Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo

Abstract

CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations.

Figure 1.

Model of cytosolic pyrimidine catabolism and salvage in Arabidopsis. kinase, Nucleoside kinase; PPase, phosphatase acting on 5′ monophosphate nucleotides (5′ nucleotidase). Dashed connections are only relevant in cda mutants.

Figure 2.

Purification of C-terminally StrepII-tagged AtCDA from leaf extracts of N. benthamiana with Streptactin affinity chromatography. Samples (10 µL) were taken at different stages of the purification and visualized by SDS gel electrophoresis followed by colloidal Coomassie Blue staining (A), silver nitrate staining (only elution fraction; B), and western blotting with Streptactin-alkaline phosphatase (AP) conjugate detection (C). Lane 1, Extract of soluble proteins; lane 2, proteins not bound after incubation with Streptactin affinity matrix; lane 3, protein in the last wash fraction; lane 4, pool of eluted protein; lane 5, protein left on the matrix after elution, released by boiling in SDS buffer.

Figure 3.

Subcellular localization of AtCDA. A to C, Confocal fluorescence microscopy images of cells at the lower leaf epidermis of N. benthamiana transiently coexpressing C-terminally AtCDA-YFP and β-UP-CFP fusion proteins. YFP (A), CFP (B), and YFP and CFP (C) detection are shown. Bars = 30 µm. D and E, Mesophyll cell protoplasts of cda-1 plants transformed with AtCDA-YFP. YFP (D) and YFP and chlorophyll (E) detection are shown. Bars = 10 µm. F, Stability test of the AtCDA-YFP fusion protein from transgenic Arabidopsis plants analyzed by an immunoblot developed with a GFP-specific antibody.

Figure 4.

Genetic characterization and seed pyrimidine metabolite profiles of two independent cda mutant lines. A, Genomic organization of the At2g19570 locus and positions of the T-DNA insertions (triangles) in cda-1 (GK645H07) and cda-2 (SALK_036597C). The box represents the coding sequence, which does not contain any introns. Approximate primer positions for N261, 448, N61, and N262 are indicated. B, PCR and reverse transcription-PCR analyses of homozygous cda-1 and cda-2 lines and the wild type Columbia-0 (Col-0). C, Analysis of seed metabolite extracts using HPLC with photometric detection of the wild type, the two cda mutants, and a cda-1 line complemented with an AtCDA-YFP transgene. mAU, Milliabsorption units. D, Quantification of metabolites accumulating in mutant seeds. Error bars indicate sd (n = 4). Different letters mark significant differences at P < 0.05. fw, Fresh weight; n.d., not detectable.

Figure 5.

Cytidine and cytosine contents in leaves during plant development. Quantification of cytidine (top) and cytosine (bottom) is shown in rosette leaves of Col-0, cda-1, cda-2, and the complementation line from 15 to 55 d after germination (dag). Error bars indicate sd (n = 4). Different letters mark significant differences at P < 0.05. fw, Fresh weight; n.d., not detectable.

Figure 6.

Pyrimidine metabolite analysis of seeds of the cda-1 nsh1-1 double mutant in comparison with the respective single mutants and the wild type. A, HPLC spectrophotometric traces of nsh1-1 and cda1 nsh1-1 seed extracts. mAU, Milliabsorption units. B, Quantification of pyrimidine metabolites in seed extracts of Col-0, cda-1, nsh1-1, and the double mutant. Error bars indicate sd (n = 4). Different letters mark significant differences at P < 0.05. n.d., Not detectable.

Figure 7.

Growth responses to 5-FD and 5-FC. A, Scheme of seed placement on agar plates with four partitions. B, Col-0, cda-1, cda-2, and the complementation line after 10 d of growth on standard medium under long-day conditions (16 h of light). C, Like B but in the presence of 5 µm 5-FC, recorded on day 15. D, Like B but in the presence of 50 µm 5-FD, recorded on day 27.

Figure 8.

Growth phenotypes of cda lines. A, Rosette diameters of Col-0, cda-1, cda-2, and the complementation line (n = 10 for each) grown 45 d under long-day conditions (16 h of light). B, Rosettes and rosette leaves of the different genotypes at 45 d after germination (dag). For documentation, the inflorescences were removed. Bars = 5 cm.