Examining the Development of Secondary Cell Walls in Arabidopsis Inflorescence Stem Using Hard and Soft X-Ray Scattering

The durable secondary cell wall gives vascular plants the structural foundation needed to grow upwards. Fundamental research of the synthesis and material properties of secondary cell wall is the first step to developing new bio-derived materials and the utilization of lignocellulosic biomass as a renewable energy source. It is a composite material composed of cellulose microfibrils, hemicellulose, and lignin. The process of how these components come together to form the secondary cell wall in the plant is currently poorly understood. To track the development of secondary cell wall formation at different stages, we use the base of <i>Arabidopsis thaliana</i> inflorescence stems harvested at different heights. The total inflorescence stem height is proportional to the progress of secondary cell wall development in the interfascicular fiber (IFF) cells in the stem base. At lower stem heights (5 cm), we see little secondary cell wall in the IFFs and at higher stem heights (34 cm), we see full lignification of IFFs. Small-angle X-ray scattering (SAXS) tracks the changes in cellulose microfibril spacing and microfibril angle distribution. Wide-angle X-ray scattering (WAXS) tracks the relative crystalline cellulose content, chain packing, and coherence length. To investigate the effects of the content and structure of xylan, a principal hemicellulose, on secondary cell wall formation, we examined the stems of irx14 mutants. We found that compared to the wild type, the irx14 mutants had altered cellulose microfibril angle distribution and coherence length, but not individual cellulose chain packing distribution. To determine the spacing of lignin domains in secondary cell wall, we applied resonant soft X-ray scattering (RSoXS). Lignin is unique from other cell wall components in that it is highly aromatic, to which the secondary cell wall owes its hydrophobicity and resistance to degradation. RSoXS probes the sample with an X-ray beam tuned around the carbon absorption edge, increasing the contrast between lignin and other cell wall components without the need for labelling. At energies where the contrast between cellulose and lignin is enhanced, we see a broad nanometer-scale feature at high stem height, but not low stem height.