Identification of Arabidopsis phospholipase Ds involved in circadian clock alterations using CRISPR/Cas9-based multiplex editing.

Reciprocal regulation between circadian clock and lipid metabolism is emerging, but its mechanistic details remain elusive in plants. We previously reported that a lipid metabolite phosphatidic acid (PA) bound to the core clock transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and chemical suppression of phospholipase D (PLD)-catalyzed PA formation perturbed the clock in Arabidopsis. Here, we investigated to identify, among 12 isoforms, specific PLD(s) important to the regulation of clock function. We generated a library of Arabidopsis plants bearing PLD s randomly mutated by multiplex CRISPR/Cas9 system. PCR and gel-based analyses showed that all PLD s, except for β2 , were effectively edited and the mutations were heritable. Screening of T2 plants identified some with an altered rhythmic expression of CCA1 and this trait was observed in many of their progenies. Genotyping by DNA sequencing revealed that at least two of six PLD s ( α1, α3 , γ1 , δ , ε and ζ2 ) were mutated in the clock-altered T3 plants. This study identifies different combinations of two or more PLDs involved in altering clock outputs, and also suggests a functional redundancy of the six PLDs for the regulation of plant circadian clock.