Clarification of the dispensability of PDX1.2 for Arabidopsis viability using CRISPR/Cas9

Abstract

BackgroundPDX1.2 has recently been shown to be a regulator of vitamin B6 biosynthesis in plants and is implicated in biotic and abiotic stress resistance. PDX1.2 expression is strongly and rapidly induced by heat stress. Interestingly, PDX1.2 is restricted to eudicota, wherein it behaves as a non-catalytic pseudoenzyme and is suggested to provide an adaptive advantage to this clade. A first report on an Arabidopsis insertion mutant claims that PDX1.2 is indispensable for viability, being essential for embryogenesis. However, a later study using an independent insertion allele suggests that knockout mutants of pdx1.2 are viable. Therefore, the essentiality of PDX1.2 for Arabidopsis viability is a matter of debate. Given the important implications of PDX1.2 in stress responses, it is imperative to clarify if it is essential for plant viability.ResultsWe have studied the previously reported insertion alleles of PDX1.2, one of which is claimed to be essential for embryogenesis (pdx1.2–1), whereas the other is viable (pdx1.2–2). Our study shows that pdx1.2–1 carries multiple T-DNA insertions, but the T-DNA insertion in PDX1.2 is not responsible for the loss of embryogenesis. By contrast, the pdx1.2–2 allele is an overexpressor of PDX1.2 under standard growth conditions and not a null allele as previously reported. Nonetheless, upregulation of PDX1.2 expression under heat stress is impaired in this mutant line. In wild type Arabidopsis, studies of PDX1.2-YFP fusion proteins show that the protein is enhanced under heat stress conditions. To clarify if PDX1.2 is essential for Arabidopsis viability, we generated several independent mutant lines using the CRISPR-Cas9 gene editing technology. All of these lines are viable and behave similar to wild type under standard growth conditions. Reciprocal crosses of a subset of the CRISPR lines with pdx1.2–1 recovers viability of the latter line and demonstrates that knocking out the functionality of PDX1.2 does not impair embryogenesis.ConclusionsGene editing reveals that PDX1.2 is dispensable for Arabidopsis viability and resolves conflicting reports in the literature on its function.

PDX proteins from Arabidopsis thaliana as novel substrates of cathepsin B: implications for vitamin B6 biosynthesis regulation

“…Arabidopsis encodes for only one PDX2 protein, while there are three homologs of PDX1 (PDX1.1, PDX1.2, and PDX1.3) [17,18]. Only PDX1.1 and PDX1.3 are catalytically active, whereas PDX1.2 is considered to be a pseudoenzyme [19] that can function as an allosteric regulator to promote activities of neighboring PDX1.1 and PDX1.3 proteins [20,21]. Subcellular localization studies find PDX proteins in the cytosol and plasma membrane [17,22].…”