Carbopac pa10 column

After centrifugation of 1 mL culture aliquots, the cell pellets were dried at 75 °C for 24 h, and directly used for PHB and glycogen sample preparation and measurement [33 (link)]. Briefly, PHB was quantified on an UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) with an Aminex HPX-87 H column (Bio-Rad Laboratories, Hercules, CA, USA). Glycogen was determined on an ICS-6000 ion chromatography system with a CarboPac PA10 column (Thermo Fisher Scientific, Waltham, MA, USA).

Three repeats for each PGN sample and three different batches of quantification were performed because as PGN is insoluble in aqueous solution, these repeats minimize potential errors in the quantification. The pure PGN suspension was left O/N stirring at RT for maximum homogenization. 20 μL of the suspensions were hydrolysed in 3 M HCl at 95°C for 2 h (150 μL reaction volume). The hydrolysed suspension was lyophilized until it was completely dried and resuspended in 500 μL of MilliQ H₂O and lyophilized O/N. Finally, the acid-free hydrolysed material was resuspended in 150 μL MilliQ H₂O.
For monosaccharide separation 10 μL of the samples were injected in a Thermo Scientific Dionex ICS-5000 system, in 18 mM NaOH constant and a gradient of 1 M NaCH₃COO and MilliQ H₂O. The MilliQ H₂O as all the eluents were in MilliQ H₂O of resistivity ≥ 18 MΩ, filtered with 0.2 μm ϕ pore filter and degassed for 15 min in an ultrasonic bath. Between the eluents pump and the injection valve it was used a BorateTrap™ column to remove borate contamination from eluents. An AminoTrap™ (Thermo Scientific Dionex AminoTrap) was used as a pre-treatment column to remove the amino acids from the samples, thus only the sugars passed to the CarboPac PA10 column (Thermo Scientific CarboPac PA10) where they were separated. Each injection was done in triplicates.

Cell wall composition analysis was performed using a stepwise acid hydrolysis method. AIR (0.3 g) was taken and acidified by using 72% sulfuric acid (3 mL) at 30 °C for 1 h and then treated at 121 °C for 1 h with the addition of 84 mL of deionized water. After cooling, it was filtered using a sand core funnel (G4). The diluted filtrate and the fine residue were filtered through 0.22 μm nylon membrane for monosaccharide composition analysis by ion chromatography. The ion chromatogram (Metrohm 940, Herisau, Switzerland) equipped with the CarboPac PA10 column (2 mm × 250 mm, Dionex, Sunnyvale, CA, USA) and coupled with a PAD detector was used for quantification. NaOH (20 mM) was added as an isocratic eluent and eluted at a flow rate of 0.5 mL/min for 20 min. The program was set to 0–75 mM NaAc 15 min for gradient elution, 200 mM NaOH 10 min for washing, and then 20 mM NaOH for re-equilibration. A calibration curve was established to calculate the quantification of the monosaccharide.
The acidified elute was diluted ten times, and the percentage of the sample content was calculated by the following formula: $\%Comtent=\frac{D\times V_1\times {10}^{-6}\times P\times 0.9}{M}\times 100\%$
where D was the dilution factor of acidified elute (10×), V₁ was the total liquid volume used for hydrolysis (V = 86.663 mL), P was the concentration determined by ion chromatography (mg/L), and the M was the weight of AIR.

High-performance anion-exchange chromatography coupled to a pulsed amperometric detector (HPAEC-PAD) was used for monosaccharide composition analysis^{20 (link),38 (link),39 (link)}. Samples (1 mg/mL) were hydrolyzed with 2 M trifluoroacetic acid at 120 °C for 90 min. After the samples were cooled down to room temperature, t-butyl alcohol was added, and the mixture was evaporated under N₂ flow. The dried samples were solubilized in water, filtered (0.45 µm) and analyzed in a DX 500 system (Dionex, Sunnyvalle, CA, USA) equipped with a CarboPac PA10 column (250 × 4 mm)^{20 (link)}. Neutral sugars analysis was performed in water (1 mL/min; 40 min). Followed by a cleaning sequence with 300 mM NaOH for 10 min with another 10 min of re-equilibration. Uronic acids analysis was performed in 150 mM NaOH (1 mL/min; 30 min) with a 0–220 mM sodium acetate gradient, followed by a cleaning step with 500 mM sodium acetate for 10 min. A post-running adjustment of 10 min with 220 mM and 10 min with 150 mM NaOH followed. Neutral sugars (arabinose (Ara), fucose, galactose (Gal), glucose, mannose, rhamnose (Rha) and xylose), and uronic acids (galacturonic acid (GalA) and glucuronic acid), were used as standards^{40 (link)}.

Glucose, sucrose and fructose were extracted from cambial zone samples, three biological replicates with three technical replicates were used for each treatment. Tissues were grounded and freeze dried for 48 h, after 1 mL of water was added in 0.2 g of dry powder and samples were kept in bath (80°C) for 1 h. Then, samples were centrifuged for 10 min, 16.000 xg, the supernatant was recovered and stored at −4°C. Summer and winter samples were analyzed using a high-performance liquid chromatography (ICS 2500, HPLC Dionex) with amperometric detection (ED50) equipped with an autosampler AS50 (Dionex). Sugars were assigned according to the retention times of standards (sucrose, glucose and fructose). A Carbopac PA-1 column (4 × 250 mm, Dionex) and a guard Carbopac PA-10 column (4 × 50 mm, Dionex) were used. To identify statistical differences between summer and winter samples two-tailed Student's t-test (P ≤ 0.05) was performed.