Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.

doi:10.21203/rs.3.rs-3110076/v1

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Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.

https://doi.org/10.21203/rs.3.rs-3110076/v1

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Background

Lignin and xylan are important determinants of a cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, we generated combinatorial cell wall mutants and studied the consequences on plant cell wall properties, biotic stress responses and plant cell wall integrity.

Results

Arabidopsis plant co-harbouring mutation in ferulate 5-hydroxylase (fah1-2) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) transgenic were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 crossed named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild-type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose content. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular processes.

Conclusions

Simultaneous modification in xylan and lignin improve cellulose content with enhanced saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose release with or without treatment after xylanase digestion. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Acetyl Xylan Esterase (AXE)

G Lignin

Saccharification

Xylose release

A bulk of lignocellulosic biomass is mainly composed of plant secondary cell walls (SCWs) which are deposited inside the primary cell walls in xylem and sclerenchyma cells when their growth stops. Cellulose is a main component of SCW. It is a water-insoluble crystallite called microfibril which is composed of linear polymers made up of glucose subunits. Cellulose provides the structural integrity to plant cell wall. Xylan, which is the second most abundant SCW polysaccharide in angiosperms, is made up of xylose units often substituted with methylated or unmethylated glucuronic acid and acetyl groups. Lignin which makes up one-fourth of biomass is a phenolic polymer mainly present in the compound middle lamella (CML) and SCW layers in cells but is rarely found in cells with primary walls. Pectins, which are polysaccharides with a high galacturonic acid content, are mainly found in the primary cell walls of CML and help to maintain cell-to-cell adhesion (Mohnen, 2008). All these above components interact either covalently or non-covalently to form a heterogeneous matrix which gives cell wall strength and rigidity. (Houston et al., 2016; Takenaka et al., 2018).

Xylan backbone is connected with β-1,4 linked xylose subunit (Liepman et al., 2010). Xylan is a branched polymer decorated with 2-O- and 3-O-acetyl and glucuronic acid or 4-O-methyl glucuronic acid which prevents self-aggregation (Qaseem & Wu, 2020). Additionally, the presence of an even substitution pattern favours the interaction between xylan and cellulose (Grantham et al. 2017). Although the direct interaction of lignin and acetyl groups is not known, it has been proposed that the presence of acetyl groups might limit the formation of ether-type of lignin-carbohydrate complexes (LCCs) (Giummarella and Lawoko 2016). Acetylation pattern also affects the pattern of glucuronosylation (Chong et al. 2014; Lee et al. 2014) suggesting that the substitution of xylan is strictly regulated. Moreover, O-acetyl decorations prevent xylan degradation by glycosyl hydrolases during the saccharification process and contribute to the recalcitrance of the lignocellulosic biomass (Wierzbicki et al., 2019). Also, during the fermentation of lignocellulosic biomass, these O-acetyl groups are present in the form of acetic acid which might reduce fermentation efficiency. Therefore, altering acetyl content is an attractive target to improve the properties of the lignocellulosic biomass (Pawar et al. 2013).

Previously, we have reported that Arabidopsis expressing fungal acetyl xylan esterase (AXE) from Aspergillus niger under the influence of constitutive promoter as well as xylem-specific promoter showed an increase in esterase activity and reduced levels of xylan acetylation in both Arabidopsis and Populus (Pawar et al., 2016; Pawar et al., 2017a). The transgenic lines showed increased xylan and cellulose digestibility with and without different pre-treatments. Populus AXE overexpressor lines had reduced S/G lignin composition and increased the lignin solubility in dioxane and water. Moreover, these lines exhibited elevated extraction of hot water-soluble lignin-carbohydrate fraction (Pawar et al., 2017b). Similarly, overexpression of AXE from Hypocrea jecorina in Populus improves cellulose as well as xylan saccharification without a major effect on plant growth parameters (Wang et al. 2020). In another approach, the downregulation of Reduced Wall Acetylation (RWA) genes reduced the overall xylan acetylation and improved saccharification (Pawar et al., 2017b). All these Populus transgenic lines were grown in the field and they showed maintained reduced acetylation after five years in the field with higher saccharification efficiency than wild-type trees (Derba-Maceluch et al., 2020; Pramod et al., 2021). All these reports suggest that in planta reduction of xylan acetylation could be a viable strategy to improve lignocellulosic biomass properties.

Lignin monomers i.e., monolignols are synthesized in the cytoplasm through the phenylpropanoid pathway, transported and polymerized in the apoplast. Predominant monolignols namely coumaroyl alcohol (H), guaiacyl alcohol (G) and syringyl alcohol (S) participate in the formation of lignin (Barros et al., 2019; Liu et al., 2018). Other non-conventional monomers can also be incorporated into lignin polymer making it a highly dynamic structure. Although lignin is well-known to hinder the bioprocessing of lignocellulosic biomass, lignin itself is valuable and can be utilized for making value-added chemicals or materials (Isikgor and Becer 2015). Many strategies of lignin modification have been tested before to improve the digestion of lignocellulosic biomass. Reducing lignin content by altering the expression of phenylpropanoid pathway genes in many cases improved digestibility but it negatively affected plant growth and total biomass yield. However, Arabidopsis lignin composition mutants having either S, G or H type of lignin did not show growth defects (Ruegger et al., 1999; Meyer et al. 1998; Bonawitz et al., 2014). Previous studies have shown that the glucose release during hot water pre-treatment is increased in both S- and G-monolignol hyperaccumulating plants compared to wild-type and mutant Arabidopsis with hyperaccumulation of H monolignol shows increased saccharification efficiency with or without pretreatment (Li et al. 2010). The glucose yield was found two times higher in H lignin plants as compared to wild-type plants (Bonawitz et al., 2014).

Xylan and lignin are cross-linked in the cell wall matrix by covalent interactions. It might occur in the cell wall by coupling of xylan to the newly generated monolignol radicles with the formation of phenyl glycoside linkages (Terrett and Dupree 2019). It could also happen before the secretion of xylan to cell wall, as recently suggested for the γ-ester linkages (Derba-Maceluch et al, 2023). The efficient conversion of lignocellulosic biomass into biofuel will be eased if the cross-linking between xylan and lignin is reduced (Min et al., 2014a; 2014b). It might be through the catalytic or enzymatic breakdown of linkages between xylan and lignin or by altering the composition of responsible components (Terrett & Dupree, 2019). Gene stacking could be a better approach to improve cell wall properties and understand the effect of polysaccharide digestibility and wall integrity. Altering the fibre-specific cell wall by gene stacking approach using the secondary cell wall-specific transcription factors to manipulate lignin biosynthesis has proved to be a better approach for improving lignocellulosic biomass (Yang et. al., 2013). Combinatorial post-synthetic modification by expressing fungal pectin and xylan acetyl esterase can alter plant immunity and cell wall integrity responses (Swaminathan et al. 2021). However, the effect on the plant cell wall and saccharification properties has not been extensively studied using such a gene stacking approach to alter both lignin and xylan.

In this study, we investigated the effect of simultaneously reducing xylan acetylation and increasing G-lignin content in Arabidopsis, by analyzing cell wall composition and saccharification efficiency in a homozygotic hybrid between a line expressing acetyl xylan esterase from Aspergillus niger (35S:AnAXE1) and fah1-2 mutant enriched in G-lignin (Chapple et al. 1992). The newly generated homozygotic line called HyperGHypoAcetylated (HrGHypAc) exhibited an increase in cell wall carbohydrate content, saccharification efficiency and xylan extractability without any adverse effects on growth or after Pseudomonas infection.

High G lignin xylan hypo acetylated lines does not affect plant growth or biotic stress responses

The fah1-2 point mutation ferulate 5-hydrxylase (F5H) gene leads to over-accumulation of guaicyl (G) lignin (Chapple et al., 1992) whereas the overexpression of acetyl xylan esterase from Aspergillus niger (35S:AnAXE1) depletes xylan acetylation level in Arabidopsis (Pawar et al., 2016). To understand the effect of simultaneous G-lignin enrichment and acetyl-hypo-accumulation on plant cell wall properties and saccharification, we crossed the fah1-2 mutant and the single-insert containing line D with 35S:AnAXE1 construct. The parent plants and their homozygous progeny were indistinguishable from the wild-type plants (Fig. 1a). To further confirm the presence of fah1-2 mutation in the F5H gene in the obtained hybrid plants, we amplified the F5H fragment with gene-specific PCWL-58 and PCWL-59 primers (Additional file: Table S1). The amplicon obtained by amplifying F5H gene was digested with MseI restriction enzyme and the banding pattern after restriction showed the expected difference between wild type and AnAXE1 showing 400 bp and 300 bp bands and fah1-2 and fah1-2/35S:AnAXE1 plants showing 300 bp, 275 bp and 125 bp bands (Additional file: Fig. S1a and S1b). Another typical chemotype of fah1-2 mutant is the absence of sinapoyl malate (Chapple 1992). Using a high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) analysis of soluble metabolites from leaves revealed undetectable levels of sinapoyl malate in fah1-2 and fah1-2/35S: AnAXE1 whereas the wild-type and 35S:AnAXE1 line D plants had detectable levels of this compound (Additional file: Fig. S1c). To further confirm the expected change in lignin composition in the hybrid, we exposed four weeks old rosette leaves to ultraviolet (UV) light and we found that fah1-2 and fah1-2/35S:AnAXE1 plants showed red colour indicative of lack of sinapoyl malate level whereas the wild-type and 35S:AnAXE1 line D plants showed blue color (Fig. 1b). To demonstrate the lack of S-lignin in hybrid plants, we stained transverse sections of their inflorescence stem with Mȧule stain. We found that the interfascicular fibres stained red (positive for S-lignin) in the wild-type and 35S:AnAXE1 plants and brown (absence of S-lignin) in fah1-2 and fah1-2/35S:AnAXE1 (Pradhan Mitra & Loqué, 2014) (Fig. 1c).

Similarly, the presence of AnAXE1 in the hybrid was confirmed by amplifying expected size band using AnAXE1 - specific primers PCWL-33 and PCWL-34 (Additional file: Table S1) whereas these amplicons were absent in for fah1-2 and wild-type (Additional file: Fig. S1d). The further analysis was performed on F2 and F3 generation plants which were positive for AnAXE1 amplicon and also confirmed its expression by qPCR (Additional file: Fig. S1e and S1f). AnAXE1 overexpressed lines showed an increase in esterase activity (Pawar et al., 2016). To evaluate this, we performed esterase activity assays on the leaves of the parent lines and the selected homozygotic hybrid line using p-nitrophenyl acetate as a substrate. Esterase activity in the wall-bound protein fraction was increased in the parent 35S:AnAXE1 line and in fah1-2/35S:AnAXE1 homozygotic plants compared to the wild type and fah1-2 mutants. (Fig. 1d). To further confirm a reduction in polysaccharide acetylation in the hybrid line because of the increase in esterase activity, we analysed cell wall acetyl content in alcohol-extracted wall residues (AIR) after saponification. As expected, we observed decreased acetyl content in 35S:AnAXE1 and fah1-2/35S:AnAXE1 plants as compared to wild type and fah1-2 mutant (Fig. 1e). These experiments confirmed expected genotypes and phenotypes in parents and fah1-2/35S:AnAXE1. We hereafter referred to these crossed plants as HyperG Hypo Acetylated line (HrGHypAc).

Morphological studies revealed that HrGHypAc plants were indistinguishable from wild-type plants. The xylem vessel cells were not collapsed (Fig. 1c) as in irx cell wall mutants with compromised growth (Turner and Somerville 1997). Often, changes in cell wall composition affect plant responses to biotic stress (Miedes et al. 2014). More specifically, cell wall-associated components are known to function as damage-associated molecular patterns (DAMPs) to induce or aggravate immunity. To evaluate innate immune responses of the HrGHypAc plants, we performed a quantitative pathogenesis assay. Individual parents were also tested similarly, with wild-type plants as controls. Fully expanded rosette leaves from the investigated plants were first infiltrated with Pseudomonas syringae DC 3000 pv. tomato strain (PstDC3000) suspension at a density of 5 x 10⁴ cfu/mL. The accumulation and growth of PstDC3000 in the infiltrated leaves were evaluated at day 0 and day 3, post-infiltration. Simultaneously, we also determined the expression changes in defence-related marker genes namely WRKY53, PR1, PAD3, LORE, and SERK in the infiltrated leaves at 0 and 3 dpi (days post-infection). Neither fah1-2, nor HrGHypAc plants showed any differences in bacterial accumulation at 3 dpi in comparison to the wild-type plants (Additional file: Fig. S1g). Interestingly, fah1-2 plants showed higher upregulation of WRKY53, PR1 and PAD3 genes at 3 dpi compared to the wild-type. Contrastingly, WRKY53 was down-regulated in 35S:AnAXE1 at day 3 dpi. In the HrGHypAc plants, the tested immune markers were wild-type comparable indicating that genetically FAH1 and over-expressed AnAXE1 may function antagonistically on WRKY53 expression (Additional file: Fig. S1h). However, since overall pathogenesis outcomes were unaffected in the tested plants, it is implied that the contribution of these genes towards defence is not significant. Taken together, these data suggest that the HrGHypAc or the parental plants are not compromised in growth or defence.

The total sugar content and Updegraff cellulose content is higher in HrGHypAc plants

To study the effect of simultaneous modification of lignin and xylan structure, we performed detailed cell wall characterization of alcohol insoluble residue (AIR) isolated from seven weeks old inflorescence stems of above plants. Using the phenol sulphuric method, we analysed the total cell wall polysaccharide composition which was higher (+ 20%) in HrGHypAC as compared to wild-type or parent plants. (Fig. 2a). Further, the content of crystalline cellulose by the Updegraff method was increased by 13% in HrGHypAc line as compared to either wild type or parent plants (Fig. 2b). Non-crystalline glucose originated from amorphous cellulose and hemicellulosic polysaccharide and other hemicellulosic sugars such as xylose, arabinose, rhamnose, fucose, galactose and mannose were similar in analysed plants (Fig. 2c). These findings suggests that the increase in total sugar content could be because of higher cellulose content. Another important component in the stem cell wall is lignin which was unchanged between investigated plants (Fig. 2d). Overall, these data suggest that HrGHypAc line contained increased total sugar, and crystalline cellulose content and with no major changes in lignin content or matrix sugar composition as compared to parent and wild type cell wall.

HrGHypAc plants show improvement in xylan and cellulose digestibility

To understand the effect of simultaneous modification in lignin monomer and xylan acetylation on biomass recalcitrance, we performed a polysaccharide digestibility assay with and without pre-treatment using AIR from the stems of parent genotypes, progeny and wild-type. After digesting the untreated AIR of wild type, fah1-2, 35S:AnAXE1 and HrGHypAc with commercial enzyme blend containing cellulases and hemicellulases and by measuring the glucose release by GOD-POD assay (Acker et al. 2016), we detected moderately higher (+ 4.38%) in the HrGHypAc compared to wild-type whereas the parent was comparable to wild type (Fig. 3a). Furthermore, after hot water pre-treatment known to increase accessibility of polysaccharide degrading enzymes, the saccharification efficiency was increased in both the parents and HrGHypAc by 19.85%, 17.41%, and 19.25% respectively as compared to the wild type (Fig. 3b). The glucose release was comparable in HrGHypAc and parent plants. Deacetylated xylan is more accessible to xylanases (Pawar et al. 2016). Therefore, AIR was digested with glycosyl hydrolase family 11 (GH11) without treatment, a higher level of xylose release was observed in parent and HrGHypAc plants as compared to the wild type. 35SAnAXE1 line exhibited a higher amount of xylose release as compared to the fah1-2 and HrGHypAc plants (Fig. 3c). This was further confirmed by running digested product on TLC as more xylose was detected in parents and cross as compared to wild type (Additional file: Fig. S2a) (Additional file: Fig. S2b). Moreover, xylobiose or conjugated xylobiose were also in higher amounts and longer chain xylotetraose or conjugated xylotetraose were detected in a lower amount in the cross than wild type. Since glucuronoyl esterase (GE) breaks the γ-ester linkage between xylan and lignin (Hüttner et al. 2017), AIR was treated with GE followed by endoxylanases and xylose released by these treatments was increased by 45%, 26% and 43% in fah1-2, 35S:AnAXE1 and HrGHypAc, respectively compared to wild-type (Fig. 3d). Also, similar amount of xylose release was detected in fah1-2 and HrGHypAc line but was higher in 35S:AnAXE1 parent as compared to above plants.

We also digested AIR with a GH67 alpha glucuronidase (which hydrolyses glucuronic acid side-group on xylan chain) followed by digestion with a GH11 endoxylanase and found the xylose release increased by 34%, 53% and 49.5% in fah1-2, 35S:AnAXE1 and HrGHypAc, respectively, compared to wild-type (Fig. 3e). We also measured glucose release in GE and alpha glucuronidase digested pellet and it was only increased in fah1-2 mutant as compared to wild type plants (Additional file: Fig. S2c).

In summary, whereas only the HrGHypAc line showed an increase in glucose yield in saccharification without pre-treatment, all three lines, both parents and the HrGHypAc line, showed improved xylan digestibility as compared to wild type by a GH11 endoxylanase after pre-treatment with hot water and either GE or alpha glucuronidase.

Altered extractability and digestibility of xylan, cellulose and lignin in cell wall-modified lines

Lignin and xylan modification can alter its interaction with pectin and consequently can influence polysaccharide digestibility (Liu et al. 2022; Broxterman and Schols 2018; Ralet et. al., 2016). To study further the effect of the genotypes (fah1-2, 35S:AnAXE1 and HrGHypAc) on the accessibility of xylan to a GH11 endoxylanase, the AIR was treated with ammonium formate to solubilise pectin and the remaining pellet was digested with pectate lyase to completely remove the residual homogalacturonan. The galacturonic acid was analysed in both pectin-rich ammonium formate extract and in pectate lyase fraction. HrGHypAc plants showed a decrease in galacturonic acid level by 14% as compared to the wild type in the ammonium formate extract but it was similar in wild type, parent lines and their progeny in the pectate lyase fraction (Fig. 4a). The de-pectinated pellet obtained in above assay was digested with a GH11 endoxylanase and the total xylose released by the enzyme was measured in the supernatant. Significantly higher xylose content than wild type (5%, 6% and 2.5% in fah1-2, 35S:AnAXE1 and the HrGHypAc respectively) were detected in this assay (Fig. 4b). Subsequently, we measured cellulose content in de-pectinized xylanase digested samples, found it significantly higher by 49%, 41% and 55.5% in fah1-2, 35S:AnAXE1 and the HrGHypAc, respectively, as compared to wild-type (Fig. 4c). To understand the effect of the genotypes on lignin content in de-pectinized samples and in de-pectinized xylanase-digested samples, we measured acetyl bromide soluble lignin (ABSL) in these samples. In de-pectinized samples, ABSL was significantly increased in the HrGHypAc line by 7% as compared to wild-type and parent lines (Additional file: Fig. S3a). In the de-pectinized xylanase-treated samples, fah1-2 and 35S:AnAXE1 lines showed 13% and 9.5% decreased lignin content, respectively, and the HrGHypAc line showed similar lignin content as wild-type plants (Additional file: Fig. S3b). We also measured ABSL content after saccharification and found that it was decreased by 5.7%, 10.4% and 15% respectively in fah1-2, 35S:AnAXE1 and HrGHypAc lines (Additional file: Fig. S3c). To further understand the effect of genotypes on xylan extractability, we sequentially extracted cell walls with ammonium formate and KOH and analysed the xylan content in 1M and 4 M KOH fractions using LM10 antibody. The xylan signal was significantly increased in fah1-2, AnAXE1, and the HrGHypAc plants, respectively, as compared to wild-type in combined 1M and 4 M KOH fractions (Additional file: Fig. S3d). The extractability was higher in 35S:AnAXE1 among all tested genotypes. All these cell wall digestibility and extractability experiments revealed that both HrGHypAc and parental plants show altered xylan digestibility and lignin extractability after sequential extraction.

HrGHypAc plants show increased expression of CESA, THESUS1 and WAK genes

To correlate the changes in plant cell wall properties in parental and HrGHypAc plants, we evaluated the expression of the key cell wall-associated genes. Cellulose Synthases (CESAs) in the rosette complexes present in the plasma membrane are responsible for the cellulose polymer biosynthesis (Li et al. 2014). When tested for relative expression levels of various CESAs transcripts, secondary cell wall-specific CESA6 and CESA8 (+ 2.6 and + 2.5-fold) mRNAs were significantly upregulated whereas CESA1 was lower (-19.64%) in HrGHypAc plants than wild type (Fig. 5). No significant change in fold change expression was found for CESA2 among tested plants. The expression of secondary cell wall biosynthesis transcriptional activator MYB83 (Ko et al. 2014) and the lignin biosynthesis pathway transcriptional activator MYB63 (Zhou et al. 2009) were also higher in the HrGHypAc line when compared to wild type (Fig. 5). Expression levels of xylan modifying genes xylan modifying enzymeβ-xylosidase 1 (BXL1) (Goujon et al. 2003) and xyloglucan-modifying enzyme XTH4 (Xyloglucan endotransglucosylase/hydrolase) (Kushwah et al., 2020) were unchanged except XTH4 in fah1-2 compared to the wild-type (Fig. 5). THESUS1 (THE1) is a receptor-like kinase involved in sensing defective cell wall growth and development, and a related gene FERONIA (FER) implicated maintenance of in pollen tube cell wall integrity sensing (Cheung and Wu 2011) were upregulated in the HrGHypAc by 78.61%, or unchanged compared to wild-type (Fig. 5). Similar to THE1 and FER, Wall Associated Kinases (WAKs) function as cell wall integrity sensing receptors associated with pectin and have an affinity for pectin oligomers which may be released during cell wall degradation (Amsbury 2020). HrGHypAc showed upregulation of several WAKs (WAK1, 3 and 4) (Fig. 5). This transcriptomic data suggested secondary cell wall-specific genes and receptor-like kinases are differentially regulated in mainly in HrGHypAc plants.

According to a recent report, the affinity of lignin to xylan is more as compared to lignin to cellulose (Kang et al., 2019). Lignin-cellulose interactions are bridged by xylan structure, suggesting the importance of lignin-xylan interaction in determining secondary cell wall structure and lignocellulosic biomass properties. Therefore, in this study, we altered the structure of xylan and lignin to elucidate the effect on plant cell wall properties. In our previous study, we reported that overexpression of AXE from carbohydrate esterase (CE) family 1 improves saccharification efficiency and ethanol production in Arabidopsis (Pawar et al., 2016). Therefore, we crossed AnAXE1 expressed line D with G lignin hyperabundant fah1-2 mutant. The homozygous hybrid HrGHypAc plants were growing normally in controlled growth chamber conditions because the xylem vessel structure was intact. This also suggests that lignin and xylan interactions in HrGHypAc plants give stable cell wall structure (Busse-Wicher et al., 2014; Crestini et al., 2011). Also, HrGHypAc cell wall showed an increase in total polysaccharide sugar content because of an increase in Updegraff cellulose content. CESAs are responsible for cellulose synthesis and the expression of two of these genes was significantly upregulated in HrGHypAc plants (Fig. 5). This increase in cellulose level probably helped to keep the xylem vessel cells of the HrGHypAc line intact leading to normal plant growth and development. Moreover, parent lines did not exhibit abnormalities in our and previous studies (Chapple et al. 1992; Pawar et al. 2016). We also tested the effect of the pathogen Pseudomonas syringae DC3000 pv. tomato to understand whether plant defense was compromised and it was not affected either in parents of HrGHypAc lines (Additional file: Fig. S1g and S1h). Also, the expression of defence-related marker genes was unchanged between the wild-type and HrGHypAc plants. All these data suggest that simultaneous alteration of xylan and lignin do not induce growth and developmental defects.

The glucose release was increased by 4.83% without pre-treatment only in HrGHypAc, this was because cellulose content was higher in HrGHypAc (Fig. 3a). Similarly, after hot water treatment, the glucose release was hiked by 19.25% in HrGHypAc plant. 35S:AnAXE1 overexpression showed 17.14% increase in saccharification efficiency after hot water treatment. Also, the fah1-2 showed 19.85% increase in saccharification (Fig. 3b). Importantly, HrGHypAc plants showed an increase in saccharification even after hot water treatment similar level to fah1-2 line. Similar results were observed by (Pawar et al. 2017) in Arabidopsis wood expressing fungal acetyl xylan esterase after hot water pre-treatment. Parents and HrGHypAc plants xylan digested more easily than wild-type plants (Fig. 3c). We also found a significant effect of genotypes on xylanase digestion after treatment with α- glucuronidase and glucuronyl esterase. We used these two enzymes because they are responsible for breaking the putative connection between xylan and lignin (Larsbrink & lo Leggio, 2023; Raji et al., 2021). Parents and HrGHypAc showed enhanced accessibility to both α-glucuronidase and glucuronyl esterase as this line showed more xylose release after xylanase treatment (Fig. 3d and 3e).HrGHypAc plants xylan is more accessible after glucuronoyl esterase and alpha glucuronidase treatment respectively (Fig. 3d and 3e). Improvement in xylanase digestibility with and without treatment can be explained in plants with lower xylan acetylation (35S:AnAXE1 and HrGHypAc) but is surprising for fah1-2 plants as it is known to be resistant for cellulase digestion. The possible explanation could be lower interaction of xylan and G lignin in fah1-2 as compared to wild-type containing tight xylan-S lignin interactions. As this interaction depends of the O-methyl group present on the aromatic phenyl ring (Kang et al., 2019). But, this needs to be further investigated by understanding G-lignin and xylanase interaction during the saccharification process with and without the pretreatment (Li and Zheng 2017; Kang et al., 2019). Further, we analyzed changes in cell wall extractability and found lower pectin galacturonic acid levels in ammonium formate fraction of HrGHypAc as compared to wild type and parents (Fig. 4a). Xylanase digested de-pectinated pellet showed an increase in xylose release both in parents and HrGHypAc (Fig. 4b). The cellulose content measured in pellets was elevated in 35S:AnAXE1, fah1-2 and HrGHypAc lines. In an independent experiment, we found that xylan epitopes were more abundant in KOH fractions of parents and HrGHypAc lines than in wild-type, indicating increased xylan extractability (Additional file: Fig. 3d). The xylan extractability was higher also in both parents as compared to parents. Lignin was reduced in xylanase and cellulase digested pellet suggesting changes in plant cell wall structure (Additional file: Fig. 3b and 3c). All these results suggest that the pectin-xylan connection is loosened in parental and HrGHypAc plants. Similarly, irregular xylem (irx) 9 and irx15 irx15L showed an increase in pectin and xylan extractability (Crowe et al., 2021). It is also well known that pectin modification improves polysaccharide digestibility and extractability (Biswal et al., 2018; Biswal, Soeno, Gandla, et al., 2014). More cell wall analytical studies are needed to dissect the mechanism of improvement in selective digestibility and extractability in both lignin and xylan-modified lines.

The changes in cell wall generally perceived by wall receptors, further transduce the signal which reinforces or weaken the cell wall or impacts the immune response which is refereed plant cell wall integrity (CWI) maintenance (Nuhse 2012). Cell wall elicitors feeding which was isolated from fah1-2 can induce the expression of defense-related genes mostly because the abundance of pectin elicitors (Gallego-Giraldo et. al., 2018). AnAXE1 plants are resistant to Hyaloperonospora arabidopsidis infection and altered defence responses (Pawar, et.al., 2016). This suggests alteration in CWI responses in parent plants. Although we did not see growth defect in the hypoacetylated G lignin-rich line, the cell wall integrity signalling has been altered as changes in cell wall extractability and digestibility are clearly observed in the above lignin and xylan-modified plants. The changes in cell wall generally perceived by wall receptors, further transduce the signal which reinforces or weaken the cell wall. In our qPCR data, we found that THESUS1 (THE1), WALL ASSOCIATED KINASES 1 (WAK1), WAK3 and WAK4 expressions are higher in the HrGHypAc line but were similar to wild type in parent plants (Fig. 5). THE1 belongs receptor-like kinase (RLK) family which was identified in suppressor screening of cellulose-deficient mutant (Hématy et al. 2007). WAK is a five-member family in Arabidopsis and WAK1 binds to oligo-galacturonides (Decreux and Messiaen 2005). Perturbation in lignin and xylan acetylation might have changed plant cell wall oligosaccharide profile which are sensed by THE1 or WAK1 or other cell wall sensors which might have led to enhanced accumulation in cellulose, effect on plant cell wall saccharification and extractability (Wolf, Hématy, and Höfte 2012, Baez, Tichá, and Hamann 2022, Gallego-Giraldo 2018). Complete transcriptomic analysis and elicitor studies in parents and HrGHypAc might reveal differential regulation of plant cell integrity sensing upon xylan and lignin modification.

Hyperaccumulation of the G lignin in combination with the post-synthetic reduction in xylan acetylation increases cellulose content. Prior digestion with or without glucuronyl esterase and α- glucuronidase treatment improves the xylan digestibility. Alterations in lignin, xylan and cellulose were observed in parents as well as HrGHypAc lines with any effect on plant development. Changes in the transcription of cell wall-associated biosynthetic and signaling genes suggest activation of plant cell wall integrity sensing which seems more prominent when both lignin and xylan structure is modified. Overall, this study revealed that simultaneous modification of xylan and lignin could be tested in other bioenergy crops to improve lignocellulosic biomass properties for different biotechnological applications.

Plant growth condition and genotyping

Homozygous plants of fah1-2 (Chapple, 1992) and 35S:AnAXE1-Line D (Pawar et al., 2016) were grown in a plant walk-in growth chamber under 16-h-light/8-h-dark photoperiod at 22°C. The mutation in fah1-2 was genotyped by amplifying the F5H gene using primers PCWL – 58 and PCWL – 59 (Additional file: Table S1) followed by digestion with MseI (R0525S, New England Biolabs, UK). AnAXE1 gene was confirmed using primers PCWL – 33 and PCWL – 34 (Additional file: Table S1). The fah1-2 mutant was further confirmed by UV phenotyping, homozygous fah1-2 mutant lacked sinapoyl malate and exhibit red colour under UV.

Detection by sinapoyl malate by HPLC

Three weeks old Arabidopsis leaf was processed with 70% methanol, incubated at 65°C for 1h and the sample were run on a C18 column (Waters, SunFire C18, 2.5µm, 3.0X75 mm column). The gradient was set from 98–80% of 0.1% formic acid for 20 minutes with acetonitrile and then reversed back to 98% up to 35 minutes on Agilent 1260 Infinity II HPLC-DAD system. The injection volume was 10 µl with a column temperature of 40˚C. The diode array detector was set at 330 nm with 1 nm bandwidth (Kim et al., 2015).

Mäule staining

Six weeks old Arabidopsis inflorescence stem were stained by Mäule reagent as described by (Pradhan Mitra & Loqué, 2014). Briefly handmade stem sections were treated with 0.5 % potassium permanganate for5 min, washed with water and dipped in 3.5 % hydrochloric acid (HCl) toremove excess colour. After removing HCl, the section was treated with ammonium hydroxide and immediately observed under a Nikon ECLIPSE Ti fluorescence microscope with 10X and 20X magnification.

Total RNA extraction and qRT-PCR

Total RNA was extracted from fresh main stems by the Trizol method (Cat#15596018, Thermo Fisher Scientific - Invitrogen). DNA contamination was removed by DNAse treatment using the kit (AM1907, Thermo Fisher Scientific, Lithuania). cDNA was synthesized from 1 µg of DNAse treated RNA with PrimeScript TAKARA cDNA synthesis kit. (Takara, 6110A). qPCR was performed using HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis Biodyne, 08-24-00001, Estonia) master mix on QuantStudio- 6 Flex Real Time PCR machine for gene of interest and reference gene (Additional file: Table S1). The expression was quantified using the ΔΔCt method.

Esterase activity assay

Esterase activity was determined by extracting soluble and wall-bound protein fractions in phosphate buffer (Biswal, Soeno, Latha Gandla, et al., 2014). The esterase activity was carried out using p- nitrophenyl acetate as a substrate (Sigma, N8130, Switzerland). The enzyme reaction mixture was incubated for 2 h at 37 ˚C and the release of p-nitrophenol (Sigma, 1048, United States) was quantified by spectrophotometry at 400 nm. The specific esterase activity was estimated by calculating nM per minute per mg of total protein using the p-nitrophenol standard curve.

Pseudomonas infection study

For Pseudomonas syringae DC 3000 pv. tomato (pstDC3000) plants were grown at short day condition i.e., 8/16-hour photoperiod with 70% humidity. Plants were grown for one month and leaves from fully expanded rosettes were used for infection. Leaves were infiltrated with bacterial suspension in 10 mM MgCl₂ at concentration of 5 × 10⁴ CFU ml^− 1 by using needle less syringe. Bacterial infection was measured on 0 and 3rd dpi by harvesting leaf discs of constant diameter and macerating them in 10 mM MgCl₂ and inoculating their serially diluted suspension on Pseudomonas agar plates with antibiotic selections. Bacterial infection in each genotype were then estimated through number of colonies and expressed as Log₁₀CFU cm^− 2. (Roy et al. 2022).

Preparation of stem powder for cell wall analysis

The inflorescence stem of 10 cm was collected from a completely dried Arabidopsis plant and homogeneous fine powder was prepared using Qiagen TissueLyserII for further cell wall analysis. The stem powder was treated for 30 min at 70 ˚C in 80% ethanol prepared in a 4 mM HEPES buffer. After treatment, sample was washed with 70% ethanol, trichloromethane: methanol (1:1) and acetone sequentially. The final pellet was dried in a desiccator overnight and this alcohol insoluble residue (AIR) was used for plant cell wall composition analysis.

Cell wall composition analysis

Quantification of acetyl content

Acetic acid content was estimated by treating 1 mg AIR with 1M NaOH. Treated samples were then neutralized with equimolar HCl. Samples were diluted and the mixture was used for acetic acid quantification by Megazyme kit (Megazyme, K-ACET, Ireland).

Total sugar content

Total sugar was estimated by the phenol sulphuric acid method. AIR was suspended in Milli-Q water at a concentration of 0.5 mg/ml. The reaction was set up in a glass tube by adding 100 µl of 5% (v/v) phenol solution and 100 µl of sample suspension. 500 µl of concentrated sulphuric acid was added to the reaction, vortex thoroughly, and incubated for 20 min in fume hood. After the completion of incubation, the absorbance of reaction mixture was taken at 490 nm. The concentration of total sugar in the samples was estimated by using glucose standard curve.

Cellulose content quantification

Cellulose estimation was performed on 1 mg AIR by performing nitric acid: acetic acid: water treatment. The treated samples were then acid hydrolysed to convert cellulose polymer into glucose monomer. The glucose content was then quantified by an Anthrone (96476, SRL, India) assay(Updegraff, 1969).

Hemicellulosic mono sugar composition analysis by GC-MS

2 mg of AIR samples were treated with 2M trifluoroacetic acid (208–02741, Wako, Japan) at 121°C for 90 min, then centrifuged at 10,000 rpm for 10 min and supernatant was collected in glass tubes. TFA was evaporated from supernatant under a stream of nitrogen gas and treated with isopropanol, three times and evaporated. Then, the dried fractions were re-dissolved in methoxyamine hydrochloride (139-18071, Wako, Japan) prepared in pyridine (163–05326, Wako, Japan) and incubated for 90 min at 37°C. These fractions were then derivatized at 37°C with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (130-17881, Wako, Japan) for 30 min. 100 µl of derivatized samples were then run on GC-MS. Inositol was used as internal standard in each sample.

Lignin content quantification

1 mg of AIR was treated with 25% acetyl bromide (135968, Sigma-Aldrich, USA) prepared in acetic acid at 50 ˚C for 2 h with intermittent shaking. 2 M NaOH and freshly prepared 0.5 M Hydroxylamine hydrochloride (159417, Sigma-Aldrich, China) were then mixed to samples. Samples were diluted with acetic acid and absorbance was taken at 280 nm. (Foster et al., 2010).

Xylose content quantification

Total cell wall xylose content was estimated by treating the 2 mg AIR with 1.3 M HCl in a dry bath pre-set at 100 ˚C for 1 h, neutralized with equimolar NaOH. Samples were diluted followed by centrifugation at 16000 rpm for 5 min and 50 µl of supernatant was used for xylose quantification by Megazyme kit (K-XYLOSE, Megazyme, Ireland).

Cell wall digestibility analysis

Saccharification of untreated, hot-water treated and xylanase-digested AIR was performed using cellulase enzyme blend (SAE0020-50ML, Sigma, China). The enzyme concentration and incubation period were first standardized for Arabidopsis stem biomass according to the dosage guidelines suggested by Novozymes (FUEL ETHANOL APPLICATION SHEET Cellic® CTec2 and HTec2-Enzymes for Hydrolysis of Lignocellulosic Materials, n.d.). 15 % w/w of cellulose enzyme was used for each sample. Samples were incubated at 50 ˚C for 48 h in a dry bath with cnstant stirring. After incubation, the samples were centrifuged at 16000 rpm speed for 5 mins. The hydrolysed supernatant was analysed for the glucose content with the help of the GOD-POD kit. (K-GLUC, Megazyme, Ireland). AIR was pre-treated with hot water at 90˚C for 30 min. After that, the sample was centrifuged and the supernatant was collected and stored. Pellet was washed with Milli Q water, acetone and dried in vacuum desiccator. Saccharification on pre-treated samples was done as described above.

Xylanase digestibility prior and after glucuronoyl esterase and α- glucuronidase treatment

Xylanase digestion was performed by mixing AIR with xylanase GH11 (SRL, 13814) prepared in reaction buffer i.e. acetate buffer (pH 4.5) equivalent to approximate 13 U/ reaction. The samples were then incubated in acetate buffer (pH 4.5) at 50 ˚C for 6 hours. After incubation the xylose content from supernatant was estimated by using megazyme kit (K-XYLOSE, Megazyme, Ireland). Digested XOS were observed by TLC separation against xylose, (X1) xylobiose (X2), xylotetraose (X4) and xylohexaose (X6) standard. For this commercial TLC plate was used (1.05554.0007, Merck, Germany) and the solvent system Chloroform: Acetic Acid: Water (6:7:1) was used. TLC for the digestion product was repeated for 2 times (Sharma et al. 2020). Separated XOS were quantified using ImageJ.

Xylanase digestibility of the AIR was checked after the removal of the glucuronic acid and the glucuronoyl ester linkages the AIR was partially treated with the glucuronoyl esterase and the α- glucuronidase separately. The reaction for the glucuronoyl esterase (Megazyme, E-GERF, Ireland) treatment was set with the 0.1 M sodium phosphate buffer (pH 6.5) and 2 µl/mg AIR enzyme. The reaction was incubated at 40 ˚C for 6 h. For α-glucuronidase (Megazyme, E-AGUBS, Ireland) treatment, the AIR was incubated with 0.1 M MOPS buffer (pH 7) containing2 µl/mg AIR α- glucuronidase enzyme at 40 ˚C for 6 h. The samples pellets were washed with MilliQ water two times, acetone and dried in the vacuum desiccator. Dried pellet was digested with the GH11 xylanase as described before for 6 h. Xylose content was analysed from the supernatant of the digestion reaction by using the xylose assay kit (K-XYLOSE, Megazyme, Ireland).

Sequential extraction of cell wall components and xylan quantification through immunodetection

For sequential extraction we followed protocol as per (Rastogi et al., 2022). The stems AIR was first treated with 50 mM ammonium formate at 37˚C for 24 h, centrifuged and the supernatant collected. Then the remaining pellet was digested with desalted pectate lyase in 50 mM tris HCl buffer (pH 8) at 40 ˚C for 24 h. Again, the samples were centrifuged and supernatant was collected. Both the ammonium formate and pectate lyase supernatants were lyophilized and re-dissolved in water. The pellet was washed with water, acetone and dried as described before. Galacturonic acid from the above two fractions were estimated by kit (Megazyme, K-URONIC, Ireland). The washed pellet was then subjected to the GH11 xylanase digestion and xylose quantification was as described earlier. Pellet was washed with water and acetone and dried in a vacuum. The cellulose content of the xylanase-digested pellet was estimated by Updegraff's method. Cellulose digestibility was estimated by saccharification assay as described before. All the remaining pellets after pectin extraction, xylanase (GH11) digestion and saccharification were analysed for the ABSL content as described.

Glycan antibody profiling

Different fractions of cell wall components were isolated ithrough sequential extraction by using ammonium oxalate, sodium carbonate, 1M KOH, and 4M KOH. Total sugar content was estimated by the phenol-sulphuric acid method as described before, and all wall extract samples were diluted to a sugar concentration of 0.25 µg/mL. in ELISA plate, and evaporated at 37°C. Nonspecific sites in the coated ELISA plates were blocked by incubating with blocking buffer and room temperature. 50 µL xylan-specific primary antibodies LM10 (1:20 dilution) (AS184206 Agrisera, Sweden) were dispensed into each well and the plate was incubated for an hour at room temperature. The primary antibody was washed with the wash buffer and 50 µL of Anti-rat secondary antibodies (1:5,000 dilution) (A9542, Sigma-Aldrich, Germany) was added to each well and incubated for an hour. Wells were washed with the washing buffer, 50 µL TMB substrate solution was added into each well and incubated for 20 min at 37°C, reaction was stopped and monitored absorbance at 488 nm. The standard curve of xylan (Birchwood) (5 to 125 ng/ml) was prepared and used for xylan estimation in samples (Pattathil et al., 2017).

Calculations and Statistics

All the calculations and statistics were performed in Microsoft excel using the student’s t-test.

Acknowledgement

We would like to thank Regional Centre for Biotechnology-Advanced Technology Platform Centre for the sequencing facility, the National Institute of Plant Genome Research (NIPGR) for GC-MS facility. We would like to acknowledge Dr Clint Chapple for providing Arabidopsis fah1 seeds.

Authors information

Authors and Affiliations

Regional Centre for Biotechnology, NCR Biotech Science Cluster 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad-121001, Haryana, India.

Department of Forest Genetics and Plant Physiology, Umea Plant Science Centre, Swedish University of Agricultural Sciences, Umea, Sweden.

Contributions

AC performed most of the experiments with help from LR, BD, AS, DS, AS, RS, SD. EM and SB reviewed the manuscript and gave insightful suggestions for experiments. AC and PP wrote the manuscript with input from all the authors. PP designed and conceptualized the manuscript.

Corresponding author

Dr Prashant Anupama-Mohan Pawar

Funding

DST-INSPIRE faculty fellowship, RCB-Core Grant

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No competing interests reported.

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Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.

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Abstract

Background

Results

Conclusions

Figures

Introduction

Results

The total sugar content and Updegraff cellulose content is higher in HrGHypAc plants

HrGHypAc plants show improvement in xylan and cellulose digestibility

Altered extractability and digestibility of xylan, cellulose and lignin in cell wall-modified lines

HrGHypAc plants show increased expression of CESA, THESUS1 and WAK genes

Discussion

Conclusions

Methods

Plant growth condition and genotyping

Detection by sinapoyl malate by HPLC

Mäule staining

Total RNA extraction and qRT-PCR

Esterase activity assay

Pseudomonas infection study

Preparation of stem powder for cell wall analysis

Cell wall composition analysis

Quantification of acetyl content

Total sugar content

Cellulose content quantification

Hemicellulosic mono sugar composition analysis by GC-MS

Lignin content quantification

Xylose content quantification

Cell wall digestibility analysis

Xylanase digestibility prior and after glucuronoyl esterase and α- glucuronidase treatment

Sequential extraction of cell wall components and xylan quantification through immunodetection

Glycan antibody profiling

Calculations and Statistics

Declarations

References

Additional Declarations

Supplementary Files

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