Multimodal imaging analysis in silver fir reveals coordination in cellulose and lignin deposition

Despite lignin being a key component of wood, the dynamics of tracheid lignification are generally overlooked in xylogenesis studies, which hampers our understanding of environmental drivers and blurs the interpretation of isotopic and anatomical signals stored in tree rings. Here, we analyzed cell wall formation in silver fir (Abies alba Mill.) tracheids to determine if cell wall lignification lags behind secondary wall deposition. For this purpose, we applied a multimodal imaging approach combining transmitted light microscopy (TLM), confocal laser scanning microscopy (CLSM), and confocal Raman microspectroscopy (RMS) on anatomical sections of wood microcores collected in northeast France on 11 dates during the 2010 growing season. Wood autofluorescence after laser excitation at 405 and 488 nm associated with the RMS scattering of lignin and cellulose, respectively, which allowed identification of lignifying cells (cells showing lignified and nonlignified wall fractions at the same time) in CLSM images. The number of lignifying cells in CLSM images mirrored the number of wall-thickening birefringent cells in polarized TLM images, revealing highly synchronized kinetics for wall thickening and lignification (similar timings and durations at the cell level). CLSM images and RMS chemical maps revealed a substantial incorporation of lignin into the wall at early stages of secondary wall deposition. Our results show that most of the cellulose and lignin contained in the cell wall undergo concurrent periods of deposition. This suggests a strong synchronization between cellulose and lignin-related features in conifer tree-ring records, as they originated over highly overlapped time frames.