The phytohormone ethylene plays an important role in various stages of plant growth and development, including senescence. Exposure to ethylene accelerates the senescence of flowers in many species, and the primary symptom of senescence induced by ethylene is petal wilting or petal (sepal) abscission (Woltering and van Doorn 1988). In many ethylene-sensitive flowers, such as carnation (Nichols 1968) and Eustoma grandiflorum (Ichimura et al. 1998), the petals wilt in response to ethylene exposure. In Digitalis (Stead and Moore 1983), geranium (Hilioti et al. 2000), and torenia flowers (Goto et al. 1999), petals abscise upon exposure to ethylene. In these flowers, ethylene production in the gynoecium or pistil increases with the progression of flower senescence. However, this finding is not observed in the petals (Stead and Moore 1983; Goto et al. 1999; Hilioti et al. 2000). Therefore, ethylene produced by the gynoecium has been considered responsible for petal abscission.
Pollination accelerates petal wilting in many ethylene-sensitive flowers, including carnation (Jones and Woodson 1997), petunia (Gillisen and Hoekstra 1984; Whitehead et al. 1984), and gentian (Shimizu-Yumoto and Ichimura 2012). Similarly, pollination induces petal abscission in Digitalis (Stead and Moore 1979, 1983), geranium (Clark et al. 1997), and torenia (Goto et al. 1999). Pollination induces climacteric-like increases in ethylene production, and ethylene inhibitors suppress pollination-induced senescence in many plants, including orchids (O’Neill et al. 1993; Porat et al. 1995), E. grandiflorum (Ichimura and Goto 2000), and torenia (Goto et al. 1999), indicating that pollination-induced petal senescence is regulated by ethylene.
The biosynthetic pathway for ethylene in higher plants has been established. 1-Aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, is produced by the conversion of S-adenosyl-l-methionine (SAM) by ACC synthase (ACS). ACC is converted to ethylene, carbon dioxide, and HCN by ACC oxidase (ACO). ACS is generally considered to be a rate-limiting enzyme for ethylene biosynthesis in plants because ACO activity is constitutive in many species (Yang and Hoffman 1984; Kende 1993). However, ACO is important for ethylene biosynthesis in some examples, including fruit maturation (Nakatsuka et al. 1998) and flower senescence (Tang et al. 1994). In cut carnation flowers, the biosynthesis of ethylene is regulated by the activities of ACS and ACO (Woodson et al. 1992; Woltering et al. 1993; Lee et al. 1997). ACS and ACO proteins are encoded by multi-gene families (Argueso et al. 2007; Lin et al. 2009), and these genes are differently regulated in response to developmental and environmental factors (Lin et al. 2009). Expression analyses of ACS and ACO genes have been performed in floral organs, using carnation (Jones and Woodson 1997, 1999; Jones 2003), petunia (Tang et al. 1994), tomato (Llop-Tous et al. 2000), and rose (Xue et al. 2008). In carnation flowers, three ACS genes, namely, DcACS1, DcACS2, and DcACS3, have been cloned, and their expression is differentially regulated (Jones and Woodson 1999). In tomato flowers, SlACS2 is upregulated by pollination (Llop-Tous et al. 2000). However, few studies have analyzed ACS and ACO gene expression in flowers displaying petal abscission. In geranium, ACS and ACO gene expression has only been semi-quantified using RNA gel blot analysis (Clark et al. 1997).
Delphinium has long spikes with flowers of various colors, such as white, blue, and purple. The genus Delphinium consists of more than 300 species, and D. elatum, D. grandiflorum and D. × belladonna, which is a hybrid of D. elatum and D. grandiflorum, are widely produced as ornamental plants. Instead of D. × belladonna, which was mainly produced approximately a decade ago, D. grandiflorum is widely produced for cut flowers. Delphinium flowers are sensitive to ethylene, and their sepals rapidly abscise upon exposure (Woltering and van Doorn 1988; Ichimura et al. 2000), which is accompanied by signs of programmed cell death (Yamada et al. 2007). In Delphinium, sepals and petals are directly connected to the receptacle, but not to the gynoecium. Thus, ethylene produced by the receptacle should directly induce the abscission of the sepals (Ichimura et al. 2009). Ethylene production in the gynoecium and receptacle increases during flower senescence, but that in the other floral organs including petals and sepals, does not increase (Ichimura et al. 2009). ACS and ACO activity in the gynoecium and receptacle increases during flower senescence (Ichimura et al. 2009). Although genes for ethylene receptors and signal transduction components have been identified in D. elatum (Kuroda et al. 2003, 2004) and D. × belladonna (Tanase and Ichimura 2006), ACS and ACO genes have not yet been cloned in Delphinium. The quantification of ACS and ACO gene expression will help to clarify the effects of ethylene on sepal abscission.
In the present study, we cloned ACS and ACO genes and determined their nucleotide sequences in D. grandiflorum. Furthermore, we investigated their expression of ACS and ACO genes in the gynoecium and receptacle during natural senescence, following exposure to ethylene, and after pollination in cut D. grandiflorum flowers.