Figure 5.

Exogenous C24:0 Promotes the Accumulation of ACO Transcripts Rapidly While Inducing Sphingolipid Biosynthesis at a Later Stage.

(A) C24:0 promoted significant accumulation of ACO transcripts but not ACS transcripts.

(B) C16:0 did not affect transcript levels of ACSs and ACOs.

(C) C24:0 promoted the accumulation of transcripts encoding several sphingolipid biosynthesis genes (LCBs) when it was supplemented in the culture for >24 h.

RNA samples were prepared from three independent ovule cultures in the presence or absence of 5.0 μM C24:0 or C16:0 for the indicated times. QRT-PCR experiments were performed using gene-specific primers, as reported in Supplemental Table 1 online. All 11 lipid biosynthesis genes available in our cotton transcriptome were included in this analysis. Medium-24 h, wild-type ovules cultured in the original medium with MTBE for 24 h.

(D) Liquid chromatography–MS analysis of o-phthaldialdehyde derivatives of cotton LCBs. Sphingolipids were extracted from 1-DPA wild-type ovules cultured in the medium (Medium-24 h), in the presence of 2 μM ACE (ACE-24 h), or in the presence of 5 μM C24:0 (C24:0-24 h) for 24 h (left panel) or 72 h (right panel). LCBs were released by hydrolysis from sphingolipids and converted to their o-phthaldialdehyde derivatives. Peak 1, t18:1(8Z)-glucose; peak 2, t18:1(8E)-glucose; peak 3, t18:1(8E); peak 4, 1,4-anhydro-t18:1(8E).