Anda di halaman 1dari 13
Article <a href=p u b s . a c s . o r g / J A F C Potential Use of a Weak Ethylene Receptor Mutant, Sletr1 ‑ 2 , 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 e ff ective in reducing ethylene sensitivity and improving fruit shelf life. In this study the e ff ect 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 e ff ects in the F1 hybrid lines. The Sletr1-2 mutation was e ff ective in improving fruit shelf life of F1 hybrid lines for 4 − 5 days longer. It was also e ff ective in improving fruit fi rmness without change in fruit size, ethylene production, respiration rate, and total soluble solids or a great reduction in fruit color, lycopene, and β -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 fl eshy fruit that is grown worldwide on an estimated total of 4.98 million hectares of land. 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 in fl uence the price/value of the fruit on the market. The tomato fruit must have high-quality attributes of appearance, performance, texture, fl avor, nutritional value, and safety. Furthermore, the shelf life of the fruit is an important characteristic for commercial markets. 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 a ff ected 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 fl avor, color, aroma, and texture, and there are also increases in ethylene production and respiration. 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 modi fi ed and partially disassembled by enzymes. 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. However, di ff erent 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 di ff erent maturation stages, especially at the mature green and/or immature green stages; instead, all 5 6 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 ). 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. Di ff erent 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 a ff ect ethylene sensitivity. In Sletr1-1 and Sletr1-2 , the mutations are located in a di ff erent transmembrane region of the ethylene receptor. P51L for Sletr1-1 and V69D for Sletr1-2 are located in the fi rst and second domains of the transmembrane region, respectively. The ethylene inhibition mutation has a positive e ff ect 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 . The use of heterozygous ethylene- insensitive mutants can contribute to the agronomic improve- ment 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. In rin mutants, the heterozygous condition delays initiation, and ripening proceeds more slowly than normal ripening, whereas heterozygous Received: January 29, 2015 Revised: July 22, 2015 Accepted: July 24, 2015 Published: July 24, 2015 © 2015 American Chemical Society 7995 DOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-0-2" src="pdf-obj-0-2.jpg">
 

Article

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 β-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. 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. 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

5

6

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 ethylene- insensitive mutants can contribute to the agronomic improve- ment 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:

January 29, 2015

Revised:

July 22, 2015

Accepted:

July 24, 2015

Published: July 24, 2015

Article <a href=p u b s . a c s . o r g / J A F C Potential Use of a Weak Ethylene Receptor Mutant, Sletr1 ‑ 2 , 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 e ff ective in reducing ethylene sensitivity and improving fruit shelf life. In this study the e ff ect 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 e ff ects in the F1 hybrid lines. The Sletr1-2 mutation was e ff ective in improving fruit shelf life of F1 hybrid lines for 4 − 5 days longer. It was also e ff ective in improving fruit fi rmness without change in fruit size, ethylene production, respiration rate, and total soluble solids or a great reduction in fruit color, lycopene, and β -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 fl eshy fruit that is grown worldwide on an estimated total of 4.98 million hectares of land. 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 in fl uence the price/value of the fruit on the market. The tomato fruit must have high-quality attributes of appearance, performance, texture, fl avor, nutritional value, and safety. Furthermore, the shelf life of the fruit is an important characteristic for commercial markets. 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 a ff ected 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 fl avor, color, aroma, and texture, and there are also increases in ethylene production and respiration. 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 modi fi ed and partially disassembled by enzymes. 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. However, di ff erent 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 di ff erent maturation stages, especially at the mature green and/or immature green stages; instead, all 5 6 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 ). 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. Di ff erent 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 a ff ect ethylene sensitivity. In Sletr1-1 and Sletr1-2 , the mutations are located in a di ff erent transmembrane region of the ethylene receptor. P51L for Sletr1-1 and V69D for Sletr1-2 are located in the fi rst and second domains of the transmembrane region, respectively. The ethylene inhibition mutation has a positive e ff ect 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 . The use of heterozygous ethylene- insensitive mutants can contribute to the agronomic improve- ment 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. In rin mutants, the heterozygous condition delays initiation, and ripening proceeds more slowly than normal ripening, whereas heterozygous Received: January 29, 2015 Revised: July 22, 2015 Accepted: July 24, 2015 Published: July 24, 2015 © 2015 American Chemical Society 7995 DOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-0-235" src="pdf-obj-0-235.jpg">

© 2015 American Chemical Society

7995

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

Journal of Agricultural and Food Chemistry

Article

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 character- istics 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. 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.

7

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 plantscultivation, 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.

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 chromato- graph (GC-8A; Shimadzu, Kyoto, Japan) equipped with a photo- ionization 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 (μg/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:

h = arctan

b ⎞ ⎝

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 β-carotene, it has become popular for human consumption. The lycopene and β-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. Absorption was measured using a spectrophotometer (Beckman Coulter DU 640

15

Journal of Agricultural and Food Chemistry Article
Journal of Agricultural and Food Chemistry
Article

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 Craigas 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, WT-

MT 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 (A 663 ),

645 (A 645 ), 505 (A 505 ), and 453 nm (A 453 ). Lycopene (C LYC ) and β-

carotene (C CAR ) were measured using the following equations:

C

LYC

=−

0.04584(

AAA

663

+

645

+

) 0.204(

) 0.372(

505

)

0.0806(

A

453

)

C

CAR

= 0.216(AA

663

1.22(

) −−

645

) 0.304(A ) + 0.452(A )

505

453

The lycopene and β-carotene contents were represented by micro-

grams per grams of fresh weight (μg/g FW).

Fruit Firmness Analysis. Firmness is an important characteristic

for the marketability of tomato fruit. To analyze the eect of the Sletr1-

2 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, Schwerzen-

bach, Switzerland). The TA was measured using pH meter methods by

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.

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 Sletr1- 1 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 1 B,C).

Journal of Agricultural and Food Chemistry

Article

Journal of Agricultural and Food Chemistry Article Figure 2. Vegetative growth of WT-MT F1, Sletr1-1 F1,DOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-3-8" src="pdf-obj-3-8.jpg">

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).

Journal of Agricultural and Food Chemistry Article Figure 2. Vegetative growth of WT-MT F1, Sletr1-1 F1,DOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-3-67" src="pdf-obj-3-67.jpg">

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.

Exogenous ethylene exposure in the Sletr1-2 F1 hybrid lines resulted in an intermediate ethylene-insensitive phenotype. Those hybrid lines expressed weaker ethylene sensitivity

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

Journal of Agricultural and Food Chemistry

Article

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 Sletr1- 1 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 character- istics 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 × M82specimens survived; however, Sletr1-1 × Aichi First, Ailsa Craig, and Moneymakersurvived 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 × M82did 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 A ect 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 M82reduced 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 Moneymakerand 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

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

Journal of Agricultural and Food Chemistry Article parent ( Figure 1 A). Treatment with 5 ppmDOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-4-231" src="pdf-obj-4-231.jpg">

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 Firstresulted 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 M82were 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 × Money- maker) (Figure 5AC). The Sletr1-2 mutant crossed with Moneymakerresulted 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 signi cantly 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 Firstresulted 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,

Journal of Agricultural and Food Chemistry

Article

Journal of Agricultural and Food Chemistry Article Figure 5. Fruit characterizations of the Sletr1-2 F1 andDOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-5-8" src="pdf-obj-5-8.jpg">

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.

Journal of Agricultural and Food Chemistry Article Figure 5. Fruit characterizations of the Sletr1-2 F1 andDOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-5-30" src="pdf-obj-5-30.jpg">

Figure 6. Representative photographs indicating tomato fruit shelf life

during storage of Sletr1-2 F1 and WT-MT F1 hybrid lines and pure-

line 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.

and Moneymakerwas 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 Moneymakerfruits, respectively), all of the Sletr1-2 F1 hybrid lines showed signi cant 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 fruitsshelf life up to 3.96, 4.71, 3.92, and 5.13 days longer than the fruit shelf life of WT- MT F1 hybrid line for the Sletr1-2 F1 hybrid lines from Aichi First, Ailsa Craig, Moneymaker, and M82background, respectively (Figure 7).

Journal of Agricultural and Food Chemistry Article Figure 5. Fruit characterizations of the Sletr1-2 F1 andDOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-5-100" src="pdf-obj-5-100.jpg">

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.

Ethylene Production and Respiration Rate of the Sletr1-2 F1 Hybrid Line Were Similar to Those of the WT- MT 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 a ect either ethylene production (Figure 8A) or respiration rate (Figure 8B) until

Journal of Agricultural and Food Chemistry

Article

Journal of Agricultural and Food Chemistry Article Figure 8. Ethylene production and respiration rate of Sletr1-2DOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-6-8" src="pdf-obj-6-8.jpg">

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 WT- MT F1 hybrid line (Figure 8A,B). At the beginning of storage, Sletr1-2 × Moneymakerhad the highest ethylene production (2.36 μg/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 μg/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 μg/g FW/h (Sletr1-2 × Aichi First) and 0.67 μg/g FW/h (Sletr1-2 × Ailsa Craig) (Figure 8A) and the respiration rate ranged between 2.53 μg/g FW/h (Sletr1-2 × M82) and 3.36 μg/g FW/h (Sletr1-2 × Ailsa Craig) (Figure 8B). Compared with the pure-line commercial cultivars, the ethylene production and respiration rate of Sletr1- 2 F1 hybrid lines varied during 30 days of storage. At 0 DPS, Sletr1-2 crossed with Moneymakerand M82had signicant increases in both ethylene production of 0.96 and 1.09 μg/g FW/h and respiration rate of 2.47 and 2.23 μg/g FW/h, respectively, whereas, at 12 DPS, all of the Sletr1-2 F1 hybrid lines except for Sletr1-2 × M82resulted in higher ethylene production than that for the pure-line cultivar; however, it had a similar respiration rate (Figure 8A,B).

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 back- grounds. Compared with the WT-MT F1 hybrid lines, the Sletr1-2 mutation signicantly increased the L* value only in Sletr1-2 crossed with Aichi Firstup 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 Moneymakerat 0 DPS with the value of 42.29 (Figure 9A). Of all the F1 hybrid lines, Sletr1-2 crossed with

Journal of Agricultural and Food Chemistry Article Figure 8. Ethylene production and respiration rate of Sletr1-2DOI: 10.1021/acs.jafc.5b02742 J. Agric. Food Chem. 2015, 63, 7995 − 8007 " id="pdf-obj-6-190" src="pdf-obj-6-190.jpg">

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.

M82had 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 Moneymakerand M82. However, in crosses with Aichi Firstand 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 Firstand Moneymaker, with values of 0.75 and 0.77, respectively, and

Journal of Agricultural and Food Chemistry Article
Journal of Agricultural and Food Chemistry
Article

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) β-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.

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 Firstand M82, the h° value signicantly increased up to 30 DPS. In Sletr1-2 × Money- maker, 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 μg/g FW (Sletr1-2 × Money- maker) and 23.25 μg/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 μg/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 μg/g FW (Figure 10A). Compared with the WT-MT F1 hybrid lines, both Sletr1-2 × Aichi Firstand Sletr1-2 × Moneymakerhad signicantly lower lycopene content up to 30 DPS, whereas in

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

Journal of Agricultural and Food Chemistry Article
Journal of Agricultural and Food Chemistry
Article

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 β-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 Money- makerand 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 M82signicantly 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 M82parent 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 Sletr1- 2 crossed with M82that showed similar level after 10 DPS (Figure 11). At 0 DPS, the pH from Sletr1-2 crossed with Aichi First, Ailsa Craig, Moneymaker, and M82was 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

Journal of Agricultural and Food Chemistry

Article

M82was 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).

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 Sletr1- 1 F1 hybrid lines were similar to those of the Sletr1-1 mutant parent, which showed an increased susceptibility to infestation

by Fusarium oxysporum. An undesirable e ect 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. 20 22 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 develop- ment. 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. 25 27 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) 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,

29

TSS content, and owers per cluster in the F1 hybrid line. 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. ,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. 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 CO 2 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

33

30

30

, 31

Journal of Agricultural and Food Chemistry

Article

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. ,13 The reduction in ethylene

  • 4 ,11

production in the rin F1 hybrid lines is due to reductions in the ACO gene expression levels. 5 , 35 1-Aminocyclopropane 1- carboxylate synthase (ACS) and 1-aminocyclopropane 1- carboxylate 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 i s 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 β-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 continu- ance 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 β-carotene and lycopene. 42 Lycopene and β-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 β-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 1- MCP 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 Caimantaparent. 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

Journal of Agricultural and Food Chemistry

Article

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 β-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.

REFERENCES

(1) Garg, N.; Cheema, D. S. Assessment of fruit quality attributes of

tomato hybrids involving ripening mutants under high temperature

conditions. Sci. Hortic. 2011, 131, 2938.

(2) Kader, A. A. Quality and safety factor: denition and evaluation

for fresh horticultural crops. In Postharvest Technology of Horticultural

Crops, 3rd ed.; Kader, A. A., Ed.; University of California, Division of

Agriculture and Natural Resources: Richmond, CA, USA, 2002; pp

279287.

(3) Opiyo, A. M.; Ying, T. J. The effects of 1-methylcyclopropene

treatment on the shelf life and quality of cherry tomato (Lycopersicon

esculentum var. cerasiforme) fruit. Int. J. Food Sci. Technol. 2005, 40,

665673.

(4) Kitagawa, M.; Ito, H.; Shiina, T.; Nakamura, N.; Inakuma, T.;

Kasumi, T.; Ishiguro, Y.; Yabe, K.; Ito, Y. Characterization of tomato

fruit ripening and analysis of gene expression in F1 hybrids of the

ripening inhibitor (rin) mutant. Physiol. Plant. 2005, 123, 331338.

(5) Alexander, L.; Grierson, D. Ethylene biosynthesis and action in

tomato: a model for climacteric fruit ripening. J. Exp. Bot. 2002, 53,

20392055.

(6) Jones, J. B. Tomato Plant Culture: in the Field, Greenhouse, And

Home Garden; CRC Press: Boca Raton, FL, USA, 2007; pp 101128.

(7) Lanahan, M. B.; Yen, H. C.; Giovannoni, J. J.; Kleea, H. J. The

never ripe mutation blocks ethylene perception in tomato. Plant Cell

1994, 6, 521530.

(8) Okabe, Y.; Asamizu, E.; Saito, T.; Matsukura, C.; Ariizumi, T.;

Bres, C.; Rothan, C.; Mizaguchi, T.; Ezura, H. Tomato TILLING

technology: development of a reverse genetic tool for the efficient

isolation of mutants from Micro-Tom mutant libraries. Plant Cell

Physiol. 2011, 52, 19942005.

(9) Chang, C.; Kwok, S. F.; Bleecker, A. B.; Meyerowitz, E. M.

Arabidopsis ethylene-response gene ETR1: similarity of product to

two-component regulators. Science 1993, 262, 539544.

(10) Wilkinson, J. Q.; Lanahan, M. B.; Yen, H. C.; Giovannoni, J. J.;

Klee, H. J. An ethylene-inducible component of signal transduction

encoded by never-ripe. Science 1995, 270, 18071809.

(11) Tigchelaar, E. C.; Mcglasson, W. B.; Franklin, M. J. Natural and

ethephon-stimulated ripening of F 1 hybrids of the ripening inhibitor

(rin) and non-ripening (nor) mutants of tomato (Lycopersicon

esculentum Mill.). Aust. J. Plant Physiol. 1978, 5, 449456.

(12) McGlasson, W. B.; Smueghy, J. B.; Morris, L. L.; McBride, R. L.;

Best, D. J.; Tigchelaar, E. C. Yield and evaluation of F 1 tomato hybrids

incorporating the non-ripening nor gene. Aust. J. Exp. Agric. 1983, 23,

106112.

(13) Kitagawa, M.; Nakamura, N.; Usuda, H.; Shiina, T.; Ito, H.;

Yasuda, J.; Inakuma, T.; Ishiguro, Y.; Kasumi, T.; Ito, Y. Ethylene

biosynthesis regulation in tomato fruit from the F 1 hybrid of the

ripening inhibitor (rin) mutant. Biosci., Biotechnol., Biochem. 2006, 70,

17691772.

(14) Murashige, T.; Skoog, F. A revised medium for rapid growth

and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15,

473497.

(15) Nagata, M.; Yamashita, I. Simple method for simultaneous

determination of chlorophyll and carotenoids in tomato fruit. Nippon

Shokuhin Kogyo Gakkaishi 1992, 39, 925928.

(16) Dalal, K. B.; Salunkhe, D. K.; Boe, A. A.; Olsen, L. E. Certain

physiological and biochemical changes in the developing tomato fruit

(Lycopersicon esculentum Mill.). J. Food Sci. 1965, 30, 504508.

(17) Crocker, W.; Knight, L. I.; Rose, R. C. A delicate seedling test.

Science 1913, 37, 380381.

(18) Guzman, ́ P.; Ecker, J. R. Exploiting the triple response of

Arabidopsis to identify ethylene-related mutants. Plant Cell 1990, 2,

513524.

(19) OMalley, R. C.; Rodriguez, F. I.; Esch, J. J.; Binder, B. M.;

ODonnell, P.; Klee, H. J.; Bleecker, A. B. Ethylene-binding activity,

gene expression levels, and receptor system output for ethylene

receptor family members from Arabidopsis and tomato. Plant J. 2005,

41, 651659.

(20) Geraats, B. P. J.; Bakker, P. A. H. M.; van Loon, L. C. Ethylene

insensitivity impairs resistance to soilborne pathogens in tobacco and

Arabidopsis thaliana. Mol. PlantMicrobe Interact. 2002, 15, 1078

1085.

(21) Geraats, B. P. J.; Bakker, P. A. H. M.; Lawrence, C. B.; Achuo, E.

A.; Höfte, M.; van Loon, L. C. Ethylene-insensitive tobacco shows

differentially altered susceptibility to different pathogens. Phytopathol-

ogy 2003, 93, 813821.

(22) ODonnell, P. J.; Schmelz, E. A.; Moussatche, P.; Lund, S. T.;

Jones, J. B.; Klee, H. J. Susceptible to intolerance a range of hormonal

actions in a susceptible Arabidopsis pathogen response. Plant J. 2003,

33, 245257.

Journal of Agricultural and Food Chemistry

Article

(23) Vidoz, M. L.; Loreti, E.; Mensuali, A.; Alpi, A.; Perata, P.

Hormonal interplay during adventitious root formation in flooded

tomato plants. Plant J. 2010, 63, 551562.

(24) Visser, E. J. W.; Voesenek, L. A. C. J. Acclimation to soil

flooding sensing and signal-transduction. Root Physiol.: Gene Funct.

2004, 254, 197214.

(25) Francia, D.; Demaria, D.; Calderini, O.; Ferraris, L.; Valentino,

D.; Arcioni, S.; Tamietti, G.; Cardinale, F. Wounding induces

resistance to pathogens with different lifestyles in tomato: role of

ethylene in cross-protection. Plant, Cell Environ. 2007, 30, 13571365.

(26) Kavroulakis, N.; Ntougias, S.; Zervakis, G. I.; Ehaliotis, C.;

Haralampidis, K.; Papadopoulou, K. K. Role of ethylene in the

protection of tomato plants against soil-borne fungal pathogens

conferred by an endophytic Fusarium solani strain. J. Exp. Bot. 2007,

58, 38533864.

(27) Cantu, D.; Blanco-Ulate, B.; Yang, L.; Labavitch, J. M. Ripening-

regulated susceptibility of tomato fruit to Botrytis cinerea requires NOR

but not RIN or ethylene. Plant Physiol. 2009, 150, 14341449.

(28) Hannan, M. M.; Biswas, M. K.; Ahmed, M. B.; Hossain, M.;

Islam, R. Combining ability analysis of yield and yield components in

tomato (Lycopersicon esculentum Mill.). Turk. J. Bot. 2007, 31, 559

563.

(29) Matsukura, C.; Aoki, K.; Fukuda, N.; Mizoguchi, T.; Asamizu,

E.; Saito, T.; Shibata, D.; Ezura, H. Comprehensive resources for

tomato functional genomics based on the miniature model tomato

Micro-Tom. Curr. Genomics 2008, 9, 436443.

(30) Pratta, G.; Zorzoli, R.; Picardi, L. A. Diallel analysis of

production traits among domestic, exotic and mutant germplasms of

Lycopersicon. Genet. Mol. Res. 2003, 2, 206213.

(31) Shalaby, T. A. Mode of gene action, heterosis and inbreeding

depression for yield and its components in tomato ( Solanum

lycopersicum L.). Sci. Hortic. 2013, 164, 540543.

(32) Buescher, R. W.; Sistruck, W. A.; Tigchelaar, E. C.; Ng, T. J.

Softening, pectolytic activity, and storage life of rin and nor tomato

hybrids. HortScience 1976, 11, 603604.

(33) Rodríguez, G. R.; Pratta, G. R.; Liberatti, D. R.; Zorzoli, R.;

Picardi, L. A. Inheritance of shelf life and other quality traits of tomato

fruit estimated from F1s, F2s and backcross generatios derived from

standard cultivar, nor homozygote and wild cherry tomato. Euphytica

2010, 176, 137147.

(34) Munns, R.; Schmidt, S.; Christine, B. Plant in Action, 2nd ed.;

(35) Thompson, A. J.; Tor, M.; Barry, C. S.; Vrebalov, J.; Orfila, C.;

Jarvis, M. C.; Giovannoni, J. J.; Grierson, D.; Seymour, G. B. Molecular

and genetic characterization of a novel pleiotropic tomato-ripening

mutant. Plant Physiol. 1999, 120, 383389.

(36) Theologis, A.; Oeller, P. W.; Wong, L. M.; Rottmann, W. H.;

Gantz, D. M. Use of a tomato mutant constructed with reverse

genetics to study fruit ripening, a complex developmental process. Dev.

Genet. 1993, 14, 282295.

(37) Barry, C. S.; Giovannoni, J. J. Ethylene and fruit ripening. J.

Plant Growth Regul. 2007, 26, 143159.

(38) Shibli, R. A.; Kushad, M.; Yousef, G. G.; Lila, M. A.

Physiological and biochemical responses of tomato microshoots to

induced salinity stress with associated ethylene accumulation. Plant

Growth Regul. 2007, 51, 159169.

(39) Nguyen, V. Q.; Ashcroft, W. J.; Jones, K. J.; McGlasson, W. B.

Evaluation of F1 hybrids incorporating the rin (ripening inhibitor)

gene to improve the storage life and fruit quality of fresh market

tomatoes (Lycopersicon esculentum Mill). Aust. J. Exp. Agric. 1991, 31,

407413.

(40) Sies, H.; Stahl, W. Lycopene: antioxidant and biological effects

and its bioavailability in the human. Exp. Biol. Med. 1998, 218, 121

124.

(41) Jeffery, D.; Smith, C.; Goodenough, P.; Prosser, I.; Grierson, D.

Ethylene-independent and ethylene-dependent biochemical changes in

ripening tomatoes. Plant Physiol. 1984, 74, 3238.

(42) Boe, A. A.; Salunkhe, D. K. Ripening tomatoes: ethylene, oxygen

and light treatments. Econ. Bot. 1967, 21, 312319.

(43) Matas, A. J.; Gapper, N. E.; Chung, M. Y.; Giovannoni, J. J.;

Rose, J. K. C. Biology and genetic engineering of fruit matura- tion for

enhanced quality and shelf-life. Curr. Opin. Biotechnol. 2009, 20, 197

203.

(44) Saladie, M.; Matas, A. J.; Isaacson, T.; Jenks, M. A.; Goodwin, S.

M.; Niklas, K. J.; Ren, X. L.; Labavitch, J. M.; Shackel, K. A.; Fernie, A.

R.; Lytovchenko, A.; ONeill, M. A.; Watkins, C. B.; Rose, J. K. C. A

reevaluation of the key factors that influence tomato fruit softening

and integrity. Plant Physiol. 2007, 144, 10121028.

(45) Dias, T. J. M.; Maluf, W. R.; Faria, M. V.; De Freitas, J. A.

Alcobaca̧ allele and genotypic backgrounds affect yield and fruit shelf

life of tomato hybrids. Sci. Agric. 2003, 60, 269275.

(46) Aly, M. A. A.; Beltagy, A. S.; Hobson, G. E. Comparison

between three tomato lines (RIN RIN, RIN rin and rin rin) in ACC

content loss in firmness and loss in weight. Acta Hortic. 1986, 190,

183190.

(47) Defilippi, B. G.; Dandekar, A. M.; Kader, A. A. Impact of

suppression of ethylene action and biosynthesis on flavor metabolites

in apple (Malus domestica Borkh) fruits. J. Agric. Food Chem. 2004, 52,

56945701.

(48) Mir, N.; Canoles, M.; Beaudry, R.; Baldwin, E.; Pal Mehla, C.

Inhibiting tomato ripening with 1-methylcyclopropene. J. Am. Soc.

Hortic. Sci. 2004, 129, 112120.

(49) Opiyo, A. M.; Ying, T. J. The effects of 1-methylcyclopropene

treatment on the shelf life and quality of cherry tomato (Lycopersicon

esculentum var. cerasiforme) fruit. Int. J. Food Sci. Technol. 2005, 40,

66573.

(50) Paulson, K. N.; Stevens, M. A. Relationships among titratable

acidity, pH and buffer composition of tomato fruits. J. Food Sci. 1974,

39, 354357.