The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway

Jaya Joshi; Manaki Mimura; Masaharu Suzuki; Shan Wu; Jesse F Gregory 3rd; Andrew D Hanson; Donald R McCarty

doi:10.3389/fpls.2021.721391

The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway

Front Plant Sci. 2021 Aug 6:12:721391. doi: 10.3389/fpls.2021.721391. eCollection 2021.

Authors

Jaya Joshi¹, Manaki Mimura², Masaharu Suzuki¹, Shan Wu¹, Jesse F Gregory 3rd³, Andrew D Hanson¹, Donald R McCarty¹

Affiliations

¹ Department of Horticultural Sciences, University of Florida, Gainesville, FL, United States.
² Plant Cytogenetics, Department of Gene Function and Phenomics, National Institute of Genetics, Mishima, Japan.
³ Department Food Science and Human Nutrition, University of Florida, Gainesville, FL, United States.

Abstract

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 (th3) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 (DXS1), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway.

Keywords: 5-deoxyxylulose-phosphate synthase; biotic stress response; methylerythritol 4-phosphate pathway; retrograde signaling; thiamin; thiamin requiring.