Distinctive features and differential regulation of the DRTS genes of Arabidopsis thaliana

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.

在植物和原生生物中，二氢叶酸还原酶（DHFR）和胸苷酸合酶（TS）共同构成了一种双功能酶（DRTS），该酶能够高效地循环利用由TS活性产生的二氢叶酸。拟南芥（Arabidopsis thaliana）含有三个DRTS基因，分别命名为AtDRTS1、AtDRTS2和AtDRTS3，它们位于sec14-like SFH基因家族三个成员的下游。在本研究中，对AtDRTS基因的表征发现了选择性剪接的转录本，这些转录本编码AtDRTS异构体，可能负责支持与DNA合成无关的单一功能DHFR酶。此外，我们发现了AtDRTS基因的复杂差异调控，证实了AtDRTS基因在细胞增殖和端粒复制中的预期作用，同时也揭示了与细胞其他活动相关的功能。AtDRTS1在分生组织和分化组织中均广泛表达，而AtDRTS2的表达几乎仅限于顶端分生组织，AtDRTS3则优先在茎尖、托叶和根冠细胞中表达。AtDRTS基因的差异调控与独特的启动子结构相关，且AtDRTS1在顶端分生组织中的表达严格依赖于包含基因第二内含子的内源区域。在发芽种子的细胞增殖激活过程中，分生细胞中AtDRTS1和AtDRTS2启动子的活性在细胞周期的G1/S期达到最大。此外，AtDRTS2和AtDRTS3的启动子通过E2F顺式作用元件进行负调控，且这两个基因（而非AtDRTS1）在过表达AtE2Fa因子的植物中下调。本研究为植物DRTS基因的功能和调控提供了新的信息，并为进一步探讨叶酸合成与特定细胞活动重要性的研究开辟了道路。