Identification of the phosphoglycerate dehydrogenase isoform EDA9 as the essential gene for embryo and male gametophyte development in Arabidopsis

Abstract

Three different pathways of serine (Ser) biosynthesis have been described in plants: the Glycolate pathway, which is part of the Photorespiratory pathway, and 2 non-Photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Ser Biosynthesis (PPSB) has been known to exist since the 1950s, but its biological relevance was not revealed until quite recently when the last enzyme of the pathway, the Phosphoserine Phosphatase, was functionally characterized. In the associated study¹, we characterized a family of genes coding for putatite phosphoglycerate dehydrogenases (PGDH, 3-PGDH, and EDA9), the first enzyme of the PPSB. A metabolomics study using overexpressing plants indicated that all PGDH family genes were able to regulate Ser homeostasis but only lacking of EDA9 expression caused drastic developmental defects. We provided genetic and molecular evidence for the essential role of EDA9 for embryo and pollen development. Here, some new insights into the physiological/molecular function of PPSB and Ser are presented and discussed.

The amino acid l-serine (Ser) participates in several essential processes for plants, which include biosynthesis of proteins, amino acids (glycine, methionine, cysteine), purines, pyrimidines, and lipids (phospholipids and sphingolipids). Ser is also the main source of one-carbon tetrahydrofolate adducts, which are critical for the regulation of cellular processes such as the methylation reactions of nucleic acids and proteins via generation of S-adenosylmethionine.² Besides, D-Ser has been assigned a signaling mechanism between male gametophyte and pistil which is similar to the amino acid-mediated communication observed in animal nervous systems.³

Three different Ser biosynthetic pathways have been described in plants. One of them is the Glycolate pathway, which is associated with photorespiration and is considered, at least quantitatively, the most important.^4-6 Two other non photorespiratory mechanisms of Ser biosynthesis (the Phosphorylated and Glycerate pathways of Ser biosynthesis) have also been described.⁷ The biological significance of the non photorespiratory Ser biosynthetic pathways has not been unravelled until recently when the essential role of the Phosphoserine Phosphatase (PSP), the last enzyme of the Phosphorylated Pathway of Ser Biosynthesis (PPSB), for embryo and pollen development, and for proper root growth in Arabidopsis was demonstrated.⁸ The PPSB involves 3 enzymes catalyzing sequential reactions: 3-phosphoglycerate dehydrogenase (PGDH), 3-phosphoserine aminotransferase (PSAT), and PSP1 (Fig. 1). In order to continue the characterization of the PPSB in Arabidopsis, a genetic, molecular and metabolomics characterization of genes coding for putative PGDH was conducted.¹ A search in databases (TAIR; http://www.arabidopsis.org) identified 2 genes (EDA9 and 3-PGDH) whose encoded proteins showed a high percentage of identity with the previously characterized PGDH.⁹ The 3 genes conserved all the residues that form the putative catalytic site and the ligand binding domain previously described in other organisms and were referred to as the Arabidopsis PGDH family. A metabolomics study using overexpressing plants indicated that all PGDH family genes are able to regulate Ser homeostasis, with PGDH being quantitatively the most important in the Ser biosynthetic process at the whole plant level. However, the phenotypic characterization of PGDH family mutants revealed that only eda9 causes drastic developmental defects. Homozygous eda9 mutants phenocopied the embryo and pollen developmental defects observed in the psp1 mutants, identifying EDA9 as the essential gene in the PGDH family. These results strongly suggest that the essentiality of EDA9 might be related to its expression in specific cell types such as embryos, anther tapetum, and pollen. The specific expression pattern of EDA9 at the organ, tissue, and cell level supports this idea.

Figure 1. Schematic representation of the Phosphorylated Pathway of Serine Biosynthesis. The enzymes participating in the pathway along with the Arabidopsis genes identified so far are indicated. If an AGI number is shown in bold, there is experimental evidence for its participation in the pathway.

The expression pattern of some PPSB genes had been previously studied¹⁰ to find that PGDH, PSAT, and PSP1 were more expressed in light-grown than in dark-grown roots. In leaves, PSP1 and PSAT were also induced in light-grown tissues as compared with dark-grown ones. These results imply that PPSB may be more important during daylight hours than at nighttime. Our results indicate that under physiological dark conditions (8-h exposures) neither the PGDH family genes nor PSP1 are repressed as compared with light conditions.^1,8 On the contrary, 3-PGDH, EDA9, and PSP1 were clearly induced by darkness in aerial parts. In the experiments performed by Ho et al. (1999),¹⁰ plants were exposed to long periods of darkness (7 d). We could also observe that PGDH family genes and PSP1 expression were repressed under 24-h exposures to darkness. We believe that results of long darkness exposures cannot be extrapolated to plants growing in natural light environments. Thus under physiological conditions, PPSB is probably more relevant at night when the photorespiratory pathway is not functioning, than during daylight hours, especially in photosynthetic organs.

Most experimental research has focused on understanding the intraregulation of plant amino acid metabolism, but the role of amino acid metabolism in the whole metabolism and the specific role of amino acids in developmental processes have rarely been assessed. We have shown that alteration of the PGDH family gene expression drastically modifies the primary metabolism of Arabidopsis. We have also demonstrated that PPSB plays an important developmental role in non photosynthetic actively dividing cells such as embryos and anthers. Both metabolism and development must be extremely well coordinated to provide flexibility to plant development and acclimation to environmental stresses. However, the connecting links between these processes remain ill-known. Thus Ser/Ser metabolism could be one of the links connecting primary metabolism with the control of some developmental processes in plants. Regarding this point, it is interesting to note the role of Ser in the PGDH family gene expression. We did not find a negative feedback of Ser in the PGDH family gene expression following a 24-h treatment.¹ It was unexpected, however, that continuous growth with exogenously supplied Ser produced an induction of the expressions of PGDH (in aerial parts and roots) and EDA9 (in aerial parts; Fig. 2). It is difficult to interpret these inductions simplistically, solely on the basis of a negative feed-back effect of Ser on the PPSB itself. Thus we think that other parameters should be taken into account. We postulate that Ser plays an important role in the transcriptional regulation of whole primary metabolism as previously demonstrated for photorespiration¹¹ which, in turn, could affect the expression of PGDH family genes irrespectively of a feedback regulatory effect. If we are right, the potential role of Ser in the regulation of transcriptional, translational, and post-translational networks should be investigated in more detail.

Figure 2. Effect of serine on the relative expression of PGDH family genes. qRT-PCR analysis of PGDH, 3-PGDH, and EDA9 in the aerial parts and roots of 18-d-old seedlings grown in one-fifth-streght Murashige and Skoog medium ± 0.1 mM serine. Values (mean ± SE; n = 3 independent biological replicates) are normalized to the expression in control medium without serine. *Significantly different as compared with the control (P < 0.05).