Figure 6.

The G92 Mutation Affects the Arabidopsis Sec24A Isoform in a Highly Conserved Amino Acid Residue, and It Is Recessive.

(A) Schematic diagram of Arabidopsis Sec24A indicating the insertion of the GABI line GK-172F03. The position of the EMS-induced mutation that results in an amino acid residue change in R693K is also indicated. Protein domains were identified using Conserved Domain Architecture Retrieval Tool: ZF, zinc finger domain Sec23/24; Sec24-like, Sec23/24 α/β trunk domain; BB, Sec23/24 β-barrel domain; HR, Sec23/24 all-helical region; GL, gelsolin-like repeat.

(B) An alignment of the amino acid region containing the Arabidopsis Sec24A R693 residue (highlighted in red) with regions of Sec24 proteins from rice (Oryza sativa, EEE60426), moss (Physcomitrella patens, XP_001785970), algae (Chlamydomonas reinhardtii, XP_001701974), yeast (Saccharomyces cerevisiae_ABSV01001191), and mammals (Homo sapiens, AAI43269) shows that this amino acid residue is highly conserved. (Source: National Center for Biotechnology Information Protein database).

(C) Complementation experiments: A comparison of RT-PCR products (25, 27, and 29 cycles; indicated below the gel) from cotyledons of G92, G92/35S:Sec24A, and G92/35S:Sec24A^R693K obtained using primers designed to amplify Sec24A (see Supplemental Table 1 online) shows higher levels of expression of wild-type and mutant Sec24A alleles in transformed lines compared with untransformed G92 seedlings. Amplification was performed on 25 ng of cDNA. Amplification of UBQ10 was used as a control. Additional evidence for plant transformation is provided in Supplemental Figure 6 online.

(D) Confocal images of cross sections of cotyledon epidermal cells of T1 G92/35S:Sec24A and G92/35S:Sec24A^R693K plants show presence of the G92 structures in the G92/35S:Sec24A^R693K sample (arrows) but not in the G92/35S:Sec24A sample, indicating that the G92 phenotype is complemented by wild-type Sec24A but not Sec24A^R693K. Bar = 20 μm.