Plants have many expansin paralogues that are closely related in sequence but they have diverse and complex spatiotemporal expression profiles often regulated by external stimuli such as hormones and light (Hong et al., 2003; Reyes et al., 2004; Jiao et al., 2005). This suggests that the expression profile may be more important than functional differences between proteins when it comes to determining the roles of individual genes (Fleming, 2006). In turn, this indicates that promoter analysis is likely to provide clues as to the function of individual expansin genes, so we started this study by cloning the promoter region of the PhEXPA1 gene and analysing its sequence. Like other expansin promoters (Lee et al., 2001; Cho & Cosgrove, 2002), the 1 kb of PhEXPA1 promoter contains response elements to gibberellin and auxin. The regulation of expansin genes by gibberellins and auxins is well documented mainly from studies on deepwater rice (Oryza sativa; Lee & Kende, 2002), tomato (Catala et al., 2000; Vogler et al., 2003), soybean (Glycine max; Downes et al., 2001), and chickpea (Cicer arietinum; Sanchez et al., 2004). PhEXPA1 promoter activity was studied using a GUS reporter assay, showing a pattern of activity consistent with the spatiotemporal profile determined by real-time RT-PCR (Zenoni et al., 2004). In flowers, GUS activity was detected in petals, sepals, ovaries, styles and stigmas, and was coincident with areas of cell expansion during limb development rather than zones of cell division as mapped by Reale et al. (2002). There was no GUS activity in anthers. In stems, GUS staining was restricted to the nodes at the base of axillary meristems, suggesting that PhEXPA1 has a role in promoting stem elongation and possibly axillary bud outgrowth. These processes are, at least partially, under the control of hormones, mainly auxin and gibberellin (Benkova et al., 2003; Reinhardt et al., 2003; Fleet & Sun, 2005). Although we have not demonstrated the direct influence of phytohormones on PhEXPA1 expression, the presence of corresponding response elements in the PhEXPA1 promoter, together with its restricted expression pattern in the stem, suggests that PhEXPA1 acts downstream of phytohormones in balancing meristem maintenance and organ initiation.
In order to determine the intracellular distribution of PhEXPA1, we generated transgenic plants constitutively expressing a PhEXPA1-eGFP fusion protein. Expansins have previously been localized to the cell wall by immunolocalization and electron microscopy (Cosgrove et al., 2002; Fudali et al., 2008). However, because EXPA-specific antibodies have yet to be developed, it is only possible to look at expansin distribution in a general context, rather than the distribution of specific gene products. Furthermore, in a recent study, Mohanty et al. (2009) showed that an EXPA-YFP fusion protein was correctly targeted to the cell wall in maize (Zea mays). We confirmed using confocal microscopy that the PhEXPA1-eGFP fluorescence signal overlapped with the cell wall-specific calcofluor signal in plasmolysed limb abaxial epidermis tissue, strongly suggesting that PhEXPA1-eGFP is localized in the cell wall. A strong GFP signal was also detected inside the protoplast, although this could reflect the high activity of the constitutive CaMV 35S promoter producing larger quantities of the protein than normally found in planta.