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Abstract
In plants and protists, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are part of a bifunctional enzyme (DRTS) that allows efficient recycling of the dihydrofolate resulting from TS activity. Arabidopsis thaliana possesses three DRTS genes, called AtDRTS1, AtDRTS2 and AtDRTS3, that are located downstream of three members of the sec14-like SFH gene family. In this study, a characterization of the AtDRTS genes identified alternatively spliced transcripts coding for AtDRTS isoforms which may account for monofunctional DHFR enzymes supporting pathways unrelated to DNA synthesis. Moreover, we discovered a complex differential regulation of the AtDRTS genes that confirms the expected involvement of the AtDRTS genes in cell proliferation and endoreduplication, but indicates also functions related to other cellular activities. AtDRTS1 is widely expressed in both meristematic and differentiated tissues, whereas AtDRTS2 expression is almost exclusively limited to the apical meristems and AtDRTS3 is preferentially expressed in the shoot apex, in stipules and in root cap cells. The differential regulation of the AtDRTS genes is associated to distinctive promoter architectures and the expression of AtDRTS1 in the apical meristems is strictly dependent on the presence of an intragenic region that includes the second intron of the gene. Upon activation of cell proliferation in germinating seeds, the activity of the AtDRTS1 and AtDRTS2 promoters in meristematic cells appears to be maximal at the G1/S phase of the cell cycle. In addition, the promoters of AtDRTS2 and AtDRTS3 are negatively regulated through E2F cis-acting elements and both genes, but not AtDRTS1, are downregulated in plants overexpressing the AtE2Fa factor. Our study provides new information concerning the function and the regulation of plant DRTS genes and opens the way to further investigations addressing the importance of folate synthesis with respect to specific cellular activities.
Highlights
Cofactors derived by the tetrahydrofolate (THF), collectively named folates or vitamin B9, are essential for all organisms and are necessary for the addition or removal of one-carbon units in key reactions of various biochemical pathways (C1-metabolism)
Increased expression of AtDRTS1 can be detected in both the AtE2FaOE lines, whereas the expression of AtDRTS2 and AtDRTS3 clearly diminished (Fig 7A). These results suggest an E2F-dependent repression of the AtDRTS2 and AtDRTS3 promoters, which could be direct targets of E2F factors, and a positive influence of AtE2Fa overexpression on the expression of AtDRTS1, which is not necessarily reflecting a direct regulation but could be linked to the increased cell proliferation observed in the AtE2FaOE lines
With this study we discovered a complex differential regulation of the AtDRTS genes that confirms their expected involvement in cell proliferation and endoreduplication, but indicates peculiar functions related to distinctive cellular activities
Summary
Cofactors derived by the tetrahydrofolate (THF), collectively named folates or vitamin B9, are essential for all organisms and are necessary for the addition or removal of one-carbon units in key reactions of various biochemical pathways (C1-metabolism). These reactions are crucial for the synthesis of a large number of compounds in many cellular processes, including amino acid and nucleic acid metabolisms. Additional roles of folates in iron—sulphur cluster metabolism have been described [7] and 5,10-methylene THF is known to be necessary for the synthesis of pantothenate (vitamin B5), which is essential for the the synthesis of CoA and ACP (acyl-carrier protein) involved in reactions of β-oxidation generating important signaling molecules such as indoleacetic, jasmonic and salicylic acids [8,9]. Because of the multiple and essential roles of THF cofactors, it is clear that the control of THF biosynthesis has important implications for plant growth and productivity, as confirmed by the embryo-lethal phenotype of biosynthetic mutants of Arabidopsis thaliana that are unable to produce functional folates [10,11]
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