DEVELOPMENTAL REGULATION OF MANNAN, ARABINOGALACTAN-PROTEIN, AND PECTIC EPITOPES IN PISTILS OF VICIA FABA (FABA BEAN) Wen Chen,* Fred L. Stoddard, y and Timothy C. Baldwin 1, * *School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1SB, United Kingdom; and y Department of Applied Biology, University of Helsinki, P.O. Box 27, FIN-00014 Helsinki, Finland Cell walls of the stigma and style are important zones for cell-cell recognition, nutrition, guidance, and protection of the pollen tube along the transmitting tract. The objective of this study was to investigate the modulation of selected cell wall epitopes during pistil development in the crop species Vicia faba L. (faba bean). An immunocytochemical investigation of pistil development was performed using a panel of four anti– cell wall/plasma membrane antibodies at both light and electron microscope levels during the 4 days leading up to anthesis. The selected antibodies recognized mannan-containing polysaccharides (antimannan antisera), b-1,4-galactan (LM5), a-1,5-L-arabinan (LM6), and arabinogalactan/arabinogalactan-protein (AG/AGP) epitopes, with GlcpUA-b-1,!3-D-Gal pUA-a-1,!2-L-Rha trisaccharide motifs (JIM13). Mannan accumulation was shown to be associated with the formation and maturation of sclerenchyma cells and xylem elements. The b-1,4-galactan and a-1,5-L-arabinan epitopes were highly expressed in the walls of parenchyma and epidermis in a developmentally regulated manner. AG/AGP epitopes were particularly abundant in the stigma and cells lining the stylar canal, together with the a-1,5-L-arabinan epitope recognized by LM6. These data suggest an association of AGs/AGPs with pollen tube growth, of mannan with strengthening and stiffening the pistil, and of b-1,4-galactan and a-1,5-L-arabinan components with tissue flexibility within the pistil. Keywords: faba bean, open style, arabinogalactan-protein, b-galactan, mannan, a-arabinan. Introduction The importance of the composition and architecture of the extracellular matrix (including cell walls and intercellular re- gions) of the style in pistil function has been demonstrated both where the style is ‘‘open,’’ e.g., Lilium longiflorum (La- barca and Loewus 1972; Dickinson et al. 1982; Janson et al. 1994; Park et al. 2000; Lord 2003), and where it is ‘‘solid,’’ e.g., Nicotiana tabacum (Cheung et al. 1993; Cheung 1995). In these model systems, specific polysaccharides (Lord and Mol- let 2002), lipids, and structural proteins have been shown to be involved in the regulation of pollen tube growth (Cheung et al. 1993; Janson et al. 1994; Lord 2003), to serve as adhe- sives (Jauh et al. 1997; Park et al. 2000; Lord 2001, 2003), nutrients (Labarca and Loewus 1972), attractants, and recog- nition molecules, and to guide pollen tube growth (Dickinson et al. 1982; Baldwin et al. 1992; Cheung et al. 1993, 1996, 2000; Wang et al. 1993; Cheung 1995; Wu et al. 1995; Wheeler et al. 2001; Minorsky 2003). Several components have been characterized, such as cysteine-rich adhesin (Jauh et al. 1997; Park et al. 2000; Lord 2001) and transmitting- tissue-specific (TTS) proteins (Cheung et al. 2000). Moreover, TTS arabinogalactan proteins (AGPs) were shown to enhance and guide pollen tube growth in the solid styles of N. tabacum and Nicotiana alata (Cheung and Wu 1999; Wu et al. 2000, 2001; de Graaf et al. 2003; Lord 2003). Grain legumes are extremely important in world agricul- ture, yet information on the structure, composition, and functioning of their solid stigma and open style is limited. The faba bean (Vicia faba L.) is a particularly suitable tool for this purpose because it produces numerous large flowers with straight styles that are easy to dissect and is partially de- pendent on bee activity for its pollination. Most other impor- tant grain legumes, including soybean, pea, common bean, chickpea, and lentil, reliably self-pollinate; in some, the flow- ers are small, and in many, the style is twisted. The faba bean stigma produces an exudate that is held within the cuti- cle (Paul et al. 1978). The requirement for mechanical stimu- lation for the release of this exudate sets this species apart from typical ‘‘wet’’-stigma species (Lord and Heslop-Harrison 1984). The apex of the style immediately beneath the stigma is initially solid and contains a transmitting tissue through which the pollen tubes extend, while the remainder of the style is open (Ghosh and Shivanna 1982; Lord and Heslop- Harrison 1984). Pollen germination and the early stages of pollen tube elongation in faba bean, therefore, may be simi- lar to those observed in other species possessing a wet stigma with a solid style (Heslop-Harrison and Heslop-Harrison 1983), such as N. tabacum (Cheung et al. 1995), while later extension of the pollen tube may resemble that in species possessing a stylar canal (such as L. longiflorum), in which proper adhesion and guidance along the surface of the stylar canal is essential (Lord and Mollet 2002; Lord 2003). We therefore investigated pistil development and floral func- tion in faba bean and the role(s) of selected cell wall–associated components in this process, using an immunocytochemical 1 Author for correspondence; telephone 44-1902-322142; fax 44-1902-322680; e-mail t.baldwin@wlv.ac.uk. Manuscript received March 2006; revised manuscript received May 2006. 919 Int. J. Plant Sci. 167(5):919–932. 2006. Ó 2006 by The University of Chicago. All rights reserved. 1058-5893/2006/16705-0001$15.00