Enhanced cell wall digestibility and immunity in Arabidopsis through targeted modification of xylan structure by heterologous expression of acetyl xylan esterase and xylanases

Enhanced cell wall digestibility and immunity in Arabidopsis through targeted modification of xylan structure by heterologous expression of acetyl xylan esterase and xylanases | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced cell wall digestibility and immunity in Arabidopsis through targeted modification of xylan structure by heterologous expression of acetyl xylan esterase and xylanases Anant Mohan Sharma, Bhagwat Prasad Dewangan, Aniket Chaudhari, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9277431/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Background Xylan, a major component of the secondary cell wall in dicots, is often recalcitrant due to its acetylation and interaction with cellulose and lignin. To fine-tune the xylan structure and improve the processing of lignocellulosic biomass, overexpression of cell wall-degrading microbial enzymes is a viable option that can enhance cell wall properties and immunity. Here, we expressed a glycosyl hydrolase (GH10) xylanase (XYL) from Aspergillus nidulans and a carbohydrate esterase (CE)1 acetyl xylan esterase (AXE) from Aspergillus niger , or both enzymes simultaneously (AXE/XYL), under the control of the constitutive Cauliflower mosaic virus 35S promoter (35S) and the woody-tissue-specific Populus trichocarpa GT43B promoter (WP) in Arabidopsis thaliana and studied their effects on the cell wall, saccharification properties, and biotic resistance. Results Transgenic WP:AXE/XYL lines exhibited an irregular xylem phenotype and compromised deposition of cell wall components, which correlated with downregulation of the responsible genes as revealed by RNA sequencing analysis. In contrast, 35S:AXE/XYL plants did not show any deformities, possibly because XYL expression was lower than in WP:AXE/XYL or due to variation in transgene expression. Biochemical analyses revealed reduced acetyl content in 35S:AXE and 35S:XYL/AXE lines, while total xylan content was increased in 35S:XYL, 35S:AXE, and 35S:XYL/AXE expressing plants. Notably, cellulose, xylan digestibility, and ethanol production were highest in the 35S:XYL/AXE line, surpassing the parental lines. Moreover, the 35S:XYL/AXE lines showed enhanced resistance to Pseudomonas syringae . Comparative RNA sequencing of xylobiose-treated and 35S:AXE/XYL plants revealed altered expression of defense-related genes. This was supported by elicitor assays, which demonstrated that xylo-oligosaccharides present in the 35S:XYL/AXE lines can promote the activation of immune marker genes along with increased accumulation of reactive oxygen species. Conclusions In summary, simultaneous hydrolysis of the xylan backbone and its acetylation represents a promising approach to boost both lignocellulosic biomass quality and plant immunity. acetyl xylan esterase xylanase xylan modification xylooligosaccharide immunity elicitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Plant cells are encased inside complex extracellular matrix known as the cell wall which is intricate organization of cellulose, hemicelluloses (including xylan, xyloglucan, and mannan), pectins, polyphenols, and structural proteins. These components determine cell growth and provide mechanical strength, flexibility, and structural integrity to the cell [1]. Cell wall remodeling also plays a vital role in biotic and abiotic stress adaptation [2]. In vascular plants, xylan is an abundant non-cellulosic polysaccharide, reaching 30% of secondary cell wall in dicots, 50% in monocots and 15% conifers [3–7]. Typically, xylan is a branched polymer of β-(1,4)-linked xylose residues substituted with acetyl, glucuronic acid (GlcA), 4-O-methylglucuronic acid (Me-GlcA), and arabinose residues, depending on species and tissues. In Arabidopsis secondary walls, there is acetylated glucuronoxylan, which xylosyl backbone is substituted with GlcA or Me-GlcA at C2 xylose position and acetyl groups at C2, C3 or both [8]. Xylan is polymerized in the Golgi apparatus by glycosyl and acetyl transferases, which when mutated, cause an irregular xylem phenotype characterized by collapsed xylem vessels [9]. Disruption of xylan structure can alter the physicochemical properties of the wall, affecting interactions among cell wall components [10, 11]. In Arabidopsis, xylan acetylation is mediated by a family of Trichome Birefringence-Like (TBL) proteins, including TBL29/ESK1, and mutation in TBL29/ESK1 causes xylan hypoacetylation, altered wall extensibility, changes in glucosinolate metabolism and stress sensitivities [12, 13]. However, the spectrum of plant responses to disruption in xylan acetylation is not well understood. Post-synthetic reduction in xylan acetylation by expressing fungal acetyl xylan esterases (AXE) in muro often enhances cell wall digestibility and extractability without affecting plant growth [14]. Moreover, AXE expressing plants have increased immunity against specific pathogens [14–16]. Since these plants do not have irregular xylem phenotypes, they are suitable genotypes to study responses to moderate deacetylation. Deacetylated xylan is prone to degradation by xylanolytic enzymes resulting in an altered pool of apoplastic xylo-oligosaccharides (XOS) that can act as a damage associated molecular patterns (DAMPs) triggering plant immunity. Plant treatment with XOS can induce pattern triggered immunity (PTI) which activates MAPK and calcium signaling, and reactive oxygen species (ROS) accumulation [17–19]. The structural features of XOS, such as degree of polymerization and side-chain patterns, influence immune potency and cell wall integrity maintenance mechanism. Xylan-derived signals could intersect with hormone signaling pathways, particularly salicylic acid and jasmonic acid signaling, shaping local and systemic defense responses [18, 20–22]. Crosstalk with lignification pathways further amplifies immunity through the deposition of phenolic polymers at infection sites [15, 23]. However, the role of apoplastic steady-flow XOS during development or after pathogen attack remains relatively unclear. Since plant endoxylanases are relatively unexplored, expression of xylanases and acetyl xylan esterase from fungal enzymes can serve as a tool to study the role of XOS in immunity and cell wall remodeling [14, 24–27]. Also, co-expression of AXE and xylanase can improve digestibility of different polysaccharide cell wall components by affecting the interaction of xylan with lignin and cellulose [15, 28–30] but such transgenic manipulation has not been to our knowledge previously attempted. Therefore, in this study, we investigated the combined effect of xylan deacetylation and backbone hydrolysis by co-expression of Aspergillus niger CE1 acetyl xylan esterase (AXE) and Aspergillus nidulans GH10 xylanase (XYL) in Arabidopsis thaliana . Since the choice of promoter can substantially influence effects of transgenes [27, 31]. In our study, we combine AXE and XYL with two promoters, the constitutive 35S promoter (35S) and the wood specific promoter (WP) active in cells depositing secondary walls [32]. Indeed, 35S lines showed no apparent morphological or xylem defects, however co-expression of AXE and XYL controlled by WP lead to several developmental and cell wall defects. Transcriptomic analysis indicated repression of secondary wall biosynthetic genes and compensatory changes in hemicellulosic sugars in WP:AXE/XYL lines. We also found that 35S:AXE/XYL lines had reduced xylan acetylation, increased xylose content, improved lignocellulosic biomass properties and saccharification. Increased sugar availability enhanced microbial fermentation efficiency, resulting in higher ethanol yields and reduced acetic acid accumulation. Additionally, 35S:AXE/XYL-mediated cell wall remodeling increased resistance to Pseudomonas syringae , associated with activation of several pathogen triggered immunity (PTI) marked possibly through elicitor activity of xylo-oligosaccharides in 35S:AXE/XYL plants. These findings revealed that coordinated xylan deacetylation and hydrolysis improve biomass digestibility while simultaneously enhancing plant immunity. Results Co-expression of microbial acetyl xylan esterase (AXE) and xylanase (XYL) enzymes alter plant development when regulated by a wood-specific promoter (WP). Our earlier studied showed that overexpressing carbohydrate esterase 1 (CE1) acetyl xylan esterase from Aspergillus niger (AnAXE1) under control of 35S or WP promoters leads to reduced xylan acetylation in Arabidopsis [14]. For making crosses with xylanase-expressing lines, we selected two highly expressing lines: line D carrying 35S:AXE construct and line E carrying WP:AXE construct as parent plants [14](Figure S1 a). We also observed that expressing GH10 endoxylanase from Aspergillus nidulans (XYL) in aspen can enhance xylan digestibility and increase drought resistance in Arabidopsis [24, 26]. The construct with XYL gene under control of the 35S promoter [24] (Figure S1 a) was used to generate stable transgenic lines which were used as hemi- or homozygous for crossing with the D line carrying 35S:AXE, and the resulting progenies were genotyped by PCR amplification (Figure S1 b). Expression of transgenes in selected progenies was verified by quantitative RT-qPCR (Figures S1 c). The 35S:AXE lines exhibited two times higher AXE expression than the 35S:AXE/XYL crossed lines (Fig. 1 a). Both parents and co-expressing lines with 35S promoter constructs showed no visible morphological differences compared to wild type plants (Fig. 1 b). Staining of inflorescence stems using phloroglucinol showed no abnormalities in xylem vessel cells, which are typically affected if xylan structure is compromised (Figure S1 d-e). Esterase activity was evaluated using both synthetic and natural acetylated xylan substrates, revealing higher activity in 35S:AXE and 35S:AXE/XYL compared to the wild type and 35S:XYL lines (Fig. 1 c, Figure S1 f). We also assessed xylanase in both parent and crossed lines, finding that 35S:XYL parents had 15 times more expression than the 35S:AXE/XYL crossed lines (Fig. 1 d). This pattern matched xylanase activity, which was significantly higher in 35S:XYL and 35S:AXE/XYL than in wild type and 35S:AXE (Fig. 1 e). To generate WP:AXE/XYL, we crossed the best expressing WP:XYL line [26] with the E line carrying WP:AXE and selected the progenies carrying both transgenes which were either homozygous or hemizygous (Figure S2 a and S2b).. AXE expression was 2.5 times higher in WP:AXE than in WP:AXE/XYL, while XYL expression was 3.5 times higher in WP:XYL than in WP:AXE/XYL (Figure S2 c-S2e). This corresponded to lower esterase and xylanase activities, reflecting the expression levels of AXE and XYL (Figure S2 f and Figure S2 g). However, WP:AXE/XYL lines were severely dwarf as compared to WT and WP parent lines (Figure S2 h) and xylem vessels were not normal shaped in WP:AXE/XYL line (Figure S2 i-2j). These dwarf phenotypes were observed in different generations which were either homozygous or hemizygous. In summary, we examined the crossed lines together with their parental and wild type plants, observing the anticipated increases in gene expression and activity. Notably, the 35S:AXE/XYL plants exhibited normal growth, whereas the WP:AXE/XYL plants were significantly stunted. Plants expressing both acetyl xylan esterase and xylanase showed decrease in acetyl content, along with compensatory changes in other cell wall components. We further analyze acetyl content in alcohol-insoluble residue (AIR) prepared from basal part of inflorescence stems of lines expressing AXE, XYL or both under control of the 35S promoter. Both the 35S:AXE and 35S:AXE/XYL lines showed reductions in acetyl content of 32% and 11%, respectively, compared to wild-type (WT) plants (Fig. 2 a). Additionally, AIR xylose content was elevated in both parental and 35S:AXE/XYL lines (Fig. 2 b). The acetyl/xylose ratio was also decreased, confirming a lower content of xylan acetylation (Fig. 2 c). All analyzed 35S transgenic lines did not differ in total carbohydrate, cellulose and lignin contents from the wild type (Fig. 2 d- 2 f). Furthermore, these constitutive promoter lines exhibited decreased levels of non-cellulosic monosaccharides in alcohol-insoluble residue prepared from inflorescence stems such as arabinose, rhamnose, fucose, and mannose (Table 1). Conversely, xylose levels were higher in 35S:AXE, 35S:XYL and 35S:AXE/XYL transgenic lines (Table 1). Above non-cellulosic sugar analysis was performed by derivatization using MS-TFA and analysis by GC-MS. We further found increase in xylose content in hydrolysed AIR1 with HCl and quantified using Megazyme K-Xylose kit (Table 1 & Fig. 2 b). However, glucose levels were comparable in wild type and transgenic lines. The acetyl content was also reduced in WP:AXE, WP:XYL and WP:AXE/XYL by 26%, 17% and 67% respectively as compared to wild type (Figure S3 a). However, acetyl to xylose ratio was reduced only in WP:AXE and xylose levels were reduced only in WP:AXE/XYL (Figure S3 b-S3c). WP:AXE/XYL plants also showed significant reductions in total carbohydrates, cellulose, and lignin compared to WT (Figure S3 c-S3f) and displayed a compensatory rise in non-cellulosic monosaccharides, including arabinose, rhamnose, fucose, mannose, and glucose (Table 1). To clarify the pronounced effects on growth and cell wall composition observed in WP:AXE/XYL plants, we conducted untargeted RNA-seq analysis comparing wild-type (WT) and WP:AXE/XYL 6-week-old stems. This revealed 264 genes upregulated and 269 genes downregulated, with a significant false discovery rate (FDR) adjusted p-value of ≤ 0.05 and a Log 2 FoldChange of ± 1 (Figure S4a). Further analysis showed substantial downregulation of key biological processes, including xylan biosynthesis, the phenylpropanoid pathway, and cell wall polysaccharide metabolism (Figure S4b-S4d, Supplementary Data 1). This led to reductions in essential cell wall components, such as xylan, and lignin. The transcriptomic data were consistent with cell wall chemotyping results for WP:AXE/XYL, which ultimately resulted in abnormal xylem vessel development and dwarfism in the plants. Co-expression of AXE and XYL driven by a constitutive promoter enhances lignocellulose saccharification and boosts ethanol yield Due to the dwarf phenotype observed in WP:AXE/XYL plants either because of increased in xylanase or esterase activity levels or tissue specific activity, we next focused on lines driven by the 35S promoter. To evaluate how co-expression of AXE and XYL affects the enzymatic saccharification of lignocellulose, we performed assays using untreated samples, hot water, alkali, and xylanase pretreatments, followed by incubation of the stem material with a cellulase mixture. After cellulase digestion, the 35S:AXE/XYL lines exhibited a 29% increase in glucose release compared to wild type, with 35S:XYL and 35S:AXE lines also outperforming the control in the absence of pretreatment (Fig. 3 a). When AIR was pretreated with hot water and then digested with cellulases, 35S:AXE/XYL plants again showed the highest increase in glucose release compared to parent and wild type plants (Fig. 3 b). A similar trend was seen after pretreatment with 0.4 NaOH and xylanase, where 35S:AXE/XYL plants demonstrated an 18% and 33% increase in xylose release, respectively, over wild type (Fig. 3 c- 3 d). We also digested AIR with xylanase alone and in combination with glucuronyl esterase. Xylose release after xylanase treatment was increased significantly in all 35S lines (Fig. 3 e). Similarly simultaneous digestion with glucuronyl esterase and xylanase enhanced xylose release by 14–35% in parental lines and by 50% in crossed line (Fig. 3 f). Modifying xylan can influence its interactions with other cell wall components, such as lignin, cellulose, and pectin, thereby affecting their extractability [15]. To investigate this, we sequentially extracted the cell wall using ammonium oxalate (AOE), sodium carbonate (SCE), 1 M KOH, and 4 M KOH, and assessed the extracts with cell wall-specific antibodies (Fig. 4 ). The 35S:AXE line, which has reduced acetyl content, displayed increased LM10 signals and decreased LM15 signals in the 4M-KOH fraction, reflecting increased epitopes of unsubstituted xylan and reduced epitopes of xyloglucan. In contrast, the 35S:AXE/XYL co-expression line did not show such changes. However, both parental (35S:AXE and 35S:XYL) and crossed (35S:AXE/XYL) lines exhibited reduced signals of rhamnogalacturonan-1derived fragments in the AOE and 1M KOH extracts as detected by CCRC-M7 antibody (Fig. 4 ). We further examined how elevated glucose and xylose release influenced fermentation efficiency in 35S:AXE/XYL plants compared to wild type, using an E. coli diauxic strain. Under anaerobic conditions with standard media conditions, native E. coli strain ferments 1 g of glucose to 0.2 g of ethanol. Therefore, to understand how the genetic changes in plants affect fermentation, we evaluated ethanol production using modified plants as carbon source. First, we digested AIR1 with CTec2 and measured glucose which was more in 35S:AXE/XYL as compared to WT and parent (Fig. 5 a). The digested saccharide mixture from plants was supplemented separately as a carbon source to the E. coli culture. Upon complete utilization of glucose, ethanol titer and yield was estimated. As expected 35S:AXE and 35S:AXE/XYL plants support higher ethanol production compared to wild type (Fig. 5 b- 5 c). Interestingly, only 35S:AXE/XYL reached theoretical maxima, suggesting minimal interference from the inhibitors present in the biomass. We also measured acetic acid level in the cultures and found it was reduced in 35S:AXE and 35S:AXE/XYL at 0 h and 24 h (Fig. 5 d). This strain can also use xylose during fermentation and at 0 h, xylose was increased and decreased after 24 h in 35S:AXE/XYL as compared to wild type (Fig. 5 e). Overall, co-expression of AXE and XYL under the 35S promoter improves cell wall digestibility, resulting in increased glucose and xylose release and enhanced ethanol yield. 35S:AXE/XYL plants exhibited enhanced immune responses and altered the expression of multiple genes associated with immunity and cell wall structure. Alterations in cell wall composition influenced plant responses to biotic and abiotic stresses, with decreased xylan acetylation correlating with enhanced pathogen resistance [14] and enhanced xylan hydrolysis with increased drought resistance [26]. To assess the immune response in the 35S:AXE/XYL line, a pathogenesis assay was conducted using wild-type plants as controls. Four-week-old fully expanded rosette leaves were infiltrated with a suspension of Pseudomonas syringae pv. Tomato DC 3000 (pstDC3000) and bacterial proliferation was measured at 0- and 3-days post-inoculation (dpi). The 35S:AXE/XYL leaves showed reduced bacterial accumulation compared to wild type at 3 dpi, indicating an increased resistance (Fig. 6 a). Correspondingly, expression of defense-related marker genes, including PATHOGEN-RELATED 1 (PR1), WRKY33, and WRKY53 , was upregulated at 3 dpi and expression of WRKY33 and WRKY53 was already increased at 0 dpi in this line (Fig. 6 b). Expression of other analyzed genes including PAD3 involved in carotene biosynthesis and FLAGELLIN SENSITIVE 22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1) associated with leaf senescence as well as WRKY30 and WRKY40 transcription factors remained unchanged (Fig. 6 b). To investigate how AXE and XYL overexpression affects cell wall remodeling and defense, RNA-seq analysis was performed in inflorescence stem tissue revealing 635 genes upregulated and 108 downregulated (Fig. 7 a). The GO-Biological process analyses revealed many genes responsive to several stimulus; related to reactive oxygen species (ROS) generation were upregulated (Fig. 7 b). The GO-molecular function and GO-cellular component analysed indicated upregulation genes involved in cell wall metabolism (Fig. 7 c- 7 d). Among the upregulated genes, 67 were linked to cell wall related processes determined by ShinyGo tool (Table S1 ). Notably, several members of the xyloglucan endotransglucosylase/hydrolase (XTH) family were induced, including XTH18 (AT4G30280), XTH8 (AT1G11545) , XTH9 (AT4G03210) , XTH19 (AT4G30290) , and XTH16 (AT3G23730) . In addition, several expansin genes such as EXPA5 (AT3G29030) , EXPA6 (AT2G28950) , EXPA4 (AT2G39700) , EXPA11 (AT1G20190) , EXPA14 (AT5G56320) , and EXPB1 (AT2G20750) were upregulated. Genes involved in pectin modification and degradation were also prevalent. These included PECTIN METHYLESTERASE 5 (PME5; AT5G47500) , PME1 (AT1G53840) , various pectin lyase-like superfamily proteins ( AT1G10640, AT4G23820) , and POLYGALACTURONASE 2 (PG2; AT1G70370) . Collectively, these findings indicate active xyloglucan and pectin remodeling in 35S:AXE/XYL plants. In addition, differential expression analysis identified 72 upregulated defense-related genes (Table S2 ), signifying robust activation of stress and defense-associated transcriptional programs. The most elevated gene was cytochrome P450 CYP94B3 (AT3G48520) , a critical enzyme in jasmonate catabolism (Koo et al., 2011). Consistently, multiple JASMONATE ZIM-DOMAIN (JAZ) repressors—including JAZ10, JAZ7, JAZ8, JAZ5 , and JAZ1 were significantly upregulated, indicating activation of the jasmonic acid (JA) signaling pathway with feedback regulation [33–35]. Upregulation of MYB96 , WRKY53 , WRKY40 , and WRKY18 further supports the transcriptional reprogramming in response to biotic and abiotic stress [36–38]. In summary, the 35S:AXE/XYL lines exhibited enhanced immunity against Pseudomonas and elevated expression of immunity-related marker genes as well as genes related to primary wall modification. Integrated transcriptomic and oligosaccharide elicitor analyses revealed that xylo-oligosaccharides trigger immune activation. We hypothesized the gene activation stems from increased in vivo production of xylo-oligosaccharides from xylan affected by the combined action of AXE and XYL. To explore this, AIR of inflorescence stems was digested with GH11 xylanase and released xylo-oligosaccharides were analyzed using MALDI-TOF. Results showed that xylobiose without acetyl groups (Xyl2) was more abundant in the 35S:AXE/XYL line than in wild type (Figure S5), and fewer acetylated Xyl4 XOS were present in this line. The xylanase digested fraction was further analyzed using ion chromatography, which revealed increase in xylose, xylobiose and xylotriose (Table S3 ). We also checked level of these soluble XOS in 70% ethanol fraction and found accumulation of xylose, xylobiose and xylotetratose and xylohexaose (Table S4). These results suggest that 35S:AXE/XYL lines have steady flow of XOS in the cell which may act as signalling molecules [18]. Our earlier study revealed that xylobiose treatment can trigger immune response and change cell wall composition. Therefore, we compared RNA-seq data from xylobiose treated plants [18] and 35S:AXE/XYL plants, finding 51 upregulated and 4 downregulated genes in common (Fig. 8 a). The upregulated genes included WRKY53 and WRKY18 , key modulators of pathogen-responsive gene expression, indicating a shift toward heightened defense and PEROXIDASE 2 , suggesting increased reactive oxygen species (ROS) metabolism—a hallmark of plant defense [39, 40]. We also found several of the JAZ1 (JAZ10, JAZ7, JAZ8, JAZ5, AND JAZ1) were upregulated in comparative RNA sequencing analysis. To further validate the hypothesis that in vivo generation of xylooligosaccharides (XOS) through co-expression of AXE and XYL initiates a defense response, we infiltrated XOS obtained from GH11 xylanase digestion into healthy 4-week-old wild-type leaves. Leaves treated with 35S:AXE/XYL-derived XOS exhibited a greater accumulation of reactive oxygen species compared to those treated with XOS from wild-type plants or mock controls (Figure S9a & 9a). Subsequent RT-qPCR analysis demonstrated upregulation of genes related to cell wall remodeling (such as XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 24 and CELLULOSE SYNTHASE-LIKE A11 ), pattern-triggered immunity (PTI) markers (including PATHOGEN-RELATED 1 and FLAGELLIN SENSITIVE 22-INDUCED RECEPTOR-LIKE KINASE 1 ), as well as genes linked to different stress responses ( WRKY22, WRKY30, WRKY33, PAD3, MYB24 , and MYB87 ) in leaves treated with 35S:AXE/XYL XOS (Fig. 9 b). Collectively, these findings indicate that 35S:AXE/XYL plants exhibit enhanced immune activation and altered cell wall composition, likely mediated by XOS-induced defense pathways. Discussion Plant secondary cell walls are a highly organized structure in which xylan acts as a critical hemicellulosic matrix, tethering cellulose microfibers to lignin [4, 41]. Post-synthetic targeted modification of plant polysaccharides by overexpressing microbial hydrolases to improve secondary cell wall properties is a promising strategy. Acetyl substitutions on the xylan backbone limit enzymatic accessibility, whereas backbone depolymerization shortens polymer length and disrupts its interaction with cellulose and lignin [42, 43]. Previous work showed that expression of fungal acetyl xylan esterase in Arabidopsis decreases xylan acetylation and improves saccharification without major growth defects [14]. However, the combined in-planta effects of deacetylation and the simultaneous hydrolysis of xylan had not been systematically explored. Here, co-expression of AXE and XYL provides direct evidence that coordinated modification of substitution pattern and polymer length synergistically alters cell wall architecture, digestibility, and immunity signaling. Distinct developmental consequences of xylem-specific expression of xylanases and acetyl xylan esterase AXE and XYL were expressed individually or together in plants; most of the resulting lines did not display any noticeable growth defects (Fig. 1 b). The lines co-expressing both enzymes exhibited higher enzymatic activity than the wild type, though lower than the parent lines expressing a single gene. This reduction in activity among co-expressing lines is likely due to the use of the same promoter, selection marker genes or gene dosage effect. Despite this, the WP:AXE/XYL line developed a dwarf phenotype, and both acetyl and xylose contents dropped by over 60% compared to wild type (Figure S3 a-S3c). Typically, plants compensate for such changes by increasing other components, but the WP:AXE/XYL line showed a significant decrease in cellulose and lignin content. This observation was reinforced by the transcriptional repression of genes involved in secondary cell wall formation in the WP:AXE/XYL line. Members of the TRICHOME BIREFRINGENCE-LIKE (TBL) family TBL35 and TBL27, responsible for xyloglucan acetylation [44–47], and TBL29/ESK1, TBL3 and TBL31, responsible for xylan acetylation, along with glucuronoxylan 4-O-methyltransferase-like proteins, were notably downregulated (Figure S4c) [13, 48–50]. The diminished expression of these genes implies disrupted hemicellulose patterning, which destabilizes the cellulose-xylan network. Reduced glucuronoxylan methylation is also known to compromise cell wall structure [51]. Genes from the phenylpropanoid pathway, including PHENYLALANIE AMMONIA LYASE ( PAL), 4-COUMARATE CoA LIGASE (4CL)1 and 4CL2, FERULATE-5-HYDROLASE , and the MYB58 transcription factor, were suppressed, resulting in less lignin deposition and reduced monolignol production (Figure S4d) [52, 53]. Furthermore, several XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH ) genes, which are involved in xyloglucan remodeling and the integration of cellulose microfibrils, were also downregulated [54, 55]. The formation of secondary cell walls requires the precise deposition of cellulose, xylan, and lignin [56]; disruption of xylan structure likely weakens the scaffold necessary for lignin polymerization and cellulose crystallization, which explains the overall reduction in wall polymers. This transcriptomic suppression of cell wall biosynthesis genes indicates feedback from wall damage, aligning with plant cell wall integrity signaling pathways [57, 58]. Reduced recalcitrance and improved saccharification efficiency in 35S:AXE/XYL plants Because of normal growth phenotype, we focused our further study on the 35S:AXE/XYL line. The parent 35S:AXE line showed a higher reduction in cell wall acetyl content than the 35S:AXE/XYL line, in agreement with a lower esterase activity in the latter (Fig. 1 c). A decreased acetyl-to-xylose ratio confirmed that AXE-driven deacetylation facilitated XYL-mediated backbone cleavage. MALDI-TOF profiling further demonstrated increased release of short, neutral xylo-oligosaccharides especially xylobiose and a reduction in acetylated oligomers, confirming that xylan in the 35S:AXE/XYL plants is less acetylated and more accessible to xylanase. (Figure S5) Saccharification assays revealed a significant increase in xylan and cellulose hydrolysis in the co-expressing lines compared to the parent lines (Fig. 3 a- 3 f). The enhanced xylan hydrolysis after xylanase and glucuronyl esterase digestion primarily resulted from xylan deacetylation; 35S:AXE lines exhibited a 35% increase in xylose release, while 35S:XYL lines showed a 14% increase as compared to wild type (Fig. 3 f). This indicates that deacetylation could be a key factor limiting xylan hydrolysis and suggests it may be a more effective strategy than altering the xylan backbone itself. However, the combined 35S:AXE/XYL line displayed superior saccharification efficiency compared to either parent alone, indicating that the combinatorial approach is more effective—even though esterase activity was lower in 35S:AXE/XYL than in 35S:AXE or 35S:XYL (Fig. 3 a-e). This may be because esterase activity exposes the xylan backbone, while xylanase shortens it, together weakening the matrix that links cellulose and lignin. Notably, the lignin and cellulose content in the transgenic lines remained comparable to wild type plants. As a result, the improved saccharification efficiency led to increased ethanol yields in both 35S:AXE and 35S:AXE/XYL lines compared to 35S:XYL and wild-type plants (Fig. 5 b) [14]. The enhancement in saccharification is likely result of reduced acetic acid levels; lower acetate concentrations stem from decreased acetyl ester hydrolysis during microbial processing, which is advantageous since acetate can inhibit microbial growth and reduce fermentation efficiency (Fig. 5 e) [14, 59, 60]. Additionally, the consumption of xylose during fermentation supports the conclusion that overexpressing AXE and XYL together enhances both saccharification efficiency and ethanol production (Fig. 5 d). As a result, controlled reduction in xylan acetylation presents a promising approach for enhancing the conversion of lignocellulosic biomass into bioethanol. This strategy could be applied to bioenergy crops by engineering endogenous genes that regulate xylan acetylation, thereby improving biomass digestibility and biofuel yields. Xylooligosaccharides (XOS) elicitors can activate plant immunity Biotic stress, such as pathogen infection, can cause damage to plant cell walls, leading to the release of wall molecules that function as damage-associated molecular patterns (DAMPs), which are perceived by pattern recognition receptors (PRRs) present in the cell wall vicinity to activate pattern-triggered immunity (PTI) and help to boost immunity against pathogens [39]. Plants release or synthesize these self-driven damage-associated molecular patterns in response to pathogen infection [61]. Plant immunity by damage associated molecular patterns (DAMPs) leads to changes in cell wall chemistry are perceived by cell wall-associated receptors located at the plasma membrane–cell wall interface, such as Wall-Associated Kinase 1 (WAK1), THESEUS1 (THE1), FERONIA (FER), and MIK2 [62–64]. These receptors alone or in complexes may detect perturbations in cell wall status and activate signaling pathways that modulate disease resistance, either enhancing immunity or increasing susceptibility depending on the nature of the modification. Furthermore, in planta expression of cell wall–degrading enzymes (CWDEs), such as those targeting pectin, cellulose, or hemicellulose, can mimic pathogen-induced wall damage [65]. This leads to the generation of damage-associated molecular patterns (DAMPs), which are recognized by the plant immune system and trigger defence responses. However, excessive or uncontrolled degradation may compromise wall integrity, facilitate pathogen invasion and thereby tipping the balance toward susceptibility. Recent advances highlight that cellulose derived oligosaccharides (cellodextrins) released during enzymatic hydrolysis of cellulose, function as potent DAMPs capable of inducing immune responses such as reactive oxygen species (ROS) production and transcriptional reprogramming [20, 65]. Similarly, hemicellulose-derived fragments, including mannose-rich oligosaccharides from glucomannans and other polysaccharides like xyloglucans, have been shown to act as elicitors of defense signaling, indicating that diverse wall polysaccharides contribute to DAMP-mediated immunity [39, 66]. Among all DAMPs, pectin-derived oligogalacturonides (OGs) are the most extensively characterized. These fragments are generated through the activity of cell wall–degrading enzymes such as polygalacturonases, often secreted by pathogens, and their accumulation is tightly regulated by plant-derived polygalacturonase-inhibiting proteins (PGIPs) [65, 67]. OGs with a degree of polymerization of ~ 10–15 are particularly active in triggering immune responses, including MAPK activation, ROS burst, and defense gene expression, while also modulating growth-defense trade-offs [68]. Also, xylan acetylation reduction due to ESKIMO1 impairment has been shown plant resistance to several pathogens, including P. Cucumerina . [69]. So far, fewer DAMPs from xylooligosacharides and their receptors, derived from plants have been identified [70–72]. Plant pathogens and their hosts release cell wall-degrading enzymes (CWDEs) to degrade the cell walls of opponents during interactions [73]. DAMPS are produced by xylan polysaccharides hydrolysis by CWDE, like endo-1,4-beta xylanase, which belong to the GH10 and GH11 families [74]. GH11 b-xylanase from N. pariciarum able to release structures such as XA3XX and XA2XX and perceives as Xylan -derived DAMPS [75, 76]. Here we showed, 35S:AXE/XYL expressing plants displayed accumulation of xylo-oligosaccharide in vivo or after xylanases digestion (Figure S5 & Table S4). Although, we could not detect substituted XOS in probably because low amount in apoplastic space. However, after inducing XOS by digestion with xylanase released different XOS as compared to wild type plants (Table S3 ). These XOS or DAMPs could be possibly responsible for induction in basal plant immunity and XOS induced immunity. As we found accumulation ROS in AXE/XYL plants along with increase expression of several PTI markers after 0-dpi and 3 dpi (Fig. 6 b). To supplement our findings, we performed transcriptomic overlap between AXE/XYL plants and xylobiose-treated samples, which were previously available in our lab that supports the induction of stress-protective proteins, such as the late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein (AT2G27080), suggesting enhanced cellular protection in 35S:AXE/XYL expressing plants similar to Xylobiose treatment. Multiple JAZ family members were upregulated, reflecting activation and fine-tuning of JA-mediated defense responses (Fig. 8 c). It is known that pectin-derived oligogalacturonides (OGs) can selectively influence the jasmonic acid (JA) and salicylic acid pathways, both of which are crucial for initiating immune responses in plants [77]. Similar effects were observed in the 35S:AXE/XYL line, where there was an upregulation of PR1, PEP1 RECEPTOR 1 and NON-EXPRESSOR OF PR1-LIKE 3 (NPR3) , markers for the salicylic acid pathway, along with several genes associated with JA-related pathways (Fig. 7 b, Fig. 8 c & Table 3). To further clarify the significance of these responses, future studies could systematically measure hormone levels following XOS treatment or in plants with modified xylan to gain deeper insights into how XOS induces plant immunity. Additionally, just as the active OG machinery has been shown to play a role in defense responses, the function and potential activation and inactivation of the XOS machinery should be explored in upcoming research [78]. Overall, these shared transcriptional changes strongly suggest activation of JA-dependent defense pathways, WRKY-mediated transcription regulation, and ROS signaling in both the 35S:AXE/XYL line and the xylobiose treatment, indicating coordinated priming of innate immunity. In nutshell, transgenic WP:AXE/XYL lines showed abnormal xylem development and reduced cell wall deposition. In contrast, 35S:AXE/XYL plants had no growth deformities, higher cellulose and xylan digestibility after different chemical and enzymatic treatment, and improved resistance to Pseudomonas syringae . Comparative RNA sequencing and elicitor-based assays indicated improvement in the immunity is possibly through more XOS levels in 35S:AXE/XYL plants. Overall, controlled breaking down both the xylan backbone and its acetyl groups can improve biomass quality and disease resistance. Materials and Methods Plant growth conditions and genotyping Arabidopsis Plants were grown under a 16-h light/8-h dark photoperiod at 22°C. The cloning and generation of parent plants expressing acetyl esterase and xylanases were described in our previous reports [15, 79]. Acetyl xylan esterase (AXE) and Xylanases (XYL) expressing homozygous parents and a hemizygous cross were genotyped using PCWL-33, PCWL-34, and PCWL-49, PCWL-50 primers, respectively (Supplementary file 1). Phloroglucinol-HCl (Wiesner) Staining Six-week-old inflorescence stem sections were stained with 3% phenol-HCl (P3502-25G, Sigma-Aldrich, USA)[80] and imaged by a Nikon fluorescence microscope under 10X and 40X magnification. Total RNA isolation and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated from the six-week-old side stem using TRIzol (15596026, Thermo Fisher Scientific Invitrogen) and treated with RNase-free DNase I (EN0521, Thermo Fisher Scientific Invitrogen). cDNA was synthesized from 1 µg DNase-treated RNA by iScript™ cDNA Synthesis kit (1708891, Bio-Rad, USA) used for RT-qPCR, performed on Quantstudio-6 Flex Real-time machine for the gene of interest and reference gene using HOT FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne, 08240001). The relative fold change was calculated by the ΔΔCt method. Protein extraction and enzyme activities Six weeks of side stem tissue was ground in liquid nitrogen to a fine powder and stirred for 1 h at 4°C in buffer A (50 mM sodium phosphate buffer, pH 6.8, containing 2.5 mM EDTA, 2% PVP, and 1 mM DTT). The supernatant was collected as a soluble protein fraction by centrifuging at 10000 rpm for 10 min. The pellet was resuspended in buffer A with 1 M NaCl, stirred for 1 h, and collected as a wall-bound fraction [81]. The protein concentration was determined by the Bradford assay and used to assess esterase and xylanase activity. Esterase activity with acetylated xylan as a substrate Esterase activity was performed with partially acetylated xylan (P-ACXYL, Megazyme, Ireland) as substrate, incubated at 37°C for 6 h, and the released acetic acid was quantified by the acetic acid kit (K-ACET, Megazyme, Ireland) [82]. Xylanase activity Xylanase activity was performed using the Xylanase Assay kit (K-XylX6-2V, Megazyme, Ireland) by incubating the XylX6 substrate at 40°C for 10 min. The released 4-nitrophenol was quantified at 400 nm, and the specific xylanase activity was calculated in nmol min −1 mg − 1 of total protein using the 4-nitrophenol release by Trichoderma sp. endo-1,4-β xylanase standard curve. Alcohol insoluble residue (AIR) Preparation for cell wall analysis 10 cm inflorescence stems of completely were dried, and homogeneous fine powder was prepared using Qiagen TissueLyserII for AIR preparation. The stem powder was stirred with 4 mM HEPES in 70% ethanol for 1 h at 70°C. After treatment, the sample was sequentially washed with chloroform: methanol (1:1) and acetone. The final pellet was dried overnight in a desiccator, and the resulting alcohol-insoluble residue (AIR) was used for analysis of plant cell wall composition. Acetyl content 1 mg of the AIR sample was incubated with 1 M NaOH for 1 h, then neutralized with 1 M HCl. The final volume was made up to 1 ml with MilliQ water and centrifuged at 1500g for 10 min. The supernatant was analyzed using an acetic acid kit (K-ACET, Megazyme, Ireland). Xylose content 2 mg of the AIR sample was incubated with 100 µl of 1.3 M HCL at 100°C for 1 h. Then, the sample was neutralized with 100 µL of 1.3 M NaOH, the final volume was adjusted to 1 mL with MilliQ water, and the mixture was centrifuged at 1500g for 10 min. The supernatant was quantified by a D-Xylose assay kit (K-XYLOSE, Megazyme, Ireland). Total carbohydrate (phenol sulfuric method) 100 µl of AIR suspension prepared from 0.5 mg/ml AIR was treated with 100 µl of 5% (v/v) phenol and 500 µl concentrated sulfuric acid, vortexed thoroughly, and incubated for 20 min. The total sugar concentration was estimated using a glucose standard curve by reading absorbance at 490 nm. Cellulose content 3 mg of AIR was treated with Updegraff reagent (acetic acid, nitric acid, and water in an 8:1:2 ratio, respectively) and heated for 30 min at 100°C. The tube centrifuge was then cooled to maximum rpm for 10 min, and the pellet was washed with water, then with acetone. The pellet was dried overnight in the desiccator. A pellet was used to determine glucose content by Anthrone reagent (0.2% Anthrone in 92% sulphuric acid) [83]. Acetyl Bromide Soluble Lignin (ABSL) AIR samples were incubated at 50 C for 2 h with freshly prepared 25% acetyl bromide (135968-500G, Sigma) in glacial acetic acid. The supernatant was diluted with 2 M NaOH and 0.5 M hydroxylamine hydrochloride (159417-100G) freshly prepared. The absorbance was measured at 280 nm, and lignin content was expressed as a percentage of AIR [84]. Hemicellulosic monosaccharide composition analysis by GC-MS 2 mg AIR sample was hydrolyzed by incubation at 121°C for 90 min with 2 M trifluoroacetic acid (TFA) (76-05-1, SRL). The supernatant was collected in a glass tube, evaporated under a stream of nitrogen gas, and washed three times with isopropanol. Then, the dried fraction was dissolved in methoxyamine hydrochloride in pyridine and incubated for 90 minutes at 37°C. These fractions were then derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) for 30 min at 37°C, and GC-MS analyzed 100 µL of the derivatized samples. Inositol was used as an internal standard for normalization [18]. Determination of xylo-oligosaccharides using mass spectroscopy 3 mg of stem tissue AIR was digested with GH11 Endo-1,4-β-Xylanase (1U/mg of AIR-1) (13814, SRL). The resulting hydrolysate was purified with Hypersep Hypercarb Porous Graphitized Carbon (PGC) columns and separated into neutral and acidic fractions using 50% acetonitrile and 50% acetonitrile containing 0.05% of trifluoroacetic acid, respectively. Eluents were freeze-dried and resuspended in 30 µl HPLC-grade water; the resuspended sample was mixed with DHB in a 1:1 ratio and applied to a 384-well metal target plate (Opti-TOP LC/MALDI insert (123 x 81 mm) part no. 1018497) and air-dried. The data were acquired over the m/z range of 300–1500. Soluble sugar isolation and profiling Fresh stem and leaf were homogenized in liquid nitrogen and treated with 1 ml of 80% ethanol for 1 h at 80°C. The supernatant was centrifuged, vacuum-dried, and dissolved in 300 µL of Milli-Q water. Soluble sugars were normalized to total sugar and run on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Glycan antibody profiling for cell wall extractability Different fractions of cell wall components were isolated through sequential extraction using 50 mM ammonium oxalate, 50 mM sodium carbonate, 1 M KOH, and 4 M KOH. All wall extracts were loaded (50 µL of 500 ng/mL) into 96-well ELISA plates (Coster 3599, Corning Life Science, Acton, MA) and incubated for 12 h at 37°C. Nonspecific sites were blocked by incubating with blocking buffer at room temperature, followed by aspiration of blocking buffer and incubation with 50 µl of primary antibody (1:50) and secondary antibody (1:5000) for 1 h at room temperature (Table S6). The plate was washed with 300 µl wash buffer in between transition for primary antibody to the secondary antibody. After that 50 µl of 3,3,5,5-Tetramethylbenzidine (TMB) (860336-1G, Sigma-Aldrich, India) substrate solution was prepared in 0.1M citrate-acetate buffer, added into each well and incubated for 5 min at 37°C. The reaction was stopped by adding 0.5 N sulfuric acid, measuring net OD values of color formation at 450 nm, and subtracting the background reading at 655 nm [85]. Saccharification analysis Xylanase pretreatment The AIR sample was digested with GH11 Endo-1,4-β-Xylanase (1U/mg of AIR-1) (13814, SRL) at 60°C for 6 h, and centrifuged at 1500 g for 10 min. The supernatant was used for the quantified D-Xylose assay kit (K-XYLOSE, Megazyme, Ireland); the digested pellet was washed with acetone, dried, and used for further for saccharification assays. Chemical pretreatment AIR samples were treated with water and 0.4N NaOH at 90°C for 30 minutes [14]. Then it was centrifuged, washed with water and acetone, dried overnight, and used for glucose release. Glucose release estimation Pre-treated and untreated AIR samples were incubated with Cellulase, enzyme blend – Cellic CTec2 (0.1 U for 1 mg AIR-1) in 0.1 M Sodium acetate buffer (pH- 4.8) at 50°C for 24 h, then centrifuged, and glucose was estimated in the supernatant by D-Glucose Assay kit (GOPOD Format, K-GLUC) [86]. Xylanase digestibility with glucuronyl esterase The AIR sample was incubated with GH11 Endo-1,4-β-Xylanase (1U/mg of AIR), supplemented with glucuronyl esterase (1U/mg of AIR) in sodium acetate buffer (pH 4.5) at 50°C for 4 h, and centrifuged at 1500 g for 10 min. The supernatant was used for the quantified D-Xylose assay kit (K-XYLOSE, Megazyme, Ireland). Fermentation analysis Autoclaved 50 mg sample digested with 10% (g/g) Cellic CTec2 at 50 C for 6 h. The digested samples were used as glucose sources, with 1 mL of glucose (20 g/L) added to a final reaction volume of 20 mL. Autoclaved LB broth was used in fermentation jars, which were then sparged with nitrogen gas to create an anaerobic environment. 1.3 ml of E. coli culture was added to the media; 1 ml of the culture was taken at 0 h, and the jars were incubated at 37°C for 24 h. After 24 h, samples were checked for Ethanol using the Megazyme kit (K - ETOH, Megazyme, Ireland). Pseudomonas infection assay Four-week-old Arabidopsis rosette leaves were infiltrated with Pseudomonas syringae DC 3000 pv. tomato (pstDC3000) suspension in 10 mM MgCl 2 at concentration of 5 × 10 4 by needleless syringe. Bacterial infection was measured at 0 and 3 dpi by harvesting 5 mm leaf discs, macerating them in 10 mM MgCl2, and inoculating their serially diluted suspension onto a Pseudomonas agar plate with antibiotic selection. Bacterial infection was estimated through the number of colonies and expressed as Log 10 CFU cm − 2 [87]. RNA sequencing analyses RNA was isolated from the 6-week side stem of the old side stem using TRIzol (15596026, Thermo Fisher Scientific Invitrogen), and RNA sequencing was done on the Illumina Novaseq 6000 Platform. Significantly differentially expressed genes were analyzed by the DSEq2 tool with Log2Fold change1, p-value < 0.05. Gene ontology analysis was done using ShinyGO 0.77 ( http://bioinformatics.sdstate.edu/go/ ). Xylo oligosaccharides infiltration and estimation of reactive oxygen species (ROS) Four-week-old Arabidopsis rosette leaves were infiltrated with supernatant from 200 µg/ml xylanase (GH11)- digested stem samples, calibrated to total sugar 200 µg/ml. Treated leaves were collected at 30 min, checked for ROS accumulation using the diaminobenzidine (DAB) assay, and checked for the following genes (Table S5). Briefly, mock- and treated-leaves were incubated in DAB (17076, SRL, India) staining solution (1 mg/ml in 10 mM Na 2 HPO 4 ) for 8 h. After that, the DAB staining solution was replaced with a bleaching solution (ethanol: acetic acid: glycerol, 3:1:1), and the sample was placed in boiling water for 15 min. Then, the old bleaching solution was replaced by a fresh bleaching solution, and DAB accumulation was quantified using Fiji ( https://fiji.sc/ ) [88]. Declarations Ethics approval and consent to participate Not applicable Consent for publication All authors gave consent to publish the work Availability of data and materials All the data are provided in the manuscript, and we have deposited transcriptomic data on IBDC webserver (https://ibdc.dbt.gov.in/) with accession ID - INRP000631. Funding We would like to thank Regional Centre for Biotechnology and DST-INSPIRE faculty for funding Authors contributions AMS performed, most of the experiments compiled and analyzed all the data. BPD and RKS performed total sugar and xylose content analysis respectively. NA helped in fermentation analysis and interpretation. EM provided xylanases expressing lines, involved discussion and writing of the manuscript. AMS and PA-MP wrote the manuscript with input from all the authors. PA-MP conceptualized, designed and secured the funding for the project. All authors read and agree to publish the manuscript. Acknowledgment We would like to thank National Institute of Plant Genome Research, New Delhi for Metabolomic Facility, Regional Centre for Biotechnology, Faridabad and Advanced Technology Platform Centre, Faridabad for genomics and mass spectroscopy facility. Conflict of Interest Authors declare no conflict of interest. References Chebli, Y. and A. Geitmann, Cellular growth in plants requires regulation of cell wall biochemistry , in Current Opinion in Cell Biology . 2017, Elsevier Ltd. p. 28-35. Bellincampi, D., F. Cervone, and V. Lionetti, Plant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions , in Frontiers in Plant Science . 2014, Frontiers Media S.A. Timell, T.E., Recent progress in the chemistry of wood hemicelluloses. Wood Science and Technology March 1967. Volume 1 : p. 45–70. Scheller, H.V. and P. Ulvskov, Hemicelluloses. Annual Review of Plant Biology, 2010. 61 : p. 263-289. Aspinall, G.O., Chemistry of Cell Wall Polysaccharides , in Carbohydrates: Structure and Function . 1980, Elsevier. p. 473-500. Gao, Y., et al., A grass-specific cellulose–xylan interaction dominates in sorghum secondary cell walls. Nature Communications, 2020. 11 (1). Saijonkari-Pahkala, K., Non-wood plants as raw material for pulp and paper . 2001. Rennie, E.A. and H.V. Scheller, Xylan biosynthesis , in Current Opinion in Biotechnology . 2014. p. 100-107. Peña, M.J., et al., Arabidopsis irregular xylem8 and irregular xylem9: Implications for the complexity of glucuronoxylan biosynthesis. Plant Cell, 2007. 19 (2): p. 549-563. Giummarella, N. and M. Lawoko, Structural Basis for the Formation and Regulation of Lignin-Xylan Bonds in Birch. ACS Sustainable Chemistry and Engineering, 2016. 4 (10): p. 5319-5326. Grantham, N.J., et al., An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls. Nature Plants, 2017. 3 (11): p. 859-865. Singh, D., et al., Characterization of Arabidopsis eskimo1 reveals a metabolic link between xylan O-acetylation and aliphatic glucosinolate metabolism. Physiologia Plantarum, 2024. 176 (6). Xiong, G., K. Cheng, and M. Pauly, Xylan O-acetylation impacts xylem development and enzymatic recalcitrance as indicated by the arabidopsis mutant tbl29 , in Molecular Plant . 2013, Oxford University Press. p. 1373-1375. Pawar, P.M.A., et al., Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose. Plant Biotechnology Journal, 2016. 14 (1): p. 387-397. Chaudhari, A.A., et al., Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility. Biotechnology for Biofuels and Bioproducts, 2024. 17 (1). Pogorelko, G., et al., Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens. Plant Physiology, 2013. 162 (1): p. 9-23. Sun, G., et al., Oligosaccharide elicitors in plant immunity: Molecular mechanisms and disease resistance strategies. Plant Communications, 2025. 6 (12): p. 101469. Dewangan, B.P., et al., Xylobiose treatment triggers a defense-related response and alters cell wall composition. Plant Molecular Biology, 2023. Melida, H., et al., Arabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating Disease Resistance. Front Plant Sci, 2020. 11 : p. 1210. de Azevedo Souza, C., et al., Cellulose-derived oligomers act as damage-associated molecular patterns and trigger defense-like responses. Plant Physiology, 2017. 173 (4): p. 2383-2398. Rzemieniewski, J. and M. Stegmann, Regulation of pattern-triggered immunity and growth by phytocytokines , in Current Opinion in Plant Biology . 2022, Elsevier Ltd. Wang, H., et al., Transcriptomic Insights into the Degree of Polymerization-Dependent Bioactivity of Xylo-Oligosaccharides. Plants, 2025. 14 (19). Zhao, Q. and R.A. Dixon, Altering the cell wall and its impact on plant disease: From forage to bioenergy. Annual Review of Phytopathology, 2014. 52 : p. 69-91. Sivan, P., et al., Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification. Plant Biotechnology Journal, 2025. 23 (1): p. 174-197. Swaminathan, S., et al., Coexpression of fungal cell wall-modifying enzymes reveals their additive impact on arabidopsis resistance to the fungal pathogen, Botrytis cinerea. Biology, 2021. 10 (10). Barbut, F.R., et al., Integrity of xylan backbone affects plant responses to drought. Frontiers in Plant Science, 2024. 15 . Donev, E.N., et al., Glucuronoyl Esterase of Pathogenic Phanerochaete carnosa Induces Immune Responses in Aspen Independently of Its Enzymatic Activity. Plant Biotechnology Journal, 2025. Marasinghe, S.D., et al., Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass. Microbial Cell Factories, 2021. 20 (1). Várnai, A., et al., Effects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions. Biotechnology for Biofuels, 2014. 7 (1). Zhang, J., et al., The role of acetyl xylan esterase in the solubilization of xylan and enzymatic hydrolysis of wheat straw and giant reed. Biotechnology for Biofuels, 2011. 4 . Urbancsok, J., et al., Wood-specific modification of glucuronoxylan can enhance growth in Populus. Journal of Experimental Botany, 2026. 77 (2): p. 445-462. Ratke, C., et al., Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification. Plant Biotechnology Journal, 2015. 13 (1): p. 26-37. Chini, A., et al., The JAZ family of repressors is the missing link in jasmonate signalling. Nature, 2007. 448 (7154): p. 666-671. Hoo, S.C., et al., Regulation and function of arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory. Plant Physiology, 2008. 146 (3): p. 952-964. Thines, B., et al., JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature, 2007. 448 (7154): p. 661-665. Pandey, S.P. and I.E. Somssich, The role of WRKY transcription factors in plant immunity. Plant Physiology, 2009. 150 (4): p. 1648-1655. Seo, P.J. and C.M. Park, MYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis. New Phytologist, 2010. 186 (2): p. 471-483. Xu, X., et al., Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell, 2006. 18 (5): p. 1310-1326. Molina, A., et al., Plant cell walls: source of carbohydrate-based signals in plant-pathogen interactions , in Current Opinion in Plant Biology . 2024, Elsevier Ltd. Zang, H., et al., Mannan oligosaccharides trigger multiple defence responses in rice and tobacco as a novel danger-associated molecular pattern. Molecular Plant Pathology, 2019. 20 (8): p. 1067-1079. Busse-Wicher, M., et al., The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana. Plant Journal, 2014. 79 (3): p. 492-506. Kang, X., et al., Lignin-polysaccharide interactions in plant secondary cell walls revealed by solid-state NMR. Nature Communications, 2019. 10 (1). Simmons, T.J., et al., Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nature Communications, 2016. 7 . Yuan, Y., et al., Roles of Arabidopsis TBL34 and TBL35 in xylan acetylation and plant growth. Plant Science, 2016. 243 : p. 120-130. Yuan, Y., et al., The Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan. Plant and Cell Physiology, 2013. 54 (7): p. 1186-1199. Zhu, X.F., et al., TRICHOME BIREFRINGENCE-LIKE27 Affects Aluminum Sensitivity by Modulating the O-Acetylation of Xyloglucan and Aluminum-Binding Capacity in Arabidopsis Plant Physiology, 2014. 166 (1): p. 181-189. Zhong, R., D. Cui, and Z.-H. Ye, Regiospecific Acetylation of Xylan is Mediated by a Group of DUF231-Containing O-Acetyltransferases. Plant and Cell Physiology, 2017. 58 (12): p. 2126-2138. Yuan, Y., et al., TBL3 and TBL31, Two Arabidopsis DUF231 Domain Proteins, are Required for 3- O -Monoacetylation of Xylan. Plant and Cell Physiology, 2016. 57 (1): p. 35-45. Xiong, G., K. Cheng, and M. Pauly, Xylan O-Acetylation Impacts Xylem Development and Enzymatic Recalcitrance as Indicated by the Arabidopsis Mutant tbl29. Molecular Plant, 2013. 6 (4): p. 1373-1375. Morcillo, R.J.L., et al. Plant Transcriptome Reprograming and Bacterial Extracellular Metabolites Underlying Tomato Drought Resistance Triggered by a Beneficial Soil Bacteria . Metabolites, 2021. 11 , 369 DOI: 10.3390/metabo11060369. Urbanowicz, B.R., et al., 4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109 (35): p. 14253-14258. Vanholme, R., et al., Lignin biosynthesis and structure. Plant Physiology, 2010. 153 (3): p. 895-905. Boerjan, W., J. Ralph, and M. Baucher, Lignin Biosynthesis , in Annual Review of Plant Biology . 2003. p. 519-546. Kushwah, S., et al., Arabidopsis XTH4 and XTH9 contribute to wood cell expansion and secondary wall formation1[Open]. Plant Physiology, 2020. 182 (4): p. 1946-1965. Rose, J.K.C., et al., The XTH Family of Enzymes Involved in Xyloglucan Endotransglucosylation and Endohydrolysis: Current Perspectives and a New Unifying Nomenclature , in Plant Cell Physiol . 2002. p. 1421-1435. Zhong, R. and Z.H. Ye, Secondary cell walls: Biosynthesis, patterned deposition and transcriptional regulation. Plant and Cell Physiology, 2015. 56 (2): p. 195-214. Hamann, T., The plant cell wall integrity maintenance mechanism - Concepts for organization and mode of action , in Plant and Cell Physiology . 2015, Oxford University Press. p. 215-223. Hamann, T., The plant cell wall integrity maintenance mechanism - A case study of a cell wall plasma membrane signaling network , in Phytochemistry . 2015, Elsevier Ltd. p. 100-109. Jönsson, L.J., B. Alriksson, and N.O. Nilvebrant, Bioconversion of lignocellulose: Inhibitors and detoxification , in Biotechnology for Biofuels . 2013. Pawar, P.M.A., et al., Acetylation of woody lignocellulose: Significance and regulation , in Frontiers in Plant Science . 2013, Frontiers Research Foundation. De Lorenzo, G. and F. Cervone, Plant immunity by damage-associated molecular patterns (DAMPs). Essays in Biochemistry, 2022. 66 (5): p. 459-469. Hématy, K., et al., A Receptor-like Kinase Mediates the Response of Arabidopsis Cells to the Inhibition of Cellulose Synthesis. Current Biology, 2007. 17 (11): p. 922-931. Kohorn, B.D. and S.L. Kohorn, The cell wall-associated kinases, WAKs, as pectin receptors , in Frontiers in Plant Science . 2012, Frontiers Research Foundation. Van der Does, D., et al., The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses. PLoS Genetics, 2017. 13 (6). Pontiggia, D., et al., Dampening the DAMPs: How Plants Maintain the Homeostasis of Cell Wall Molecular Patterns and Avoid Hyper-Immunity , in Frontiers in Plant Science . 2020, Frontiers Media S.A. Claverie, J., et al., The cell wall-derived xyloglucan is a new DAMP triggering plant immunity in vitis vinifera and arabidopsis thaliana. Frontiers in Plant Science, 2018. 871 . Ferrari, S., et al., Oligogalacturonides: Plant damage-associated molecular patterns and regulators of growth and development , in Frontiers in Plant Science . 2013, Frontiers Research Foundation. Degli Esposti, C., et al., Cell wall bricks of defence: the case study of oligogalacturonides , in Frontiers in Plant Science . 2025, Frontiers Media SA. Escudero, V., et al., Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein. Plant Journal, 2017. 92 (3): p. 386-399. Choi, H.W. and D.F. Klessig, DAMPs, MAMPs, and NAMPs in plant innate immunity , in BMC Plant Biology . 2016, BioMed Central Ltd. Duran-Flores, D. and M. Heil, Sources of specificity in plant damaged-self recognition , in Current Opinion in Plant Biology . 2016, Elsevier Ltd. p. 77-87. Molina, A., et al., Arabidopsis cell wall composition determines disease resistance specificity and fitness. 2021. 118 (5). Rovenich, H., A. Zuccaro, and B.P.H.J. Thomma, Convergent evolution of filamentous microbes towards evasion of glycan-triggered immunity , in New Phytologist . 2016, Blackwell Publishing Ltd. p. 896-901. McCleary, B.V., et al., Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-l-arabinofuranosidase and β-xylosidase. Carbohydrate Research, 2015. 407 : p. 79-96. Mélida, H., et al., Arabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating Disease Resistance. Frontiers in Plant Science, 2020. 11 . Vardakou, M., et al., Understanding the Structural Basis for Substrate and Inhibitor Recognition in Eukaryotic GH11 Xylanases. Journal of Molecular Biology, 2008. 375 (5): p. 1293-1305. Degli Esposti, C., et al., Cell wall bricks of defence: the case study of oligogalacturonides. Frontiers in Plant Science, 2025. Volume 16 - 2025 . Salvati, A., et al., Berberine bridge enzyme-like oxidases orchestrate homeostasis and signaling of oligogalacturonides in defense and upon mechanical damage. The Plant Journal, 2025. 122 (1): p. e70150. Rastogi, L., et al., Arabidopsis GELP7 functions as a plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency. Plant Molecular Biology, 2022. 109 (6): p. 781-797. Pradhan Mitra, P. and D. Loqué, Histochemical staining of Arabidopsis thaliana secondary cell wall elements. Journal of Visualized Experiments, 2014(87). Biswal, A.K., et al., Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield. Biotechnology for Biofuels, 2014. 7 (1). Rastogi, L., et al., Arabidopsis At GELP53 modulates polysaccharide acetylation and defense response through oligosaccharide-mediated signaling . 2024. Updegraff, D.M., Semimicro Determination of Cellulose in Biological Materials . 1969. p. 420-424. Foster, C.E., T.M. Martin, and M. Pauly, Comprehensive compositional analysis of plant cell walls (Lignocellulosic biomass) part I: Lignin. Journal of Visualized Experiments, 2010(37). Pattathil, S., et al., Immunological approaches to plant cell wall and biomass characterization: Glycome profiling. Methods in Molecular Biology, 2012. 908 : p. 61-72. Acker, R.V., et al., Saccharification Protocol for Small-scale Lignocellulosic Biomass Samples to Test Processing of Cellulose into Glucose , in Iss . 1701. Roy, A., et al., Kappaphycus alvarezii-derived formulations enhance salicylic acid-mediated anti-bacterial defenses in Arabidopsis thaliana and rice. Journal of Applied Phycology, 2022. 34 (1): p. 679-695. Daudi, A., J.A. O'Brien, and B.P. Author, Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves HHS Public Access Author manuscript , in Bio Protoc . 2012. Tables Table-1. Non-cellulosic Monosaccharide composition (mol %) through derivatization followed by Gas Chromatography-Mass Spectrometry. Data presented as mean ± standard deviation (SD) from 3 biological replicates. Asterisk represents significant differences using Student’s t-test at *p ≤ 0.1, ** p ≤ 0.05, ***p ≤ 0.001. xylose arabinose rhamnose fucose mannose glucose WT 46.84 ± 0.8 10.28 ± 0.47 4.61 ± 0.31 2.62 ± 0.2 12.66 ± 0.16 6.51 ± 0.65 35S:AXE 50.42 ± 0.47 * 9.01 ± 0.15 * 4.07 ± 0.08 * 2.2 ± 0.05 * 11.92 ± 0.07 * 5.87 ± 0.12 35S:XYL 54.88 ± 1.87 * 9.01 ± 0.53 * 3.79 ± 0.11 * 2.21 ± 0.13 * 10.74 ± 0.35 * 6.09 ± 0.25 35S:AXE/XYL 49.12 ± 0.39 9.3 ± 0.13 * 3.81 ± 0.29 * 2.19 ± 0.14 * 9.9 ± 0.28 * 6.23 ± 0.23 WP:AXE 55.99 ± 2.09 * 6.87 ± 0.89 * 2.64 ± 1.37 * 2.81 ± 0.2 9.59 ± 0.86 * 6.7 ± 1.03 WP:XYL 44.83 ± 2.45 9.57 ± 0.66 5.53 ± 2.57 3.39 ± 0.17 * 12.18 ± 1.57 9.14 ± 1.14 * WP:AXE/XYL 34.5 ± 0.9 *** 14.58 ± 2 * 10.27 ± 2.67 * 5.21 ± 0.24 *** 15.21 ± 0.41 * 10.25 ± 0.28 ** Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiguresBMC31032026.pptx Supplementary Figure 1. Schematics of construct, genotyping, gene expression and anatomical studies Supplementary Figure 2. Morphology, gene expression, enzyme activity, and anatomical analysis Supplementary Figure 3. Plant cell wall composition on AIR samples prepared from Arabidopsis inflorescence stem. Supplementary Figure 4. RNA sequencing analysis of WT and WP:AXE/XYL plants. Supplementary Figure 5. Xylo-oligosaccharide analysis by MALDI-TOF on AIR of Arabidopsis inflorescence stem through xylanase (GH11) digestion. Supplementary Figure 6. Picture of Arabidopsis leaf after DAB staining. SupplementryData1BMC31032026.xlsx Supplementary File SupplemntaryTableBMC310032026.docx Supplementary Table 1. Differentially expressed cell wall-related processes genes in 35S:AXE/XYL. Supplementary Table 2. Differentially expressed defense-related genes in 35S:AXE/XYL. Supplementary Table 3. Xylooligosaccharide-profiling after digestion with xylanase (GH11) digestion and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Supplementary Table 4. Xylooligosaccharide -profiling (ug/ml) in alcohol soluble fraction and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Supplementary Table 5. Primer used for Genotyping and RT-qPCR. Supplementary Table 6.Antibodies Used for Glycome Profiling. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 11 May, 2026 Reviews received at journal 08 May, 2026 Reviews received at journal 03 May, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers invited by journal 09 Apr, 2026 Editor assigned by journal 09 Apr, 2026 Submission checks completed at journal 09 Apr, 2026 First submitted to journal 09 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9277431","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620971750,"identity":"f4ca62ac-13da-48a2-aa44-56abd515ebf8","order_by":0,"name":"Anant Mohan Sharma","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Anant","middleName":"Mohan","lastName":"Sharma","suffix":""},{"id":620971751,"identity":"6f127e26-216a-4a0d-9a87-6deb335139be","order_by":1,"name":"Bhagwat Prasad Dewangan","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Bhagwat","middleName":"Prasad","lastName":"Dewangan","suffix":""},{"id":620971752,"identity":"c28336a0-245d-40d5-a1bb-b9e320526b29","order_by":2,"name":"Aniket Chaudhari","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Aniket","middleName":"","lastName":"Chaudhari","suffix":""},{"id":620971753,"identity":"256094a5-1a6b-495f-9cf9-28a21501b408","order_by":3,"name":"Rajan Kumar Sah","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Rajan","middleName":"Kumar","lastName":"Sah","suffix":""},{"id":620971754,"identity":"52b4b78a-860f-4b6c-bedf-83593941023f","order_by":4,"name":"Nidhi Adlakha","email":"","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Nidhi","middleName":"","lastName":"Adlakha","suffix":""},{"id":620971755,"identity":"00ef87eb-0a52-42a6-8331-061c3b3d932e","order_by":5,"name":"Ewa Mellerowicz","email":"","orcid":"","institution":"Swedish University of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Mellerowicz","suffix":""},{"id":620971756,"identity":"cad60226-991a-43c5-bd6b-388f9e4dc6f9","order_by":6,"name":"Prashant Anupama-Mohan Pawar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYJACZiS2DQODBITFQ0gLVB1DGulaDiOYuIC59OHHnwtq7tQxSDc/k/i553w+/+wGtg8f/jDImOPQYtmXZiY949gzCQaZY2aSPc9uW864c4B55sw2Bh7LBuxaDM4wmDHzsB2WYJBIMLvBc+C2gYFEAjMzbwMDj8EBXFrYP3/m+QfSkv7t5p8D5yBa/vzBp4XHQJq3DaQlx+w2z4EDEC0MbLi1WPbwlEnz9h2WbJM5U/5b5kCygcSdg82MvW0SOLWY87Bv/szz7TA/v3T7ZsM3B+wM+Gc3H2b48cfGHqfDYAw2RHQwNjDgix24FoIxOApGwSgYBSMXAABT6lLMFtuXlQAAAABJRU5ErkJggg==","orcid":"","institution":"Regional Centre for Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Prashant","middleName":"Anupama-Mohan","lastName":"Pawar","suffix":""}],"badges":[],"createdAt":"2026-03-31 09:10:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9277431/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9277431/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107179068,"identity":"8c8a2b02-a698-44d7-9739-8333771c8f32","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology, gene expression, and enzyme activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Relative gene expression levels in inflorescence stem tissue of \u003cem\u003eAXE\u003c/em\u003e were calculated by normalizing to the 35S:AXE/XYL (b) representative image illustrating the morphology of a six-week-old plant expressing acetyl xylan esterase (AXE) and xylanases (XYL) under the 35S promoter with WT. Bar = 5 cm. \u003cem\u003eActin\u003c/em\u003ewas used as a reference gene (c) esterase activity was measured on an acetylated Birchwood xylan substrate, and the released acetic acid was quantified using the Megazyme K-ACET kit (d) relative gene expression levels in the inflorescence stem tissue of XYL were calculated by normalizing to the 35S:AXE/XYL. \u003cem\u003eActin\u003c/em\u003e was used as a reference gene (e) xylanase activity was assessed using XylX6 as a substrate (from Xylanase Assay kit K-XylX6-2V, Megazyme, Ireland), and the 4-nitrophenol released was measured at 400 nm. Data presented as mean ± standard deviation (SD) from 2-4 biological replicates. Statistical significance determined by Student’s t-test at ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. nd – not detected.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/a3c51e8ec22d49b2b915eff9.png"},{"id":107483142,"identity":"5322f1ad-e745-4afd-8ba5-802721ae718b","added_by":"auto","created_at":"2026-04-22 02:26:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlant cell wall composition on AIR samples prepared from Arabidopsis inflorescence stem\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) AIR was saponified using NaOH and acetic acid released was quantified by K-ACET kit (b) xylose content was hydrolyzed by 1.3M HCl Acid and quantified using K-Xylose kit (c) acetic acid and xylose content was used to calculate acetyl to xylose ratio (d) total carbohydrate content estimated by Phenol-Sulphuric acid method (e) cellulose content by Updegraff’s method (f) lignin content by acetyl bromide soluble lignin method. Data presented as mean ± standard deviation (SD) from 3-4 biological replicates. Statistical significance determined by Student's t-test at ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/39dbd3b812bda57b05980315.png"},{"id":107179069,"identity":"c5075690-ad3b-49a4-b685-1767ce93185f","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSaccharification on the AIR of the Arabidopsis inflorescence stem\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSaccharification by using the CTec2 commercial enzyme (a) without any pretreatment (b) hot water pretreatment (c) 0.4M NaOH pretreatment (d) and xylanase (GH11) pretreatment. The glucose content was estimated through Megazyme GOD-POD assay Megazyme kit (K-GLUC) (e) xylose release after xylanase (GH11) digestion (f) xylose release after xylanase (GH11) and glucuronyl esterase digestion, respectively measured using Megazyme K-Xylose kit. Data presented as mean ± standard deviation (SD) from 3-4 biological replicates. Statistical significance determined by Student's t-test at ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. % difference representation is compared to wild-type plants.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/ca61a27e869d85784aed68ad.png"},{"id":107179071,"identity":"36e97936-5268-4069-8c2b-1c07e5f3053b","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlycome Profile of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inflorescence stem cell walls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequential extracts of cell walls from the inflorescence stem of 8-week-old Arabidopsis thaliana plants were prepared from AIR and probed by ELISA with an array of cell wall glycan-directed mAbs shown in the bottom panel. [LM10 (Heteroxylan), LM11 (arabinoxylan and xylan), LM15 (Xyloglucan), LM28 (Glucuronoxylan), CCRC-M7 (Rhamnogalacturonan-I / Arabinogalactan)].\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/3620c9e965312eeb3e302ec2.png"},{"id":107179080,"identity":"74e100a4-4fb2-45f1-b536-881e635f7778","added_by":"auto","created_at":"2026-04-17 16:35:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFermentation analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Glucose release at the start of inoculation measured by D-Glucose Assay K-GLUC Megazyme kit (b) ethanol produced after 24 h in mg/ml by K-ETOH, Megazyme kit (c) Ethanol produced after 24 h (in gram per gram of glucose) (d) change in acetic acid at 0 and 24 h by using K-ACET, Megazyme kit (e) change in Xylose at 0 and 24 h by K-XYLOSE, Megazyme kit. Data represent mean ± SD, n = 3 technical replicates, Student’s t-test at ***p ≤ 0.001 **p ≤ 0.01, *p ≤ 0.05.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/3efd9e1dc5c7ef389d7c3847.png"},{"id":107179072,"identity":"92fb69ce-c571-49ab-9269-6aef240579d8","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunity assays against \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePseudomonas syringae\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(a) Quantification of bacterial growth using leaf infiltration method using \u003cem\u003ePseudomonas syringae DC 3000 pv. tomato (pstDC3000)\u003c/em\u003e infiltrated at bacterial density 5 *10\u003csup\u003e4\u003c/sup\u003e CFU/ml with respect to the wild infiltrated leaves. (b) the expression of key genes involved in perception, defense, and cell wall remodeling by RT-qPCR. \u003cem\u003eACTIN \u003c/em\u003ewas used as reference gene. Data represents SE, n = 4 replicates, Student’s t-test at ***p ≤ 0.001 **p ≤ 0.01, *p ≤ 0.05 applied between genotypes.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/f5e8b266018d56435ac40a6d.png"},{"id":107179076,"identity":"50eda488-5245-4e62-a0b1-60162791377d","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":88876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA sequencing analysis in 35S:AXE/XYL plants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) RNA-seq analysis was performed on six-week-old stems of WT (as control) and 35S:AXE/XYL (b) Volcano plot shows the distribution of gene expression between WT and WP:AXE/XYL. The Y-axis illustrates −log\u003csub\u003e10\u003c/sub\u003e p-values (FDR), and the X-axis corresponds to a log\u003csub\u003e2 \u003c/sub\u003efold gene expression. N represents the number of genes significantly upregulated and downregulated. (c) Top 10 upregulated GO term analysis.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/bf162b4be364169b432a9f9f.png"},{"id":107179074,"identity":"3e290ec6-685c-463b-a334-2eb0d60f315c","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":120180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative RNA-seq analysis on 35S:AXE/XYL plants and 30 min xylobiose-treated Arabidopsis four-week-old rosette leaves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Venn diagram represents the common up- and down-regulated genes generated using Venny 2.1 https://bioinfogp.cnb.csic.es/tools/venny/index2.0.2.html. (b) Heat map represents the expression pattern of common genes. (c) Differentially expressed defense-related genes in 35S:AXE/XYL common to 30_min_XB-treated Arabidopsis four-week-old rosette leaves.\u003c/p\u003e","description":"","filename":"Slide8.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/75ca317e51e7eee8917737de.png"},{"id":107179075,"identity":"0537b1ef-79a1-4bdd-b780-8f05320196e9","added_by":"auto","created_at":"2026-04-17 16:35:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":62269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFour-week-old Arabidopsis rosette leaves were infiltrated with mixture of xylo-oligosaccharides from WT, 35S:AXE/XYL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) represents H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation measured by DAB staining at 30 min by infiltration of GH11 digested AIR samples and (b) relative quantification of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is calculated based on the Image J tool. (c) the expression of key genes involved in perception, defense, and cell wall remodeling by RT-qPCR. Data represent mean ± SD, n = 3 biological replicates. Each biological replicate represents a pool of 5 plants. Student’s t-test at ***p ≤ 0.01, **p ≤ 0.05, * p ≤ 0.1.\u003c/p\u003e","description":"","filename":"Slide9.png","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/0e3c3b0657603f20a53e87ff.png"},{"id":108490768,"identity":"374993c0-9260-4d02-9772-c0ccef96b8c2","added_by":"auto","created_at":"2026-05-05 09:48:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1162927,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/de6a68c7-ef41-40a6-8db7-08fb3a8d0fe2.pdf"},{"id":107483287,"identity":"6f17ac94-8f4c-44be-ae4b-c96f19d2acc4","added_by":"auto","created_at":"2026-04-22 02:27:10","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2792010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. \u003c/strong\u003eSchematics of construct, genotyping, gene expression and anatomical studies\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. \u003c/strong\u003eMorphology, gene expression, enzyme activity, and anatomical analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. \u003c/strong\u003ePlant cell wall composition on AIR samples prepared from Arabidopsis inflorescence stem.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. \u003c/strong\u003eRNA sequencing analysis of WT and WP:AXE/XYL plants\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5. \u003c/strong\u003eXylo-oligosaccharide analysis by MALDI-TOF on AIR of Arabidopsis inflorescence stem through xylanase (GH11) digestion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 6. \u003c/strong\u003ePicture of Arabidopsis leaf after DAB staining.\u003c/p\u003e","description":"","filename":"SupplementaryFiguresBMC31032026.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/521d9578806651189a5eec4e.pptx"},{"id":107481787,"identity":"85fba275-86a1-4d58-afc0-655e907952c4","added_by":"auto","created_at":"2026-04-22 02:19:59","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":309540,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary File\u003c/p\u003e","description":"","filename":"SupplementryData1BMC31032026.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/673a311b7b7df06605dee097.xlsx"},{"id":107705238,"identity":"f1ae0797-ea24-4f96-9d63-be6f0d0b1653","added_by":"auto","created_at":"2026-04-24 09:09:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":39485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1.\u003c/strong\u003e Differentially expressed cell wall-related processes genes in 35S:AXE/XYL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 2. \u003c/strong\u003eDifferentially expressed defense-related genes in 35S:AXE/XYL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 3. \u003c/strong\u003eXylooligosaccharide-profiling after digestion with xylanase (GH11) digestion and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 4. \u003c/strong\u003eXylooligosaccharide -profiling (ug/ml) in alcohol soluble fraction and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 5\u003c/strong\u003e. Primer used for Genotyping and RT-qPCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 6.\u003c/strong\u003eAntibodies Used for Glycome Profiling.\u003c/p\u003e","description":"","filename":"SupplemntaryTableBMC310032026.docx","url":"https://assets-eu.researchsquare.com/files/rs-9277431/v1/c886105320f6f167dfa07fee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhanced cell wall digestibility and immunity in Arabidopsis through targeted modification of xylan structure by heterologous expression of acetyl xylan esterase and xylanases","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant cells are encased inside complex extracellular matrix known as the cell wall which is intricate organization of cellulose, hemicelluloses (including xylan, xyloglucan, and mannan), pectins, polyphenols, and structural proteins. These components determine cell growth and provide mechanical strength, flexibility, and structural integrity to the cell [1]. Cell wall remodeling also plays a vital role in biotic and abiotic stress adaptation [2]. In vascular plants, xylan is an abundant non-cellulosic polysaccharide, reaching 30% of secondary cell wall in dicots, 50% in monocots and 15% conifers [3\u0026ndash;7]. Typically, xylan is a branched polymer of β-(1,4)-linked xylose residues substituted with acetyl, glucuronic acid (GlcA), 4-O-methylglucuronic acid (Me-GlcA), and arabinose residues, depending on species and tissues. In Arabidopsis secondary walls, there is acetylated glucuronoxylan, which xylosyl backbone is substituted with GlcA or Me-GlcA at C2 xylose position and acetyl groups at C2, C3 or both [8]. Xylan is polymerized in the Golgi apparatus by glycosyl and acetyl transferases, which when mutated, cause an irregular xylem phenotype characterized by collapsed xylem vessels [9]. Disruption of xylan structure can alter the physicochemical properties of the wall, affecting interactions among cell wall components [10, 11]. In Arabidopsis, xylan acetylation is mediated by a family of Trichome Birefringence-Like (TBL) proteins, including TBL29/ESK1, and mutation in \u003cem\u003eTBL29/ESK1\u003c/em\u003e causes xylan hypoacetylation, altered wall extensibility, changes in glucosinolate metabolism and stress sensitivities [12, 13]. However, the spectrum of plant responses to disruption in xylan acetylation is not well understood. Post-synthetic reduction in xylan acetylation by expressing fungal acetyl xylan esterases (AXE) \u003cem\u003ein muro\u003c/em\u003e often enhances cell wall digestibility and extractability without affecting plant growth [14]. Moreover, AXE expressing plants have increased immunity against specific pathogens [14\u0026ndash;16]. Since these plants do not have irregular xylem phenotypes, they are suitable genotypes to study responses to moderate deacetylation. Deacetylated xylan is prone to degradation by xylanolytic enzymes resulting in an altered pool of apoplastic xylo-oligosaccharides (XOS) that can act as a damage associated molecular patterns (DAMPs) triggering plant immunity. Plant treatment with XOS can induce pattern triggered immunity (PTI) which activates MAPK and calcium signaling, and reactive oxygen species (ROS) accumulation [17\u0026ndash;19]. The structural features of XOS, such as degree of polymerization and side-chain patterns, influence immune potency and cell wall integrity maintenance mechanism. Xylan-derived signals could intersect with hormone signaling pathways, particularly salicylic acid and jasmonic acid signaling, shaping local and systemic defense responses [18, 20\u0026ndash;22]. Crosstalk with lignification pathways further amplifies immunity through the deposition of phenolic polymers at infection sites [15, 23]. However, the role of apoplastic steady-flow XOS during development or after pathogen attack remains relatively unclear. Since plant endoxylanases are relatively unexplored, expression of xylanases and acetyl xylan esterase from fungal enzymes can serve as a tool to study the role of XOS in immunity and cell wall remodeling [14, 24\u0026ndash;27]. Also, co-expression of AXE and xylanase can improve digestibility of different polysaccharide cell wall components by affecting the interaction of xylan with lignin and cellulose [15, 28\u0026ndash;30] but such transgenic manipulation has not been to our knowledge previously attempted.\u003c/p\u003e \u003cp\u003eTherefore, in this study, we investigated the combined effect of xylan deacetylation and backbone hydrolysis by co-expression of \u003cem\u003eAspergillus niger\u003c/em\u003e CE1 acetyl xylan esterase (AXE) and \u003cem\u003eAspergillus nidulans\u003c/em\u003e GH10 xylanase (XYL) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Since the choice of promoter can substantially influence effects of transgenes [27, 31]. In our study, we combine AXE and XYL with two promoters, the constitutive 35S promoter (35S) and the wood specific promoter (WP) active in cells depositing secondary walls [32]. Indeed, 35S lines showed no apparent morphological or xylem defects, however co-expression of AXE and XYL controlled by WP lead to several developmental and cell wall defects. Transcriptomic analysis indicated repression of secondary wall biosynthetic genes and compensatory changes in hemicellulosic sugars in WP:AXE/XYL lines. We also found that 35S:AXE/XYL lines had reduced xylan acetylation, increased xylose content, improved lignocellulosic biomass properties and saccharification. Increased sugar availability enhanced microbial fermentation efficiency, resulting in higher ethanol yields and reduced acetic acid accumulation. Additionally, 35S:AXE/XYL-mediated cell wall remodeling increased resistance to \u003cem\u003ePseudomonas syringae\u003c/em\u003e, associated with activation of several pathogen triggered immunity (PTI) marked possibly through elicitor activity of xylo-oligosaccharides in 35S:AXE/XYL plants. These findings revealed that coordinated xylan deacetylation and hydrolysis improve biomass digestibility while simultaneously enhancing plant immunity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCo-expression of microbial acetyl xylan esterase (AXE) and xylanase (XYL) enzymes alter plant development when regulated by a wood-specific promoter (WP).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur earlier studied showed that overexpressing carbohydrate esterase 1 (CE1) acetyl xylan esterase from \u003cem\u003eAspergillus niger\u003c/em\u003e (AnAXE1) under control of 35S or WP promoters leads to reduced xylan acetylation in Arabidopsis [14]. For making crosses with xylanase-expressing lines, we selected two highly expressing lines: line D carrying 35S:AXE construct and line E carrying WP:AXE construct as parent plants [14](Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). We also observed that expressing GH10 endoxylanase from \u003cem\u003eAspergillus nidulans\u003c/em\u003e (XYL) in aspen can enhance xylan digestibility and increase drought resistance in Arabidopsis [24, 26]. The construct with XYL gene under control of the 35S promoter [24] (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea) was used to generate stable transgenic lines which were used as hemi- or homozygous for crossing with the D line carrying 35S:AXE, and the resulting progenies were genotyped by PCR amplification (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb). Expression of transgenes in selected progenies was verified by quantitative RT-qPCR (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). The 35S:AXE lines exhibited two times higher AXE expression than the 35S:AXE/XYL crossed lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth parents and co-expressing lines with 35S promoter constructs showed no visible morphological differences compared to wild type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Staining of inflorescence stems using phloroglucinol showed no abnormalities in xylem vessel cells, which are typically affected if xylan structure is compromised (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed-e). Esterase activity was evaluated using both synthetic and natural acetylated xylan substrates, revealing higher activity in 35S:AXE and 35S:AXE/XYL compared to the wild type and 35S:XYL lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ef). We also assessed xylanase in both parent and crossed lines, finding that 35S:XYL parents had 15 times more expression than the 35S:AXE/XYL crossed lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). This pattern matched xylanase activity, which was significantly higher in 35S:XYL and 35S:AXE/XYL than in wild type and 35S:AXE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eTo generate WP:AXE/XYL, we crossed the best expressing WP:XYL line [26] with the E line carrying WP:AXE and selected the progenies carrying both transgenes which were either homozygous or hemizygous (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea and S2b).. \u003cem\u003eAXE\u003c/em\u003e expression was 2.5 times higher in WP:AXE than in WP:AXE/XYL, while \u003cem\u003eXYL\u003c/em\u003e expression was 3.5 times higher in WP:XYL than in WP:AXE/XYL (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec-S2e). This corresponded to lower esterase and xylanase activities, reflecting the expression levels of AXE and XYL (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ef and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eg). However, WP:AXE/XYL lines were severely dwarf as compared to WT and WP parent lines (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eh) and xylem vessels were not normal shaped in WP:AXE/XYL line (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ei-2j). These dwarf phenotypes were observed in different generations which were either homozygous or hemizygous. In summary, we examined the crossed lines together with their parental and wild type plants, observing the anticipated increases in gene expression and activity. Notably, the 35S:AXE/XYL plants exhibited normal growth, whereas the WP:AXE/XYL plants were significantly stunted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePlants expressing both acetyl xylan esterase and xylanase showed decrease in acetyl content, along with compensatory changes in other cell wall components.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further analyze acetyl content in alcohol-insoluble residue (AIR) prepared from basal part of inflorescence stems of lines expressing AXE, XYL or both under control of the 35S promoter. Both the 35S:AXE and 35S:AXE/XYL lines showed reductions in acetyl content of 32% and 11%, respectively, compared to wild-type (WT) plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Additionally, AIR xylose content was elevated in both parental and 35S:AXE/XYL lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The acetyl/xylose ratio was also decreased, confirming a lower content of xylan acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). All analyzed 35S transgenic lines did not differ in total carbohydrate, cellulose and lignin contents from the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Furthermore, these constitutive promoter lines exhibited decreased levels of non-cellulosic monosaccharides in alcohol-insoluble residue prepared from inflorescence stems such as arabinose, rhamnose, fucose, and mannose (Table\u0026nbsp;1). Conversely, xylose levels were higher in 35S:AXE, 35S:XYL and 35S:AXE/XYL transgenic lines (Table\u0026nbsp;1). Above non-cellulosic sugar analysis was performed by derivatization using MS-TFA and analysis by GC-MS. We further found increase in xylose content in hydrolysed AIR1 with HCl and quantified using Megazyme K-Xylose kit (Table\u0026nbsp;1 \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). However, glucose levels were comparable in wild type and transgenic lines.\u003c/p\u003e \u003cp\u003eThe acetyl content was also reduced in WP:AXE, WP:XYL and WP:AXE/XYL by 26%, 17% and 67% respectively as compared to wild type (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea). However, acetyl to xylose ratio was reduced only in WP:AXE and xylose levels were reduced only in WP:AXE/XYL (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eb-S3c). WP:AXE/XYL plants also showed significant reductions in total carbohydrates, cellulose, and lignin compared to WT (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec-S3f) and displayed a compensatory rise in non-cellulosic monosaccharides, including arabinose, rhamnose, fucose, mannose, and glucose (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eTo clarify the pronounced effects on growth and cell wall composition observed in WP:AXE/XYL plants, we conducted untargeted RNA-seq analysis comparing wild-type (WT) and WP:AXE/XYL 6-week-old stems. This revealed 264 genes upregulated and 269 genes downregulated, with a significant false discovery rate (FDR) adjusted p-value of \u0026le;\u0026thinsp;0.05 and a Log\u003csub\u003e2\u003c/sub\u003e FoldChange of \u0026plusmn;\u0026thinsp;1 (Figure S4a). Further analysis showed substantial downregulation of key biological processes, including xylan biosynthesis, the phenylpropanoid pathway, and cell wall polysaccharide metabolism (Figure S4b-S4d, Supplementary Data 1). This led to reductions in essential cell wall components, such as xylan, and lignin. The transcriptomic data were consistent with cell wall chemotyping results for WP:AXE/XYL, which ultimately resulted in abnormal xylem vessel development and dwarfism in the plants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-expression of\u003c/b\u003e \u003cb\u003eAXE\u003c/b\u003e \u003cb\u003eand XYL driven by a constitutive promoter enhances lignocellulose saccharification and boosts ethanol yield\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDue to the dwarf phenotype observed in WP:AXE/XYL plants either because of increased in xylanase or esterase activity levels or tissue specific activity, we next focused on lines driven by the 35S promoter. To evaluate how co-expression of AXE and XYL affects the enzymatic saccharification of lignocellulose, we performed assays using untreated samples, hot water, alkali, and xylanase pretreatments, followed by incubation of the stem material with a cellulase mixture. After cellulase digestion, the 35S:AXE/XYL lines exhibited a 29% increase in glucose release compared to wild type, with 35S:XYL and 35S:AXE lines also outperforming the control in the absence of pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). When AIR was pretreated with hot water and then digested with cellulases, 35S:AXE/XYL plants again showed the highest increase in glucose release compared to parent and wild type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). A similar trend was seen after pretreatment with 0.4 NaOH and xylanase, where 35S:AXE/XYL plants demonstrated an 18% and 33% increase in xylose release, respectively, over wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). We also digested AIR with xylanase alone and in combination with glucuronyl esterase. Xylose release after xylanase treatment was increased significantly in all 35S lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Similarly simultaneous digestion with glucuronyl esterase and xylanase enhanced xylose release by 14\u0026ndash;35% in parental lines and by 50% in crossed line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eModifying xylan can influence its interactions with other cell wall components, such as lignin, cellulose, and pectin, thereby affecting their extractability [15]. To investigate this, we sequentially extracted the cell wall using ammonium oxalate (AOE), sodium carbonate (SCE), 1 M KOH, and 4 M KOH, and assessed the extracts with cell wall-specific antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The 35S:AXE line, which has reduced acetyl content, displayed increased LM10 signals and decreased LM15 signals in the 4M-KOH fraction, reflecting increased epitopes of unsubstituted xylan and reduced epitopes of xyloglucan. In contrast, the 35S:AXE/XYL co-expression line did not show such changes. However, both parental (35S:AXE and 35S:XYL) and crossed (35S:AXE/XYL) lines exhibited reduced signals of rhamnogalacturonan-1derived fragments in the AOE and 1M KOH extracts as detected by CCRC-M7 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further examined how elevated glucose and xylose release influenced fermentation efficiency in 35S:AXE/XYL plants compared to wild type, using an \u003cem\u003eE.\u003c/em\u003e coli diauxic strain. Under anaerobic conditions with standard media conditions, native \u003cem\u003eE. coli\u003c/em\u003e strain ferments 1 g of glucose to 0.2 g of ethanol. Therefore, to understand how the genetic changes in plants affect fermentation, we evaluated ethanol production using modified plants as carbon source. First, we digested AIR1 with CTec2 and measured glucose which was more in 35S:AXE/XYL as compared to WT and parent (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The digested saccharide mixture from plants was supplemented separately as a carbon source to the \u003cem\u003eE. coli\u003c/em\u003e culture. Upon complete utilization of glucose, ethanol titer and yield was estimated. As expected 35S:AXE and 35S:AXE/XYL plants support higher ethanol production compared to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Interestingly, only 35S:AXE/XYL reached theoretical maxima, suggesting minimal interference from the inhibitors present in the biomass. We also measured acetic acid level in the cultures and found it was reduced in 35S:AXE and 35S:AXE/XYL at 0 h and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). This strain can also use xylose during fermentation and at 0 h, xylose was increased and decreased after 24 h in 35S:AXE/XYL as compared to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Overall, co-expression of AXE and XYL under the 35S promoter improves cell wall digestibility, resulting in increased glucose and xylose release and enhanced ethanol yield.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e35S:AXE/XYL plants exhibited enhanced immune responses and altered the expression of multiple genes associated with immunity and cell wall structure.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAlterations in cell wall composition influenced plant responses to biotic and abiotic stresses, with decreased xylan acetylation correlating with enhanced pathogen resistance [14] and enhanced xylan hydrolysis with increased drought resistance [26]. To assess the immune response in the 35S:AXE/XYL line, a pathogenesis assay was conducted using wild-type plants as controls. Four-week-old fully expanded rosette leaves were infiltrated with a suspension of \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. Tomato DC 3000 (pstDC3000) and bacterial proliferation was measured at 0- and 3-days post-inoculation (dpi). The 35S:AXE/XYL leaves showed reduced bacterial accumulation compared to wild type at 3 dpi, indicating an increased resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Correspondingly, expression of defense-related marker genes, including \u003cem\u003ePATHOGEN-RELATED 1 (PR1), WRKY33, and WRKY53\u003c/em\u003e, was upregulated at 3 dpi and expression of \u003cem\u003eWRKY33\u003c/em\u003e and \u003cem\u003eWRKY53\u003c/em\u003e was already increased at 0 dpi in this line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Expression of other analyzed genes including \u003cem\u003ePAD3\u003c/em\u003e involved in carotene biosynthesis and \u003cem\u003eFLAGELLIN SENSITIVE 22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1)\u003c/em\u003e associated with leaf senescence as well as \u003cem\u003eWRKY30 and WRKY40\u003c/em\u003e transcription factors remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate how AXE and XYL overexpression affects cell wall remodeling and defense, RNA-seq analysis was performed in inflorescence stem tissue revealing 635 genes upregulated and 108 downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The GO-Biological process analyses revealed many genes responsive to several stimulus; related to reactive oxygen species (ROS) generation were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The GO-molecular function and GO-cellular component analysed indicated upregulation genes involved in cell wall metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Among the upregulated genes, 67 were linked to cell wall related processes determined by ShinyGo tool (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, several members of the xyloglucan endotransglucosylase/hydrolase (XTH) family were induced, including \u003cem\u003eXTH18 (AT4G30280), XTH8 (AT1G11545)\u003c/em\u003e, \u003cem\u003eXTH9 (AT4G03210)\u003c/em\u003e, \u003cem\u003eXTH19 (AT4G30290)\u003c/em\u003e, and \u003cem\u003eXTH16 (AT3G23730)\u003c/em\u003e. In addition, several expansin genes such as \u003cem\u003eEXPA5 (AT3G29030)\u003c/em\u003e, \u003cem\u003eEXPA6 (AT2G28950)\u003c/em\u003e, \u003cem\u003eEXPA4 (AT2G39700)\u003c/em\u003e, \u003cem\u003eEXPA11 (AT1G20190)\u003c/em\u003e, \u003cem\u003eEXPA14 (AT5G56320)\u003c/em\u003e, and \u003cem\u003eEXPB1 (AT2G20750)\u003c/em\u003e were upregulated. Genes involved in pectin modification and degradation were also prevalent. These included \u003cem\u003ePECTIN METHYLESTERASE 5 (PME5; AT5G47500)\u003c/em\u003e, \u003cem\u003ePME1 (AT1G53840)\u003c/em\u003e, various pectin lyase-like superfamily proteins (\u003cem\u003eAT1G10640, AT4G23820)\u003c/em\u003e, and \u003cem\u003ePOLYGALACTURONASE 2 (PG2; AT1G70370)\u003c/em\u003e. Collectively, these findings indicate active xyloglucan and pectin remodeling in 35S:AXE/XYL plants. In addition, differential expression analysis identified 72 upregulated defense-related genes (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), signifying robust activation of stress and defense-associated transcriptional programs. The most elevated gene was cytochrome \u003cem\u003eP450 CYP94B3 (AT3G48520)\u003c/em\u003e, a critical enzyme in jasmonate catabolism (Koo et al., 2011). Consistently, multiple JASMONATE ZIM-DOMAIN (JAZ) repressors\u0026mdash;including \u003cem\u003eJAZ10, JAZ7, JAZ8, JAZ5\u003c/em\u003e, and \u003cem\u003eJAZ1\u003c/em\u003e were significantly upregulated, indicating activation of the jasmonic acid (JA) signaling pathway with feedback regulation [33\u0026ndash;35]. Upregulation of \u003cem\u003eMYB96\u003c/em\u003e, \u003cem\u003eWRKY53\u003c/em\u003e, \u003cem\u003eWRKY40\u003c/em\u003e, and \u003cem\u003eWRKY18\u003c/em\u003e further supports the transcriptional reprogramming in response to biotic and abiotic stress [36\u0026ndash;38]. In summary, the 35S:AXE/XYL lines exhibited enhanced immunity against \u003cem\u003ePseudomonas\u003c/em\u003e and elevated expression of immunity-related marker genes as well as genes related to primary wall modification.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIntegrated transcriptomic and oligosaccharide elicitor analyses revealed that xylo-oligosaccharides trigger immune activation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe hypothesized the gene activation stems from increased \u003cem\u003ein vivo\u003c/em\u003e production of xylo-oligosaccharides from xylan affected by the combined action of AXE and XYL. To explore this, AIR of inflorescence stems was digested with GH11 xylanase and released xylo-oligosaccharides were analyzed using MALDI-TOF. Results showed that xylobiose without acetyl groups (Xyl2) was more abundant in the 35S:AXE/XYL line than in wild type (Figure S5), and fewer acetylated Xyl4 XOS were present in this line. The xylanase digested fraction was further analyzed using ion chromatography, which revealed increase in xylose, xylobiose and xylotriose (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). We also checked level of these soluble XOS in 70% ethanol fraction and found accumulation of xylose, xylobiose and xylotetratose and xylohexaose (Table S4). These results suggest that 35S:AXE/XYL lines have steady flow of XOS in the cell which may act as signalling molecules [18]. Our earlier study revealed that xylobiose treatment can trigger immune response and change cell wall composition. Therefore, we compared RNA-seq data from xylobiose treated plants [18] and 35S:AXE/XYL plants, finding 51 upregulated and 4 downregulated genes in common (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The upregulated genes included \u003cem\u003eWRKY53\u003c/em\u003e and \u003cem\u003eWRKY18\u003c/em\u003e, key modulators of pathogen-responsive gene expression, indicating a shift toward heightened defense and \u003cem\u003ePEROXIDASE 2\u003c/em\u003e, suggesting increased reactive oxygen species (ROS) metabolism\u0026mdash;a hallmark of plant defense [39, 40]. We also found several of the \u003cem\u003eJAZ1 (JAZ10, JAZ7, JAZ8, JAZ5, AND JAZ1)\u003c/em\u003e were upregulated in comparative RNA sequencing analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the hypothesis that \u003cem\u003ein vivo\u003c/em\u003e generation of xylooligosaccharides (XOS) through co-expression of AXE and XYL initiates a defense response, we infiltrated XOS obtained from GH11 xylanase digestion into healthy 4-week-old wild-type leaves. Leaves treated with 35S:AXE/XYL-derived XOS exhibited a greater accumulation of reactive oxygen species compared to those treated with XOS from wild-type plants or mock controls (Figure S9a \u0026amp; 9a). Subsequent RT-qPCR analysis demonstrated upregulation of genes related to cell wall remodeling (such as \u003cem\u003eXYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 24\u003c/em\u003e and \u003cem\u003eCELLULOSE SYNTHASE-LIKE A11\u003c/em\u003e), pattern-triggered immunity (PTI) markers (including \u003cem\u003ePATHOGEN-RELATED 1\u003c/em\u003e and \u003cem\u003eFLAGELLIN SENSITIVE 22-INDUCED RECEPTOR-LIKE KINASE 1\u003c/em\u003e), as well as genes linked to different stress responses (\u003cem\u003eWRKY22, WRKY30, WRKY33, PAD3, MYB24\u003c/em\u003e, and \u003cem\u003eMYB87\u003c/em\u003e) in leaves treated with 35S:AXE/XYL XOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Collectively, these findings indicate that 35S:AXE/XYL plants exhibit enhanced immune activation and altered cell wall composition, likely mediated by XOS-induced defense pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant secondary cell walls are a highly organized structure in which xylan acts as a critical hemicellulosic matrix, tethering cellulose microfibers to lignin [4, 41]. Post-synthetic targeted modification of plant polysaccharides by overexpressing microbial hydrolases to improve secondary cell wall properties is a promising strategy. Acetyl substitutions on the xylan backbone limit enzymatic accessibility, whereas backbone depolymerization shortens polymer length and disrupts its interaction with cellulose and lignin [42, 43]. Previous work showed that expression of fungal acetyl xylan esterase in Arabidopsis decreases xylan acetylation and improves saccharification without major growth defects [14]. However, the combined \u003cem\u003ein-planta\u003c/em\u003e effects of deacetylation and the simultaneous hydrolysis of xylan had not been systematically explored. Here, co-expression of AXE and XYL provides direct evidence that coordinated modification of substitution pattern and polymer length synergistically alters cell wall architecture, digestibility, and immunity signaling.\u003c/p\u003e\n\u003ch3\u003eDistinct developmental consequences of xylem-specific expression of xylanases and acetyl xylan esterase\u003c/h3\u003e\n\u003cp\u003eAXE and XYL were expressed individually or together in plants; most of the resulting lines did not display any noticeable growth defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The lines co-expressing both enzymes exhibited higher enzymatic activity than the wild type, though lower than the parent lines expressing a single gene. This reduction in activity among co-expressing lines is likely due to the use of the same promoter, selection marker genes or gene dosage effect. Despite this, the WP:AXE/XYL line developed a dwarf phenotype, and both acetyl and xylose contents dropped by over 60% compared to wild type (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea-S3c). Typically, plants compensate for such changes by increasing other components, but the WP:AXE/XYL line showed a significant decrease in cellulose and lignin content. This observation was reinforced by the transcriptional repression of genes involved in secondary cell wall formation in the WP:AXE/XYL line. Members of the TRICHOME BIREFRINGENCE-LIKE (TBL) family TBL35 and TBL27, responsible for xyloglucan acetylation [44\u0026ndash;47], and TBL29/ESK1, TBL3 and TBL31, responsible for xylan acetylation, along with glucuronoxylan 4-O-methyltransferase-like proteins, were notably downregulated (Figure S4c) [13, 48\u0026ndash;50]. The diminished expression of these genes implies disrupted hemicellulose patterning, which destabilizes the cellulose-xylan network. Reduced glucuronoxylan methylation is also known to compromise cell wall structure [51]. Genes from the phenylpropanoid pathway, including \u003cem\u003ePHENYLALANIE AMMONIA LYASE\u003c/em\u003e (\u003cem\u003ePAL), 4-COUMARATE CoA LIGASE (4CL)1\u003c/em\u003e and \u003cem\u003e4CL2, FERULATE-5-HYDROLASE\u003c/em\u003e, and the \u003cem\u003eMYB58\u003c/em\u003e transcription factor, were suppressed, resulting in less lignin deposition and reduced monolignol production (Figure S4d) [52, 53]. Furthermore, several \u003cem\u003eXYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH\u003c/em\u003e) genes, which are involved in xyloglucan remodeling and the integration of cellulose microfibrils, were also downregulated [54, 55]. The formation of secondary cell walls requires the precise deposition of cellulose, xylan, and lignin [56]; disruption of xylan structure likely weakens the scaffold necessary for lignin polymerization and cellulose crystallization, which explains the overall reduction in wall polymers. This transcriptomic suppression of cell wall biosynthesis genes indicates feedback from wall damage, aligning with plant cell wall integrity signaling pathways [57, 58].\u003c/p\u003e\n\u003ch3\u003eReduced recalcitrance and improved saccharification efficiency in 35S:AXE/XYL plants\u003c/h3\u003e\n\u003cp\u003eBecause of normal growth phenotype, we focused our further study on the 35S:AXE/XYL line. The parent 35S:AXE line showed a higher reduction in cell wall acetyl content than the 35S:AXE/XYL line, in agreement with a lower esterase activity in the latter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). A decreased acetyl-to-xylose ratio confirmed that AXE-driven deacetylation facilitated XYL-mediated backbone cleavage. MALDI-TOF profiling further demonstrated increased release of short, neutral xylo-oligosaccharides especially xylobiose and a reduction in acetylated oligomers, confirming that xylan in the 35S:AXE/XYL plants is less acetylated and more accessible to xylanase. (Figure S5)\u003c/p\u003e \u003cp\u003eSaccharification assays revealed a significant increase in xylan and cellulose hydrolysis in the co-expressing lines compared to the parent lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The enhanced xylan hydrolysis after xylanase and glucuronyl esterase digestion primarily resulted from xylan deacetylation; 35S:AXE lines exhibited a 35% increase in xylose release, while 35S:XYL lines showed a 14% increase as compared to wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). This indicates that deacetylation could be a key factor limiting xylan hydrolysis and suggests it may be a more effective strategy than altering the xylan backbone itself. However, the combined 35S:AXE/XYL line displayed superior saccharification efficiency compared to either parent alone, indicating that the combinatorial approach is more effective\u0026mdash;even though esterase activity was lower in 35S:AXE/XYL than in 35S:AXE or 35S:XYL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-e). This may be because esterase activity exposes the xylan backbone, while xylanase shortens it, together weakening the matrix that links cellulose and lignin. Notably, the lignin and cellulose content in the transgenic lines remained comparable to wild type plants.\u003c/p\u003e \u003cp\u003eAs a result, the improved saccharification efficiency led to increased ethanol yields in both 35S:AXE and 35S:AXE/XYL lines compared to 35S:XYL and wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) [14]. The enhancement in saccharification is likely result of reduced acetic acid levels; lower acetate concentrations stem from decreased acetyl ester hydrolysis during microbial processing, which is advantageous since acetate can inhibit microbial growth and reduce fermentation efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) [14, 59, 60]. Additionally, the consumption of xylose during fermentation supports the conclusion that overexpressing AXE and XYL together enhances both saccharification efficiency and ethanol production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). As a result, controlled reduction in xylan acetylation presents a promising approach for enhancing the conversion of lignocellulosic biomass into bioethanol. This strategy could be applied to bioenergy crops by engineering endogenous genes that regulate xylan acetylation, thereby improving biomass digestibility and biofuel yields.\u003c/p\u003e\n\u003ch3\u003eXylooligosaccharides (XOS) elicitors can activate plant immunity\u003c/h3\u003e\n\u003cp\u003eBiotic stress, such as pathogen infection, can cause damage to plant cell walls, leading to the release of wall molecules that function as damage-associated molecular patterns (DAMPs), which are perceived by pattern recognition receptors (PRRs) present in the cell wall vicinity to activate pattern-triggered immunity (PTI) and help to boost immunity against pathogens [39]. Plants release or synthesize these self-driven damage-associated molecular patterns in response to pathogen infection [61]. Plant immunity by damage associated molecular patterns (DAMPs) leads to changes in cell wall chemistry are perceived by cell wall-associated receptors located at the plasma membrane\u0026ndash;cell wall interface, such as Wall-Associated Kinase 1 (WAK1), THESEUS1 (THE1), FERONIA (FER), and MIK2 [62\u0026ndash;64]. These receptors alone or in complexes may detect perturbations in cell wall status and activate signaling pathways that modulate disease resistance, either enhancing immunity or increasing susceptibility depending on the nature of the modification.\u003c/p\u003e \u003cp\u003eFurthermore, in planta expression of cell wall\u0026ndash;degrading enzymes (CWDEs), such as those targeting pectin, cellulose, or hemicellulose, can mimic pathogen-induced wall damage [65]. This leads to the generation of damage-associated molecular patterns (DAMPs), which are recognized by the plant immune system and trigger defence responses. However, excessive or uncontrolled degradation may compromise wall integrity, facilitate pathogen invasion and thereby tipping the balance toward susceptibility.\u003c/p\u003e \u003cp\u003eRecent advances highlight that cellulose derived oligosaccharides (cellodextrins) released during enzymatic hydrolysis of cellulose, function as potent DAMPs capable of inducing immune responses such as reactive oxygen species (ROS) production and transcriptional reprogramming [20, 65]. Similarly, hemicellulose-derived fragments, including mannose-rich oligosaccharides from glucomannans and other polysaccharides like xyloglucans, have been shown to act as elicitors of defense signaling, indicating that diverse wall polysaccharides contribute to DAMP-mediated immunity [39, 66]. Among all DAMPs, pectin-derived oligogalacturonides (OGs) are the most extensively characterized. These fragments are generated through the activity of cell wall\u0026ndash;degrading enzymes such as polygalacturonases, often secreted by pathogens, and their accumulation is tightly regulated by plant-derived polygalacturonase-inhibiting proteins (PGIPs) [65, 67]. OGs with a degree of polymerization of ~\u0026thinsp;10\u0026ndash;15 are particularly active in triggering immune responses, including MAPK activation, ROS burst, and defense gene expression, while also modulating growth-defense trade-offs [68]. Also, xylan acetylation reduction due to ESKIMO1 impairment has been shown plant resistance to several pathogens, including \u003cem\u003eP. Cucumerina\u003c/em\u003e. [69]. So far, fewer DAMPs from xylooligosacharides and their receptors, derived from plants have been identified [70\u0026ndash;72]. Plant pathogens and their hosts release cell wall-degrading enzymes (CWDEs) to degrade the cell walls of opponents during interactions [73]. DAMPS are produced by xylan polysaccharides hydrolysis by CWDE, like endo-1,4-beta xylanase, which belong to the GH10 and GH11 families [74]. GH11 b-xylanase from \u003cem\u003eN. pariciarum\u003c/em\u003e able to release structures such as XA3XX and XA2XX and perceives as Xylan -derived DAMPS [75, 76].\u003c/p\u003e \u003cp\u003eHere we showed, 35S:AXE/XYL expressing plants displayed accumulation of xylo-oligosaccharide \u003cem\u003ein vivo or\u003c/em\u003e after xylanases digestion (Figure S5 \u0026amp; Table S4). Although, we could not detect substituted XOS in probably because low amount in apoplastic space. However, after inducing XOS by digestion with xylanase released different XOS as compared to wild type plants (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These XOS or DAMPs could be possibly responsible for induction in basal plant immunity and XOS induced immunity. As we found accumulation ROS in AXE/XYL plants along with increase expression of several PTI markers after 0-dpi and 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). To supplement our findings, we performed transcriptomic overlap between AXE/XYL plants and xylobiose-treated samples, which were previously available in our lab that supports the induction of stress-protective proteins, such as the late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein (AT2G27080), suggesting enhanced cellular protection in 35S:AXE/XYL expressing plants similar to Xylobiose treatment. Multiple JAZ family members were upregulated, reflecting activation and fine-tuning of JA-mediated defense responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eIt is known that pectin-derived oligogalacturonides (OGs) can selectively influence the jasmonic acid (JA) and salicylic acid pathways, both of which are crucial for initiating immune responses in plants [77]. Similar effects were observed in the 35S:AXE/XYL line, where there was an upregulation of \u003cem\u003ePR1, PEP1 RECEPTOR 1\u003c/em\u003e and \u003cem\u003eNON-EXPRESSOR OF PR1-LIKE 3 (NPR3)\u003c/em\u003e, markers for the salicylic acid pathway, along with several genes associated with JA-related pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec \u0026amp; Table\u0026nbsp;3). To further clarify the significance of these responses, future studies could systematically measure hormone levels following XOS treatment or in plants with modified xylan to gain deeper insights into how XOS induces plant immunity. Additionally, just as the active OG machinery has been shown to play a role in defense responses, the function and potential activation and inactivation of the XOS machinery should be explored in upcoming research [78]. Overall, these shared transcriptional changes strongly suggest activation of JA-dependent defense pathways, WRKY-mediated transcription regulation, and ROS signaling in both the 35S:AXE/XYL line and the xylobiose treatment, indicating coordinated priming of innate immunity.\u003c/p\u003e \u003cp\u003eIn nutshell, transgenic WP:AXE/XYL lines showed abnormal xylem development and reduced cell wall deposition. In contrast, 35S:AXE/XYL plants had no growth deformities, higher cellulose and xylan digestibility after different chemical and enzymatic treatment, and improved resistance to \u003cem\u003ePseudomonas syringae\u003c/em\u003e. Comparative RNA sequencing and elicitor-based assays indicated improvement in the immunity is possibly through more XOS levels in 35S:AXE/XYL plants. Overall, controlled breaking down both the xylan backbone and its acetyl groups can improve biomass quality and disease resistance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth conditions and genotyping\u003c/h2\u003e \u003cp\u003eArabidopsis Plants were grown under a 16-h light/8-h dark photoperiod at 22\u0026deg;C. The cloning and generation of parent plants expressing acetyl esterase and xylanases were described in our previous reports [15, 79]. Acetyl xylan esterase (AXE) and Xylanases (XYL) expressing homozygous parents and a hemizygous cross were genotyped using PCWL-33, PCWL-34, and PCWL-49, PCWL-50 primers, respectively (Supplementary file 1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhloroglucinol-HCl (Wiesner) Staining\u003c/h3\u003e\n\u003cp\u003eSix-week-old inflorescence stem sections were stained with 3% phenol-HCl (P3502-25G, Sigma-Aldrich, USA)[80] and imaged by a Nikon fluorescence microscope under 10X and 40X magnification.\u003c/p\u003e\n\u003ch3\u003eTotal RNA isolation and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from the six-week-old side stem using TRIzol (15596026, Thermo Fisher Scientific Invitrogen) and treated with RNase-free DNase I (EN0521, Thermo Fisher Scientific Invitrogen). cDNA was synthesized from 1 \u0026micro;g DNase-treated RNA by iScript\u0026trade; cDNA Synthesis kit (1708891, Bio-Rad, USA) used for RT-qPCR, performed on Quantstudio-6 Flex Real-time machine for the gene of interest and reference gene using HOT FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne, 08240001). The relative fold change was calculated by the ΔΔCt method.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and enzyme activities\u003c/h2\u003e \u003cp\u003eSix weeks of side stem tissue was ground in liquid nitrogen to a fine powder and stirred for 1 h at 4\u0026deg;C in buffer A (50 mM sodium phosphate buffer, pH 6.8, containing 2.5 mM EDTA, 2% PVP, and 1 mM DTT). The supernatant was collected as a soluble protein fraction by centrifuging at 10000 rpm for 10 min. The pellet was resuspended in buffer A with 1 M NaCl, stirred for 1 h, and collected as a wall-bound fraction [81]. The protein concentration was determined by the Bradford assay and used to assess esterase and xylanase activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEsterase activity with acetylated xylan as a substrate\u003c/h2\u003e \u003cp\u003eEsterase activity was performed with partially acetylated xylan (P-ACXYL, Megazyme, Ireland) as substrate, incubated at 37\u0026deg;C for 6 h, and the released acetic acid was quantified by the acetic acid kit (K-ACET, Megazyme, Ireland) [82].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eXylanase activity\u003c/h2\u003e \u003cp\u003eXylanase activity was performed using the Xylanase Assay kit (K-XylX6-2V, Megazyme, Ireland) by incubating the XylX6 substrate at 40\u0026deg;C for 10 min. The released 4-nitrophenol was quantified at 400 nm, and the specific xylanase activity was calculated in nmol min\u003csup\u003e\u0026minus;1\u003c/sup\u003emg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of total protein using the 4-nitrophenol release by Trichoderma sp. endo-1,4-β xylanase standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAlcohol insoluble residue (AIR) Preparation for cell wall analysis\u003c/h2\u003e \u003cp\u003e10 cm inflorescence stems of completely were dried, and homogeneous fine powder was prepared using Qiagen TissueLyserII for AIR preparation. The stem powder was stirred with 4 mM HEPES in 70% ethanol for 1 h at 70\u0026deg;C. After treatment, the sample was sequentially washed with chloroform: methanol (1:1) and acetone. The final pellet was dried overnight in a desiccator, and the resulting alcohol-insoluble residue (AIR) was used for analysis of plant cell wall composition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAcetyl content\u003c/h2\u003e \u003cp\u003e1 mg of the AIR sample was incubated with 1 M NaOH for 1 h, then neutralized with 1 M HCl. The final volume was made up to 1 ml with MilliQ water and centrifuged at 1500g for 10 min. The supernatant was analyzed using an acetic acid kit (K-ACET, Megazyme, Ireland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eXylose content\u003c/h2\u003e \u003cp\u003e2 mg of the AIR sample was incubated with 100 \u0026micro;l of 1.3 M HCL at 100\u0026deg;C for 1 h. Then, the sample was neutralized with 100 \u0026micro;L of 1.3 M NaOH, the final volume was adjusted to 1 mL with MilliQ water, and the mixture was centrifuged at 1500g for 10 min. The supernatant was quantified by a D-Xylose assay kit (K-XYLOSE, Megazyme, Ireland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTotal carbohydrate (phenol sulfuric method)\u003c/h2\u003e \u003cp\u003e100 \u0026micro;l of AIR suspension prepared from 0.5 mg/ml AIR was treated with 100 \u0026micro;l of 5% (v/v) phenol and 500 \u0026micro;l concentrated sulfuric acid, vortexed thoroughly, and incubated for 20 min. The total sugar concentration was estimated using a glucose standard curve by reading absorbance at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCellulose content\u003c/h2\u003e \u003cp\u003e3 mg of AIR was treated with Updegraff reagent (acetic acid, nitric acid, and water in an 8:1:2 ratio, respectively) and heated for 30 min at 100\u0026deg;C. The tube centrifuge was then cooled to maximum rpm for 10 min, and the pellet was washed with water, then with acetone. The pellet was dried overnight in the desiccator. A pellet was used to determine glucose content by Anthrone reagent (0.2% Anthrone in 92% sulphuric acid) [83].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAcetyl Bromide Soluble Lignin (ABSL)\u003c/h2\u003e \u003cp\u003eAIR samples were incubated at 50 C for 2 h with freshly prepared 25% acetyl bromide (135968-500G, Sigma) in glacial acetic acid. The supernatant was diluted with 2 M NaOH and 0.5 M hydroxylamine hydrochloride (159417-100G) freshly prepared. The absorbance was measured at 280 nm, and lignin content was expressed as a percentage of AIR [84].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHemicellulosic monosaccharide composition analysis by GC-MS\u003c/h2\u003e \u003cp\u003e2 mg AIR sample was hydrolyzed by incubation at 121\u0026deg;C for 90 min with 2 M trifluoroacetic acid (TFA) (76-05-1, SRL). The supernatant was collected in a glass tube, evaporated under a stream of nitrogen gas, and washed three times with isopropanol. Then, the dried fraction was dissolved in methoxyamine hydrochloride in pyridine and incubated for 90 minutes at 37\u0026deg;C. These fractions were then derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) for 30 min at 37\u0026deg;C, and GC-MS analyzed 100 \u0026micro;L of the derivatized samples. Inositol was used as an internal standard for normalization [18].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of xylo-oligosaccharides using mass spectroscopy\u003c/h2\u003e \u003cp\u003e3 mg of stem tissue AIR was digested with GH11 Endo-1,4-β-Xylanase (1U/mg of AIR-1) (13814, SRL). The resulting hydrolysate was purified with Hypersep Hypercarb Porous Graphitized Carbon (PGC) columns and separated into neutral and acidic fractions using 50% acetonitrile and 50% acetonitrile containing 0.05% of trifluoroacetic acid, respectively. Eluents were freeze-dried and resuspended in 30 \u0026micro;l HPLC-grade water; the resuspended sample was mixed with DHB in a 1:1 ratio and applied to a 384-well metal target plate (Opti-TOP LC/MALDI insert (123 x 81 mm) part no. 1018497) and air-dried. The data were acquired over the m/z range of 300\u0026ndash;1500.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSoluble sugar isolation and profiling\u003c/h2\u003e \u003cp\u003eFresh stem and leaf were homogenized in liquid nitrogen and treated with 1 ml of 80% ethanol for 1 h at 80\u0026deg;C. The supernatant was centrifuged, vacuum-dried, and dissolved in 300 \u0026micro;L of Milli-Q water. Soluble sugars were normalized to total sugar and run on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003eGlycan antibody profiling for cell wall extractability\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eDifferent fractions of cell wall components were isolated through sequential extraction using 50 mM ammonium oxalate, 50 mM sodium carbonate, 1 M KOH, and 4 M KOH. All wall extracts were loaded (50 \u0026micro;L of 500 ng/mL) into 96-well ELISA plates (Coster 3599, Corning Life Science, Acton, MA) and incubated for 12 h at 37\u0026deg;C. Nonspecific sites were blocked by incubating with blocking buffer at room temperature, followed by aspiration of blocking buffer and incubation with 50 \u0026micro;l of primary antibody (1:50) and secondary antibody (1:5000) for 1 h at room temperature (Table S6). The plate was washed with 300 \u0026micro;l wash buffer in between transition for primary antibody to the secondary antibody. After that 50 \u0026micro;l of 3,3,5,5-Tetramethylbenzidine (TMB) (860336-1G, Sigma-Aldrich, India) substrate solution was prepared in 0.1M citrate-acetate buffer, added into each well and incubated for 5 min at 37\u0026deg;C. The reaction was stopped by adding 0.5 N sulfuric acid, measuring net OD values of color formation at 450 nm, and subtracting the background reading at 655 nm [85].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSaccharification analysis\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eXylanase pretreatment\u003c/strong\u003e \u003cp\u003eThe AIR sample was digested with GH11 Endo-1,4-β-Xylanase (1U/mg of AIR-1) (13814, SRL) at 60\u0026deg;C for 6 h, and centrifuged at 1500 g for 10 min. The supernatant was used for the quantified D-Xylose assay kit (K-XYLOSE, Megazyme, Ireland); the digested pellet was washed with acetone, dried, and used for further for saccharification assays.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eChemical pretreatment\u003c/strong\u003e \u003cp\u003eAIR samples were treated with water and 0.4N NaOH at 90\u0026deg;C for 30 minutes [14]. Then it was centrifuged, washed with water and acetone, dried overnight, and used for glucose release.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGlucose release estimation\u003c/strong\u003e \u003cp\u003ePre-treated and untreated AIR samples were incubated with Cellulase, enzyme blend \u0026ndash; Cellic CTec2 (0.1 U for 1 mg AIR-1) in 0.1 M Sodium acetate buffer (pH- 4.8) at 50\u0026deg;C for 24 h, then centrifuged, and glucose was estimated in the supernatant by D-Glucose Assay kit (GOPOD Format, K-GLUC) [86].\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eXylanase digestibility with glucuronyl esterase\u003c/strong\u003e \u003cp\u003eThe AIR sample was incubated with GH11 Endo-1,4-β-Xylanase (1U/mg of AIR), supplemented with glucuronyl esterase (1U/mg of AIR) in sodium acetate buffer (pH 4.5) at 50\u0026deg;C for 4 h, and centrifuged at 1500 g for 10 min. The supernatant was used for the quantified D-Xylose assay kit (K-XYLOSE, Megazyme, Ireland).\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eFermentation analysis\u003c/h2\u003e \u003cp\u003eAutoclaved 50 mg sample digested with 10% (g/g) Cellic CTec2 at 50 C for 6 h. The digested samples were used as glucose sources, with 1 mL of glucose (20 g/L) added to a final reaction volume of 20 mL. Autoclaved LB broth was used in fermentation jars, which were then sparged with nitrogen gas to create an anaerobic environment. 1.3 ml of E. coli culture was added to the media; 1 ml of the culture was taken at 0 h, and the jars were incubated at 37\u0026deg;C for 24 h. After 24 h, samples were checked for Ethanol using the Megazyme kit (K\u003cb\u003e-\u003c/b\u003eETOH, Megazyme, Ireland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ePseudomonas infection assay\u003c/h2\u003e \u003cp\u003eFour-week-old Arabidopsis rosette leaves were infiltrated with \u003cem\u003ePseudomonas syringae DC 3000 pv. tomato (pstDC3000)\u003c/em\u003e suspension in 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e at concentration of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e by needleless syringe. Bacterial infection was measured at 0 and 3 dpi by harvesting 5 mm leaf discs, macerating them in 10 mM MgCl2, and inoculating their serially diluted suspension onto a \u003cem\u003ePseudomonas\u003c/em\u003e agar plate with antibiotic selection. Bacterial infection was estimated through the number of colonies and expressed as Log\u003csub\u003e10\u003c/sub\u003eCFU cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e [87].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eRNA sequencing analyses\u003c/h2\u003e \u003cp\u003eRNA was isolated from the 6-week side stem of the old side stem using TRIzol (15596026, Thermo Fisher Scientific Invitrogen), and RNA sequencing was done on the Illumina Novaseq 6000 Platform. Significantly differentially expressed genes were analyzed by the DSEq2 tool with Log2Fold change1, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene ontology analysis was done using ShinyGO 0.77 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eXylo oligosaccharides infiltration and estimation of reactive oxygen species (ROS)\u003c/h2\u003e \u003cp\u003eFour-week-old Arabidopsis rosette leaves were infiltrated with supernatant from 200 \u0026micro;g/ml xylanase (GH11)- digested stem samples, calibrated to total sugar 200 \u0026micro;g/ml. Treated leaves were collected at 30 min, checked for ROS accumulation using the diaminobenzidine (DAB) assay, and checked for the following genes (Table S5). Briefly, mock- and treated-leaves were incubated in DAB (17076, SRL, India) staining solution (1 mg/ml in 10 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) for 8 h. After that, the DAB staining solution was replaced with a bleaching solution (ethanol: acetic acid: glycerol, 3:1:1), and the sample was placed in boiling water for 15 min. Then, the old bleaching solution was replaced by a fresh bleaching solution, and DAB accumulation was quantified using Fiji (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://fiji.sc/\u003c/span\u003e\u003cspan address=\"https://fiji.sc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [88].\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors gave consent to publish the work\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data are provided in the manuscript, and we have deposited transcriptomic data on IBDC webserver (https://ibdc.dbt.gov.in/) with accession ID -\u003cstrong\u003eINRP000631.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Regional Centre for Biotechnology and DST-INSPIRE faculty for funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAMS performed, most of the experiments compiled and analyzed all the data. BPD and RKS performed total sugar and xylose content analysis respectively. NA helped in fermentation analysis and interpretation. EM provided xylanases expressing lines, involved discussion and writing of the manuscript. AMS and PA-MP wrote the manuscript with input from all the authors. PA-MP conceptualized, designed and secured the funding for the project. All authors read and agree to publish the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank National Institute of Plant Genome Research, New Delhi for Metabolomic Facility, Regional Centre for Biotechnology, Faridabad and Advanced Technology Platform Centre, Faridabad for genomics and mass spectroscopy facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChebli, Y. and A. Geitmann, \u003cem\u003eCellular growth in plants requires regulation of cell wall biochemistry\u003c/em\u003e, in \u003cem\u003eCurrent Opinion in Cell Biology\u003c/em\u003e. 2017, Elsevier Ltd. p. 28-35.\u003c/li\u003e\n\u003cli\u003eBellincampi, D., F. Cervone, and V. Lionetti, \u003cem\u003ePlant cell wall dynamics and wall-related susceptibility in plant-pathogen interactions\u003c/em\u003e, in \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e. 2014, Frontiers Media S.A.\u003c/li\u003e\n\u003cli\u003eTimell, T.E., \u003cem\u003eRecent progress in the chemistry of wood hemicelluloses.\u003c/em\u003e Wood Science and Technology March 1967. \u003cstrong\u003eVolume 1\u003c/strong\u003e: p. 45\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003eScheller, H.V. and P. Ulvskov, \u003cem\u003eHemicelluloses.\u003c/em\u003e Annual Review of Plant Biology, 2010. \u003cstrong\u003e61\u003c/strong\u003e: p. 263-289.\u003c/li\u003e\n\u003cli\u003eAspinall, G.O., \u003cem\u003eChemistry of Cell Wall Polysaccharides\u003c/em\u003e, in \u003cem\u003eCarbohydrates: Structure and Function\u003c/em\u003e. 1980, Elsevier. p. 473-500.\u003c/li\u003e\n\u003cli\u003eGao, Y., et al., \u003cem\u003eA grass-specific cellulose\u0026ndash;xylan interaction dominates in sorghum secondary cell walls.\u003c/em\u003e Nature Communications, 2020. \u003cstrong\u003e11\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eSaijonkari-Pahkala, K., \u003cem\u003eNon-wood plants as raw material for pulp and paper\u003c/em\u003e. 2001.\u003c/li\u003e\n\u003cli\u003eRennie, E.A. and H.V. Scheller, \u003cem\u003eXylan biosynthesis\u003c/em\u003e, in \u003cem\u003eCurrent Opinion in Biotechnology\u003c/em\u003e. 2014. p. 100-107.\u003c/li\u003e\n\u003cli\u003ePe\u0026ntilde;a, M.J., et al., \u003cem\u003eArabidopsis irregular xylem8 and irregular xylem9: Implications for the complexity of glucuronoxylan biosynthesis.\u003c/em\u003e Plant Cell, 2007. \u003cstrong\u003e19\u003c/strong\u003e(2): p. 549-563.\u003c/li\u003e\n\u003cli\u003eGiummarella, N. and M. Lawoko, \u003cem\u003eStructural Basis for the Formation and Regulation of Lignin-Xylan Bonds in Birch.\u003c/em\u003e ACS Sustainable Chemistry and Engineering, 2016. \u003cstrong\u003e4\u003c/strong\u003e(10): p. 5319-5326.\u003c/li\u003e\n\u003cli\u003eGrantham, N.J., et al., \u003cem\u003eAn even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls.\u003c/em\u003e Nature Plants, 2017. \u003cstrong\u003e3\u003c/strong\u003e(11): p. 859-865.\u003c/li\u003e\n\u003cli\u003eSingh, D., et al., \u003cem\u003eCharacterization of Arabidopsis eskimo1 reveals a metabolic link between xylan O-acetylation and aliphatic glucosinolate metabolism.\u003c/em\u003e Physiologia Plantarum, 2024. \u003cstrong\u003e176\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eXiong, G., K. Cheng, and M. Pauly, \u003cem\u003eXylan O-acetylation impacts xylem development and enzymatic recalcitrance as indicated by the arabidopsis mutant tbl29\u003c/em\u003e, in \u003cem\u003eMolecular Plant\u003c/em\u003e. 2013, Oxford University Press. p. 1373-1375.\u003c/li\u003e\n\u003cli\u003ePawar, P.M.A., et al., \u003cem\u003eExpression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.\u003c/em\u003e Plant Biotechnology Journal, 2016. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 387-397.\u003c/li\u003e\n\u003cli\u003eChaudhari, A.A., et al., \u003cem\u003eModifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility.\u003c/em\u003e Biotechnology for Biofuels and Bioproducts, 2024. \u003cstrong\u003e17\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003ePogorelko, G., et al., \u003cem\u003eArabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens.\u003c/em\u003e Plant Physiology, 2013. \u003cstrong\u003e162\u003c/strong\u003e(1): p. 9-23.\u003c/li\u003e\n\u003cli\u003eSun, G., et al., \u003cem\u003eOligosaccharide elicitors in plant immunity: Molecular mechanisms and disease resistance strategies.\u003c/em\u003e Plant Communications, 2025. \u003cstrong\u003e6\u003c/strong\u003e(12): p. 101469.\u003c/li\u003e\n\u003cli\u003eDewangan, B.P., et al., \u003cem\u003eXylobiose treatment triggers a defense-related response and alters cell wall composition.\u003c/em\u003e Plant Molecular Biology, 2023.\u003c/li\u003e\n\u003cli\u003eMelida, H., et al., \u003cem\u003eArabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating Disease Resistance.\u003c/em\u003e Front Plant Sci, 2020. \u003cstrong\u003e11\u003c/strong\u003e: p. 1210.\u003c/li\u003e\n\u003cli\u003ede Azevedo Souza, C., et al., \u003cem\u003eCellulose-derived oligomers act as damage-associated molecular patterns and trigger defense-like responses.\u003c/em\u003e Plant Physiology, 2017. \u003cstrong\u003e173\u003c/strong\u003e(4): p. 2383-2398.\u003c/li\u003e\n\u003cli\u003eRzemieniewski, J. and M. Stegmann, \u003cem\u003eRegulation of pattern-triggered immunity and growth by phytocytokines\u003c/em\u003e, in \u003cem\u003eCurrent Opinion in Plant Biology\u003c/em\u003e. 2022, Elsevier Ltd.\u003c/li\u003e\n\u003cli\u003eWang, H., et al., \u003cem\u003eTranscriptomic Insights into the Degree of Polymerization-Dependent Bioactivity of Xylo-Oligosaccharides.\u003c/em\u003e Plants, 2025. \u003cstrong\u003e14\u003c/strong\u003e(19).\u003c/li\u003e\n\u003cli\u003eZhao, Q. and R.A. Dixon, \u003cem\u003eAltering the cell wall and its impact on plant disease: From forage to bioenergy.\u003c/em\u003e Annual Review of Phytopathology, 2014. \u003cstrong\u003e52\u003c/strong\u003e: p. 69-91.\u003c/li\u003e\n\u003cli\u003eSivan, P., et al., \u003cem\u003eModification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification.\u003c/em\u003e Plant Biotechnology Journal, 2025. \u003cstrong\u003e23\u003c/strong\u003e(1): p. 174-197.\u003c/li\u003e\n\u003cli\u003eSwaminathan, S., et al., \u003cem\u003eCoexpression of fungal cell wall-modifying enzymes reveals their additive impact on arabidopsis resistance to the fungal pathogen, Botrytis cinerea.\u003c/em\u003e Biology, 2021. \u003cstrong\u003e10\u003c/strong\u003e(10).\u003c/li\u003e\n\u003cli\u003eBarbut, F.R., et al., \u003cem\u003eIntegrity of xylan backbone affects plant responses to drought.\u003c/em\u003e Frontiers in Plant Science, 2024. \u003cstrong\u003e15\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eDonev, E.N., et al., \u003cem\u003eGlucuronoyl Esterase of Pathogenic Phanerochaete carnosa Induces Immune Responses in Aspen Independently of Its Enzymatic Activity.\u003c/em\u003e Plant Biotechnology Journal, 2025.\u003c/li\u003e\n\u003cli\u003eMarasinghe, S.D., et al., \u003cem\u003eCharacterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass.\u003c/em\u003e Microbial Cell Factories, 2021. \u003cstrong\u003e20\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eV\u0026aacute;rnai, A., et al., \u003cem\u003eEffects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions.\u003c/em\u003e Biotechnology for Biofuels, 2014. \u003cstrong\u003e7\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eZhang, J., et al., \u003cem\u003eThe role of acetyl xylan esterase in the solubilization of xylan and enzymatic hydrolysis of wheat straw and giant reed.\u003c/em\u003e Biotechnology for Biofuels, 2011. \u003cstrong\u003e4\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eUrbancsok, J., et al., \u003cem\u003eWood-specific modification of glucuronoxylan can enhance growth in Populus.\u003c/em\u003e Journal of Experimental Botany, 2026. \u003cstrong\u003e77\u003c/strong\u003e(2): p. 445-462.\u003c/li\u003e\n\u003cli\u003eRatke, C., et al., \u003cem\u003ePopulus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification.\u003c/em\u003e Plant Biotechnology Journal, 2015. \u003cstrong\u003e13\u003c/strong\u003e(1): p. 26-37.\u003c/li\u003e\n\u003cli\u003eChini, A., et al., \u003cem\u003eThe JAZ family of repressors is the missing link in jasmonate signalling.\u003c/em\u003e Nature, 2007. \u003cstrong\u003e448\u003c/strong\u003e(7154): p. 666-671.\u003c/li\u003e\n\u003cli\u003eHoo, S.C., et al., \u003cem\u003eRegulation and function of arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory.\u003c/em\u003e Plant Physiology, 2008. \u003cstrong\u003e146\u003c/strong\u003e(3): p. 952-964.\u003c/li\u003e\n\u003cli\u003eThines, B., et al., \u003cem\u003eJAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling.\u003c/em\u003e Nature, 2007. \u003cstrong\u003e448\u003c/strong\u003e(7154): p. 661-665.\u003c/li\u003e\n\u003cli\u003ePandey, S.P. and I.E. Somssich, \u003cem\u003eThe role of WRKY transcription factors in plant immunity.\u003c/em\u003e Plant Physiology, 2009. \u003cstrong\u003e150\u003c/strong\u003e(4): p. 1648-1655.\u003c/li\u003e\n\u003cli\u003eSeo, P.J. and C.M. Park, \u003cem\u003eMYB96-mediated abscisic acid signals induce pathogen resistance response by promoting salicylic acid biosynthesis in Arabidopsis.\u003c/em\u003e New Phytologist, 2010. \u003cstrong\u003e186\u003c/strong\u003e(2): p. 471-483.\u003c/li\u003e\n\u003cli\u003eXu, X., et al., \u003cem\u003ePhysical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors.\u003c/em\u003e Plant Cell, 2006. \u003cstrong\u003e18\u003c/strong\u003e(5): p. 1310-1326.\u003c/li\u003e\n\u003cli\u003eMolina, A., et al., \u003cem\u003ePlant cell walls: source of carbohydrate-based signals in plant-pathogen interactions\u003c/em\u003e, in \u003cem\u003eCurrent Opinion in Plant Biology\u003c/em\u003e. 2024, Elsevier Ltd.\u003c/li\u003e\n\u003cli\u003eZang, H., et al., \u003cem\u003eMannan oligosaccharides trigger multiple defence responses in rice and tobacco as a novel danger-associated molecular pattern.\u003c/em\u003e Molecular Plant Pathology, 2019. \u003cstrong\u003e20\u003c/strong\u003e(8): p. 1067-1079.\u003c/li\u003e\n\u003cli\u003eBusse-Wicher, M., et al., \u003cem\u003eThe pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.\u003c/em\u003e Plant Journal, 2014. \u003cstrong\u003e79\u003c/strong\u003e(3): p. 492-506.\u003c/li\u003e\n\u003cli\u003eKang, X., et al., \u003cem\u003eLignin-polysaccharide interactions in plant secondary cell walls revealed by solid-state NMR.\u003c/em\u003e Nature Communications, 2019. \u003cstrong\u003e10\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eSimmons, T.J., et al., \u003cem\u003eFolding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR.\u003c/em\u003e Nature Communications, 2016. \u003cstrong\u003e7\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eYuan, Y., et al., \u003cem\u003eRoles of Arabidopsis TBL34 and TBL35 in xylan acetylation and plant growth.\u003c/em\u003e Plant Science, 2016. \u003cstrong\u003e243\u003c/strong\u003e: p. 120-130.\u003c/li\u003e\n\u003cli\u003eYuan, Y., et al., \u003cem\u003eThe Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan.\u003c/em\u003e Plant and Cell Physiology, 2013. \u003cstrong\u003e54\u003c/strong\u003e(7): p. 1186-1199.\u003c/li\u003e\n\u003cli\u003eZhu, X.F., et al., \u003cem\u003eTRICHOME BIREFRINGENCE-LIKE27 Affects Aluminum Sensitivity by Modulating the O-Acetylation of Xyloglucan and Aluminum-Binding Capacity in Arabidopsis \u003c/em\u003ePlant Physiology, 2014. \u003cstrong\u003e166\u003c/strong\u003e(1): p. 181-189.\u003c/li\u003e\n\u003cli\u003eZhong, R., D. Cui, and Z.-H. Ye, \u003cem\u003eRegiospecific Acetylation of Xylan is Mediated by a Group of DUF231-Containing O-Acetyltransferases.\u003c/em\u003e Plant and Cell Physiology, 2017. \u003cstrong\u003e58\u003c/strong\u003e(12): p. 2126-2138.\u003c/li\u003e\n\u003cli\u003eYuan, Y., et al., \u003cem\u003eTBL3 and TBL31, Two Arabidopsis DUF231 Domain Proteins, are Required for 3- O -Monoacetylation of Xylan.\u003c/em\u003e Plant and Cell Physiology, 2016. \u003cstrong\u003e57\u003c/strong\u003e(1): p. 35-45.\u003c/li\u003e\n\u003cli\u003eXiong, G., K. Cheng, and M. Pauly, \u003cem\u003eXylan O-Acetylation Impacts Xylem Development and Enzymatic Recalcitrance as Indicated by the \u0026lt;em\u0026gt;Arabidopsis\u0026lt;/em\u0026gt; Mutant \u0026lt;em\u0026gt;tbl29\u0026lt;/em\u0026gt;.\u003c/em\u003e Molecular Plant, 2013. \u003cstrong\u003e6\u003c/strong\u003e(4): p. 1373-1375.\u003c/li\u003e\n\u003cli\u003eMorcillo, R.J.L., et al. \u003cem\u003ePlant Transcriptome Reprograming and Bacterial Extracellular Metabolites Underlying Tomato Drought Resistance Triggered by a Beneficial Soil Bacteria\u003c/em\u003e. Metabolites, 2021. \u003cstrong\u003e11\u003c/strong\u003e, 369 DOI: 10.3390/metabo11060369.\u003c/li\u003e\n\u003cli\u003eUrbanowicz, B.R., et al., \u003cem\u003e4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein.\u003c/em\u003e Proceedings of the National Academy of Sciences of the United States of America, 2012. \u003cstrong\u003e109\u003c/strong\u003e(35): p. 14253-14258.\u003c/li\u003e\n\u003cli\u003eVanholme, R., et al., \u003cem\u003eLignin biosynthesis and structure.\u003c/em\u003e Plant Physiology, 2010. \u003cstrong\u003e153\u003c/strong\u003e(3): p. 895-905.\u003c/li\u003e\n\u003cli\u003eBoerjan, W., J. Ralph, and M. Baucher, \u003cem\u003eLignin Biosynthesis\u003c/em\u003e, in \u003cem\u003eAnnual Review of Plant Biology\u003c/em\u003e. 2003. p. 519-546.\u003c/li\u003e\n\u003cli\u003eKushwah, S., et al., \u003cem\u003eArabidopsis XTH4 and XTH9 contribute to wood cell expansion and secondary wall formation1[Open].\u003c/em\u003e Plant Physiology, 2020. \u003cstrong\u003e182\u003c/strong\u003e(4): p. 1946-1965.\u003c/li\u003e\n\u003cli\u003eRose, J.K.C., et al., \u003cem\u003eThe XTH Family of Enzymes Involved in Xyloglucan Endotransglucosylation and Endohydrolysis: Current Perspectives and a New Unifying Nomenclature\u003c/em\u003e, in \u003cem\u003ePlant Cell Physiol\u003c/em\u003e. 2002. p. 1421-1435.\u003c/li\u003e\n\u003cli\u003eZhong, R. and Z.H. Ye, \u003cem\u003eSecondary cell walls: Biosynthesis, patterned deposition and transcriptional regulation.\u003c/em\u003e Plant and Cell Physiology, 2015. \u003cstrong\u003e56\u003c/strong\u003e(2): p. 195-214.\u003c/li\u003e\n\u003cli\u003eHamann, T., \u003cem\u003eThe plant cell wall integrity maintenance mechanism - Concepts for organization and mode of action\u003c/em\u003e, in \u003cem\u003ePlant and Cell Physiology\u003c/em\u003e. 2015, Oxford University Press. p. 215-223.\u003c/li\u003e\n\u003cli\u003eHamann, T., \u003cem\u003eThe plant cell wall integrity maintenance mechanism - A case study of a cell wall plasma membrane signaling network\u003c/em\u003e, in \u003cem\u003ePhytochemistry\u003c/em\u003e. 2015, Elsevier Ltd. p. 100-109.\u003c/li\u003e\n\u003cli\u003eJ\u0026ouml;nsson, L.J., B. Alriksson, and N.O. Nilvebrant, \u003cem\u003eBioconversion of lignocellulose: Inhibitors and detoxification\u003c/em\u003e, in \u003cem\u003eBiotechnology for Biofuels\u003c/em\u003e. 2013.\u003c/li\u003e\n\u003cli\u003ePawar, P.M.A., et al., \u003cem\u003eAcetylation of woody lignocellulose: Significance and regulation\u003c/em\u003e, in \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e. 2013, Frontiers Research Foundation.\u003c/li\u003e\n\u003cli\u003eDe Lorenzo, G. and F. Cervone, \u003cem\u003ePlant immunity by damage-associated molecular patterns (DAMPs).\u003c/em\u003e Essays in Biochemistry, 2022. \u003cstrong\u003e66\u003c/strong\u003e(5): p. 459-469.\u003c/li\u003e\n\u003cli\u003eH\u0026eacute;maty, K., et al., \u003cem\u003eA Receptor-like Kinase Mediates the Response of Arabidopsis Cells to the Inhibition of Cellulose Synthesis.\u003c/em\u003e Current Biology, 2007. \u003cstrong\u003e17\u003c/strong\u003e(11): p. 922-931.\u003c/li\u003e\n\u003cli\u003eKohorn, B.D. and S.L. Kohorn, \u003cem\u003eThe cell wall-associated kinases, WAKs, as pectin receptors\u003c/em\u003e, in \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e. 2012, Frontiers Research Foundation.\u003c/li\u003e\n\u003cli\u003eVan der Does, D., et al., \u003cem\u003eThe Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses.\u003c/em\u003e PLoS Genetics, 2017. \u003cstrong\u003e13\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003ePontiggia, D., et al., \u003cem\u003eDampening the DAMPs: How Plants Maintain the Homeostasis of Cell Wall Molecular Patterns and Avoid Hyper-Immunity\u003c/em\u003e, in \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e. 2020, Frontiers Media S.A.\u003c/li\u003e\n\u003cli\u003eClaverie, J., et al., \u003cem\u003eThe cell wall-derived xyloglucan is a new DAMP triggering plant immunity in vitis vinifera and arabidopsis thaliana.\u003c/em\u003e Frontiers in Plant Science, 2018. \u003cstrong\u003e871\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eFerrari, S., et al., \u003cem\u003eOligogalacturonides: Plant damage-associated molecular patterns and regulators of growth and development\u003c/em\u003e, in \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e. 2013, Frontiers Research Foundation.\u003c/li\u003e\n\u003cli\u003eDegli Esposti, C., et al., \u003cem\u003eCell wall bricks of defence: the case study of oligogalacturonides\u003c/em\u003e, in \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e. 2025, Frontiers Media SA.\u003c/li\u003e\n\u003cli\u003eEscudero, V., et al., \u003cem\u003eAlteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the \u0026beta;-subunit of the heterotrimeric G-protein.\u003c/em\u003e Plant Journal, 2017. \u003cstrong\u003e92\u003c/strong\u003e(3): p. 386-399.\u003c/li\u003e\n\u003cli\u003eChoi, H.W. and D.F. Klessig, \u003cem\u003eDAMPs, MAMPs, and NAMPs in plant innate immunity\u003c/em\u003e, in \u003cem\u003eBMC Plant Biology\u003c/em\u003e. 2016, BioMed Central Ltd.\u003c/li\u003e\n\u003cli\u003eDuran-Flores, D. and M. Heil, \u003cem\u003eSources of specificity in plant damaged-self recognition\u003c/em\u003e, in \u003cem\u003eCurrent Opinion in Plant Biology\u003c/em\u003e. 2016, Elsevier Ltd. p. 77-87.\u003c/li\u003e\n\u003cli\u003eMolina, A., et al., \u003cem\u003eArabidopsis cell wall composition determines disease resistance specificity and fitness.\u003c/em\u003e 2021. \u003cstrong\u003e118\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eRovenich, H., A. Zuccaro, and B.P.H.J. Thomma, \u003cem\u003eConvergent evolution of filamentous microbes towards evasion of glycan-triggered immunity\u003c/em\u003e, in \u003cem\u003eNew Phytologist\u003c/em\u003e. 2016, Blackwell Publishing Ltd. p. 896-901.\u003c/li\u003e\n\u003cli\u003eMcCleary, B.V., et al., \u003cem\u003eHydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by \u0026beta;-xylanase, \u0026alpha;-l-arabinofuranosidase and \u0026beta;-xylosidase.\u003c/em\u003e Carbohydrate Research, 2015. \u003cstrong\u003e407\u003c/strong\u003e: p. 79-96.\u003c/li\u003e\n\u003cli\u003eM\u0026eacute;lida, H., et al., \u003cem\u003eArabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating Disease Resistance.\u003c/em\u003e Frontiers in Plant Science, 2020. \u003cstrong\u003e11\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eVardakou, M., et al., \u003cem\u003eUnderstanding the Structural Basis for Substrate and Inhibitor Recognition in Eukaryotic GH11 Xylanases.\u003c/em\u003e Journal of Molecular Biology, 2008. \u003cstrong\u003e375\u003c/strong\u003e(5): p. 1293-1305.\u003c/li\u003e\n\u003cli\u003eDegli Esposti, C., et al., \u003cem\u003eCell wall bricks of defence: the case study of oligogalacturonides.\u003c/em\u003e Frontiers in Plant Science, 2025. \u003cstrong\u003eVolume 16 - 2025\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eSalvati, A., et al., \u003cem\u003eBerberine bridge enzyme-like oxidases orchestrate homeostasis and signaling of oligogalacturonides in defense and upon mechanical damage.\u003c/em\u003e The Plant Journal, 2025. \u003cstrong\u003e122\u003c/strong\u003e(1): p. e70150.\u003c/li\u003e\n\u003cli\u003eRastogi, L., et al., \u003cem\u003eArabidopsis GELP7 functions as a plasma membrane-localized acetyl xylan esterase, and its overexpression improves saccharification efficiency.\u003c/em\u003e Plant Molecular Biology, 2022. \u003cstrong\u003e109\u003c/strong\u003e(6): p. 781-797.\u003c/li\u003e\n\u003cli\u003ePradhan Mitra, P. and D. Loqu\u0026eacute;, \u003cem\u003eHistochemical staining of Arabidopsis thaliana secondary cell wall elements.\u003c/em\u003e Journal of Visualized Experiments, 2014(87).\u003c/li\u003e\n\u003cli\u003eBiswal, A.K., et al., \u003cem\u003eAspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield.\u003c/em\u003e Biotechnology for Biofuels, 2014. \u003cstrong\u003e7\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eRastogi, L., et al., \u003cem\u003eArabidopsis \u0026lt;i\u0026gt;At\u0026lt;/i\u0026gt; GELP53 modulates polysaccharide acetylation and defense response through oligosaccharide-mediated signaling\u003c/em\u003e. 2024.\u003c/li\u003e\n\u003cli\u003eUpdegraff, D.M., \u003cem\u003eSemimicro Determination of Cellulose in Biological Materials\u003c/em\u003e. 1969. p. 420-424.\u003c/li\u003e\n\u003cli\u003eFoster, C.E., T.M. Martin, and M. Pauly, \u003cem\u003eComprehensive compositional analysis of plant cell walls (Lignocellulosic biomass) part I: Lignin.\u003c/em\u003e Journal of Visualized Experiments, 2010(37).\u003c/li\u003e\n\u003cli\u003ePattathil, S., et al., \u003cem\u003eImmunological approaches to plant cell wall and biomass characterization: Glycome profiling.\u003c/em\u003e Methods in Molecular Biology, 2012. \u003cstrong\u003e908\u003c/strong\u003e: p. 61-72.\u003c/li\u003e\n\u003cli\u003eAcker, R.V., et al., \u003cem\u003eSaccharification Protocol for Small-scale Lignocellulosic Biomass Samples to Test Processing of Cellulose into Glucose\u003c/em\u003e, in \u003cem\u003eIss\u003c/em\u003e. 1701.\u003c/li\u003e\n\u003cli\u003eRoy, A., et al., \u003cem\u003eKappaphycus alvarezii-derived formulations enhance salicylic acid-mediated anti-bacterial defenses in Arabidopsis thaliana and rice.\u003c/em\u003e Journal of Applied Phycology, 2022. \u003cstrong\u003e34\u003c/strong\u003e(1): p. 679-695.\u003c/li\u003e\n\u003cli\u003eDaudi, A., J.A. O\u0026apos;Brien, and B.P. Author, \u003cem\u003eDetection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves HHS Public Access Author manuscript\u003c/em\u003e, in \u003cem\u003eBio Protoc\u003c/em\u003e. 2012.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable-1.\u0026nbsp;\u003c/strong\u003eNon-cellulosic Monosaccharide composition (mol %) through derivatization followed by Gas Chromatography-Mass Spectrometry. Data presented as mean \u0026plusmn; standard deviation (SD) from 3 biological replicates. Asterisk represents significant differences using Student\u0026rsquo;s t-test at *p \u0026le; 0.1, ** p \u0026le; 0.05, ***p \u0026le; 0.001.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"621\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003exylose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003earabinose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003erhamnose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003efucose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emannose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eglucose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e46.84 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e10.28 \u0026plusmn; 0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4.61 \u0026plusmn; 0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e2.62 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e12.66 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6.51 \u0026plusmn; 0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35S:AXE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e50.42 \u0026plusmn; 0.47 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.01 \u0026plusmn; 0.15 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e4.07 \u0026plusmn; 0.08 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.05 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e11.92 \u0026plusmn; 0.07 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e5.87 \u0026plusmn; 0.12\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35S:XYL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e54.88 \u0026plusmn; 1.87 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.01 \u0026plusmn; 0.53 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e3.79 \u0026plusmn; 0.11 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e2.21 \u0026plusmn; 0.13 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e10.74 \u0026plusmn; 0.35 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6.09 \u0026plusmn; 0.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35S:AXE/XYL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e49.12 \u0026plusmn; 0.39\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.3 \u0026plusmn; 0.13 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e3.81 \u0026plusmn; 0.29 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e2.19 \u0026plusmn; 0.14 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.9 \u0026plusmn; 0.28 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6.23 \u0026plusmn; 0.23\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWP:AXE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e55.99 \u0026plusmn; 2.09 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6.87 \u0026plusmn; 0.89 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e2.64 \u0026plusmn; 1.37 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e2.81 \u0026plusmn; 0.2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.59 \u0026plusmn; 0.86 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e6.7 \u0026plusmn; 1.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWP:XYL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e44.83 \u0026plusmn; 2.45\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.57 \u0026plusmn; 0.66\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e5.53 \u0026plusmn; 2.57\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e3.39 \u0026plusmn; 0.17 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e12.18 \u0026plusmn; 1.57\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e9.14 \u0026plusmn; 1.14 *\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWP:AXE/XYL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e34.5 \u0026plusmn; 0.9 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e14.58 \u0026plusmn; 2 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e10.27 \u0026plusmn; 2.67 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e5.21 \u0026plusmn; 0.24 ***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e15.21 \u0026plusmn; 0.41 *\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 89px;\"\u003e\n \u003cp\u003e10.25 \u0026plusmn; 0.28 **\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acetyl xylan esterase, xylanase, xylan modification, xylooligosaccharide, immunity, elicitor","lastPublishedDoi":"10.21203/rs.3.rs-9277431/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9277431/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eXylan, a major component of the secondary cell wall in dicots, is often recalcitrant due to its acetylation and interaction with cellulose and lignin. To fine-tune the xylan structure and improve the processing of lignocellulosic biomass, overexpression of cell wall-degrading microbial enzymes is a viable option that can enhance cell wall properties and immunity. Here, we expressed a glycosyl hydrolase (GH10) xylanase (XYL) from \u003cem\u003eAspergillus nidulans\u003c/em\u003e and a carbohydrate esterase (CE)1 acetyl xylan esterase (AXE) from \u003cem\u003eAspergillus niger\u003c/em\u003e, or both enzymes simultaneously (AXE/XYL), under the control of the constitutive Cauliflower mosaic virus 35S promoter (35S) and the woody-tissue-specific \u003cem\u003ePopulus trichocarpa\u003c/em\u003e GT43B promoter (WP) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and studied their effects on the cell wall, saccharification properties, and biotic resistance.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTransgenic WP:AXE/XYL lines exhibited an irregular xylem phenotype and compromised deposition of cell wall components, which correlated with downregulation of the responsible genes as revealed by RNA sequencing analysis. In contrast, 35S:AXE/XYL plants did not show any deformities, possibly because XYL expression was lower than in WP:AXE/XYL or due to variation in transgene expression. Biochemical analyses revealed reduced acetyl content in 35S:AXE and 35S:XYL/AXE lines, while total xylan content was increased in 35S:XYL, 35S:AXE, and 35S:XYL/AXE expressing plants. Notably, cellulose, xylan digestibility, and ethanol production were highest in the 35S:XYL/AXE line, surpassing the parental lines. Moreover, the 35S:XYL/AXE lines showed enhanced resistance to \u003cem\u003ePseudomonas syringae\u003c/em\u003e. Comparative RNA sequencing of xylobiose-treated and 35S:AXE/XYL plants revealed altered expression of defense-related genes. This was supported by elicitor assays, which demonstrated that xylo-oligosaccharides present in the 35S:XYL/AXE lines can promote the activation of immune marker genes along with increased accumulation of reactive oxygen species.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, simultaneous hydrolysis of the xylan backbone and its acetylation represents a promising approach to boost both lignocellulosic biomass quality and plant immunity.\u003c/p\u003e","manuscriptTitle":"Enhanced cell wall digestibility and immunity in Arabidopsis through targeted modification of xylan structure by heterologous expression of acetyl xylan esterase and xylanases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-17 16:35:26","doi":"10.21203/rs.3.rs-9277431/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-11T19:03:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T03:44:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T02:27:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51670514206264507567412145516693521206","date":"2026-04-10T12:34:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281337043226561385261698101848560451521","date":"2026-04-10T10:51:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180669290928275613235407944382515061489","date":"2026-04-10T04:00:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-10T03:54:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-10T03:32:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-09T18:23:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-04-09T17:39:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"437261f4-24e7-4b7c-9001-a6ebac843f62","owner":[],"postedDate":"April 17th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-11T19:03:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T03:44:54+00:00","index":27,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T02:27:21+00:00","index":25,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T19:10:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-17 16:35:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9277431","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9277431","identity":"rs-9277431","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}