Natural variation in tetrapyrrole biosynthetic enzymes and their regulation modify the chlorophyll mutant Oy1-N1989

Tetrapyrroles are macrocyclic compounds present in and required for all life on Earth. Mutants with defects in tetrapyrrole pathway enzymes can be used to uncover natural variation in this pathway and study pathway regulation. We report here the effects of the Oy1-N1989/+ mutation, a semi-dominant allele of subunit I in the Mg-chelatase enzyme with reduced chlorophyll biosynthesis, on global gene expression and morphological traits. Coordinate regulation of the tetrapyrrole pathway was observed as transcriptional feedback regulation of genes in the tetrapyrrole pathway in Oy1-N1898/+ mutants. Natural variation in the wild-type allele at oy1 modulated the severity of the impact of Oy1-N1989/+ on gene expression. We also studied the effects of previously identified cis-acting expression variation at oy1 in wild-type plants. The seventy transcripts encoding biosynthetic enzymes in the tetrapyrrole pathway exhibited similar transcriptional co-regulation in response to cis-variation in oy1 transcript accumulation in wild-type plants as observed in the RNAseq of Oy1-N1989/+ mutants. This demonstrated that the coordinate regulation of the pathway also occurs during physiologically-relevant variation in OY1 abundance. Cis variants at seven tetrapyrrole pathway genes were linked to variation in chlorophyll accumulation in Oy1-N1989/+ mutant or wild-type plants. Analysis of trans-acting transcriptional variation by eGWAS detected multiple transcriptional hotspots, which disproportionately affected the expression of a subset of tetrapyrrole pathway genes, indicating that some genes are repeated targets of transcriptional regulation. The trans-regulatory hotspots coordinately regulated this pathway and may work to limit the accumulation of phototoxic intermediates. Author SummaryThe mutant allele and interactions with the genetic background determine the severity of mutant phenotypes. We used Oy1-N1989/+ mutants, affecting Mg-chelatase subunit I, to identify natural variants in the tetrapyrrole pathway affecting transcriptional regulation and variation in mutant traits. By integrating GWAS and transcriptomics, we identified similar feedback regulation of porphyrin and chlorophyll metabolism in mutants as well as in wild-type plants affected by natural variation. This is the first demonstration of natural variation affecting transcriptional feedback in the tetrapyrrole pathway under normal conditions. Using GWAS as a regulatory discovery tool, we identified unexplored regulatory mechanisms in a pathway that is critical for energy capture from the sun and chemical energy utilization in all living things and multiple human diseases.