Nanosecond pulsed electric fields trigger cell differentiation in Chlamydomonas reinhardtii

Nanosecond pulsed electric fields (nsPEFs) have great potential for biotechnological and medical applications. However, the biological mechanisms causing the cellular responses are still far from understood. We used the unicellular green algae Chlamydomonas reinhardtii as experimental model to dissect the immediate consequences of electroporation from the developmental cellular responses evoked by nsPEFs. We observe that nsPEFs induce a short-term permeabilization of the membrane, accompanied by swelling and oxidative burst. These response are transient, but are followed, several days later, by a second wave of oxidative burst, arrested cell division, stimulated cell expansion, and the formation of an immobile palmella stage. This persistent oxidative burst can be suppressed by specific inhibitor diphenyl iodonium (DPI), but not by the unspecific antioxidant ascorbic acid (Asc). Treated with natural and artificial auxins allow to modulating the cell cycle and cell expansion, and natural auxin can suppress the spontaneous formation of palmella stages. However, when administered prior to the nsPEFs treatment, auxin cannot mitigate the elevated formation of palmella stages induced by nsPEFs. We interpret our findings in terms of a model, where nsPEFs generate a developmental signal that persists, although the other immediate responses remain transient. This signal will initiate, several days later, a developmental programme comprising halted cell cycle, stimulation of cell expansion, a persistent activation of NADPH oxidase activity causing a second wave of oxidative burst, and the irreversible initiation of palmella stages. Thus, a short transient nsPEFs treatment can initiate a stable response of cellular differentiation in Chlamydomonas reinhardtii.