Natural variation in tetrapyrrole biosynthetic enzymes and their regulation modifies the maize chlorophyll mutant <i>Oy1-N1989</i>

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 the effects of the Oil yellow1 mutation, Oy1-N1989, a semi-dominant allele of subunit I in the Mg-chelatase enzyme with reduced chlorophyll biosynthesis in maize (Zea mays), on global gene expression and chlorophyll content. In Oy1-N1989/+ mutants, coordinate feedback regulation of the tetrapyrrole pathway was observed as transcriptional feedback regulation of genes encoding steps in the tetrapyrrole pathway. Natural variation in the wild-type allele at oy1 modulated the severity of the impact of Oy1-N1989/+ on gene expression. Previously identified cis-acting expression variation at oy1 in wild-type plants affected similar transcriptional co-regulation of genes encoding steps in the tetrapyrrole pathway as observed in the RNA-seq 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 7 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 affected the expression of a subset of tetrapyrrole pathway genes, indicating that these genes are repeated targets of transcriptional regulation. The hotspots were encoded at locations with no known regulators of the tetrapyrrole pathway, indicating as yet undiscovered molecular mechanisms of feedback regulation operating in natural populations. The trans-regulatory hotspots coordinately regulate this pathway and may work to limit the accumulation of phototoxic intermediates.