Identification of poplar and spruce GDP-mannose pyrophosphorylases involved in mannan and ascorbic acid biosynthesis

Abstract

Mannans are hemicellulosic polysaccharides integral to plant growth and development, yet their functions in trees and the regulation of their synthesis remain poorly understood. Here, we investigated the biosynthesis of guanosine diphosphate (GDP)-mannose, the primary sugar donor required for mannan synthesis. Using phylogenetic analyses, protein domain characterization, and RNA sequencing-based expression profiling, putative GDP-mannose pyrophosphorylase (GMP) candidates were identified in poplar and spruce. Functional validation of candidate genes was performed by complementation of the Arabidopsis vtc1-1 mutant, and by generating CRISPR/Cas9 knockout and ectopic overexpression lines in poplar. Our results showed that poplar PtaGMP1 and spruce PgGMP1 successfully complemented the vtc1-1 mutant, restoring mannan content to wild-type levels. In poplar, CRISPR/Cas9 knockouts of PtaGMP1 reduced mannan by 20% and ascorbic acid by 47%, whereas ectopic overexpression of PgGMP1 increased mannan by 14% without significantly affecting ascorbic acid content. In both cases, these changes did not significantly affect tree growth. Overall, our study provides new insights into the regulation of mannan biosynthesis in trees and the contribution of GMPs to both mannan and ascorbic acid production in poplar.

Introduction

Mannans are hemicelluloses broadly distributed throughout the plant kingdom, present in both the primary and secondary cell walls of many species, with abundance and composition varying across plant lineages and developmental stages (Scheller and Ulvskov 2010, Terrett et al. 2019). In gymnosperms such as spruce trees, galactoglucomannan is the predominant hemicellulose, whereas in angiosperms like poplar, glucomannan is present but less abundant, with xylan being the major hemicellulose (Scheller and Ulvskov 2010, Terrett et al. 2019). Mannans play an important role in plant biology, as they contribute to embryo development (Goubet et al. 2003, Goubet et al. 2009), seed mucilage architecture (Voiniciuc et al. 2015), and the structural integrity of some tissues via interactions with the primary load bearing carbohydrate, cellulose (Voiniciuc et al. 2015, Yu et al. 2018, Nishigaki et al. 2021). Although the biosynthetic pathway of this ubiquitous polysaccharide has been well characterized in Arabidopsis, our knowledge of its synthesis in trees remains limited. In the current study, we identified genes involved in mannan biosynthesis in poplar and spruce, as they represent two evolutionary distant plant lineages with contrasting mannan profiles, have distinct cell-wall architectures, and play major roles in global biomass production. In particular, we functionally characterized genes involved in the production of guanosine diphosphate (GDP)-mannose (GDP-Man), the primary nucleotide sugar donor required for the synthesis of the mannan backbone.

Mannan is synthesized from GDP-Man, which is produced in the cytosol by GDP-mannose pyrophosphorylase (GMP, EC 2.7.7.22; Figure 1). GMP catalyzes the reversible formation of GDP-Man from mannose-1-phosphate (Man1P; Conklin et al. 1999, Zhang et al. 2022), and its activity is enhanced by the KONJAC proteins (KJC; Sawake et al. 2015). GDP-Man serves as the main substrate for Cellulose Synthase-Like A (CSLA) proteins to synthesize the mannan backbone in the Golgi (Goubet et al. 2009, Guevara-Rozo et al. 2025), where it can subsequently be decorated with acetyl and galactosyl groups (Voiniciuc et al. 2015, Zhong et al. 2018, Yu et al. 2022). In addition to serving as the main sugar donor for mannan biosynthesis, GDP-Man is a precursor of other nucleotide sugars, including GDP-l-fucose (GDP-l-Fuc; Bonin and Reiter 2000), GDP-l-galactose (GDP-l-Gal; Qi et al. 2017), and GDP-l-gulose (GDP-l-Gul; Wolucka and Van Montagu 2003). These activated sugars contribute to the side chains that decorate various cell wall polymers, including xyloglucan, rhamnogalacturonan II, and arabinogalactan proteins (AGPs; Zablackis et al. 1996, Pauly et al. 2001, Van Hengel and Roberts 2002, Voxeur et al. 2011). GDP-Man also plays a role in the Dolichol Pathway, contributing to the N-glycosylation of glycoproteins (Conklin et al. 1999). In addition, GDP-Man produced by GMP enters the d-Man/l-Gal pathway (also known as the Smirnoff-Wheeler pathway) where it is converted by GDP-mannose-3′,5′-epimerase (GME) into GDP-l-Gul and GDP-l-Gal, ultimately leading to the production of ascorbic acid, an essential antioxidant involved in plant growth, development, and protection against oxidative stress (Barth et al. 2010, Jadid et al. 2011).

Figure 1.

Biosynthesis of GDP-mannose (GDP-Man) and its role in mannan synthesis. Mannose-1-phosphate (Man1P) is converted to GDP-Man by GDP-mannose pyrophosphorylases (GMP/VTC1) in the cytosol (Conklin et al. 1999, Zhang et al. 2022), a process enhanced by KONJAC (KJC) proteins (Sawake et al. 2015). GDP-Man acts as the primary sugar donor for mannan synthesis via CSLA enzymes (Goubet et al. 2009, Guevara-Rozo et al. 2025). It also serves as a precursor of GDP-l-fucose (GDP-l-Fuc) via GDP-mannose-3,6-dehydratase (GMD; Bonin and Reiter 2000) and GDP-l-galactose (GDP-l-Gal) and GDP-l-gulose (GDP-l-Gul) via GME (Qi et al. 2017, Wolucka and Van Montagu 2003). These additional sugar donors contribute to the biosynthesis of other cell wall carbohydrates, including xyloglucan, rhamnogalacturonan II (RGII), and arabinogalactan proteins (AGPs) (Zablackis et al. 1996, Pauly et al. 2001, Van Hengel and Roberts 2002, Voxeur et al. 2011) and play an important role in L-ascorbate formation. Finally, GDP-Man is involved in the N-glycosylation of glycoproteins (Dolichol Pathway; Conklin et al. 1999, Barth et al. 2010, Jadid et al. 2011). GTP, guanosine triphosphate; PPi, pyrophosphate; green circles, mannose; blue circles, glucose; yellow circles, galactose; red circles, acetyl groups. Light gray dotted arrows, poorly understood mechanisms; black dotted arrows, multistep processes.

In Arabidopsis, GMP1, also known as Vitamin C Defective 1 (VTC1), has been characterized as the principal enzyme responsible for the synthesis of GDP-Man (Conklin et al. 1997, 1999, 2000). The vtc1-1 mutant carries a Pro22Ser substitution that imparts an approximately 50% reduction in enzyme activity (Conklin et al. 1996, 1997, 2000). This mutant also exhibits approximately 50% of wild-type mannose content and 30% of wild-type ascorbate content and is ozone sensitive as well as less tolerant to stress conditions such as salinity (Conklin et al. 1996, Conklin et al. 1997, Conklin et al. 1999, Conklin et al. 2000, Huang et al. 2005). If VTC1 is truncated, as is the case in the cyt1 mutant, the mutation is embryo lethal (Lukowitz et al. 2001). GMPs have also been characterized in other plant species, including tomato and rice, and their genetic modification has proven advantageous for plant stress tolerance (Wang et al. 2011, Qin et al. 2016a, b). For example, overexpression of GMPs in tobacco and rice manifests in higher mannose and ascorbic acid contents and enhances tolerance to temperature and salinity stress (Wang et al. 2011, Qin et al. 2016b). Thus, understanding how GDP-Man is synthesized in trees will not only provide better tools to engineer mannan in woody species, but may also offer an alternative strategy to enhance plant stress tolerance.

In the present study, we identified GMP genes responsible for GDP-Man production in poplar and spruce (PtaGMP1 and PgGMP1) by functionally characterizing them in Arabidopsis vtc1-1 mutants and poplar trees. We show that manipulating the expression of these genes in poplar significantly alters mannan content and levels of the antioxidant ascorbate without substantially affecting plant growth. Overall, this study broadens our understanding of mannan regulation in woody species and provides valuable insights for mannan engineering in trees, as well as for modulating antioxidant levels that could potentially contribute to stress resilience.

Materials and methods

BLAST searches and phylogenetics of putative GMPs in poplar and spruce

To identify putative functional GMPs in poplar (Populus trichocarpa Torr. and A. Gray ex Hook) and spruce (Picea engelmannii Parry ex Engelm. × Picea glauca (Moench) Voss × Picea sitchensis (Bong) Carrière; clone PG29), the Arabidopsis VTC1 (At2g39770) sequence was used as a query in BLAST searches against the transcriptomes of both species (P. trichocarpa, v3 and PG29, v3, respectively) available through the National Center for Biotechnology Information (NCBI). Searches were performed with the following parameters: maximum target sequences set to 500, expect threshold of 0.05, word size of 28, maximum matches in a query range set to 0, and linear gap costs. The presence of conserved protein domains associated with functionally characterized GMPs in other plant species (PF02020 (W2 domain), PF00132 (Hexapep transferase domain), PF00483 (Nucleotidyl transferase domain) and CL11394 (Glycosyltransferase domain); Wang et al. 2023) was evaluated in the retrieved sequences using InterPro (Blum et al. 2025). Full-length protein sequences of the candidate GMP genes from poplar and spruce were then aligned with previously identified GMPs from a range of plant species, including Apium graveolens (UZH44860; Liu et al. 2022), Corymbia citriodora (CcGMP4; Wang et al. 2023), Fragaria × ananassa (FaGMP4; Lin et al. 2021), Malpighia glabra (A0EJL9; Badejo et al. 2009), Solanum lycopersicum (NP001234025; Zou et al. 2006) and Oryza sativa (NP001404413; Qin et al. 2016a) using MUSCLE in MEGA-X (Kumar et al. 2018). The multiple sequence alignment was then used to construct a phylogenetic tree in IQ-Tree with an ultrafast bootstrap with 1000 replicates using the LG+G4 substitution model (Nguyen et al. 2015) to evaluate sequence relationships.

RNA sequencing analysis of putative GMPs from poplar and spruce

We evaluated the transcript abundance of the candidate GMP genes in poplar and spruce xylem by employed available RNA sequencing (RNA-seq) expression data. In poplar, we retrieved the expression data of the most plausible candidates from a previously reported RNA-seq expression database (hereafter GolS transcriptome database) generated by Unda et al. (2017). This expression database was derived from transgenic poplar trees expressing the Arabidopsis galactinol synthase (GolS3) gene that manifested in higher cellulose content and a significantly reduced lignin and mannose content compared with wild-type trees, making the data set ideal for identifying genes that are likely involved in mannan biosynthesis.

For spruce, we used RNA-seq reads from NCBI (Accession Number: SRX318121) derived from PG29 xylem tissue. The raw reads were pre-processed by removing adapters and low-quality sequences using Trim-Galore V. 0.6.0 (Krueger 2021) with default setting and adapter auto detection. Read quality was assessed before and after trimming using FastQC (Andrews 2010). The processed reads were mapped against the PG29 v 5.0 (downloaded from https://www.bcgsc.ca/downloads/btl/Spruce/) genome assembly and indexed with STAR v.2.5.2b (Dobin et al. 2013). A custom annotation of PgGMP1 was added to the annotation file used during the indexing and mapping. FeatureCounts version 2.0.6 (Liao et al. 2014) was used to calculate gene expression values as raw fragment counts. The number of FPKM (Fragments Per Kilobase of transcript per Million mapped fragments) was determined using the function count2FPKM from the Recount package (Collado-Torres et al. 2017) in R v 4.3.1.

Gene isolation from plant material

Xylem tissue from spruce (provided by the Bohlmann Lab at UBC) and poplar 717 (Populus tremula L. × Populus alba L., INRA 717-1B4) was collected by scraping debarked stems of approximately 2-m-tall trees using a sterile razor blade. The collected tissue was immediately flash-frozen in liquid nitrogen and stored at −80 °C prior to RNA extraction. Total RNA was extracted using the CTAB-based protocol (Kolosova et al. 2004). RNA samples were then treated with DNase using the Turbo DNA-free kit (Life Technologies, Carlsbad, CA, USA), and subsequently used for the synthesis of cDNA with the iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA).

GMP plant expression constructs

The coding region of PtaGMP1 (Potri.010G198800) and PgGMP1 (BT102800) was amplified using BestTaq polymerase (Applied Biological Materials, Richmond, BC, Canada) with the primers PtaGMP1_FWD_attB and PtaGMP1_REV_attB for PtaGMP1, and PgGMP1_FWD_attB and PgGMP1_REV_attB for PgGMP1 (Table S1 available as Supplementary Data at Tree Physiology Online). The coding sequences were then cloned into the pDONR221 (Invitrogen, Carlsbad, CA, USA) Gateway entry vector by BP clonase recombination, and subsequently transferred into the pk7WG2 plant expression vector driven by the Arabidopsis secondary tissue specific CESA7 promoter (AtCESA7; Karimi et al. 2002, Smith et al. 2015) using LR recombination. Chemically competent Agrobacterium tumefaciens cells (strains GV3101 and EHA105) were transformed with each of the expression vectors carrying the putative genes following the freeze–thaw protocol (Wise et al. 2006).

gRNA design

A database with all possible gRNAs targeting PtaGMP1 in poplar was created using the variant-sensitive Aspen CRISPR designer pipeline (Tsai and Xue 2015). From this database, the gRNA2 (5′-TTAGGGACACTGAGAGTCAA-3′) targeting the first exon of PtaGMP1 was selected for subsequent experiments. The total number of predicted off-targets for this gRNA was 21 all carrying at least three mismatches and being present in the seed region.

CRISPR/Cas9 cloning

Knockout constructs were generated by Gibson assembly following the protocol of Jacobs and Martin (2016). Briefly, the Medicago truncatula U6 promoter and the scaffold fragment were amplified from the pUC gRNA shuttle plasmid (Addgene plasmid #47024) using the primers SwaI.MtU6F and MtU6R, and SpeI.hpR and hpF, respectively (Table S1 available as Supplementary Data at Tree Physiology Online). Then, these two fragments were cloned alongside the selected gRNA into the p201N::Cas9 plasmid (Addgene plasmid # 59175) using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs). Following transformation of TOP10 E. coli (Invitrogen) competent cells with 3 μL of the Gibson reaction, PCR-positive colonies were confirmed by Sanger sequencing (Genewiz) of two different fragments, the first using the primers: SpeI.hpR and gRNA2.SF.R-GMPs, and the second one using the primers: 2X35Shyb and gRNA2.U6.F-GMPs (Table S1 available as Supplementary Data at Tree Physiology Online). Confirmed vectors were then transferred into A. tumefaciens strain EHA105 for poplar transformation.

Agrobacterium-mediated transformation of Arabidopsis

Transformation of Arabidopsis vtc1 mutant was completed following the floral dip method (Clough and Bent 1998) using Agrobacterium strain GV3101 carrying the expression vectors. Seeds were plated on 1/2 concentration Murashige and Skoog basal medium (MS media; Murashige and Skoog 1962) plates containing 50 μg mL⁻¹ kanamycin, stratified at 4 °C in the dark for 2 days, and subsequently germinated under constant light. Germinated seeds were transferred to soil and grown for 6 weeks under long-day conditions (16 h light/8 h dark) (Zhang et al. 2006).

Agrobacterium-mediated transformation of poplar

Poplar leaf discs were transformed following the protocol of Wilkerson et al. (2014). Briefly, discs were cut from 5-week-old poplar 717 leaves and co-cultivated for 1 h at 28 °C with A. tumefaciens strains (EHA105; OD_600nm = 0.1–0.2) transformed with the expression vectors. Leaf discs were placed abaxial-side-up on Woody Plant Media (WPM; 0.1 μM NAA, 0.1 μM BA and 0.1 μM TDZ) and incubated in the dark for 2 days. Then, leaf discs were transferred to WPM media (0.1 μM NAA, 0.1 μM BA and 0.1 μM TDZ) containing 250 μg mL⁻¹ cefotaxime and 500 μg mL⁻¹ carbenicillin to eliminate A. tumefaciens, and maintained in darkness for 2 days. Afterward, leaf discs were placed on WPM (0.1 μM NAA, 0.1 μM BA and 0.1 μM TDZ) selection media containing 50 μg mL⁻¹ of kanamycin for 5 weeks. Leaf discs were then transferred to WPM shooting media (0.01 μM BA, 250 μg mL⁻¹ cefotaxime, 500 μg mL⁻¹ carbenicillin and μg mL⁻¹ kanamycin) for 4 weeks, and subsequently moved to WPM rooting media (0.01 μM NAA, 250 μg mL⁻¹ cefotaxime, 500 μg mL⁻¹ carbenicillin and 50 μg mL⁻¹ kanamycin). After 5 weeks of growth, genomic DNA was used to screen for positive transgenics using the gene-specific primers (Table S1 available as Supplementary Data at Tree Physiology Online), and positive transformants were propagated in antibiotic-free WPM media (0.01 μM NAA). Apical stem pieces from propagated transformants (eight biological replicates per line) were grown for 4 weeks and then the subsequently rooted plantlets were transferred to the soil. This protocol was concomitantly followed using wild-type poplar 717 leaf discs as a control, with the exception that all media were antibiotics-free.

Genomic screening of Agrobacterium-transformed Arabidopsis plants

Genomic DNA was extracted from 7-week-old leaf tissue from Arabidopsis complemented mutants. Leaf tissue was ground in a mortar and pestle with liquid nitrogen and 400 μL of DNA extraction buffer containing 25 mM NaEDTA, 250 mM NaCl, 200 mM Tris HCl and 0.5% Sodium Dodecyl Sulfate. Samples were centrifuged at 13,000 r.p.m. for 3 min, and the supernatant was placed in a new 1.5 mL tube. DNA precipitation was achieved by adding 300 μL of isopropanol for 10 min followed by centrifugation at 13,000 r.p.m. for 5 min. The supernatant was discarded, and the pellet was washed by adding 500 μL of 70% ethanol. After centrifugation at 13,000 r.p.m. for 2 min, the pellet was dried and then resuspended in sterile distilled water. Genomic screening was performed using the following gene-specific primers on the extracted DNA: PtaGMP1_FWD and PtaGMP1_REV, and PgGMP1_FWD and PgGMP1_REV (Table S1 available as Supplementary Data at Tree Physiology Online).

Arabidopsis and poplar cell wall composition

Cell wall composition was quantified using secondary acid hydrolysis (Coleman et al. 2008). A modified protocol was used to determine the total carbohydrate content of Arabidopsis stems (Glass et al. 2015). Briefly, the bottom 13 cm of 7-week-old Arabidopsis stems were collected and dried at 50 °C for 48 h. For one biological replicate, seven inflorescent stems isolated from seven plants were pooled. Three to four biological replicates were generated from each line and wild-type plants. Each pool of seven stems was ground in a 2000 Geno/Grinder® (SPEX® SamplePrep, USA) and extracted overnight with hot acetone using a Soxhlet apparatus. A total of 10 mg of extracted ground stem tissue was treated with 100 μL of 72% sulphuric acid for 5 min, then vortexed and briefly centrifuged on a bench-top centrifuge. Samples were incubated at 30 °C for 1 h at 500 r.p.m., and then 2  mL of nanopure water was added. Next, samples were vortexed and incubated at 121 °C for 75 min followed by centrifugation at 13,000 r.p.m. for 5 min. Finally, the supernatant was collected and diluted (1:10) with nanopure water. Carbohydrate content was determined by high-performance anion exchange liquid chromatography (HPAEC) of the filtered hydrolysates. A dilution series of external standards (arabinose, rhamnose, galactose, glucose, xylose, and mannose) was used to create standard curves and determine sugar concentrations in each sample. The samples and standards were run on a Dionex Dx-600 anion-exchange High-Performance Liquid Chromatography (HPAEC; Dionex, Sunnyvale, CA, USA) fit with a CarboPac PA1 column with distilled deionized water as an eluant at a flow rate of 0.8 mL/min. Detection was achieved with a pulsed amperometric detector fit with a gold electrode and post-column addition of 0.2 M sodium hydroxide at a flow of 0.5 mL/min. Meso-erythritol was used as an internal standard.

For poplar, 200 mg of 40-mesh wood powder was subjected to Soxhlet extraction. The dried samples were then treated with 3 mL of 72% sulphuric acid for 2 h with constant manual stirring with a glass rod every 10 min. Subsequently, 112 mL of water was added, and samples were autoclaved at 121 °C for 75 min. After autoclaving, the samples were filtered through pre-weighed coarseness crucibles, and the remaining residues were rinsed with 150 mL of water, dried overnight at 105 °C, and weighed to determine acid-insoluble lignin. The filtered aliquot was used to determine the acid-soluble lignin by measuring its UV absorbance at 205 nm. Carbohydrate content was determined by HPAEC as previously described, using fucose as an internal standard.

Genomic screening of poplar

Genomic DNA was extracted from the leaves of wild-type and transformed poplar in tissue culture following the same procedure employed for Arabidopsis. The target genes were detected using gene-specific primers for PtaGMP1 and PgGMP1 (Table S1 available as Supplementary Data at Tree Physiology Online). In the case of the poplar lines transformed with the p201NCas9::gRNA2 construct, the genomic screening targeted the Cas9 gene using the Cas9_p59175_FWD and Cas9_p59175_REV primers (Table S1 available as Supplementary Data at Tree Physiology Online). For these trees, only positive lines were subsequently analyzed by Sanger sequencing to confirm the mutations.

Gene expression analysis of GMPs in transformed poplar

Total RNA was isolated from poplar xylem scrapings using the PureLinkTM RNA Mini Kit (Invitrogen). RNA was treated with DNase using the Turbo DNA-free kit (Ambion, Life Technologies), and then employed to synthesize cDNA with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression levels of PtaGMP1, PtaGMP2, and PgGMP1 were quantified using BlasTaq 2X qPCR MasterMix (Applied Biological Materials, Richmond, BC, Canada; Table S1 available as Supplementary Data at Tree Physiology Online). PCR consisted of 5 μL BlasTaq 2X master mix, 0.3 μL of reverse and forward primers (10 μM), 1 μL cDNA, and deionized water to a volume of 10 μL. Three technical replicates were used per sample. qPCR parameters were as follows: 5 min at 95 °C, 39 cycles of 95 °C for 5 s and 60 °C for 30 s, then 1 cycle of 94 °C for 10 s, followed by a melt curve cycle of 56 °C to 95 °C at 0.5 °C increments for 5 s. Average expression of the reference gene PtrEF1β (Potri.009G018600; primers: PtEF1β-qPCR-FWD and PtEF1β-qPCR-REV) was used to calculate the relative expression of the target genes (Table S1 available as Supplementary Data at Tree Physiology Online). Relative expression of target genes in the CRISPR-edited lines was calculated relative to wild-type levels, whereas in lines expressing the spruce gene, expression was calculated relative to the poplar line exhibiting the highest expression.

Determination of mutant spectrums in CRISPR poplar lines

Using genomic DNA isolated from lines that proved positive after the genomic screening of Cas9, a PCR was performed using the primers CRISPR_PtaGMP1_FWD and CRISPR_PtaGMP1_REV to amplify the PtaGMP1 sequence (Table S1 available as Supplementary Data at Tree Physiology Online). PCR products were Sanger sequenced using the same primers employed for amplification and analyzed for possible mutations via TIDE (Brinkman et al. 2014). Confirmation of a biallelic or monoallelic mutation of the gene was completed by analyzing the chromatogram and comparing it with the known gene sequence of each possible allele. To confirm PtaGMP2 was not edited, we also sequenced the targeted region with the CRISPR_PtaGMP2_FWD and CRISPR_PtaGMP2_REV primers.

Poplar growth conditions and measurement

The apical meristems of 6-week-old, transformed plants and corresponding controls were transferred to two-gallon pots containing perennial soil mix (50% peat, 25% fine bark, and 25% pumice; pH 6.0). Trees were grown on flooding tables under 16 h of light at the UBC greenhouse for 5 months. At the time of harvest, stem height, stem diameter, and weight of fresh biomass were recorded. Height was measured from root collar to tree apex, while the diameter was measured 10 cm above root collar with digital calipers. The weight of fresh biomass was recorded using the whole tree cut 10 cm above the root collar. Developing xylem was collected by scraping the debarked stems with a sterile razor blade, and subsequently flash-frozen in liquid nitrogen and stored at −80 °C. Debarked stems were air-dried for 2 weeks, and then a 10-cm piece originating from the base of the cut stem was ground using a Wiley mill to pass a 40-mesh sieve.

Total ascorbic acid measurement

The content of total ascorbic acid in the xylem tissue of transgenic and wild-type poplars was assessed following the protocol described by Lemmens et al. (2020) with some modifications. Briefly, 1.5 mL of 0.1% metaphosphoric acid was added to 100 mg of fresh xylem tissue and incubated at 4 °C for 1 h with constant shaking. Three technical replicates were extracted for each biological replicate. Next, samples were centrifuged at 8000 r.p.m. for 10 min at 4 °C and the supernatant decanted and filtered through 0.45 μm filters. Then, 50 μL of 200 mM dithiothreitol in 400 mM Tris base was added to 100 μL of the filtered ascorbic acid containing supernatant and incubated for 30 min at room temperature in dark. Then, 50 μL of 8.5% orthophosphoric acid was added to the sample and incubated for 5 min on ice. Finally, 6 μL of each sample was analyzed on an Agilent 1290 Infinity II UPLC system (Agilent Technologies, CA, USA) equipped with an Eclipse Plus C18 column with a mobile phase of 0.1% metaphosphoric acid and acetonitrile (98:2, v/v) and a flow rate of 0.5 mL/min. Ascorbic acid was detected with a Diode Array Detector at 243.3 nm. The total ascorbic acid concentration was calculated by comparing area to those obtaining fitting a standard curve made with a commercial ascorbic acid standard.

Statistical analysis

A Kruskal–Wallis test followed by Dunn’s post-hoc analysis with a Bonferroni correction was used to assess statistical differences. Significance was evaluated using an experiment-wise error rate of 0.05. All analyses were performed in the R software environment (version 4.3.1).

Results

Identification of GMP gene candidates in poplar and spruce

Three spruce genes (BT102800, BT107981, and GCHX01355040) and four poplar genes (Potri.010G198800, Potri.008G060100, Potri.006G090300, and Potri.008G006700) were identified as putative GMP candidates based on BLAST searches (Figure 2a). All candidate genes contained the conserved PF02020, PF00132, PF00483, and CL11394 protein domains and showed a high degree of similarity with VTC1 (Figure S1 available as Supplementary Data at Tree Physiology Online). The protein sequence identity to Arabidopsis VTC1 was 85.60% (BT102800), 86.15% (BT107981), and 77.44% (GCHX01355040) in spruce, and 91.97% (Potri.010G198800), 90.03% (Potri.008G060100), 88.92% (Potri.006G090300), and 82.66% (Potri.008G006700) in poplar. Analysis of poplar expression data from the GolS transcriptome database revealed that Potri.010G198800 (hereafter PtaGMP1) was the most highly expressed homolog in wild-type trees, with an FPKM of 292, followed by Potri.008G060100 (hereafter PtaGMP2), which had an FPKM of 119 (Figure 2b). In addition, PtaGMP1 exhibited the strongest downregulation in transgenic trees overexpressing the galactinol synthase gene (GolS) which displayed a decrease of nearly 2 log₂ fold-change in trees that exhibited significantly reduced mannan content in their secondary cell walls (Table S2 available as Supplementary Data at Tree Physiology Online; Unda et al. 2017). In spruce, BT102800 (hereafter PgGMP1) and BT107981 (hereafter PgGMP2) had the shortest phylogenetic distance with previously identified GMPs, while GCHX01355040 (hereafter PgGMP3) was less similar (Figure 2a). Our spruce xylem RNA-seq analysis indicated that PgGMP2 was more highly expressed in this tissue than PgGMP1 and PgGMP3 (Figure 2b). Based on these expression patterns and phylogenetic relationships, PtaGMP1 and PgGMP1 were selected for in planta functional characterization.

Figure 2.

Phylogenetic tree and expression profiles of putative poplar and spruce orthologs of Arabidopsis VTC1. (a) Four putative poplar orthologs and three candidate spruce orthologs were identified via BLAST searches. All candidates contained the conserved protein domains previously associated with GMP enzymes (PF02020, PF00132, PF00483 and CL11394). (b) Expression patterns from RNA-seq data (FPKM) for poplar and spruce GMP candidates. In poplar, PtaGMP1 and PtaGMP2 exhibited the highest expression levels in xylem tissue, while in spruce, PgGMP1 and PgGMP3 showed the strongest expression. Of the three spruce candidates, PgGMP1 and PgGMP2 clustered most closely with previously identified GMPs. Node labels represent UFBoot support values. The scale bar indicates phylogenetic distance. The tree was generated using the LG + G4 substitution model.

GMPs from spruce and poplar increase mannose content in the Arabidopsis vtc1-1 mutant

To determine whether the candidate genes encode functional GMPs, PtaGMP1 and PgGMP1 were expressed in the Arabidopsis vtc1-1 mutant, which is impaired in GDP-Man biosynthesis. Complemented plants expressing AtCESA7::PtaGMP1 or AtCESA7::PgGMP1 were compared with wild-type and vtc1-1 mutants to assess the restoration of stem cell wall carbohydrate composition. Expression of PtaGMP1 restored mannose content in the vtc1-1 mutant, with line 2 reaching mannose levels comparable to wild-type plants (Figure 3). In these lines, no significant changes were observed in other structural sugars when compared with wild-type and the vtc1-1 mutant (Table S3 available as Supplementary Data at Tree Physiology Online). Similarly, plants complemented with the AtCESA7::PgGMP1 construct showed increased mannose content, with line 1 displaying levels comparable to wild type and significantly higher levels than the vtc1-1 mutant (P-value = 0.023; Figure 3). For other structural sugars, only line 1 exhibited a significant decrease in xylose content when compared with vtc1-1 (P-value = 0.0178; Table S4 available as Supplementary Data at Tree Physiology Online), while no significant differences were observed for the remaining neutral sugars. Overall, these results support the involvement of PtaGMP1 and PgGMP1 in mannan biosynthesis and suggest that they encode GMPs.

Figure 3.

Stem mannose composition of wild-type Arabidopsis, vtc1-1 mutants and vtc1-1 mutants complemented with PtaGMP1 and PgGMP1. Independent whole cell wall analyses were performed to determine the mannose content in the complemented mutant lines expressing the AtCESA7::PtaGMP1 and AtCESA7::PgGMP1 constructs. Mutant lines complemented with PtaGMP1 showed higher mannose content compared with vtc1-1 (Kruskal–Wallis and post-hoc analysis, P-value >0.05, n = 3–4 for each line, different letters indicate significant differences). In lines complemented with PgGMP1, mannose content was also higher, and line 1 was significantly different from vtc1-1 (Kruskal–Wallis and post-hoc analysis, P-value <0.05, n = 3–4 for each line, different letters indicate significant differences). Error bars indicate the SE. Values are represented in μg/mg of extracted tissue. GMP, GDP-mannose pyrophosphorylase; WT, wild-type Col-0 Arabidopsis.

Generation of CRISPR/Cas9-mediated PtaGMP1 knockout poplar lines

To confirm the activity of PtaGMP1 in mannan biosynthesis, poplar knockouts were created via CRISPR/Cas9 targeting the first exon of the candidate gene using gRNA2 (3′-TTAGGGACACTGAGAGTCAA-5′; Figure 4a). This gRNA was designed to target a SNP-free region using the Aspen CRISPR design pipeline (Tsai and Xue 2015). Following tree regeneration, the presence of the Cas9 gene was confirmed by PCR, and only positive lines were subjected to Sanger sequencing. Three lines with indels in PtaGMP1 were obtained, all carrying biallelic frameshift mutations caused by thymine insertions in the target sequence (Table S5 available as Supplementary Data at Tree Physiology Online). Due to the high similarity between PtaGMP1 and PtaGMP2, we also sequenced the targeted region in PtaGMP2 and confirmed that it had not been edited by the selected gRNA (Table S5 available as Supplementary Data at Tree Physiology Online). Lines carrying indels in PtaGMP1 were designated as PtaGMP1 knockout lines (PtaGMP1 KO) and clonally propagated and transferred to soil for growth in the greenhouse.

Figure 4.

Characterization of PtaGMP1 single-knockout poplar lines. (a) Using gRNA2 targeting the first exon of PtaGMP1, we generated three single knockout (KO) lines. In all cases, indels were single-nucleotide insertions causing frameshift mutations. Chromatograms of the targeted region of WT and line 2 are shown, where PtaGMP1 KO carries a thymine (T) insertion. Biallelic mutations were confirmed by verifying that both alleles were amplified and visible in the chromatograms. (b) Mannose content (μg/mg of extracted tissue) in WT and PtaGMP1 KO lines. Line 2 and line 3 displayed a significant reduction in mannose relative to WT (Kruskal–Wallis with post-hoc analysis, P-value <0.05, n = 4-5 per line). (c) Ascorbic acid content (μmol/g of FW) was significantly reduced in line 3 compared with wild-type control (Kruskal–Wallis with post-hoc analysis, P-value <0.05, n = 3–4 per line). Overall, all three knockout lines showed reduced ascorbic acid. Different letters indicate significant differences. WT, wild-type.

Loss of PtaGMP1 does not induce compensatory expression of PtaGMP2 in poplar

To determine whether the CRISPR-induced mutations affected PtaGMP1 expression or triggered compensation by other GMP genes, xylem tissue was collected from PtaGMP1 KO and wild-type trees after 5 months of greenhouse growth and analyzed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). Expression of PtaGMP1 was slightly reduced in all CRISPR-edited lines compared to wild-type trees, although these differences were not statistically significant (Figure S2 available as Supplementary Data at Tree Physiology Online). To assess potential compensation by other GMPs, we measured PtaGMP2 expression. The results showed that PtaGMP2 expression was similar to that of wild-type trees, with a slight and non-significant increase in line 3 (Figure S3 available as Supplementary Data at Tree Physiology Online). Overall, these results suggest that PtaGMP2 is unlikely to compensate for the putative loss of PtaGMP1 activity.

Mutation of PtaGMP1 in hybrid poplar does not affect plant growth

Height, diameter, and biomass were recorded at the time of harvest in the wild-type and CRISPR-edited trees grown in the greenhouse. Collectively, our results showed that PtaGMP1 KO lines did not exhibit any significant changes in the growth traits assessed compared with wild-type trees (Figure S4 available as Supplementary Data at Tree Physiology Online; Table S6 available as Supplementary Data at Tree Physiology Online).

PtaGMP1 KO lines exhibit a reduced mannan and ascorbic acid content

Xylem tissue from PtaGMP1 KO lines and wild-type trees was analyzed to determine the impact of PtaGMP1 disruption on cell wall composition. Total lignin content, including soluble and insoluble lignin, did not differ significantly between the PtaGMP1 KO lines and wild-type controls (Table S6 available as Supplementary Data at Tree Physiology Online). However, analysis of individual neutral cell wall carbohydrates by HPAEC revealed a reduction in mannose content in all knockout lines, with line 2 and line 3 showing significant decreases compared to wild-type trees (P-value <0.001 and P-value = 0.031, respectively; Figure 4b). In these lines, mannose content was reduced by approximately 20% relative to wild type (Figure 4b), while no significant differences were observed for other structural sugars (Table S7 available as Supplementary Data at Tree Physiology Online).

Given that GDP-Man is also a precursor for ascorbic acid biosynthesis, we next evaluated the content of this antioxidant in the xylem of the PtaGMP1 KO lines. Our results indicated that PtaGMP1 KO lines displayed reduced ascorbic acid levels (Figure 4c). When compared with wild-type trees, line 1 showed a 17% reduction in ascorbate content, while line 2 and line 3 showed decreases of 49 and 48%, respectively. Among these lines, only line 3 differed significantly from wild type (P-value = 0.043; Figure 4c). Overall, our results confirmed that PtaGMP1 functions as a GMP pyrophosphorylase in poplar and contributes to both mannan biosynthesis and ascorbic acid production.

PgGMP1 overexpression in poplar

To confirm the function of the spruce PgGMP1 as a putative GMP, we expressed this gene in poplar by transforming wild type trees with the AtCESA7::PgGMP1 construct. Two positive poplar transgenic lines carrying this construct were propagated in tissue culture and then transferred to the greenhouse, where they were permitted to grow for 5 months before being harvested.

To corroborate the expression of PgGMP1 in poplar, we collected fresh xylem tissue at harvest and subsequently evaluated PgGMP1 expression by RT-qPCR. Our results indicated that line 2 had the highest expression between the two lines analyzed (Figure 5a). We did not observe major developmental differences in height, diameter, or biomass when comparing the transgenic lines to wild-type trees (Figure S4  available  as available as Supplementary Data at Tree Physiology Online; Table S8 available as Supplementary Data at Tree Physiology Online). In general, these results show that PgGMP1 is expressed in poplar and that its expression did not alter plant growth.

Figure 5.

Characterization of poplar trees expressing PgGMP1. (a) Expression analysis showing that PgGMP1 is expressed in the two transgenic poplar lines evaluated, with line 2 exhibiting the highest expression (RT-qPCR; Kruskal–Wallis and post-hoc analysis, P-value <0.05, n = 3–4 for each line). (b) Mannose content (μg/mg extracted tissue) in wild-type (WT) and PgGMP1-expressing poplar lines. Line 2 showed a significant increase in mannose relative to WT (Kruskal–Wallis with post-hoc analysis, P-value < 0.05, n = 3–4 per line). (c) Ascorbic acid content (μmol/g FW) in PgGMP1 poplar expressing lines. A slight increase was observed in both lines, although not statistically significant (Kruskal–Wallis with post-hoc analysis, P-value >0.05, n = 3–4 per line). Different letters indicate significant differences.

PgGMP1 alters mannan and ascorbic acid content in poplar stems

Similarly, as described above, xylem cell wall composition was quantified in all poplar lines carrying the AtCESA7::PgGMP1 construct. We did not observe a significant difference in total, insoluble- or acid-soluble lignin content between the transgenic lines and the wild-type trees (Table S8 available as Supplementary Data at Tree Physiology Online). When comparing the structural polysaccharides, we observed that line 2, the line with the highest gene expression, showed a significant increase in the content of mannose (P-value = 0.035; Figure 5b). In this line, the mannose content displayed a 14% increase in comparison with wild-type trees, increasing from 25 μg/mg of extracted tissue in wild-type trees to 28.5 μg/mg in the transgenic line. No changes were observed in other polysaccharides (Table S9 available as Supplementary Data at Tree Physiology Online).

Finally, we also evaluated whether PgGMP1 expression caused an increase in ascorbic acid. Although the results were not statistically significant, ascorbic acid content increased by 26.3% in line 1 and 13.4% in line 2 when compared with wild type (Figure 5c; from 8.4 to 10.7 μmol/g FW and 9.6 μmol/g FW, respectively). Overall, these findings confirmed that PgGMP1 is a functional GMP and has the capacity to alter mannan content in trees.

Discussion

Understanding how mannans are synthesized and regulated in plants is essential for elucidating their biological functions and for tailoring their production for industrial applications. Beyond their roles in plant growth and development, mannans are valuable polymers with significant potential for use in bio-based materials, food additives, and biofuels (Mudgil et al. 2014, Singh et al. 2018, Yong et al. 2022). The chemical and physical properties that make mannans suitable for these applications are closely linked to their structure, particularly their side-chain composition (Berglund et al. 2020a, 2020b). Recent work has demonstrated that tailoring the side chains to produce mannans with specific structures is feasible, and these alterations can impact mannan–cellulose interactions, ultimately positively affecting mannan extractability (Yoshimi et al. 2025). However, the limited understanding of mannan biosynthesis in trees restricts the translation of these findings to industrial applications using lignocellulosic biomass. Therefore, this study aimed to expand our understanding of GMP-mediated regulation of mannan biosynthesis in trees and establish a foundation for future strategies to modify mannan content in woody species.

Identification and functional characterization of GMPs in poplar

Our bioinformatic analyses identified four putative poplar orthologs of Arabidopsis VTC1 (PtaGMP1–PtaGMP4) involved in GDP-Man biosynthesis. Functional analyses confirmed PtaGMP1 as a GMP showing that its expression in the Arabidopsis vtc1-1 mutant restored stem mannan content, while CRISPR disruption in poplar reduced mannan and ascorbic acid levels in the xylem. Notably, these metabolic changes did not result in detectable tree growth defects.

Similar observations have been reported in other plant systems when GMPs have been disrupted. For example, in rice, knockdown of OsVTC1-1 reduced mannose and galactose abundance, decreased ascorbic acid content, and altered leaf anatomy (Qin et al. 2016a, Lamanchai et al. 2022). In potato, suppression of GMPase activity using an antisense cDNA construct led to marked reductions in both mannose and ascorbate (Keller et al. 1999). In these plants, mannose content was reduced to 30–50% of wild-type levels whereas other monosaccharides remained largely unaffected (Keller et al. 1999). Ascorbate levels also declined, with leaf ascorbate dropping to 44–72% of wild-type levels and tubers retaining approximately 71% of normal content. The transgenic plants showed phenotypical changes when transferred from tissue culture to soil, where they developed dark lesions on both the leaves and stems after 10 weeks, while tubers showed no visible abnormalities (Keller et al. 1999). Similarly, in tomato, silencing of SIGMP3 substantially reduced ascorbic acid levels and caused leaf lesions associated with increased H₂O₂ accumulation (Zhang et al. 2013). In the current study, we showed that the disruption of PtaGMP1 was associated with significant reductions in both mannose and ascorbic acid content. Interestingly, unlike the reported developmental defects observed in other plant species with impaired GMP activity, PtaGMP1 KO lines showed no visible growth abnormalities, suggesting a tolerance to moderate reduction in mannose and ascorbic acid content in poplar.

In our previous work, we achieved complete elimination of stem mannan by knocking out CSLA genes and likewise observed no major growth effects (Guevara-Rozo et al. 2025). This is consistent with the present findings, where a partial reduction in mannan did not lead to noticeable growth consequences. Regarding ascorbic acid, substantial decreases without major growth effects have also been reported in the Arabidopsis vtc2-4 mutant, which maintains only ~ 20–30% of wild-type ascorbate levels (Lim et al. 2016). VTC2 catalyzes the conversion of GDP-l-galactose to l-galactose 1-phosphate, which is subsequently converted into l-ascorbate. This step is considered the primary control point in the ascorbate biosynthetic pathway in plants (Fenech et al. 2021, Smirnoff and Wheeler 2024). Based on the vtc2-4 phenotype, it has been proposed that ascorbate levels must drop below 20% of wild type levels (i.e., a reduction greater than 80%) before substantial growth defects emerge due to insufficient antioxidant capacity (Lim et al. 2016). This, combined with the fact that we only modified this pathway in secondary cell wall tissue specifically, may explain why we did not observe major growth changes in the CRISPR-edited poplar lines that showed only half the levels of the wild type ascorbate content.

Overall, our findings indicate that PtaGMP1 contributes to GDP-Man biosynthesis in xylem, affecting mannan accumulation and, to some extent, ascorbic acid production. Future studies should investigate the roles of PtaGMP2, PtaGMP3, and PtaGMP4 in this pathway, as the incomplete loss of mannan in the PtaGMP1 KO lines suggests that the other GMP isoforms are likely participating in GDP-Man biosynthesis in poplar.

Spruce GMPs in poplar

We identified and functionally characterized PgGMP1 a spruce ortholog of Arabidopsis VTC1. Similar to PtaGMP1, we showed that PgGMP1 can restore Arabidopsis vtc1-1 mannan phenotype and demonstrated that its expression in poplar contributes to the synthesis of this polysaccharide specifically in the secondary cell wall. We also showed that the ectopic expression of this gene slightly increased ascorbic acid content in the xylem and identified two additional putative GMP candidates: BT10798 (PgGMP2) and GCHX01496356 (PgGMP3).

Manipulating GDP-Man biosynthesis in trees is not only relevant for mannan biotechnology but also provides an opportunity to better understand how ascorbic acid is produced in woody species. Ascorbic acid acts as an efficient scavenger of reactive oxygen species, particularly H₂O₂ generated during photosynthesis and photorespiration (Buettner and Schafer 2004, Foyer and Noctor 2011). Since oxidative stress can inhibit key enzymes responsible for CO₂ fixation in the chloroplast, ascorbic acid has been shown to play an important role in protecting and maintaining photosynthetic efficiency (Foyer 2004). This metabolite also plays a crucial role in iron (Fe) homeostasis by preventing Fe over-oxidation in iron-deficient conditions (Zaharieva et al. 1999, Ramírez et al. 2013) and reducing Fe³⁺ to Fe²⁺ in vacuoles for proper storage and cytosolic transport (Grillet et al. 2014). In general, high levels of ascorbate in plants seem to increase tolerance to stress conditions such as temperature fluctuations, high salinity, and metal deficiency (Foyer and Halliwell 1977, Kaiser 1979). Moreover, ascorbate has substantial industrial relevance, being widely used in vitamin C production and as a preservative in food and beverage processing (Hancock and Viola 2002, Running et al. 2004). Therefore, having a better fundamental understanding of how this biosynthetic pathway is regulated is relevant to both tree resilience and tree biotechnology.

Although GMPs are not the main regulators of ascorbic acid biosynthesis in plants (Fenech et al. 2021), increased GMP expression has shown to positively affect the accumulation of this metabolite and improve plant stress tolerance (Conklin et al. 1997, Conklin et al. 1999). For example, in tobacco, overexpression of a tomato GMPase (ID DQ449030) increased ascorbic acid content fourfold in leaves and improved tolerance to both low and high temperatures (Wang et al. 2011). These plants displayed higher net photosynthetic rate and greater maximal photochemical efficiency of PSII compared with wild-type plants when grown under temperature stress (Wang et al. 2011). In rice, OsVTC1 expression was highly induced under salt stress, and the suppression of this gene greatly reduced seedling survival and grain production under these conditions (Qin et al. 2016b). In OsVTC1 RNAi rice transgenic lines seedling survival was only 20%, whereas wild-type plants showed 65% survival (Qin et al. 2016b). Similarly, when Arabidopsis vtc1-1 mutants, known to be salt-sensitive (Huang et al. 2005), were complemented with OsVTC1 they performed better under salt stress conditions (Qin et al. 2016b).

In this study, we manipulated the GDP-Man pathway in poplar by expressing PgGMP1 in xylem. Our results showed that the expression of this gene increased mannan content by up to 14%, representing a slight improvement over our previous work in which expression of the spruce PgCSLA1 gene in poplar resulted in a 11% increase in mannose content (Guevara-Rozo et al. 2025). These findings clearly support a role for PgGMP1 in GDP-Man biosynthesis and demonstrate that its expression is a viable strategy to enhance mannan production.

Similarly, our results showed that although PgGMP1-expressing lines displayed a slight increase in ascorbate, the difference was not significant. This may be due to carbon being preferentially directed toward the mannan biosynthetic pathway, or because VTC2 is the primary regulatory control point for ascorbate biosynthesis (Fenech et al. 2021, Smirnoff and Wheeler 2024). Since VTC2 expression was not altered, increased GDP-Man availability, suggested by the higher mannose content observed in these trees, does not necessarily translate into greater ascorbate production in poplar, as has been observed in other plant species. However, it remains to be determined whether the modest increase in this metabolite is sufficient to influence tree stress tolerance.

In this study, we provide new insights into important genes regulating the production of GDP-Man, the primary substrate for mannan biosynthesis, in poplar and spruce. Our results clearly indicate that PtaGMP1 from poplar and PgGMP1 from spruce function as GMPs that contribute not only to mannan biosynthesis but also, to some extent, to ascorbic acid production. Moreover, through functional analysis we show that disruption of PtaGMP1, via CRISPR/Cas9, led to coordinated reductions in both metabolites without major growth penalties. The successful expression of PgGMP1 in poplar showed the feasibility of enhancing mannan content by increasing the concentration of GDP-Man, while its impact on ascorbate levels remained modest compared with responses reported in other plants. Together, these findings provide a foundation for future strategies aimed at modifying mannan content in woody biomass and exploring the relationship between GDP-Man metabolism and ascorbic acid biosynthesis. Further exploration of additional GMP isoforms, as well as combinatorial manipulation of GMP and CSLA genes, will be important to better understand how mannan biosynthesis is regulated in poplar.

Author contributions

S.G.-R. and S.D.M. conceived the study. S.G.-R. performed all bioinformatic analyses and carried out all laboratory experiments. S.G.-R. wrote the manuscript and S.D.M. contributed to the final version.

Conflict of interest

None declared.

Funding

This project was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants to S.D.M. (RGPIN-2024-04385), and in part, with support from the UBC Four Year Doctoral Fellowship (4YF) program to S.G.-R.

Data availability

All data supporting the findings of this study are either in the main document or contained in the Supplementary data.