Nanosecond pulsed electric fields modulate the expression of the astaxanthin biosynthesis genes psy, crtR-b and bkt 1 in Haematococcus pluvialis

Nanosecond pulsed electric fields (nsPEFs) have been extensively studied with respect to cellular responses. Whether nsPEFs can regulate gene expression and to modulate the synthesis of valuable compounds, has so far been only tested in the context of apoptosis in cancer cells. We used the unicellular algae Haematococcus pluvialis as system to test, whether nsPEFs could alter gene expression and to promote the biosynthesis of astaxanthin. We find that nsPEFs induce a mild, but significant increase of mortality up to about 20%, accompanied by a moderate increase of astaxanthin accumulation. Steady-state transcript levels of three key genes psy, crtR-b and bkt 1 were seen to increase with a maximum at 3 d after PEF treatment at 50 ns. Pulsing at 25 ns reduce the transcripts of psy, crtR-b from around day 2 after the pulse, while those of bkt 1 remain unchanged. By blocking the membrane-located NADPH oxidase RboH, diphenylene iodonium by itself increased both, the levels of astaxanthin and transcripts of all three biosynthetic genes, and this increase was added up to that produced by nsPEFs. Artificial calcium influx by an ionophore did not induce major changes in the accumulation of astaxanthin, nor in the transcript levels, but amplified the response of crtR-b to nsPEFs at 25 ns, while decreased in 50 ns treatment. When Ca(2+) influx was inhibited by GdCl(3), the transcript of psy and bkt 1 were decreased for both 25 ns and 50 ns treatments, while crtR-b exhibited an obvious increase for the 25 ns treatment. We interpret these data in a working model, where nsPEFs permeabilise plasma and chloroplast membrane depending on pulse duration leading to a differential release of plastid retrograde signaling to the nucleus.