Visualization of plant cell wall lignification using fluorescence‐tagged monolignols

Abstract

Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development.

Hydroxystilbenes Are Monomers in Palm Fruit Endocarp Lignins

“…Assignment details are described in the Supplemental Material. caffeyl alcohol or 5-hydroxyconiferyl alcohol, and particularly with the trans-isomer (Chen et 214 al., 2012;Chen et al, 2013;Tobimatsu et al, 2013), and are clearly different from the 215 benzodioxane structure of homo-coupled piceatannols P b . The benzodioxane structures do not 216 result from the incorporation of caffeyl alcohol, nor from the related 5-hydroxyconiferyl alcohol, 217 both of which may be evidenced in the spectra of lignins from O-methyltransferase-deficient 218 plants (Ralph et al, 2001;Wagner et al, 2011), as the expected monomers from such 219 involvement in lignins were not observed here by DFRC and the characteristic aromatic signals 220 from such catechols were also not evident; all of the peaks in the HSQC spectra ( Fig.…”

Laccases Direct Lignification in the Discrete Secondary Cell Wall Domains of Protoxylem

“…The tools to assess this prediction were recently developed, in the form of fluorescently tagged monolignol analogs, such as g-nitrobenzofuran (NBD)-tagged coniferyl alcohol (CA), that become polymerized into lignin via oxidase-mediated radical coupling (Tobimatsu et al, 2011). When applied in planta, these NBD-tagged probes were incorporated into the lignifying tissues in Arabidopsis and Pinus radiata plants (Tobimatsu et al, 2013).…”

“…Labeling cell wall lignin using NBD-CA was carried out as follows: a mixture of 0.5 mM NBD-CA (Tobimatsu et al, 2011(Tobimatsu et al, , 2013 and 5 mM CA (Sigma) was added to liquid one-half-strength MS medium to a final concentration of 1 and 10 mM, respectively, together with 10 mM dexamethasone (Sigma) for VND7 induction. VND7-VP16-GR seedlings were incubated in this induction solution for 4 to 48 h, and they were gently shaken under regular growth conditions (listed above).…”

Re-constructing our models of cellulose and primary cell wall assembly

“…While antibodies and carbohydrate binding modules have proved informative [11,12], epitope masking can be problematic [13]. Click labeling of pectins has given insights into pectin secretion and its aftermath [14*] while fluorescent monolignols enable new ways to monitor the process of lignification [15*,16]. …”