Catechyl-lignin tissues in Vanilla orchid and Candlenut: structure/property studies
In 2012, a new type of lignin, catechyl (C)-lignin was found in the seed coat of vanilla orchid (Vanilla planifolia) and Melocactus cacti, and later in the nutshell of Aleurites moluccana (candlenut). This caffeyl alcohol homopolymer is the exclusive lignin in vanilla seed coat but separated in time and/or location with guaiacyl (G)-lignin in candlenut. Unlike conventional guaiacyl/syringyl (G/S-lignins) with alkyl-aryl ether linkages, intermonomer linkages in C-lignin are connected by benzodioxane linkages which are stiffer than alkyl-aryl ether linkages. C-lignin is unusually stable against acid-catalyzed cleavage. Tissues with C-lignin are expected to exhibit high glass transition temperature (Tg) compared to tissues with G/S/H-lignin. C-lignin also probably shows high crystallinity due to its highly linear-homopolymer structure. The ability of some seed coats/nutshells in angiosperms to synthesize a new type of lignin is another level of lignin evolution. However, the role of C-lignin related to the function of the seed coat is unclear while it exhibits different behaviors to the regular G/S/H-lignin. These points motivated us to conduct cell-wall structure/property studies in the context of plant evolution, using microscopy, X-ray diffraction (XRD) and dynamic mechanical analysis (DMA). Light and electron microscopes were used to identify cell's size and type of intact and macerated vanilla seed coat and candlenut shell. Vanilla seeds are tiny, sized approximately 300μm and the surface is covered with dark-colored seed coat. Candlenut is slightly smaller than walnut, with uneven, hard, dark brown shell covering the nut. Microscopy observations indicated that both seed coat and nutshell are dominated by highly lignified cells, known as sclereids. The types of sclereids in vanilla seed coat and candlenut shell are different; vanilla seed coat has ostoesclereid-type cells, while candlenut shell has macrosclereid-type cells. XRD was used to study tissue with C-lignin crystallinity by comparing diffractograms of vanilla seed coat and candlenut shell to Southern Yellow Pine wood diffractograms. The Southern Yellow Pine wood diffractogram corresponds to a typical native cellulose in higher plants, that is cellulose I allomorph. Diffractogram XRD analysis on vanilla seed coat and candlenut shell shows similarities to Southern Yellow Pine native cellulose, suggesting that cellulose is the contributor for crystallinity in seed coat and nutshell, and this also indicated that tissues with C-lignin is not crystalline. Crystallinities of vanilla seed coat and candlenut shell determined using peak deconvolution methods were about half of Southern Yellow Pine crystallinity. DMA was used to measure Tg in vanilla seed coat and candlenut shell. Measurements were conducted in solvent-submersion mode using organic plasticizers to reduce the Tg to non-damaging temperatures. DMA measurement of vanilla seed coat and candlenut shell is challenging due to specimen size and shape. Specimen preparation for DMA measurement included seed coat purification for vanilla and cutting/milling for candlenut shell followed by specimen saturation in plasticizers. Compressive-torsion DMA was used to allow tiny specimens gripping. Vanilla seed coats exhibited higher glass transition temperature compared to wood, while candlenut shells exhibited various Tgs depending on specimen type/size.