Secondary cell wall characterization in a BY-2 inductive system

Plant secondary cell walls (SCW) are likely to serve as major sources of sugars for biofuel production. Yet, current knowledge of SCW biosynthesis and modifications is far from complete. Cell culture has been suggested as an ideal system for identifying and characterizing cell wall biosynthetic genes. This knowledge will underpin strategies to improve bioenergy feedstocks, and their subsequent fermentation. We previously established a glucocorticoid receptor (GR)-mediated post-translational inducible system of VASCULAR-RELATED NAC DOMAIN7 (VND7) fused to the activation domain of the herpes virus VP16 in tobacco Bright Yellow-2 (BY-2) cells. VND7 is a master transcription factor that controls xylem vessel element differentiation. Its activation by dexamethasone (DEX) resulted in the induction of vessel element formation including cell wall thickening and cell death at high frequency. Here we investigate the kinetics of SCW-related genes after DEX induction in tobacco. Homologues of Arabidopsis cellulose and xylan biosynthetic genes were cloned, and used to conduct kinetic RT-qPCR. Results show an increase in SCW gene expression in BY-2 cells 10 h after DEX induction, with a maximum observed at 24 h. Biochemical analyses of the DEX-induced lines reveal increased cellulose to non-cellulosic polysaccharide ratio, as well as time-dependent increases in characteristic SCW polymers, such as xylan and mannan, indicating the deposition of true SCW. Therefore, we conclude that the BY2::VND7-VP16-GR cell suspension culture is a convenient in vitro system that can be used as an efficient tool to elucidate the regulation of SCW formation through RNA transcript and biochemical analysis.