Figure 7.
Analysis of ACO Enzyme Activity and Transcript Expression in Specific Seed Tissues of L. sativum during Germination.
The framed box shows symbols used in (A) and (E) to (G). Mean values ± se ([A] to [C]) or +se (qRT-PCR; [E] to [G]) are presented for three ([A] to [C]) or four ([E] to [G]) biologically independent samples each with 50 seeds (C), 100 to 200 seed parts ([A] and [B]) or 1000 endosperm caps or 100 radicles ([E] to [G]) from seeds with ruptured testa but intact endosperm ([A], [B], and [E] to [G]). L. sativum FR14 ([A] to [D]) or FR1 ([E] to [G]) seeds were incubated at 24°C in continuous light. Note that the qRT-PCR and the microarrays were performed as independent experiments.
(A) Time course of in vivo ACO enzyme activities in endosperm caps and radicles of seeds incubated without (CON) and with 10 μM ABA; for comparison, the kinetics of endosperm rupture are presented. In vivo ACO enzyme activities of endosperm caps and radicles dissected from seeds at different time points were measured by subsequent organ incubation in medium with ACC (plus ABA for the ABA series).
(B) The effect of ambient and reduced oxygen atmospheres on the in vivo ACO enzyme activities of isolated seed parts compared with intact seeds. Note that only seeds in the testa rupture (TR) state were selected for the measurements (i.e., seeds with intact endosperm). No wound-induced ethylene production by the radicle/embryo was evident.
(C) The effect of oxygen on the promotion of endosperm rupture by ACC and the reversion of the ABA inhibition of endosperm rupture by ACC.
(D) Molecular phylogenetic analysis of Lepidium ACO and Arabidopsis ACO cDNA sequences. The bar (0.07) defines the number of substitutions per 100 amino acids. Lepidium ACO cDNA sequences and comparisons to the Arabidopsis orthologs are presented in Supplemental Figure 3 online.
(E) to (G) LesaACO transcript expression pattern determined by qRT-PCR in endosperm cap and radicle during incubation on medium without (CON) or with 10 μM ABA added. Relative ΔΔCt expression values based on the comparison with validated constitutive transcripts are presented.