Comparative genomics and the identification of GT2 1314PDglucan synthases

Another biologically and commercially important group of GT2 enzymes that defied characterization until the emergence of functional genomics and associated technologies is the group containing the (1,3;1,4)-P-d-glucan synthases. These were expected to be GT2 enzymes and it was further anticipated that the corresponding genes would be found in one of the Csl subgroups, given the structural similarities between cellulose and (1,3;1,4)-P-d-glucans. During the original compilation of the phylogenetic tree of the cellulose synthase gene superfamily (Richmond & Somerville 2000; Fig. 5.4), it was noted that two of the Csl subgroups, namely CslF and CslH, were found exclusively in the grasses. Given that the (1,3;1,4)-P-d-glucans are also distributed almost exclusively in the grasses (Trethewey et al. 2005), members of the CslF and CslH gene families immediately became candidate genes for (1,3;1,4)-p-d-glucan synthases (Hazen et al. 2002). Reverse genetic approaches were therefore adopted by a number of research groups, but progress towards proving the functions of the CslF and CslH genes was relatively slow.

Using a combination of forward genetics and comparative genomics, Burton et al. (2006) showed that the CslF genes of rice mediate the synthesis of cell wall (1,3;1,4)-p-d-glucans. The forward genetic component had been undertaken previously by Han et al. (1995), who had explored natural variation in a barley mapping population to identify quantitative trait loci (QTLs) for the amount of (1,3;1,4)-P-d-glucan in ungerminated barley grain. The impetus for the analysis of QTLs for grain (1,3;1,4)-P-d-glucan content in barley had been provided by the importance of these polysaccharides in the malting and brewing processes (Bamforth 1993). Although it would have been possible at that stage to pursue the forward genetic approach further through fine mapping and eventually map-based cloning of the genes under the QTLs, Burton et al. (2006) chose to adopt a comparative genomic approach to short-cut the identification of the (1,3;1,4)-P-d-glucan synthase genes. One major QTL was located in a large chromosomal interval (more than 10cM) on barley chromosome 2H (Han et al. 1995), but in the absence of a genome sequence for barley it was not possible to identify the genes under the QTL that might be involved in (1,3;1,4)-P-d-glucan synthesis.

However, the common evolutionary origin of the cereals is reflected in a well-established property known as synteny (Gale & Devos 1998; Kota et al. 2007), which describes the conservation of gene order along corresponding sections of chromosomes in different cereal species. Syntenous regions in different genomes can be fragmented or located on different chromosomes, and in some instances the colinearity breaks down at the 'micro' level (Pourkheirandish et al. 2007). Nevertheless, it was possible to identify the region of the rice genome that was syntenous to the region of the barley chromosome 2H where the QTL for grain (1,3;1,4)-P-d-glucan was located, using the sequences of DNA molecular markers that flank the barley QTL. The sequences corresponding to those barley markers were found on rice chromosome 7, where they defined a region of about 3 Mb (Burton et al. 2006). The availability of the annotated rice genome sequence allowed this region of rice chromosome 7 to be scanned for candidate genes for (1,3;1,4)-P-d-glucan synthesis. A cluster of six OsCslF genes and two truncated OsCslF pseudogenes was detected in a region of about 120 kb near one end of the 3 Mb section on rice chromosome 7 (Fig. 5.5). The observation that this gene

OsCslF4

OsCslF2

OsCslF9

OsCslF3 OsCslFI OsCslF8

Barley chr 2H

OsCslF cluster

Rice chr 7 (partial)

OsCslF4

OsCslF2

OsCslF9

OsCslF3 OsCslFI OsCslF8

Barley chr 2H

OsCslF cluster

Rice chr 7 (partial)

155 cH

Adh8 ABG019

Figure 5.5 The CslF family was identified as the prime candidate for genes encoding (1,3;1,4)-P-d-glucan synthases. A major QTL for (1,3;1,4)-P-d-glucan content of unger-minated barley grains was identified on barley chromosome 2H by Han et al. (1995) and the 'likelihood of differences ' (LOD) scores shown here were derived from that work. Markers flanking the estimated position of the barley chromosome 2H QTL, (Adh8, ABG019 and Bmy2), were used to identify a syntenic region of about 3.5 Mb on rice chromosome 7. Examination of the rice genome sequence in this region revealed a group of six OsCslF genes, close to the Bmy2 marker; the six genes were clustered within an interval of about 118 kb. From Burton et al. (2006), reprinted with permission from AAAS.

155 cH

Adh8 ABG019

Figure 5.5 The CslF family was identified as the prime candidate for genes encoding (1,3;1,4)-P-d-glucan synthases. A major QTL for (1,3;1,4)-P-d-glucan content of unger-minated barley grains was identified on barley chromosome 2H by Han et al. (1995) and the 'likelihood of differences ' (LOD) scores shown here were derived from that work. Markers flanking the estimated position of the barley chromosome 2H QTL, (Adh8, ABG019 and Bmy2), were used to identify a syntenic region of about 3.5 Mb on rice chromosome 7. Examination of the rice genome sequence in this region revealed a group of six OsCslF genes, close to the Bmy2 marker; the six genes were clustered within an interval of about 118 kb. From Burton et al. (2006), reprinted with permission from AAAS.

cluster in rice was located in a position syntenous to the grain (1,3;1,4)-P-d-glucan QTL on barley chromosome 2H, coupled with the fact that the CslF subgroup is a so- called monocot- specific group in the cellulose synthase gene superfamily (Richmond & Somerville 2000), immediately increased our confidence that these genes might indeed mediate (1,3;1,4)-P-d-glucan synthesis in cereals and grasses.

Forward genetics and comparative genomics had delivered strong candidate genes, but it remained necessary to prove the functions of the CslF genes in (1,3;1,4)-P-d-glucan synthesis. The rice OsCslF genes were therefore inserted into Arabidopsis, which normally does not contain CslF genes and does not have (1,3;1,4)-P-d-glucans in its walls. (1,3;1,4)-P-d-Glucan was subsequently detected in walls of the transgenic plants, using specific monoclonal antibodies and enzymatic analyses (Burton et al. 2006 ). These experiments provided direct, gain-of-function evidence for the participation of rice OsCslF genes in (1,3;1,4)-P-d-glucan biosynthesis. However, Burton et al. (2006) pointed out that the observations did not preclude a requirement for other enzymes, proteins or cofactors in (1,3;1,4)-P-d-glucan synthesis. The relatively low levels of (1,3;1,4)-P-d-glucan in walls of the transgenic Arabidopsis plants, where OsCslF transcript levels were often high, was consistent with limiting levels of other components that might be required for high-level synthesis of the polysaccharide or its transfer to the cell wall (Burton et al. 2006). While there are two 'monocot-specific ' subgroups (CslF and CslH) in the cellulose synthase gene superfamily, there is only type of polysaccharide, namely (1,3;1,4)-P-d-glucan, that is found almost exclusively in the commelinoid monocotyledons (Trethewey et al. 2005 ) and whether the CslH and CslF proteins might act in concert or mediate the synthesis of (1,3;1,4)-P-d-glucans with different fine structures remains to be demonstrated. Whether the CslH and CslF proteins might act in concert or mediate the synthesis of (1,3;1,4)-P-d-glucans with different fine structures remains to be demonstrated. It has been suggested that the biosynthesis of (1,3;1,4)-P-d-glucans might require the concerted action of multiple enzymes that form large multimeric complexes (Buckeridge et al. 1999; Buckeridge et al. 2004), as observed for cellulose biosynthesis (Doblin et al. 2002 ).

In describing the enzymic apparatus for (1,3;1,4)-P-d-glucan synthesis, one must also ask how the fine structure of the polysaccharide is effected, in particular with respect to the insertion of (1,3)-P -d- glucopyranosyl residues at irregular intervals along the backbone chain, and hence to the arrangement of the cellotriose, cellotetraose and longer (1,4)-P-d-oligoglucoside blocks described above.

Was this article helpful?

0 0

Post a comment