This article describes a methodology for detailed mapping of the lignification capacity of plant cell walls that we have called “REPRISAL” for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G* and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labelling strategy based on the sequential use of 3 main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence (AI) segmentation algorithm is developed that assigns fluorescent image pixels to 3 distinct cell wall zones corresponding to cell corners (CC), compound middle lamella (CML) and secondary cell walls (SCW). The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method 1) and proportions (ratiometric method 2) within the different cell wall zones. We firstly describe the use of this methodology to map developmentally-related changes in the lignification capacity of wild-type Arabidopsis (Arabidopsis thaliana) interfascicular fiber cells. We then apply REPRISAL to analyze the Arabidopsis peroxidase (PRX) mutant prx64 and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. In addition, we also demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar (Populus tremula × P. alba), flax (Linum usitatissimum) and maize (Zea mays). Finally, we show that the methodology can be used to map the incorporation of a fucose reporter into non-cellulosic cell wall polymers.

Reactive species causing oxidative stress are unavoidable by-products of various plant metabolic processes, such as photosynthesis, respiration or photorespiration. In leaves, flavonoids scavenge reactive species produced during photosynthesis and protect plant cells against deleterious oxidative damages. Their biosynthesis and accumulation are therefore under tight regulation at the cellular level. Glycosylation has emerged as an essential biochemical reaction in the homeostasis of various specialized metabolites such as flavonoids. This article provides a functional characterization of the Populus tremula x P. alba (poplar) UGT72A2 coding for a UDP-glycosyltransferase that is localized in the chloroplasts. Compared to the wild type, transgenic poplar lines with decreased expression of UGT72A2 are characterized by reduced growth and oxidative damages in leaves, as evidenced by necrosis, higher content of glutathione and lipid peroxidation products as well as diminished soluble peroxidase activity and NADPH to NADP+ ratio under standard growing conditions. They furthermore display lower pools of phenolics, anthocyanins and total flavonoids but higher proanthocyanidins content. Promoter analysis revealed the presence of cis-elements involved in photomorphogenesis, chloroplast biogenesis and flavonoid biosynthesis. UGT72A2 is regulated by the poplar MYB119, a transcription factor known to regulate the flavonoid biosynthesis pathway. Phylogenetic analysis and molecular docking suggest that UGT72A2 could glycosylate flavonoids, however the actual substrate(s) was(ere) not consistently evidenced with in vitro assays nor analyses of glycosylated products in leaves of transgenic poplar overexpressing or downregulated for UGT72A2. This article provides elements highlighting the importance of flavonoid glycosylation regarding protection against oxidative stress in poplar leaves and raises new questions about the link between this biochemical reaction and regulation of the redox homeostasis system.

In this paper, we present the use of multiplex click/bioorthogonal chemistry combined with super-resolution Airyscan microscopy to track biomolecules in living systems with a focus on studying lignin formation in plant cell walls. While laser scanning confocal microscopy (LSCM) provided insights into the tissue-scale dynamics of lignin formation and distribution in our previous reports, its limited resolution precluded an in-depth analysis of lignin composition at the unique cell wall or substructure level. To overcome this limitation, we explored the use of Airyscan microscopy, which, among the super-resolution techniques available, offers an optimal balance between performance, cost, accessibility, and ease of implementation. Our study demonstrates that a triple labeling strategy using copper-catalyzed azide–alkyne cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), and inverse electronic-demand Diels–Alder cycloaddition (IEDDA) to label modified lignin metabolic precursors can be combined with Airyscan microscopy to reveal the zones of active lignification at the single cell level with improved sensitivity and resolution. This approach enables insights into the lignin composition in wall substructures, such as pits or in wall layers that are otherwise not distinguishable by classical LSCM. Our work emphasizes the importance of studying lignin formation in plant cell walls and demonstrates the potential of combining bioorthogonal chemistry and super-resolution microscopy techniques for studying biomolecules in living systems.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software which makes possible the quantification of lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.