Effect of overexpression of plastid glyceraldehyde-3-phosphate dehydrogenase1 on fruit ripening and metabolites in strawberry

Abstract:【Objective】Plastid glyceraldehyde-3-phosphate dehydrogenase (GAPCp) is a key enzyme in glycolysis. Besides its catalytic function, GAPCp participates in the regulation of plant stress response, growth and development. However, its role in strawberry fruit ripening has been rarely men- tioned. Through overexpression of FaGAPCp1 gene in the fruits, combined with metabonomics, the ef- fect of FaGAPCp1 gene on fruit ripening and metaboconlites of strawberry was studied.【Methods】The strawberry (Fragaria × ananassa‘Benihoppe’) plants were grown in greenhouse in Chengdu, Chi- na. Fruits at de-greening (DG, 18 d after anthesis) stage were chosen for RNA extraction and FaGAP- Cp1 transient gene expression. Total RNA was extracted using a modified CTAB protocol. Approxi- mately 1 μg of total RNA was reverse-transcribed to cDNA cloning using a SMARTTM RACE cDNA Synthesis Kit (TaKaRa), and primers were designed for FaGAPCp1 cloning. For transient overexpres- sion, the cDNA fragments of FaGAPCp1 were inserted into the vector Eco RI-XbaI-cut pCAMBIA1301, and the recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method. A 5 mL culture of a single Agrobacterium colony was inoculated on LB medium (containing20μg·mL-1 Rif,40μg·mL-1 Genand50μg·mL-1 Kan)andculturedovernightat28°C.The product was then transferred to 50 mL LB medium (containing 20 μg·mL-1 Rif, 40 μg·mL-1 Gen and 50μg · mL- 1 Kan) and cultured at 28 °C. After the culture medium was turbid, the cells were collected by centrifugation (5 000 × g, 5 min, 20 °C ), and then resuspended in infiltration buffer (containing 10 mmol·L-1 MgCl2, 10 mmol·L-1 MES, 200 mmol·L-1 acetosyringone), the OD600 of cells reached 1.0-2.0. The sterilized 1 mL syringe was used to inject bacterial liquid into the fruits of DG stage. Ten similar- sized fruits were used for the infiltration experiment. The fruits injected with empty vector were used as control. Each fruit was a biological repeat. The fruits were collected on the 3rd day after transformation, and the phenotype was photographed. Then, the fruits were quickly frozen with liquid nitrogen. The expression of FaGAPCp1 gene was detected by real-time PCR using synthesized cDNA as template andFaActin as the reference gene. Next, metabolite profiling was performed using a widely targeted metab- olome method by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). The freeze-dried FaGAP- Cp1 overexpressing fruits were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. 100 mg powder was weighted and extracted overnight at 4 °C with 1.0 mL 70% aque- ous methanol. After centrifugation at 10 000× g for 10 min, the extracts were absorbed and filtered and then were analyzed using an LC-electrospray ionization (ESI)-MS/MS system. The metabolites were quantified using the multiple reactions monitoring (MRM) method. The results were obtained from Wu- han Metware Biotechnology Co., Ltd. (Wuhan, China) and the metabolites related to fruit ripening were visualized as heat maps by Tbtools (1.6.1) software.【Results】The qRT-PCR results showed that theFaGAPCp1 had higher expression in the FaGAPCp1 overexpression fruits compared with that of the control. In addition, overexpressed FaGAPCp1 delayed the coloring of strawberry fruits on the 3 d afterAgrobacterium injection. Furthermore, metabonomics analysis showed that 108 differential metabolites were detected in the FaGAPCp1 overexpressed fruits compared with those of the control. 50 up-regulat- ed differential metabolites mainly belonged to nucleotide and their derivates, proanthocyanidins, vita- mins and hydroxycinnamoyl derivatives, and 58 down- regulatded differential metabolites contained amino acids and their derivatives, anthocyanins, flavonoids, organic acids and their derivatives. Among the 12 detected amino acids and their derivatives, except for the relative contents of L-(+)-Lysine and D- Alanyl-D-Alanine, the relative contents of S-(5’-Adenosyl)-L-methionine and other amino acids were significantly decreased in the FaGAPCp1-overexpression fruits. The relative contents of 9 anthocyanins in the FaGAPCp1-overexpression fruits were significantly decreased, including the main anthocyanins of strawberry fruits, that is, cyanidin 3-O-glucoside, pelargonidin 3- O -beta-D-glucoside and pelargonidin 3- O -malonylhexside. The relative content of procyanidin A3, which was closely related to anthocyanin, was significantly increased in the FaGAPCp1-overexpression fruits. Meanwhile, 9 organic acids and their derivatives were detected, except for trans-Muconic acid, the relative contents of 7 organic ac- ids such as 2-isopropylmalate, 3-hydroxybutyrate, 2-hydroxyisocaproic acid and rosmarinic acid were significantly decreased compared with those of the control. Among the vitamins, the relative contents of riboflavin and nicotinamide-N-oxide were up-regulated, while the relative content of methyl nicotinate was down-regulated. Except for p-coumaraldehyde and cinnamic acid, the relative contents of 5 hydroxycinnamoyl derivatives were significantly up-regulated in the FaGAPCp1-overexpression fruits. Furthermore, 23 flavonoids were detected, including 7 flavonoids, 5 flavonols, 4 flavone C-glycosides, 4 flavanones and 3 isoflavones. Flavonols accounted for a large proportion of the detected flavonoids, among them the relative contents of methylquercetin O-hexoside, isorhamnetin O-hexoside and isorh- amnetin 5-O-hexoside were significantly increased compared with those of the control. The relative con- tents of flavone, flavone C-glycosides, flavanone and isoflavone were significantly decreased in theFaGAPCp1-overexpression fruits.【Conclusion】The GAPCp1 was a negative regulator for fruit ripen- ing in strawberry, which is regulated by affecting the metabolic process of fruit ripening.