Chaperone-Mediated Autophagy

Cytokinins (zeatin, Z, and zeatin riboside, ZR), indole-3-acetic acid (IAA), and abscisic acid (ABA) were extracted and purified according to the method of Dobrev and Kaminek (2002) (link). One gram of fresh plant material (leaf or root) was homogenized in liquid nitrogen and placed in 5 ml of cold (–20 °C) extraction mixture of methanol/water/formic acid (15/4/1 by vol., pH 2.5). After overnight extraction at –20 °C solids were separated by centrifugation (20 000 g, 15 min) and re-extracted for 30 min in an additional 5 ml of the same extraction solution. Pooled supernatants were passed through a Sep-Pak Plus †C₁₈ cartridge (SepPak Plus, Waters, USA) to remove interfering lipids and plant pigments and evaporated to dryness. The residue was dissolved in 5 ml of 1 M formic acid and loaded on an Oasis MCX mixed mode (cation-exchange and reverse phase) column (150 mg, Waters, USA) preconditioned with 5 ml of methanol followed by 5 ml of 1 M formic acid. To separate different CK forms (nucleotides, bases, ribosides, and glucosides) from IAA and ABA, the column was washed and eluted stepwise with different appropriate solutions indicated in Dobrev and Kaminek (2002) (link). ABA and IAA were analysed in the same fraction. After each solvent was passed through the columns, they were purged briefly with air. Solvents were evaporated at 40 °C under vacuum. Samples then dissolved in a water/acetonitrile/formic acid (94.9:5:0.1 by vol.) mixture for HPLC/MS analysis. Analyses were carried out on a HPLC/MS system consisting of an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a μ-well plate autosampler and a capillary pump, and connected to an Agilent Ion Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) using an electrospray (ESI) interface. Prior to injection, 100 μl of each fraction extracted from tissues or a similar volume of xylem sap were filtered through 13 mm diameter Millex filters with 0.22 μm pore size nylon membrane (Millipore, Bedford, MA, USA). 8 μl of each sample, dissolved in mobile phase A, was injected onto a Zorbax SB-C18 HPLC column (5 μm, 150×0.5 mm, Agilent Technologies, Santa Clara, CA, USA), maintained at 40 °C, and eluted at a flow rate of 10 μl min⁻¹. Mobile phase A, consisting of water/acetonitrile/formic acid (94.9:5:0.1 by vol.), and mobile phase B, consisting of water/acetonitrile/formic acid (10:89.9:0.1 by vol.), were used for the chromatographic separation. The elution programme maintained 100% A for 5 min, then a linear gradient from 0% to 6% B in 10 min, followed by another linear gradient from 6% to 100% B in 5 min, and finally 100% B maintained for another 5 min. The column was equilibrated with the starting composition of the mobile phase for 30 min before each analytical run. The UV chromatogram was recorded at 280 nm with a DAD module (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was operated in the positive mode with a capillary spray voltage of 3500 V, and a scan speed of 22 000 m/z s⁻¹ from 50–500 m/z. The nebulizer gas (He) pressure was set to 30 psi, whereas the drying gas was set to a flow of 6.0 l min⁻¹ at a temperature of 350 °C. Mass spectra were obtained using the DataAnalysis program for LC/MSD Trap Version 3.2 (Bruker Daltonik GmbH, Germany). For quantification of Z, ZR, ABA, and IAA, calibration curves were constructed for each component analysed (0.05, 0.075, 0.1, 0.2, and 0.5 mg l⁻¹) and corrected for 0.1 mg l⁻¹ internal standards: [²H₅]trans-zeatin, [²H₅]trans-zeatin riboside, [²H₆]cis,trans-abscisic acid (Olchemin Ltd, Olomouc, Czech Republic), and [¹³C₆]indole-3-acetic acid (Cambridge Isotope Laboratories Inc., Andover, MA, USA). Recovery percentages ranged between 92% and 95%.
ACC (1-aminocyclopropane-1-carboxylic acid) was determined after conversion into ethylene by gas chromatography using an activated alumina column and a FID detector (Konik, Barcelona, Spain). ACC was extracted with 80% (v/v) ethanol and assayed by degradation with alkaline hypochlorite in the presence of 5 mM HgCl₂ (Casas et al., 1989 ). A preliminary purification step was performed by passing the extract through a Dowex 50W-X8, 50–100 mesh, H⁺-form resin and later recovered with 0.1 N NH₄OH. The conversion efficiency of ACC into ethylene was calculated separately by using a replicate sample containing 2.5 nmol of ACC as an internal standard and used for the correction of data.

X-ray photoelectron spectroscopy (XPS) measurements were performed on a Kratos AXIS Ultra DLD surface analysis instrument. Al Kα radiation (1,486.71 eV) at 15 kV was used as the excitation source. Areas of the measured peaks were calculated by the software Origin (2019b, OriginLab Corporation). The relative sensitivity factors (RSF) were used to scale the areas of the measured peaks in calculating element ratios in the samples so that the normalized peak areas represent the relative amount of the element in the sample (56 ). RSF for Fe³⁺, O, N, and C in the present study were determined as 16.4, 2.93, 1.8, and 1, respectively (CasaXPS, Casa Software Ltd). Subsequently, the ratio between Fe³⁺ and N was calculated using the following equation: $$\text{ r = }\frac{{\text{A}}_{\text{Fe}}/\mathrm{\text{16}}\text{.4}}{{\text{A}}_{\text{N}}/\mathrm{\text{1}}\text{.8}}\text{,}$$
where A_Fe and A_N are the peak areas of the Fe and N elements in the XPS spectrum, respectively. Water content in the Fe³⁺-doped COF samples was determined by calculating the ratio between O and Fe elements. In this case, the O element in the pure COF sample was taken into consideration. Assuming that N content is not changed by metal ion-doping, the adsorbed water of Fe³⁺ in COF nanochannel was calculated with the following equations: $$\begin{array}{c}{\text{r}}_{\text{1}}\text{ = }\frac{{\text{A}}_{\text{O1}}/\mathrm{\text{2}}\text{.93}}{{\text{A}}_{\text{N1}}/\mathrm{\text{1}}\text{.8}}{\text{, r}}_{\text{2}}\text{ = }\frac{{\text{A}}_{\text{O2}}/\mathrm{\text{2}}\text{.93}}{{\text{A}}_{\text{N2}}/\mathrm{\text{1}}\text{.8}}{\text{, r}}_{\text{3}}\text{ = }\frac{{\text{A}}_{\text{Fe2}}/\mathrm{\text{16}}\text{.4}}{{\text{A}}_{\text{N2}}/\mathrm{\text{1}}\text{.8}}{\text{, r}}_{\text{4}}\text{ = }\frac{{\text{r}}_{\text{2}}{\text{ − r}}_{\text{1}}}{{\text{r}}_{\text{3}}}\text{,}\hfill \end{array}$$
where A_O1 and A_N1 are the XPS peak areas of O and N elements in the pure COF, A_O2 and A_N2 are XPS peak areas of O and N elements in the metal ion doped-COF, A_Fe2 is the XPS peak area of Fe in the metal ion-doped-COF, and r₄ is the ratio between water and Fe³⁺ in the metal ion-doped COF.