The AkCSLA3-AkMSR1 module mediates Konjac glucomannan biosynthesis in Amorphophallus konjac

The AkCSLA3-AkMSR1 module mediates Konjac glucomannan biosynthesis in Amorphophallus konjac | 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 The AkCSLA3-AkMSR1 module mediates Konjac glucomannan biosynthesis in Amorphophallus konjac Yumei Shi, Dongbao Li, Honglong Chu, Xaiosong Gu, Changxin Luo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9274000/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Konjac glucomannan (KGM) is a high-viscosity, water-soluble dietary fiber that accumulates predominantly in the corms of Amorphophallus konjac ( A. konjac ). Due to its excellent gel-forming and water-retention properties, KGM is widely used in the food industry. The biosynthesis of KGM involves the coordinated action of cellulose synthase-like (CSL) enzymes and mannan synthesis-related (MSR) proteins. However, the regulatory mechanisms and functional interactions between these components remain poorly understood in A. konjac. Results In this study, we systematically characterized the AkCSLA gene family in A. konjac and identified 11 family members. Transcriptomic analysis revealed that AkCSLA2 and AkCSLA3 were highly expressed during the corm expansion and maturation stages, which correspond to the periods of active KGM accumulation. Subcellular localization assays in Nicotiana benthamiana showed that both AkCSLA2 and AkCSLA3 proteins localized to the plasma membrane, endoplasmic reticulum, and Golgi apparatus. Protein interaction analyses using yeast two-hybrid and co-immunoprecipitation assays demonstrated that AkMSR1, a homologue of Arabidopsis MSR1, specifically interacted with AkCSLA3, but not with AkCSLA2. Functional studies in Pichia pastoris revealed that co-expression of AkCSLA3 and AkMSR1 significantly increased the production of mannose and glucose, with mannose content elevated more than threefold compared to expression of AkCSLA3 alone. Conclusion This study identifies the AkCSLA3-AkMSR1 module as a crucial regulatory mechanism in KGM biosynthesis, wherein AkMSR1 acts as a specific cofactor to enhance the glucomannan synthesis mediated by AkCSLA3. This research provides novel insights into the molecular regulation of KGM biosynthesis and presents potential targets for the genetic enhancement of KGM content in A. konjac. Konjac glucomannan Cellulose synthase-like 3 A. konjac Mannan synthesis-related Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Glucomannan is a soluble dietary fiber polysaccharide known for its high viscosity. It is predominantly found in the roots, stems, leaves, and other parts of plants, and also in the cell walls of microorganisms, including bacteria, fungi, and yeasts. Konjac glucomannan (KGM) is the main constituent of konjac polysaccharide, and the most extensively studied form of glucomannan to date [ 1 , 2 ]. KGM is primarily synthesized and accumulates in the corms of Amorphophallus. It is an edible, perennial herbaceous plant belonging to the genus Amorphophallus within the Araceae family. The corms of Amorphophallus grow underground and represent the main edible parts of the plants. The corms are rich in KGM, with contents exceeding 50% by weight [ 3 ]. Dissection of the corm tissues of Amorphophallus reveals the presence of numerous distinct, colorless, and bright particles, with diameters of approximately 1 mm, known as glucomannan cells. The deposition of KGM within these glucomannan cells is subject to temporal regulation, with its expression increasing towards the end of each vegetative growth cycle [ 4 ]. KGM is a water-soluble, non-ionic polysaccharide that is primarily composed of D-mannose and D-glucose units linked via β-1, 4-glycosidic bonds in a 1:1.6 ratio. The high molecular weight of KGM and the interactions between its monosaccharide units contribute to its high viscosity, which enhances its gel-formation and water retention properties, making it suitable for applications in the food sector [ 5 , 6 ]. KGM is a high-density, branched macromolecular polymer, and its interactions with starch can potentially modulate its properties. Blending with starch significantly enhances the viscoelasticity of KGM, retards the retrogradation of starch, and promotes the formation of stable gels. Moreover, KGM is capable of decreasing the dimensions of gelatinized starch granules. Earlier research utilizing low-field nuclear magnetic resonance has shown that KGM can impede the swelling and gelatinization processes of native starch, competing with corn starch for water absorption [ 7 – 9 ]. Research has further shown that KGM improves the consistency and network density of mixtures containing pea and potato starch, as demonstrated by analyses using scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). A variety of studies have clarified the functions of KGM in maintaining the texture of starch-based food items and prolonging their shelf life [ 9 , 10 ]. However, current research on KGM primarily focuses on its structure, properties, and pharmacological characteristics, placing less emphasis on the regulatory mechanisms underlying KGM biosynthesis in A . konjac . The pathway of KGM biosynthesis is related to the pathways of sucrose metabolism and nucleotide sugar conversion, and is regulated by over 90 putative genes and their respective expression patterns [ 3 , 11 ]. Sucrose serves as a precursor for the synthesis of KGM and is degraded into fructose (Fru) and glucose (Glc) through catalysis by invertase (INV). A portion of the glucose undergoes glycolysis, while the remaining glucose and fructose units are converted into mannose-6-phosphate (Man-6-P). The phosphomannomutase (PMM) enzyme then converts Man-6-P to mannose-1-phosphate (Man-1-P), which is subsequently converted to its active GDP-mannose (GDP-Man) form by GDP-mannose pyrophosphorylase (GMPP). GDP-Man finally enters the Golgi apparatus, which mediates the synthesis and elongation of polysaccharide chains catalyzed by the cellulose synthase-like (CSL) enzymes, CSLA/CSLD [ 12 ]. CSL enzymes belong to the glycosyltransferase 2 (GT2) superfamily and catalyze the biosynthesis of plant hemicelluloses [ 13 ] (Richmond and Somerville 2000). They play critical roles in plant growth, immune responses to pathogens, and the enhancement of plant biomass [ 14 ]. Whole-genome studies on the CSL gene family in various plants have been conducted to date, including rice [ 15 ], flax [ 16 ], tea [ 17 ], strawberry [ 18 ], pineapple [ 19 ], and tomato [ 20 ]. The CSL gene family in plants consists of 30–50 members and is further categorized into nine subfamilies, namely, CSLA , CSLB , CSLC , CSLD , CSLE , CSLF , CSLG , CSLH , and CSLJ [ 21 – 25 ]. The gene subfamilies CSLA, CSLC, and CSLD are frequently identified across all terrestrial plants, whereas the other subfamilies are confined to particular plant groups. For example, the subfamilies CSLB and CSLG predominantly occur in non-grass species, while the CSLF, CSLH, and CSLJ subfamilies are exclusive to grasses [ 21 , 26 ]. In Arabidopsis thaliana ( Arabidopsis ), the model plant, researchers have identified a minimum of six subfamilies within the CSL gene family, namely CSLA, CSLB, CSLC, CSLD, CSLE, and CSLG, totaling 29 distinct members [ 13 , 15 ]. It has been demonstrated that CSLA proteins possess mannan and glucomannan synthase activities, and catalyze the biosynthesis of the main chains of mannan and glucomannan in plants [ 27 , 28 ]. A total of nine AtCSLA genes have been identified in Arabidopsis to date. Depending on the substrate, CSLA can synthesize different types of mannan molecules [ 27 , 29 – 31 ]. For instance, AtCSLA2, AtCSLA3, and AtCSLA9 are essential for the synthesis of glucomannan in the stems of Arabidopsis [ 30 ]. Previous studies have demonstrated that the disruption of AtCSLA2 reduced the mucus halo in the seeds of Arabidopsis , and the contents of mannosyl and glucose in the mucus decreased by 30% compared to those of the wild type (WT) [ 32 , 33 ]. The disruption of the CSLA7 gene leads to abnormal pollen development and embryonic lethality in Arabidopsis , suggesting that mannan polysaccharides have critical structural or signaling functions in plant embryonic development [ 29 ]. It has been previously demonstrated that AtCSLA9 localizes to the Golgi apparatus in Pichia sp., with its active site facing the Golgi lumen. Additionally, mutations in the AtCSLA9 gene have been shown to prevent Agrobacterium -mediated root transformations in rat4 Arabidopsis mutants [ 34 , 35 ]. Additionally, the putative GTs associated with mannan synthesis-related (MSR) enzymes are implicated in the synthesis of (gluco)mannan [ 36 , 37 ]. It has been reported that the AtMSR enzymes of Arabidopsis likely generate a primer to initiate the synthesis of heteromannan, enhance the activity of CSLA enzymes through glycosylation, or support the stability and functionality of CSLA enzymes via glycosylation-independent interactions [ 37 , 38 ]. The AtMSR1 and AtMSR2 enzymes of Arabidopsis have been shown to localize to the Golgi body. It has been reported that the levels of mannosyl reduced by approximately 40% and 50% in single and double Atmsr1 mutants, respectively, compared to those in the WT. In contrast, single Atmsr2 mutants exhibit no significant differences in the contents of mannosyl compared to those in the WT [ 37 ]. It has been reported that AtCSLA2 alone produces mannan, while the co-expression of AtCSLA2 and AtMSR1 synthesizes glucomannan in Pichia strains [ 30 , 32 , 33 , 38 ]. By analyzing monosaccharide and glycosidic linkages, a previous study demonstrated that the co-expression of AtMSR1 and the AkCSLA3 protein of Pichia upregulated the levels of glucomannan in the heteromannan-enriched fraction of Pichia by at least 70%, compared to that in the presence of AkCSLA3 alone [ 38 ]. To clarify the regulatory mechanism of KGM biosynthesis and the functional roles of key enzymes, this study investigated the expression patterns and subcellular localizations of AkCSLA2, AkCSLA3, and AkMSR1 in A. konjac . It mainly analyzed the interaction relationships between AkCSLA2, AkCSLA3 and AkMSR1, and further explored their roles in the process of KGM synthesis. The research results indicated that AkMSR1 could significantly promote the synthesis of KGM by AkCSLA3, thus laying a foundation for the study of KGM biosynthesis. Materials and methods Plant materials and growth conditions In this study, we utilized A. konjac and N. benthamiana as the experimental materials. The seeds of both species were obtained from our laboratory [ 39 ]. Initially, the A. konjac and N. benthamiana seeds were cultivated on Murashige and Skoog (MS) agar medium containing 0.8% agar for approximately 5 to 7 days, after which they were transplanted into soil. Throughout this process, the seedlings were maintained under a photoperiod of 16 hours of light and 8 hours of darkness at a temperature of 23°C and a relative humidity of 60%. Additionally, the seedlings were exposed to a light intensity of approximately 100 µmol·m⁻²·s⁻¹ for a duration of 3 to 4 weeks. RNA sequencing (RNA-seq), analysis of gene expression, and Phylogenetic analysis The protocols for RNA-seq and analysis of gene expression data were derived from the study by Gao et al. (2022) [ 3 ]. Briefly, the key genes involved in KGM biosynthesis were identified by time-course RNA-seq using samples obtained from the four developmental stages of corm, including the dormancy stage (stage 1), “changing head” stage (stage 2), corm expansion stage (stage 3), and maturity stage (stage 4). Each biological replicate consisted of 3–4 individuals, sampled from each developmental stage. The total RNA was extracted using an RNA extraction kit (Huayueyang, Beijing, China), and the RNA-seq libraries were constructed with a library preparation kit. Sequencing was performed using an Illumina HiSeq 4000 platform. The clean reads were mapped to the reference genome following quality control, and the gene expression levels were quantified based on the values of FPKM (Fragments Per Kilobase of exon model per Million mapped fragments). The DESeq2 software was used to analyze differential gene expression and perform principal component analysis (PCA), while the heatmaps were generated using TBtools, and gene ontology (GO) enrichment analysis was conducted using the clusterProfiler program. The gene expression profiles and phylogenetic analysis were analyzed as previously described [ 39 ]. Plasmid construction and analysis of subcellular localization The coding sequences (CDSs) for AkCSLA2, and AkCSLA3, along with their respective stop codons, were amplified and cloned into a pCAMBIA1300 vector, which also contained an eGFP fluorescent marker and the UBQ10 promoter sequence. Subsequently, the resulting plasmid was introduced into the GV3101 strain of Agrobacterium tumefaciens . The pCAMBIA1300-UBQ10-eGFP-AkCSLA2 and pCAMBIA1300-UBQ10-eGFP-AkCSLA3 constructs were subsequently introduced into the leaves of 4-week-old N . benthamiana plants using an infiltration buffer consisting of 10 mM MgCl 2 and 100 µM acetosyringone respectively. The N. benthamiana plants that were infiltrated were incubated under low light conditions for 48 hours. Following this incubation period, the fluorescent signals emitted by eGFP were observed using a confocal laser scanning microscope (CLSM600; SOPTOP). Yeast two-hybrid (Y2H) assays The AkMSR1 gene was inserted into the pMetYCgate destination vector for investigating the protein-protein interactions using Y2H assays. The AkCSLA2 and AkCSLA3 genes were cloned into the pPR3N vector [ 40 ]. Various combinations of bait and prey vectors, as well as the positive and negative control plasmids, were co-transformed into the NMY51 strain of yeast. The successfully co-transformed clones were capable of growing on the selective medium with moderate stringency lacking tryptophan and leucine (-Trp/-Leu). However, only the positive control and clones expressing the interacting proteins were able to grow on the high-stringency selective medium lacking histidine, tryptophan, leucine, and adenine (-His/-Trp/-Leu/-Ade). Protein extraction and co-immunoprecipitation (Co-IP) studies The leaves of N . benthamiana co-expressing the pCAMBIA1300-UBQ10-eGFP-AkCSLA3 and pBIB-UBQ10-AkMSR1-Flag constructs were collected 72 h post-infiltration. The total protein was extracted from these N . benthamiana leaves using an extraction buffer containing 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl 2 , 1% Triton, 1 mM EDTA, and a 1:100 dilution of a complete protease inhibitor cocktail (catalog number: 04693132001; Roche). The samples were solubilized with the extraction buffer, vortexed for 5 minutes, and subsequently centrifuged at 16,000 × g for 10 minutes at 4°C to remove the debris and obtain a clear solution. The resulting supernatant was then mixed with anti-FLAG beads (catalog number: A2220; Sigma) and incubated for 4 h at 4°C with gentle agitation. The beads were subsequently collected and washed five times with an extraction buffer containing 0.5% Triton X-100. The proteins bound to the beads were then boiled in 1×SDS loading buffer for 5 minutes, and subsequently analyzed by immunoblotting using anti-GFP (catalog number: SAB4301138; Sigma) and anti-FLAG-tag-HRP (catalog number: M20026M; Abmart) antibodies. Pichia Transformation The codon-optimized AkMSR1 and AkCSLA3 genes from A . konjac were synthesized by Jinkairui Biological Engineering Co., Ltd. (Wuhan, China). The genes were subsequently cloned into the donor helper plasmids using digestion/ligation and/or Gibson assembly methods. The gRNAs were designed using the Benchling CRISPR tool ( https://benchling.com ), and subsequently cloned into pHARS-gRNA digested with Bsa I. The DH5α gene of Escherichia coli was cultured in Luria-Bertani (LB) medium supplemented with 100 mg/L ampicillin. The GS115 strain of Pichia pastoris ( P . pastoris ) was used as the parent strain for studying the genomic integrations of two genes, and was routinely cultured in Yeast Extract Peptone Dextrose (YPD) medium containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose. The recombinant P . pastoris strains were screened in solid YPD medium supplemented with 100 mg/L Zeocin. A single colony was selected and inoculated into 5 mL of the YPD medium and incubated at 30°C with shaking at 250 rpm for 18 h to produce heteromannan. The culture was then transferred to a 500 mL shake flask containing 100 mL of YPD medium containing 20 g/L glucose. After incubation at 30°C for 24 h, methanol was added to the culture to achieve a final concentration of 2%. Fermentation was allowed to continue for an additional 72 h, following which the cell cultures were subjected to sugar metabolism profiling analysis at Shanghai Biotree Biomedical Technology Co., Ltd. Results AkCSLA genes were predominantly expressed in the corms KGM is primarily synthesized in the corms of A . konjac , which have four stages of development, including the dormancy stage (stage 1), “head-changing” stage (stage 2), corm expansion stage (stage 3), and maturity stage (stage 4), according to the vegetative growth cycle of the plant. Previous studies have reported that 97 genes are likely involved in the pathway of KGM biosynthesis [ 3 , 11 , 41 ]. The corms harvested during the four different stages of vegetative growth were subjected to RNA-seq analysis, and the expression patterns of the 97 genes involved in KGM biosynthesis were analyzed. The findings revealed that the majority of these genes were highly expressed in stages 2 and 3 of development (Fig. 1 A). The AkCSLA family comprises 11 members, which can be categorized into four distinct clades. Clade I is uniquely represented by AkCSLA13. Clade II includes AkCSLA6, AkCSLA8, and AkCSLA3. Clade III consists of AkCSLA1 and AkCSLA2, whereas Clade IV contains AkCSLA5, AkCSLA9, AkCSLA11, and AkCSLA14 (Fig. 1 B). This family of CSL enzymes plays a critical role in regulating the biosynthesis of KGM [ 3 , 39 ]. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis of the expression levels of AkCSLA genes in the corms of A . konjac revealed that the expression level of AkCSLA2 was highest, while the expression level of AkCSLA12 was lowest. The expression levels of the other AkCSLA genes in the corms of A . konjac were relatively moderate (Fig. 2 ). Altogether, the findings revealed that AkCSLA genes are predominantly expressed in the corms of A . konjac and play key roles in the biosynthesis of KGM within the corms of A . konjac . Subcellular localization of AkCSLA2 and 3 Analysis of the phylogenetic relationships of the 11 AkCSLA proteins demonstrated that these proteins grouped into 4 distinct clusters (I–IV). Of these, the AkCSLA2 protein, with the highest level of expression, and the AkCSLA3 enzyme, with a moderate level of expression, clustered in groups Ⅱ and Ⅲ, respectively (Fig. 1 B). In silico analysis of the structures and subcellular localization of AkCSLA2 and AkCSLA3 revealed the presence of five transmembrane domains. It was predicted that the proteins primarily localized to the plasma membrane (PM), endoplasmic reticulum (ER), and Golgi apparatus (Figs. S1, S2). The full-length CDSs of AkCSLA2 and AkCSLA3 , including the stop codon, were subsequently cloned to determine the pattern of subcellular localization of the AkCSLA proteins. We constructed the pCAMBIA1300-UBQ10-eGFP-AkCSLA2 and pCAMBIA1300-UBQ10-eGFP-AkCSLA3 vectors, which are driven by the UBQ10 promoter. The vectors were transferred into the GV3101 strain of Agrobacterium tumefaciens and introduced into N . benthamiana leaves for instantaneous expression. The research results indicate that the empty pCAMBIA1300 vector, devoid of any inserted genes, is localized in the plasma membrane and nucleus of the epidermal cells of N . benthamiana . Both AkCSLA2 and AkCSLA3 exhibit similar subcellular localization patterns. In addition to their presence in the plasma membrane, they display a punctate localization pattern reminiscent of the Golgi apparatus and endoplasmic reticulum (Fig. 3 ). These findings indicate that AkCSLA2 and AkCSLA3 are primarily localized to the plasma membrane, Golgi apparatus, and endoplasmic reticulum. AkCSLA3 physically interacts with AkMSR1 The TfMSR protein of Trigonella foenum-graecum L. and its two homologues, AtMSR1 and AtMSR2, from Arabidopsis , are involved in the biosynthesis of mannan [ 37 ]. AtMSR1 can promote the synthesis of glucomannan by AtCSLA2 and AkCSLA3. The AtMSR1 protein enables AtCSLA2 to use mannan as a substitute, thereby facilitating the production of glucomannan [ 38 ]. Previous studies have demonstrated that MSR is a critical cofactor in the biosynthesis of glucomannan by CSLA [ 38 , 42 ], and that the AkMSR1 protein of A . konjac is a homologue of AtMSR1 [ 3 ]. The results of pairwise sequence alignment revealed that the sequence identity was 54.85% between AkMSR1 and AtMSR1, and 53.30% between AkMSR1 and AtMSR2 (Fig. S3). It was predicted that AkMSR1 possesses a transmembrane domain, and primarily localized to the extracellular compartment, ER, and Golgi apparatus (Fig. S4). We speculated that AkCSLA may form a complex with AkMSR to catalyze the synthesis of KGM. The dual-membrane yeast two-hybrid (Y2H) experiment showed that AkMSR1 interacts with AkCSLA3, but not with AkCSLA2 (Fig. 4 A). Co-IP assays conducted with N. benthamiana leaves, utilizing flag-tagged AkMSR1 and eGFP-tagged AkCSLA3, demonstrated that AkMSR1 co-immunoprecipitated with AkCSLA3 (Fig. 4 B). Collectively, these biochemical analyses indicate that AkCSLA3 interacts with AkMSR1 within the KGM biosynthesis pathway. AkMSR1 enhanced AkCSLA3-induced glucomannan synthesis Glucomannan is a hydrocolloidal polysaccharide that consists of mannose and glucose units connected via β-1,4 linkages [ 43 ]. Therefore, in order to elucidate the effects of AkMSR1 on AkCSLA3-mediated glucomannan synthesis, the benchling CRISPR tool was used to design gRNAs. The AkCSLA3 and AkMSR1 genes were cloned individually as well as in combination into a Bsa I-digested pARS-gRNA vector, and the transgene was efficiently integrated into the genome of the GS115 strain of P . pastoris (Fig. S5). The targeted detection of the monosaccharides and disaccharides in the GS115 strain of P . pastoris revealed that the co-expression of AkCSLA3 and AkMSR1 significantly upregulated the contents of L-arabinose, D(-)-fructose, D-(+)-mannose, D-galactose, D-glucuronic acid, and sucrose, but significantly downregulated the contents of xylose, D-sorbitol, D-(+)-galacturonic acid monohydrate, inositol, D-glucose-6-phosphate, maltose, D-(+)-trehalose, and trehalose-6-phosphate, compared to those following the expression of AkCSLA3 or AkMSR1 alone (Fig. 5 ). Notably, In P. pastoris , the co-expression of AkMSR1 and AkCSLA3 significantly enhanced the mannose content, reaching a level that was at least three times that observed in the presence of AkCSLA3 alone. Concurrently, glucose levels increased by approximately 13% (Fig. 6 ). Consistently, these results indicate that AkMSR1 physically associates with AkCSLA3 to augment its enzymatic activity and stability, thereby substantially promoting the biosynthesis of mannose residues—the critical building blocks for glucomannan assembly. Discussion KGM is a type of soluble dietary fiber polysaccharide with a high viscosity that is predominantly found in the corms of A . konjac . The biosynthesis of KGM is mediated by multiple gene families, including the CSLA , CSLD , and MSR families. These genes function cooperatively during the synthesis of KGM, forming a complex regulatory network [ 12 ]. Of these, the CSLA family plays a critical role in the synthesis of KGM. In this study, transcriptomic analysis of A . konjac during the different stages of growth revealed that the genes related to the biosynthesis of KGM were primarily expressed in the corms. Their expression levels increased gradually in the later stages of corm development, which was consistent with the pattern of KGM accumulation in the corms [ 3 , 4 ]. This complex regulatory network of genes ensures the efficient synthesis and accumulation of KGM, which is critical for the biological functions of A . konjac . The CSL gene family is extensively found in plants, consisting of 30–50 members that are classified into nine subfamilies [ 21 , 22 ]. The enzymes encoded by these genes catalyze the synthesis of hemicelluloses in plant cell walls, and it has been demonstrated that the CSLA enzyme subfamily possesses mannan and glucomannan synthase activities [ 27 , 28 , 38 ]. By analyzing the expression patterns of the CSLA gene family in A . konjac , the present study revealed that AkCSLA2 and AkCSLA3 were highly expressed in the corms, and that their expression levels closely correlated with the synthesis of KGM [ 3 , 39 ]. The subcellular localization analysis results revealed that when expressed in N. benthamiana leaves, both AkCSLA2 and AkCSLA3 exhibited similar subcellular distribution patterns. Specifically, these two proteins were primarily localized to the PM, while also displaying punctate distribution patterns resembling the ER and Golgi apparatus—consistent with their predicted subcellular localization and the known role of the Golgi apparatus in polysaccharide chain synthesis and elongation [ 12 , 34 ]. The endoplasmic reticulum and Golgi apparatus are key organelles involved in the synthesis, modification, and transport of polysaccharides. The localization of AkCSLA2 and AkCSLA3 in these organelles supports their direct participation in the assembly and secretion of KGM chains. Although AkCSLA2 and AkCSLA3 share similar subcellular localization patterns, their distinct expression levels in A. konjac corms (with AkCSLA2 showing the highest expression among all AkCSLA members and AkCSLA3 displaying moderate expression) suggest potential functional differentiation in KGM biosynthesis. This functional divergence may be related to their different phylogenetic clade affiliations—AkCSLA2 belongs to Clade III and AkCSLA3 to Clade II of the AkCSLA family—wherein different clades may be responsible for distinct steps in KGM chain synthesis or modification. Additionally, the subsequent interaction experiments revealed that AkCSLA3, but not AkCSLA2, physically interacts with AkMSR1, a homologue of AtMSR1 that enhances glucomannan synthesis by CSLA enzymes [ 37 , 38 ]. This differential interaction further supports the functional specialization of AkCSLA2 and AkCSLA3, with AkCSLA3 playing a more direct role in AkMSR1-mediated KGM synthesis. Collectively, these findings highlight the critical roles of AkCSLA2 and AkCSLA3 in KGM biosynthesis, with their subcellular localization in the PM, ER, and Golgi apparatus providing the spatial basis for their enzymatic activity. The differential interaction with AkMSR1 and distinct expression levels further suggest that these two CSLA members may coordinate to regulate KGM synthesis in A. konjac corms, laying a foundation for further elucidating the molecular mechanism underlying KGM biosynthesis and providing candidate genes for the genetic improvement of KGM content in A. konjac . Despite significant advancements in dissecting the foundational framework of glucomannan biosynthesis, critical gaps in our mechanistic understanding of this pathway persist. While key synthetases (e.g., CSLA subfamily members) and cofactors (such as MSR family proteins) have been identified as core regulators of glucomannan synthesis [ 38 ], the precise molecular mechanisms underpinning their enzymatic catalysis, the spatiotemporal dynamics of their physical and functional interactions, and the intricate layers of transcriptional and post-translational modulation governing their activity remain largely unelucidated. For example, the structural basis by which CSLA enzymes recognize GDP-mannose and GDP-glucose substrates, and the exact nature of post-translational modifications (e.g., glycosylation) that drive MSR-mediated enhancement of CSLA catalytic efficiency, represent fundamental unresolved questions. Addressing these gaps is not only essential for delineating the core glucomannan biosynthetic pathway but also critical for explaining the quantitative and qualitative disparities in glucomannan accumulation across plant species and tissues. Notably, our findings in A. konjac demonstrate that AkMSR1 exerts a profound, substrate-specific regulatory effect on AkCSLA3, driving a more than threefold increase in mannose residue synthesis and a moderate elevation in glucose levels during glucomannan biosynthesis. This observation aligns with and extends prior studies in Arabidopsis and other model plants, which established that MSR homologs physically interact with CSLA enzymes to facilitate glucomannan synthesis [ 37 , 38 ]. However, within these regulatory networks, significant species-specific differences exist. In A. konjac , AkMSR1 interacts with AkCSLA3 but not with AkCSLA2. Conversely, the AtMSR1 gene been demonstrated to regulate the activities of both AtCSLA2 and AkCSLA3 [ 38 ]. This functional specificity underscores the adaptive diversification of glucomannan biosynthetic genes across plant taxa and implies that the regulatory networks underlying polysaccharide synthesis have evolved to meet the unique metabolic and physiological demands of individual species. Systematic characterization of the conservation and divergence of CSLA-MSR interactions, transcriptional regulatory elements, and post-translational modification machineries across diverse plant species will enable us to unravel these evolutionary and adaptive processes. Ultimately, these comparative insights will deepen our fundamental understanding of plant polysaccharide metabolism and provide a rational, mechanistically informed framework for the biotechnological improvement of glucomannan content and quality in A. konjac and other agronomically and economically important crops. Conclusion In this study, we systematically investigated the roles of AkCSLA2, AkCSLA3, and AkMSR1 in the biosynthesis of KGM in A. konjac (Fig. 7 ). Transcriptomic analyses revealed that AkCSLA genes are predominantly expressed in the corms during the key stages of KGM accumulation, with AkCSLA2 exhibiting the highest expression level. Subcellular localization experiments demonstrated that both AkCSLA2 and AkCSLA3 localize to the plasma membrane, endoplasmic reticulum, and Golgi apparatus, consistent with their functions in polysaccharide synthesis and transport. Notably, protein interaction assays confirmed that AkMSR1 physically interacts with AkCSLA3, but not with AkCSLA2. Functional studies in Pichia pastoris further demonstrated that co-expression of AkMSR1 and AkCSLA3 significantly enhances mannose and glucose levels, with mannose content increasing more than threefold compared to expression of AkCSLA3 alone, indicating that AkMSR1 acts as a critical cofactor that promotes AkCSLA3-mediated glucomannan synthesis. Collectively, these findings elucidate a key regulatory mechanism in KGM biosynthesis, highlighting the functional specialization among AkCSLA members and the essential role of AkMSR1 in enhancing the catalytic efficiency of AkCSLA3. This work provides a molecular foundation for understanding KGM biosynthesis and offers potential targets for genetic improvement of KGM content in A. konjac . Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The transcriptomic data is available in the National Center for Biotechnology Information (NCBI) Bioproject database under the accession number: PRJNA734512. The annotation files of A. konjac genome are available at figshare: https://doi.org/10.6084/m9.figshare.15169578. The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by grants from the National Natural Science Foundation of China (grant numbers 32460457 and 32560077), the Yunnan Fundamental Research Projects (grant number 202501AU070172), the Yunnan Provincial Department of Education Science Research Fund Project (grant number 2024J0939), and the Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau & Qujing Normal University (grant numbers KJLH2024ZD04 and KJLH2023YB08). Authors' contributions Conceptualization: S.Y.M., and L.C.X.; Data curation: S.Y.M., G.X.S., and L.C.X.; Methodology: L.D.B., C.H.L., and G.X.S.; Validation: S.Y.M., and L.C.X.; Project administration: L.C.X.; Resources: S.Y.M., and L.C.X.; Supervision: S.Y.M., Writing original draft: S.Y.M., and L.C.X.; Writing review & editing: S.Y.M., L.D.B., C.H.L., G.X.S., and L.C.X.; Funding acquisition: S.Y.M., and L.C.X. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable. Conflict of interest The authors declare that they have no conflict of interest. References Zhang Y, Xie B, Gan X. Advance in the applications of konjac glucomannan and its derivatives. Carbohydr Polym. 2005;60:27–31. https://doi.org/10.1016/j.carbpol.2004.11.003 . Li Y, Kang Y, Du Y, Chen M, Guo L, Huang X, et al. Effects of konjaku flour on the gut microbiota of obese patients. Front Cell Infect Microbiol. 2022a;12:771748. https://doi.org/10.3389/fcimb.2022.771748 . 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Supplementary Files Supplementaryfile.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 05 May, 2026 Reviews received at journal 01 May, 2026 Reviews received at journal 29 Apr, 2026 Reviews received at journal 14 Apr, 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 08 Apr, 2026 Editor invited by journal 03 Apr, 2026 Editor assigned by journal 02 Apr, 2026 Submission checks completed at journal 02 Apr, 2026 First submitted to journal 30 Mar, 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. 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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-9274000","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623159587,"identity":"37270d9a-1f6a-458b-aa4c-9fcc36403400","order_by":0,"name":"Yumei Shi","email":"","orcid":"","institution":"Qujing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yumei","middleName":"","lastName":"Shi","suffix":""},{"id":623159588,"identity":"dbb30875-61a5-45c2-91f6-537c7396b33e","order_by":1,"name":"Dongbao Li","email":"","orcid":"","institution":"Liaocheng People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dongbao","middleName":"","lastName":"Li","suffix":""},{"id":623159589,"identity":"d5d31894-8689-4319-b65f-d10403ecd3a3","order_by":2,"name":"Honglong Chu","email":"","orcid":"","institution":"Qujing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Honglong","middleName":"","lastName":"Chu","suffix":""},{"id":623159590,"identity":"4ae3f019-e5ec-40d4-9e62-177eec76bdf2","order_by":3,"name":"Xaiosong Gu","email":"","orcid":"","institution":"Hubei Province Key Lab Yeast Function","correspondingAuthor":false,"prefix":"","firstName":"Xaiosong","middleName":"","lastName":"Gu","suffix":""},{"id":623159591,"identity":"ad3cae5f-016a-4686-8060-0b0d329642f5","order_by":4,"name":"Changxin Luo","email":"data:image/png;base64,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","orcid":"","institution":"Qujing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Changxin","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2026-03-31 03:53:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9274000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9274000/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107480572,"identity":"c4372745-2206-44de-ab85-b26e05677e42","added_by":"auto","created_at":"2026-04-22 02:12:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3386553,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of gene expression heatmap related to KGM biosynthesis and Phylogenetic analysis of AkCSLAs protein family. \u003cstrong\u003eA\u003c/strong\u003e Heatmaps of expression of KGM biosynthesis-related genes in stage 1, 2, 3, and 4. \u003cstrong\u003eB\u003c/strong\u003e A maximum phylogenetic tree of 11 AkCSLAs proteins from \u003cem\u003eA. konjac\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/7dda658f9d2721138ccfcef7.png"},{"id":107009767,"identity":"d5283fe7-8406-4b01-87c9-2c9203aad8d6","added_by":"auto","created_at":"2026-04-15 17:20:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5819630,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of \u003cem\u003eAkCSLA\u003c/em\u003egenes. \u003cstrong\u003eA\u003c/strong\u003e Representative \u003cem\u003eA\u003c/em\u003e.\u003cem\u003e konjac \u003c/em\u003eplant. \u003cstrong\u003eB\u003c/strong\u003e Expression profiles of \u003cem\u003eAkCSLA\u003c/em\u003egenes in the corms of \u003cem\u003eA\u003c/em\u003e.\u003cem\u003e konjac\u003c/em\u003e as determined by RT-qPCR. The data are presented as the mean ± standard deviation (SD) of results obtained from three independent experiments. The expression levels are depicted relative to those of the reference gene, \u003cem\u003eAkEIF4A\u003c/em\u003e. Scale bar = 5 cm.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/3472ef848dffdde29e1da8ab.png"},{"id":107009766,"identity":"728181a5-a812-46d4-8bbc-b4af33fda991","added_by":"auto","created_at":"2026-04-15 17:20:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26624142,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of AkCSLA2 and AkCSLA3 in the epidermal cells of\u003cem\u003e N\u003c/em\u003e.\u003cem\u003e benthamiana\u003c/em\u003e leaves. The intracellular eGFP signals were detected by confocal microscopy. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/207ded2622d60edf17278203.png"},{"id":107009771,"identity":"be757ab4-03b8-4bba-8716-fe597a4864a3","added_by":"auto","created_at":"2026-04-15 17:20:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5336698,"visible":true,"origin":"","legend":"\u003cp\u003eAkCSLA3 physically interacts with AkMSR1. \u003cstrong\u003eA \u003c/strong\u003eThe full-length AkMSR1 protein interacted with the full-length AkCSLA3 protein but not with AkCSLA2, as revealed by dual-membrane Y2H assays. Yeast cells harboring the specified plasmids were either cultured on a growth medium lacking leucine and tryptophan (-Leu/-Trp) or on a selective medium lacking leucine, tryptophan, histidine, and adenine (-Leu/-Trp/-His/-Ade). The experiments were conducted independently in triplicate. \u003cstrong\u003eB \u003c/strong\u003eThe interactions between AkCSLA3 and AkMSR1 in the foliar epidermal cells of \u003cem\u003eN\u003c/em\u003e.\u003cem\u003e benthamiana \u003c/em\u003ewere determined by Co-IP assays. The eGFP-AkCSLA3 construct was transiently co-expressed with AkMSR1-Flag in the epidermal cells of \u003cem\u003eN\u003c/em\u003e.\u003cem\u003e benthamiana\u003c/em\u003e leaves. Plants transiently expressing eGFP-AkCSLA3 alone were used as the negative control. Proteins were extracted from the infiltrated leaves and analyzed using anti-FLAG and anti-GFP antibodies. The immunoprecipitated (IP) AkMSR1-Flag proteins were subsequently probed with an anti-GFP antibody for detecting the Co-IP of eGFP-AkCSLA3.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/aaf8905326a443766e9a3721.png"},{"id":107009769,"identity":"78b8ed12-6c46-4634-8d9b-a0e7eacb9b98","added_by":"auto","created_at":"2026-04-15 17:20:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1709799,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolite profiling and content analysis of differentially expressed metabolites in \u003cem\u003ePichia pastoris \u003c/em\u003estrains.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/e518df5fbaca965ad8cff5c7.png"},{"id":107009772,"identity":"9ac5b7e1-dd63-49e6-a55e-d62d1e84e2ef","added_by":"auto","created_at":"2026-04-15 17:20:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":490401,"visible":true,"origin":"","legend":"\u003cp\u003eRelative contents of D-(+)-mannose and D-glucose linkages in the GS115 strain of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epastoris\u003c/em\u003e. The data are presented as the mean ± SD of results obtained from at least three technical replicates. The different letters denote significant differences determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/1eb68f211a75df26df277a2d.png"},{"id":107480934,"identity":"5174ef3a-eccd-4491-b373-c248496df567","added_by":"auto","created_at":"2026-04-22 02:14:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14292280,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic model of the AkCSLA3-AkMSR1 module illustrates its regulatory role in the biosynthesis of KGM in\u003cem\u003e A. konjac\u003c/em\u003e. Within the corm cells of A. konjac, AkCSLA2 and AkCSLA3 are localized in the PM, ER, and Golgi apparatus, where they contribute to the fundamental synthesis of KGM. Notably, AkMSR1 specifically interacts with AkCSLA3 (but not with AkCSLA2) to form a protein complex that significantly enhances the catalytic activity of AkCSLA3. This interaction facilitates the synthesis of D-(+)-mannose and D-glucose, which are the key monosaccharide units of KGM, thereby accelerating the assembly and accumulation of KGM polysaccharides.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/6aff5adcc1138edb260a9a60.png"},{"id":107705297,"identity":"01a4195a-fe1d-4c6a-be46-1fba80e94759","added_by":"auto","created_at":"2026-04-24 09:11:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":52186299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/fe9ad828-f36a-4860-887a-04e763596e61.pdf"},{"id":107009773,"identity":"f2a5ad91-91ec-4ef3-aa42-259339496f04","added_by":"auto","created_at":"2026-04-15 17:20:14","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":3435157,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-9274000/v1/aa32c306f84f88e6d549a442.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe AkCSLA3-AkMSR1 module mediates Konjac glucomannan biosynthesis in \u003cem\u003eAmorphophallus konjac\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlucomannan is a soluble dietary fiber polysaccharide known for its high viscosity. It is predominantly found in the roots, stems, leaves, and other parts of plants, and also in the cell walls of microorganisms, including bacteria, fungi, and yeasts. Konjac glucomannan (KGM) is the main constituent of konjac polysaccharide, and the most extensively studied form of glucomannan to date [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. KGM is primarily synthesized and accumulates in the corms of \u003cem\u003eAmorphophallus.\u003c/em\u003e It is an edible, perennial herbaceous plant belonging to the genus \u003cem\u003eAmorphophallus\u003c/em\u003e within the Araceae family. The corms of \u003cem\u003eAmorphophallus\u003c/em\u003e grow underground and represent the main edible parts of the plants. The corms are rich in KGM, with contents exceeding 50% by weight [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Dissection of the corm tissues of \u003cem\u003eAmorphophallus\u003c/em\u003e reveals the presence of numerous distinct, colorless, and bright particles, with diameters of approximately 1 mm, known as glucomannan cells. The deposition of KGM within these glucomannan cells is subject to temporal regulation, with its expression increasing towards the end of each vegetative growth cycle [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. KGM is a water-soluble, non-ionic polysaccharide that is primarily composed of D-mannose and D-glucose units linked via β-1, 4-glycosidic bonds in a 1:1.6 ratio. The high molecular weight of KGM and the interactions between its monosaccharide units contribute to its high viscosity, which enhances its gel-formation and water retention properties, making it suitable for applications in the food sector [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. KGM is a high-density, branched macromolecular polymer, and its interactions with starch can potentially modulate its properties. Blending with starch significantly enhances the viscoelasticity of KGM, retards the retrogradation of starch, and promotes the formation of stable gels. Moreover, KGM is capable of decreasing the dimensions of gelatinized starch granules. Earlier research utilizing low-field nuclear magnetic resonance has shown that KGM can impede the swelling and gelatinization processes of native starch, competing with corn starch for water absorption [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Research has further shown that KGM improves the consistency and network density of mixtures containing pea and potato starch, as demonstrated by analyses using scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). A variety of studies have clarified the functions of KGM in maintaining the texture of starch-based food items and prolonging their shelf life [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, current research on KGM primarily focuses on its structure, properties, and pharmacological characteristics, placing less emphasis on the regulatory mechanisms underlying KGM biosynthesis in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe pathway of KGM biosynthesis is related to the pathways of sucrose metabolism and nucleotide sugar conversion, and is regulated by over 90 putative genes and their respective expression patterns [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Sucrose serves as a precursor for the synthesis of KGM and is degraded into fructose (Fru) and glucose (Glc) through catalysis by invertase (INV). A portion of the glucose undergoes glycolysis, while the remaining glucose and fructose units are converted into mannose-6-phosphate (Man-6-P). The phosphomannomutase (PMM) enzyme then converts Man-6-P to mannose-1-phosphate (Man-1-P), which is subsequently converted to its active GDP-mannose (GDP-Man) form by GDP-mannose pyrophosphorylase (GMPP). GDP-Man finally enters the Golgi apparatus, which mediates the synthesis and elongation of polysaccharide chains catalyzed by the cellulose synthase-like (CSL) enzymes, CSLA/CSLD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. CSL enzymes belong to the glycosyltransferase 2 (GT2) superfamily and catalyze the biosynthesis of plant hemicelluloses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] (Richmond and Somerville 2000). They play critical roles in plant growth, immune responses to pathogens, and the enhancement of plant biomass [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Whole-genome studies on the \u003cem\u003eCSL\u003c/em\u003e gene family in various plants have been conducted to date, including rice [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], flax [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], tea [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], strawberry [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], pineapple [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and tomato [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eCSL\u003c/em\u003e gene family in plants consists of 30\u0026ndash;50 members and is further categorized into nine subfamilies, namely, \u003cem\u003eCSLA\u003c/em\u003e, \u003cem\u003eCSLB\u003c/em\u003e, \u003cem\u003eCSLC\u003c/em\u003e, \u003cem\u003eCSLD\u003c/em\u003e, \u003cem\u003eCSLE\u003c/em\u003e, \u003cem\u003eCSLF\u003c/em\u003e, \u003cem\u003eCSLG\u003c/em\u003e, \u003cem\u003eCSLH\u003c/em\u003e, and \u003cem\u003eCSLJ\u003c/em\u003e [\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The gene subfamilies CSLA, CSLC, and CSLD are frequently identified across all terrestrial plants, whereas the other subfamilies are confined to particular plant groups. For example, the subfamilies CSLB and CSLG predominantly occur in non-grass species, while the CSLF, CSLH, and CSLJ subfamilies are exclusive to grasses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eArabidopsis\u003c/em\u003e), the model plant, researchers have identified a minimum of six subfamilies within the CSL gene family, namely CSLA, CSLB, CSLC, CSLD, CSLE, and CSLG, totaling 29 distinct members [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It has been demonstrated that CSLA proteins possess mannan and glucomannan synthase activities, and catalyze the biosynthesis of the main chains of mannan and glucomannan in plants [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A total of nine \u003cem\u003eAtCSLA\u003c/em\u003e genes have been identified in \u003cem\u003eArabidopsis\u003c/em\u003e to date. Depending on the substrate, CSLA can synthesize different types of mannan molecules [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For instance, AtCSLA2, AtCSLA3, and AtCSLA9 are essential for the synthesis of glucomannan in the stems of \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previous studies have demonstrated that the disruption of \u003cem\u003eAtCSLA2\u003c/em\u003e reduced the mucus halo in the seeds of \u003cem\u003eArabidopsis\u003c/em\u003e, and the contents of mannosyl and glucose in the mucus decreased by 30% compared to those of the wild type (WT) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The disruption of the \u003cem\u003eCSLA7\u003c/em\u003e gene leads to abnormal pollen development and embryonic lethality in \u003cem\u003eArabidopsis\u003c/em\u003e, suggesting that mannan polysaccharides have critical structural or signaling functions in plant embryonic development [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It has been previously demonstrated that AtCSLA9 localizes to the Golgi apparatus in \u003cem\u003ePichia\u003c/em\u003e sp., with its active site facing the Golgi lumen. Additionally, mutations in the \u003cem\u003eAtCSLA9\u003c/em\u003e gene have been shown to prevent \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated root transformations in \u003cem\u003erat4 Arabidopsis\u003c/em\u003e mutants [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, the putative GTs associated with mannan synthesis-related (MSR) enzymes are implicated in the synthesis of (gluco)mannan [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It has been reported that the AtMSR enzymes of \u003cem\u003eArabidopsis\u003c/em\u003e likely generate a primer to initiate the synthesis of heteromannan, enhance the activity of CSLA enzymes through glycosylation, or support the stability and functionality of CSLA enzymes via glycosylation-independent interactions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The AtMSR1 and AtMSR2 enzymes of \u003cem\u003eArabidopsis\u003c/em\u003e have been shown to localize to the Golgi body. It has been reported that the levels of mannosyl reduced by approximately 40% and 50% in single and double \u003cem\u003eAtmsr1\u003c/em\u003e mutants, respectively, compared to those in the WT. In contrast, single \u003cem\u003eAtmsr2\u003c/em\u003e mutants exhibit no significant differences in the contents of mannosyl compared to those in the WT [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It has been reported that AtCSLA2 alone produces mannan, while the co-expression of AtCSLA2 and AtMSR1 synthesizes glucomannan in \u003cem\u003ePichia\u003c/em\u003e strains [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. By analyzing monosaccharide and glycosidic linkages, a previous study demonstrated that the co-expression of AtMSR1 and the AkCSLA3 protein of \u003cem\u003ePichia\u003c/em\u003e upregulated the levels of glucomannan in the heteromannan-enriched fraction of \u003cem\u003ePichia\u003c/em\u003e by at least 70%, compared to that in the presence of AkCSLA3 alone [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo clarify the regulatory mechanism of KGM biosynthesis and the functional roles of key enzymes, this study investigated the expression patterns and subcellular localizations of AkCSLA2, AkCSLA3, and AkMSR1 in \u003cem\u003eA. konjac\u003c/em\u003e. It mainly analyzed the interaction relationships between AkCSLA2, AkCSLA3 and AkMSR1, and further explored their roles in the process of KGM synthesis. The research results indicated that AkMSR1 could significantly promote the synthesis of KGM by AkCSLA3, thus laying a foundation for the study of KGM biosynthesis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eIn this study, we utilized \u003cem\u003eA. konjac\u003c/em\u003e and \u003cem\u003eN. benthamiana\u003c/em\u003e as the experimental materials. The seeds of both species were obtained from our laboratory [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Initially, the \u003cem\u003eA. konjac\u003c/em\u003e and \u003cem\u003eN. benthamiana\u003c/em\u003e seeds were cultivated on Murashige and Skoog (MS) agar medium containing 0.8% agar for approximately 5 to 7 days, after which they were transplanted into soil. Throughout this process, the seedlings were maintained under a photoperiod of 16 hours of light and 8 hours of darkness at a temperature of 23\u0026deg;C and a relative humidity of 60%. Additionally, the seedlings were exposed to a light intensity of approximately 100 \u0026micro;mol\u0026middot;m⁻\u0026sup2;\u0026middot;s⁻\u0026sup1; for a duration of 3 to 4 weeks.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA sequencing (RNA-seq), analysis of gene expression, and Phylogenetic analysis\u003c/h3\u003e\n \u003cp\u003eThe protocols for RNA-seq and analysis of gene expression data were derived from the study by Gao et al. (2022) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Briefly, the key genes involved in KGM biosynthesis were identified by time-course RNA-seq using samples obtained from the four developmental stages of corm, including the dormancy stage (stage 1), \u0026ldquo;changing head\u0026rdquo; stage (stage 2), corm expansion stage (stage 3), and maturity stage (stage 4). Each biological replicate consisted of 3\u0026ndash;4 individuals, sampled from each developmental stage. The total RNA was extracted using an RNA extraction kit (Huayueyang, Beijing, China), and the RNA-seq libraries were constructed with a library preparation kit. Sequencing was performed using an Illumina HiSeq 4000 platform. The clean reads were mapped to the reference genome following quality control, and the gene expression levels were quantified based on the values of FPKM (Fragments Per Kilobase of exon model per Million mapped fragments). The DESeq2 software was used to analyze differential gene expression and perform principal component analysis (PCA), while the heatmaps were generated using TBtools, and gene ontology (GO) enrichment analysis was conducted using the clusterProfiler program. The gene expression profiles and phylogenetic analysis were analyzed as previously described [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePlasmid construction and analysis of subcellular localization\u003c/h3\u003e\n\u003cp\u003eThe coding sequences (CDSs) for AkCSLA2, and AkCSLA3, along with their respective stop codons, were amplified and cloned into a pCAMBIA1300 vector, which also contained an eGFP fluorescent marker and the UBQ10 promoter sequence. Subsequently, the resulting plasmid was introduced into the GV3101 strain of \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. The pCAMBIA1300-UBQ10-eGFP-AkCSLA2 and pCAMBIA1300-UBQ10-eGFP-AkCSLA3 constructs were subsequently introduced into the leaves of 4-week-old \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e plants using an infiltration buffer consisting of 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 100 \u0026micro;M acetosyringone respectively. The \u003cem\u003eN. benthamiana\u003c/em\u003e plants that were infiltrated were incubated under low light conditions for 48 hours. Following this incubation period, the fluorescent signals emitted by eGFP were observed using a confocal laser scanning microscope (CLSM600; SOPTOP).\u003c/p\u003e\n\u003ch3\u003eYeast two-hybrid (Y2H) assays\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eAkMSR1\u003c/em\u003e gene was inserted into the pMetYCgate destination vector for investigating the protein-protein interactions using Y2H assays. The \u003cem\u003eAkCSLA2\u003c/em\u003e and \u003cem\u003eAkCSLA3\u003c/em\u003e genes were cloned into the pPR3N vector [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Various combinations of bait and prey vectors, as well as the positive and negative control plasmids, were co-transformed into the NMY51 strain of yeast. The successfully co-transformed clones were capable of growing on the selective medium with moderate stringency lacking tryptophan and leucine (-Trp/-Leu). However, only the positive control and clones expressing the interacting proteins were able to grow on the high-stringency selective medium lacking histidine, tryptophan, leucine, and adenine (-His/-Trp/-Leu/-Ade).\u003c/p\u003e\n\u003ch3\u003eProtein extraction and co-immunoprecipitation (Co-IP) studies\u003c/h3\u003e\n\u003cp\u003eThe leaves of \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e co-expressing the pCAMBIA1300-UBQ10-eGFP-AkCSLA3 and pBIB-UBQ10-AkMSR1-Flag constructs were collected 72 h post-infiltration. The total protein was extracted from these \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e leaves using an extraction buffer containing 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1% Triton, 1 mM EDTA, and a 1:100 dilution of a complete protease inhibitor cocktail (catalog number: 04693132001; Roche). The samples were solubilized with the extraction buffer, vortexed for 5 minutes, and subsequently centrifuged at 16,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes at 4\u0026deg;C to remove the debris and obtain a clear solution. The resulting supernatant was then mixed with anti-FLAG beads (catalog number: A2220; Sigma) and incubated for 4 h at 4\u0026deg;C with gentle agitation. The beads were subsequently collected and washed five times with an extraction buffer containing 0.5% Triton X-100. The proteins bound to the beads were then boiled in 1\u0026times;SDS loading buffer for 5 minutes, and subsequently analyzed by immunoblotting using anti-GFP (catalog number: SAB4301138; Sigma) and anti-FLAG-tag-HRP (catalog number: M20026M; Abmart) antibodies.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePichia Transformation\u003c/h2\u003e \u003cp\u003eThe codon-optimized \u003cem\u003eAkMSR1\u003c/em\u003e and \u003cem\u003eAkCSLA3\u003c/em\u003e genes from \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e were synthesized by Jinkairui Biological Engineering Co., Ltd. (Wuhan, China). The genes were subsequently cloned into the donor helper plasmids using digestion/ligation and/or Gibson assembly methods. The gRNAs were designed using the Benchling CRISPR tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://benchling.com\u003c/span\u003e\u003cspan address=\"https://benchling.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and subsequently cloned into pHARS-gRNA digested with \u003cem\u003eBsa\u003c/em\u003eI.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eDH5α\u003c/em\u003e gene of \u003cem\u003eEscherichia coli\u003c/em\u003e was cultured in Luria-Bertani (LB) medium supplemented with 100 mg/L ampicillin. The GS115 strain of \u003cem\u003ePichia pastoris\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e. \u003cem\u003epastoris\u003c/em\u003e) was used as the parent strain for studying the genomic integrations of two genes, and was routinely cultured in Yeast Extract Peptone Dextrose (YPD) medium containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose. The recombinant \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epastoris\u003c/em\u003e strains were screened in solid YPD medium supplemented with 100 mg/L Zeocin. A single colony was selected and inoculated into 5 mL of the YPD medium and incubated at 30\u0026deg;C with shaking at 250 rpm for 18 h to produce heteromannan. The culture was then transferred to a 500 mL shake flask containing 100 mL of YPD medium containing 20 g/L glucose. After incubation at 30\u0026deg;C for 24 h, methanol was added to the culture to achieve a final concentration of 2%. Fermentation was allowed to continue for an additional 72 h, following which the cell cultures were subjected to sugar metabolism profiling analysis at Shanghai Biotree Biomedical Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAkCSLA\u003c/b\u003e \u003cb\u003egenes were predominantly expressed in the corms\u003c/b\u003e\u003c/p\u003e \u003cp\u003eKGM is primarily synthesized in the corms of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, which have four stages of development, including the dormancy stage (stage 1), \u0026ldquo;head-changing\u0026rdquo; stage (stage 2), corm expansion stage (stage 3), and maturity stage (stage 4), according to the vegetative growth cycle of the plant. Previous studies have reported that 97 genes are likely involved in the pathway of KGM biosynthesis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The corms harvested during the four different stages of vegetative growth were subjected to RNA-seq analysis, and the expression patterns of the 97 genes involved in KGM biosynthesis were analyzed. The findings revealed that the majority of these genes were highly expressed in stages 2 and 3 of development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The AkCSLA family comprises 11 members, which can be categorized into four distinct clades. Clade I is uniquely represented by AkCSLA13. Clade II includes AkCSLA6, AkCSLA8, and AkCSLA3. Clade III consists of AkCSLA1 and AkCSLA2, whereas Clade IV contains AkCSLA5, AkCSLA9, AkCSLA11, and AkCSLA14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This family of CSL enzymes plays a critical role in regulating the biosynthesis of KGM [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis of the expression levels of \u003cem\u003eAkCSLA\u003c/em\u003e genes in the corms of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e revealed that the expression level of \u003cem\u003eAkCSLA2\u003c/em\u003e was highest, while the expression level of \u003cem\u003eAkCSLA12\u003c/em\u003e was lowest. The expression levels of the other \u003cem\u003eAkCSLA\u003c/em\u003e genes in the corms of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e were relatively moderate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Altogether, the findings revealed that \u003cem\u003eAkCSLA\u003c/em\u003e genes are predominantly expressed in the corms of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e and play key roles in the biosynthesis of KGM within the corms of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSubcellular localization of\u003c/b\u003e \u003cb\u003eAkCSLA2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of the phylogenetic relationships of the 11 AkCSLA proteins demonstrated that these proteins grouped into 4 distinct clusters (I\u0026ndash;IV). Of these, the AkCSLA2 protein, with the highest level of expression, and the AkCSLA3 enzyme, with a moderate level of expression, clustered in groups Ⅱ and Ⅲ, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). \u003cem\u003eIn silico\u003c/em\u003e analysis of the structures and subcellular localization of AkCSLA2 and AkCSLA3 revealed the presence of five transmembrane domains. It was predicted that the proteins primarily localized to the plasma membrane (PM), endoplasmic reticulum (ER), and Golgi apparatus (Figs. S1, S2). The full-length CDSs of \u003cem\u003eAkCSLA2\u003c/em\u003e and \u003cem\u003eAkCSLA3\u003c/em\u003e, including the stop codon, were subsequently cloned to determine the pattern of subcellular localization of the AkCSLA proteins. We constructed the pCAMBIA1300-UBQ10-eGFP-AkCSLA2 and pCAMBIA1300-UBQ10-eGFP-AkCSLA3 vectors, which are driven by the \u003cem\u003eUBQ10\u003c/em\u003e promoter. The vectors were transferred into the GV3101 strain of \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e and introduced into \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e leaves for instantaneous expression. The research results indicate that the empty pCAMBIA1300 vector, devoid of any inserted genes, is localized in the plasma membrane and nucleus of the epidermal cells of \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e. Both AkCSLA2 and AkCSLA3 exhibit similar subcellular localization patterns. In addition to their presence in the plasma membrane, they display a punctate localization pattern reminiscent of the Golgi apparatus and endoplasmic reticulum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings indicate that AkCSLA2 and AkCSLA3 are primarily localized to the plasma membrane, Golgi apparatus, and endoplasmic reticulum.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAkCSLA3 physically interacts with AkMSR1\u003c/h3\u003e\n\u003cp\u003eThe TfMSR protein of \u003cem\u003eTrigonella foenum-graecum\u003c/em\u003e L. and its two homologues, AtMSR1 and AtMSR2, from \u003cem\u003eArabidopsis\u003c/em\u003e, are involved in the biosynthesis of mannan [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. AtMSR1 can promote the synthesis of glucomannan by AtCSLA2 and AkCSLA3. The AtMSR1 protein enables AtCSLA2 to use mannan as a substitute, thereby facilitating the production of glucomannan [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Previous studies have demonstrated that MSR is a critical cofactor in the biosynthesis of glucomannan by CSLA [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and that the AkMSR1 protein of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e is a homologue of AtMSR1 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The results of pairwise sequence alignment revealed that the sequence identity was 54.85% between AkMSR1 and AtMSR1, and 53.30% between AkMSR1 and AtMSR2 (Fig. S3). It was predicted that AkMSR1 possesses a transmembrane domain, and primarily localized to the extracellular compartment, ER, and Golgi apparatus (Fig. S4). We speculated that AkCSLA may form a complex with AkMSR to catalyze the synthesis of KGM. The dual-membrane yeast two-hybrid (Y2H) experiment showed that AkMSR1 interacts with AkCSLA3, but not with AkCSLA2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Co-IP assays conducted with \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, utilizing flag-tagged AkMSR1 and eGFP-tagged AkCSLA3, demonstrated that AkMSR1 co-immunoprecipitated with AkCSLA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Collectively, these biochemical analyses indicate that AkCSLA3 interacts with AkMSR1 within the KGM biosynthesis pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAkMSR1 enhanced AkCSLA3-induced glucomannan synthesis\u003c/h2\u003e \u003cp\u003eGlucomannan is a hydrocolloidal polysaccharide that consists of mannose and glucose units connected via β-1,4 linkages [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, in order to elucidate the effects of AkMSR1 on AkCSLA3-mediated glucomannan synthesis, the benchling CRISPR tool was used to design gRNAs. The \u003cem\u003eAkCSLA3\u003c/em\u003e and \u003cem\u003eAkMSR1\u003c/em\u003e genes were cloned individually as well as in combination into a \u003cem\u003eBsa\u003c/em\u003eI-digested pARS-gRNA vector, and the transgene was efficiently integrated into the genome of the GS115 strain of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epastoris\u003c/em\u003e (Fig. S5). The targeted detection of the monosaccharides and disaccharides in the GS115 strain of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epastoris\u003c/em\u003e revealed that the co-expression of AkCSLA3 and AkMSR1 significantly upregulated the contents of L-arabinose, D(-)-fructose, D-(+)-mannose, D-galactose, D-glucuronic acid, and sucrose, but significantly downregulated the contents of xylose, D-sorbitol, D-(+)-galacturonic acid monohydrate, inositol, D-glucose-6-phosphate, maltose, D-(+)-trehalose, and trehalose-6-phosphate, compared to those following the expression of AkCSLA3 or AkMSR1 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Notably, In \u003cem\u003eP. pastoris\u003c/em\u003e, the co-expression of AkMSR1 and AkCSLA3 significantly enhanced the mannose content, reaching a level that was at least three times that observed in the presence of AkCSLA3 alone. Concurrently, glucose levels increased by approximately 13% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Consistently, these results indicate that AkMSR1 physically associates with AkCSLA3 to augment its enzymatic activity and stability, thereby substantially promoting the biosynthesis of mannose residues\u0026mdash;the critical building blocks for glucomannan assembly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eKGM is a type of soluble dietary fiber polysaccharide with a high viscosity that is predominantly found in the corms of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e. The biosynthesis of KGM is mediated by multiple gene families, including the \u003cem\u003eCSLA\u003c/em\u003e, \u003cem\u003eCSLD\u003c/em\u003e, and \u003cem\u003eMSR\u003c/em\u003e families. These genes function cooperatively during the synthesis of KGM, forming a complex regulatory network [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Of these, the \u003cem\u003eCSLA\u003c/em\u003e family plays a critical role in the synthesis of KGM. In this study, transcriptomic analysis of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e during the different stages of growth revealed that the genes related to the biosynthesis of KGM were primarily expressed in the corms. Their expression levels increased gradually in the later stages of corm development, which was consistent with the pattern of KGM accumulation in the corms [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This complex regulatory network of genes ensures the efficient synthesis and accumulation of KGM, which is critical for the biological functions of \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eCSL\u003c/em\u003e gene family is extensively found in plants, consisting of 30\u0026ndash;50 members that are classified into nine subfamilies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The enzymes encoded by these genes catalyze the synthesis of hemicelluloses in plant cell walls, and it has been demonstrated that the CSLA enzyme subfamily possesses mannan and glucomannan synthase activities [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. By analyzing the expression patterns of the \u003cem\u003eCSLA\u003c/em\u003e gene family in \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ekonjac\u003c/em\u003e, the present study revealed that AkCSLA2 and AkCSLA3 were highly expressed in the corms, and that their expression levels closely correlated with the synthesis of KGM [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The subcellular localization analysis results revealed that when expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, both AkCSLA2 and AkCSLA3 exhibited similar subcellular distribution patterns. Specifically, these two proteins were primarily localized to the PM, while also displaying punctate distribution patterns resembling the ER and Golgi apparatus\u0026mdash;consistent with their predicted subcellular localization and the known role of the Golgi apparatus in polysaccharide chain synthesis and elongation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The endoplasmic reticulum and Golgi apparatus are key organelles involved in the synthesis, modification, and transport of polysaccharides. The localization of AkCSLA2 and AkCSLA3 in these organelles supports their direct participation in the assembly and secretion of KGM chains.\u003c/p\u003e \u003cp\u003eAlthough AkCSLA2 and AkCSLA3 share similar subcellular localization patterns, their distinct expression levels in \u003cem\u003eA. konjac\u003c/em\u003e corms (with AkCSLA2 showing the highest expression among all AkCSLA members and AkCSLA3 displaying moderate expression) suggest potential functional differentiation in KGM biosynthesis. This functional divergence may be related to their different phylogenetic clade affiliations\u0026mdash;AkCSLA2 belongs to Clade III and AkCSLA3 to Clade II of the AkCSLA family\u0026mdash;wherein different clades may be responsible for distinct steps in KGM chain synthesis or modification. Additionally, the subsequent interaction experiments revealed that AkCSLA3, but not AkCSLA2, physically interacts with AkMSR1, a homologue of AtMSR1 that enhances glucomannan synthesis by CSLA enzymes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This differential interaction further supports the functional specialization of AkCSLA2 and AkCSLA3, with AkCSLA3 playing a more direct role in AkMSR1-mediated KGM synthesis. Collectively, these findings highlight the critical roles of AkCSLA2 and AkCSLA3 in KGM biosynthesis, with their subcellular localization in the PM, ER, and Golgi apparatus providing the spatial basis for their enzymatic activity. The differential interaction with AkMSR1 and distinct expression levels further suggest that these two CSLA members may coordinate to regulate KGM synthesis in A. konjac corms, laying a foundation for further elucidating the molecular mechanism underlying KGM biosynthesis and providing candidate genes for the genetic improvement of KGM content in \u003cem\u003eA. konjac\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eDespite significant advancements in dissecting the foundational framework of glucomannan biosynthesis, critical gaps in our mechanistic understanding of this pathway persist. While key synthetases (e.g., CSLA subfamily members) and cofactors (such as MSR family proteins) have been identified as core regulators of glucomannan synthesis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], the precise molecular mechanisms underpinning their enzymatic catalysis, the spatiotemporal dynamics of their physical and functional interactions, and the intricate layers of transcriptional and post-translational modulation governing their activity remain largely unelucidated. For example, the structural basis by which CSLA enzymes recognize GDP-mannose and GDP-glucose substrates, and the exact nature of post-translational modifications (e.g., glycosylation) that drive MSR-mediated enhancement of CSLA catalytic efficiency, represent fundamental unresolved questions. Addressing these gaps is not only essential for delineating the core glucomannan biosynthetic pathway but also critical for explaining the quantitative and qualitative disparities in glucomannan accumulation across plant species and tissues. Notably, our findings in \u003cem\u003eA. konjac\u003c/em\u003e demonstrate that AkMSR1 exerts a profound, substrate-specific regulatory effect on AkCSLA3, driving a more than threefold increase in mannose residue synthesis and a moderate elevation in glucose levels during glucomannan biosynthesis. This observation aligns with and extends prior studies in Arabidopsis and other model plants, which established that MSR homologs physically interact with CSLA enzymes to facilitate glucomannan synthesis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, within these regulatory networks, significant species-specific differences exist. In \u003cem\u003eA. konjac\u003c/em\u003e, AkMSR1 interacts with AkCSLA3 but not with AkCSLA2. Conversely, the AtMSR1 gene been demonstrated to regulate the activities of both AtCSLA2 and AkCSLA3 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This functional specificity underscores the adaptive diversification of glucomannan biosynthetic genes across plant taxa and implies that the regulatory networks underlying polysaccharide synthesis have evolved to meet the unique metabolic and physiological demands of individual species. Systematic characterization of the conservation and divergence of CSLA-MSR interactions, transcriptional regulatory elements, and post-translational modification machineries across diverse plant species will enable us to unravel these evolutionary and adaptive processes. Ultimately, these comparative insights will deepen our fundamental understanding of plant polysaccharide metabolism and provide a rational, mechanistically informed framework for the biotechnological improvement of glucomannan content and quality in \u003cem\u003eA. konjac\u003c/em\u003e and other agronomically and economically important crops.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we systematically investigated the roles of AkCSLA2, AkCSLA3, and AkMSR1 in the biosynthesis of KGM in \u003cem\u003eA. konjac\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Transcriptomic analyses revealed that AkCSLA genes are predominantly expressed in the corms during the key stages of KGM accumulation, with AkCSLA2 exhibiting the highest expression level. Subcellular localization experiments demonstrated that both AkCSLA2 and AkCSLA3 localize to the plasma membrane, endoplasmic reticulum, and Golgi apparatus, consistent with their functions in polysaccharide synthesis and transport. Notably, protein interaction assays confirmed that AkMSR1 physically interacts with AkCSLA3, but not with AkCSLA2. Functional studies in Pichia pastoris further demonstrated that co-expression of AkMSR1 and AkCSLA3 significantly enhances mannose and glucose levels, with mannose content increasing more than threefold compared to expression of AkCSLA3 alone, indicating that AkMSR1 acts as a critical cofactor that promotes AkCSLA3-mediated glucomannan synthesis. Collectively, these findings elucidate a key regulatory mechanism in KGM biosynthesis, highlighting the functional specialization among AkCSLA members and the essential role of AkMSR1 in enhancing the catalytic efficiency of AkCSLA3. This work provides a molecular foundation for understanding KGM biosynthesis and offers potential targets for genetic improvement of KGM content in \u003cem\u003eA. konjac\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptomic data is available in the National Center for Biotechnology Information (NCBI) Bioproject database under the accession number: PRJNA734512. The annotation files of A. konjac genome are available at figshare: https://doi.org/10.6084/m9.figshare.15169578.\u0026nbsp;The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (grant numbers 32460457 and 32560077), the Yunnan Fundamental Research Projects (grant number 202501AU070172), the Yunnan Provincial Department of Education Science Research Fund Project (grant number 2024J0939), and the Special Basic Cooperative Research Innovation Programs of Qujing Science and Technology Bureau \u0026amp; Qujing Normal University (grant numbers KJLH2024ZD04 and KJLH2023YB08).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: S.Y.M., and L.C.X.; Data curation: S.Y.M., G.X.S., and L.C.X.; Methodology: L.D.B., C.H.L., and G.X.S.; Validation: S.Y.M., and L.C.X.; Project administration: L.C.X.; Resources: S.Y.M., and L.C.X.; Supervision: S.Y.M., Writing original draft: S.Y.M., and L.C.X.; Writing review \u0026amp; editing: S.Y.M., L.D.B., C.H.L., G.X.S., and L.C.X.; Funding acquisition: S.Y.M., and L.C.X. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang Y, Xie B, Gan X. Advance in the applications of konjac glucomannan and its derivatives. Carbohydr Polym. 2005;60:27\u0026ndash;31. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.carbpol.2004.11.003\u003c/span\u003e\u003cspan address=\"10.1016/j.carbpol.2004.11.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Kang Y, Du Y, Chen M, Guo L, Huang X, et al. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://commons.emich.edu/honors/565/\u003c/span\u003e\u003cspan address=\"https://commons.emich.edu/honors/565/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlonso-Sande M, Teijeiro-Osorio D, Remu\u0026ntilde;\u0026aacute;n-L\u0026oacute;pez C, Alonso MJ. Glucomannan, a promising polysaccharide for biopharmaceutical purposes. Eur J Pharm Biopharm. 2009;72:453\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejpb.2008.02.005\u003c/span\u003e\u003cspan address=\"10.1016/j.ejpb.2008.02.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Konjac glucomannan, Cellulose synthase-like 3, A. konjac, Mannan synthesis-related","lastPublishedDoi":"10.21203/rs.3.rs-9274000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9274000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKonjac glucomannan (KGM) is a high-viscosity, water-soluble dietary fiber that accumulates predominantly in the corms of \u003cem\u003eAmorphophallus konjac\u003c/em\u003e (\u003cem\u003eA. konjac\u003c/em\u003e). Due to its excellent gel-forming and water-retention properties, KGM is widely used in the food industry. The biosynthesis of KGM involves the coordinated action of cellulose synthase-like (CSL) enzymes and mannan synthesis-related (MSR) proteins. However, the regulatory mechanisms and functional interactions between these components remain poorly understood in\u003cem\u003e A. konjac.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we systematically characterized the AkCSLA gene family in \u003cem\u003eA. konjac \u003c/em\u003eand identified 11 family members. Transcriptomic analysis revealed that AkCSLA2 and AkCSLA3 were highly expressed during the corm expansion and maturation stages, which correspond to the periods of active KGM accumulation. Subcellular localization assays in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e showed that both AkCSLA2 and AkCSLA3 proteins localized to the plasma membrane, endoplasmic reticulum, and Golgi apparatus. Protein interaction analyses using yeast two-hybrid and co-immunoprecipitation assays demonstrated that AkMSR1, a homologue of \u003cem\u003eArabidopsis\u003c/em\u003eMSR1, specifically interacted with AkCSLA3, but not with AkCSLA2. Functional studies in Pichia pastoris revealed that co-expression of AkCSLA3 and AkMSR1 significantly increased the production of mannose and glucose, with mannose content elevated more than threefold compared to expression of AkCSLA3 alone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study identifies the AkCSLA3-AkMSR1 module as a crucial regulatory mechanism in KGM biosynthesis, wherein AkMSR1 acts as a specific cofactor to enhance the glucomannan synthesis mediated by AkCSLA3. This research provides novel insights into the molecular regulation of KGM biosynthesis and presents potential targets for the genetic enhancement of KGM content in \u003cem\u003eA. konjac.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"The AkCSLA3-AkMSR1 module mediates Konjac glucomannan biosynthesis in Amorphophallus konjac","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-15 17:20:07","doi":"10.21203/rs.3.rs-9274000/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T03:53:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T06:25:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T01:54:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T18:34:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250214271505736993294083874646601571559","date":"2026-04-10T11:03:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284600034727926288340024501254850030404","date":"2026-04-10T10:56:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152190216024259800409291864476466545445","date":"2026-04-10T06:24:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T10:21:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-03T06:38:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T14:00:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-02T13:59:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-03-31T03:43:15+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":"be24c528-dc8d-4c3a-8b52-fe9907e8f16b","owner":[],"postedDate":"April 15th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T03:53:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T06:25:05+00:00","index":34,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T01:54:35+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T04:08:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-15 17:20:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9274000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9274000","identity":"rs-9274000","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}