Figure 3.

The Sha and Sha-like NCED3 haplotypes have an altered pattern of NCED3 apparent molecular mass. A, Immunoblot detection of NCED3 in Ler, Sha, and nced3 at 10 h after transfer to –1.2 MPa. In each lane, 40 μg of protein was loaded. The numbers 1 to 5 and red arrows mark the five bands of NCED3 detected. After NCED3 detection, blots were stripped and reprobed to detect HSC70 as a loading control. B, Immunoblot detection of NCED3 in accessions with the Sha NCED3 haplotype (Sha, Old-1, and Sorbo), Sha-like NCED3 haplotype (Spr1-6, Dra-iV, and Tdr3), or Ler/Col-0 NCED3 haplotype (Ler, Kas-1, and Nd-0). Samples were collected 10 h after transfer to –1.2 MPa and 40 μg of protein was loaded. C, Immunoblot detection of NCED3 in arbitrarily selected accessions with the Ler/Col-0 NCED3 haplotype. D, Complementation of nced3 with NCED3 site-directed mutants (SDMs) expressed under the control of the Ler NCED3 promoter. ABA data for the SDM lines are combined from two independent transgenic lines for each construct (n = 19–40 combined from two independent experiments) and are shown as percentages of the Col wild type (W.T.) assayed in the same experiments. Formatting of the graph is as described for Figure 1C. Asterisks indicate significant differences from Sha NCED3-HA. An expanded graph showing data for individual transgenic lines separately is shown in Supplemental Figure S3B. The Sha NCED3-HA data are replotted from Figure 1E for comparison. Immunoblotting was performed using the same conditions described for A. Note that because of the HA epitope tag, the Sha NCED3-HA lane should only be used to compare protein amount and not molecular masses.