Figure 6.

Transmission genetics showing that a triple KO of aca1/2/7 has a pollen deficiency that can be rescued through pollen-specific expression of transgenes encoding ACA1, 2, or 7. All crosses were conducted using transgenic lines harboring a single hemizygous transgene. All significance testing was done with a Pearson’s χ² with a minimum threshold set at P < 0.05. Not significant denoted as (ns). TE_rs were scored in F1 progeny as # of resistant/# of sensitive seedlings for selection markers associated with T-DNA insertions as indicated (Basta^R, Kan^R, Sulf^R). A, Parts 1–5 show segregation analyses of single KO alleles for aca1-7 (1), aca2-2 (2), aca2-5 (3), aca7-8 (4), aca7-9 (5). Part 6 shows segregation analyses for aca1-2(−/−)/7-9(−/−)/2-2(+/−) in which the segregation of the hemizygous aca2-2 allele was scored by a Basta^R marker. B, Bar graph showing TE_rs associated with pollen transmission of hemizygous rescue constructs. For rescue constructs, TE_rs were calculated based on the transmission of the Hyg^R marker associated with each transgene. For the “WT” ACA2, the TE_r shown represents the endogenous wild-type ACA2 segregating in competition with the aca2-2 allele, as quantified in part A-6 (inverse of the 0.38 TE_r calculated for aca2-2). A dotted line shows the expected TE_r of 1 for normal Mendelian transmission. The average TE_r is shown for the best rescue lines for each of the two triple KO combinations, with open triangles representing rescues in aca1-7(−/−)/2-3(−/−)/7-5(−/−) (Col background), and closed triangles representing rescues in aca1-2(−/−)/2-2(−/−)/7-9(−/−) (Col/WS mixed background). Corresponding ss numbers and ps are ACA9::YFP (ss2497, ps532 for Col background, ss2224 for Col/WS mixed background), ACA1 (ss2220–2221, ps1295 for Col background, ss2222 for Col/WS mixed background), ACA2 (ss2253 and 2255, ps585 for Col background, ss2498–2500 for Col/WS background), and ACA7 (ss2501, ps1960 for Col background, ss2249–2252 for Col/WS mixed background).