Generation of phylogenetic tree
Arabidopsis 103 amino acid sequences were fetched from superfamily database (https://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/) using SGNH as a query. Rice BS1 (LOC_Os02g15230.1) and DARX1 (LOC_Os05g06720.) sequences were fetched from rice genome annotation project (http://rice.plantbiology.msu.edu/). The phylogenetic tree was generated by MEGA-X tool using Neighbor-Joining method with 250 bootstraps.
Cloning of AtGELP7 and MYB46
AtGELP7 (AT1G28580) and MYB46 (AT5G12870) were amplified using complete attB flanking primers PCWL41, and PCWL42 and partial attB flanking primers PCWL119, and PCWL120, respectively from Arabidopsis stem cDNA (Supplementary table S1) and cloned into pDONRTM 207 using the Gateway BP Clonase II enzyme mix (11789, Invitrogen, Canada). This pENTR-AT1G28580 and pENTR-AT5G12870 were further subcloned into destination vectors pCC0995 containing constitutive 35S promoter (35S::AT1G28580, 35S::AT5G12870) (Weng et al. 2010) and pSITE-2CA (35S:GFP-AT1G28580) (Chakrabarty et al. 2007) containing N-terminal tagged GFP vector using the Gateway LR Clonase II enzyme mix (11791, Invitrogen, Canada). These expression clones were transformed into Agrobacterium GV3101 strain, colonies were confirmed by PCR and used for stable and transient expression in plant.
Agrobacterium containing 35S::GFP-AT1G28580 and plasma membrane marker CD3-1007 (Nelson et al. 2007) was incubated at 28°C, the culture was centrifuged at 6000 rpm, the pellet was resuspended in a mixture of 10mM MES buffer (RM1128, HiMedia, India) and 10mM MgCl2 (MB237, HiMedia, India) pH- 5.6 and diluted to 0.4 OD. 100 mM Acetosyringone was added to the suspension and kept overnight at room temperature. The leaves of Nicotiana benthamiana were then infiltrated with the suspension (Betsuyaku and Sawa 2009) and at 2nd or 3rd day infiltrated leaf sections were analyzed in LEICA SP8 confocal microscope. For plasmolysis experiment, infiltrated leaf sections were dipped in 30% glycerol for 30 min under vacuum.
Genotyping of T-DNA mutant
SALK_030805 line of AT1G28580 was obtained from Arabidopsis Biological Resource Centre (ABRC) and homozygous lines were isolated by genotyping using LP(PCWL99), RP (PCWL100), and BP(PCWL29) primers (Supplementary table S1).
The total RNA extraction using Trizol method and quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Fresh tissue was collected from Arabidopsis rosette leaf and stem and ground in liquid nitrogen. RNA was extracted using Trizol method (15596018, Invitrogen, Canada) and extracted RNA was treated with TURBO DNA-free kit (AM1907, Invitrogen, Lithuania). cDNA synthesis of the DNAse treated 1 mg RNA samples was done using iScript cDNA Synthesis kit (1708891, BioRad, USA). Using ACTIN2 (AT3G18780) as reference gene, the expression of MYB46 (AT5G12870), PAL2 (AT3G53260), and AtGELPs from clade Id and Ic were measured using qPCR primers listed in supplementary table S1. The relative fold change was calculated by ΔΔCt method.
Generation of Arabidopsis transgenic lines
The Agrobacterium culture containing 35S::AT5G12870 (MYB46) and 35S:AT1G28580 (AtGELP7) plasmids were grown at 28°C for 2 days, pelleted at 6000 rpm for 15 min and suspended in transformation medium consisting of 5% sucrose (GRM3063, HiMedia, India) and 0.05% silwet-77 (PCT1554, HiMedia, India) suspension. The floral dip transformation was performed as explained in (Clough and Bent 1998). When the plants were completely dried, seeds were collected and selected against glufosinate ammonium (BASTA) (C45520, Sigma-Aldrich, Switzerland). The homozygous lines from T3 generation were used for further experiments.
Staining and imaging of Arabidopsis stem sections
Wild-type and transgenic Arabidopsis stem sections were stained with Toluidine Blue O (T3260, Sigma-Aldrich, USA) and imaged under Nikon DS-Qi2 ECLIPSE Ti fluorescence microscope under 10X and 40X magnification (Pradhan Mitra and Loqué 2014).
The soluble and wall bound proteins were extracted from stem and leaf tissue as explained by (Biswal et al. 2014). The total protein concentration was determined by Bradford assay. Esterase activity was performed by p-Nitrophenyl Acetate (18432, SRL, India) as substrate, incubated at 37°C for 2 h, released 4-nitrophenol quantified at 400 nm and specific esterase activity was calculated in nmol per min per mg of total protein using 4-nitrophenol standard curve.
The soluble and wall bound proteins were isolated from Nicotiana leaf tissue as described by (Gupta et al. 2002). Lipase activity was performed with 4-Nitrophenyl palmitate (N2752, Sigma-Aldrich, Mongolia) as substrate, incubated at 50°C for 5 h, and released 4-nitrophenol quantified at 410 nm. Then using the BSA standard curve, the specific lipase activity was calculated in nmol of product releases per min per mg of total protein using 4-nitrophenol standard curve.
Preparation of Alcohol Insoluble Residue (AIR)
The dried plant tissue was ground in Qiagen TissueLyser II, treated with 80% ethanol in 4 mM HEPES buffer (MB016, HiMedia, India) for 30 mins at 70°C. The mixture was then centrifuged and pellet was sequentially washed with 800 ul of 70% ethanol, chloroform:methanol (1:1) and acetone. After removing acetone, the pellet was dried overnight.
Cell wall acetyl content analysis
1 mg of AIR sample was saponified using 1M NaOH, and neutralized with 1M HCl, and saponified supernatant was analyzed by acetic acid kit (K-ACET, Megazyme, Ireland).
Sequential extraction of pectin, xyloglucan, and xylan
Leaf AIR was incubated with 50mM ammonium formate (50504, SRL, India) buffer at 37°C for 24 h in shaker dry bath. The supernatant ammonium formate fraction was collected, and pellet was digested with pectate lyase (E-PCLYAN2, Megazyme, Ireland). Both ammonium formate and pectate lyase fractions were freeze dried and the pellet was dissolved in water. After pectin extraction, the pellet containing xyloglucan and xylan was digested with endoglucanse i.e., cellulase (C1184-5KU, Sigma-Aldrich, USA) to remove xyloglucan and the remaining pellet rich in xylan was freeze dried. Then pectin fraction, combined xyloglucan and xylan-rich fraction, and xylan-rich fraction were analyzed for acetic acid (K-ACET, Megazyme, Ireland), galacturonic acid (K-URONIC, Megazyme, Ireland), and xylose (K-XYLOSE, Megazyme, Ireland) content.
DMSO extraction of xylan
AIR samples were delignified using 1.3% sodium hypochlorite (Q27905, Thermo Fisher Scientific, India), incubated at 75°C for 2 h with shaking in dry bath. Samples were centrifuged, pellet was washed with acetone and dried overnight. DMSO was added to above holocellulosic fraction and incubated at 60°C for 24 h in shaking dry bath. Supernatant collected after centrifugation was precipitated in ethanol:methanol:water (7:2:1) mixture by keeping at 4°C for 2 days. Precipitated xylan was centrifuged, pellet was washed with acetone and dried overnight. This DMSO extracted xylan was used for further analysis.
Fourier Transform Infrared (FTIR) spectroscopy analysis
Qiagen TissueLyser II milled dried stem powder and AIR from fresh leaf tissues were placed on ZnSe ATR crystal in Bruker Tensor27 and scanned 16 times to get the absorbance peaks of different functional groups at wavenumbers ranging from 800 cm-1 to 2000 cm-1. Acetyl linkages were detected at 1240 cm-1, and 1740 cm-1 and xylose linkages were detected at 976 cm-1, 1037 cm-1, and 1073 cm-1 by analyzing birchwood xylan (partially acetylated) (P-ACXYL, Megazyme, Ireland) and beechwood xylan (P-XYLNBE, Megazyme, Ireland) as controls.
XOS analysis by MALDI-TOF-MS
3 mg of AIR from dried stem tissue was digested with GH11 endo-1,4-β-Xylanase (E-XYLAA, Megazyme, Ireland) at 60°C for 2 days and the remaining hydrolysate was purified as explained in (Chong et al. 2011). Both neutral and acidic XOS fractions were subjected to MALDI analysis separately.
Cell wall composition
Xylose content: 2 mg of AIR sample was treated with 100 µl of 1.3M HCL, incubated in dry bath at 100°C for 1 hr. The samples were neutralized with 100 µl of 1.3M NaOH and final volume was made up to 1ml by adding MilliQ water, centrifuged at 1500g for 10 mins. 50 µl supernatant was used for xylose analysis and analyzed by Megazyme Kit (K-XYLOSE, Megazyme, Ireland).
Cellulose content: AIR sample was digested with α-amylase (A3176, Sigma-Aldrich, USA) at 45°C to remove starch. Destarched cell wall residue was used to generate glucose monomer which was analyzed by anthrone assay (Updegraff 1969).
Acetyl Bromide Soluble Lignin (ABSL): AIR samples were incubated with freshly prepared 25% acetyl bromide (135968, Sigma-Aldrich, Mongolia) containing acetic acid at 50°C for 2 h The supernatant was diluted with 2M NaOH and freshly prepared hydroxylamine hydrochloride (159417, Sigma-Aldrich,). The absorbance was taken at 280 nm and lignin content was represented in mg per g of AIR (Foster et al. 2010).
Alkali pre-treatment: AIR sample was treated with 0.4 M NaOH at 90°C for 30 min. The supernatant was removed by centrifugation, the pellet was washed with water and acetone and dried for further analysis.
Xylanase pre-treatment: AIR from dried stem tissue was digested with GH11 endo-1,4-β-Xylanase (E-XYLAA, Megazyme, Ireland) at 60°C for 48 hrs. The pellet was washed with acetone, dried and used for further analysis.
Preparation of enzyme mix for saccharification: Equimolar enzyme solutions (40U/ml) were prepared for both cellulase (C1184-5KU, Sigma-Aldrich, USA) and β-glucosidase (E-BGLUC, Megazyme, Ireland). The enzyme solutions were then mixed with 0.1 M acetic acid buffer (pH- 4.8) in ratio 1:2 separately. The individual solution desalted through PD 10 column (17085101, GE Healthcare, UK). The desalted cellulase and β-glucosidase was then mixed in 8:2 ratio to prepare enzyme mix. Finally, the enzyme mix was diluted ten times with 0.1 M acetic acid buffer (pH- 4.8) to make a diluted enzyme mix for saccharification.
Enzymatic hydrolysis: The untreated or treated cell wall powder was suspended in reaction mixture containing 0.1 M acetic acid buffer (pH- 4.8) and enzyme mix (8:2) of cellulase and β- glucosidase. All the samples were incubated at 50°C for 96 h, centrifuged, the glucose in supernatant was analyzed by Glucose oxidase (GOD) - Peroxidase (POD) assay (Acker et al. 2016). To analyze the glucose content, the sample collected after saccharification was incubated with GOD-POD solution at 37°C for 30 mins. This GOD-POD mixture was prepared by mixing glucose oxidase (61788, SRL, India), Peroxidase ex. Horseradish (73292, SRL, India), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (A9941, Sigma-Aldrich, USA) in 0.1M acetic acid buffer (pH 4.5). The absorbance was measured after incubation at 405 nm. Glucose content was calculated using standard curve.
The p value was calculated using Student’s t-test in Office Excel 365.