The time-course study of the hemp hypocotyl aged between 6 and 20 days highlighted the roles and expression dynamics of several players involved in lignin and lignan biosynthesis. Two developmental stages, i.e. H6 and H9 versus H15 and H20, were discerned at the chemical (lignin accumulation and composition), protein (cell wall biogenesis and monolignol biosynthesis) and transcript levels (transcription factor related to secondary cell wall thickening, dirigent- and dirigent-like proteins, candidates involved in lignin polymerisation).

In this study, we have focused on two families of lignin-related genes, namely the DIR and PLR/PRR. In Arabidopsis, 25 DIR have been found. Since we have retrieved only 9 DIR genes in hemp, several other DIR-genes may be missing in our analysis. Two main groups of hemp DIR are clearly distinct from the phylogenetic analysis (Fig. 3). CsaDIR6A and CsaDIR6B belong to the same group as the Arabidopsis, Schisandra chinensis, Forsythia x intermedia and flax dirigent proteins (Group I). In those species, these DIRs are involved in the stereoselective initial coupling step of coniferyl alcohol to yield either (+)-pinoresinol or (−)-pinoresinol, a G(8–8)G lignan, in the presence of laccases [16]. We found that these hemp DIRs were more expressed at later stages of the hypocotyl development (H15 and H20) compared to H6 and H9 (Fig. 4). In Arabidopsis, PRR reduces (+)-pinoresinol to lariciresinol [32]. However, the patterns of expression of hemp DIR6A, DIR6B, PLR and PRR1 did not overlap, since PLR and PRR1 were more expressed in H6 and H9. It is therefore plausible that, in hemp, DIR6A and DIR6B may be involved in other biochemical reactions, such as those occurring in the ellagitannin or sesquiterpenoid pathways [33]. Various terpenes, including sesquiterpenes, have been detected in both medicinal and fibre varieties of C. sativa [34]. Several squalene epoxidases, key-enzymes of terpenoids biosyntheses, are expressed in the thale cress hypocotyl [35]. Therefore, the higher abundance of hemp DIR6A and DIR6B might be related to the biosynthesis of terpenes. Further analyses are required to determine the DIR involved in the stereoconformation of the lignans in hemp. Candidates may be found in the DIR-like proteins subfamilies, which regroup proteins whose functions are not yet clearly established [14]. The expression pattern of CsaDLP4 is compatible with a role in lignan biosynthesis, as it is coexpressed with PLR and PRR1. The comparison with the co-expression data of genes related to AtPRR1 may support this hypothesis. Indeed, the closest ortholog of CsaDLP4 (AtDIR18) according to the phylogenetic analysis (Fig. 3) is co-expressed with AtPRR1 (p-value <1E-03) (atted.jp). AtPRR1 is solely responsible for lignan biosynthesis in the stem [10]. However, in contrast with the expression of hemp PRR1, AtPRR1 is co-expressed with many genes involved in secondary cell wall deposition such as MYB46, SND1, CESA7 or LAC17. In a previous RNA-Seq-based study, the genes involved in secondary cell wall biosynthesis were more expressed either in H15 or in H20 [24]. It remains to be investigated whether the expression of PLR / PRR in our hemp system is transient and linked to the young developmental stage, or if it also applies to older phases.

Lignans positively (e.g., syringaresinol) or negatively (e.g., sesamin) regulate root and shoot lengths [4, 36]. A disruption in (+)-secoisolariciresinol diglucoside (SDG) biosynthesis by RNAi of flax PLR resulted in higher concentrations of dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) without compromising growth [37]. DCG promotes cell division, possibly by transducing the cytokinin signal [5]. This may indicate that the plant compensates the lack of SDG induced by the down-regulation of PLR by the biosynthesis of DCG. A detailed analysis of lignans present at the different time points of the hemp hypocotyl system may shed additional light on the exact functions of this class of molecules.

PLR expression and thus the synthesis and accumulation of lignans may also be involved in the response to oxidative stress. In F. intermedia, PLR mRNA is more abundant in young stems as compared to more mature stems and is localised in the vascular cambium and developing xylem [8]. During the early stages of xylem parenchyma cell development, the activity of PLR results in the synthesis of lignans, while lignification occurs in tracheary elements and fibres [8]. Xylem parenchyma cells, tracheary elements and fibres which did not yet complete their programmed cell death have to cope with oxidative intracellular conditions because of the production of H₂O₂ by xylem parenchyma cells [38]. H₂O₂ is able to diffuse from cell to cell, and may be used as a substrate for the activity of peroxidases involved in lignification. Niculaes and colleagues [39] propose that class I peroxidases catalyse the oxidation of monolignols and their subsequent dimerization into dilignols such as pinoresinol to prevent damages to the cells that have not completed their programmed cell death. The suggestion of a PLR protective function against oxidative stress in the developing xylem cells may explain its high expression in young hemp hypocotyls, besides its potential implication in the regulation of plant growth. Later in the development, the abundance of PCBER was higher (H15-H20, Fig. 6). In vitro, PCBER reduces dehydrodiconiferyl alcohol (DDC) to isodihydrodehydrodiconiferyl alcohol (IDDDC), a G(8–5)G neolignan. In living cells, such reduced products are oxidised by a peroxidase consuming H₂O₂, providing a protection against oxidative stress during lignification [39]. The authors suggest that PCBER and pinoresinol reductases prevent oxidative damage by producing radical scavenging molecules such as reduced phenylpropanoid coupling products. At the time points where PCBER was more abundant, one can speculate that neolignans are excreted to the secondary cell walls to cope with the oxidative stress accompanying lignin polymerization [9, 10]. Alternatively, PCBER may also be involved in xylem lignification by reducing the arylglycerol of S(8–5)G glycoside, whose product is finally used for scavenging H₂O₂ in the oxidative conditions found in lignifying tissues [39].

At the transcriptomic level, the ortholog of Arabidopsis NST1, a transcription factor involved in secondary cell wall deposition and lignification [7] was significantly more expressed in H15 and H20 as compared to H6 and H9 (Fig. 4). Consequently, most of the genes of the monolignol and lignin biosynthetic pathways were more expressed in older hypocotyls. The increased expression of NST1 and genes of the monolignol-lignin biosynthesis is in accordance with the data retrieved in our previous RNA-Seq experiment [24]. During the proteome study, the abundance of enzymes involved in the generation of methyl donors was identified as significantly changing: METS, SAMS, SAHH and MTHFR were differentially abundant within the time course experiment (Fig. 6). The two proteins identified as the orthologs of AtMETS2 displayed different trends: the first was highly abundant at all the time points, with small but significant fold-change, while the second was following the same trend as the proteins of the monolignol pathway, with massive fold-change variations [Additional file 3]. This may point to different functions of these two isoforms. The same observation applies to the abundance of three isoforms of SAM synthases, the orthologs of AtSAMS2, AtSAMS3 and AtSAMS4, with SAMS2 and SAMS3 abundances remaining almost constant and SAMS4 being more abundant as the hypocotyl ages. Such changes in the abundance / expression profiles of the proteins / genes involved in the biosynthesis of methyl donors have also been documented in the flax hypocotyl, where genes of the S-adenosylmethionine pathway were more expressed at 15 days as compared to 6 days [40]. The methyl donors are involved in many biochemical reactions, including the methylation of G and S monolignol [41, 42].

The lignin content and composition depends on the activity of ca. ten enzymes, from PAL to COMT [43] (Fig. 1). Perturbations in the enzymatic activities of those enzymes lead to changes in lignin content, lignin composition, or both [44]. Accordingly, the higher abundances of PAL1, PAL2, 4CL2, CAD4, HCT, CCoAOMT and COMT in H15 and H20 correlate with the rise in lignin content (Fig. 6). The abundance pattern of the proteins involved in lignification is consistent with the transcriptomics data obtained with the RNA-Seq analysis of the same time points (Correlation plot in Additional file 3) [24]. The lignin composition also depends on the cell type (e.g., vessels versus fibres) and age (deposition and maturation of the secondary cell wall). H lignin accumulates in the middle lamella of vascular cells [16], prior to G and S lignin deposition in the S1 sublayer. H lignin is believed to be a factor determining the shape of the vascular cells [44]. Lignin rich in H units is more condensed because this subunit is capable of forming condensed units at the 3 and 5 positions [45]. The pectic nature of the middle lamella may explain why lignin is present in a more condensed substructure with a high H unit content [46, 47]. Indeed, the loose structure of the pectic matrix sterically favours the formation of the condensed lignin and accommodates its bulky organisation [48]. Noncellulosic polysaccharides such as galactan and xylan have roles in controlling the cellulose microfibril orientation, resulting in qualitative and quantitative changes in the lignification of the middle lamella and cell walls [47, 49]. In alfalfa, the middle lamella lignification occurs in specific spots where pectin, peroxidase activity and H2O2 are present [50].

During the hypocotyl elongation, the expression of several genes involved in lignification was shown to be under control of the circadian clock [51]. Among them, C4H, COMT and CCoAOMT show circadian-dependent expression in thale cress. More specifically, their transcripts are more abundant 4-8 h before dawn, when cell elongation is slowed down or has stopped. These results may be linked to the availability of metabolizable sugars for lignification [52]. In support of this hypothesis, these authors have shown that sex1, a mutant impaired in starch turnover resulting in a reduced pool of available carbon, accumulates less lignin than the wild type. In addition, the lignin of the sex1 mutant was completely depleted of H unit because of the higher C3’H activity relative to other enzymes of the phenylpropanoid/monolignol pathway [52]. Moreover, sucrose supplied to dark-grown hypocotyl induces lignification [52]. Sucrose may be a signalling molecule to induce the activity of the lignin biosynthetic pathway. As a result, sucrose may be considered both as a source of carbon-rich skeletons for lignification, and as a signalling molecule regulating a suite of developmental processes, including the differentiation of xylem cells [52]. One may speculate that the deposition of H lignin in the elongating hypocotyl may be (partially) tuned by sugar availability. Several enzymes involved in sucrose, glucose and fructose metabolism (e.g. SUS4, INV, FRK3; Additional file 3 and Fig. 1) were more abundant in H15 and H20. Increased sucrose synthase activity leads to higher cellulose content, as it provides UDP-glucose, the precursor used by the cellulose synthase complex [53]. Invertase hydrolyses sucrose into glucose and fructose. Glc-6-P may be converted in erythrose-4-P to be shunted to the shikimate pathway, producing phenylalanine, a precursor for the biosynthesis of lignols (Fig. 1) [54]. Specifically in H20, most of the proteins associated with downstream phenylalanine metabolism, especially monolignol biosynthesis (PAL1, PAL2, 4CL2, CAD4, HCT, COMT and CCoAOMT) and lignin polymerisation (orthologs of AtPRX3, AtPRX52 and AtPRX54), reached their maximum abundance [Additional file 3]. Fructose is likely phosphorylated by FRK3 to avoid feedback inhibition of SUS4 and invertase, therefore contributing to cellulose biosynthesis. Fructose may also be converted to UDP-glucose and finally sucrose [55].

As the hypocotyl ages, increased CCoAOMT and COMT abundances (Fig. 6) result in lignin richer in G and S subunits (Table 1). We therefore show a consistent link between proteomics data and lignin monomeric composition. The lignification of the secondary cell wall begins with deposition of G units in the S1 sub-layer [16] in discrete domains where LAC4 and LAC17 are present [20]. Despite the lack of a precise localisation of these two enzymes in the hemp hypocotyl system, we may however assume that LAC4 and LAC17 activities lead to a decrease in the relative proportion of H lignin (Table 1). This was previously observed in hemp bast fibres: the H lignin proportion of apical fibres was systematically higher than in basal fibres, irrespective of the stage of development [56]. Using thioacidolysis, the same authors found out that the S/G molar ratio was also higher in the basal fibres. Primary xylem is almost completely devoid of S lignin, as shown by the brown coloration of the Mäule staining (Fig. 2) and as previously described [57]. Lignin in the secondary xylem is progressively enriched in S units [57]. This rise in the S/G ratio was also observed during lignification of mature secondary cell walls in woody Angiosperms, mainly due to S lignin polymerisation [58]. The development of fibres in both xylem and phloem likewise contributes to this increase: fibres, as compared to tracheary elements, are richer in S lignin to provide mechanical strength. In Quercus suber, the xylem lignin is enriched in S units because of the large proportion of fibres (S/G of 1.2, [58]). The monomeric composition of lignin also depends on the carbohydrate composition of the cell wall where the polymerization occurs [46]. In secondary cell walls, elongated patches of lignin are deposited between the cellulose microfibrils, acting as a template to guide the lignification [48, 49]. Because secondary cell walls are richer in hemicellulose, e.g. xylan in the xylem cells of hemp [24, 59], than the middle lamella, lignification mainly occurs with polymerization of G and S subunits [46]. By controlling the orientation of the cellulose microfibrils in the secondary cell wall, xylan favours the formation of the microfibril matrix [48]. This, in turn, favours the formation of non-condensed lignin (mainly made of G and S subunits) and its extended conformation adapted to the tight volume available between the cellulose microfibrils [48]. For example, the irx8 mutant, which is disturbed in the xylan architecture of the secondary cell wall, has less lignin because of the lower amount in G subunits [47]. In old cell walls, lignin and xylan are covalently linked to form the lignin-carbohydrate complex (LCC) by the addition of nucleophilic groups (hydroxyl or carboxylic groups of hemicelluloses) on the transient quinone methide intermediate, determining the final step of cell wall construction [60]. In hemp, primary and secondary phloems are rich in extraxylary fibres, which are already lignified in the hypocotyl aged 20 days (Fig. 2). However, the rise in the S/G ratio in the hypocotyl time course system differs from the trend observed in the outer tissues of adult plants, where the S/G ratio in the same stem fragment (apical or basal) does not change significantly when it gets older [56]. The authors suggest that the high amount of crystalline cellulose of the bast fibres may impair the polymerisation process. Since in our hypocotyl system the vascular tissue of the xylem is more developed than the sclerenchyma bast fibre tissue, we can observe an increase in the S/G ratio throughout time. Laser capture microdissection was found to be a reliable method to study the lignin composition of specific cell types in herbaceous species, where the manual separation of specific tissues is cumbersome [44] and may provide important data complementing the Mäule staining.

The synthesized monolignols are excreted to the cell wall where they are polymerised into lignin under the activities of laccases and class III peroxidases [47], with the possible intervention of DIR. The involvement of peroxidases in the lignification process is obvious when considering the abundances of both transcripts (orthologs of AtPRX49, AtPRX52 and AtPRX72) and proteins (orthologs of AtPRX3, AtPRX52 and AtPRX54), which are in general higher at older stages of development (Figs. 4 and 6). The three transcripts were more abundant in H15, while the three proteins were more abundant in H20. Peroxidase activity is essential for fibre lignification in several species including Arabidopsis [21, 61] and flax [62] and is required for S lignin polymerisation, since laccases seem not to be able to catalyse this polymerisation [63]. Likewise, S lignin staining and peroxidase activity were overlapping in the bast fibres (Figs. 2 and 7). Moreover, the increase in the peroxidase activity observed during tracheary element differentiation (close to the cambial region) and lignification (Fig.7d, h) was previously described in Zinnia [63]. During the hemp hypocotyl development, secondary xylem cells are visible 12 days after sowing [24] and lignification is ongoing at least until day 20 (Table 1). The role of the two laccases LAC11 and LAC17 is more difficult to define, as there is no significant change in their expression when determined by RT-qPCR (Fig. 4). However, we have previously described the expression pattern of putative orthologs of A. thaliana LAC17 using RNA-Seq in the same hypocotyl system, showing that one isoform of this gene was strongly upregulated in H20 [24]. As several isoforms of LAC17 were found using RNA-Seq, it is plausible that the transcripts detected by the two methods are different isoforms. One may also speculate that there is a basal level of laccase activity until the hypocotyl reaches its final diameter, i.e. when all fibres and vessels have differentiated from the cambium and started their initial lignification with G units. Laccases are involved in secondary cell wall lignification of protoxylem tracheary elements in young elongating tissues [20]. In H15 and H20, no laccase activity signal was observed in the primary xylem (Fig. 8), but a strong peroxidase signal was instead visible (Fig. 7c). This may suggest that a peroxidase-driven lignification occurs after the polymerisation of monolignols by laccases. Berthet and colleagues [19] suggested that laccases are expressed at the beginning of the lignification and involved in the polymerisation of G-rich lignin in fibres. In flax, three orthologs of AtLAC4 and AtLAC17 were more abundant in the upper region of inner stem tissues, containing the xylem, while five peroxidases were more expressed in the lower region of inner stem tissues [9]. The proposed mechanism of lignification suggested by these two studies is compatible with the increase in the S/G ratio along the time course.

The role of the DIR in lignification is limited. Since lignin polymerisation is performed through end-wise radical coupling of phenols to the free-phenolic end of the growing polymer and not by enzymatic control [17], a direct role of DIR in this process is unlikely. However, they may play a role in lignin localisation in specific regions of the cell wall. By silencing AtDIR10 (ESB1), perturbations in the organisation of Casparian strips have been observed [12]. The authors suggest that ESB1 plays a role in the localisation of lignin. In the phylogenetic analysis (Fig. 3), CsaDLP4 and CsaDLP5 fall into the same cluster as ESB1, possibly suggesting a role in lignification. The expression pattern of DLP5 is in line with such a function, as it is more expressed in old hypocotyls and differently from DLP4. The hemp genes DLP2, DLP20A and DLP20B were more expressed in H15 and H20 (Fig. 4). These DLPs belong to another subgroup, from which no proteins have been functionally characterised yet. The need of such studies is obvious to understand the precise role of the DIR-like proteins.