Plant growth conditions
Arabidopsis thaliana (Columbia-0) was used for all experiments and grown under 14 h light/10 h dark cycle conditions at 22°C. To perform experiments on A. thaliana either 4-weeks old or 10-day old seedlings were used for the xylobiose (XB) treatment.
Treatment of XB
A four-week-old Arabidopsis plant's rosette leaves were infiltrated with 100 µM XB (O-XBI, Megazyme, Ireland) and distilled water (mock). Control (T0), XB-treated, and mock-treated leaves were collected after 0 min, 30 min, 3 h, 6 h, 12 h, and 24 h and used for further analysis.
Measurement of reactive oxygen species using diaminobenzidine (DAB) assay
Treated and mock Arabidopsis leaf were dipped into a 12-well plate containing 1 mg/ml diaminobenzidine (DAB) (17076, SRL, India) which was prepared in 10 mM Na2HPO4 (TC507M, Himedia, India). The plate was wrapped in aluminium foil and left at room temperature for 8 h on the shaker. After that DAB staining solution was replaced with a bleaching solution (ethanol: acetic acid: glycerol; 3:1:1), and placed on a boiling water bath for 15 min. The old bleaching solution was replaced with the fresh bleaching solution, leaf photographs were taken and DAB accumulation was quantified using Fiji (image analysis tool) (https://fiji.sc/)(Daudi and O'brien 2012).
Immunoblot analysis of MAPK activation
10-day-old Arabidopsis seedlings were treated with 100 µM of XB. Control (T0) and XB-treated samples were collected after 30 min, 3 h, 6 h, 12 h, and 24 h, and homogenized into the liquid nitrogen. Protein was isolated in extraction buffer containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM EDTA, 1 mM sodium molybdate, 10% (v/v) glycerol, 0.1% (v/v) Tween-20, 1 mM 1,4-dithiothreitol, 1 mM phenylmethylsulfonyl fluoride. The total extracted protein was quantified by the Bradford assay (5000006, Bio-Rad, USA) and mixed with 4X Laemmli buffer containing 0.1M Tris, pH 6.8, 20% v/v glycerol, 4% w/v SDS, 10 mM DTT, and 0.001% w/v bromophenol blue) and heated at 95˚C for 10 min, separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane by wet transfer procedure. This followed membrane blocking using 5% (w/v) skimmed milk powder in 1 X phosphate buffer saline with 0.1% w/v Tween®20 detergent buffer for 1 h at room temperature. Membranes were incubated overnight at 4°C in 1 × PBST containing phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (4370, Cell Signaling Technology, USA) (1:1000) and anti-AtMPK6 (1:10000) antibodies (A7104, Sigma-Aldrich, Germany) or anti-GAPDH (PAB932Mu01, Cloud-Clone Corp., USA) primary antibody. Membranes were washed with PBS containing 0.1% Tween-20 and incubated with horseradish peroxidase (HRP)-conjugated Goat Anti-Rabbit antibody (BB-SAB01A, BioBharti LifeSciences Pvt.Ltd, India). Blots were developed using Luminata™ Forte western HRP substrate (WBLUF0100, Merck Millipore, Germany), and imaged under Image Quant LAS 4000 (GE Healthcare, USA) and fold change was quantified with reference to GAPDH (AT1G13440) (Rebaque et al. 2021).
Reverse Transcription-PCR and qRT-PCR
The total RNA was isolated from the homogenized control (T0), XB-infiltrated, and mock-infiltrated Arabidopsis leaves by Trizol method (15596018, Invitrogen, Canada), treated with DNase I (EN0521, Thermo Scientific), and 500 µg of RNA per 10 µL reaction was used to generate first-strand cDNA using iScript™ cDNA Synthesis Kit (1708891, Bio-Rad, USA). Using ACTIN2 (AT3G18780) as a reference gene, the expression of genes was measured using qPCR primers listed in Table S1 on Thermo QuantStudio 6 Flex Real-Time PCR System, and the relative fold change was calculated by ΔΔCt method.
Isolation of soluble sugars
200 mg of homogenized dried powder was treated with 1 ml of 80% ethanol for 1 h at room temperature. The supernatant was separated by centrifugation, vacuum dried and dissolved in 200 µl of water.
Estimation of total sugar and xylose
A microplate assay with phenol and sulfuric acid was used to estimate the total amount of sugar. 50 µl of the soluble fraction containing sugar was mixed with 50 µl of 5.0% (v/v) phenol (Q26355, Thermo) in a glass test tube. Next, 250 µl of concentrated sulfuric acid was added drop by drop. Following incubation for 20 min at RT, 250 µl of the reaction mixture was transferred to the Costar 3598 ELISA, and absorbance was measured at 490 nm. A standard curve of D-glucose (5 µg/ml to 50 µg/ml) was prepared to determine the concentration of total sugar. The xylose content of the isolated soluble fraction was tested using a D-xylose kit (K-XYLOSE, Megazyme, Ireland). For the extracted soluble fraction, the total xylose concentration was determined using a standard curve. The total amount of xylose was calculated and normalized with the total amount of sugar.
Xylosidase activity
The soluble proteins were extracted from the leaf tissue with sodium phosphate buffer pH-6.8 (containing 2% PVP, and 2.5mM EDTA), and incubated with 2 mM of para-nitrophenyl xylopyranoside (18432, Sigma-Aldrich, Germany) at 37°C for 2 h. The released 4-nitrophenol was measured at 400 nm using spectrophotometer and the specific xylosidase activity was calculated in nmol/min/mg of total protein using a 4-nitrophenol standard curve.
Checking purity of XB and xylose by HPLC
The 100 µM of XB (O-XBI, Megazyme, Ireland) and xylose (GRM111, Himedia, India) were dissolved in water ,and filtered using a 0.2 µm syringe tip filter (GE Healthcare Life Sciences Uniflo™ Buckinghamshire, UK), and 10 µL filtered samples were injected on Agilent 1260 Infinity II (Agilent cross lab, Santa Clara, CA, USA) HPLC operating at a flow rate of 0.6 mL/min (0.005 N sulphuric acid in water) on the Rezex™ ROA-Organic Acid H + column and detected using refractive index (RI) detector. The purity of the standard was checked on the basis of the specific retention time of XB and xylose. (McGinley 2008).
Transcriptomic analysis
RNA was isolated from the 100 µM XB (O-XBI, Megazyme, Ireland) and mock-infiltrated four-week-old Arabidopsis leaves and RNA sequencing was done on Illumina NovaSeq 6000 platform. Upregulated and downregulated genes were analyzed through DSEq2 tool with Log2FoldChange 1, p-value < 0.05, and overlap genes were analyzed by Venn diagrams using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/). Gene ontology analysis was done with differentially expressed genes (DEGs) using ShinyGO 0.77 (http://bioinformatics.sdstate.edu/go/). Selected genes were validated by qRT-PCR.
Callose deposition
10-day-old Arabidopsis seedlings were fixed in 1 mL of 95% ethanol at room temperature, chlorophyll was removed using multiple changes of 95% ethanol. 95% ethanol was replaced with 1 mL of 50% ethanol and incubated for 60 minutes on a rocker at room temperature. The seedlings were rinsed with 1 mL of 67 mM K2HPO4, pH-12 followed by stained with 1 mL of 0.01% (w/v) aniline blue (B8563, Sigma-Aldrich, Germany), incubated on a rocker at room temperature for 60 min. Samples were rinsed and washed with 1 mL of 67mM K2HPO4 for 60 min on a rocker at RT. Seedlings were mounted on the slide with 100–150 µl of a mounting solution containing 67 mM K2HPO4 (pH 12) and 70% glycerol. Mounted seedlings were examined in a LEICA SP5 confocal microscope with a DAPI filter to detect callose. Images of callose deposition were captured and measured using Fiji (image analysis tool) (https://fiji.sc/) (Mason, Ekanayake, and Heese 2020).
Preparation of Alcohol Insoluble Residue (AIR)
Homogenized tissue was treated with 80% ethanol prepared in 4 mM HEPES buffer (MB016, HiMedia, India) for 30 mins at 70°C. The mixture was then centrifuged, and the supernatant was discarded. The pellet was sequentially washed with 800 µl of 70% ethanol, chloroform: methanol (1:1), and acetone. After removing the acetone, the pellet was dried overnight in a vacuum dryer. This AIR was further used for cell wall analysis.
Fourier Transform Infrared (FTIR) spectroscopy analysis.
AIR powder from fresh leaf tissues was placed on ZnSe ATR crystal in Bruker Tensor27 and scanned the polysaccharides standards avicell cellulose (11365, Sigma-Aldrich, Germany), xylan (P-XYLNBE, Megazyme, Ireland), xyloglucan (O-XGHDP, Megazyme, Ireland), acetylated xylan (P-ACXYL, Megazyme, Ireland), and pectin (P9135, Sigma-Aldrich, Germany) and AIR from samples for 16 times to get the absorbance peaks of different functional groups at wavenumbers ranging from 800 cm− 1 to 1800 cm− 1. Data was analyzed by Principal component analysis (PCA) using the MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml).
Cellulose content
AIR was treated with Updegraff reagent (Acetic acid: nitric acid: water, 8:1:2) at 100°C for 30 min, and centrifuged at the 18000 rcf for 10 min. The pellet was washed with water, vacuum dried, and hydrolyzed using 72% sulphuric acid. The hydrolysate containing glucose was measured by anthrone assay (Updegraff 1969).
Mono-sugar composition analysis
20 µL of an inositol solution (5 mg/ml) was added to a 2 mg AIR sample as an internal control. The sample was hydrolyzed with 250 µl of 2M trifluoroacetic acid (TFA), which was incubated for 90 minutes at 121˚C on heating block, and chilled on ice. The hydrolysate was centrifuged, the supernatant was evaporated under a gentle stream of nitrogen gas. The remaining lyophilized powder was washed with 300 µl of 2-Propanol, vortexed, and evaporated at 25˚C. The dried sugar was re-dissolved in 40 µL of 20 mg/ml methoxamine hydrochloride in pyridine and incubated for 90 min at 37˚C. It was further derivatized with 60 µL of MSTFA (N-methyIN-(trimethylsilyl) trifuoroacetamide C.100 µL of the derivatized sample was transferred to glass vials and analyzed on Gas Chromatography-Mass spectrometry (GC-MS/MS) (TQ8050 NX, Shimadzu, Japan).
Isolation and estimation of pectin fraction
1 mg AIR was digested with pectate lyase (Megazyme, E-PCLYAN) at the 50 mM tris HCl buffer (pH − 8) for 24 h at 40°C and the enzyme was deactivated at 100°C for 15 min. The supernatant fraction was isolated after centrifuging at 18000Xg for 10 min and quantified with the biphenyl method. 250 µg of AIR + 100ul water or 10 µl of pectate lyase fraction + 90 µl of water was mixed with 10µl of sulphamate reagent and 300 µl borate buffer, incubated at 100°C for 5 min followed by the addition of 5 µl of biphenyl solution (0.15% 3-Hydroxybiphenyl in 0.5% NaOH). The 200 µl was transferred to the 96 well plates and absorbance was measured at 525 nm. Total uronic acid and pectin were quantified using the standard graph of pectin (P9135, Sigma-Aldrich, Germany).
Defense hormone analysis
25 mg of the lyophilized leaf sample was mixed with 1 ml of methanol containing 40 ng mL-1 of D6-jasmonic acid (HPC Standards GmbH, Cunners dorf, Germany), 40 ng mL-1 D4-salicylic acid, 40 ng mL-1 D6-abscisic acid, and 8 ng mL-1 of jasmonic acid- [13C6] isoleucine conjugate as internal standards, vortexed for 2 minutes while keeping the sample at 4˚C. After that, the sample was put into a tube rotor at 4˚C, with shaking for 20 min. After that, the sample was centrifuged for 15 min at 4˚C for 12,000 x g. The supernatant was vacuum dried, redissolved in 500 µl of methanol and analyzed on Liquid Chromatography-Mass spectrometry (LC- MS/MS) (QTRAP 6500+, SCIEX, USA) (Vadassery et al. 2012).
Growth hormone analysis
25 mg of lyophilized leaf sample was treated with 1 ml of ice-cold extraction buffer (MeOH: H2O: HCOOH, 15:4:0.1) containing 25 ng of trans- [ 2H5] Zeatin and D2-indole-3-acetic acid (D2-IAA) as internal standards, vortexed for 8 min (and centrifuged for 10 min at 12,000 g at 4 ˚C. The supernatant was passed through the C18 RP SPE column, conditioned with 1 ml of methanol and 0.1% of formic acid in water. The hormone was eluted with 1 ml of ice-cold 0.1% HCOOH in acetonitrile. The eluted fraction was vacuum-dried, resuspended in 50 µl of 5% MeOH and analyzed by Liquid Chromatography-Mass spectrometry (LC-MS/MS) (QTRAP 6500+, SCIEX, USA) (Šimura et al. 2018).