Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella

Abstract

Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome, might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant bryophytes. Whereas angiosperms repair DNA double strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in bryophytes homologous recombination (HR) is the main DNA-DSB repair pathway facilitating the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in the moss Physcomitrella using haploid plants and autodiploid plants, generated via an artificial duplication of the whole genome. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the whole genome duplication event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p<0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis

“…These precautions were made as the transformation procedure may generate plants with multiple integrations 80 , possibly leading to off-target effects. Further, the transformation procedure may lead to diploid plants with altered gene expression 81 . We used haploid lines with a single integration of the KO construct (Fig.…”

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention

“…Flow cytometry analysis was performed according to Heck et al (2021) to determine ploidy of the transgenic lines (pinC#10, pinC#29, pinC#69, pinCPromCit). As there is a risk of creating diploid plants during PEG-mediated transformation of protoplasts, which could alter gene expression compared with haploid plants (Rempfer et al, 2022), only haploid transgenics were analysed further.…”

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention

“…Flow cytometry analysis was performed according to Heck et al (2021) to determine ploidy of the transgenic lines (pinC#10,pinC#29,pinC#69,pinCPromCit). As there is a risk of creating diploid plants during PEG-mediated transformation of protoplasts, which could alter gene expression compared to haploid plants (Rempfer et al, 2022), only haploid transgenics were analysed further.…”