Insitupro machine

Four-to 6-d-old seedlings were incubated on 24-well cell culture plates for 1 h in 50 mM BFA (Invitrogen, Thermo Fisher Scientific) containing liquid growth medium (half-strength MS medium and 1% [w/v] Suc, pH 5.8) at 23°C and then fixed for 1 h in 4% (w/v) formaldehyde in microtubulestabilizing buffer at room temperature. Whole-mount immunofluorescence staining was performed manually as described (Lauber et al., 1997) or with an InsituPro machine (Intavis; Müller et al., 1998) . All antibodies were diluted in 13 PBS. The following antisera were used for immunofluorescence staining: mouse anti-c-Myc mAb 9E10 (Santa Cruz Biotechnology) diluted 1:600; rabbit anti-ARF1 (Agrisera) diluted 1:1000; rabbit anti-AtgCOP (Agrisera) diluted 1:1000; anti-mouse Alexa488 (Invitrogen) and antirabbit CY3 (Dianova)-conjugated secondary antibodies diluted 1:600. Nuclei were stained with 49,6-diamidino-2-phenylindole (1:600 dilution).

Riboprobes were either generated from linearized plasmid clones or from cDNA fragments amplified by RT-PCR as described previously (Bildsoe et al., 2009; Loebel et al., 2011) (Supplementary Table S2). The AmpliScribe T7-flash transcription kit (Epicentre), modified to allow incorporation of DIG-11-UTP, was used for Riboprobe generation.
Automated whole mount in situ hybridization was carried out using an Intavis In situ Pro machine as described previously (Bildsoe et al., 2009; Bildsoe et al., 2013; Loebel et al., 2004) . Alternatively, in situ hybridization was carried out manually using a modification of the published protocol (Wilkinson and Nieto, 1993) . Embryos were incubated in pre-hybridization buffer at 70 °C for 1 h and hybridization carried out overnight, at 70 °C. Post hybridization washes were in pre-hybridization buffer (2 times), 2 Â SSC, pH4.5 (2 times) and 0.2 Â SSC, pH4.5, (2 times). Antibody detection and staining with BM Purple was performed as described previously (Loebel et al., 2004) .
Embryos were imaged using a Leica dissecting microscope and SPOT digital camera (SciTech). Embryos to be sectioned were washed in 0.1% Tween20 and refixed in 4% PFA and processed for wax histology. The embedded embryos were sectioned at 8 mm thickness, dried overnight, and counterstained with nuclear fast red.