Insights into the molecular regulation of monolignol-derived product biosynthesis in the growing hemp hypocotyl

Abstract

Background: Lignin and lignans are both derived from the monolignol pathway. Despite the similarity of their building blocks, they fulfil different functions in planta. Lignin strengthens the tissues of the plant, while lignans are involved in plant defence and growth regulation. Their biosyntheses are tuned both spatially and temporally to suit the development of the plant (water conduction, reaction to stresses). We propose to study the general molecular events related to monolignol-derived product biosynthesis, especially lignin. It was previously shown that the growing hemp hypocotyl (between 6 and 20 days after sowing) is a valid system to study secondary growth and the molecular events accompanying lignification. The present work confirms the validity of this system, by using it to study the regulation of lignin and lignan biosynthesis. Microscopic observations, lignin analysis, proteomics, together with in situ laccase and peroxidase activity assays were carried out to understand the dynamics of lignin synthesis during the development of the hemp hypocotyl.

Results: Based on phylogenetic analysis and targeted gene expression, we suggest a role for the hemp dirigent and dirigent-like proteins in lignan biosynthesis. The transdisciplinary approach adopted resulted in the gene- and protein-level quantification of the main enzymes involved in the biosynthesis of monolignols and their oxidative coupling (laccases and class III peroxidases), in lignin deposition (dirigent-like proteins) and in the determination of the stereoconformation of lignans (dirigent proteins).

Conclusions: Our work sheds light on how, in the growing hemp hypocotyl, the provision of the precursors needed to synthesize the aromatic biomolecules lignin and lignans is regulated at the transcriptional and proteomic level.

Keywords: Gene expression; Hemp; Hypocotyl; Laccase; Lignan; Lignin; Monolignols; Peroxidase; Proteomics.

Fig. 1

Monolignol and cellulose pathways. The molecules (in black) and enzymes (in blue) of the two pathways are indicated. Cellulose synthase (CESA) is membrane bound. Cinnamate-4-hydroxylase (C4H) and coumarate 3-hydroxylase (C3H) localise in the endoplasmic reticulum; hydroxycinnamoyl transferase (HCT) is partially associated with the endoplasmic reticulum [71]. All the other enzymes are active in the cytosol. 4CL 4-coumarate ligase, CAD cinnamyl alcohol dehydrogenase, CCoAOMT caffeoyl-CoA 3-O-methyltransferase, CCR cinnamoyl CoA reductase, COMT caffeate O-methyltransferase, F5H ferulate 5-hydroxylase, FRK fructokinase, G6PI glucose-6-phosphate isomerase, HK hexokinase, INV invertase, PAL phenylalanine ammonia lyase, Phe phenylalanine, SUS sucrose synthase

Fig. 2

Lignification of the hemp hypocotyl between 6 and 20 days. a to d, fixed cross sections of H6 (a), H9 (b), H15 (c) and H20 (d) stained with FASGA. Lignification of the bast fibres is illustrated in the insets of c and d. e to h, fresh cross-sections of H15 (e and g) and H20 (f and h) stained with Mäule reagent. Higher magnifications of the bast fibres are shown in g and h. The primary xylem cells and tracheary elements of the secondary xylem are indicated with arrow heads, while fibres of the secondary xylem are indicated with an arrow in e and f. Scale bar = 100 μm in the main pictures and 25 μm in the insets

Fig. 3

Phylogenetic analysis of DIR and DIR-like proteins (DLP). Cannabis sativa (Csa), A. thaliana (At), Forsythia x intermedia, Populus trichocarpa, Schisandra chinensis and Linum usitatissimum (Lu). Neighbour-joining tree calculated with 1000 bootstraps replicates with bioNJ algorithm (phylogeny.fr; [72]). Scale bar: expected numbers of amino acid substitutions per site. The sequences are in the Additional file 1

Fig. 4

Heatmap hierarchical clustering showing the expression of genes assessed by RT-qPCR. Values represent Calibrated Normalized Relative Quantities (CNRQ) calculated with qbase⁺. DLP dirigent-like protein, LAC laccase, PRR1 pinoresinol reductase 1, DIR dirigent protein, NST1 NAC secondary cell wall thickening 1, MET1 methionine synthase 1, SAM S-adenosylmethionine synthase, PRX peroxidase, PLR pinoresinol-lariciresinol reductase. The colour bar indicates the expression values represented as an increasing intensity gradient. The numbers refer to the Pearson correlation coefficients between the lengths of two branches. The CNRQ data are given in Additional file 2

Fig. 5

Clustering of the proteome profiles of hypocotyls at different ages. H6 green dots, H9 blue dots, H15 orange dots, H20 red dots. a: Principal component analysis based on the gel-based proteome study. b: Independent component analysis of LC-MS/MS based proteome profiles. In both panels, the significance of the coordinates in the two main axes was assessed using a Tukey post-hoc test, different letters within one column indicate that the proteome profiles are significantly different

Fig. 6

NSAF relative quantities of proteins involved in cell wall biogenesis assessed by LC-MS. The parameters of the hierarchical clustering are indicated in the Methods section. The values are given in Additional file 3. Abbreviations are as in the text. For each group, the average of the abundances as calculated for the hierarchical clustering was plotted (± standard deviation)

Fig. 7

Peroxidase activity in H15 and H20. a to d, H15; E to H, H20. Details of the bast fibres and xylem regions are shown in b and f and d and h, respectively. Blue arrows indicate peroxidase activity in the middle lamella and cell corners of the bast fibres; blue arrowheads indicate peroxidase activity in the xylem vessels and fibres. i: negative control without DAB in H20. j: negative control with salicylhydroxamic acid as inhibitor of peroxidase activity in H20. bf1 primary bast fibre, bf2 secondary bast fibre, c cambial zone, xf xylem fibre, xv xylem vessel. Scale bar: 100 μm (g, i, j), 50 μm (a, c, e); 25 μm (d, f, h); 10 μm (b)

Fig. 8

Laccase activity in H15 and H20. a to d, H15; e to h, H20. Details of the xylem regions and bast fibres are shown in b and f and d and h, respectively. Orange colour indicates the presence of laccase activity. Blue arrowheads indicate the absence of laccase activity in the lignified primary xylem. i: negative control without DAB in H20. j: negative control with sodium azide as inhibitor of laccase activity in H20. bf1 primary bast fibre, bf2 secondary bast fibre. Scale bar: 100 μm (50 μm in the insets)