Oxidative stress in chloroplasts and mitochondria are communicated via multiple retrograde signalling pathways to the nucleus to alter expression of nuclear-encoded genes. However, less is understood about whether and how retrograde signals participate in additional regulatory layers on these genes, such as alternative splicing. Furthermore, there is emerging evidence that some of these retrograde pathways are interconnected, but mechanisms enabling these connections are enigmatic.
Here we show that the SAL1-PAP chloroplast signalling pathway modulates large-scale alternative splicing changes. Differential splicing analysis on RNA sequencing of Arabidopsis treated with exogenous PAP revealed widespread PAP-associated alternative splicing (AS) across >1600 genes, many of which also undergo AS during abiotic stress. PAP-associated AS predicted to change protein sequences were significantly enriched in gene ontologies related to signal transduction and hormonal responses, suggesting that PAP-mediated AS could alter the cellular proteome during stress.
Interestingly, one of the strongest PAP-induced AS was in Radical-Induced Cell Death (RCD1), a negative regulator of the RCD1-NAC017 mitochondrial signalling pathway. PAP treatment induced expression of an intron-retained RCD1 isoform containing premature termination codons, decreased RCD1 protein abundance, and activated signalling events normally suppressed by RCD1 such as induction of NAC017-regulated genes. Thus, PAP might influence mitochondrial signalling at least in part by altering RCD1 AS.
Our results suggest that organelle signalling pathways can fine-tune stress acclimation, and intersect with each other, by influencing AS of genes involved in the cellular response to oxidative stress. This opens new avenues to understanding how organelles contribute to, and fine-tune, their cellular environment.