Lipid-binding Protein COQ9 In Plant Coenzyme Q Biosynthetic Pathway

Coenzyme Q(CoQ)is a lipid-soluble quinone which is ubiquitous in prokaryotes and eukaryotes,and functions as electron transporter in the respiratory chain.In Saccharomyces cerevisiae,at least 11 proteins have been found to directly participate in the biosynthesis of Co Q(Coq1p-Coq11p).In addition to the enzymes that catalyze the chemical reactions within the Co Q biosynthetic pathway,other proteins are also required for Co Q biosynthesis.Yeast Coq9 p is a lipid-binding protein,is an important member of complex Q(Co Q-synthome),and is necessary for Coq6 p and Coq7 p to perform the hydroxylation function.In human,COQ9 mutations lead to severe metabolic disorders.Loss of COQ9 results in decreased levels of COQ7 and high accumulation of demethoxy coenzyme Q(DMQ),the substrate of COQ7.It has been reported that COQ9 from Arabidopsis thaliana failed to complement the corresponding Schizosaccharomyces pombe mutant.Thus,it is still not clear whether plant COQ9 is involved in the biosynthesis of Co Q.In this study,we investigate the function of AtCOQ9:1.The sequence alignment and phylogenetic analysis show that COQ9 proteins are highly conserved from prokaryotes to eukaryotes.2.Arabidopsis plants transformed with a construct in which the AtCOQ9 promoter was fused to GUS revealed that GUS activity is detected in 7-day-old seedlings,14-day-old seedlings,4-week-old rosettes,and inflorescences.The expression level of AtCOQ9 in six different tissues(seedlings,leaves,stems,inflorescences,siliques,and seeds)was further determined by q RT-PCR.AtCOQ9 is more highly expressed in seeds,consistent with the distribution of Co Q.3.Subcellular localization experiments found that AtCOQ9 is localized in mitochondria,indicating that it may be involved in the biosynthesis of Co Q.In addition,AtCOQ9 was also found in chloroplasts,suggesting an unknown function of AtCOQ9 in chloroplasts.4.We found that expression of AtCOQ9 rescues the growth of the yeast coq9temperature-sensitive mutant by increasing Co Q biosynthesis,indicating the functional conservation of COQ9 between yeast and plants.5.To further study the function of AtCOQ9 in plants,knockout lines were generated using CRISPR/Cas9 technology.The leaves of three independent knockout lines showed a decrease in Co Q content compared with wild-type plants,thus providing genetic evidence that AtCOQ9 is involved in Co Q biosynthesis in Arabidopsis.In summary,our data demonstrate that the function of COQ9 is conserved between Arabidopsis and yeast,and COQ9 is involved in the biosynthesis of Co Q in plants.Our findings contribute to the understanding of the Co Q biosynthetic pathway in plants.