Xylan epitope profiling: an enhanced approach to study organ development-dependent changes in xylan structure, biosynthesis, and deposition in plant cell walls

Cell walls perform a number of important roles in plant growth and development including providing shape to different cell types, act as an interface between adjacent cells, intercellular communication, and defence responses against pathogenic attack. Cell walls in mature plant cells are structurally divided into primary cell walls, which surround expanding cells and secondary cell walls that are thickened structures containing lignin in order to provide structural support to the plant [1]. The major components of plant secondary walls are cellulose, hemicellulose (that include xylan, xyloglucan, and glucomannan), and lignin [2]. One of the major hemicellulose components in secondary cell walls, xylan, is a linear cell wall polymer that consists of a backbone made of β-(1,4)-linked xylose residues that is generally substituted with arabinose, acetyl, glucuronic acid (GlcA), and 4-O-methylglucoronic acid residues at varying degrees depending on plant species [3]. In dicots, the type I primary cell wall predominantly consists of glucuronoxylan (GX) with a linear backbone of β-(1,4)-linked xylose (Xyl) residues that are substituted with α-(1,2)-linked glucuronic acid (GlcA). These GXs occur as either binding tightly to exposed faces of glucan chains in cellulose microfibrils, and spanning the distance between adjacent microfibrils or with other GXs to space and lock cellulose microfibrils into place [4, 5]. In contrast, type II primary walls, characteristic of monocots are composed of glucuronoarabinoxylans (GAX) which attach to cellulose microfibrils in a similar fashion to type I walls. The type I and type II primary walls differ from the secondary cell walls of dicots including Arabidopsis, where xylan is the major hemicellulosic polymer present in the wall where they are actively being biosynthesised [6, 7]. Along with cellulose and lignin, xylan represents one of the main structural components in xylem vessels that facilitate the rapid movement of water while maintaining its structural integrity despite the negative pressure that occurs. In interfascicular fibres, xylan contributes to the thickness of the wall in fibre cells which allows them to maintain the mechanical strength of the stem [8, 9].The importance of xylan as a major constituent of the secondary cell wall in stems is well-emphasised by xylan-deficient mutants exhibiting weakened stems [10]. Xylan has been shown to exhibit varying structural features in plant cell wall throughout the development. For example, in wheat seedlings; specifically, arabinoxylan content changed from a high degree of arabinose substitution to a much lower degree of substitution, highlighting the complex manner in which high- and low-substituted arabinoxylans are deposited in primary and secondary cell walls in different cell types and at different development stages [11]. In developing willow stems, it was observed during stem maturation that xylan content and the degree of methylation of its GlcA side chains increase. Additionally, immunolabelling of xylan distribution using LM10 monoclonal antibody demonstrated increased labelling of unsubstituted and substituted xylan epitopes as the secondary xylem developed [12].

Since xylans are the second most abundant cell wall polymer after cellulose, they are a major non-cellulosic component of plant biomass. The xylose monomer units in xylan, however, are incompatible for fermentation into biofuels and other bio-products by organisms such as yeast due to their pentose structure [3]. Xylan also has a contributing role in cell wall recalcitrance by (1) cross-linking with lignin through ester bonds to GlcA and ether bonds with Xyl or Ara and (2) the ferulic acid dimerisation in grass xylans results in cross-linking of adjacent xylan chains or to lignin [3, 13, 14]. Additionally, these ferulic acid esters negatively impact xylan and cellulose hydrolysis [3]. Understanding xylan composition, structure, and biosynthesis is of key significance in designing strategies for engineering xylan in plants with improved properties such as decreased recalcitrance for biofuel production.

Advances have been made in gaining insights into the co-expression analysis of genes directly or indirectly associated with xylan biosynthesis including putative glycosyltransferases [15]. Several recent studies have focused on xylan structural variations during primary and secondary cell wall formation in Arabidopsis (see review by Hao and Mohnen [16]); however, there are only a handful of information on the structural dynamics of xylan in primary and secondary cell walls especially as a function of developmental stages of plant organs [17]. Moreover, despite early attempts to map the spatial and temporal distribution of xylans depending on developmental stages [12, 18], there are still no comprehensive studies on development-dependent variations pertaining to xylan sub-structures during stem maturation process. Understanding the differentiation of xylan deposition in a developmental context would provide a clearer picture of spatio-temporal regulation of xylan biosynthetic process and hence overall cell wall biosynthetic process in plant organs.

Plant cell wall glycan-directed monoclonal antibodies (mAbs) are highly specific probes used in plant cell wall analyses [19]. mAbs can bind monospecifically to glycan sub-structures, also known as glycan epitopes. This monospecificity feature provides mAbs with distinct advantages: it can bind to glycans whose structures are frequently repetitive and found in multiple macromolecular contexts (for example, arabinogalactan epitopes that are present in glycoproteins and pectic polysaccharides such as rhamnogalacturonan-I) and more importantly, their epitope binding specificity can be determined unambiguously. These mAbs can be used through either (1) in vitro detection through glycome profiling and (2) in situ visualisation by immunohistochemical methods. Glycome profiling (GP) involves sequential extraction of cell wall samples using a series of reagents increasing in harshness and then ELISA-screening these isolated extracts with mAbs in order to determine and monitor the glycan epitopes released by each extraction. Sequential extractions allow cell wall glycans to be isolated in extracts on the basis of the relative tightness with which they are integrated to the final wall structure. GP provides data regarding cell wall composition as well as the difficulty in extractability of these components in the wall (thereby revealing the relative tightness of their integration to the wall) [20]. GP has been extensively used in analysing cell walls for functional characterisation of genes involved in cell wall biosynthetic process, pretreated and bio-converted plant biomass, and comparative glycomics of different plant phylogenies based on their cell wall composition [21–23]. However, the limitations with GP are that firstly, low-molecular-weight glycans released by cell wall extractions do not bind to solid supports such as nitrocellulose, glass slides, or multiwell plastic plates. The lower limit of the glycan size while not definitively determined, is greater than 10 kDa. Secondly, certain glycans may get modified by the harsh alkaline extraction conditions, for example, the loss of acetylation or methylation [24].

In situ visualisation by immunolabelling techniques requires the fixation, embedding, and sectioning of biomass samples which are then probed by mAbs followed by a fluorescently tagged secondary antibody allowing an in situ visualisation of glycan epitope distribution under a fluorescence microscope [25]. Immunolabelling has aided in determining epitope distribution of cell wall biosynthetic mutants such as WRKY transcription factor knockouts, xylan-deficient mutants, and compared distribution of glycan epitopes in plant biomass before and after various methods of pretreatments to reduce cell wall recalcitrance [26, 27]. A major disadvantage of in situ visualisation is that any glycan epitopes that are buried within the cell walls (masked) are not exposed by the sectioning process and therefore, are not visualised [25]. In situ immunolocalisation studies along with GP could thus be significantly more powerful as these two methods together provide complementary information [19].

Recent epitope characterisation of xylan-directed mAbs through automated oligosaccharide synthesis provides a library of xylan structural epitopes specifically recognised by monoclonal antibodies (mAbs) [6]. Therefore, with use of different mAbs, it is now possible to monitor abundance throughout most major structural regions of xylans that vary based on their degree of polymerisation (DP), arabinosylation, acetylation, and MeGlcA substitution [6].

Studies with complementary approaches of full-scale GP, immunolabelling, and incorporating the details of above-mentioned recent advances in xylan epitope characterisations provide a powerful method in studying the patterning of xylan deposition in different stages of stem development. Our study, for the first time, describes xylan deposition and its spatio-temporal distribution over different stages of Arabidopsis stem development/maturation through GP, xylan epitope monitoring, and immunolabelling. Moreover, xylan patterns that we identified were supported by in silico gene expression analysis. Our results from GP, xylan epitope monitoring, immunolabelling, and expression data depict that xylan patterning increases throughout stem development. Interestingly, certain xylan epitope structure abundance displays degrees of specificity with regards to certain developmental stages of the stem. Lastly, GP illustrated that xylan epitope abundance and distribution may also influence other patterning and distributions of non-xylan epitopes such as pectins and xyloglucan across stem development. Therefore, using an epitope-directed approach may be useful in further characterising xylan biosynthetic mutants based on their specific xylan epitope phenotypes across developmental gradients of organs.

Plants cell walls are structurally highly complex, heterogeneous, and vary significantly across species, organs, and development. The studies herein analysed variations in cell wall makeup during Arabidopsis stem maturation process, especially focussing on xylan integration into the walls. Previous studies have demonstrated organ development-dependent variations in cell wall makeup in diverse phylogenetic classes of plants such as willow (S. purpurea) and switchgrass [12, 18]. However, more comprehensive and rapid approaches employing advanced and reliable cell wall analytical tools that can operate in a medium-to-high-throughput manner have always been in demand. In the current study, we developed, validated, and report such an advanced immunological approach for higher plant xylan analyses employing a comprehensive collection of fully characterised xylan-directed mAbs. Agreeing with the above-mentioned studies on willow and switchgrass, the whole glycome profiles (Fig. 1) of cell walls isolated from different development stages of stems revealed changes in the composition and extractabilities of most major non-cellulosic cell wall matrix glycans revealing unique cell wall makeup for different developmental stages of an organ. Of the comprehensive suite of cell wall directed mAbs used in the glycome profiling analysis, monospecific epitope-level structural information is currently available for most major xylan-directed antibodies [6]. Together, these xylan-directed antibodies could monitor five groups of xylan epitope structural regions namely small DP (DP, 3–5) homoxylan, large DP (DP, 4–8) homoxylan, single arabinosyl-substituted regions, double arabinosyl-substituted regions, and 4-O-MeGlcA-substituted regions. This allows monitoring of most major structural regions of complex xylans in higher plants including unsubstituted, arabinosyl-substituted, and glucurono/methylglucurono-substituted xylan structures. Given this extent of monospecific epitope-level information available for such a large group of xylan-directed antibodies, a unique opportunity opens up towards a molecular level understanding of xylan formation and integration into cell walls under diverse spatio-temporal stages of plant organs. In this work, we wanted to take advantage of the above well-characterised xylan-directed probes to study Arabidopsis stem development, validate the same, and develop an advanced tool for rapid and reliable characterisation of xylan formation and integration to the wall in higher plants.

Glycome profiling results demonstrated that all five xylan epitope categories are significantly present in 1 M and 4 M KOH extracts of cell walls from all developmental regions. These high alkali-extracted xylan components collectively represent tightly integrated xylan classes. These tightly integrated classes of xylan also contain unsubstituted and substituted regions (indicated by the binding of all xylan-4 through 7 clades of mAbs). All epitopes of tightly integrated xylan are abundantly present in all developmental stages including the youngest (D1) stage revealing lesser degree of variation of these classes of xylan across development. However, for tightly integrated xylan classes, subtle variations in patterns were observed in 1 M KOH extracts in that an increasing abundance of both unsubstituted and substituted regions of xylan was noted with the increasing stem maturation. In contrast, 4 M KOH extracts displayed decreasing abundance of these xylan epitopes as stem development progressed. This can be attributed to the corresponding increase in the proportion of xyloglucan epitopes in 4 M KOH extracts as shown in Fig. 1.

Immunolabelling analysis conducted in our study largely agreed with the glycome profiling results in that an increased intensity of labelling was observed during stem maturation. However, our results contradict those previously gathered by Kim and Daniel using xylan-directed LM10 and LM11 mAbs. They obtained different xylan epitope distributions compared to our study [33]. Their study selected LM10, which displays specificity towards unsubstituted or low-substituted xylans, and LM11, a wheat arabinoxylan and unsubstituted xylan-specific antibody [34]. Using these antibodies, their results displayed no differences in immunolabelling intensities in vessels and fibres at the apical and basal stem portions of Arabidopsis. Our results on the other hand, clearly demonstrated that certain homo-xylan epitopes (recognised by CCRC-M137, CCRC-M138, and CCRC-M150) displayed increasing intensities as stem development progressed. Additionally, epitopes that bind to CCRC-M114 and CCRC-M119 mAbs are absent in early (D1) and intermediate (D2) stages, but appear only in mature stem segments (D4). There are critical differences between these two studies, in our study, we surveyed a more comprehensive representation of stem developmental gradients including four regions namely apical (D1), lower apical (D2), upper basal (D3), and basal (D4). Again, in our current study, we used a more comprehensive repertoire of mAbs whose epitopes are well-defined. Hence, a direct comparison between these two studies may not be feasible. Lastly, our whole glycome profiling, xylan epitope-directed profiling, and in silico expression analysis of xylan genes [29] are also contradictory to the transcriptomic studies performed by Minic et al. [35], suggesting that xylan deposition starts to occur at the intermediate and late stages of stem development.

We represented the gravimetric amounts of material extracted out during each extraction step and the amounts of carbohydrate materials decreased with increasing stem maturity. This is because, as the stem matures, the secondary cell wall formation increases with increasing lignification processes. Hence, the extractability of non-cellulosic matrix glycans is reduced due to the increased abundance of more structurally rigid secondary walls. One of the main objectives of this study is to optimise a tool for dissecting xylan epitope extractability from different developmental zones of stem and use this tool to conduct comparative glycomics analysis of comparable developmental regions of organs originating from cell wall biosynthetic mutants in Arabidopsis and/or conduct such studies across plants belonging to diverse phylogenetic classes. Studies using this tool would thus allow us to compare and correlate abundancies of diverse xylan epitope structures among cell wall extracts prepared from comparable organ developmental zones. The two approaches employed here provide complementary information on the cell wall epitope abundance monitoring in vitro and in vivo changes. The two approaches (glycome profiling and immunolabelling) measure glycan epitopes differently. Glycome profiling monitors the chemically extracted glycan epitopes which are mostly not masked by other cell wall components. However, in immunolabelling, in vivo distribution of the epitopes is monitored which still could be conformationally masked (even after 0.1 M KOH treatment) hence a direct correlation between glycome profiling and immunolabelling data is not expected. Additionally, subjecting the cell wall to chemical fractionation may cause modification/s of some glycans resulting in the loss of some epitope structures [19, 20]. For example, in glycome profiling, CCRC-M114 displays increasing epitope abundance in all stages. However, the immunolabelling data for this specific epitope show signals only at the basal stage of the stem. Additionally, the similar increasing pattern of epitope abundance specific to CCRC-M147 in glycome profiling and immunolabelling is not significantly correlated. Thus, complementary information provided by these two approaches become invaluable when conducting comparative glycomic studies [19, 20].

Our systematic approach allows the comprehensive monitoring of the spatial distribution of specific xylan structures that occurs during stem development. By combining glycome profiling, immunolabelling techniques, and the available xylan epitope characterisation data, we can apply such an approach in order to explain certain behaviours in wood formation, an important potential source of raw materials for biofuel production [2, 36]. Applying our approach to surveying the structural and compositional differences observed in xylan biosynthetic mutants can reveal new insights into the functional characterisation of the complete spectrum of genes required for xylan biosynthesis/modification in the context of stem development [37]. Furthermore, our approach is a rapid, cost effective, and is a high-throughput method to determine the effects of pretreatments on xylan and other hemicellulosic structures in biofuel crops without requiring time-consuming and sophisticated computational methods involved in NMR and FTIR approaches [38–40].

Our method of xylan epitope-directed glycome profiling complemented by in situ visualisation using immunolabelling provides a viable approach to characterise specific xylan epitopes that are deposited in the cell wall at different stages of stem development. Furthermore, our approach can monitor the changes in xylan epitope composition throughout stem development that are caused by specific mutations among xylan biosynthetic/modifying genes. The approach could also be a powerful tool in an evolutionary context in that phylogenetic analyses of variations in xylan structures across species, organ, and developmental stages can be performed in a rapid and medium--to-high-throughput manner. This approach also opens up the possibility to employ other cell wall glycan-directed mAbs for monitoring other important cell wall glycans such as pectins, arabinogalactans, and xyloglucans.