Title: Maize Brittle Stalk2 Encodes a Cobra-like Protein Expressed in Early Organ Development but Required for Tissue Flexibility at Maturity

University of Florida funds to purchase a pyrolysis mass spectrometer (to W.V.). Abstract The maize brittle stalk2 (bk2) is a recessive mutant, the aerial parts of which are easily broken. The bk2 phenotype is developmentally regulated and appears four weeks after planting, at about the fifth-leaf stage. Before this time, mutants are indistinguishable from wild-type siblings. Afterwards, all organs of the bk2 mutants turn brittle, even the pre-existing ones, and they remain brittle throughout the life of the plant. Leaf tension assays and bend tests of the internodes show that the brittle phenotype does not result from loss of tensile strength but from loss in flexibility that causes the tissues to snap instead of bend. The Bk2 gene was cloned by a combination of transposon tagging and a candidate gene approach and found to encode a COBRA-like protein similar to rice BC1 and Arabidopsis COBL4. The outer periphery of the stalk has fewer vascular bundles, and the sclerids underlying the epidermis possess thinner secondary walls. Relative cellulose content is not strictly correlated with the brittle phenotype. Cellulose content in mature zones of bk2 mature stems is lowered by 40% but is about the same as wild type in developing stems. Although relative cellulose content is lowered in leaves after the onset of the brittle phenotype, total wall mass as a proportion of dry mass is either unchanged or slightly increased indicating a compensatory increase in non-cellulosic carbohydrate mass. Fourier transform infrared spectra indicated an increase in phenolic ester content in the walls of bk2 leaves and stems. Total content of lignin is unaffected in bk2 juvenile leaves before or after appearance of the brittle phenotype, but bk2 mature and developing stems are markedly enriched in lignin compared to wild-type stems. Despite increased lignin in bk2 stems, loss of staining with phloroglucinol and UV autofluorescence is observed in vascular bundles and sclerid layers. Consistent with the infrared analyses, levels of saponifiable hydroxycinnamates are elevated in bk2 leaves and stems. As Bk2 is highly expressed during early development, well before the onset of the brittle phenotype, we propose that Bk2 functions in a patterning of lignin-cellulosic interactions that maintains organ flexibility rather than having a direct role in cellulose biosynthesis.