Szx16

Brightfield images were acquired using Olympus SZX16 or Zeiss Axiovert 200 M microscopes with Cell^F or Zeiss Axiovision image analysis software. Sprouting area and explant area were manually quantified using ImageJ freehand tool and explant area subtracted from overall area. Statistical differences between vehicle- and drug-treated samples were determined by one-way ANOVA with Dunnett’s post-hoc test. Statistical analyses were performed with PRISM 5 software and significance accepted where P≤0.05.

The promoter sequences of SnRK1.1 (At3g01090) and SnRK1.2 (At3g29160) were analyzed using tools available from the web site Plant Cis-Acting Regulatory Element (P.L.A.C.E.) (Higo et al., 1999 (link)). We focused on using the native, intergenic regions of SnRK1.1 and SnRK1.2 genes, which is approximately 0.8 and 4.3 kB of the SnRK1.1 and SnRK1.2 5′ upstream sequences (i.e., promoters, respectively). These were amplified from CS60000 genomic DNA by PCR using the primers indicated in Supplemental Table 1, and were cloned via the Gateway system into plasmid pBGWFS7 (Karimi et al., 2002 (link)) containing an eGFP:uidA gene fusion. GUS constructs were transformed into Arabidopsis using Agrobacterium transformation (Bechtold et al., 1993 (link)). Homozygous lines were obtained through BASTA resistance screening. GUS staining of 3–10-day old grown on 0.5× MS agar plates + indicated sugars or of plant tissues from soil grown plants has been described (Styer et al., 2004 (link)), and staining was observed using an Olympus SZX16 and Zeiss Axiophot microscope. At least three biological replicates of different developmental stage and sugar-treated seedlings were performed.

A standard protocol was followed for in situ hybridization of embryos after fixation and dechorionation at the indicated stages. Probes were prepared by in vitro transcription. Whole-mount in situ hybridization was performed using Digoxigenin labelled antisense RNA probe and visualized using anti-Digoxigenin Fab fragments conjugated with alkaline phosphatase (Roche Molecular Biochemicals) as described [17 (link), 18 (link)]. Embryos were processed and hybridized as described [19 (link)], except that 10 mg/ml of proteinase K in PBS/0.1% Tween-20 was used for 10 to 30 minutes depending on the age of the collected embryos.
For histology, zebrafish embryos were fixed in 4% paraformaldehyde and dehydrated prior embedding in JB-4 (Polysciences). 5-μm sections were cut using Leica RM2255 microtome (Leica, Wetzlar, Germany), dried, and stained with Hematoxylin and Eosin as described [20 ]. Still images were taken with an Olympus SZX 16 and Zeiss stereomicroscope (AxioSkop 2 plus).