Regulation of secondary cell wall biosynthesis by a NAC transcription factor from Miscanthus

Abstract Cell wall recalcitrance is a major limitation for the sustainable exploitation of lignocellulosic biomass as a renewable resource. Species and hybrids of the genus Miscanthus have emerged as candidate crops for the production of lignocellulosic feedstock in temperate climates, and dedicated efforts are underway to improve biomass yield. However, nothing is known about the molecular players involved in Miscanthus cell wall biosynthesis to facilitate breeding efforts towards tailored biomass. Here, we identify a Miscanthus sinensis transcription factor related to SECONDARY WALL‐ASSOCIATED NAC DOMAIN1 (SND1), which acts as a master switch for the regulation of secondary cell wall formation and lignin biosynthesis. MsSND1 is expressed in growth stages associated with secondary cell wall formation, together with its potential targets. Consistent with this observation, MsSND1 was able to complement the secondary cell wall defects of the Arabidopsis snd1 nst1 double mutant, and ectopic expression of MsSND1 in tobacco leaves was sufficient to trigger patterned deposition of cellulose, hemicellulose, and lignin reminiscent of xylem elements. Transgenic studies in Arabidopsis thaliana plants revealed that MsSND1 regulates, directly and indirectly, the expression of a broad range of genes involved in secondary cell wall formation, including MYB transcription factors which regulate only a subset of the SCW differentiation program. Together, our findings suggest that MsSND1 is a transcriptional master regulator orchestrating secondary cell wall biosynthesis in Miscanthus.

SCW polysaccharide network of cellulose and hemicellulose, it has become the main target for efforts aiming at decreasing cell wall recalcitrance (Marriott et al., 2014;Sibout et al., 2016;Van Acker et al., 2014;Wilkerson et al., 2014). Attempts to decrease lignin content or alter lignin composition frequently caused undesirable phenotypes such as collapsed xylem vessels, dwarfing, or increased susceptibility to pathogens (Bonawitz & Chapple, 2013). However, dwarfism in response to interference with lignin biosynthesis was in some cases caused by signaling, rather than by insufficient cell wall integrity, and plants with altered lignin content, but without growth penalty could be obtained by circumventing the signaling feedback (Bonawitz et al., 2014;Gallego-Giraldo, Escamilla-Trevino, Jackson, & Dixon, 2011a;Gallego-Giraldo, Jikumaru, Kamiya, Tang, & Dixon, 2011). This demonstrates that plant growth, in principle, tolerates alternative cell wall structures if secondary responses can be controlled. Thus, a thorough understanding of cell wall biosynthesis, maintenance, and perception can greatly facilitate tailoring biomass for broad application.
The herbaceous monocot genus Miscanthus harbors perennial C4 grasses that originate from subtropical and tropical regions in East Asia. Due to low water and modest nutrient requirements, high photosynthetic efficiency, cold and drought tolerance, and high biomass yield, Miscanthus has emerged as leading second-generation bioenergy crop for the production of lignocellulosic biomass in temperate climates (Lewandowski et al., 2016;van der Weijde et al., 2013). To improve biomass composition and yield, several global breeding programs have been initiated, aiming to capitalize on existing genetic and phenotypic variation within and between Miscanthus species (Robson et al., 2013). In addition, these efforts are complemented by in-depth cell wall profiling of various genotypes (da Costa et al., 2014(da Costa et al., , 2017. However, understanding of the molecular regulation involved in SCW formation in Miscanthus has received thus far little attention.

| Molecular cloning
Plasmid constructs were assembled via GreenGate cloning (Lampropoulos et al., 2013). The protein-coding region of the MsSND1 gene was amplified by PCR using cDNA from Miscanthus sinensis and appropriate primers with BsaI restriction site overhang (see Table S1). More details about cloning such as primers, modules, and assembled constructs for plant transformation can be found in  (Clough & Bent, 1998). For selection, plants were grown on halfstrength MS plates supplemented with 25 mg/L hygromycin or 7.5 mg/L glufosinate ammonium (Sigma-Aldrich); 10-day-old transgenic Arabidopsis seedlings of mCherry-GR-MsSND1 lines were pretreated with 10 lM cycloheximide (CHX), a protein synthesis inhibitor, for 2 h. To activate mCherry-GR-MsSND1, the seedlings were transferred to 10 lM dexamethasone (DEX) and/or 10 lM cycloheximide (CHX) solution for 4 h. Arabidopsis snd1 nst1 double mutant seeds (Mitsuda et al., 2007) were obtained from the Nottingham Arabidopsis Stock Center (NASC).

| Transient transformation of Nicotiana benthamiana leaves
For overexpression experiments, Agrobacterium tumefaciens ASE (pSOUP + ) was transformed with the respective construct. Transgenic clones were incubated in liquid LB medium containing respective antibiotics for 2 days. The bacteria were transferred to infiltration medium (10 mM MgCl 2 , 10 mM MES, 0.15 mM acetosyringone at pH 5.7). The suspension was set to OD 600 0.4 and infiltrated with a needless syringe into 4-to 6-week-old Nicotiana benthamiana leaves.
After 5 days, the leaves were embedded in 6% agarose, hand-sectioned, and stained.

| Tissue staining and microscopy
Lignin was stained with either HCl-phloroglucinol or basic fuchsin as described in Ref. (Valdivia et al., 2013). Cellulose was stained with the fluorochrome calcofluor white M2R (fluorescent brightener 28).
Hemicelluloses were detected with xylan and arabinoxylan-specific LM11 as primary (McCartney, Marcus, & Knox, 2005) and Alexa 488-conjugated donkey anti-rat IgG (H+L; Thermo Fisher) as secondary antibody according to the procedure described elsewhere (McCartney, Steele-King, Jordan, & Knox, 2003). Cell walls were stained with SCRI Renaissance 2200 staining (Musielak, Schenkel, Kolb, Henschen, & Bayer, 2015). HCl-phloroglucinol staining was imaged under brightfield with a 20.0 9 0.40 NA objective on a Leica DM IRB inverted microscope. Fluorescent images were captured with a confocal Leica TCS SP5II microscope equipped with a 40.0 9 1.25 NA objective. Basic fuchsin-and SCRI Renaissancestained tissues were excited at 561 nm and 405 nm to detect emission at 593/40 nm and with DAPI filter, respectively. Calcofluor white-stained tissues were irradiated with UV light and detected with DAPI filter. Immunolabeled tissues with LM11 and anti-rat Alexa 488 conjugate were excited using the 488-nm Argon laser line to detect emission with Alexa 488 filter settings. The fluorescent protein mCherry was imaged at 543-nm excitation and 600/80-nm detection. Orthogonal sections and scale bars were produced with ImageJ software.

| Gene expression analysis
Developing Miscanthus leaves were cut at their node, dissected, and immediately frozen in liquid nitrogen. Total RNA was isolated from ground plant material from 100 mg Arabidopsis seedlings or 30 mg  (Table S2). During the course of the leaf gradient, cytoskeleton reference genes like tubulin or actin were differentially expressed in leaf base and tip. Hence, the most stable reference genes PP2A and UBC21 were selected for normalization. Geometrical mean of three biological replicates is shown. Gene expression of Arabidopsis was normalized against clathrin adaptor subunit (At5g46630) GOLFIER ET AL. | 3 described in (Czechowski et al., 2005). The qPCR products were either sequenced or checked on acrylamide gel in cases of small amplicons.  (Barling et al., 2013) for TFs involved in SCW biosynthesis using known factors from Arabidopsis thaliana and other plants. A phylogenetic analysis of NAC TFs from different angiosperm lineages showed that the subfamily Ic may be further divided into three classes (Figure 1). Members of classes II and III are implicated in xylem differentiation (Kubo et al., 2005;Zhou, Zhong, & Ye, 2014), whereas members of class I are described to be involved in fiber differentiation (Zhong et al., 2006). In the Miscanthus transcriptome, we found various transcripts putatively encoding SCW TFs and concentrated on the Miscanthus sinensis SND1 candidate. The predicted protein shares 41.5% identity and 51.5% similarity on amino acid level with AtSND1 (Fig. S1). The structurally and functionally essential N-terminal DNA-binding motif, known as the NAC domain, shows the highest similarity with 93% within the amino acid sequence, suggesting that MsSND1 may possess similar DNA-binding characteristics as AtSND1. The more divergent C-terminus that contains the transcriptional activation domain (Duval, Hsieh, Kim, & Thomas, 2002;Zhong et al., 2006) is 63 amino acids longer in MsSND1 compared to AtSND1 that may reflect different activation properties. In summary, our analysis indicates a close relationship between MsSND1 and AtSND1. Miscanthus development, the expression profile of MsSND1 together with those of putative cell wall-related transcription factors and biosynthetic genes was determined along the developmental gradient in the leaf and visualized (Figure 2a,b, Fig. S2). The transcripts of R2R3-MYB TFs, lignin biosynthesis, and polymerization genes were identified in the Miscanthus transcriptome in the same manner as described for MsSND1. Along the leaf developmental gradient, the expression of MsSND1 was highest at the leaf sheath and decreased rapidly over the following two leaf segments (Figure 2a,b). We then analyzed, the expression pattern of putative downstream targets of SND1 related to AtMYBs involved in the SCW transcriptional network. In this case, relationship to previously described factors was more ambiguous; therefore, we named them Miscanthus sinensis This was further confirmed by basic fuchsin staining of the leaf cross-sections. Basic fuchsin was able to stain cells in the vascular tissue but was unable to stain sclerenchyma fibers (Fig. S2b-e).

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Notably, sclerenchyma cells were found to possess a distinct lignin composition in alfalfa (Vallet, Chabbert, Czaninski, & Monties, 1996), which is probably the result of different lignification mechanisms employed by xylary and extraxylary fibers (Smith et al., 2013). The high expression of MsSND1, candidate MYB TFs, and lignin biosynthesis genes that occurs concurrently with differentiation and SCW formation of xylary and extraxylary elements suggests a possible involvement of the investigated genes in this process. previous study has revealed that only partial activation of the regulatory network is already sufficient to rescue the snd1 nst1 mutant, albeit with compositional changes to the SCW (Sakamoto & Mitsuda, 2015). Therefore, we continued with a more profound functional characterization of MsSND1 to shed light on its regulatory role in SCW formation.  Figure 4). Interestingly, the patterns of ectopic SCW deposition are reminiscent of tracheary elements which has been previously observed after induced overexpression of NAC and MYB SCW master switches from several species such as Arabidopsis, poplar, Eucalyptus, switchgrass, rice, maize, and Brachypodium (Kubo et al., 2005;Zhong et al., 2007a,b;Zhong et al., 2010a;Zhong, Lee, & Ye, 2010b;Zhong et al., 2011;McCarthy et al., 2009;Valdivia et al., 2013;Yoshida et al., 2013). Cortical microtubules have long been known to play a crucial role in patterned deposition of SCW by guiding exocyst complex and secretory vesicles at the sites of SCW deposition (Baskin, 2001;Oda & Fukuda, 2013;Vukasinovic et al., 2017;Watanabe et al., 2015;Wightman & Turner, 2008).
In untreated mCherry-GR-MsSND1 seedlings, detection of the mCherry signal in root tips revealed a cytosolic localization of the fusion protein mCherry-GR-MsSND1 (Figure 6c). After 24 h of DEX treatment, the mCherry signal was exclusively detected in nuclei of root cells (Figure 6d). The sharp boundaries of the mCherry signal, excluding nuclei, in involved in SCW patterning and biosynthesis throughout vascular plants (Bomal et al., 2008;Li et al., 2012;Zhong et al., 2010a). The F I G U R E 7 Induction of mCherry-GR-MsSND1 activates expression of genes involved in SCW formation. Expression analysis of candidate genes directly and indirectly targeted by MsSND1. Ten-day-old heterozygous Arabidopsis mCherry-GR-MsSND1 seedlings (line 16) were treated with cycloheximide (CHX) and/or dexamethasone (DEX). Gene expression is normalized against clathrin adaptor subunit. Bars depict means AE SE from three technical replicates. The same experiment was performed with an independent transgenic line with similar results (Fig. S5) establishment of a precisely inducible mCherry-GR-MsSND1 Arabidopsis line allows localization and tracking of the fusion protein and corroborates the finding that MsSND1 is capable of activating the SCW program resulting in patterned SCW deposition reminiscent of xylem elements.

| Induction of mCherry-GR-MsSND1 activates expression of genes involved in SCW formation
Next, we assessed the ability of MsSND1 to act as transcriptional regulator of SCW formation. Therefore, we induced mCherry-GR-MsSND1 Arabidopsis lines with or without pretreatment with the pro-

| CONCLUSION
Cell wall recalcitrance, which is mainly conferred by incorporation of lignin into SCWs, remains a major limitation to explore lignocellulosic biomass as renewable resource. Concomitantly, considerable efforts are directed at increasing production of lignocellulosic biomass that can be valorized in a sustainable manner. However, the molecular mechanisms involved in lignification and SCW formation in Miscanthus have received only little attention. In this study, we identified and functionally characterized Miscanthus sinensis SND1 as a transcriptional master regulator orchestrating SCW formation, most likely in extraxylary fibers. In addition, we could identify other TF from the SCW transcriptional network, which appears to only regulate a subset of the differentiation program. Despite the fact that research on Miscanthus is still hampered by challenging transformation procedures, our approach shows that utilization of the increasingly available genomic resources and transfer of knowledge from model plants greatly facilitate the investigation of molecular mechanisms.
The functional characterization of MsSND1 contributes to a deeper understanding of the molecular network of TFs involved in SCW formation in Miscanthus. It is tempting to speculate that lower-tier TFs such as MsSCM4 (related to AtMYB63/AtMYB58) could affect lignin qualities more specifically, suggesting that may serve as potential targets for breeding programs. Moreover, the possible involvement of MsSND1 in extraxylary fibers differentiation may represent a target for cell wall engineering (Yang et al., 2013) as it offers the opportunity to manipulate lignin specifically in extraxylary fibers without affecting xylem elements and thereby bypassing severe growth defects. Furthermore, we are grateful to Iris Lewandowski (University of Hohenheim) for helpful discussions and for providing the Miscanthus genotypes. We thank Adrej Miotk for sharing plasmid material.

CONFLI CT OF INTEREST
The authors declare no conflict of interest.