Plant cell walls are composed of independent but interacting networks of carbohydrates, proteins, and aromatic substances (McCann and Roberts 1991, Carpita and Gibeaut 1993). Interacting with this complex matrix are several hundred enzymes and other proteins that carry out many functions, from wall assembly and disassembly to defense against would-be pathogens. In recent years, several cell-wall structural proteins and enzymes, and their respective genes, have been identified (Henrissat et al. 2001, Richmond and Somerville 2001). Cell wall polysaccharides are the most abundant organic molecules on our planet. As secondary products of metabolism, these molecules have proved to be the most elusive to a genetic approach, and consequently, only a few dozen genes involved in cell-wall biogenesis have been identified. Of the 40 or so cell types that plants make, almost all of them are identified by unique features of their cell walls. Cell wall biogenesis during cell growth and differentiation involves several thousand genes (Carpita et al. 2001). Further, a majority of the cell-wall proteins from several species that were randomly microsequenced showed no similarity with previously described sequences (Robertson et al. 1997). These studies show that only a few of the genes that encode the machinery that makes cell wall polymers have been identified, and a systematic and comprehensive identification of the function of cell wall biogenesis-related genes is barely underway.
We have developed Fourier transform infrared (FTIR) microspectroscopy as a powerful and selective screening technique to identify broad classes of cell wall biogenesis-related genes (Chen et al. 1998). In addition to the FTIR screen, we have strengthened the overall program to include other spectroscopic approaches. In addition, Steve Thomas and colleagues (NREL; Golden, CO) have developed near-infrared (NIR) spectroscopy as an ultrahigh through-put means to identify maize secondary wall mutants in plants in the field.
Our approach to identify cell wall biogenesis mutants also employs reverse-genetics to uncover mutant phenotypes resulting from insertions of transposon and T-DNA in genes that are already known to be wall biogenesis-related, such as those of the cellulose-synthase gene family, hydrolases, and transglycosylases. In contrast, the FTIR and other spectroscopic screens offer complementary strategies to identify mutations for which no prior function is known.
Maize and Arabidopsis represent genetic models with the most widely distinct cell walls among the angiosperms. Insertionally mutagenized populations for each of these species have been developed that will maximize the ability of these IR spectroscopies to identify genes that function in wall biogenesis. Efficient systematic protocols employing biochemical, spectroscopic, and cytological approaches were developed in parallel to deduce specific defects in wall metabolism that result in the IR phenotypes revealed by our screens.
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