Abstract text: In angiosperms, the composition of lignin varies significantly among the secondary cell walls of different xylem elements, yet the significance of this variation remains unclear. The secondary cell walls of the vessel elements are enriched in guaiacyl-type lignin, which is believed to contribute to enhanced stiffness and perhaps also increased hydrophobicity of the cell walls. Here we investigated the significance of lignin composition on the physiology and vessel functioning in hybrid aspen (Populus tremula × P. tremuloides).

We identified earlier in Arabidopsis thaliana that PIRIN2 functions from within the vessel-associated cells to supress the syringyl to guaiacyl lignin (S/G) ratio. An increase in the S/G ratio of the prn2 mutant correlated with increased expression of the lignin-biosynthetic genes, and could be reverted by supressing the expression of FERULATE 5-HYDROXYLASE in the PRN2 expression domain. We aimed in this study to increase the S/G ratio with the help of the PRN genes in hybrid aspen (Populus tremula x P. tremuloides) to study the effect of S/G ratio on xylem vessel functioning. Populus has two PRN genes, and promoter–reporter analyses revealed that PttPRN2 was specifically expressed in the vessel-associated cells, whereas PttPRN1 was broadly expressed in the cambial zone and differentiating xylem. Total lignin content was increased in the CRISPR Cas9 lines for PttPRN2, supporting importance of PttPRN2 in lignification. Optical phototermal microspectrocopy (OPTIR) analysis of the PttPRN2 CRISPR–Cas9 line revealed an increase in the S/G ratio specifically in the vessel–fiber cell walls. However, the increase in the S/G ratio did not seem to significantly impair functioning of the vessels in water transport or mechanical support. Even though the equilibrium moisture content of the wood powder was slightly reduced, hydraulic efficiency remained largely unchanged. Furthermore, atomic force microscopy revealed that, contrary to the expected decrease in stiffness of the cell walls, the increase in the S/G ratio correlated with an increase in the stiffness of the vessel cell walls. Together, these results demonstrate that increased S-lignin does not seem to compromise vessel functionality. We propose therefore that the vessel associated cells primarily serve to supress lignin accumulation rather than control lignin composition of the xylem vessels. Enrichment of G-lignin in the vessels might simply be a result of G-type lignin deposition being predominant during differentiation of vessels which occurs much earlier than lignification of the fibers.