Fig. 1

The two Arabidopsis synthetic systems devised to test phloem-to-SAM sRNA silencing. A The first system uses a transgenic sRNA source and an endogenous SAM target. The transgene has the SUC2 promoter (pSUC2; blue) driving phloem expression of an inverted repeat (FDi) homologous to the native FD genomic locus (red). If the phloem-expressed pSUC2::FDi can repress endogenous FD in the SAM, then the floral transition should be delayed under long day photoperiods. Top Inset: Depiction of where native FD is expressed in the SAM. The region in the first FD exon homologous to the FDi cassette is denoted by a black box. B The second system uses a transgenic sRNA source and SAM target. A site complementary to MmuMiR124 (gold) was inserted into the 3′ UTR of the CLV3 (purple) transcript. This modified CLV3 transcript was cloned with the native CLV3 promoter (pCLV3) and 3′ enhancer (eCLV3). This CLV3 transgene (pCLV3::CLV3: MmuMiR124’::eCLV3; abbreviated to “C”) was then used to rescue clv3–2 mutants. After selecting for stable homozygous C lines, a second transgene was stacked into these plants. This transgene used pSUC2 to expresses an aMiR consisting of the mature MmuMiR124 sequence in the Arabidopsis MiR319a precursor transcript (See Fig. S5 for aMiR secondary structure and the base pairing between S and C genes). If the pSUC2::aMmuMiR124 transgene (abbreviated to “S”) can silence C expression, then the stacked transgenics should show enlarged SAMs and an increase in floral organ numbers. Middle Inset: Depiction of where CLV3 is expressed in the SAM. C & D A pSUC2::GUS:GFP transcriptional reporter was used to confirm pSUC2 vein specificity in Col. Both cleared whole mounts (C) and paraffin wax embedded and sectioned tissues (D) exhibited GUS reporter staining constricted to the veins, distal to the shoot apex. The black scale bar denotes the length corresponding to 500 base pairs (bp) of DNA