The enzyme GAD (EC 18.104.22.168) is responsible for converting L-glutamic acid to gamma-aminobutyric acid (GABA) in the presence of the cofactor pyridoxal 5′-phosphate (PLP). GABA, a non-protein amino acid, is ubiquitously present in prokaryotes and eukaryotes. Plant GADs carry a C-terminal extension that binds to Ca2+/calmodulin (CaM) to modulate enzyme activity (Chen et al., 1994; Ling et al., 1994). Changes in gene expression and/or activity of GADs in plants are regulated by cytosolic levels of H+ and Ca2+ that are associated with various environmental stimuli (Bown and Shelp, 1989), including mechanical stimuli (Wallace et al., 1984) or damage (Ramputh and Bown, 1996), cold shock (Mazzucotelli et al., 2006), heat shock (Mayer et al., 1990), hypoxia (Tsushida and Murai, 1987), cytosolic acidification (Crawford et al., 1994), water stress (Bolarin et al., 1995), and phytohormones (Ford et al., 1996). Activated GADs lead to GABA accumulation that is important in regulating cytoplasmic acidity, glucose use, nitrogen storage, plant development, plant defense, and fruit ripening (Bown and Shelp, 1997; Gallego et al., 1995).
Although GAD and its product GABA have been identified in legumes decades ago, their functional role in plants, especially in ethylene biosynthesis and fruit ripening, is poorly understood (Kulkarni and Sohonie, 1956). Previous studies investigating the role of GADs in fruit ripening isolated GAD genes in several species of fruits, including tomato and citrus. These studies showed that the expression of GAD genes is changed at different ripening stages (Akihiro et al., 2008; Cercós et al., 2006; Gallego et al., 1995). Furthermore, the relationship between GABA levels and ethylene production was investigated in excised sunflower tissues to elucidate the role of GAD in ethylene biosynthesis. The results indicated that exogenous GABA caused an increase in ethylene production rate mainly by promoting GAD activity and up-regulating 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS: EC 22.214.171.124) gene expression (Kathiresan et al., 1997). However, the function of GAD in climacteric ethylene biosynthesis and postharvest fruit ripening is unclear.
The banana (Musa acuminata L. AAA group cv. Brazilian), a typical climacteric fruit, undergoes a postharvest ripening process characterized by a green storage phase followed by a burst in ethylene production that signals the beginning of the climacteric period. The banana enzymes involved in ethylene biosynthesis have been well characterized, and the related cDNAs were cloned. For example, MaACS1, encoding ACS, and MaACO1, encoding ACC oxidase (ACO: EC 1.4.3), were shown to play crucial roles in ethylene biosynthesis in banana postharvest ripening (Adams and Yang, 1979;Liu et al., 1999; Yang and Hoffman, 1984). Coincidently with the respiratory climacteric period, numerous physiological and biochemical changes occur in the banana fruit, including chlorophyll breakdown, flavochrome accumulation (Jacob-Wilk et al., 1999; Thomas and Janave, 1992), degradation of cell wall components resulting in fruit softening (Lohani et al., 2004), and degradation of stored starch into soluble sugar (Hill and Rees, 1995a, 1995b). These changes influence characteristics of the banana fruit such as its firmness, astringency, aroma, color, and shelf life. As a result, postharvest ripening plays an important role in improving the quality of the fruit as well as limiting its shelf life.
In our previous study (Xu et al., 2007), we constructed a forward suppression subtractive hybridization (SSH) cDNA library. Specifically, 265 cDNAs were isolated that were up-regulated in the ripening banana at 2 d postharvest (DPH). To investigate the effects of these differentially expressed cDNAs in the initiation and peaking of climacteric ethylene biosynthesis after harvest, cDNA microarray analysis was used to identify up- or down-regulated cDNAs at the onset of climacteric ethylene biosynthesis. A cDNA encoding glutamate decarboxylase was up-regulated during climacteric ethylene biosynthesis. Moreover, the expression level of this cDNA was the highest among the levels of all the cDNAs in the microarray on the initiation of ethylene biosynthesis (Jin et al., 2009; Xu et al., 2007). Our previous evidences implied that the glutamate decarboxylase activity might be related to ethylene biosynthesis associated with banana postharvest ripening. Therefore, we isolated the full-length cDNA of the glutamate decarboxylase gene from banana (MuGAD).
In this study, by studying changes in the ethylene production and expression of MuGAD, MaACS1, and MaACO1 of banana fruits related to ripening under various treatment conditions, we investigated the role of MuGAD in post-harvest ripening. Our results showed that MuGAD expression correlates with the expression of MaACS1 and MaACO1, key genes in ethylene biosynthesis.
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