
Such qualities determine whether the fruit is acceptable to
consumers and inuence the price/value of the fruit on the
market. The tomato fruit must have high-quality attributes of
appearance, performance, texture, avor, nutritional value, and
safety.2 Furthermore, the shelf life of the fruit is an important
characteristic for commercial markets.3
Breeding to extend the shelf life of tomato fruit extends not
only its storage time but also its marketability. Because tomato
is a climacteric fruit, shelf life is commonly aected by the
presence of ethylene. Ethylene accelerates fruit ripening, which
contributes to changes in the nutrient content and other
compounds; however, it also accelerates quality deterioration
by shortening the shelf life of the fruit. Ripening is a normal
process during fruit maturation. Several biochemical and
physiological changes occur, such as those involving avor,
color, aroma, and texture, and there are also increases in
ethylene production and respiration.4 The color change from
green to red in tomato fruits is an indication of the conversion
of chloroplasts to chromoplasts in which chlorophyll is
degraded and carotenoids accumulate. Fruit softening and
structural alterations are other physiological changes that occur
during fruit maturation; such changes are modied and partially
disassembled by enzymes.5
Most farmers harvest tomato fruit at either the mature green
or breaker stage to minimize damage and to enhance shelf life
during handling and marketing.6 However, dierent stages of
maturation result in lower homogeneity, which contributes to
decreases in fruit quality and market acceptability. The use of
an ethylene-insensitive tomato cultivar can allow farmers to
avoid harvesting fruit at dierent maturation stages, especially
at the mature green and/or immature green stages; instead, all
2015 American Chemical Society

of the fruit can be harvested at the red stage and still have a
long shelf life. Several ripening mutants conferring long shelf
life have been isolated, such as ripening-inhibitor (rin),
nonripening (nor), never ripe (NR), and alcobaca (alc).7
Recently, other tomato mutants that are capable of delaying
the normal process of ripening have been generated from the
Micro-Tom library, including Sletr1-1 and Sletr1-2. Compared
with the wild type, Sletr1-1 and Sletr1-2 mutants show lower
ethylene sensitivities, including completely and moderately
ethylene-insensitive phenotypes, respectively.8
Dierent locations of mutations in ethylene-related genes
correlate with sensitivity to ethylene. In Arabidopsis and tomato,
mutations in the transmembrane domain region of the ethylene
receptor gene dramatically aect ethylene sensitivity.9,10 In
Sletr1-1 and Sletr1-2, the mutations are located in a dierent
transmembrane region of the ethylene receptor. P51L for
Sletr1-1 and V69D for Sletr1-2 are located in the rst and
second domains of the transmembrane region, respectively.8
The ethylene inhibition mutation has a positive eect on
delaying the ripening process, as well as undesirable roles in
other fruit quality characteristics. For instance, in homozygous
mutants of Nr, nor, Sletr1-1, and Sletr1-2, the shelf life of the
fruit is extended; however, the carotenoid content is reduced,
except in Sletr1-2.8,11 The use of heterozygous ethyleneinsensitive mutants can contribute to the agronomic improvement of tomato shelf life but results in a slight reduction of the
red pigment. In nor mutants, the heterozygous condition delays
the start of ripening and increases the interval between the
breaker and the table-ripe stages.12 In rin mutants, the
heterozygous condition delays initiation, and ripening proceeds
more slowly than normal ripening,12 whereas heterozygous
Received:
Revised:
Accepted:
Published:
7995

January 29, 2015

July 22, 2015
July 24, 2015
July 24, 2015
DOI: 10.1021/acs.jafc.5b02742
J. Agric. Food Chem. 2015, 63, 79958007

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Journal of Agricultural and Food Chemistry

Sletr1-1 exhibits impaired petal abscission.8 In the ripening
process, heterozygous Nr mutants have a more pronounced
impairment than Sletr1-1 and Sletr1-2, so those mutants are
more suitable than Nr for use in breeding programs for the
development of a long-shelf life tomato. Additionally, Sletr1-2
showed prolonged fruit shelf life without a reduction in
carotenoid content.8
Crossing commercial tomato cultivars with ripening mutants
that have prolonged fruit shelf life, rin, Sletr1-1, and Sletr1-2,
may facilitate the development of an F1 hybrid line that has the
desired characteristic. Several studies have shown that F1
hybrid lines of rin exhibited prolonged shelf life.13 Hybrids of
the nor mutant locus have an extended shelf life and
intermediate fruit color but no dierences in pH, soluble solids
content, or fruit size.12 However, not all ethylene-insensitive
mutants can be used for improving the postharvest characteristics of tomato in breeding programs; for instance, Nr has an
incomplete ripening phenotype, even in the heterozygous form
(Nr/nr), and also has insucient red coloration.7
High-quality tomato fruit, such as fruit with a long shelf life
and high nutrient content, is attractive for consumers.
Generating new F1 hybrid lines from Sletr1-1 and Sletr1-2 by
crossing these mutants with large commercial tomato cultivars
will improve tomato fruit quality by combining the prolonged
shelf life of these mutants with other desired characteristics
from commercial tomato cultivars. The objective of this study
was to evaluate the eect of the Sletr1-1 and Sletr1-2 mutation
on the plant development and fruit quality characteristics of F1
hybrid lines of Sletr1-1 and Sletr1-2 crossed with four
commercial pure-line tomato cultivars, Aichi First, Ailsa
Craig, Moneymaker, and M82.

The seedling triple response was investigated by measuring the

hypocotyl and root lengths.
Fruit Characteristic Analysis. The harvested fruits were evaluated
to elucidate fruit characterization. All of the tomato fruits were
harvested at 6 days after the breaker stage (Br+6), which was
characterized by >60% and <90% of the fruit surface having changed
to a red color or when the fruit was at the light red stage. The
harvesting time was initiated as 0 days post-storage (DPS) and was
used as the initial time of fruit storage for the shelf life analysis. The
evaluated characteristics were fruit diameter, length, weight, locule
number, and pericarp thickness.
Evaluation of Fruit Shelf Life. The Br date was tagged to
determine a fruit-harvesting time and the stage of fruit maturation.
Investigated fruits were stored on the laboratory bench under the
following conditions: temperature, 20 2 C; humidity, 80%. To
evaluate shelf life, fruits were harvested at the same maturation stage
(Br+6), which was initiated at 0 DPS when the harvested fruit was at
the light red stage and its fruits would be acceptable to consumers in
the market. The shelf life was determined by counting the days from
the beginning of storage until the fruit quality was lost, which was
indicated by the appearance of black spots or wrinkling of >10% of the
fruit surface.
Determination of Ethylene Production and Respiration
Rate. To elucidate the ethylene production and respiration rate in
F1 hybrid lines of Sletr1-2, fruits at the same stage of maturation (Br
+6) were analyzed. The analysis was repeated every 4 DPS for a total
of 12 days. The harvested fruits were kept for 23 h before
measurement to minimize the contamination of ethylene production
from the wound caused by harvesting the fruit from the stalk. Two
fruits were stored in a 440 mL sealed plastic chamber at 25 2 C for
60 min. A 1 mL gas sample was taken from the headspace of the
chamber using a syringe to determine ethylene production and
respiration rate. For ethylene production, the gas sample was analyzed
using a gas chromatograph (GC-18A; Shimadzu, Kyoto, Japan)
equipped with a Porapak Q (mesh 60/80) column and ame
ionization detector, with helium (He) as the carrier gas and injector,
cooling, and detector temperatures of 100, 130, and 100 C,
respectively. The respiration rate was analyzed using a gas chromatograph (GC-8A; Shimadzu, Kyoto, Japan) equipped with a photoionization detector, with He as the carrier gas and 180 and 70 C
detector/injector temperature and column temperature, respectively.
Ethylene production and respiration rate were represented as
micrograms per gram of fresh weight per hour (g/g FW/h).
Fruit Color Analysis. Fruit color was evaluated to assess the eects
of the mutations in the SlETR1 gene of the Sletr1-2 allele on color
during the ripening process. Fruit color was evaluated during storage
and every 10 days until fruit quality was lost, to a maximum of 30 DPS.
The measurement of fruit color was performed according to the
CIELAB color space analysis. Color measurements were taken from
three points on the surface of individual fruits (the middle, base, and
ends of the fruit) using a Minolta Color Reader CR-10 (Konica
Minolta Sensing, Inc., Osaka, Japan). Three color parameters were
represented as the lightness of the color/brightness (L*), the red to
green scale (a*), and the yellow to blue scale (b*). a* and b* were
used to measure the hue (h), which indicates the pure spectrum
colors or the gradation of color, using the following equation:

MATERIALS AND METHODS

Plant Materials and Cultivation. Two ethylene receptor

(SlETR1) mutant alleles, Sletr1-1 and Sletr1-2, and the wild-type
Micro-Tom (WT-MT) were crossed with four pure-line cultivars
(Aichi First, Ailsa Craig, Moneymaker, and M82). Tested plants
were cultivated with four biological replications using the nutrient lm
technique (NFT) cultivation system in the greenhouse of the
Agricultural and Forestry Research Center, University of Tsukuba,
Japan, during the winter season from October 2013 to February 2014.
At the beginning of the plants cultivation, seeds were sown in paper
pots 10 cm in diameter lled with commercial coir coco peat as a
growing medium and were fertilized with a commercial nutrient
solution (OAT house A, OAT Agrio Co., Ltd., Tokyo, Japan) that had
an electrical conductivity (EC) level of 11.2 dS/m. After plants
produced 56 true leaves, they were transplanted into the NFT
cultivation system and irrigated with a commercial nutrient solution
(OAT house A) with an EC level of 22.5 dS/m. Four trusses per
plant were maintained for obtaining a test-fruit sample. When the
fruits were showing a change of <10% in the surface fruit color from
green to yellow, pink, or red, they were tagged as being at the breaker
stage (Br). Some parameters were investigated during plant growth
and development, for example, number of leaves under the rst truss,
time to Br, number of owers per truss, and number of fruits per truss.
Ethylene Triple Response. To investigate the ethylene sensitivity
of the seedlings, we investigated all of the F1 hybrid lines via the
addition of exogenous ethylene at dierent concentrations (0, 0.1, 1,
2.5, and 5 ppm). Seeds were sterilized for 20 min by being soaked in
10% commercial bleach including a detergent (Kitchen Haiter, Kao,
Tokyo, Japan) and then rinsed with sterilized water three times for 5
min. Three sterilized seeds were grown in a 50 mL glass bottle
containing 10 mL of 1/2 Murashige and Skoog (MS) and sealed with
rubber septum caps.8,14 Using a syringe, exogenous ethylene at desired
concentrations was injected into the sealed glass bottles containing
seeds. The seeds were grown and kept in the dark at 25 C for 7 days.

b
h = arctan
a
An increased L* value indicated that the fruit had more brightness, and
a decreased h indicated that the fruit color had changed to red.
Carotenoid Analysis. Because tomato contains high levels of
carotenoids, especially lycopene and -carotene, it has become popular
for human consumption. The lycopene and -carotene contents were
analyzed every 10 days from the beginning of storage until fruit quality
was lost, to a maximum of 30 DPS. Frozen fruit was ground into a ne
powder in liquid nitrogen, and 300 mg of tomato powder was
extracted with 3 mL of acetone/hexane (4:6 v/v), resulting in a clear
supernatant as described by Nagata and Yamashita.15 Absorption was
measured using a spectrophotometer (Beckman Coulter DU 640
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Figure 1. Seedling responses of WT-MT F1, Sletr1-1 F1, and Sletr1-2 F1 hybrid lines and pure-line commercial cultivars to exogenous ethylene
exposure. (A) Seedling phenotypes of WT-MT, Sletr1-1, and Sletr1-2 crossed with Ailsa Craig as an ethylene response at dierent concentrations
(05 ppm). Bar = 1 cm. The seedling quantications are represented by (B) hypocotyl length and (C) root length of pure-line cultivar parents, WTMT F1, Sletr1-1 F1, and Sletr1-2 F1, on each pure-line cultivar background, Aichi First, Ailsa Craig, Moneymaker, and M82. Mean values with
the same storage time tags followed by the same letters are not signicantly dierent according to the TukeyKramer test at p < 0.05.
spectrophotometer, Fullerton, CA, USA) at absorbances of 663 (A663),
645 (A645), 505 (A505), and 453 nm (A453). Lycopene (CLYC) and carotene (CCAR) were measured using the following equations:

titration of 0.1 N sodium hydroxide up to a pH of 8.1 as described by

Dalal et al.16
Data Analysis. This experiment was conducted in a completely
randomized design with four replicates. For statistical data analysis, to
test the normality of the data we used the KolmogorovSmirnov test.
On the basis of this test, we found the data to have normal
distribution. Therefore, one factor analysis of variance (ANOVA) was
conducted to analyze the data followed by the TukeyKramer test at p
< 0.05 to compare dierences among investigated lines.

C LYC = 0.04584(A 663) + 0.204(A 645) + 0.372(A505)

0.0806(A453)

CCAR = 0.216(A 663) 1.22(A 645) 0.304(A505) + 0.452(A453)

The lycopene and -carotene contents were represented by micrograms per grams of fresh weight (g/g FW).
Fruit Firmness Analysis. Firmness is an important characteristic
for the marketability of tomato fruit. To analyze the eect of the Sletr12 mutation, we tested the rmness of the fruits. Br+6 fruits were tested
using an Ultrasonic Hardness Tester model FHR-5 (Nippon Optical
Works Co., Ltd., Tokyo, Japan). The measurement was repeated every
10 days until 30 DPS.
Analysis of the Total Soluble Solids (TSS), pH, and Titratable
Acidity (TA). To evaluate the eect of the Sletr1-2 mutation on four
Sletr1-2 F1 hybrid lines, we measured the TSS, pH and TA. The TSS
was used to estimate the sugar level, and TA was used to estimate the
organic acid level. The TSS was measured using a refractometer PAL-J
(Atago, Tokyo, Japan). The pH and TA was measured using a pH
meter SevenCompact pH/Ion S220 (Mettler-Toledo AG, Schwerzenbach, Switzerland). The TA was measured using pH meter methods by

RESULTS

Under Heterozygous Conditions, Sletr1-1 and Sletr1-2

Expressed Ethylene-Insensitive Phenotypes. All of the
Sletr1-1 F1 and Sletr1-2 F1 hybrid lines showed a similar
tendency with ethylene exogenous exposure, that is, reduced
hypocotyl and root lengths. These measurements were
signicantly longer than in the WT-MT F1 hybrid lines and
the commercial pure-line cultivars (Figure 1A). All of the Sletr11 F1 hybrid lines resulted in a strong ethylene-insensitive
phenotype. Exogenous ethylene exposure of 5 ppm was able to
reduce the hypocotyl and root lengths of all of the investigated
plants. However, the reductions in the Sletr1-1 F1 hybrid lines
were no more than 19.4% (Sletr1-1 Moneymaker) and 5.4%
(Sletr1-1 Ailsa Craig), respectively (Figure 1B,C).
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Figure 2. Vegetative growth of WT-MT F1, Sletr1-1 F1, and Sletr1-2 F1 hybrid lines and pure-line commercial cultivars. (A) The F1 hybrid line of
Sletr1-1 (crossed with Ailsa Craig) showed wilting as a response to the transplanting process to the NFT system; the photograph was taken 3 weeks
after transplanting. (B) Injured roots of all of the Sletr1-1 F1 hybrid lines from crosses with four dierent pure-line background cultivars: Aichi First
(a), Ailsa Craig (b), Moneymaker (c), and M82 (d). (C) Representative photographs of the root systems of the hybrid lines of MT-WT F1 (1),
Sletr1-1 F1 (2), and Sletr1-2 F1 (3) (crossed with Ailsa Craig); the photographs were taken 2 weeks after transplanting to the NFT system. (D)
Plant survival of two F1 hybrid mutant lines, Sletr1-1 and Sletr1-2, compared with WT-MT F1. Data were taken 4 weeks after transplanting to the
NFT system (n = 8).

Figure 3. Vegetative characteristics of Sletr1-2 F1 and WT-MT F1 hybrid lines and pure-line commercial cultivars. (A) The number of leaves under
the rst truss is the average number of leaves from eight plants. (B) The number of owers per truss and (C) the number of fruits per truss are the
average numbers of owers and fruits from four trusses per plant. (D) The time to breaker stage for the investigated fruits was observed as the
number of days from seed sowing until the rst fruit of one plant reached the BR stage or the fruit color changed to pink or yellow. Mean values with
the same storage time tags followed by the same letters are not signicantly dierent according to the TukeyKramer test at p < 0.05.

phenotypes compared with the Sletr1-1 F1 hybrid lines;

however, these lines were more ethylene-sensitive than the
WT-MT F1 hybrid lines and the pure-line commercial cultivar

Exogenous ethylene exposure in the Sletr1-2 F1 hybrid lines

resulted in an intermediate ethylene-insensitive phenotype.
Those hybrid lines expressed weaker ethylene sensitivity
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this F1 hybrid line is most likely correlated with its pure-line

cultivar parent, Ailsa Craig, which reached the breaker stage
earlier than the other three commercial pure-line cultivars at
approximately 122 days after sowing (Figure 3D).
Sletr1-2 F1 Hybrid Resulted in Fruit Characteristics
Similar to Its WT-MT F1 Hybrids. The appearance of the
fruits from all of the Sletr1-2 F1 hybrid lines was similar to that
of fruit from the WT-MT F1 hybrid lines, which showed an
intermediate fruit size (smaller than the pure-line cultivar
parents and bigger than the Sletr1-2 and WT-MT parents).
Moreover, the fruit shapes were slightly similar to those of the
Micro-Tom parents (Figure 4). Quantication analysis revealed

parent (Figure 1A). Treatment with 5 ppm of exogenous

ethylene reduced hypocotyl length by 61.7% (Sletr1-2 Aichi
First) and root length by 29.5% (Sletr1-2 Aichi First)
(Figure 1B,C).
Sletr1-1 F1 Hybrid Line Shows Weak Phenotype
Characteristics during Stress Conditions. Two weeks
after being transplanted to the NFT system, all of the Sletr11 F1 hybrid lines showed stress response, which was
characterized by leaf wilting and disease sensitivity. However,
the Sletr1-2 F1 hybrid lines did not show any stress responses
(Figure 2A). Plant wilting in Sletr1-1 F1 hybrid lines was caused
by damage to the root system. All of the roots were browning,
the root tips were injured, and some of the roots were rotting
(Figure 2B). However, in all of the Sletr1-2 F1 hybrid lines, the
roots appeared healthy and were white in color, and no roots
were rotting (Figure 2C), which was similar to the characteristics of the WT-MT F1 hybrid line and the pure-line
commercial cultivars background. Furthermore, only a few
new Sletr1-1 F1 hybrid lines roots were formed for the 2 weeks
after transplanting. At 4 weeks after transplantation, no Sletr1-1
M82 specimens survived; however, Sletr1-1 Aichi First,
Ailsa Craig, and Moneymaker survived at percentages of less
than 13, 50, and 63%, respectively (Figure 2D). Because all of
the Sletr1-1 F1 hybrid lines had the lowest rates of plant
survival, even Sletr1-1 M82 did not survive (Figure 2D).
Thus, the Sletr1-1 F1 hybrid lines were not used for further
investigation in this study.
Mutation of the SlETR1 Gene of the Sletr1-2 Mutant
Does Not Aect the Vegetative and Generative
Characteristics of Its F1 Hybrid Lines. Crossing all of the
pure-line cultivars with WT-MT and Sletr1-2 resulted in similar
tendencies in reducing the number of leaves under the rst
truss in the F1 hybrid lines, which were signicantly dierent
from those of the pure-line cultivar parent but did not show any
dierences between the Sletr1-2 F1 and WT-MT F1 hybrid
lines. The greatest number of leaves under the rst truss was
observed on Sletr1-2 Aichi First, whereas the fewest were
observed on Sletr1-2 M82 (Figure 3A). Crossing Sletr1-2
with Aichi First, Ailsa Craig, Moneymaker, and M82
reduced the average of the number of leaves under the rst
truss by approximately 3.13, 4.00, 3.63, and 2.38, respectively,
compared with the pure-line cultivar parent (Figure 3A). The
plant growth type in the hybrid lines was similar to that of the
commercial pure-line cultivar parent, which included the
indeterminate growth type for Aichi First, Ailsa Craig, and
Moneymaker and the determinate growth type for M82. The
data showed that all of the Sletr1-2 F1 hybrid lines resulted in
numbers of owers and fruits per truss similar to those of the
WT-MT F1 hybrid line; however, the numbers were
signicantly higher than those for the pure-line cultivar parents,
for which the average numbers of owers per truss were 9.34,
9.88, 10.06, and 8.16 and the numbers of fruits per truss were
8.16, 8.38, 9.03, and 7.19 for Sletr1-2 crossed with Aichi First,
Ailsa Craig, Moneymaker, and M82, respectively (Figure
3B,C).
The time to the breaker stage for all of the Sletr1-2 F1 hybrid
lines was similar to that for the WT-MT F1 hybrid lines;
however, they were signicantly faster than the pure-line
cultivar parents. The rst fruit to reach the breaker stage was
Sletr1-2 Ailsa Craig, with an average time of 104 days after
sowing, and the last to reach it was Sletr1-2 Aichi First, with
an average time of 119 days after sowing, or 15 days later than
Sletr1-2 Ailsa Craig (Figure 3D). The early breaker stage of

Figure 4. Fruit appearance of Sletr1-2 F1 and WT-MT F1 hybrid lines

and pure-line commercial cultivars. The photograph was taken at Br+6
of the fruit maturation stage. All of the F1 hybrid lines resulted in an
intermediate fruit size. The Sletr1-2 F1 and WT-MT F1 hybrid lines
appear to have similar fruit sizes and an almost identical red fruit color.
Bar = 1 cm.

that the fruit diameter, fruit length, fruit weight, and pericarp
thickness of all of Sletr1-2 F1 hybrid lines were signicantly
smaller than those of the pure-line cultivar parents. However,
they did not show any signicant dierences compared with
WT-MT F1 hybrid line (Figure 5). The Sletr1-2 F1 hybrid line
from Aichi First resulted in a large fruit that had a greater
diameter, length, and weight compared with the other three F1
hybrid lines with average values of 39.73 mm, 34.87 mm, and
32.81 g, respectively (Figure 5AC). On the other hand, the
diameter, length, and weight of the Sletr1-2 F1 hybrid line from
Ailsa Craig, Moneymaker, and M82 were similar to each
other, with the length ranging from 29.87 mm (Sletr1-2
M82) to 30.85 mm (Sletr1-2 Ailsa Craig), the diameter
ranging from 32.53 mm (Sletr1-2 M82) to 34.01 mm
(Sletr1-2 Ailsa Craig), and the weight per fruit ranging from
20.67 g (Sletr1-2 M82) to 22.00 g (Sletr1-2 Moneymaker) (Figure 5AC). The Sletr1-2 mutant crossed with
Moneymaker resulted in the highest reduction in average
pericarp thickness compared with the pure-line cultivar parents,
with a reduction of approximately 36% (Figure 5E). The locule
numbers of all of the Sletr1-2 F1 hybrid lines from all of the
dierent pure-line cultivar parents were not signicantly
dierent from those of the WT-MT F1 hybrid lines. However,
the signicance varied compared with the pure-line cultivar
parent. Among all of the Sletr1-2 F1 hybrid lines, Sletr1-2
Aichi First resulted in the highest number of fruit locules
(3.71); the lowest locule number was observed on Sletr1-2
Moneymaker (2.46) (Figure 5D).
Sletr1-2 Mutant Has the Potential To Be an F1 Hybrid
Parent for Improving Fruit Shelf Life. In all of the WT-MT
F1 hybrid lines, wrinkles appeared on the fruit surface earlier
than on the Sletr1-2 F1 hybrid lines. At 30 DPS, >50% of the
total fruit surface of the WT-MT F1 hybrid line was wrinkled,
whereas in Sletr1-2 F1 hybrid lines, only some of the fruit
surface was wrinkled (Figure 6). Although the fruit shelf life of
the three Sletr1-2 F1 hybrid lines from Aichi First, Ailsa Craig,
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DOI: 10.1021/acs.jafc.5b02742
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Figure 5. Fruit characterizations of the Sletr1-2 F1 and WT-MT F1 hybrid lines and pure-line commercial cultivars. Fruit characteristics are
represented by (A) diameter, (B) length, (C) weight, (D) locule number, and (E) pericarp thickness. These characteristics were measured from six
fruits per replicate at the (Br+6) fruit maturation stage. Mean values with the same storage time tags followed by the same letters are not signicantly
dierent according to the TukeyKramer test at p < 0.05.

Figure 6. Representative photographs indicating tomato fruit shelf life

during storage of Sletr1-2 F1 and WT-MT F1 hybrid lines and pureline commercial cultivars. The fruits were stored for 40 days under
room conditions (20 + 2 C) until wrinkling or black spots appeared
on the fruit surface. Bar = 1 cm.

Figure 7. Fruit shelf life of Sletr1-2 F1 and WT-MT F1 hybrid lines

and pure-line commercial cultivars. Shelf life was evaluated under
room conditions until wrinkling appeared on the fruit surface. Mean
values with the same storage time tags followed by the same letters are
not signicantly dierent according to the TukeyKramer test at p <
0.05.

and Moneymaker was signicantly reduced compared with the

pure-line cultivar parent (8.46, 11.33, and 8.63 days shorter
than the shelf life for Aichi First, Ailsa Craig, and
Moneymaker fruits, respectively), all of the Sletr1-2 F1 hybrid
lines showed signicant improvement in fruit shelf life
compared with the WT-MT F1 hybrid lines (Figure 7). In
the four genetic backgrounds of pure-line cultivars, the Sletr1-2
mutation eectively extended the fruits shelf life up to 3.96,
4.71, 3.92, and 5.13 days longer than the fruit shelf life of WTMT F1 hybrid line for the Sletr1-2 F1 hybrid lines from Aichi
First, Ailsa Craig, Moneymaker, and M82 background,
respectively (Figure 7).

Ethylene Production and Respiration Rate of the

Sletr1-2 F1 Hybrid Line Were Similar to Those of the WTMT F1 Hybrid Line in All of the Pure-Line Cultivar
Parents. Our data indicate that ethylene production and the
respiration rate of the investigated fruits decreased during
storage until 12 DPS. The mutation in the SlETR1 gene of the
Sletr1-2 F1 hybrid lines did not aect either ethylene
production (Figure 8A) or respiration rate (Figure 8B) until
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Fruit Color Varied among All of the Sletr1-2 F1 Hybrid

Lines from Dierent Commercial Pure-Line Cultivar
Parents. Generally, the L* and h values of all of the Sletr1-2
F1 hybrid lines varied from those of the WT-MT F1 hybrid
lines and the pure-line commercial cultivar parents. In all of the
Sletr1-2 F1 and WT-MT F1 hybrid lines, L* and h
dramatically decreased at 10 DPS, and then their values were
stable. However, in the pure-line cultivar, the values were
similar during postharvest storage up to 30 DPS. Our statistical
analysis of the data showed dierent signicance during
postharvest storage under dierent pure-line cultivar backgrounds. Compared with the WT-MT F1 hybrid lines, the
Sletr1-2 mutation signicantly increased the L* value only in
Sletr1-2 crossed with Aichi First up to 20 DPS (42.13 at 0
DPS, 37.02 at 10 DPS, and 35.70 at 20 DPS) and in Sletr1-2
crossed with Moneymaker at 0 DPS with the value of 42.29
(Figure 9A). Of all the F1 hybrid lines, Sletr1-2 crossed with

Figure 8. Ethylene production and respiration rate of Sletr1-2 F1 and

WT-MT F1 hybrid lines and pure-line commercial cultivars. Ethylene
production (A) and respiration rate (B) were measured every 4 days
for 12 DPS. Mean values with the same storage time tags followed by
the same letters are not signicantly dierent according to the Tukey
Kramer test at p < 0.05.

12 DPS, which was similar to the ndings for WT-MT F1

hybrid lines. Although ethylene production in Sletr1-2 crossed
with all four pure-line cultivar parents was not signicantly
dierent from that of the WT-MT F1 hybrid lines, there was a
tendency for the ethylene production and respiration rate of all
the Sletr1-2 F1 hybrid lines, except for the respiration rate of
Sletr1-2 crossed with M82, to be higher than those of the WTMT F1 hybrid line (Figure 8A,B). At the beginning of storage,
Sletr1-2 Moneymaker had the highest ethylene production
(2.36 g/g FW/h) (Figure 8A), whereas the highest respiration
rates were reached by both Sletr1-2 and WT-MT crossed with
M82, with values of 6.69 and 7.07 g/g FW/h, respectively.
However, the other F1 hybrid lines showed almost the same
values for ethylene production and respiration rate until 12
DPS, at which point the ethylene production of Sletr1-2 F1
hybrid lines ranged between 0.31 g/g FW/h (Sletr1-2 Aichi
First) and 0.67 g/g FW/h (Sletr1-2 Ailsa Craig) (Figure
8A) and the respiration rate ranged between 2.53 g/g FW/h
(Sletr1-2 M82) and 3.36 g/g FW/h (Sletr1-2 Ailsa
Craig) (Figure 8B). Compared with the pure-line commercial
cultivars, the ethylene production and respiration rate of Sletr12 F1 hybrid lines varied during 30 days of storage. At 0 DPS,
Sletr1-2 crossed with Moneymaker and M82 had signicant
increases in both ethylene production of 0.96 and 1.09 g/g
FW/h and respiration rate of 2.47 and 2.23 g/g FW/h,
respectively, whereas, at 12 DPS, all of the Sletr1-2 F1 hybrid
lines except for Sletr1-2 M82 resulted in higher ethylene
production than that for the pure-line cultivar; however, it had a
similar respiration rate (Figure 8A,B).

Figure 9. Fruit color of Sletr1-2 F1 and WT-MT F1 hybrid lines and

pure-line commercial cultivars. Color was represented by (A) the L*
value as the lightness of the color, where (L* = 0 indicates black and
L* = 100 indicates white), and (B) the h value, the pure spectrum or
the gradation of color (e.g., red, purple, blue). Mean values with the
same storage time tags followed by the same letters are not
signicantly dierent according to the TukeyKramer test at p < 0.05.

M82 had the highest values of L* (44.71 at 0 DPS and 39.7 at

30 DPS) (Figure 9A). Compared with the pure-line cultivar,
the clear signicance in increasing the L* value of Sletr1-2 F1
hybrid lines up to 30 DPS was observed from the Sletr1-2
crossed with Moneymaker and M82. However, in crosses
with Aichi First and Ailsa Craig, it was observed only at 0
DPS, after which they were similar (Figure 9A).
The h value was measured to refer to the pure color. The
signicant eect of the Sletr1-2 mutation on the h value was
observed at 0 DPS only in Sletr1-2 crossed with Aichi First and
Moneymaker, with values of 0.75 and 0.77, respectively, and
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Figure 10. Carotenoid content and fruit rmness of Sletr1-2 F1, WT-MT F1 hybrid lines, and pure-line commercial cultivars. (A) Lycopene content
and (B) -carotene content represent the carotenoid content of the fruits during postharvest storage. (C) Fruit rmness represents the fruit hardness
during the 30 days of storage. Mean values with the same storage time tags followed by the same letters are not signicantly dierent according to
the TukeyKramer test at p < 0.05.

Sletr1-2 Ailsa Craig and Sletr1-2 M82, no signicant

dierences in lycopene content were detected after 20 and 10
DPS, respectively (Figure 10A). Compared to the pure-line
cultivar parent, Sletr1-2 F1 hybrid lines showed variations in the
lycopene content depending on the pure-line cultivar background. At 0 DPS, both Sletr1-2 Aichi First and Sletr1-2
Moneymaker had signicantly lower lycopene content;
however, after 20 DPS, the value was signicantly higher,
whereas in Sletr1-2 Ailsa Craig and Sletr1-2 M82, no
signicant dierences in lycopene content were detected after
10 DPS (Figure 10A).
In addition to the lycopene content, the -carotene content
increased during 30 days of storage. In Sletr1-2 M82, the carotene content did not increase as much as in the three other
Sletr1-2 F1 hybrid lines; the value was only increased 6.05 g/g
FW, whereas in the other Sletr1-2 F1 hybrid lines, it was
increased >10 g/g FW (Figure 10B). The signicant eect of
the SlETR1 gene mutation of the Sletr1-2 F1 hybrid lines on the
-carotene content was detected only in Sletr1-2 Aichi First
and Sletr1-2 Moneymaker and occurred at 10 and 0 DPS,
respectively, whereas at the end of storage, it was similar to that
of the WT-MT F1 hybrid lines (Figure 10B). At the beginning
of storage, Sletr1-2 Aichi First and Sletr1-2 Moneymaker
had reductions of 21 and 16%, respectively, compared with the
WT-MT F1 hybrid lines. The changes in -carotene in all
Sletr1-2 F1 hybrid lines diered from those of the pure-line
cultivar parent. Both Sletr1-2 Aichi First and Sletr1-2
Moneymaker had signicantly lower -carotene content at the
beginning of storage; however, after 20 DPS, it was signicantly

after 10 DPS, there were no signicant dierences from the

WT-MT F1 hybrid lines. (Figure 9B). Although the h value of
the Sletr1-2 F1 hybrid lines generally was not signicantly
dierent from that of WT-MT F1 hybrid lines during storage
time up to 30 DPS, it varied from its pure-line cultivar parent.
In Sletr1-2 crossed with Aichi First and M82, the h value
signicantly increased up to 30 DPS. In Sletr1-2 Moneymaker, the h value was signicantly lower during the storage
time except at 0 DPS, whereas in Sletr1-2 Ailsa Craig, the h
value was similar except at 0 and 10 DPS (Figure 9B).
Carotenoid Content of the Sletr1-2 F1 Hybrid Line Is
Slightly Aected by Mutation in the SlETR1 Gene. All of
the Sletr1-2 F1 hybrid lines synthesized slightly less lycopene
than the WT-MT F1 hybrid lines but varied from the pure-line
cultivar parent. On the basis of the statistical analyses of the
data, at 0 DPS, there was signicantly lower lycopene content
in all of the Sletr1-2 F1 hybrid lines than in the WT-MT F1
hybrid lines and the pure-line cultivar parent, with the average
value varying between 11.75 g/g FW (Sletr1-2 Moneymaker) and 23.25 g/g FW (Sletr1-2 M82) (Figure 10A).
The lycopene content increased gradually during storage in all
of the F1 hybrid lines up to 30 DPS; however, its amount in the
pure-line cultivar remained stable after 10 DPS. At 30 DPS, >60
g/g FW of lycopene was detected in the three Sletr1-2 F1
hybrid lines (crossed with Aichi First, Ailsa Craig, and
Moneymaker) but in Sletr1-2 M82, at only 50.05 g/g FW
(Figure 10A). Compared with the WT-MT F1 hybrid lines,
both Sletr1-2 Aichi First and Sletr1-2 Moneymaker had
signicantly lower lycopene content up to 30 DPS, whereas in
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Figure 11. Eect of Sletr1-2 mutation on fruit taste quality of its F1 hybrid line as represented by (A) total soluble solids, (B) pH, and (C) titratable
acidity. Mean values with the same storage time tags followed by the same letters are not signicantly dierent according to the TukeyKramer test
at p < 0.05.

higher. In Sletr1-2 Ailsa Craig, it was signicantly higher

than that of the pure-line cultivar parent up to 20 DPS, whereas
in Sletr1-2 M82, no signicant dierences in -carotene
content were detected (Figure 10B).
Fruit of the Sletr1-2 F1 Hybrid Lines Is Harder than
That of the F1 Hybrid Lines. The hardness of the fruit is
indicated by a rmness value, which is an important
characteristic for fresh tomato fruit. The rmness of all the
investigated fruits decreased during storage up to 30 DPS. The
statistical data analysis revealed that, from 0 until 10 DPS for
Sletr1-2 crossed with Aichi First, Ailsa Craig, and Moneymaker and until 20 DPS for Sletr1-2 M82, the fruits were
signicantly harder compared with the WT-MT F1 hybrid lines,
with rmness values 0.15, 0.12, 0.14, and 0.12 kgf higher than
those for the WT-MT F1 hybrid lines, respectively, at 0 DPS.
However, no signicant dierences were detected at the end of
storage, with values of 0.95, 1.13, 1.06, and 1.31 kgf for Sletr1-2
crossed with Aichi First, Ailsa Craig, Moneymaker, and
M82, respectively (Figure 10C). Compared with the pure-line
cultivar parent, signicant improvements in the fruit hardness
of the Sletr1-2 F1 hybrid lines occurred up to 30 DPS, except
for Sletr1-2 M82, which showed a lower fruit rmness at the
beginning of storage and a similar one at 30 DPS (Figure 10C).
A greater improvement in rmness compared with the pure-line
cultivar parents was observed in Sletr1-2 Aichi First, with an
improved rmness of 0.26 kgf (Figure 10C).
Mutation in the Sletr1-2 Allele Aects the Fruit pH
and Titratable Acidity (TA) but Does Not Aect the
Total Soluble Solids (TSS). During 30 days of storage, all of

the Sletr1-2 F1 hybrid lines had TSS levels similar to those for
the WT-MT F1 hybrid lines; however, these levels diered
from those for the pure-line cultivar. At 0 DPS, the TSS from
the four Sletr1-2 F1 hybrid lines (Sletr1-2 crossed with Aichi
First, Ailsa Craig, Moneymaker, and M82) were 4.78, 4.83,
4.60, and 4.13 Brix, respectively, whereas at 30 DPS, they were
5.00, 5.30, 4.48, and 3.88 Brix (Figure 11A). Crossing both
WT-MT and Sletr1-2, with M82 signicantly increased the
TSS values of their F1 hybrid lines and resulted in values of
1.10, 0.68, 0.63, and 0.50 Brix higher than the M82 parent at
0, 10, 20, and 30 DPS, respectively (Figure 11A).
The analyzed data indicate that Sletr1-2 aected the pH and
TA of the fruit; however, the eect was dependent on the
genetic background of the pure-line cultivar parent. All of the
Sletr1-2 F1 hybrid lines showed the lowest fruit pH and the
highest fruit TA during the 30 days of storage compared with
the WT-MT F1 hybrid line, except for the pH and TA of Sletr12 crossed with M82 that showed similar level after 10 DPS
(Figure 11). At 0 DPS, the pH from Sletr1-2 crossed with Aichi
First, Ailsa Craig, Moneymaker, and M82 was 3.81, 3.77,
3.79 and 3.80, respectively, whereas at 30 DPS it was 4.06, 4.08,
4.11, and 4.09 (Figure 11B). In all of the investigated fruits, the
pH increased during the 30 days of storage; however, the TA
decreased. This study showed that the fruits of the Sletr1-2 F1
hybrid lines with the lowest pH contained the highest TA levels
that were signicantly dierent from the levels of the WT-MT
F1 hybrid line and the pure-line cultivar parents during the 30
days of storage (Figure 11C). At 0 DPS, the TA from Sletr1-2
crossed with Aichi First, Ailsa Craig, Moneymaker, and
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M82 was 0.67, 0.70, 0.65, and 0.62%, respectively, whereas at
30 DPS, it was 0.43, 0.50, 0.46, and 0.49% (Figure 11C).

by Fusarium oxysporum. An undesirable eect of ETR1

mutation was also detected in Arabidopsis Atetr1-1, which
exhibited an increase in disease infection by pathogens such as
Botrytis cinerea, Fusarium solani, Fusarium oxysporum f. sp.
matthiolae, Xanthomonas campestris pv. Campestris, and
Pythium spp.2022 Our results are consistent with the Nr
mutant, which does not have the capacity to produce
adventitious roots under waterlogging stress conditions.
Ethylene has an important role during ooding stress because
it initiates and is involved in adventitious root development.23,24 Therefore, it plays an important role in mediating
plant responses, which may enable the plant to survive. Our
data suggest that the Sletr1-1 F1 hybrid lines exhibited a strong
ethylene-insensitive phenotype and a strong sensitivity to biotic
and abiotic stresses. Therefore, Sletr1-1 is not appropriate for
breeding. However, Sletr1-2 may have potential for breeding
because its F1 hybrid lines did not show any undesirable eects
under those stresses. Other studies have reported that only the
rin mutant is suitable for use in breeding programs, whereas Nr
is not suitable because it increases susceptibility to some
pathogens.2527 Moreover, it has insucient red coloration.3,14
Through breeding programs, the desired characteristics can
be obtained. Hence, to improve the growth and yield quality of
tomato plants, hybrid breeding can be used eciently.28 The
vegetative and generative characters of Sletr1-2 F1 hybrid lines
were comparable to those of WT-MT F1 hybrid lines even
though these lines were the result of crosses with the four
genetic backgrounds of pure-line cultivars (Figures 35).
Vegetative and generative performance in the Sletr1-2 F1 and
WT-MT F1 hybrid lines was indicated mostly by the
intermediate characteristics resulting from the Sletr1-2 or
WT-MT parent (initially from the Micro-Tom cultivar
background having both a small plant size (1520 cm) and a
small fruit size)29 and the commercial pure-line cultivar parents.
A similar study on the intermediate characteristics of F1 hybrid
line yielded the same ndings for rin and nor F1 hybrid lines,
although rin had a smaller eect than nor.4 Another study found
intermediate average mean values for fruit set, shape, weight,
TSS content, and owers per cluster in the F1 hybrid line.30,31
On the basis of this study, we conclude that mutation in the
SlETR1 gene of the Sletr1-2 mutant allele did not aect
vegetative and generative performance, although mutation did
improve fruit shelf life of its F1 hybrid lines (Figure 7). Similar
results have been shown for fruits of the heterozygous rin and
nor F1 hybrid lines that had improved shelf life.30,32 These
results suggest that the eect of the Sletr1-2 allele on fruit shelf
life is a dominant genetic action and is not dependent on
genetic background, although the nor gene is dependent on
genetic background.33 In the present study, the shelf life of the
Sletr1-2 F1 hybrid line was 45 days longer than that of the
WT-MT F1 hybrid lines (Figure 7), which was enough to
maintain the freshness of the tomato fruit. Prolonged fruit shelf
life is important for transportation to distant markets, fruit
storage, and handling, especially at markets and for the
consumer.
During the ripening process, increases in ethylene production
and respiration rate occur, and the peaks are reached in the
breaker stage when the rst red coloration is visible. Ethylene
production and respiration then decline when fruit senescence
is reached.34 In our study, no peaks of ethylene and CO2 were
detected for the investigated fruit during storage because red
fruits were used. There were similar declines in ethylene
production and respiration rate during storage time until the

DISCUSSION
Fruit shelf life is an important trait in fresh tomato. Generating
new cultivars with longer fruit shelf life may be an interesting
approach to improving the postharvest quality of tomato.
Mutations in the Sletr1 mutant alleles, Sletr1-1 and Sletr1-2,
resulted in ethylene-insensitive phenotypes in the F1 hybrid
lines. However, these F1 hybrid lines showed dierent ethylene
sensitivities at the seedling stage, with the Sletr1-1 F1 hybrid
lines exhibiting less ethylene sensitivity than the Sletr1-2 F1
hybrid lines (Figure 1). The ethylene-insensitive phenotype of
the Sletr1-1 F1 hybrid line was inherited from the Sletr1 mutant
alleles, which exhibited dominant inheritance.8 The eect of
ethylene on seedling development is characterized by hypocotyl
elongation inhibition, hypocotyl base expansion, and primary
root elongation inhibition.17,18 All of the Sletr1-1 F1 hybrid
lines responded slightly to the application of 5 ppm of
exogenous ethylene, whereas the Sletr1-2 F1 hybrid lines
responded even at low concentrations (1 ppm) (Figure 1). On
the basis of these data, the ethylene sensitivities of Sletr1-1 and
Sletr1-2 were reduced in the heterozygous condition compared
with the homozygous condition, whereas under homozygous
condition, the Sletr1-1 mutant did not show any response to
ethylene exposure up to 10 ppm.8 Reduction of ethylene
sensitivity in Sletr1-1 and Sletr1-2 mutants and these F1 hybrid
lines was due to the mutations in the rst and second domains
of transmembrane region of the ethylene receptor gene
(SlETR1), which is an important region for ethylene binding.
Amino acid substitution of P51L in the Sletr1-1 mutant as a
result of mutation in the rst domain of the transmembrane
region is similar to the amino acid substitution of P36L in Nr
and Arabidopsis etr2-1, whereas in Sletr1-2, the amino acid
substitution of V69D is located in the second domain of the
transmembrane region (V69D).8 In Arabidopsis and tomato,
mutations in these regions of ethylene receptor genes aect
ethylene binding activity and ethylene sensitivity.9,10,19
Improving agronomic characteristics in F1 hybrid lines is an
important issue in a breeding program. Because favorable allelic
interactions at heterozygous loci in F1 hybrid lines improve
performance, crossing two homozygous lines that have the
characteristics desired for making an F1 hybrid line is a tool
used in breeding programs to obtain new and improved
agronomic characteristics. Sletr1 mutant alleles could make
interesting contributions to a breeding program because these
mutants exhibit dominant inheritance in improving tomato fruit
shelf life.8 However, undesirable characteristics most likely have
been expressed in F1 hybrid lines. We detected several
undesirable eects of Sletr1-1 mutation in the Sletr1-1 F1
hybrid lines; however, these eects were not detected in the
Sletr1-2 F1 hybrid lines (Figure 2). The low survival rate of the
Sletr1-1 F1 hybrid line is most likely due to the fact that this
line does not have the capacity to develop and recover new
roots when the old roots are injured. Prolonged root damage
without recovery caused an inhibition of water uptake that was
detrimental to all of the processes involved in plant growth and
development. The inability of all of the Sletr1-1 F1 hybrid lines
to recover from root injuries and develop new roots quickly
resulted in secondary diseases, such as fungi that attack the root
system and the stem neck. The disease symptoms of the Sletr11 F1 hybrid lines were similar to those of the Sletr1-1 mutant
parent, which showed an increased susceptibility to infestation
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fruits reached senescence at 30 DPS for all of the Sletr1-2 F1,
WT-MT F1 hybrid lines, and pure-line cultivar parent (Figure
8). Mutation in the Sletr1-2 mutant allele did not have
signicant eects on ethylene production and respiration rate of
its F1 hybrid line, even when this mutant was crossed with the
dierent genetic background of the pure-line cultivar parents.
The respiration rates of the Sletr1-2 F1 hybrid lines were similar
to those found in the F1 hybrid line of rin mutant which
showed that the rin F1 mutant had no eect on respiration
rate.11 For ethylene biosynthesis, their result for the rin F1
mutant conicted with ours. The rin F1 hybrid lines had
signicantly lower ethylene production, with the peak reduced
by approximately 50% in single rin mutant hybrids and by 25%
in double-mutant hybrids.4,11,13 The reduction in ethylene
production in the rin F1 hybrid lines is due to reductions in the
ACO gene expression levels.5,35 1-Aminocyclopropane 1carboxylate synthase (ACS) and 1-aminocyclopropane 1carboxylate oxidase (ACO) are involved in and important for
the ethylene biosynthesis pathway. Therefore, during the
ripening process, the amount of ethylene produced increases.
For ethylene biosynthesis, ACS activity is a key step, whereas
ACO activity is constitutive.36 The same level of ethylene
production was measured for the Sletr1-2 F1 and WT-MT F1
hybrid lines, indicating that no changes were caused by the
SlETR1 gene mutation in the ethylene biosynthesis pathway.
The fruit ripening process is associated with several
processes, such as increases in respiration rate and ethylene
biosynthesis and changes in metabolic properties, including
changes in fruit color, softening, texture, aroma, volatiles, sugar
content, and pathogen susceptibility.37 Color performance is an
important external characteristic in tomatoes as an indicator of
fruit ripeness and postharvest life.38 Color change is easily
detected when the onset of the ripening process occurs due to
chlorophyll degradation and the accumulation of carotenoids,
which cause the fruit to change to a red color.5 In our study,
fruit color is represented by L* and h, which indicate fruit
brightness and color purity, respectively. Both the L* and h
values of the Sletr1-2 F1 hybrid lines varied according to the
dierent genetic backgrounds of the pure-line cultivar parent,
and these values were mostly higher than those for the WT-MT
F1 hybrid lines and the pure-line cultivar parents at the
beginning of storage (Figure 9). We suggest that the high L*
and h values indicate that the ripening process was delayed or
inhibited. Moreover, we believe that the eect of the Sletr1-2
mutation on the fruit color was dominant or negative over
dominance, depending on the genetic background. Our results
are consistent with other studies that have shown that the eect
of the nor gene on the chroma and L* indices of color was
dependent on the analyzed cross or genetic background.33
Although the L* and h values were signicantly dierent
between the Sletr1-2 F1 hybrid lines and the WT-MT F1 hybrid
lines, according to visual inspection, they had almost identical
red fruit colors, indicating that the fruit was red-ripened (Figure
6). Similar results for rin F1 hybrid lines have also shown the
development of red color.39 Contrasting results have been
reported for the nor F1 hybrid line, which is incapable of
developing a red fruit during ripening.12
The color change of the fruit from green to red is due to the
transformation of chlorophyll to chromoplasts as a result of the
deposition of lycopene and -carotene in the fruit during the
ripening process.40 Because Sletr1-2 has a dominant inheritance
pattern,8 Sletr1-2 F1 hybrid lines exhibited reductions in
ethylene sensitivity phenotypes, such as a delay in the ripening

process, that aect other physiological changes, such as

lycopene biosynthesis. The synthesis of lycopene was seemingly
intimately dependent on ethylene for initiation and continuance of the response.41 In the present study, there was a
relationship between fruit color and lycopene content. Fruit
with higher L* and h values resulted in a lower lycopene
content (Figures 9 and 10). Another study showed that the
increase in color was related to increases in both -carotene and
lycopene.42 Lycopene and -carotene in the F1 hybrid line of
the Sletr1-2 mutant were lower than in the WT-MT F1 hybrid
line; however, this eect was dependent on the genetic
background of the parent (Figure 10A,B). Although the
Sletr1-2 F1 hybrid line had a lower lycopene content compared
with the WT-MT F1 hybrid line, its fruit had the capacity to
develop red-ripened fruit (Figures 4 and 6). We believe that
Sletr1-2 has potential for use in breeding for improved tomato
fruit shelf life because the lycopene and -carotene contents
and the fruit color of the Sletr1-2 F1 hybrid lines were nearly
the same as those of the wild-type F1 hybrid lines.
Firmness is an important characteristic for both indicating
and helping to enhance postharvest fruit quality and shelf life,
in addition to water status and cuticle structure.43,44 Our results
showed that the Sletr1-2 F1 hybrid lines had the highest
rmness values (Figure 10C), which means that softening of
those fruits was delayed compared with the WT-MT F1 hybrid
line and the pure-line cultivar parent. The delayed softening
observed in the Sletr1-2 F1 fruits was characterized by increased
rmness during the early fruit maturation stages when the fruits
were harvested. Our results are consistent with ndings for the
nor Ca F1 hybrid line and alc Mospomorist, which both
showed improved fruit rmness compared with their
parents.33,45 Other studies have reported that the F1 hybrid
line of rin has a lower softening value and that the F1 nor line
has delayed fruit ripening.32,46 From these results, we conclude
that the mutation eect of the Sletr1-2 allele on fruit rmness is
a dominant genetic action in dierent genetic backgrounds.
Because the Sletr1-2 F1 hybrid line exhibits delayed fruit
softening, Sletr1-2 may be an interesting contributor to plant
breeding programs for improving the postharvest quality of
tomatoes.
For consumers, both shelf life and avor quality inuence the
acceptability of fruits. TSS and TA are used in tomato fruits
primarily to estimate sugar and organic acids, respectively,
which are commonly associated with fruit sweetness and
sourness, respectively.47 During 30 days of postharvest storage,
mutation in Sletr1-2 did not aect the TSS in the Sletr1-2 F1
hybrid lines, and the TSS of the Sletr1-2 F1 hybrid lines was
comparable to those of the WT-MT F1 hybrid lines. Generally,
the eect of the Sletr1-2 mutation was similar to the eect of 1MCP in inhibiting ethylene action at the receptor level. It can
be seen from other authors who studied 1-MCP that it does not
aect the TSS of treated tomato fruits.48,49 Although the TSS in
Sletr1-2 F1 hybrid line was not aected by the Sletr1-2
mutation, the TSS was signicantly dierent from that of the
commercial pure-line cultivar parent (Figure 11A). This result
is consistent with the study on the F1 hybrid line of the nor
mutant that had a dierent TSS value from that of the
Caimanta parent.33
There was detected a relationship between pH and TA in all
investigated fruit during storage, when fruit with a lower pH
contained higher TA (Figure 11B,C). Another study also found
a close relationship between total acidity and pH in tomato.50
The Sletr1-2 mutation had a signicant eect on the pH and TA
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Journal of Agricultural and Food Chemistry

(4) Kitagawa, M.; Ito, H.; Shiina, T.; Nakamura, N.; Inakuma, T.;
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of all of the F1 hybrid lines. Inhibition of the ethylene action as

a result of mutation in the SLETR1 gene was eective at
inhibiting the loss of fruit acidity in Sletr1-2 F1 hybrid lines;
however, the eect was dependent on the genetic background
of the pure-line cultivar parent. Thus, all Sletr1-2 F1 hybrid
fruits had a higher TA content. Our results are consistent with
other studies that showed that the inhibition of ethylene action
by 1-MCP signicantly increased the TA in tomato fruits.49 On
the basis of these results, pH and TA are ethylene-dependent
eects. Many studies have stated that the reduction of organic
acid degradation caused by the suppression of the ethylene
biosynthesis and action correlated with the reduction of
respiration rate because organic acids are substrates for
respiration.47 This result is in contrast to our study, which
showed that even though the respiration rates of the Sletr1-2 F1
hybrid lines were similar to those in the WT-MT F1 hybrid
lines and the pure-line cultivar parent (Figure 8B), the TA and
pH were dierent (Figure 11B,C).
In conclusion, all of the results show that Sletr1-2 exhibited
strong inheritance patterns that improved the fruit shelf life of
hybrid lines when crossed with dierent genetic backgrounds.
Moreover, the hybrids did not show many undesirable
characteristics, such as negative responses to stress conditions
and highly reduced lycopene content and red fruit color. The
plants provided similar levels of -carotene and TSS and higher
levels of TA and fruit rmness. All of these characteristics are
important for the tomato fruit quality traits that appeal to the
consumer. We conclude that the Sletr1-2 mutant has potential
as a contributor to breeding programs for the development of
new tomato cultivars with improved fruit shelf life.

AUTHOR INFORMATION

Corresponding Author

*(H.E.) Phone/fax: +81-29-853-7263. E-mail: ezura@gene.

tsukuba.ac.jp.
Funding

This work was supported by a grant-in-aid for scientic research

category A (No. 25252008) to H.E. from the Ministry of
Education, Science, Sports, and Technology of Japan.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
We thank the National BioResearch Project (NBRP), MEXT,
Japan for providing the seeds of S. lycopersicum cv. Micro-Tom,
Sletr1-1, Sletr1-2, Aichi First, Ailsa Craig, Moneymaker, and
M82. We also thank all members of our laboratory for helpful
discussions throughout the work.

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