Abstract：Abscisic acid (ABA) can promote the strawberry (Fragaria × ananassa) fruit development and ripening, and inactive ABA glucose ester (ABA-GE) can be converted into free ABA by β-glucosidases (BG), therefore, BG is involved in the fruit development process. But there is no genetic evidence to reveal the mechanism of BG involved in fruit development. To understand the function of BG in the strawberry developmental stages, in this study, Sweet Charlie strawberry was used as test materials, and three genes encoding BG that were FaBG1, FaBG2 and FaBG3 were isolated through molecular cloning of PCR. Firstly, bioinformatics and temporal expression analysis of FaBGs were performed during strawberry fruit development stages, and the ABA contents were also measured. Secondly, molecular regulation of FaBG3 gene expression levels affected fruit endogenous BG activity, and fruit phenotypic changes, physiological indexes such as ABA content, pigments content, soluble solids content, and the fruit hardness were analyzed. In addition, ABA signal pathway, the pigment metabolism and cell wall metabolism related gene expression levels were analyzed. The results showed that BG protein in strawberry contained a region of BglB region, which was a specific region could hydrolyze other complexes. Three genes encoding the BG protein in strawberry were found, and had different expression levels. During the ripening of strawberry fruits, ABA content increased gradually, the expression level of FaBG1 gene was very low, in general the expression amount of FaBG2 gene was decreased, and only FaBG3 gene expression level was consistent with the ABA content and fruit development. Therefore, FaBG3 played a more important role in the regulation of ABA content. Using a technique of RNA interference to reduce the gene expression level of FaBG3 in strawberry fruit also downregulated FaBG1 and FaBG2 genes expression level, suggesting that ABA content was downregulated by FaBG3-interfere, and FaBG1 and FaBG2 expression were also influenced. FaBG3 gene expression levels down-regulation led to the reduction of fruit endogenous beta-glucosidases activity, which influenced the ABA accumulation. Meantime, ABA content changes also affected the physiological changes in strawberry fruit, such as pigment and soluble solid content were reduced, and the fruit hardness were up-regulated, which finally influenced the fruit ripening process. On the contrary, FaBG3 overexpression promoted the strawberry fruit ripening process. In FaBG3-RNAi fruit, the genes expression levels that related to pigment metabolism, such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonoid-3-hydroxy-lase (F3H), UDP Glc-flavonoid 3-O-glucosyl transferase (UFGT) and dihydroflavo-nol-4-reductase (DFR) genes, fruit softening, such as pectin methylesterase (PE), expansin (EXP), polygalacturonase (PG) and pectate lyases (PL) genes, fruit aroma metabolism, such as quinone oxidoreductase (QR) and alcohol acyltransferase (AT), and ABA signal pathway, such as pyrabatin resistance (PYR), ABA insensitive 1 (ABI1), ABI3, ABI4 and ABI5 genes were also down-regulated, however, a negative factor of ABI1 which involved in ABA signaling pathway was up-regulated. Due to the expression levels changes of these key genes related to fruit development and ripening, at last, the fruit development process were delayed. The fruit physiological and molecular index changes could reveal the mechanism of BG in regulation of strawberry fruit development.
[1]Alexander L, Grierson D.Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening[J][J].Journal of Experimental Botany, 2002, 53(377):2039-2055
[2]Audran C, Borel C, Frey A, et al.Expression studies of the zeaxanthin epoxidase gene in Nicotiana plumbaginifolia[J][J].Plant Physiology, 1998, 118(3):1021-1028
[3]Ban T, Ishimaru M, Kobayashi S, et al. Abscisic acid and 2, 4-dichlorophenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in ‘Kyoho’ grape berries[J][J].The Journal of Horticultural Science and Biotechnology, 2003, 78(4):586-589
[4]Dietz K J, Sauter A, Wichert K, et al. Extracellular b–glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves[J]. [J].Journal of Experimental Botany, 2000, 51(346):937-944
[5]Esen A.b–Glucosidases. In b–Glucosidases: Biochemistry and Molecular Biology. ACS Symposium Series 533 (Esen, A., ed.). Washington DC: American Chemical Society[J].American Chemical Society, 1993, 261(C6-C7):1-14
[6]Giovannoni J J.Molecular biology of fruit maturation and ripening[J].[J].Annu Review of Plant Physiology and Plant Molecular Biology, 2001, 52(JUNE): 725-749
[7]Giovannoni J J. Genetic regulation of fruit development and ripening[J]. [J].Plant Cell, 2004, 16(suppl):S170-S180
[8]Himmelbach A, Yang Y, Grill E.Relay and control of abscisic acid signaling[J]. [J].Current Opinion Plant Bioloy, 2003, 6(5):470-479
[9]Jia, H F, Chai Y M, Li C L, et al.Abscisic acid plays an important role in the regulation of strawberry fruit ripening[J]. [J].Plant Physiology, 2011., 157(1):188-199
[10]Kleczkowski K, Schell J. Phytohormone conjugates: nature and function[J]. [J].Critical Reviews in Plant Science, 1995, 14(4):283-298
[11]Lee K H, Piao H L, Kim H Y, et al.Activation of glucosidase via stress-increases active pools of abscisic acid[J]. [J].Cell, 2006, 126(6):1109-1120
[12]Lee S, Chung E J, Joung Y H, et al.Non-climacteric fruit ripening in pepper: increased transcription of EIL-like genes normally regulated by ethylene[J]. [J].Function Integrative Genomics, 2010, 10(1):135-146
[13]Li Q, Li P, Sun L, et al.Expression analysis of β-glucosidase genes that regulate abscisic acid homeostasis during watermelon（Citrullus lanatus）development and under stress conditions[J]. [J].Journal Plant Physiology, 2012, 169 (1):78-85
[14]Li Q, Ji K, Sun Y F, et al.The role of FaBG3 in fruit ripening and B. cinerea fungal infection of strawberry[J]. [J].The Plant Journal, 2013, 76(1):24-35
[15]Manning K. Changes in gene expression during strawberry fruit ripening and their regulation by auxin[J]. [J].Planta, 1994, 194(1):62-68
[16]Molina-Hidalgo F J, Franco A R, Villatoro C, et al.The strawberry (Fragaria×ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae[J]. [J].Journal of Experimental Botany, 2013. , 64(6):1471-1483
[17]Morant A V, J?rgensen K, J?rgensen C, et al.β–Glucosidases as detonators of plant chemical defense[J].[J].Phytochemistry, 2008 , 69(9):1795-1813
[18]Schwarzkopf E, Miersch O.In vitro glucosylation of dihydro-jasmonic acid and abscisic acid[J].[J].Biochemie Und Physiologie der Pflanzen, 1992, 188(1): 57-65
[19]Sun L, Sun Y F, Zhang M, et al.Suppression of 9–cis-epoxycarot-enoid dioxygenase (NCED), which encodes a key enzyme in abscisic acid biosynthesis, alters fruit texture in transgenic tomatoes[J]. [J].Plant Physiology, 2012, 158(1):283-298
[20]Sun Y F, Chen P, Duan, C R, et al..Transcriptional regulation of genes encoding key enzymes of abscisic acid metabolism during melon (Cucumis melo L.) fruit development and ripening[J]. [J].Journal of Plant Growth Regulation, 2013 , 32(2):233-244
[21]王延书，李春丽，贾海锋，等.葡萄果实始熟前后 ABA 积累关键酶基因 VvNCED1 和 VvBG1 转录水平的研究[J]. [J].园艺学报, 2010, 37(5):801-804
[22]Zhang M, Leng P, Zhang G L, et al.Cloning and function analysis of 9-cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits[J]. [J].Journal of Plant Physiology, 2009 , 166(12):1241-1252
[23]Zhang J J, Wang X, Yu O, et al.Metabolic profiling of strawberry (Fragaria×ananassa Duch.) during fruit development and maturation[J].[J].Journal of Experimental Botany, 2011, 62(3):1103-1118
[24]朱海生, 温庆放, 李永平, 等.对草莓果实成熟和软化的调节[J].福建农业学报, 2012, 27(4):329-332
