Fig 4. Carotenoid pathway flux determines β-carotene accumulation in Arabidopsis callus.

A, Seed-derived calli were generated from AtPSY-overexpressing Arabidopsis lines with low (At13, AtU16) and high (At12, At22) PSY protein levels. At13 and AtU16 accumulated carotenoid levels almost like WT despite two-fold increased pathway activity concluded from the phytoene content in presence of NFZ. Further increased pathway activity as in lines At12 and At22 results in β-carotene accumulation. Different PSY activities are reflected by different PSY protein levels shown by immunoblotting using 80 μg of callus protein below. Actin levels are included as loading control. Results are mean ± SD from at least three biological replicates. Significant difference to the WT (*) and WT-NFZ (**), respectively, P<0.05. B, In vitro PSY activities were determined in isolated callus plastid membrane fractions by incubation with DMAPP, [¹⁴C]-IPP and a recombinant GGPP synthase and quantification of [¹⁴C]-phytoene levels. Results are means ± SD from three biological replicates. Significant difference to WT, *P<0.05.