Application suite 2.8.1 build software

Pre-hybridization was carried out by incubating the sections in 0.2M HCl, neutralizing them and then treating them with 1mg/ml proteinase-K (Roche Applied Science). Samples were then dehydrated in an aqueous ethanol dilution series and hybridized with sense and anti-sense digoxigenin (DIG)-labelled RNA probes, corresponding to DNA fragments (200–300bp) derived from the 3′-non coding regions of the BdMAN2, BdMAN4 and BdMAN6 genes (Supplementary Table S3), synthesized with the DIG RNA labelling mix according to the manufacturer’s specifications (Roche Applied Science). Probes were hybridized at 52ºC overnight followed by two washes in 2× SSC (150mM NaCl, 15mM Na₃-citrate) and 50% formamide for 90min at the same temperature. Incubation with the alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Applied Science) and colour detection was carried out according to the manufacturer’s instructions (Ferrandiz et al., 2000 (link)). Sections were dried and examined on a Zeiss Axiophot Microscope (Carl Zeiss, Oberkochen, Germany), and images were captured and processed with the Leica Application Suite 2.8.1 build software (Leica).

B. distachyon dry and germinating seeds were stained with 5% (w/v) toluidine blue (Merck, Darmstadt, Germany) for checking tissue integrity (Fig. 1A). Samples were stained with PAS reagent (0.5% w/v periodic acid-Schiff reagent) (Merck) to detect polysaccharides and with 1% (w/v) Naphthol Blue Black (Sigma-Aldrich) for proteins (Iglesias-Fernández and Matilla, 2010 ). Visualization was done on a Zeiss Axiophot Microscope (Carl Zeiss) and the images were captured and processed with the Leica Application Suite 2.8.1 build software (Leica).

Plant sections were incubated in 0.2 M HCl and, after washing, digested with 1 mg/ml proteinase-K (Hoffmann-La Roche). Samples were then dehydrated in an aqueous ethanol dilution series and hybridized with sense and anti-sense digoxigenin (DIG)-labelled RNA probes, corresponding to DNA fragments (200–300 bp) derived from the specific 3’-non coding regions of the HvMAN1 gene (Supplementary Table S3), synthesized according to the manufacturer’s instructions (Hoffmann-La Roche). Probes were hybridized at 52°C overnight followed by two washes in 2X SSC (150 mM NaCl, 15 mM Na₃ Citrate) and 50% formamide for 90 min at the same temperature. Incubation with the alkaline phosphatase-conjugated anti-digoxigenin antibody (Hoffmann-La Roche) and color detection was carried out according to the manufacturer’s instructions. Sections were dried and examined on a Zeiss Axiophot Microscope (Carl Zeiss, Oberkochen, Germany), images were captured and processed with the Leica Application Suite 2.8.1 build software (Leica, Wetzlar, Germany).