title: Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit.
abstract: The C-terminal extension region of SlGAD3 is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits. γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid with several health-promoting benefits. In many plants including tomato, GABA is synthesized via decarboxylation of glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), which generally contains a C-terminal autoinhibitory domain. We previously generated transgenic tomato plants in which tomato GAD3 (SlGAD3) was expressed using the 35S promoter/NOS terminator expression cassette (35S-SlGAD3-NOS), yielding a four- to fivefold increase in GABA levels in red-ripe fruits compared to the control. In this study, to further increase GABA accumulation in tomato fruits, we expressed SlGAD3 with (SlGAD3OX) or without (SlGAD3ΔCOX) a putative autoinhibitory domain in tomato using the fruit ripening-specific E8 promoter and the Arabidopsis heat shock protein 18.2 (HSP) terminator. Although the GABA levels in SlGAD3OXfruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔCOXfruits increased by 11- to 18-fold compared to control plants, indicating that removing the autoinhibitory domain increases GABA biosynthesis activity. Furthermore, the increased GABA levels were accompanied by a drastic reduction in glutamate and aspartate levels, indicating that enhanced GABA biosynthesis affects amino acid metabolism in ripe-fruits. Moreover, SlGAD3ΔCOXfruits exhibited an orange-ripe phenotype, which was associated with reduced levels of both carotenoid and mRNA transcripts of ethylene-responsive carotenogenic genes, suggesting that over activation of GAD influences ethylene sensitivity. Our strategy utilizing the E8 promoter and HSP terminator expression cassette, together with SlGAD3 C-terminal deletion, would facilitate the production of tomato fruits with increased GABA levels.
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Takayama
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Matsukura
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Ariizumi
authors.affiliation: Faculty of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki, 305-8572, Japan. ezura@gene.tsukuba.ac.jp.
authors.forename: Hiroshi
authors.identifier: http://orcid.org/0000-0001-9393-3229
authors.initials: H
authors.lastname: Ezura
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002338:Carotenoids / Q000096:biosynthesis; D004789:Enzyme Activation; D005030:Ethylenes / Q000378:metabolism; D005638:Fruit / Q000201:enzymology*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D010858:Pigmentation / Q000235:genetics; D030821:Plants, Genetically Modified; D000072417:Protein Domains; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005030:Ethylenes; D012333:RNA, Messenger; D002338:Carotenoids; D005680:gamma-Aminobutyric Acid; C036216:ethylene; D005968:Glutamate Decarboxylase
keywords: E8 promoter; Fruit ripening; GABA; HSP terminator; Tomato