Pyrimidine degradation influences germination seedling growth and production of Arabidopsis seeds

Abstract

PYD1 (dihydropyrimidine dehydogenase) initiates the degradation of pyrimidine nucleobases and is located in plastids. In this study, a physiological analysis of PYD1 employing T-DNA knockout mutants and overexpressors was carried out. PYD1 knockout mutants were restricted in degradation of exogenously provided uracil and accumulated high uracil levels in plant organs throughout development, especially in dry seeds. Moreover, PYD1 knockout mutants showed delayed germination which was accompanied by low invertase activity and decreased monosaccharide levels. Abscisic acid (ABA) is an important regulator of seed germination, and ABA-responsive genes were deregulated in PYD1 knockout mutants. Together with an observed increased PYD1 expression in wild-type seedlings upon ABA treatment, an interference of PYD1 with ABA signalling can be assumed. Constitutive PYD1 overexpression mutants showed increased growth and higher seed number compared with wild-type and knockout mutant plants. During senescence PYD1 expression increased to allow uracil catabolism. From this it is concluded that early in development and during seed production PYD1 is needed to balance pyrimidine catabolism versus salvage.

Fig. 1.

Relative expression of PYD1 in Arabidopsis tissues. PYD1 expression was monitored by quantitative RT-PCR using EF1α as the reference gene.

Fig. 2.

PYD1 transcript accumulation in overexpression mutants (35S:PYD1) and pyrimidine degradation in PYD1 knockout and overexpression lines. (A) Expression analysis of PYD1 overexpression mutants by northern blot hybridization and (B) quantitative RT-PCR. Values represent means ±SE of three biological replicates. (C–E) Catabolism of ¹⁴C-labelled uridine, uracil, and dihydrouracil in PYD1 mutants. Degradation was measured as released [¹⁴C]CO₂ based on import of the corresponding substrate. Values represent means ±SE of at least five biological replicates. The asterisks indicate significant differences between the wild type and mutants, based on a one-way ANOVA test.

Fig. 3.

Growth analysis of wild type (WT), PYD1 knockout, and PYD1 overexpressor plants. (A) Germination was monitored on soil after 2 d and 3 d (B) Germination was monitored for 2 d and 3 d on wet paper. (C) Six-week-old plants grown on soil. (D) Typical appearance of PYD1 mutants at the time of bolting. (E–G) Seeds and siliques were harvested from plants grown for 4 weeks under short day conditions and subsequently for 6 weeks under long day conditions. Values represent means ±SE of at least 10 biological replicates. The asterisks indicate significant differences between the WT and mutants, based on a one-way ANOVA test.

Fig. 4.

Germination of seeds was monitored and shown as endosperm rupture. (A) Seeds were incubated for the indicated times and monitored for endosperm rupture without stratification. (B) Seeds were stratified for 48 h in the dark at 4 °C. Five plates with 50 seeds each were analysed and the mean values ±SE were calculated.

Fig. 5.

Relative transcript level of PYD1 in response to ABA treatment and relative expression of ABI and ABF4 in 2-day-old PYD1 mutants. Expression was monitored by quantitative RT-PCR using EF1α as the reference gene. Values represent means ±SE of at least three biological replicates. The asterisks indicate significant differences between the wild type and mutants, based on a one-way ANOVA test.

Fig. 6.

Metabolite levels and invertase activity (as indicated) determined in dry seeds, and 2- and 3-day-old seedlings. Seeds were sown on wet paper in trays and incubated for 48 h in the dark at 4 °C for imbibition. The trays were then transferred to a growth chamber (see Materials and methods) and plant material was harvested after 48 h or 72 h (4 h after lights on). Values represent means ±SE of at least five biological replicates. The asterisks indicate significant differences between the wild type and mutants, based on a one-way ANOVA test.

Fig. 7.

Relative PYD1 transcript accumulation monitored at different stages of leaf sensescence and uracil levels in senescent leaves from PYD1 mutants. (A) Samples were taken from plants undergoing natural senescence, (B) plants with covered leaves, and (C) detached leaves. Expression was monitored by quantitative RT-PCR using EF1α as the reference gene. (D) Uracil was determined in leaves covered for 8 d. Values represent means ±SE of at least three biological replicates. The asterisks indicate significant differences between the wild type and mutants, based on a one-way ANOVA test