The gaseous phytohormone ethylene is involved in many aspects of plant growth and development, including seed germination, flower and leaf senescence and abscission, and fruit ripening. It also functions as an important modulator in plant responses to biotic and abiotic stimuli, like pathogen attack, flooding, chilling, and mechanical damage (Johnson and Ecker, 1998; Bleecker and Kende, 2000).
In higher plants, the ethylene biosynthesis pathway has been well defined as Yang's Cycle (Yang and Hoffman, 1984). In this pathway, AdoMet (S-adenosylmethionine) is converted to ACC (1-aminocyclopropane-1-carboxylic acid) by ACS (ACC synthase), and then ACC is oxidized to ethylene catalysed by ACO (ACC oxidase) (Yang and Hoffman, 1984; Kende, 1993). ACS is encoded by a medium-sized multigene family and expression of ACS genes is regulated by internal developmental and external stress cues (Barry et al., 2000; Nakano et al., 2003). ACO is encoded by a small multigene family and ACO genes are also regulated differently in response to developmental and environmental events (Clark et al., 1997; Nakatsuka et al., 1997, 1998). ACS is the rate-limiting enzyme in the biosynthetic pathway and ethylene production is modulated mainly via regulation of expression of ACS genes (Kende, 1993; Wang et al., 2002). However, in some cases, ACO is also the key regulator restricting plant ethylene production (Vriezen et al., 1999; Wagstaff et al., 2005).
During past decades, much effort has been made to understand the ethylene signal transduction pathway, and a framework of ethylene perception and signal transduction has been established through molecular genetic research in Arabidopsis (Guo and Ecker, 2004). Ethylene is perceived by a membrane-associated receptor family (the ETR/ERS genes) which is similar to the bacterial two-component histidine kinase receptors (Bleecker, 1999; Stepanova and Ecker, 2000). CTR1 is down-stream of these receptors and encodes a protein whose sequence is similar to the Raf family of Ser/Thr protein kinases (Kieber et al., 1993). The receptors and CTR1 all act as negative regulators, and binding of ethylene results in inactivation of the receptors and CTR1 sequentially (Hua and Meyerowitz, 1998). Receptor and CTR1 proteins form a complex via protein–protein interaction, and the complex is located in the endoplasmic reticulum; this association and location are required for CTR1 function (Chen et al., 2002; Gao et al., 2003; Huang et al., 2003). EIN2, an Nramp-like protein, acts as a down-stream component of CTR1 and is activated by inactivation of CTR1 (Alonso et al., 1999). The activated EIN2 signals to the nucleus and activates EIN3/EIL. As transcription factors, EIN3/EIL trigger transcription of down-stream genes, and induce the ethylene response (Wang et al., 2002).
Flower opening is a crucial developmental event for phanerogams. Ethylene has been demonstrated to influence several aspects of flower development, including flower sex determination (Rudich et al., 1972; Yamasaki et al., 2001), flower opening (Reid et al., 1989; Yamamoto et al., 1994), pollination-induced petal senescence (O'Neill et al., 1993; Tang and Woodson, 1996; Clark et al., 1997; Jones and Woodson, 1997; Bui and O'Neill, 1998; Llop-Tous et al., 2000), and petal abscission (van Doorn and Stead, 1997; van Doorn, 2002). Ethylene affects pollination-induced petal senescence primarily through temporal- and spatial-specific expression of ACS and ACO genes, while ethylene receptor gene expression is also involved in this event (Shibuya et al., 2002). However, to date, little is known about the function of ethylene in flower opening pre-pollination.
In modern cut roses, flower opening is a gradual and slow process. Previous studies showed that flower opening of cut roses was mostly sensitive to ethylene, but the response to ethylene varied among cultivars (Reid et al., 1989; Yamamoto et al., 1994; Cai et al., 2002). Wang et al. (2004) reported isolation of an ACS gene, RKacc7, and its expression increased at the onset of petal senescence in cut rose. In miniature potted roses, seven genes—four ethylene receptor (RhETR1, 2, 3, 4), two CTR1-like (RhCTR1 and 2), and one EIN3 (RhEIN3)—have been isolated. The expression of RhETR3, and RhCTR1 and 2 were up-regulated by exogenous ethylene, and expression of RhETR3 and RhCTR1 increased during flower senescence (Müller et al., 2000a, b, 2002, 2003). Until now, however, an important question remained unanswered: does ethylene regulate flower opening of roses through its biosynthesis, or signalling pathway, or both?
In the present work an attempt was made to understand the effect of ethylene on flower pre-pollination opening, and to identify key regulatory components in ethylene biosynthesis and signalling pathways in cut roses. For this purpose, flowers of cut rose cv. Samantha were treated at stage 2 (completely opened bud; Wang et al., 2004; Ma et al., 2005) with ethylene and 1-MCP (1-methylcyclopropene), an ethylene action inhibitor (Sisler et al., 1999). Morphological changes were observed and ethylene production and expression of ethylene biosynthesis and signal transduction genes were determined after treatment. The results demonstrate that ethylene is involved in the induction of full flower opening; transcriptional regulation of an ethylene receptor and CTR genes were involved in this induction process.