Direction des sciences du vivant

Tetrahydrofolate (THF) derivatives, collectively termed folates or vitamin B9, are involved in almost all the metabolic processes that require the addition or removal of one-carbon units (C1 metabolism). C1-substituted folate coenzymes are thus involved in several major cellular processes, including the synthesis of nucleotides (purines and thymidylate) and amino acids (methionine, serine, glycine). Methionine serves as a methyl group donor through conversion to S-adenosylmethionine, the key biological methylating agent involved in dozens methyltransferase reactions. Plants, fungi and most micro-organisms are able to synthesize THF de novo whereas humans and animals in general lack this capacity and thus rely on dietary intake of vitamin B9.
During plant development, the enzymatic capacity for THF synthesis, the pool of folates, and the demands for C1-units vary importantly. However, little is known about how folate homeostasis is controlled to match the supply of C1-units, and how C1-units are accurately distributed between the different anabolic routes. In this work, we identified different regulatory mechanisms that allow plants to respond to a limitation of the folate pool.
First, we analyzed the genome-wide and metabolic response of Arabidopsis cells to folate depletion induced by the antifolate methotrexate. Surprisingly, no significant change in the expression of genes involved in cofactor synthesis, degradation or trafficking was observed. One major response to folate limitation concerned the composition of the cofactor pool. Thus, the transcriptional regulation of a limited number of genes coding enzymes manipulating C1-moieties in plastids was associated with a re-orientation of C1-units towards the synthesis of purines and thymidylate. These data suggest that the metabolic priority of Arabidopsis cells in response to folate limitation is to shuttle the available folate derivatives to the synthesis of nucleotides at the expense of methylation reactions.
Second, because the efficiency of methylation reactions are likely affected by folate depletion, we investigated the relationships between the folate status, the methyl cycle activity, and the rate of chlorophyll synthesis, which relies at one step on a methylation reaction catalyzed by Mg protoporphyrin IX methyltransferase. Etiolated pea leaves treated with methotrexate displayed a reduced folate pool and a marked decrease in the methyl index. This resulted in a metabolic regulation (inhibition) of the Mg-protoporphyrin IX methyltransferase activity and to a decreased rate of chlorophyll synthesis. These results point out that an even moderate change in the folate status may affect essential physiological processes such as chloroplast biogenesis.
Last, the analysis of Arabidopsis cells exposed to a long-term folate starvation revealed an original post-translational regulation of methionine synthesis. This process consists in the proteolytic cleavage of the N-terminal domain of cystathionine g-synthase, the first specific enzyme for methionine synthesis. We suggest that an effector, to date unidentified, can modulate enzyme activity in vivo through an interaction with the N-terminal domain and that removal of this domain in folate-deficient cells can suppress this regulation.