High serine:glyoxylate aminotransferase activity lowers leaf daytime serine levels, inducing the phosphoserine pathway in Arabidopsis

Abstract

Serine:glyoxylate aminotransferase (SGAT) converts glyoxylate and serine to glycine and hydroxypyruvate during photorespiration. Besides this, SGAT operates with several other substrates including asparagine. The impact of this enzymatic promiscuity on plant metabolism, particularly photorespiration and serine biosynthesis, is poorly understood. We found that elevated SGAT activity causes surprisingly clear changes in metabolism and interferes with photosynthetic CO2 uptake and biomass accumulation of Arabidopsis. The faster serine turnover during photorespiration progressively lowers day-time leaf serine contents and in turn induces the phosphoserine pathway. Transcriptional upregulation of this additional route of serine biosynthesis occurs already during the day but particularly at night, efficiently counteracting night-time serine depletion. Additionally, higher SGAT activity results in an increased use of asparagine as the external donor of amino groups to the photorespiratory pathway but does not alter leaf asparagine content at night. These results suggest leaf SGAT activity needs to be dynamically adjusted to ensure (i) variable flux through the photorespiratory pathway at a minimal consumption of asparagine and (ii) adequate serine levels for other cellular metabolism.

Keywords: Arabidopsis thaliana; phosphoserine pathway; photorespiration; serine; serine biosynthesis; serine:glyoxylate aminotransferase.

Fig. 1.

Impact of excess SGAT activity on the photorespiratory pathway and photorespiratory nitrogen cycling. Red arrows indicate excess ‘SGAT’ activities including entry nodes for asparagine nitrogen. Red numbers are catalytic efficiencies of the individual ‘SGAT’ reactions according to Zhang et al. (2013). Also shown are potential serine production from hydroxypyruvate at night (grey background) and the recycling of 2OS via ω-amidase at day and night. Abbreviations: 2OG, 2-oxoglutarate; 2OS, 2-oxosuccinamate; Asp-AT, aspartate aminotransferase; ASNS, asparagine synthetase; Fd-GltS, glutamate synthase; GDC, glycine decarboxylase; GGAT, glutamate:glyoxylate aminotransferase; GLYK, glycerate 3-kinase; GOX, glycolate oxidase; GS2, glutamine synthetase; HPR1, hydroxypyruvate reductase; PGLP, phosphoglycolate phosphatase; SHMT, serine hydroxymethyltransferase.

Fig. 2.

Leaf SGAT activities in the wild type and in three transgenic lines. Enzyme activities were measured in rosette leaves from (A) seedlings at stage 1.04 according to Boyes et al. (2001) and (B) mature plants at stage 5.1 at the end of the day and end of the night. Values are means±SD from four biological replicates. Asterisks indicate that the difference from the wild-type control is significant based on Student’s t-test (*P<0.05).

Fig. 3.

Photosynthetic parameters of SGAT overexpressor leaves at growth stage 5.1 and growth over 8 weeks. (A) Net CO₂ uptake rates (A) at 390 µL L^–1 CO₂, 21% and 40% O₂. (B) CO₂ compensation points Γ at 21% and 40% oxygen. (C) Oxygen inhibition of A and (D) oxygen response of Γ (γ). Values for A–D are means±SD from at least four biological replicates. Asterisks indicate significant differences from the wild type based on Student’s t-test (P<0.05; n.s., not significant). (E) Leaf areas at five time points after germination. Values given are means±SD (n = 6). Asterisks indicate significant differences from the wild type at the respective time point based on Student’s t-test (*P<0.05).

Fig. 4.

Representative photographs of SGAT overexpressor plants and the wild type grown in normal air and air with 1% CO₂. Plants were grown under a photoperiod of 10/14 h and 20/18 °C in the day/night cycle, 75% relative humidity, at a light intensity of ~120 µmol photons m^–2 s^–1 for 8 weeks in normal air (about 390 µL L^–1 CO₂) and in 1% CO₂, respectively.

Fig. 5.

Day and night leaf amino acid contents at growth stage 5.1. Rosette leaf samples were harvested after 9 h illumination (EoD) and after 13 h darkness (EoN). Values are means±SD (n>4) in µmol g^–1 fresh weight. Asterisks indicate significant differences from the wild type based on Student’s t-test (*P < 0.05; n.s., not significant). For the complete list see Supplementary Tables S1 and S2; high-CO₂ data are shown in Supplementary Table S3.

Fig. 6.

Relative leaf contents of selected metabolites at growth stage 5.1. Leaf samples were harvested (A) after 9 h illumination at the end of the day and (B) after 13 h darkness at the end of the night. Fold values are means±SD of the respective GC-MS data from at least four biological replicates. Asterisks indicate that the difference from the wild type control is significant based on Student’s t-test (*P < 0.05). The complete list is provided as Supplementary Table S5. Abbreviations: 3HP, hydroxypyruvate; GABA, γ-aminobutyric acid.

Fig. 7.

Transcript levels of genes encoding enzymes of the phosphoserine pathway. (A) Rosette leaves of mature plants grown in soil to stage 5.1 were harvested at the middle of the day (MoD) and the middle of the night (MoN). Fold values are mean±SD (n=3) relative to the respective wild type value, which was arbitrarily set to 1. Asterisks indicate significant changes compared with the wild type value. Pluses indicate significant changes compared with the corresponding MoD value of the same genotype. (B) Rosette leaves from six seedlings per genotype grown on MS to stage 1.04 were pooled at four time points during the day/night cycle (EoN, MoD, EoD, MoN). Fold values are mean±SD (n=3) relative to the individual mean EoN values, which were arbitrarily set to 1. Asterisks indicate significant changes compared with the corresponding wild type time point. Pluses indicate significant changes compared with the EoN value of the same genotype. Comparisons are based on Student’s t-test (*^,+P < 0.05).

Fig. 8.

Expression of marker genes of the photorespiratory pathway. Wild type and SGAT overexpressors were grown on MS to stage 1.04. Cytosolic glucose-6-phosphate dehydrogenase (cG6PD) was used as a control. See Fig. 7B for further details.