create metabolite analysis assay and add protocols

assays/MetaboliteAnalysis/protocols/AnionsAnalysis.md

+## Anions
+
+For the measurement of phosphate, nitrate, and sulfate anions, 1 ml of sterile H2O was added to ~20 mg of homogenized shoot material, shaken for 1 h at 4 °C and heated to 95 °C for 15 min. The samples were centrifuged at maximum speed for 15 min at 4 °C, and 200 μl of the supernatants were transferred to an ion chromatography vial. Standard curves were generated using 0.5, 1, and 2 mM KH2PO4, KNO3, and K2SO4. The inorganic anions were measured with the Dionex ICS-1100 chromatography system and separated using a Dionex IonPac AS22 RFIC 43 250 mm analytic column (Thermo Scientific). The running buffer was made up of 4.5 mM NaCO3 and 1.4 mM NaHCO3 as described in Dietzen et al. (2020).
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assays/MetaboliteAnalysis/protocols/GlucosinolatesAnalysis.md

+## Glucosinolates
+
+GSLs were extracted from ~20 mg of homogenized plant material using 500 ml of hot 70% (v/v) methanol, and 10 μl of sinigrin was added as internal standard. The extract was vortexed and incubated at 70 °C for 45 min, vortexing twice during the incubation. The samples were left to cool and centrifuged at maximum speed for 5 min at room temperature. The supernatant was transferred to prepared columns containing 0.5 ml of DEAE Sephadex A-25, washed twice with 0.5 ml of sterile H2O, and subsequently twice again with 0.5 ml of 0.02 M sodium acetate buffer. With a new tube placed underneath each column, a layer of 75 μl of sulfatase was placed onto the column. The samples were left at room temperature overnight, and the resulting desulfo-GSLs were eluted twice with 0.5 ml of sterile H2O, followed by a final elution by 0.25 ml. The eluates were combined, vortexed, centrifuged for 5 min, and 200 μl of the supernatant were transferred to HPLC vials. The desulfo-GSLs were resolved by HPLC (Spherisorb ODS2, 250 3 4.6 mm, 5 μm; Waters) using a gradient of acetonitrile in water and detected by UV absorption at 229 nm. The GSLs were quantified using the internal standard and response factors as described in Dietzen et al. (2020).
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assays/MetaboliteAnalysis/protocols/ThiolsAnalysis.md

+## Thiols
+
+To analyze low molecular weight thiols (Cys and GSH), ~20 mg of homogenized plant material was extracted with 0.1 M HCl at a 1:10 ratio (w/v) and subsequently centrifuged at maximum speed at 4 °C. To reduce the thiols in the samples, 60 μl of the supernatant was transferred to a new tube and 100 μl of 0.25 M CHES-NaOH (pH 9.4) was added. Thereafter, 35 μl of 10 mM DTT was added, and the tubes were vortexed and incubated for 40 min at room temperature. A 5 μl aliquot of 25 mM monobromobimane was added to the reduced extracts, and the samples were vortexed and incubated in darkness for 15 min at room temperature. The reaction was stopped by adding 110 μl of 100 mM methansulfonic acid and vortexing. After centrifugation at 4 °C for 20 min, 200 μl of supernatant was transferred into HPLC vials. Standards, ranging from 0 mM to 100 mM, were prepared using 2 mM l-Cys and GSH stocks. The conjugated thiols were resolved using reverse phase (RP)-HPLC (Eurospher 100-3 C18, 150 × 4 mm; Knauer) and a gradient of 90% (v/v) methanol and 0.25% (v/v) acetic acid, pH 4.1 in 10% (v/v) methanol and 0.25% (v/v) acetic acid, pH 4.1, and detected fluorimetrically with a 474 detector with an excitation wavelength at 380 nm and emission wavelength at 470 nm. The flow rate was constant at 1 ml min−1.
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