Figure 5.

Changes in endogenous IAA levels in leaf protoplast-derived cells. A, Transient expression of auxin responsive promoters in alfalfa leaf protoplasts cultured under embryogenic/nonembryogenic conditions. Leaf protoplasts were transfected with plasmid DNAs carrying chimeric GUS genes under the control of parA, parB, and GH3 promoters known to be auxin dependent. The protoplast-derived cells were cultured for 3 d in the presence of 1 μm 2,4-D, 10 μm 2,4-D, or 1 μm 2,4-D + 1 mm Fe-EDTA, collected by centrifugation, extracted, and the activity of the GUS enzyme has been determined fluorometrically as nanomoles of methylumbelliferon produced per milligram of protein per hour. The data of experiments in duplicate are presented as relative GUS activity considering the activity of 1 μm 2,4-D-cultured protoplasts as 100%. B through G, Dynamics of endogenous IAA accumulation in the protoplast-derived alfalfa cells as measured by microliquid chromatography with column switch coupled to electrospray tandem mass spectrometry after solid phase extraction. Frequency of cell division (B and C) and the changes in the cellular levels of endogenous conjugated (D and E) and free (F and G) forms of IAA have been determined during the first 4 to 5 d of protoplast culture as indicated. Cells were cultured under different conditions: in the presence of 1 and 10 μm 2,4-D as well as 1 μm 2,4-D with Fe stress (B–F) and in the presence of 10 μm 2,4-D with or without buffering of the medium with 10 mm MES (C–G), respectively.