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Insights Into the Molecular Regulation of Monolignol-Derived Product Biosynthesis in the Growing Hemp Hypocotyl Marc Behr1,2, Kjell Sergeant1, Céline C

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Insights Into the Molecular Regulation of Monolignol-Derived Product Biosynthesis in the Growing Hemp Hypocotyl Marc Behr1,2, Kjell Sergeant1, Céline C Behr et al. BMC Plant Biology (2018) 18:1 DOI 10.1186/s12870-017-1213-1 RESEARCHARTICLE Open Access Insights into the molecular regulation of monolignol-derived product biosynthesis in the growing hemp hypocotyl Marc Behr1,2, Kjell Sergeant1, Céline C. Leclercq1, Sébastien Planchon1, Cédric Guignard1, Audrey Lenouvel1, Jenny Renaut1, Jean-Francois Hausman1, Stanley Lutts2 and Gea Guerriero1* 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 Background herbaceous plants also undergo lignification, particularly The monolignol-derived products lignin and lignans are in the xylem of roots and the hypocotyl [1]. Lignans are important plant specialized (secondary) metabolites. formed by the stereospecific oxidative coupling of They are involved in crucial events related to plant de- hydroxycinammyl alcohols mediated by a dirigent velopment, such as plant defence, growth regulation, sap protein and an oxidase, typically a laccase [2, 3]. Their conduction and erect growth habit. Lignin strengthens role in plant defence is known since more than a decade mechanically the xylem by impregnating the secondary [3], but their plant growth regulatory activity is still cell wall of both tracheary elements and fibres. This under investigation [4]. Very few studies have addressed phenomenon is particularly obvious in trees, but how this growth regulatory activity is mediated in planta [5, 6]. Lignin and lignans originate from the same build- ing blocks, i.e. the monolignols, but have very different * Correspondence: [email protected] functions. Therefore, the allocation of the building 1Environmental Research and Innovation Department (ERIN), Luxembourg Institute of Science and Technology (LIST), L-4362 Esch/Alzette, Luxembourg blocks to either biosyntheses has to be precisely tuned, Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Behr et al. BMC Plant Biology (2018) 18:1 Page 2 of 18 both temporally and spatially. As for the majority of Following monolignol biosynthesis, lignin polymerisa- genes involved in secondary cell wall deposition, the tion is performed through end-wise radical coupling of expression of lignin biosynthetic genes is regulated by phenols to the free-phenolic end of the growing polymer master regulators belonging to the NAC and MYB tran- [17]. The formation of the radical is catalysed by either scription factor families [7]. By contrast, the expression laccase or class III peroxidase and mainly takes place in of pinoresinol lariciresinol reductase (PLR), a key gene the apoplast. In Angiosperms, the relative proportion of of lignan biosynthesis, was found to be higher in the p-coumaryl (H), guaiacyl (G) and syringyl (S) units of young stem as compared to older stems in Forsythia x the lignin polymer depends on the tissue, the stage of intermedia [8], suggesting that lignan biosynthesis may development and the subcellular compartment. Starting be somehow independent from secondary cell wall de- from phenylalanine, the biosynthesis of H, G and S units position. However, a role has been ascribed to lignans in requires the activities of 5, 8 and 10 enzymes, respect- secondary cell wall-forming tissues, where they may par- ively (Fig. 1). ticipate in the maintenance of the cell redox homeostasis In Angiosperms, the water-conducting cells of the during lignification [9, 10]. The genes coding for en- xylem are enriched in G-lignin, while structural fibres zymes involved in lignan biosynthesis may thus be differ- (both from xylem and phloem) have a high S-lignin con- ent according to the stage of development and tissue. tent [18]. Lignification begins with the deposition of the This is illustrated by the expression patterns of the G units, notably in the secondary cell wall of xylem cells pinoresinol reductases AtPRR1 and AtPRR2. The former [19, 20], under the activity of laccases (LAC). H units is coexpressed with several secondary cell wall genes in are targeted to the middle lamella, while G units are ini- the lignified internodes, while the latter is highly tially deposited in the S1 sub-layer of both Gymno- expressed in the growing hypocotyl [7]. Lignan and lig- sperms and Angiosperms [16]. Less is known about the nin biosyntheses may be intertwined, as a triple laccase role of enzymes partaking in the oxidation and depos- mutant which displays strongly reduced lignin content ition of the lignin macromolecule. It has been suggested shows a higher transcript level of PRR2 [11]. that laccases (LAC) and peroxidases (PRX) do not func- Some dirigent proteins (DIR) are putatively involved in tion redundantly in lignin polymerisation in the vascular lignan biosynthesis, while others are related to lignin de- tissues of A. thaliana [11]. Indeed, a knock-out mutant position. For example, AtDIR10/ESB1 plays an essential of AtPRX52 has shown a decrease in the S units in the role in the formation of the Casparian strip in Arabidopsis interfascicular fibres [21], suggesting that laccases do not by targeting lignin polymerization at specific extracellular compensate for the loss of peroxidase activity. By con- sites [12] and AtDIR6 confers the (−) stereoconformation trast, the lignin of AtPRX2, AtPRX25 and AtPRX71 to pinoresinol [13]. The phylogenetic analysis of DIR helps knock-out mutants is richer in S units than wild type to differentiate between those DIR involved in lignan [22], showing that a lower peroxidase activity is not syn- biosynthesis (e.g. AtDIR6) and those to which other onymous to a decrease in S unit. The silencing of two functions, such as lignin deposition, are assigned and genes coding for laccases (AtLAC4, AtLAC17) induces referred to as DIR-like. According to Ralph [14], proteins an increase in the fibre S/G ratio [19]. AtLAC17 is spe- of the DIR-a subgroup (AtDIR5, AtDIR6, AtDIR12 and cifically involved in the deposition of G lignin in fibres, AtDIR13) are wound- or insect-induced while proteins while the specific activity of AtLAC4 is less clear. More from the DIR-b subgroup are part of the constitutive de- recently, the regulation of flax laccase expression by the fence of the plant. Since most of DIR have not yet been microRNA miR397 has been shown [23]. The question functionally characterised, phylogenetic analysis is a help- thus arising relates to the activity of these enzymes to- ful tool that complements gene coexpression analysis for wards the oxidation of specific lignin subunits. functional prediction [15]. Between 6 and 20 days after sowing, the hemp hypo- Because lignin is racemic and since there is no cotyl was shown to be a suitable system to study the mass spectral evidence about the occurrence of oligo- molecular events underlying secondary growth and lignol stereoisomers, the proposed model of protein- secondary cell wall deposition [24]. The late stages of driven control and template replication of lignin hypocotyl development are characterised by the up- polymerisation [16], has failed to prove its robustness regulation of transcription factors and genes involved in [17]. Therefore, the currently known functions of
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