Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development, and adaptation to environmental stresses. BRs act in a dose-dependent manner and they do not travel over long distances, hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown and its contribution to control of endogenous BR levels remains unexplored. Here, we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BRs content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

or less was achieved with the described instrumentation.Merck silica gel Kieselgel 60 (230-400 mesh) was used for column chromatography.Reagents and solvents were purchased (Sigma-Aldrich) and were not purified.
Scheme 2.1.Synthesis of CSA.The solvents methanol, glacial acetic acid, pyridine, acetonitrile, and chloroform, were all purchased from Fisher Scientific at anhydrous high-performance liquid chromatography (HPLC) quality and used without further purification.

Chromatography material
Reactions were monitored by thin-layer chromatography (TLC), with SIL G25 UV254 TLC plates with 0.25 mm thick silica gel.The TLC plates were developed with an anisaldehyde stain solution  Step 1: synthesis of CSCMO For the synthesis of CSCMO, the reported procedure cited above was followed exactly with 4.01 mg of castasterone (CS, 8.63 μmol) and 4.31 mg of O-(carboxymethyl)hydroxylamine hemichloride (CMHA, 39.6 μmol, 4.6 equivalents) in 0.3 mL of anhydrous pyridine.After overnight agitation, another 2.0 mg of CMHA was added and the reaction was heated to 60°C for 2 h to assure complete CS consumption.Volatiles were removed in vacuo and the residue was directly purified by means of normal-phase column chromatography over silica gels.The column was eluted with a 20:1 mixture of chloroform and methanol, containing 0.5% (v/v?) glacial acetic acid, allowing obtention of the title compound in pure form after coevaporating three times with acetonitrile (approximately 4.8 mg; the maximum theoretical yield is 4.64 mg, i.e. near quantitative yield).The compound was used without further purification in the next step.This compound had a proton NMR spectrum identical to that reported previously.
Copy of 1 H-{ 13 C} HSQC NMR of CSA in CDCl3 (700 MHz), still containing ~10% unevaporated N-methyl-morpholine (also seen in LC-MS at m/z 102).The obtained data files were treated in Excel.In each case, the regions of interest (ROIs) below and above a specific threshold were discarded, because they represent noise or large artefacts.The total integrated density of all ROIs in one root were summed up and presented in the graph grouped by genotype and treatment.

1 H
Nuclear Magnetic Resonance (NMR) spectra were recorded with a resolution of 700, 400, or 300 MHz.The chemical shifts (δ) are expressed in ppm and the residual solvent peak is used as the internal standard (CDCI3: 1 H = 7.26 ppm; 13 C = 77.00ppm; (CD3)2CO: 1 H = 2.05 ppm; 13 C = 29.92ppm).The multiplicity of the signals were designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; br broadened; band, several overlapping signals.Mass spectra (MS) were run with an Agilent ESI single quadrupole type detector VL.High resolution mass spectra (HRMS) are recorded on an Agilent Accurate-Mass Quadrupole Time-of-Flight mass spectrometer.
Copy of LC-MS analysis of compound after further drying in vacuo: LCMS (ESI) retention time (m/z): 1.18 (m/z 102.1), 5.99 min (m/z 575.3) Due to the dearth of material, extraction of all carbon resonance directly from the onedimensional 13 C NMR spectrum proved difficult, but all other analyses were consistent with the expected product.The two-dimensional heteronuclear NMR (HSQC and HMBC) experiments provided the complete list of carbon resonances.The obtained data are fully consistent with the proposed structure.The spectra recorded on the sample included one-dimensional 1 H, towdimensional 1 H-{ 1 H} COSY, 1 H-{ 13 C} HSQC, 1 H-{ 13 C} HMBC, and 1 H-{ 1 H} NOESY.All carbon and proton resonances were found and assigned based on the observed two dimensional correlations.Conventional steroid numbering was used to indicate the castasterone carbons and protons (see formula).Chemical shifts for unresolved (overlapping) proton resonances are cited for the center of the observed two-dimensional correlation peaks.
The castasterone-O-(carboxymethyl)oxime (CSCMO) obtained above (4.64 mg according to maximal theoretical yield, or 8.63 μmol) was redissolved in 0.464 mL of anhydrous methanol.From weight based on NMR integration).Approximately 2.6 mg of the compound was obtained (maximum theoretical yield of 2.14 mg), and was used in biological assays after thorough drying in vacuo to remove all N-methyl-morpholine that had coeluted to some degree with the compound.