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Successful modification of plant cell-wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but this can be very challenging when the target genes are members of multigene families. 4-coumarate:CoA ligase (4CL) catalyzes the formation of 4-coumaroyl CoA, a precursor of both flavonoids and monolignols, and is an attractive target for transgenic down-regulation aimed at improving agro-industrial properties. Inconsistent phenotypes of transgenic plants have been attributed to variable levels of down-regulation of multiple 4CL genes. Phylogenetic analysis of the sorghum genome revealed 24 4CL(-like) proteins, five of which cluster with bona fide 4CLs from other species. Using a map-based cloning approach and analysis of two independent mutant alleles, the sorghum brown midrib2 (bmr2) locus was shown to encode 4CL. In vitro enzyme assays indicated that its preferred substrate is 4-coumarate. Missense mutations in the two bmr2 alleles result in loss of 4CL activity, probably as a result of improper folding as indicated by molecular modeling. Bmr2 is the most highly expressed 4CL in sorghum stems, leaves and roots, both at the seedling stage and in pre-flowering plants, but the products of several paralogs also display 4CL activity and compensate for some of the lost activity. The contribution of the paralogs varies between developmental stages and tissues. Gene expression assays indicated that Bmr2 is under auto-regulatory control, as reduced 4CL activity results in over-expression of the defective gene. Several 4CL paralogs are also up-regulated in response to the mutation.
Saballos et al. (Tue,) studied this question.