CTP Synthase 2 From Arabidopsis thaliana Is Required for Complete Embryo Development

Pyrimidine de novo synthesis is an essential pathway in all organisms. The final and rate-limiting step in the synthesis of the nucleotide cytidine triphosphate (CTP) is catalyzed by CTP synthase (CTPS), and Arabidopsis harbors five isoforms. Single mutant lines defective in each one of the four isoforms do not show apparent phenotypical alterations in comparison to wild-type plants. However, Arabidopsis lines that contain T-DNA insertions in the CTPS2 gene were unable to produce homozygous offspring. Here, we show that CTPS2 exhibits a distinct expression pattern throughout embryo development, and loss-of-function mutants are embryo lethal, as siliques from +/ctps2 plants contained nearly 25% aborted seeds. This phenotype was rescued by complementation with CTPS2 under control of its endogenous promoter. CTPS2::GFP lines revealed expression only in the tip of columella cells in embryo root tips of the heart and later stages. Furthermore, CTPS2 expression in mature roots, most pronounced in the columella cells, shoots, and vasculature tissue of young seedlings, was observed. Filial generations of +/ctps2 plants did not germinate properly, even under external cytidine supply. During embryo development, the CTPS2 expression pattern resembled the established auxin reporter DR5::GFP. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element, and auxin supply increased CTPS2 expression in a cell-type-specific manner. Thus, we conclude that CTPS2 is essential for CTP supply in developing embryos, and loss-of-function mutants in CTPS2 are embryo lethal.

CTP-Synthase 2 from Arabidopsis thaliana is required for complete embryo development

Pyrimidine de novo synthesis is an essential pathway in all organisms. The final and rate limiting step in the synthesis of the nucleotide CTP is catalyzed by CTP-Synthase (CTPS) and Arabidopsis harbors five isoforms. Single knockouts of each of these do not show apparent phenotypical alterations with the exception of CTPS2. T-DNA insertion lines for this isoform were unable to produce homozygous offspring. Here we show that CTPS2 exhibits a distinct expression pattern throughout embryo development and loss of function mutants were embryo lethal, as siliques from +/ctps2 plants contained nearly 25 % aborted seeds. This phenotype was rescued by complementation with CTPS2 under control of its endogenous promoter. Reporter lines revealed CTPS2 expression only in the tip of columella cells in embryos of the heart and later stages. Furthermore CTPS2 expression in roots, most pronounced in the columella cells, shoots and vasculature tissue of young seedlings was observed. Filial generations of +/ctps2 plants did not germinate properly, even under external cytidine supply. During embryo development CTPS2 expression was similar to the well known auxin reporter DR5. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element. Thus, we conclude that CTPS2 is essential for CTP supply in the developing embryo and a knockout of CTPS2 is embryo lethal.

Antagonistic and auxin-dependent phosphoregulation of columella PIN proteins controls lateral root gravitropic setpoint angle in Arabidopsis

Lateral roots of many species are maintained at non-vertical angles with respect to gravity. These gravitropic setpoint angles (GSAs) are intriguing because their maintenance requires that roots are able to effect gravitropic response both with and against the gravity vector. Here we have used the Arabidopsis lateral root in order to investigate the molecular basis of the maintenance of non-vertical GSAs. We show that gravitropism in the lateral root is angle-dependent and that both upward and downward graviresponse requires auxin transport and the generation of auxin asymmetries consistent with the Cholodny-Went model. We show that the symmetry in auxin distribution in lateral roots growing at GSA can be traced back to a net, balanced polarization of PIN3 and PIN7 auxin transporters in the columella cells. Further, upward and downward graviresponse in lateral roots correlates with corresponding changes in PIN3 and PIN7 polar localisation. Finally, we show that auxin, in addition to driving tropic growth in the lateral root, acts within the columella to regulate GSA via the PIN phosphatase subunit RCN1 in a PIN3-dependent and PIN7-independent manner. Together, these findings provide a molecular framework for understanding gravity-dependent nonvertical growth in Arabidopsis lateral roots.