Utilization of a Genome-Edited Tomato (Solanum lycopersicum) with

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Utilization of a genome-edited tomato (Solanum lycopersicum) with high gamma aminobutyric acid content in hybrid breeding Jeongeun Lee, Satoko Nonaka, Mariko Takayama, and Hiroshi Ezura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05171 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Journal of Agricultural and Food Chemistry

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Utilization of a genome-edited tomato (Solanum lycopersicum) with high gamma

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aminobutyric acid content in hybrid breeding

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Jeongeun Lee1, Satoko Nonaka2,3, Mariko Takayama2, and Hiroshi Ezura2,3*

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1: Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1

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Tennodai, Tsukuba, Ibaraki 305-8572, Japan

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2: Tsukuba Plant Innovation Research Center, University of Tsukuba, 1-1-1 Tennodai,

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Tsukuba, Ibaraki 305-8572, Japan

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3: Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai,

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Tsukuba, Ibaraki 305-8572, Japan

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* Corresponding author: Tel/Fax: +81298537263; E-mail: [email protected]

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Abstract

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γ-Aminobutyric acid (GABA) is non-proteogenic amino acid with health-promoting

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functions. Although tomato fruits have a relatively high GABA content compared with other

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crops, levels must be further increased to effectively confer the health-promoting functions.

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In this study, we evaluated the potential of the genome-edited tomato as a breeding material

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for producing high-GABA hybrid tomatoes. Hybrid lines were produced by crossing the

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genome-edited tomato with a pure line tomato cultivar, ‘Aichi First’, and were evaluated for

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GABA accumulation and other fruit traits. The hybrid lines showed high GABA

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accumulation in the fruits, which was sufficiently high for expecting health-promoting

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functions and had minimal effects on other fruit traits, suggesting that the high GABA is a

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dominant trait and that the genome-edited tomato would be useful as a parental line of hybrid

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cultivars. These results also indicate that genome editing technology is useful for the rapid

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breeding of high-GABA hybrid tomato cultivars.

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Key words: γ-Aminobutyric acid, Solanum lycopersicum, Health-promoting function,

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Genome-edited, Dominant trait

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Introduction

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γ-Amino butyric acid (GABA) is non-proteogenic amino acid present in bacteria, fungi,

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animals, and plants. GABA is also known to be an inhibitory neurotransmitter in the central

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nervous system.1 GABA is effective in the prevention of certain life style-related diseases,

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including hypertension2-6 and diabetes7, and also has antistress properties.8 In the initial

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treatment of patients with life style-related diseases, dietary therapy is preferred to

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medication. Several studies on humans and experimental animals have shown that

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administration of exogenous GABA is effective in lowering blood pressure in patients with

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mild high blood pressure or high normal blood pressure.2-6 GABA also decreases the glucose

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level in diabetics.7 Intake of GABA through the daily diet helps to prevent “life style-related

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diseases” from becoming serious. Although several foods, such as tea, rice, and fermented

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products (i.e., fermented milk, tempeh, yogurt, and soy sauce), contain GABA,9-13 the

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concentration of GABA is generally insufficient for preventing life style-related diseases, and

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thus GABA enrichment of food is necessary to confer a health-promoting effect.

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Since GABA has the potential function of promoting human health, and also plays

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important roles in the plant life cycle, many scientists have attempted to clarify the pathways

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of GABA synthesis and metabolism. In higher plants, GABA is predominantly metabolized

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through a pathway referred to as the “GABA shunt,” which bypasses two steps of the

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tricarboxylic acid (TCA) cycle. In this pathway, GABA is mainly produced from glutamate

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by an irreversible reaction catalyzed by the cytosolic enzyme glutamate decarboxylase

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(GAD).14,15 A secondary pathway of GABA synthesis may be via polyamine (putrescine and

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spermidine) degradation.15,16 Under oxidative stress, a non-enzymatic conversion from

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proline has also been reported.17 GABA is degraded by the action of GABA transaminase

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(GABA-T) in the mitochondrial matrix. In this reaction, succinic semi-aldehyde (SSA) is 3 ACS Paragon Plus Environment


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produced with the possible participation of several amino acceptors, such as α-ketoglutarate,

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pyruvate, or glyoxylate.18,19 Subsequently, SSA is converted by SSA dehydrogenase

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(SSADH) to succinate in the mitochondria,19,20 and then SSA re-enters the TCA cycle.

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Alternatively, SSA can be converted to γ-hydroxybutyric acid (GHBA) via the action of GHB

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dehydrogenase (GHBDH).21,22

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Tomato contains relatively higher levels of GABA than other plants and/or crops.23 In

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addition, tomato is one of the most extensively produced vegetables in the world and widely

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consumed in the daily diet. In cultivated tomato, GABA accumulates to a high degree in

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mature green fruits (comprising up to 50% of the total free amino acids); however, when the

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fruits ripen, GABA levels decrease to less than 20% of the total free amino acids.24 To

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understand the molecular mechanisms underlying the changes in GABA accumulation in

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tomato fruits, our previous study isolated GABA metabolism-related genes from the tomato

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cultivar ‘Micro-Tom’.25 Three SlGAD genes (SlGAD1, SlGAD2, and SlGAD3), which are

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involved in GABA synthesis, three SlGABA-T genes (SlGABA-T1, SlGABA-T2, and

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SlGABA-T3) related to GABA degradation, and an SlSSADH gene were identified.25

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Several studies indicated that regulation of SlGADs was deeply involved in GABA

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accumulation. Suppression of SlGAD2 and/or SlGAD3 reduced GABA accumulation in

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mature green fruits and red ripe fruit.26 Conversely, over-expression of SlGAD3 driven by the

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E8 promoter (a fruit-specific promoter) increased the GABA content in red ripe fruit without

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affecting plant growth.27 In contrast, although inhibition of SlGABA-T gene expression

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increased GABA accumulation, a strong inhibition of plant growth was observed.28 These

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results indicate that upregulation of SlGAD gene expression and/or enzymatic activity is more

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effective in increasing GABA accumulation in the fruit than downregulation of SlGABA-T.

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Plant GADs generally contain an autoinhibitory domain at the C terminus. This domain

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is composed of 30–50 amino acids and it also functions as the calmodulin (CaM)-binding 4 ACS Paragon Plus Environment

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domain (CaMBD). It is considered that this domain inhibits GAD activity at physiological pH

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by folding its active site. This autoinhibition is suppressed when the active site is unfolded by

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conformational changes promoted via an acidic pH and/or by the binding of Ca2+/CaM to the

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CaMBD.29 Thus, it is assumed that removal of the C terminus containing this autoinhibitory

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domain allows the enzyme to be constitutively active, resulting in high GABA accumulation.

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Therefore, modification of the C terminus of SlGADs would appear to represent an effective

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approach in breeding tomatoes with high GABA accumulation. Indeed, in our previous study,

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we used a transgenic approach to truncate the C terminus of SlGAD3, which resulted in

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increased GABA levels.27 Furthermore, we succeeded in breed a new high-GABA content

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tomato line via removal of the C terminus of SlGAD3 using site-specific targeted mutagenesis

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(genome editing; CRISPR/Cas9) technology in the dwarf experimental cultivar ‘Micro-Tom’,

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which we named TG3C37#21-19 and TG3C37#21-7.30

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Similar to other vegetable crops, the commercially distributed tomatoes sold in markets

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are mainly hybrid cultivars, because hybrid tomatoes generally combine the good

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characteristics of both parents, and have considerably better marketability as a consequence

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of their high fruit quality.31 Many trials to improve the productivity and attractive appearance,

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including color and size, of hybrid tomatoes have been carried out over a number of years;

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however, the most important consideration for producing hybrids is to identify or develop a

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valuable parental line for hybrid breeding. Recently, there has been a trend to develop

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tomatoes with regard not only to the appearance of fruit but also to their nutrient value.31

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After removal of the transformation cassette, mutations remained in two TG3C37 lines

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(TG3C37#21-19 and TG3C37#21-7) that had an effect on GABA content. We decided to

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cross TG3C37s (TG3C37#21-19 and/or TG3C37#21-7) to the commercial cultivar ‘Aichi

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First’ to evaluate the effect of TG3C37s (TG3C37#21-19 and TG3C37#21-7) used as a single

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parent for producing an F1 hybrid. In the present study, we analyzed two tomato hybrid lines 5 ACS Paragon Plus Environment


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derived from TG3C37(TG3C37#21-19 and TG3C37#21-7) containing a single allele with a

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truncated C terminus and evaluated their fruit quality. Finally, we evaluate the genome of

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edited lines as parental lines for hybrid breeding.

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Materials and Methods

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Plant material

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Two T1 generation tomato lines, TG3C37#21-7 and TG3C37#21-19, containing

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mutations generated via CRISPR/Cas9 at the C terminus of the SlGAD3 gene (Table 1),30 and

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the wild-type of ‘Micro-Tom’ (WT-MT) were cultivated in a culture room under fluorescent

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light with a 16 h light (60 µmol/m2/s)/8 h dark photoperiod at 25°C, using standard nutrient

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solution (Otsuka A; Otsuka Chemical Co., Ltd., Osaka, Japan). For producing F1 generation

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plants, pollen from the wild type of the commercial pure line cultivar ‘Aichi First’ was used

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to pollinate the emasculated pistils of the two TG3C37 lines and WT-MT before flower

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opening (F1 lines; F1-WT, F1#21-7, and F1#21-19). F1 seeds were germinated on wet filter

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paper and transferred to rockwool at 2 weeks after germination. Seedlings were cultured in a

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culture room as described above for 1 month (to the 5 to 6 true leaf stage), and then

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transferred to a semi-containment greenhouse in the Gene Research Center of the University

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of Tsukuba, Japan. All plants were fertilized using an NFT cultivation system and irrigated

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with Otsuka standard nutrient solution at an EC level of 1.8–2.2 dS/m. Cultivation for this

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experiment was carried out from April to August 2017. Pollination of F1 plants was carried

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out up to the 6th truss from the bottom of each plant and additional trusses from the 7th were

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discarded. After observing enlargement of three fruits per truss, other flowers on the same

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truss were discarded. Fruits were harvested at three stages: the Mature green, Breaker, and

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Red stages. The “Mature green (MG)” stage fruits are the fruits pollinated at the same time to 6 ACS Paragon Plus Environment

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-2 days before the BR fruits, but have yet to break. The “Breaker (BR)” stage is that in which

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the fruit color changes from green to yellow. The “red (RED)” fruits are harvested 10 days

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after the BR day. The time to BR was counted during cultivation.

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Genetic analysis

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The F1 generation was confirmed using the dCAPS method and sequencing. More

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specifically, genomic DNA was extracted using a modified CTAB method.32 Leaf material

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(200 mg) was ground to a fine powder and pre-warmed CTAB at 65°C was added to each

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sample. After adding the same volume of chloroform/isoamyl alcohol (24:1, v/v) followed by

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inverting the contents a few times, all samples were centrifuged at 13,000 rpm for 15 min at

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4°C. The resulting clear supernatant was transferred to a new tube and the same volume of

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iso-propanol was added. After maintaining at -20°C for 2 h, the mixture was centrifuged at

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13,000 rpm for 15 min at 4°C and the supernatant was carefully discarded so as not to lose

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the DNA pellet. The pellet was rinsed using 70% (v/v) ethanol. After briefly drying at room

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temperature, the pellet was re-suspended in 50 µl of TE (10 mM Tris, 1 mM EDTA, pH 8.0)

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containing 40 µg/ml of RNase A (Amerson).

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For dCAPS analysis, PCR was carried out using SlGAD3 C-terminal-specific primers 5

′

-GATGAAGGTATACCCTTGGTGG-3

′

(forward

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-CACTCTCGCCTCTATCGCTTAT-3′) using 100 ng of gDNA. A 50-µg aliquot of the

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amplicon was incubated with BstXI (NEB) for 3 h at 37°C, and loaded onto a 2% agarose gel.

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Selected F1 plants were sequenced for more precise confirmation. The PCR product was

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diluted 50 folds with distilled water and used as a template. The forward primer used for

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gene-specific PCR was used for sequencing.

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reverse

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′

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Fruit characteristic analysis

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The diameter, length, weight, and color of harvested fruits at the MG, BR, and RED

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stages were measured to investigate the effect of the mutated SlGAD3 allele of TG3C37 on F1

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fruit appearance compared with the WT F1 hybrid. Fruit diameter was measured twice and

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length was measured once. The fruits were weighed after removing old sepals and the

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pedicle. For fruit size comparison, averages of the diameter, length, and weight of 20 fruits

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were used. Color was measured three times on the surface of the middle of fruits using a

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

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parameters assessed were the lightness, indicating the range from black (0) to white (100)

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(L*), and color direction, indicating the red to green scale (a*) and the yellow to blue scale

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(b*). For color calculation, the average of the L*, a*, and b* values was used from 20 red

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fruits of each line. Chroma, the saturation of color, was calculated as (a2+b2)1/2. Hue

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classification of red, yellow, blue, and green, was calculated by arctan(b/a). Statically, the

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results obtained for each F1 line were compared with those for the WT F1 hybrid at P < 0.05.

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Carotenoid analysis

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β-carotene and lycopene contents were analyzed according to previously described

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methods.33 Three milliliters of acetone/hexane (4:6 v/v) was added to 300 mg of frozen fruit

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powder. After brief vortexing, the clear supernatant was used for analysis. Absorption was

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measured at 663 nm (A663), 645 nm (A645), 505 nm (A505), and 453 nm (A453) using a

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Beckman Coulter DU 640 spectrophotometer (Fullerton, CA, USA). Lycopene and

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β-carotene contents were calculated according to following equations:

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

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β-carotene = 0.216(A663) – 1.22(A645) – 0.304(A505) + 0.452(A453)


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The values for lycopene and β-carotene compared with those for the WT F1 hybrid were

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analyzed statistically at P < 0.05.

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Total soluble solids (TSS) and titratable acidity (TA)

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TSS and TA were measured to estimate sugar and organic acid levels, respectively.

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TSS were measured from 300 mg of frozen fine powder of red-stage fruits using a PAL-J

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refractometer (Atago, Tokyo, Japan). TA was measured using a pH meter as described

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previously.34 One gram of red fruit powder was mixed with 10 ml of distilled water, and

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titrated using 0.1 N sodium hydroxide up to pH 8.1. Nine red fruits were used for TSS

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analysis and five were used for TA analysis. Measured TSS and TA values were compared

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statistically at P < 0.05.

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GABA and free amino acid contents

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Fruits at the MG, BR, and RED stages were harvested and jelly tissue and seeds were

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removed. The fruits were immediately frozen and crushed in liquid nitrogen. Free amino acid

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was extracted from 50–70 mg of the fine powder with 500 µl of 8% trichloroacetic acid

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(w/v). After brief shaking, the mixture was centrifuged at 12,000 rpm for 20 min. Three

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hundred microliters of the resulting supernatant were transferred to a new tube, to which 400

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µl of di-ethyl ether was added. After 10 min of vigorous shaking, the mixture was centrifuged

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at 12,000 rpm for 10 min. After removing the di-ethyl ether, this step was repeated once more.

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Thereafter, the di-ethyl ether was evaporated in a chamber for 30 min at room temperature.

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Sixty microliters of extracted amino acids (extracted as described above) was purified. The

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amino acid solution was dried at 60°C under vacuum. The resulting pellet was dissolved in

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150 µl of distilled water and subsequently re-dried. After repeating this step again, the pellet

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was dissolved in 300 µl of 0.1 N HCl, and analyzed using a JLC-500/V2 amino acid analyzer 9 ACS Paragon Plus Environment


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(Japan Electron Optics Laboratory). Analysis condition followed the instruction of

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JLC-500/V2: using citric acid lithium buffer, ion exchange resin column, the ninhydrin color

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development method, and absorbance of 570nm and 440nm.

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Statistical analysis

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Measurement of fruit length, diameter, weight and color were conducted in a

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completely randomized design with twenty replicates. Nine fruits for TSS and 5 fruits for

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lycopene, β-carotene and TA from each line were used. All data were analyzed using Tukey’s

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HSD test at P < 0.05 and expressed as mean value ± SD to compare differences among

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investigated lines.

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Results

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Genetic confirmation of the C-terminal region of F1 generation SlGAD3

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Using the dCAPS method and sequence analysis, we checked the genotypes of the F1

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lines ‘Micro-Tom’ TG3C37#21-19 × ‘Aichi First’ WT (F1#21-19) and ‘Micro-Tom’

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TG3C37#21-7 × ‘Aichi First’ WT (F1#21-7) (Figure 1). In TG3C37, the C terminus of

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SlGAD3 contains a mutation upstream of the autoinhibitory domain in the region of the

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Ca2+/calmodulin-binding domain (Figure 1a), resulting in a loss of the autoinhibitory domain

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region. TG3C37#21-19 and TG3C37#21-7 have 1-nucleotide and 53-nucleotide deletions and

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an introduced stop codon upstream of the autoinhibitory domain, respectively [Figure 1d and

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e (left), Table 1].30 The target site of the WT possesses a BstXI site, whereas in

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TG3C37#21-19 and TG3C37#21-7 this restriction enzyme recognition site is lost (Figure 1b).

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Accordingly, although the WT of ‘Micro-Tom’ and ‘Aichi First’ were clearly digested into

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two bands, the two TG3C37 lines (T1) were not. Using dCAPS analysis, F1#21-19 and 10 ACS Paragon Plus Environment

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F1#21-7 showed three bands, representing the two digested bands from ‘Aichi First’ WT and

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an un-digested band from ‘Micro-Tom’ TG3C37 (the parental lines). This result confirmed

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the heterozygous mutation in F1#21-19 and F1#21-7 (Figure 1c and Figure S1). For a more

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precise confirmation, sequences at the target site were also analyzed. In F1 generation plants,

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two peaks were detected downstream of the mutation site (Figure 1d and e).

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GABA content in F1 lines

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To determine whether the heterozygous mutation of SlGAD3 was effective in

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promoting high GABA accumulation in the F1 generation plants, GABA accumulation in

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F1#21-19, F1#21-7, and F1-WT were measured by HPLC. Three fruits from each line were

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used for this analysis at three fruit development stages (MG, BR, and RED). In all of the F1

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lines, the highest GABA accumulation was observed in MG fruit, and decreased during

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ripening (Figure 2). Compared with the F1-WT, a higher GABA level was detected

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throughout the entire fruiting stage of F1#21-19 and F1#21-7. In F1#21-19, the GABA

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concentration was 33.58 ± 6.35, 26.80 ± 1.66, and 18.50 ± 2.09 µmol/gFW (mean ± SD, n =

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3) at the MG, BR, and RED stages, respectively. F1#21-19 contained 2.1, 2.5, and 3.6 times

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higher GABA than F1-WT at these stages, respectively. Similarly, the GABA concentration

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in F1#21-7 was 31.13 ± 0.24, 21.56 ± 4.24, and 12.76 ± 1.08 µmol/gFW (mean ± SD, n = 3)

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at the MG, BR, and RED stages, respectively, which were 2.3-, 2.1-, and 3.0-fold higher than

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those in the corresponding stages of F1-WT. In both F1#21-19 and F1#21-7, the reduction

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ratio of GABA was lower than that of F1-WT. The reduction ratios (RED/MG) of GABA in

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F1-WT, F1#21-19, and F1#21-7 were 66.7%, 44.9%, and 59%, respectively. These results

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indicate that the heterozygous mutation in SlGAD3 was effective in promoting an

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up-regulation of GABA accumulation in the F1 generation, and the high GABA phenotype

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was a dominant trait. 11 ACS Paragon Plus Environment


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Content of other free amino acids in F1 lines

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The extremely high accumulation of GABA in fruit has previously been shown to affect

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the levels of other free amino acids.27 Therefore, we investigated whether the high

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accumulation of GABA in F1 generation fruit affects amino acid metabolism (Figure 3, Table

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S1). Free amino acids were measured by HPLC at three stages of fruit development (MG,

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BR, and RED). In F1#21-19 and F1#21-7, the levels of Ala, Asp, and Glu were increased,

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whereas GABA, Ser, and Val decreased during the fruit ripening process. Other free amino

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acids showed the same level from MG to RED. The same tendency was observed in F1-WT.

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Although the concentrations of Asp and Glu in F1#21-19 and F1#21-7 at the MG stage were

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lower than those in F1-WT, in the RED stage, the concentrations of all other free amino acids

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were similar among F1-WT, F1#21-19, and F1#21-7, and, with the exception of GABA, no

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statistically significant differences were observed. These results indicate that higher GABA

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concentrations in F1#21-19 and F1#21-7 did not have any significant effects on the levels of

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other free amino acids in RED tomato fruit.

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Color of the red fruit of F1 lines

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To investigate whether the mutated SlGAD3 allele and high GABA accumulation were

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associated with fruit color, coloration was observed during the fruit ripening process (Figure

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4a). No differences were observed in coloration among the F1 lines. To estimate in more

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detail, color was measured using a color difference meter in 20 red-stage fruits (Breaker + 10

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days) of F1 lines. In the evaluation of fruit color (Table 2), there were no significant

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differences in any values among F1 lines. The value of Chr (chroma) in F1#21-7 were slightly

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higher than F1#21-19 and F1-WT. These results indicate that F1#21-7 had a slightly stronger

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yellow color than the other F1 lines. Since a slight difference was found in the color of the 12 ACS Paragon Plus Environment

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red-stage fruit of F1#21-7, the levels of lycopene and β-carotene among F1 lines were

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compared. The content of lycopene (red pigment) in F1-WT, F1#21-19, and F1#21-7 was 36.2

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± 8.1, 45.5 ± 10.2, and 38.1 ± 9.0 µg/g FW (mean ± SD, n = 5), respectively. Although the

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lycopene level tended to be higher in F1#21-19, the difference was not significant (Figure 4b).

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The contents of β-carotene (yellow color pigment) were almost the same among the F1 lines

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(Figure 4c), with concentrations of 7.7 ± 1.2, 7.1, ± 1.9, and 7.1 ± 1.7 µg/g FW (mean ± SD,

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n = 5) in the F1-WT, F1#21-19, and F1#21-7 lines, respectively. Although F1#21-7, which

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contained the highest GABA levels, showed slightly stronger yellow color, β-carotene

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concentration was similar in F1-WT. These result suggest that “the slightly stronger yellow

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color” in F1#21-7 is not meaningful differences caused by the yellow color pigment.

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Therefore, the mutated SlGAD3 allele with high GABA accumulation seemed not to affect

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colorization.

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Total soluble solids and titratable acid in F1 lines

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Since taste is one of the most important fruit traits, we examined whether higher GABA

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accumulation affects the taste of red ripe fruit. Total soluble solids (TSS, Brix) and titratable

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acid (TA) were measured in the red-stage fruit (Breaker + 10 days) of F1 lines. The values of

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TSS and TA are indicators of sugar and organic acid concentrations, respectively. The Brix

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values of F1#21-19 and F1#21-7 were found to be slightly lower than that of F1-WT (Figure

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5a): 4.9 ± 0.30, 4.4 ± 0.31, and 4.3 ± 0.27 (mean ± SD, n = 9) for F1-WT, F1#21-19, and

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F1#21-7, respectively. In contrast, the TA levels of F1#21-19 and F1#21-7 were increased by

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20% and 10%, respectively, compared with that of F1-WT (Figure 5b): 0.49 ± 0.02, 0.60 ±

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0.04, and 0.55 ± 0.02 for F1-WT, F1#21-19, and F1#21-7 (mean ± SD, n = 5), respectively.

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These results suggest that higher GABA accumulation influences fruit taste.

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Effect of the mutated SlGAD3 allele on fruit size and weight

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We further examined whether the mutated SlGAD3 allele had any effects on fruit size

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and weight. To evaluate differences in fruit size, RED fruit diameters (horizontal length and

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vertical length) were measured. The size of all F1#21-19 and F1#21-7 fruits was similar to

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that of F1-WT. The average horizontal lengths of the fruits of F1-WT, F1#21-19, and F1#21-7

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were 52.3 ± 7.3, 52.6 ± 5.5, and 51.5 ± 6.2 mm (mean ± SD, n = 20), respectively (Figure 6a).

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The corresponding vertical lengths of fruit were 43.9 ± 3.8, 45.7 ± 3.1, and 46.2 ± 4.1 mm

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(mean ± SD, n =20). No statistically significant differences in fruit size were observed among

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the lines (P < 0.05) (Figure 6b). The average weights of RED fruit in F1-WT, F1#21-19, and

316

F1#21-7 were 76.2 ± 29.2, 77.8 ± 22.0, and 75.7 ± 21.4 g (mean ± SD, n = 20), respectively.

317

Although there was a relatively large variation in fruit weight within lines, no statistically

318

significant differences were observed in the fruit weight of different lines (Figure 6c). These

319

results indicated that although mutation of SlGAD3 increased the GABA content in red fruit,

320

the mutation of this allele did not affect fruit size or weight.

321 322

Discussion

323

In this study, we used a dwarf experimental cultivar, ‘Micro-Tom’, containing a

324

SlGAD3∆C allele mutated using the CRISPR/Cas9 system, and the commercialized pure line

325

cultivar ‘Aichi First’ as parental lines to produce two hybrid F1 lines (F1#21-19 and F1#21-7).

326

These two lines harbor a heterozygous mutated SlGAD3∆C allele and showed higher GABA

327

accumulation in red ripe fruit (Breaker + 10 days) than F1-WT (Figure 2). The gene

328

expression level of SlGAD3 in F1#21-7 was lower than other two lines at mature green stage.

329

On the other hand, it was almost same among three lines at breaker and red ripe stage (Figure

330

S2). Previous study, we showed truncation of C-terminal of SlGAD3 increased GAD activity,

331

resulting in higher GABA accumulation. Therefore, GABA content correlated with SlGAD3 14 ACS Paragon Plus Environment

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activity, not gene expression. Compared with the TG3C37 line of the dwarf cultivar

333

‘Micro-Tom’, which contains a homozygous SlGAD3∆C,30 the level of GABA in F1#21-19

334

and F1#21-7 was higher. Moreover, F1#21-19 and F1#21-7 showed the same GABA

335

accumulation as the GABA-rich cultivar ‘DG03-9’.35 This indicates that the heterozygous

336

SlGAD3∆C is linked to an increase in GABA accumulation, even in fruit of larger size than

337

that of ‘Micro-Tom’. A daily GABA intake of 10 to 20 mg has been demonstrated to

338

effectively reduce blood pressure in adults with mild hypertension.2,3,36,37 In F1#21-19 and

339

F1#21-7, the GABA concentration was up to 18.50 µmol/gFW and 12.76 µmol/gFW, which

340

are equivalent to 190.8 ± 21.6 mg and 131.6 ± 11.1 mg/100gFW tomato fruit, respectively.

341

Thus, the requisite amount of GABA for health-promoting effects can be obtained in 10 to 20

342

gFW of F1#21-19 and F1#21-7, which corresponds to one-eighth to one-quarter of F1 fruits,

343

which have an average weight approximately 75 g. These results indicate that the elevated

344

accumulation of GABA in the F1 hybrid lines examined in this study would appear to be

345

sufficient to have a health-promoting function.

346

Our results indicated that the increased GABA concentration in red ripe fruit (131.6

347

mg/100gFW and 190.8 mg/100gFW in F1#21-7 and F1#21-19, respectively) did not influence

348

either free amino acid accumulation (Figure 3, Table S1) or coloration during the ripening

349

process (Figure 4). In contrast, a previous transgenic study using the dwarf cultivar

350

‘Micro-Tom’ grown in a growth room showed that 268 mg/100g FW of GABA affects free

351

amino acid accumulation and inhibited the development of fruits with a red-ripe coloration

352

from those with an orange color.27 The F1 lines (‘Micro-Tom’ × ‘Aichi First’) produced in the

353

present study were grown in semi-containment green house. Accordingly, taken together, the

354

results of the present and previous studies indicate that the level at which GABA begins to

355

show an effect on fruit colorization and free amino acid accumulation is approximately 190



356

mg/100gFW, and/or that the influence of GABA on colorization and amino acid

357

accumulation is dependent on cultivar and growth conditions.

358

Along with the indices TSS and TA, taste can be estimated from the levels of glutamate

359

(Glu) and aspartate (Asp), since these amino acids are among the taste components

360

responsible for “UMAMI,” and are important amino acids in red ripe fruit. TSS and TA are

361

primarily used to estimate the sugar and organic acid contents in tomato fruits, respectively,

362

which are commonly associated with fruit sweetness and sourness. In the GABA-rich lines

363

F1#21-19 and F1#21-7, the levels of Glu and Asp were found to be similar to those in F1-WT,

364

with the TSS value being slightly lower and the TA value being slightly higher in the red ripe

365

fruits of GABA-rich lines (Figure 5). These results indicate that only sweetness and sourness

366

were changed in these two lines. Further experiments, including sensory evaluation, are

367

required to determine whether the taste of the fruit of GABA-rich lines is altered. We

368

determined the size and weight of fruits, which are also important factors in fruit quality. No

369

differences were observed with regards to fruit diameter, length, and weight in F1-WT,

370

F1#21-19 and F1#21-7 (Figure 6). Each of the F1 lines examined in the present study had a

371

fruit size that was intermediate between that of ‘Micro-Tom’ (WT and TG3C37, average 2–3

372

cm) and the commercial pure line cultivar ‘Aichi First’.

373

Previously, it has been demonstrated that Glu and Asp accumulate as GABA

374

decreases.24,38 Although the same tendency was observed in F1-WT, F1#21-19, and F1#21-7

375

(Figure 3, Table S1), there were differences in the stage at which the accumulation of Asp

376

commenced. The BR/MG ratios for Asp in F1-WT, F1#21-19, and F1#21-7 were 2.6, 2.25,

377

and 2.0, respectively, whereas the corresponding RED/BR ratios were 3.3, 6.2, and 4.2 (Table

378

S1). These results indicate that in F1-WT, Asp increases constantly during fruit development,

379

whereas in F1#21-19 and F1#21-7 there is a marked increase in the levels of this amino acid

380

after BR. At the RED stage, however, the concentrations of Asp tended to be similar in the 16 ACS Paragon Plus Environment

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three F1 lines. In contrast, the pattern of Glu accumulation during fruit development tended to

382

be similar in the three F1 lines. It seems reasonable to assume that the Asp/Glu balance could

383

induce differences in Asp accumulation pattern, but not that of Glu, which is a precursor of

384

GABA. Asp and Glu are interconverted by the activity of aspartate aminotransferase (AST).39

385

The activity of this enzyme should be higher in F1#21-19 and F1#21-7, which contain a

386

mutated SlGAD3∆C allele, than in F1-WT,30 resulting in a deficiency of Glu in the metabolic

387

system. To compensate for this deficiency, Asp should be converted to Glu via the activity of

388

AST.

389

The GABA-rich cultivar ‘DG03-9’ was bred in a previous study.35,40 The mechanism of

390

high GABA accumulation in ‘DG03-9’ appears to differ from that observed in our F1 lines.

391

Comparative studies of ‘Micro-Tom’ and ‘DG03-9’ have clarified the differences in the

392

behavior of synthesis and degradation enzymes between these two cultivars.25 ‘DG03-9’

393

showed higher activity of GABA synthesis enzymes (SlGADs) than ‘Micro-Tom’, whereas

394

the activity of the GABA degradation enzyme GABA-TK was considerably lower than that

395

in ‘Micro-Tom’ in the breaker and red stages. Since the Asp and citrate contents in ‘DG03-9’

396

did not increase significantly after the breaker stage, and the Glu content was not elevated

397

immediately after the breaker stage, this previous study also suggests that ‘DG03-9’ has

398

mutations not only in genes that are implicated in GABA degradation but also in genes that

399

are related to the synthesis or degradation of citrate, Glu, and Asp. In contrast, the effect of

400

SlGAD3∆C allele on Asp and Glu accumulation in red ripe fruit appeared to be small.

401

In tomato breeding, F1 hybrids are predominantly used. In this study, we examined the

402

utilization of a tomato line mutated via CRISPR/Cas9 for breeding. We found that F1 hybrid

403

lines containing a heterozygous mutated SlGAD3∆C allele showed an increase in GABA

404

accumulation in red ripe fruit (Breaker + 10 days) as a dominant trait, without any negative



405

effects on other tomato fruit traits. Either parental line with a truncated GAD C terminus

406

introduced by CRSIRP/Cas9 can be effectively used for breeding tomatoes with a high

407

GABA content. The removal of the C terminus in GADs has been demonstrated to increase

408

the activity of these enzymes in many plant species, including rice and apple.41,42 Therefore,

409

the results of the present study indicate that the utilization of lines mutated using the

410

CRISPR/Cas9 system as parental lines would be effective in breeding high-GABA

411

accumulation varieties of a wide range of crops.

412 413

Associated content

414

Supporting Information

415

The Supporting Information is available free of charge on the ACS Publications website at

416

DOI:

417

Supporting data was shown in Figure S1, Figure S2, and Table S1. Figure S1 is dCAPS

418

analysis of all of F1 lines. Figure S2 is estimation of GAD3 gene expression level. Table S1 is

419

raw data of Figure 3. (PDF)

420 421

Acknowledgments

422 423

We thank the National BioResearch Project (NBRP), MEST, Japan, for providing the seeds

424

of Solanum lycopersicum ‘Micro-Tom’ and ‘Aichi First’.

425 426

Funding

427 428

This work was supported by the Cabinet Office, Government of Japan, Cross-ministerial

429

Strategic Innovation Promotion Program, ‘Technologies for creating next-generation 18 ACS Paragon Plus Environment

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agriculture, forestry and fisheries’ grant (funding agency: Bio-oriented Technology Research

431

Advancement Institution, NARO) to H.E., a Grant-in-Aid for Scientific Research (C) (Grant

432

Number 15K07253) from JSPS KAKENHI to S.N., and a Cooperative Research Grant from

433

the Gene Research Centre of the University of Tsukuba to H.E. and S.N.

434 435

Author Contributions

436 437

S.N. and H.E. conceived the experiments, and J. L., M.T, and S.N. conducted the experiments.

438

All authors analyzed the results and contributed to writing and reviewing the manuscript.

439 440

References

441 442 443 444

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2. Inoue, K.; Shirai, T.; Ochiai, H.; Kasao, M.; Hayakawa, K.; Kimura, M.; Sansawa, H. Blood-pressure-lowering effect of a novel fermented milk containing γ-aminobutyric acid (GABA) in mild hypertensives. Eur. J. Clin. Nutr. 2003, 57, 490–495.

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3. Nishimura M.; Yoshida, S.; Haramoto, M.; Mizuno, H.; Fukuda, T.; Kagami-Katsuyama, H.; Tanaka, A.; Ohkawara, T.; Sato, Y.; Nishihira, J. Effects of white rice containing enriched gamma-aminobutyric acid on blood pressure. J. Tradit. Complement Med. 2015, 6, 66-71. 4. Eliott, K. A. C.; Hobbiger, F. Gamma Aminobutyric acid; circulatory and respiratory effects in different species; re-investigation of the anti-strychnine action in mice. J. Physiol. 1959, 146, 70–84. 5. Takahashi, H.; Sumi, M.; Koshino, F. Effect of gamma-aminobutyric acid (GABA) on normotensive or hypertensive rats and men. Jpn. J. Physiol. 1961, 11, 89–95. 6. Shizuka, F.; Kido, Y.; Nakazawa, T.; Kitajima, H.; Aizawa, C.; Kayamura, H.; Ichijo, N. Antihypertensive effect of γ-amino butyric acid enriched soy products in spontaneously hypertensive rats. BioFactors. 2004, 22, 165–167. 19 ACS Paragon Plus Environment


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7. Tian, J.; Dang, H.N.; Yong, J.; Chui, W.S.; Dizon, M.P.; Yaw, C.K.; Kaufman, D.L. Oral treatment with γ-aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in high fat diet-fed mice. PLoS One. 2011;6(9):e25338. 8. Abdou, A.M.; Higashiguchi, S.; Horie, K.; Kim, M.; Hatta, H.; Yokogoshi, H. Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans. Biofactors. 2006, 26, 201-208. 9. Tsushida, T.; Murai, T. Conversion of glutamic acid to γ-aminobutyric acid in tea leaves under anaerobic conditions. Agric. Biol. Chem. 1987, 51, 2865–2871. 10. Saikusa, T.; Horino, T.; Mori, Y. Distribution of free amino acids in the rice kernel and kernel fractions and the effect of water soaking on the distribution. J. Agric. Food Chem. 1994, 42, 1122–1125. 11. Aoki, H.; Uda, I.; Tagami, K.; Furuya, Y.; Endo, Y.; Fujimoto, K. The production of a new tempeh-like fermented soybean containing a high level of gamma-aminobutyric acid by anaerobic incubation with Rhizopus. Biosci. Biotechnol. Biochem. 2003, 67, 1018-1023. 12. Park, K. B.; Oh, S. H. Production of yogurt with enhanced levels of gamma-aminobutyric acid and valuable nutrients using lactic acid bacteria and germinated soybean extract. Bioresour. Technol. 2007, 98, 1675–1679. 13. Yamakoshi, J.; Fukuda, S.; Satoh, T.; Tsuji, R.; Saito, M.; Obata, A.; Matsuyama, A.; Kikuchi, M.; Kawasaki, T. Antihypertensive and natriuretic effects of less-sodium soy sauce containing γ-aminobutyric acid in spontaneously hypertensive rats. Biosci. Biotechnol. Biochem. 2007, 71, 165–173. 14. Baum, G.; Chen, Y.; Arazi, T.; Takatsuji, H.; Fromm, H. A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis. J Biol. Chem. 1993, 268, 19610-19617. 15. Fait, A.; Fromm, H.; Walter, D.; Galili, G.; Fernie, AR. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant Sci. 2008, 13, 14-19. 16. Shelp, B.J.; Bozzo, G.G.; Trobacher, C.P.; Zarei, A.; Deyman, K.L.; Brikis, C.J. Hypothesis/review: contribution of putrescine to 4-aminobutyrate (GABA) production in response to abiotic stress. Plant Sci. 2012, 193-194, 130-135. 17. Signorelli, S.; Dans, P.D.; Coitiño, E.L.; Borsani, O.; Monza, J. Connecting proline and γ-aminobutyric acid in stressed plants through non-enzymatic reactions. PLoS One. 2015, 10: e0115349. 18. Clark, S.M.; Di Leo, R.; Van Cauwenberghe, O.R.; Mullen, R.T.; Shelp, B.J. Subcellular localization and expression of multiple tomato gamma-aminobutyrate transaminases that utilize both pyruvate and glyoxylate. J. Exp. Bot. 2009, 60, 3255-3267. 19. Shelp, B.J.; Mullen, R.T.; Waller, J.C. Compartmentation of GABA metabolism raises intriguing questions. Trends Plant Sci. 2012, 17, 57-59. 20 ACS Paragon Plus Environment

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20. Bouché, N.; Fait, A.; Bouchez, D.; Møller, S.G.; Fromm, H. Mitochondrial succinic-semialdehyde dehydrogenase of the gamma-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants. Proc. Natl. Acad. Sci. USA. 2003, 100: 6843-6848. 21. Andriamampandry, C.; Siffert, J.C.; Schmitt, M.; Garnier, J.M.; Staub, A.; Muller, C.; Gobaille, S.; Mark, J.; Maitre, M. Cloning of a rat brain succinic semialdehyde reductase involved in the synthesis of the neuromodulator gamma-hydroxybutyrate. Biochem. J. 1998, 15, 43-50. 22. Breitkreuz, K.E.; Allan, W.L.; Van Cauwenberghe, O.R.; Jakobs, C.; Talibi, D.; Andre, B.; Shelp B.J. A novel gamma-hydroxybutyrate dehydrogenase: identification and expression of an Arabidopsis cDNA and potential role under oxygen deficiency. J Biol. Chem. 2003, 278, 41552-41556. 23. Ramesh, S.A.; Tyerman, S.D.; Gilliham, M.; Xu, B. γ-aminobutyric acid (GABA) signaling in plants. Cell Mol Life Sci. 2017, 74, 1577-1603. 24. Rolin, D.; Baldet, P.; Just, D.; Chevalier, C.; Biran, M.; Raymond, P. NMR study of low subcellular pH during the development of cherry tomato fruit. Aust J Plant Physiol. 2000, 27, 61–69. 25. Akihiro, T.; Koike, S.; Tani, R.; Tominaga, T.; Watanabe, S.; Iijima, Y.; Aoki, K.; Shibata, D.; Ashihara, H.; Matsukura, C.; Akama, K.; Fujimura, T.; Ezura, H. Biochemical mechanism on GABA accumulation during fruit development in tomato. Plant Cell Physiol. 2008, 49, 1378-1389. 26. Takayama, M.; Koike, S.; Kusano, M.; Matsukura, C.; Saito, K.; Ariizumi, T.; Ezura, H. Tomato glutamate decarboxylase genes SlGAD2 and SlGAD3 play key roles in regulating γ-aminobutyric acid levels in tomato (Solanum lycopersicum). Plant Cell Physiol. 2015, 56, 1533-1545. 27. Takayama, M.; Matsukura, C.; Ariizumi, T.; Ezura, H. Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit. Plant Cell Rep. 2017, 36, 103-116. 28. Koike, S.; Matsukura, C.; Takayama, M.; Asamizu, M.; Ezura, H. Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.). Plant Cell Physiol. 2013, 54, 793–807. 29. Gut, H.; Dominici, P.; Pilati, S.; Astegno, A.; Petoukhov, M.V.; Svergun, D.I.; Grütter, M.G.; Capitani, G. A common structural basis for pH- and calmodulin-mediated regulation in plant glutamate decarboxylase. J Mol. Biol. 2009, 392, 334-351. 30. Nonaka, S.; Arai, C.; Takayama, M.; Matsukura, C.; Ezura, H. Efficient increase of 21 ACS Paragon Plus Environment


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35. Saito, T.; Matsukura, C.; Sugiyama, M.; Watahiki, A.; Ohshima, I.; Iijima, Y.; Konishi, C.; Fujii, T.; Inai, S.; Fukuda, N.; Nishimura, S.; Ezura, H. Screening for γ-aminobutyric acid (GABA)-rich tomato varieties. J. Japan. Soc. Hort. Sci. 2008, 77, 242-250.

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39. Mehta, P.K.; Hale, T.I.; Christen, P. Aminotransferases: demonstration of homology and division into evolutionary subgroups. Eur J Biochem. 1993, 214, 549-561.

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ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci. Rep. 2017, 7(1), 7057. 31. Bai, Y.; Lindhout, P. Domestication and breeding of tomatoes: what have we gained and what can we gain in the future? Ann. Bot. 2007, 100, 1085-1094. 32. Kim S.H.; Hamada T. Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam. Biotechnol Lett 2005, 27, 1841-1845. 33. Nagata, M.; Yamashita, I. Simple method for simultaneous determination of cholorophll and carotenoids in tomato fruit. Nippon shokuhin Kogyo Gakkaishi. 1992, 39, 925-928. 34. 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, 504-508.

36. Fukuwatari, Y.; Sato, N.; Kawamori, R.; Watanabe, Y.; Yoshioka, K.; Ying, R.; Matsuda, K.; Fuji, A.; Uzawa, M.; Sato, R. A study on the antihypertensive action and safety of tablets containing γ-aminobutyric acid (GABA). Eastern Med. 2001, 17, 1–7. 37. Kazami, D.; Ogura, N.; Fukuchi, T.; Tsuji, K.; Anzawa, M,; Maeda, H. Antihypertensive effect of Japanese taste seasoning containing γ-amino butyric acid on mildly hypertensive and high-normal blood pressure subjects and normal subjects. Nippon Shokuhin Kagaku Kogaku Kaishi 2002, 49, 409–415. 38. Carrari, F.; Baxter, C.; Usadel, B.; Urbanczyk-Wochniak, E.; Zanor, M.I.; Nunes-Nesi, A.; Nikiforova, V.; Centero, D.; Ratzka, A.; Pauly, M.; Sweetlove, L.J.; Fernie, A.R. Integrated analysis of metabolite and transcript levels reveals the metabolic shifts that underlie tomato fruit development and highlight regulatory aspects of metabolic network behavior. Plant Physiol. 2006, 142, 1380-1396.

40. Yoshimura, M.; Toyoshi, T.; Sano A.; Izumi, T.; Fujii, T.; Konishi, C.; Inai, S.; Matsukura, C.; Fukuda, N.; Ezura, H.; Obata, A. Antihypertensive effect of a gamma-aminobutyric acid rich tomato cultivar 'DG03-9' in spontaneously hypertensive rats. J Agric. Food Chem. 2010, 58, 615-619. 41. Akama, K.; Takaiwa, F. C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells. J Exp. Bot. 2007, 58, 2699-2707. 22 ACS Paragon Plus Environment

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42. Trobacher, C.P.; Zarei, A.; Liu, J.; Clark, S.M.; Bozzo, G.G.; Shelp, B.J. Calmodul

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in-dependent and calmodulin-independent glutamate decarboxylases in apple fruit. B

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MC Plant Biol. 2013, 13, 144.

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615

Legends to Figures

616 617

Figure 1. Diagram and sequencing chromatograph of SlGAD3.

618

(a) Structure of SlGAD3 and SlGAD3∆C. Upper and lower are diagram of WT and

619

truncated C terminal (TG3C37#21-7 and TG3C37#21-19). In WT SlGAD3 contain

620

autoinhibitory domain in C terminal (deeper blue box). Blue character means autoinhibitory

621

domain, respectively. (b) Structur of the SlGAD3 gene and dCAPS strategy. Black boxes and

622

gray boxes are cording and UTR region, respectively. The target site of CRISPR/Cas9 (red

623

line) include BstXI (red character). Black triangles indicate PCR primers and green line is

624

amplified DNA fragment. The amplified fragments were digested by BstXI. Two bands

625

would be appeared in WT genotype with BstXI site, (0.3kb and 0.7kb). In contract, one band

626

would be appeared in mutant (1.0 kb). (c) A representative result of dCAPS analysis. M:

627

marker (Nippon gene ladder fast 1), 1: ‘Micro-Tom’ WT, 2: ‘Aichi First’ WT, 3:

628

TG3C37#21-7 (T1 generation), and 4: F1 between ‘Aichi First’ (WT) and TG3C37#21-19. “-”

629

indicates no restriction enzyme treatment and “+” indicates restriction enzyme treated. (d)

630

Sequence chromatogram of T1 of TG3C37 #21-7 (left, red asterisk indicates the position

631

where one nucleotide is inserted), wild-type ‘Aichi First’ (middle), and the F1 of

632

TG3C37#21-7 and ‘Aichi First’ (WT) (F1#21-7). (e) Sequence chromatogram of T1 of

633

TG3C37#21-19 (left, green asterisk indicates the position at which a 53-nucleotide deletion

634

begins), wild-type ‘Aichi First’ (middle) and the F1 (right) between TG3C37#21-19 and

635

‘Aichi First’ (WT) (F1#21-19).

636 637 638

Figure 2. GABA contents of the fruits of F1#21-19, F1#21-7, and F1-WT at three stages of

639

development. 24 ACS Paragon Plus Environment

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640

GABA contents were measured at the three stages by HPLC. MG, BR, and RED indicate the

641

Mature green stage, Breaker stage, and Red ripe stage (10 days after the breaker stage),

642

respectively. Data represent the means ± standard deviation (n = 3). Different lower-case

643

letters indicate statistically significant differences by Tukey’s HSD test (P < 0.05). F-WT,

644

F21-19, and F21-7 denote F1-WT, F1#21-19, and F1#21-7, respectively.

645 646

Figure 3. Free amino acids at the MG (a), BR (b), and RED (c) stages of fruit development.

647

Free amino acid contents were measured at the three stages by HPLC. MG, BR, and RED

648

indicate the Mature green stage, Breaker stage, and Red ripe stage (Breaker stage + 10 days),

649

respectively. Bards indicates standard deviation (n = 3).

650

Different lower-case letters indicate statistically significant differences by Tukey’s HSD test

651

(P < 0.05). ‘a’ was deleted to avoid confusing, ‘b’ and ‘c’ are only shown in this figure to

652

indicate the significant differences. For more information, refer Table S1.

653

F-WT, F21-19, and F21-7 denote F1-WT, F1#21-19, and F1#21-7, respectively.

654 655

Figure 4. Coloration and carotenoid content at the Red ripe stage of fruit development.

656

(a) Coloration from mature green to red stage. Scale bar indicates 1 cm. (b) Lycopene content

657

and (c) β-carotene content at the Red ripe stage (Breaker stage + 10 days). Bars means

658

standard deviation (n = 5). Different lower-case letters indicate statistically significant

659

differences by Tukey’s HSD test (P < 0.05). MG, BR, and RED indicate the Mature green

660

stage, Breaker stage, and Red ripe stage (Breaker stage+ 10 days), respectively. F-WT,

661


662 663

Figure 5. Total soluble solids and titratable acidity in red-stage tomato fruits. 25 ACS Paragon Plus Environment


664

(a) Total soluble solids content. Bars means standard deviation (n=9). (b) Titratable acidity at

665

the Red ripe stage fruits (Breaker stage + 10 days). Bars indicates standard deviation (n=5).

666

Different lower-case letters indicate statistically significant differences by Tukey’s HSD test

667

(P < 0.05). F-WT, F21-19, and F21-7 denote F1-WT, F1#21-19, and F1#21-7, respectively.

668 669

Figure 6. Fruit characteristics of F1#21-19, F1#21-7, and F1-WT lines.

670

Fruit characteristics are represented by (a) Horizontal diameter, (b)Vertical length, and (c)

671

Weight. The mean values ± SD of 20 biological replicates are shown. Different lower-case

672

letters indicate statistically significant differences by Tukey’s HSD test (P < 0.05). F-WT,

673


674


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Parent / F1

ID

Zygosity

Genotype

Amino acid sequence

Parent (F)

‘Micro-Tom’

Homo-

WT

…VKVLHELPNAKKVEDNLMINNEKKTEIEVQRAIAEFWKKYVLARKASIC*

Parent (F)

TG3C37#21-7

Homo-

1i

…VKVLHELPNAKKVGG*

Parent (F)

TG3C37#21-19

Homo-

53d

…VKVLHELPN*

Parent (M)

‘Aichi First’

Homo

WT

…VKVLHELPNAKKVEDNLMINNEKKTEIEVQRAIAEFWKKYVLARKASIC*

F1

F1-WT

Homos

WT

F1

F1#21-7

Hetero

1i WT

F1

F1#21-19

Hetero

53d WT

675 676

Table 1. SlGAD3 genotype and translated SlGAD3 sequence of F1 lines.

677

Amino acid sequence of C-terminal (F1 lines). The same sequence as Wild Type is expressed in Bold. The changed sequence by mutation

678

via CRISPR/Cas9 is in Regular. Bold with underline is the putative autoinhibitory domain and CaMB domain. The target site is written in

679

Italic. * is stop. Genotype of WT, 1i, and 53d are wild-type, 1 nucleotide insertion, 53 nucleotides deletion, respectively. “Homo” and “Hetero”

680

in zygosity means homozygous and heterozygous, respectively. F1 lines:

681

‘Micro-Tom’ TG3C37#21-7 × ‘Aichi First’ WT; F1#21-19, ‘Micro-Tom’ TG3C37#21-19 × ‘Aichi First’. “F” and “M” means Female and

682

Male as parent, respectively.

F1-WT, ‘Micro-Tom’ WT × ‘Aichi First’ WT; F1#21-7,

683 27

ACS Paragon Plus Environment


684

Page 28 of 36

Table 2. Fruit color of F1 lines. 685

L*

a*

b*

Chr

hue

F1-WT

38.22 ± 1.89a

27.42 ± 3.13a

28.99 ± 3.25a

39.93 ± 4.30a

a 0.841 ± 0.068686

F1#21-19

38.68 ± 2.25a

27.68 ± 1.97a

29.61 ± 3.04a

40.56 ± 3.27ab

a 0.849 ± 0.077687

F1#21-7

39.09 ± 1.92a

29.20 ± 2.47a

31.34 ± 3.29b

42.88 ± 3.53b

a 0.852 ± 0.096688

689 690

The mean values ± SD of L*, a*, b* parameters, Chr and hue functions from 20 replicates are shown. Different lower-case letters indicate

691

statistically significant differences by Tukey’s HSD test (P < 0.05).

28


Page 29 of 36


692



Page 30 of 36

KKVEDNLMINNEKKTEIEVQRAIAEFWKKYVLARKASIC

(a)

SlGAD3

WT

C terminal

SlGAD3DC

TG3C37 (b) SlGAD3

WT (No mutation) with BstXI site is divided in two bands

Amplified by PCR Target site with BstXI c.a.700bp

c.a.300bp

c.a.300bp

Digestion with BstXI

c.a.700bp

Mutated without BstXI site shows one bands c.a.1,000bp

CCCGAATCCCGAATGCCAAAAAAGTGG (c) M 2kb

1

-

+

2

3

- + - +

4

- +

1kb 0.5kb

0.1kb (d) *

(e)

TG3C37#21-7 (T1)

‘Aichi First’ WT

TG3C37#21-7 ☓‘Aichi First’ (F1)

‘Aichi First’ WT

TG3C37#21-19 ☓ ‘Aichi First’ (F1)

* TG3C37#21-19 (T1)

Figure 1. Diagram and sequencing chromatograph of SlGAD3.


Page 31 of 36


50 b

GABA (μmol/gFW)

40

b b

30

F-WT

b c

20

a

F21-19 b

a 10

F21-7

a

0 MG

BR

RED

Figure 2. GABA contents of the fruits of F1#21-19, F1#21-7, and F1-WT at three stages of development.



(a)

3

50

Free amino acid (µmol/gFW)

Page 32 of 36

b

40

2

b

30 1

20 0 Ala Arg Asn Asp Gly His Ile Leu Lys Phe Ser Thr Tyr Val

10 b 0

(b)


50

3

40

2

b 30

b 1

20 0

10

Ala Arg Asn Gln Gly His Ile Leu Lys Phe Ser Thr Tyr Val

0

(c)


50

3

40

2

30 1

c 20

b 0 Ala Arg Asn Gln Gly His Ile Leu Lys Phe Ser Thr Tyr Val

10 0 F-WT F1-WT

F21-19 F1#21-19

F21-7 F1#21-7

Figure 3. Free amino acids at the MG (a), BR (b), and RED (c) stages of fruit development. ACS Paragon Plus Environment

Page 33 of 36


(a)

BR

MG

RED

F-WT

F21-19

F21-7

(b)

(c) a

50

a

a

40 30 20 10

10

β-carotene (μg/g FW)

Lycopene (μg/g FW)

60

a

a

a

8 6 4 2 0

0 F-WT F21-19 F21-7

F-WT F21-19 F21-7

Figure 4. Coloration during fruit ripening and carotenoid content at the Red ripe fruits.



(b)

Total soluble solid (°Brix)

6

a b

b

4

2

Titratable acidity (%)

(a)

Page 34 of 36

b

0.7 0.6

a

a

0.5 0.4 0.3 0.2 0.1

0

0.0 F-WT

F21-19

F21-7

F-WT

F21-19

F21-7

Figure 5. Total soluble solids and titratable acidity in red-stage tomato fruits.


Page 35 of 36


a

a 60

40

a

a

40

20

20

0 F-WT F21-19 F21-7

120

a

Weight (g)

a 60

Vertical length (mm)

Horizontal diameter (mm)

(c)

(b)

(a)

0

a

a

a

90 60 30 0

F-WT F21-19 F21-7

F-WT F21-19 F21-7

Figure 6. Fruit characteristics of F1#21-19, F1#21-7, and F1-WT lines. .



Page 36 of 36

Genome editing technology is effective for the rapid breeding of high-GABA hybrid tomato.! ‘Micro-Tom’ with SlGAD3ΔC homozygous allele" High GABA content line"

γ – Amino Butyric Acid" (GABA)"

‘Aichi First’ WT"

1cm"

Health-promoting function for hypertension and diabetes "

F1 hybrid with SlGAD3ΔC heterozygous allele"

GABA (μmol/gFW)"

1cm"

50" 40" 30" 20" 10" 0" MG" F1-WT"

BR" F1#21-19"

The high GABA is a dominant trait and the genome-edited tomato is useful as a parental line of hybrid cultivars."

RED" F1#21-7"

Higher GABA accumulation was observed in F1 hybrid with SlGAD3ΔC heterozygous allele (F1#21-19 and F1#21-7)."

1cm"


Utilization of a Genome-Edited Tomato (Solanum lycopersicum) with

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