The essential role of the phosphorylated pathway of serine biosynthesis in Arabidopsis

Abstract

In plants, 3 different pathways of serine biosynthesis have been described: the Glycolate pathway, which is associated with photorespiration, and 2 non-photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been known since the 1950s, but has been studied relatively little, probably because it was considered of minor significance as compared with the Glycolate pathway. In the associated study¹, we described for the first time in plants the in vivo functional characterization of the PPSB, by targeting the phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Following a gain- and loss-of-function approach in Arabidopsis, we provided genetic and molecular evidence for the essential role of PSP1 for embryo and pollen development, and for proper root growth. A metabolomics study indicated that the PPSB affects glycolysis, the Krebs cycle, and the biosynthesis of several amino acids, which suggests that this pathway is an important link connecting metabolism and development. The mechanisms underlying the essential functions of PSP1 are discussed.

In addition to forming part of proteins, the amino acid l-serine (Ser) participates in several essential processes for plants, which include biosynthesis of amino acids (glycine, methionine, cysteine), nitrogenous bases, phospholipids, and sphingolipids (Fig. 1). Ser is also the main source of one-carbon units for the methylation reactions of nucleic acids and proteins via generation of S-adenosylmethionine² (Fig. 1). Furthermore, the L-Ser derivate D-Ser has been assigned a signaling role in male gametophyte-pistil communication.³ In plants, different Ser biosynthetic pathways have been described which complicate the understanding of its metabolic regulation (Fig. 1). One of them is the Glycolate pathway, which is associated with photorespiration.^4-6 Alternative non-photorespiratory mechanisms of serine biosynthesis (the Phosphorylated and the Glycerate pathways of serine biosynthesis) have also been postulated.⁷ The biological significance of the coexistence of several serine biosynthetic pathways in plants is still not understood. Besides, the functional relevance of each pathway in different organs and under distinct environmental conditions is not yet known. The PPSB synthesizes Ser in the plastids from 3-phosphoglycerate (3-PGA) and involves 3 enzymes catalyzing sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT) and PSP1 (Fig. 1). Although the PPSB has been known since the 1950s, genetic evidence for the functions of the pathway enzymes was not provided until very recently. Our group targeted PSP1 by a gain- and loss-of-function approach in Arabidopsis.¹ PSP1 displays a highly specific cell type expression pattern being expressed in embryos, root meristems, root cap columella, anther tapetum, and pollen grains. psp1 homozygous mutants arrest embryo development at early stages, producing aborted embryos that can be classified as early curled cotyledons according to the SeedGenes database (http://www.seedgenes.org). We demonstrated that PSP1 expression in anthers is required for mature pollen development. A metabolomic study of lines overexpressing and underexpressing PSP1 indicated that subtle changes in the PPSB activity are able to regulate the glycolytic flux, affecting the Krebs cycle and the biosynthesis of amino acids, especially that of tryptophan, which in turn, affect sugar metabolism. In previous works, we hypothesized that the main role of plastidial glyceraldehyde 3-phosphate dehydrogenase (GAPCp) activity was to supply the precursor 3-PGA for the PPSB in non-photosynthetic organs⁸ (Fig. 1). Lack of PSP1 activity causes the same root and pollen phenotypes as those found in the gapcp1gapcp2 double mutants,^8,9 thus corroborating our hypothesis stating the importance of the metabolic link between GAPCp and the PPSB, which is critical for mature pollen and root development. gapcp1gapcp2 double mutants do not display the embryo lethal phenotype observed in psp1 mutants. Unlike psp1 mutants, we postulate that the PPSB is not completely inactivated in gapcp1gapcp2 double mutants because 3-PGA could be supplied at low levels in the plastid of these mutants either by the cytosolic pool through the triose-phosphate translocator or through the reverse reactions of the 2 plastidial glycolytic reactions downstream of phosphoglycerate kinase, that is, the phosphoglycerate mutase and enolase. However, the reverse activity of these enzymes and that of the triose phosphate transporter in embryo plastids still need to be demonstrated.

Figure 1. Pathways of serine biosynthesis in plants and simplified scheme showing the main metabolites synthesized from serine. The metabolic link between plastidial glycolytic GAPCp and the Phosphorylated Pathway is also illustrated. The enzymes participating in each pathway are as follows. Photorespiratory pathway (Glycolate pathway): GDC, glycine decarboxylase; SHMT, serine hydroxymethyltransferase. Glycerate pathway: PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, alanine-hydroxypyruvate aminotransferase. Phosphorylated pathway: PGDH, 3-phosphoglycerate dehydrogenase; PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Plastidial glycolysis: GAPCp, plastidial glyceraldehyde 3-phosphate dehydrogenase; PGKp, plastidial phosphoglycerate kinase. Broken lines indicate several enzymatic reactions. Abbreviations used for metabolites: THF, tetrahydrofolate. 5,10-CH₂-THF, 5,10-methylene-tetrahydrofolate. 3-PGA, 3-phosphoglycerate. 3-PHP, 3-phosphohydroxypyruvate. 3-PS, 3-phosphoserine; GAP, glyceraldehyde phosphate; 1,3BisPGA, 1,3 Bisphosphoglycerate.

From our results, 2 new questions arise: 1) why is a “minor” Ser biosynthetic pathway so important for plant development?, and 2) which is the mechanism underlying the drastic developmental defects in PSP1 deficient mutants? We postulated that the essential role of PSP1, and thus of PPSB, might be related to its expression in very specific cell types, such as tapetum, embryo, and root meristems, in which Ser supplies would originate exclusively from intracellular PPSB biosynthesis. Regarding the mechanisms responsible for the developmental defects observed in psp1 mutants, our first hypothesis was that PSP1 deficiency may affect the one-carbon metabolism. Ser serves as the major intracellular source of one-carbon tetrahydrofolate adducts, which donate carbon for the synthesis de novo of purine and pyrimidine nucleotide bases. It has been shown that mutants deficient in the tetrahydrofolate metabolism display defects in root and embryo development.^10,11 However, it was not possible to rescue the root growth of conditional psp1mutants by 5-formyl-tetrahydrofolate supplementation to the growing medium, as rescued in tetrahydrofolate deficient mutants (Fig. 2).¹¹ Thus, the essential role of Ser in root development cannot be ascribed, at least solely, to tetrahydrofolate metabolism but might well be due to several, or a combination of several processes in which serine is involved. One of them could be its participation in sphingolipid or phospholipid biosynthesis, which are known to be essential for plant viability.^12,13 In addition, a role of Ser or Ser derivatives (D-Ser) in a plant signaling mechanism or in regulating transcripcional and translational networks should also be taken into account.^3,14

Figure 2. (A) Root length of 10-d-old wild type (WT) and conditional psp1 mutant seedlings (psp1 mutant transformed with a PSP1-GFP construct under the control of the inducible heat shock promoter HS18.2; lines ProHSP:PSP1 L1 and ProHSP:PSP1 L2) grown in a MS 1/5 medium ± 5-formyl-tetrahydrofolate (5-CHO-THF) at 0.5 or 1 mM as indicated. (B) Picture showing the phenotype of conditional psp1 mutants supplemented or not with 5-CHO-THF. Values are the mean ± SE; n ≥ 20 independent biological replicates. * Significantly different as compared with controls (P < 0.05).

Finally, it could be argued that PSP1 performs additional functions, as has been demonstrated for other metabolic enzymes in plants. Along these lines, Bachelor et al. (2011)¹⁷ found that PSP1 overexpression in mammal cells abrogates squamous cell carcinoma proliferation independently of serine levels, thus postulating additional roles for the enzyme, such as additional enzymatic substrates or novel non-enzymatic interacting proteins, which may be important for carcinogenesis. Some appealing possibilities for PSP1 functions beyond the serine metabolism include protein/lipid phosphorylation or modulation of cell signaling pathways by non-enzymatic means. Further research on this subject is needed to unravel the mechanism connecting PPSB with plant development.