Comparison between tracheary element lignin formation and extracellular lignin-like substance formation during the culture of isolated Zinnia elegans mesophyll cells

Abstract

Lignin is synthesized not only during morphogenesis of vascular plants but also in response to various stresses. Isolated Zinnia elegans mesophyll cells can differentiate into tracheary elements (TEs), and deposit lignin into cell walls in TE-inductive medium (D medium). Meanwhile isolated mesophyll cells cultured in hormone-free medium (Co medium) accumulate stress lignin-like substance during culture. Therefore this culture system is suitable for study of lignin and lignin-like substance formation. In D medium lignin was deposited in TEs, but in Co medium, extracellular lignin-like substance accumulated. Analysis of the culture media indicated the presence of dilignols in D culture, but not in Co culture. To investigate the fate of lignin precursors, we added coniferyl alcohol (CA) in each culture. In Co medium, CA was polymerized into dilignols rapidly but they were present only temporarily, and in D medium CA was polymerized into dilignols relatively slowly but their content increased continually. Meanwhile, in Co culture, peroxidase activity in the medium was much higher than the peroxidase activity bound ionically to the cell walls. In D culture, ionically bound peroxidase activity was higher than that in the medium. These results may suggest that lignin deposition in TEs is related to ionically bound peroxidases in D culture, and lignin-like substance deposition in the medium is related to peroxidases in the medium in Co culture.

Formation of plant tracheary elements in vitro – a review

“…This work will help refine current cambium models, both the in vitro discontinuous and in planta continuous model (Pesquet and Tuominen 2011). Finally, further analysis using in vitro TE systems could help provide additional insight into stress-responses and the complex mechanisms of lignin formation during xylem differentiation (Pesquet et al 2010;Sato et al 2011;Wagner et al 2011). New genomic technologies are well suited to identify the basic biological networks underlying cambial functions and wood formation (Fenning and Gershenzon 2002;Fenning et al 2008;Du and Groover 2010;Groover 2007).…”

“…Observation of secondary wall thickening under a light microscope allows TEs to be easily detected (Sato et al 2011). Tracheary elements can be analysed qualitatively using morphological features and quantitatively by determining the rate of differentiation using cell counting methodologies.…”

“…Phloroglucinol staining can be used to monitor the level of lignification (McCann 1997;Sato et al 2011). Safranin also reacts with lignin by staining it red.…”

“…These include: a thioglycolic acid lignin assay (Möller et al 2003); Klason lignin analysis (Möller et al 2005b); thioacidolysis (Möller et al 2005a(Möller et al , 2005bWagner et al 2013); two dimensional nuclear magnetic resonance (Wagner et al 2007); an acetyl bromide lignin assay (Wagner et al 2007); alkalinenitrobenzene oxidation (Sato et al 2011); pyrolysis-gas chromatography coupled mass spectroscopy (Pillai et al 2011); and Fourier-transform infrared (FTIR) spectroscopy (Pillai et al 2011). In some cases, similar methods are applicable to the analysis of cell-wall polysaccharides, e.g.…”

“…Pesquet and Tuominen (2011) demonstrated the strict dependence of TE differentiation on ethylene biosynthesis and a stimulatory effect of ethylene on the rate of TE differentiation in vitro. Sato et al (2011) compared TE lignification and the formation of stress-induced, lignin-like substances using the Z. elegans in vitro system to understand their mechanism of formation. The authors suggested that in the Z. elegans lignin formation in TEs is regulated by the localisation and activity levels of peroxidases.…”