Potential Use of a Weak Ethylene Receptor Mutant, Sletr1-2, as

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Journal of Agricultural and Food Chemistry

The potential use of a weak ethylene receptor mutant Sletr1-2 as a 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

ACS Paragon Plus Environment

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ABSTRACT

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Mutations in the ethylene receptor gene (SlETR1), Sletr1-1 and Sletr1-2, are effective in reducing

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ethylene sensitivity and improving fruit shelf life. In this study the effect of Sletr1-1 and Sletr1-2

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mutations was investigated in the F1 hybrid lines. These two mutants and control were crossed

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with four commercial pure-line tomatoes. The Sletr1-1 mutation showed undesirable pleiotropic

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effects in the F1 hybrid lines. The Sletr1-2 mutation was effective in improving fruit shelf life of

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the F1 hybrid lines for 4 to 5 days longer. It was also effective in improving fruit firmness

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without the change in fruit size, ethylene production, respiration rates and total soluble solids, as

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well as the great reduction in fruit color, lycopene and β-carotene; although the titratable acidity

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was increased by Sletr1-2 mutation. These results indicate that the Sletr1-2 mutant allele has the

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potential to improve fruit shelf-life via incorporation in tomato breeding programs.

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Keywords: ethylene, fruit shelf-life, Sletr1-1, Sletr1-2, tomato


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INTRODUCTION

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Tomato (Solanum lycopersicum L.) is a popular fleshy fruit that is grown worldwide on

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an estimated total of 4.98 million hectares of land.1 The qualities of the tomato are important

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whether the tomato fruit is sold on the market or to industry. Such qualities determine whether

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the fruit is acceptable to consumers and influence the price/value of the fruit on the market. The

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tomato fruit must have high-quality attributes of appearance, performance, texture, flavor,

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nutritional value and safety.2 Furthermore, the shelf-life of the fruit is an important characteristic

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for commercial markets.3

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Breeding to extend the shelf-life of tomato fruit extends not only its storage time but also

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its marketability. Because tomato is a climacteric fruit, shelf-life is commonly affected by the

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presence of ethylene. Ethylene accelerates fruit ripening, which contributes to changes in the

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nutrient content and other compounds; however, it also accelerates quality deterioration by

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shortening the shelf-life of the fruit. Ripening is a normal process during fruit maturation.

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Several biochemical and physiological changes occur, such as those involving flavor, color,

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aroma, and texture, and there are also increases in ethylene production and respiration.4 The

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color change from green to red in tomato fruits is an indication of the conversion of chloroplasts

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to chromoplasts in which chlorophyll is degraded and carotenoids accumulate. Fruit softening

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and structural alterations are other physiological changes that occur during fruit maturation; such

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changes are modified and partially disassembled by enzymes.5

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Most farmers harvest tomato fruit at either the mature green or breaker stage to minimize

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damage and to enhance shelf-life during handling and marketing.6 However, different stages of

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maturation result in lower homogeneity, which contributes to decreases in fruit quality and

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market acceptability. The use of an ethylene-insensitive tomato cultivar can allow farmers to


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avoid harvesting fruit at different maturation stages, especially at the mature green and/or

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immature green stages; instead, all the fruit can be harvested at the red stage and still have a long

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shelf-life. Several ripening mutants conferring long shelf-life have been isolated, such as

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ripening-inhibitor (rin), non-ripening (nor), never ripe (NR), and alcobaca (alc).7 Recently,

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other tomato mutants that are capable of delaying the normal process of ripening have been

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generated from the Micro-Tom library, including Sletr1-1 and Sletr1-2. Compared with the wild

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type, Sletr1-1 and Sletr1-2 mutants show lower ethylene sensitivities, including completely and

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moderately ethylene-insensitive phenotypes, respectively.8

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Different locations of mutations in ethylene-related genes correlate with sensitivity to

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ethylene. In Arabidopsis and tomato, mutations in the transmembrane domain region of the

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ethylene receptor gene dramatically affect ethylene sensitivity.9,10 In Sletr1-1 and Sletr1-2, the

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mutations are located in a different transmembrane region of the ethylene receptor. P51L for

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Sletr1-1 and V69D for Sletr1-2 are located in the first and second domains of the transmembrane

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region, respectively.8 The ethylene inhibition mutation has a positive effect on delaying the

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ripening process, as well as undesirable roles in other fruit quality characteristics. For instance,

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in homozygous mutants of Nr, nor, Sletr1-1, and Sletr1-2, the shelf-life of the fruit is extended;

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however, the carotenoid content is reduced, except in Sletr1-2.8,11 The use of heterozygous

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ethylene-insensitive mutants can contribute to the agronomic improvement of tomato shelf-life

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but results in a slight reduction of the red pigment. In nor mutants, the heterozygous condition

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delays the start of ripening and increases the interval between the breaker and the table-ripe

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stages.12 In rin mutants, the heterozygous condition delays initiation, and ripening proceeds more

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slowly than normal ripening,12 whereas heterozygous Sletr1-1 exhibits impaired petal

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abscission.8 In the ripening process, heterozygous Nr mutants have a more pronounced


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impairment than Sletr1-1 and Sletr1-2, so those mutants are more suitable than Nr for use in

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breeding programs for the development of a long-shelf-life tomato. Additionally, Sletr1-2

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showed prolonged fruit shelf-life without a reduction in carotenoid content.8

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Crossing commercial tomato cultivars with ripening mutants that have prolonged fruit

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shelf-life, rin, Sletr1-1, and Sletr1-2, may facilitate the development of an F1 hybrid line that has

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the desired characteristic. Several studies have shown that F1 hybrid lines of rin exhibited

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prolonged shelf-life.13 Hybrids of the nor mutant locus have an extended shelf-life and

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intermediate fruit color but no differences in pH, soluble solids content or fruit size.12 However,

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not all ethylene-insensitive mutants can be used for improving the postharvest characteristics of

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tomato in breeding programs; for instance, Nr has an incomplete ripening phenotype, even in the

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heterozygous form (Nr/nr), and also has insufficient red coloration.7

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High-quality tomato fruit, such as fruit with a long shelf-life and high nutrient content, is

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attractive for consumers. Generating new F1 hybrid lines from Sletr1-1 and Sletr1-2 by crossing

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these mutants with large commercial tomato cultivars will improve tomato fruit quality by

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combining the prolonged shelf-life of these mutants with other desired characteristics from

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commercial tomato cultivars. The objective of this study was to evaluate the effect of the Sletr1-

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1 and Sletr1-2 mutation on the plant development and fruit quality characteristics of F1 hybrid

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lines of Sletr1-1 and Sletr1-2 crossed with four commercial pure-line tomato cultivars: ‘Aichi

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First’, ‘Ailsa Craig’, ‘Moneymaker’, and ‘M82’.

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MATERIALS AND METHODS


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Plant materials and cultivation

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Two ethylene receptor (SlETR1) mutant alleles, Sletr1-1 and Sletr1-2, and the wild-type

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Micro-Tom (WT-MT) were crossed with four pure-line cultivars (‘Aichi First’, ‘Ailsa Craig’,

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‘Moneymaker’, and ‘M82’). Tested plants were cultivated with four biological replications using

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the nutrient film technique (NFT) cultivation system in the greenhouse of the Agricultural and

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Forestry Research Center, University of Tsukuba, Japan, during the winter season from October

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2013 to February 2014. At the beginning of the plants’ cultivation, seeds were sown in paper

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pots 10 cm in diameter filled with commercial coir coco peat as a growing medium and were

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fertilized with a commercial nutrient solution (OAT house A, OAT Agrio Co., Ltd, Tokyo,

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Japan) that had an electric conductivity (EC) level of 1-1,2 dS/m. After plants produced 5–6 true

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leaves, they were transplanted into the NFT cultivation system and irrigated with a commercial

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nutrient solution (OAT house A, OAT Agrio, Tokyo, Japan) with an EC level of 2–2.5 dS/m.

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Four trusses per plant were maintained for obtaining a test-fruit sample. When the fruits were

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showing a change of less than 10% in the surface fruit color from green to yellow, pink or red,

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they were tagged as being at the breaker stage (Br). Some parameters were investigated during

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plant growth and development, e.g., number of leaves under the 1st truss, time to BR, number of

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flowers per truss, and number of fruits per truss.

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Ethylene triple response

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To investigate the ethylene sensitivity of the seedlings, we investigated all of the F1

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hybrid lines via the addition of exogenous ethylene at different concentrations (0, 0.1, 1, 2.5, and

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5 ppm). Seeds were sterilized for 20 minutes by being soaked in 10% commercial bleach

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including a detergent (Kitchen Haiter, Kao, Tokyo, Japan), and then rinsed with sterilized water


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three times for 5 minutes. Three sterilized seeds were grown in a 50-mL glass bottle containing

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10 mL of 1/2 Murashige and Skoog (MS) and sealed with rubber septum caps.8,14 Using a syringe,

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exogenous ethylene at desired concentrations were injected into the sealed glass bottles

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containing seeds. The seeds were grown and kept in the dark at 25 °C for 7 days. The seedling

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triple response was investigated by measuring the hypocotyl and root lengths.

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Fruit characteristic analysis

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The harvested fruits were evaluated to elucidate fruit characterization. All of the tomato

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fruits were harvested at 6 days after the breaker stage (Br + 6), which was characterized by more

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than 60% and less than 90% of the fruit surface having changed to a red color or when the fruit

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was at the light red stage. The harvesting time was initiated as 0 days post-storage (DPS) and

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was used as the initial time of fruit storage for the shelf-life analysis. The evaluated

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characteristics were fruit diameter, length, weight, locule number, and pericarp thickness.

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Evaluation of fruit shelf-life

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The Br date was tagged to determine a fruit harvesting time and the stage of fruit

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maturation. Investigated fruits were stored on the laboratory bench under the following

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conditions: temperature 20 ± 2 °C and humidity 80%. To evaluate shelf-life, fruits were

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harvested at the same maturation stage (Br + 6), which was initiated at 0 DPS when the harvested

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fruit was at the light red stage and its fruits would be acceptable to consumers in the market. The

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shelf-life was determined by counting the days from the beginning of storage until the fruit

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quality was lost, which was indicated by the appearance of black spots or wrinkling of more than

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10% of the fruit surface.


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Determination of ethylene production and respiration rate

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To elucidate the ethylene production and respiration rate in F1 hybrid lines of Sletr1-2,

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fruits at the same stage of maturation (Br + 6) were analyzed. The analysis was repeated every

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four DPS for a total of 12 days. The harvested fruits were kept for 2 to 3 hours before

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measurement to minimize the contamination of ethylene production from the wound caused by

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harvesting the fruit from the stalk. 2 fruits were stored in a 440-mL sealed plastic chamber at 25

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± 2 °C for 60 minutes. A 1-mL gas sample was taken from the headspace of the chamber using a

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syringe to determine ethylene production and the respiration rate. For ethylene production, the

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gas sample was analyzed using a gas chromatograph (GC-18A; Shimadzu, Kyoto, Japan)

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equipped with a Porapak Q (Mesh 60/80) column and flame ionization detector, with Helium

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(He) as the carrier gas, and injector, cooling, and detector temperatures of 100 °C, 130 °C, and

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100 °C, respectively. The respiration rate was analyzed using a gas chromatograph (GC-8A;

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Shimadzu, Kyoto, Japan) equipped with a photoionization detector, with He as the carrier gas

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and a 180 and 70 °C detector/injector temperature and column temperature, respectively.

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Ethylene production and respiration rate were represented by µg/g FW/h.

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Fruit color analysis

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Fruit color was evaluated to assess the effects of the mutations in the SlETR1 gene of the

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Sletr1-2 allele on color during the ripening process. Fruit color was evaluated during storage and

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every 10 days until fruit quality was lost, to a maximum of 30 DPS. The measurement of fruit

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color was performed according to the CIELAB color space analysis. Color measurements were

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taken from three points on the surface of individual fruits (the middle, base, and ends of the fruit)


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using a Minolta Color Reader CR-10 (Konica Minolta Sensing, Inc., Osaka, Japan). Three color

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parameters were represented as the lightness of the color/brightness (L*), the red to green scale

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(a*), and the yellow to blue scale (b*). a* and b* were used to measure the hue (h°), which

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indicates the pure spectrum colors or the gradation of color, using the following equation:

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b h = arctan  a

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An increased L* value indicated that the fruit had more brightness, and a decreased h° indicated

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that the fruit color had changed to red.

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Carotenoid analysis

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Because tomato contains high levels of carotenoids, especially lycopene and β-carotene,

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it has become popular for human consumption. The lycopene and β-carotene content were

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analyzed every 10 days from the beginning of storage until fruit quality was lost, to a maximum

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of 30 DPS. Frozen fruit was ground into a fine powder in liquid nitrogen, and 300 mg of tomato

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powder was extracted with 3 mL acetone:hexane (4:6/v:v), resulting in a clear supernatant as

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described by Nagata and Yamashita.15 Absorption was measured using a spectrophotometer

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(Beckman Coulter DU 640 Spectrophotometer, Fullerton, CA, USA) at absorbances of 663

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(A663), 645 (A645), 505 (A505) and 453 nm (A453). Lycopene (CLYC) and β-carotene (CCAR) were

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measured using the following equation:

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CLYC = -0.04584(A663) + 0.204(A645) + 0.372 (A505) – 0.0806(A453)

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CCAR = 0.216(A663) - 1.22(A645) - 0.304 (A505) + 0.452(A453)

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The lycopene and β-carotene content was represented by µg/g FW.

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Fruit firmness analysis


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Firmness is an important characteristic for the marketability of tomato fruit. To analyze

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the effect of the Sletr1-2 mutation, we tested the firmness of the fruits. Br+6 fruits were tested

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using an Ultrasonic Hardness Tester Model FHR-5 (Nippon Optical Works Co., Ltd, Tokyo,

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Japan). The measurement was repeated every 10 days until 30 DPS.

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Analysis of the total soluble solids (TSS), pH and titratable acidity (TA)

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To evaluate the effect of the Sletr1-2 mutation on four Sletr1-2 F1 hybrid lines, we

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measured the TSS, pH and TA. The TSS was used to estimate the sugar level, and TA was used

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to estimate the organic acid level. The TSS was measured using a refractometer PAL-J (Atago,

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Tokyo, Japan). The pH and TA was measured using a pH meter SevenCompactTM pH/Ion S220

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(Mettler-Toledo AG, Schwerzenbach, Switzerland). The TA was measured using pH meter

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methods by titration of 0.1 N sodium hydroxide up to a pH of 8.1 as described by Dalal et al.

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(1965).16

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Data analysis

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This experiment was conducted in a completely randomized design with four replicates.

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For statistical data analysis, to test the normality of the data we used Kolmogorov-Smirnov test.

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Based on this test, we found out the data have normal distribution. Therefore, one factor analysis

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of variance (ANOVA) was conducted to analyze the data followed by Tukey-Kramer test at p

Potential Use of a Weak Ethylene Receptor Mutant, Sletr1-2, as

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