The role of APT4 in LCPA biosynthesis and cell wall morphogenesis of T. pseudonana
Diatoms are unicellular eukaryotic algae that produce intricately structured cell walls made of nanopatterned silica (SiO2). Long-chain polyamines (LCPAs) are intimately associated with diatom biosilica and believed to be involved in silica biogenesis. They consist of a propyleneimine backbone with a variable degree of N-methylation and chain length. There are many enzymes that potentially catalyse LCPA biosynthesis, but experimental proof is lacking. One of these candidates is APT4. In this study I used CRISPR/Cas9 to knockout APT4. Further I investigated the LCPA composition, quantified the biosilica and analysed the cell wall morphology of knockout mutants.
The knockout resulted in 1. shorter LCPAs, 2. same amount of biosilica per cell, 3. unchanged morphology in SEM analysis.
This demonstrates that APT4 is indeed involved in LCPA biosynthesis, but not essential for silica biogenesis. More detailed investigations into biosilica morphology with TEM and quantitative image analysis are required. Further in depth determination of LCPA structure from APT4 KO mutants are required to further characterize the role of APT4 in LCPA biosynthesis.