The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase (2023)

Plant Signal Behav. 2016 Mar; 11(3): e1128614.

Published online 2016 Mar 8. doi:10.1080/15592324.2015.1128614

PMCID: PMC4883961

Armand Djoro Anoman,^a,^b María Flores-Tornero,^a,^b Sara Rosa-Telléz,^a,^b Jesús Muñoz-Bertomeu,^a Juan Segura,^a,^b and Roc Ros^a,^b,^*

ABSTRACT

The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development.

KEYWORDS: glyceraldehyde-3-phosphate dehydrogenase, phosphorylated pathway of serine biosynthesis, plastidial glycolysis, serine, 2-oxoglutarate

Abbreviations

GAPDH: glyceraldehyde-3-phosphate dehydrogenase
GAPCp: plastidial glyceraldehyde-3-phosphate dehydrogenase
PPSB: phosphorylated pathway of serine biosynthesis
3-PGA: 3-phosphoglycerate
PGK: phosphoglycerate kinase
GAP: glyceraldehyde-3-phosphate
GOGAT: glutamate synthase
TCA: tricarboxylic acid
GABA: gamma-aminobutyric acid

GAPCp is not relevant in photosynthetic cells

The main function of glycolysis is to oxidize hexoses to provide ATP, reducing power and pyruvate, and to produce precursors for anabolism.¹ In plants, this metabolic process occurs in the cytosol and plastids of both photosynthetic and non-photosynthetic organs. However, the requirements of glycolytic energy and precursors may be completely different in autotrophic and heterotrophic cells. In this sense, the presence of a complete glycolytic plastidial pathway in chloroplasts, specifically its lower part catalyzed by phosphoglycerate mutase and enolase, has even been questioned.^2-4 The reactions of plastidial/chloroplastic phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are particularly interesting because different isoforms of the same enzyme catalyze the same reactions, but in the opposite direction, of the plastidial glycolysis and Calvin cycle. Plastidial glycolytic GAPDH (GAPCp) converts glyceraldehyde-3-phosphate (GAP) into 1,3-bisphosphoglycerate (1,3-bisPGA), which is then converted into 3-phosphoglycerate (3-PGA) by plastidial PGK (PGKp). On the other hand, assimilation of CO₂ during photosynthesis leads to 3-PGA production, which is then converted into triose phosphates by the sequential reactions of the photosynthetic isoforms of PGK and GAPDH (GAPA/GAPB). 3-PGA production through glycolysis would then be a wasteful process in chloroplasts at least during the day. For this reason, it was postulated that, in chloroplasts, glycolysis would occur in the dark when the Calvin cycle is not operative.

Lack of GAPCp activity impairs primary root growth and microspore development,^5,6 but also reduces growth of the aerial part (AP), suggesting a role of the enzyme in this organ. To study the function of GAPCp in the aerial part, we specifically expressed the enzyme in photosynthetic cells of a GAPCp double mutant (gapcp1gapcp2). The metabolite profile and growth parameters of these plants were similar to that of the gapcp1gapcp2 under day and night conditions, or in the presence of high concentrations of sucrose which inhibits photosynthesis.⁷ We could conclude that GAPCp expression is irrelevant in photosynthetic cells under all the assayed conditions. Most importantly, since GAPCp is expressed, we could hypothesize that a post-translational mechanism could be inhibiting the activity of the enzyme in photosynthetic cells.

But GAPCp is essential in heterotrophic cells

gapcp1gapcp2 expressing GAPCp under the control of a promoter specific for heterotrophic cells, namely the promoter of the phosphate transporter PHT1.2, showed a totally different behavior to that of the mutant. In the associated study,⁷ we were able to show that (i) GAPCp activity in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and that (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism.

In plastids, 3-PGA is converted into acetyl-coA, which is used for the biosynthesis of fatty acids. It is also the precursor of serine in the phosphorylated pathway of serine biosynthesis (PPSB) and of other amino acids, as well as the precursor for the synthesis of 6-carbon compounds in the Calvin cycle. It has been assumed that 3-PGA is in equilibrium between the cytosol and plastids through highly selective transporters present in the inner plastid membrane^8,9 (Fig.1). Although this may be true for photosynthetic cells, we postulate that heterotrophic cells rely only on plastidial glycolysis for their 3-PGA supply. The fact that GAPCp expression in photosynthetic cells of gapcp1gapcp2 did not complement the growth arrest of the aerial parts of the mutant plants led us to suggest that it is the lack of GAPCp activity in epidermal cells what restricts leaf growth. GAPCp would then play the same role in heterotrophic leaf cells as in roots, that is, producing the precursors for anabolic processes needed for organ growth and development.

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Figure 1.

Proposed model for metabolic pathways interacting with GAPCp activity. Lack of GAPCp activity reduces the 3-PGA precursor for the PPSB which provides Ser to specific non-photosynthetic cells but also 2-oxoglutarate required for ammonium assimilation through the GS/GOGAT pathway and/for anaplerotic reactions into the TCA cycle. The reduced activity of the PPSB would affect the GS/GOGAT pathway which would increase glutamine and would divert L-glutamate to the biosynthesis of GABA. This amino acid could play a role in balancing carbon and nitrogen metabolism in gapcp1gapcp2 by contributing to anaplerotic flux of glutamine-derived carbon into TCA cycle when PPSB is restricted. The enzymes participating in each biosynthetic pathway are as follows: Phosphorylated pathway (PPSB): PGDH, 3-phosphoglycerate dehydrogenase; PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Ammonium assimilation pathway (GS/GOGAT): GS, glutamine synthetase, GOGAT, glutamate synthase. Other enzymes: GAD, glutamate decarboxylase; GAPC, glyceraldehyde-3-phosphate dehydrogenase; ENO, enolase; npGAPC, non-phosphorylating GAPC; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; p at the end of the enzyme name stands for plastidial. Translocators: GPT, glucose phosphate/P_i translocator; PPT, phosphoenolpyruvate/P_i translocator; TPT, triose phosphate/P_i translocator. Abbreviations used for metabolites: DHAP, dihydroxyacetone phosphate; Fru-1,6P₂, fructose 1,6 bisphosphate; Fru-6P, fructose 6 phosphate; GABA, gamma-aminobutyric acid; GAP, glyceraldehyde-3-phosphate; Glc-6P, glucose 6 phosphate; PEP, phosphoenolpyruvate; 1,3-bisPGA, 1,3-bisphosphoglycerate; 2-PGA, 2-phosphoglycerate; 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; 3-PGA, 3-phosphoglycerate.

We demonstrated that the main sink of 3-PGA in heterotrophic tissues would be the PPSB since serine supplementation is able to complement most of the phenotypic alterations of gapcp1gapcp2.⁶ In mammals, serine has been shown to be required for cell division and growth, and to play an important role in the control of cell proliferation in cancer cells.^10,11 The connection between GAPCp and the PPSB could give clues about the possible mechanism involved in the growth inhibition of gapcp1gapcp2. In addition to synthesising serine, the PPSB provides 2-oxoglutarate in a reaction catalyzed by the second enzyme of the pathway, phosphoserine aminotransferase (PSAT), which uses L–glutamate as co-substrate (Fig.1). The high glutamine levels found in gapcp1gapcp2 may be a direct consequence of arrested PPSB activity, since the second enzyme of the NH₄⁺ assimilation pathway, the glutamate synthase (GOGAT), uses 2-oxoglutarate as co-substrate along with glutamine, to produce L-glutamate. Therefore, if 2-oxoglutarate supply is restricted, GOGAT activity would be inhibited and glutamine would accumulate. Possemato etal. (2011)¹¹ found that the PPSB could play an important role in anaplerosis of glutamine-derived carbon into the tricarboxylic acid (TCA) cycle in mammalian cells. Proliferating cells use the TCA cycle intermediates, such as 2-oxoglutarate, as biosynthetic precursors, and up-regulate anaplerotic reactions that drive glutamine-derived carbon units into the TCA cycle, counterbalancing biosynthetic efflux. Thus, if the PPSB is blocked, TCA cycle intermediates would decrease and cell proliferation would stop. In this respect, Possemato etal. (2011)¹¹ also found that suppression of the PPSB inhibits cell proliferation, even in the presence of externally added serine, which would corroborate the important role of the pathway in supplying TCA intermediates.

Although serine supplementation restored aerial part growth of gapcp1gapcp2, this supplementation did not completely restore the mutant metabolite profile and primary root growth. Besides, externally supplied serine was also unable to complement gapcp1gapcp2 male sterile phenotype,¹² suggesting that, at least in heterotrophic cells, the PPSB control of plant growth and development could occur through the supply of both serine and 2-oxoglutarate. Accordingly, we postulated a metabolic model in which lack of GAPCp1 activity reduced the PPSB flux that provides serine to some specific cells located in heterotrophic tissues, but also supplies 2-oxoglutarate required for the GS/GOGAT pathway and for anaplerotic reactions of TCA (Fig.1). Diminished GOGAT activity would increase glutamine, and L-glutamate would be diverted to GABA biosynthesis. This metabolite could balance the carbon and nitrogen metabolism in gapcp1gapcp2 by contributing to anaplerotic flux of glutamine-derived carbon into the TCA cycle when the PPSB is restricted.

Glycolysis is a central metabolic pathway that provides energy and generates precursors for the synthesis of primary metabolites to plants. Both plastidial and cytosolic glycolysis must be finely coordinated to provide flexibility to plant development and acclimation to environmental stresses. This coordination is probably performed in a cell-specific manner. Although information about the function of different glycolytic enzymes and pathways in specific cell types is still lacking, our data indicate that there could be a glycolysis “a la carte” for each individual plant cell type.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work has been funded by the Spanish Government and the European Union (Fondo Europeo de Desarrollo Regional grant no. BFU2012–31519, Formación de Personal Investigador fellowship to S.R.-T), by the Valencian Regional Government (grant nos. PROMETEOII/2014/052), and by the University of Valencia (Atracció de Talent fellowship to M.F.-T).

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