Opinion
Serine in plants: biosynthesis, metabolism, and functions Roc Ros1, Jesu´s Mun˜oz-Bertomeu1*, and Stephan Krueger2 1
ERI de Biotecnologia i Biomedicina, Departament de Biologia Vegetal, Facultat de Farma`cia, Universitat de Vale`ncia, 46100 Burjassot (Valencia), Spain 2 Botanical Institute II, Cologne Biocenter, University of Cologne, D-50674 Cologne, Germany
Serine (Ser) has a fundamental role in metabolism and signaling in living organisms. In plants, the existence of different pathways of Ser biosynthesis has complicated our understanding of this amino acid homeostasis. The photorespiratory glycolate pathway has been considered to be of major importance, whereas the nonphotorespiratory phosphorylated pathway has been relatively neglected. Recent advances indicate that the phosphorylated pathway has an important function in plant metabolism and development. Plants deficient in this pathway display developmental defects in embryos, male gametophytes, and roots. We propose that the phosphorylated pathway is more important than was initially thought because it is the only Ser source for specific cell types involved in developmental events. Here, we discuss its importance as a link between metabolism and development in plants. Serine functions in animals and plants In addition to forming part of proteins and performing catalytic functions in many enzymes, L-Ser participates in the biosynthesis of several biomolecules required for cell proliferation, including amino acids, nitrogenous bases, phospholipids, and sphingolipids (Figure 1). L-Ser also has an indispensable role in several cellular processes, such as the metabolism of one-carbon (C1) units [1], or in signaling mechanisms, where it is one of the three amino acids that are phosphorylated by kinases. Recent major advances in our understanding of specific Ser functions have been achieved in mammals. L-Ser is essential for normal embryonic development, especially for brain morphogenesis [2,3]. It also has a pivotal role in controlling cell proliferation, having been implicated in cancer progression [2,4–9]. L-Ser is an allosteric activator of enzymes like pyruvate kinase M2, which is overexpressed in cancer cells [5]. It also induces metabolic remodeling in cancer cells dependent on the tumor suppressor protein p53, which led researchers to suggest the potential role of L-Ser depletion in the treatment of p53-deficient tumors [7]. Corresponding author: Ros, R. ([email protected]). Keywords: serine biosynthesis; plants; phosphorylated serine biosynthesis pathway. * Current address: Instituto de Biologı´a Molecular y Celular de Plantas (IBMCP), UPVCSIC, 46022 Valencia, Spain 1360-1385/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.06.003
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In plants, deficiencies in molecules derived from L-Ser have drastic consequences. For instance, deficiency in phosphatidylserine, a relatively minor plant cell lipid, leads to alterations in microspore development and to a high embryo abortion rate in Arabidopsis (Arabidopsis thaliana) [10]. Mutants lacking Ser palmitoyltransferase, the enzyme participating in the first committed sphingolipid biosynthesis step by condensation of L-Ser with palmitoyl-CoA, display embryo and male gametophyte lethality [11,12]. Adult plants with reduced sphingolipid content present altered mineral ion homeostasis and are unable to survive [12,13]. L-Ser is also crucial for the regulation of methyl group transfer by providing tetrahydrofolate metabolism with C1 units [14,15]. Folate metabolism has proven essential for embryogenesis, post-embryonic root development, and photorespiration [16,17]. Finally, some evidence suggests that L-Ser is involved in the plant response to biotic and abiotic stresses [18–20]. In mammals and plants, additional non-metabolic functions for Ser have been postulated. L-Ser is the precursor of D-Ser, a well-known neuromodulator [2,21]. In plants, DSer has been assigned a signaling role between the male gametophyte and pistil communication, similar to that observed in animal nervous systems [22]. Arabidopsis knockout mutants for Ser racemase, the enzyme that converts L-Ser into D-Ser, display decreased glutamate receptor-like activity in pistils, affecting Ca2+ influx across the plasma membrane and, in turn, pollen tube growth and morphogenesis [22]. In most organisms, L-Ser is primarily synthesized by the so-called ‘phosphorylated pathway’. In plants, L-Ser biosynthesis proceeds by different pathways (Figure 1), one that is associated with photorespiration, the glycolate pathway [23–27] and two nonphotorespiratory pathways, the phosphorylated and the glycerate pathways [28]. Given its fundamental role in plant metabolism and development, Ser homeostasis is expected to be strictly regulated. However, the coexistence of several L-Ser biosynthetic pathways complicates the understanding of the regulation of this essential process. L-Ser production through the glycolate pathway has been considered the most important [24,26]. No relevant information about the biological significance of the nonphotorespiratory pathways was available until recently [18,29,30], probably because they were considered of minor importance. In this review, we focus on recent findings on the biological function of the phosphorylated pathway of L-Ser biosynthesis (PPSB).
Opinion
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Plasdial glycolysis Starch Glc-6P Fru-6P Fru-1,6P2 GAP
DHAP
NAD+ GAPCp NADH
NAD+ NADH
Phosphorylated pathway
Glycerate pathway
Glycolate pathway
Calvin cycle
Cytosolic glycolysis
Photorespiraon
1,3-BisPGAP ADP
ADP PGKp ATP
ATP
PGDH
NADH PEP
H2O
PGAP
PSAT 2-oxoglutarate Anapleroc reacons
NADH
THF NAD+
SHMT
GDH
NADH 3-PS
H2O
CO2
Glycerate NAD+
L-glutamate
Isocitrate
GDC
Pi 3-PHP
Pyruvate
OAA
2-Glycine NH3
NAD+
2-PGA
Citrate
3-PGA
3-PGA
3-PGA
Hydroxypyruvate
5,10-CH2THF
Alanine
PSP
Pi
NADH
AH-AT Pyruvate
2-oxoglutarate NADH
Serine pool
Malate NADH Fumarate
FADH2
Suc-CoA Proteins
Succinate
One-carbon metabolism
L-glutamate
TCA cycle
Phospholipids sphingolipids
SAM
Proline Purines Pyrimidines
X Methylaon reacons X-CH3 SAH
Amino acids
Ornine
Glycine Tryptophan Methionine
Auxin
Homocysteine TRENDS in Plant Science
Figure 1. Schematic representation of serine (Ser) biosynthesis in plants and connecting metabolic pathways. The enzymes participating in each pathway are as follows: phosphorylated pathway: PGDH, 3-phosphoglycerate dehydrogenase; PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Glycerate pathway: PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, alanine-hydroxypyruvate aminotransferase. Photorespiratory pathway (glycolate pathway): GDC, glycine decarboxylase; SHMT, serine hydroxymethyltransferase. Plastidial glycolysis: emphasis is given to the reactions catalyzed by the glyceraldehyde-3phosphate dehydrogenase (GAPCp) and phosphoglycerate kinase (PGKp). Broken lines indicate several enzymatic reactions. Abbreviations: DHAP, dihydroxyacetone phosphate; Fru-1,6P2, fructose-1,6-bisphosphate; Fru-6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; Glc-6P, glucose-6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; SAH, S-adenosylhomocysteine; SAM, S-adenosymethionine; Suc-CoA, succinil Coenzyme A; TCA, tricarboxylic acid; THF, tetrahydrofolate; 1,3BisPGAP, 1,3-bisphosphoglycerate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; 5,10-CH2-THF, 5,10-methylene-tetrahydrofolate.
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The L-Ser biosynthetic pathways in plants: why several pathways? Some of the most important reactions in photorespiration take place within the mitochondria matrix and lead to the biosynthesis of L-Ser through the glycolate pathway (Figure 1). In these reactions, one glycine molecule is decarboxylated and deaminated by the glycine decarboxylase complex (GDC), with the formation of CO2 and NH3 and the concomitant reduction of NAD+ to NADH [24] (Figure 1). The remaining methylene carbon of glycine is transferred to tetrahydrofolate (THF) to form methyleneTHF, which reacts with a second glycine to form L-Ser in a reaction catalyzed by Ser hydroxymethyltransferase (SHMT). The glycerate pathway, which synthesizes L-Ser by dephosphorylation of 3-phosphoglyceric acid (3-PGA) [28], includes the reverse sequence of part of the photorespiratory cycle, from 3-PGA to L-Ser (3-PGA-glycerate-hydroxypyruvate-Ser) (Figure 1). These reactions are catalyzed by enzymes, such as 3-PGA phosphatase (PGAP; catalyzing the conversion of 3-PGA to glycerate), glycerate dehydrogenase (GDH, catalyzing the production of hydroxypyruvate from glycerate), alanine-hydroxypyruvate aminotransferase and glycine hydroxypyruvate aminotransferase (AHAT and GH-AT catalyzing the last conversion of hydroxypyruvate into L-Ser) acting either in the cytosol (i.e., PGAP) or peroxisomes (i.e., GDH, AH-AT, and GH-AT). The activity of the glycerate pathway enzymes in plants has been demonstrated [28] and some of the genes encoding these enzymes have been cloned [31]. However, the functional significance of this pathway remains unknown. PPSB is conserved in animals, plants, and bacteria [19,32]. Plant PPSB synthesizes L-Ser from 3-PGA in the plastids, where it defines a branching point for 3PGA from plastidial glycolysis (Figure 1). It comprises three sequential reactions catalyzed by 3-PGA dehydrogenase (PGDH, EC 1.1.1.95), 3-phosphoserine aminotransferase (PSAT, EC 2.6.1.52), and 3-phosphoserine phosphatase (PSP, EC 3.1.3.3; Figure 1). The precursor
3-PGA is oxidized by PGDH utilizing NAD+ as cofactor to form 3-phosphohydroxypyruvate (3-PHP), which is converted to 3-phosphoserine by PSAT in a transamination reaction using L-glutamate as an amino donor and liberating 2-oxoglutarate. The last step is the conversion of 3phosphoserine into L-Ser in a reaction catalyzed by PSP. Biochemical evidence to support the activity of PPSB enzymes in plants emerged during the 1960–1970s [33– 35]. However, genetic and physiological evidence has been lacking until recently, when the functional characterization of the first and last enzymes of the pathway was achieved [18,29,30]. In the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/), three genes are described to encode putative PGDHs (At1g17745, PGDH; At3g19480, 3-PGDH; and At4g34200, EDA9), two encoding PSAT (At4g35630, PSAT1, and At2g17630, PSAT2) and one encoding PSP (At1g18640). During the 1990s, one PGDH (At1g17745) one PSAT (At4g35630), and the PSP genes were cloned and the encoded proteins demonstrated to have activity in vitro [19,36–38]. Recently, two additional PGDH isoforms (At3g19480 and At4g34200) were also demonstrated to have in vitro enzyme activity [18]. All three PGDH isoforms characterized show a high percentage of identity at the amino acid level, and phylogenetic analysis indicates that they are probably the only members of the PGDH family in Arabidopsis [30]. Therefore, we propose renaming them as PGDH1 (At4g34200, formerly known as EDA9), PGDH2, (At1g17745, formerly known as PGDH), and PGDH3 (At3g19480, formerly known 3-PGDH) (Table 1). In some organisms, PGDH activity is controlled by L-Ser feedback inhibition. In plants, only PGDH1 and PGDH3 are sensitive to L-Ser inhibition, whereas PGDH2 is insensitive. The reason for the different regulation of the PGDH enzymes in plants is not known. However, it might be that, in plants, PPSB functions not only in the biosynthesis of L-Ser, but also in the provision of 2-oxoglutarate, as recently described for human cells [9]. In such a scenario, the PGDH2 enzyme would guarantee a flux through the PPSB pathway even under high Ser concentrations.
Table 1. The PSBP in Arabidopsis thaliana: nomenclature, mutants, and phenotypes Locus
Gene name
At4g34200
Proposed PGDH1
Other EDA9
Mutant name
At1g17745
PGDH2
PGDH
At3g19480
PGDH3
3-PGDH
At4g35630 At2g17630 At1g18640
PSAT1 PSAT2 PSP1
-
Proposed pgdh1-1 pgdh1-2 pgdh1-3 pgdh1-4 pgdh2-1 pgdh2-2 pgdh2-3 pgdh3-1 pgdh3-2 psp1-1 psp1-2
a
Refers to pgdh1-2 mutant.
b
Refers to eda9.1 mutant.
c
Refers to pgdh2-2 mutant.
d
Refers to pgdh2 mutant.
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Other eda9.1 eda9.2 eda9 pgdh2 pgdh1 3-pgdh1 3-pgdh2 psp-1 psp1.1 psp-2
Line SAIL_209G08 GK_155B09 GK_867A04 EDA9 SALK_069543 SALK_149747 SALK_048256 SM_337584 GK_877F12 SALK_062391 SALK_062391 SAIL_658A06
Mutant phenotype
Refs
Embryo lethal Embryo lethal Non-obvious Embryo lethal Non-obvious Non-obvious Non-obvious Non-obvious Non-obvious Embryo lethal Embryo lethal Embryo lethal
[18] [18a,30] b [30] [48] [18] [18c,30] d [30] [30] [30]
[18] [29] [30]
Opinion Given the magnitude of photorespiration (rates of glycine metabolism by isolated leaf mitochondria can exceed 1200 nmol glycine converted to L-Ser per mg of protein per min) [24], L-Ser production through the glycolate pathway has been considered the most important, at least in photosynthetic organs [24,26]. Moreover, L-Ser is easily transported through the phloem [39–41], which would imply that L-Ser synthesized through photorespiration in photosynthetic cells could be supplied to nonphotosynthetic organs. So, why do plants need other L-Ser biosynthetic pathways? One possible explanation is that the activity of the glycolate pathway is restricted to daylight hours, suggesting that alternative L-Ser biosynthetic pathways are required at least during dark periods. A second nonexclusive explanation implies that the L-Ser produced by the glycolate pathway does not reach all plant cell types, either because some cells are devoid of L-Ser transporters or others are not conveniently connected to the vasculature, or indeed a combination of both these hypotheses. PPSB may be important in both photosynthetic and nonphotosynthetic organs PPSB genes are expressed in the shoot and root-apical meristem, vasculature, embryos, anthers, stigma, and pollen grains [18,29,30]. Most of the cells in these tissues lack functional chloroplasts and, therefore, the ability to synthesize L-Ser by the glycolate pathway. According to the hypothesis stated above, this may indicate that PPSB is the only source of L-Ser for these cells. PPSB genes are also expressed in photosynthetic organs, where their expression is regulated by light–dark transitions. For instance, PGDH1 and PGDH3 are induced by 8-h darkness exposure [30]. PGDH1 expression is also upregulated in plants grown at high CO2 concentrations and the L-Ser content is reduced in PGDH1-silenced plants after transfer into a high CO2 atmosphere [18]. These results suggest that, in photosynthetic organs, PPSB is more relevant at night and under conditions when the photorespiratory pathway does not function, than during the daylight hours. However, in nonphotosynthetic organs, such as roots, PGDH family gene expression occurs similarly under light and dark conditions, which would indicate that, in these organs, PPSB is equally important during both day and night. The tight transcriptional control of PGDH family genes at the organ level contrasts with that of PSP1, which is expressed similarly in all organs studied [29,30]. This may be due to the presence of a unique gene compared with the three genes in the PGDH family, but may also suggest that the bottleneck regulating the pathway at transcriptional level is the first reaction of the pathway. Regulating the PGDH activity might be also a checkpoint, because the equilibrium of the reaction catalyzed by the PGDH enzyme lies far on the side of the substrate 3-PGA and represents the rate-limiting step in the PPSB pathway. PPSB is essential for embryo, pollen, and postembryonic root development The first indirect genetic evidence of the biological significance of PPSB in plants came from the phenotypic characterization of mutants of the plastidial glyceraldehyde
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3-phosphate dehydrogenase (GAPCp) from Arabidopsis [42–44]. gapcp1gapcp2 double mutants show a drastic developmental phenotype, including arrested primary root growth and defects in pollen development. It was shown that gapcp1gapcp2 mutant roots displayed a L-Ser deficiency and that L-Ser supplementation to the growing medium recovered the root growth in the mutants [44]. These results suggested that developmental defects observed in gapcp1gapcp2 mutants are caused by the absence of precursor 3-PGA for PPSB supplied by GAPCp activity in plastids (Figure 1). This hypothesis was later confirmed by the molecular and genetic characterization of PPSB genes, indicating that the pathway is essential for embryo, male gametophyte, and postembryonic root development in Arabidopsis [18,29,30]. In psp1 and pgdh1 homozygous mutants, embryo development is arrested at early stages [18,29,30]. Reduction in PSP1 and PGDH1 activity also inhibits postembryonic root growth and leads to an arrest of microspore development at the polarized stage, rendering pollen unviable [18,29,30]. When plants with reduced PGDH activity were grown at high CO2 conditions to prevent photorespiratory L-Ser biosynthesis, leaf initiation was completely inhibited [18], indicating that PPSB is also important for proper shoot apical meristem function under nonphotorespiratory conditions. The root developmental arrest observed in PSP1- and PGDH1-deficient lines may relate to a defect in auxin biosynthesis. The reduced auxin levels found in PGDH1-silenced lines [18] might be achieved through impairment of the biosynthesis of the auxin precursor tryptophan that, in turn, is synthesized by condensation of L-Ser and indole [45,46] (Figure 1). Accordingly, expression of the PPSB genes PGDH1 and PSAT1 is upregulated by MYB51 and MYB34, two transcriptional activators of tryptophan biosynthesis. However, the short root phenotype of PGDH1-silenced lines was not rescued by tryptophan supplementation to the growing medium [18]. PPSB deficiency may also affect C1 metabolism because L-Ser serves as the major intracellular source of C1 tetrahydrofolate adducts (Figure 1). Mutants that are deficient in tetrahydrofolate metabolism display defects in root and embryo development [16,17]. However, neither was the root growth of conditional psp1 mutants rescued by 5formyl-tetrahydrofolate supplementation to the growing medium [47]. Thus, the essential role of L-Ser in root development cannot be ascribed, at least solely, to tetrahydrofolate or auxin metabolism, but might well be due to several, or a combination of several, processes in which LSer is involved. PPSB may connect metabolism, development, and plant responses to environmental stresses Modifications of PPSB expression in Arabidopsis mutants and overexpressing lines significantly altered glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid biosynthesis [29,30], indicating the central role of this pathway in plant primary metabolism. Possemato et al. found that human PPSB, in addition to L-Ser production, may have a key role in anaplerosis of glutamine-derived carbon into the TCA cycle [9]. Specifically, the pathway provides aketoglutarate in the reaction catalyzed by PSAT (Figure 1). 567
Opinion Proliferating cells use TCA-cycle intermediates, such as 2-oxoglutarate, as biosynthetic precursors, and upregulate the anaplerotic reactions that drive glutamine-derived carbon into the TCA cycle to counterbalance biosynthetic efflux. Possemato et al. found that PPSB suppression inhibited cell proliferation even in the presence of externally added L-Ser, which may corroborate the important role of the pathway in supplying TCA intermediaries [9]. A similar mechanism might act in actively dividing plant cells, such as embryos, anthers, or root meristems. If this idea is correct, it opens up the possibility of a conserved signaling mechanism between animals and plants. PPSB has been postulated to have an important role in plants under environmental stresses [19]. L-Ser accumulation in plants grown under low temperature and elevated salinity conditions has been reported ([19] and references therein). Analysis of the promoter region of the Arabidopsis PSP1 and PGDH1 genes showed significant enrichment in sequences present in genes responding to abiotic stresses [29,30]. Hence, overexpression of a PGDH from the cyanobacterium Aphanothece halophytica into Arabidopsis plants enhanced their salt and cold tolerance [20]. By contrast, a function of PPSB in plant–pathogen interactions was recently suggested [18]. The expression of PPSB pathway genes is induced in plants upon infection with necrotrophic pathogens such as Botrytis cinerea. Infection of plants with Botrytis induces the biosynthesis of the phytoalexin camalexin, a tryptophan-derived secondary metabolite. In addition, the content of a major indolic glucosinolate is significantly diminished in roots of plants with reduced PGDH activity. In both cases, the PPSB pathway might be essential to provide L-Ser for the synthesis of tryptophan, the common precursor for the biosynthesis of camalexin and indolic glucosinolate. Concluding remarks and outlook Recent advances in the functional characterization of LSer biosynthetic pathways indicate that L-Ser biosynthesis from the glycolate pathway and subsequent transport through the phloem does not suffice to meet the L-Ser requirements of all cells. PPSB is essential for plant metabolism and development probably because it provides LSer to specific cell types, such as the anther tapetum or meristematic cells. PPSB is a central pathway participating in carbon and nitrogen metabolism and may be one of the missing links connecting metabolism with development in plants, as has been shown in animals. Further research is needed to determine the exact contribution of each L-Ser biosynthetic pathway in different species and metabolisms, for instance, in C4 plants where, due to the low photorespiratory rates, the glycolate pathway can be restricted and the function of the PPSB may be even more important than in C3 plants, or under different environmental conditions. The coordination and connecting links between the different pathways also require further investigation. Specifically, how photorespiration and PPSB interact to regulate L-Ser homeostasis in plants, how the changing environmental conditions will affect these interactions, and the exact consequences for plants, are issues that need answering in the near future. 568
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Acknowledgments This work has been funded by the Spanish Government and the European Union: FEDER/BFU2012-31519, JdlC to J.M-B, and the Valencian Regional Government: PROMETEO/2009/075 and the Deutsche Forschungsgemeinschaft (Grant Kr4245/1-1).
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Serine in plants: biosynthesis, metabolism, and functions Roc Ros1, Jesu´s Mun˜oz-Bertomeu1*, and Stephan Krueger2 1
ERI de Biotecnologia i Biomedicina, Departament de Biologia Vegetal, Facultat de Farma`cia, Universitat de Vale`ncia, 46100 Burjassot (Valencia), Spain 2 Botanical Institute II, Cologne Biocenter, University of Cologne, D-50674 Cologne, Germany
Serine (Ser) has a fundamental role in metabolism and signaling in living organisms. In plants, the existence of different pathways of Ser biosynthesis has complicated our understanding of this amino acid homeostasis. The photorespiratory glycolate pathway has been considered to be of major importance, whereas the nonphotorespiratory phosphorylated pathway has been relatively neglected. Recent advances indicate that the phosphorylated pathway has an important function in plant metabolism and development. Plants deficient in this pathway display developmental defects in embryos, male gametophytes, and roots. We propose that the phosphorylated pathway is more important than was initially thought because it is the only Ser source for specific cell types involved in developmental events. Here, we discuss its importance as a link between metabolism and development in plants. Serine functions in animals and plants In addition to forming part of proteins and performing catalytic functions in many enzymes, L-Ser participates in the biosynthesis of several biomolecules required for cell proliferation, including amino acids, nitrogenous bases, phospholipids, and sphingolipids (Figure 1). L-Ser also has an indispensable role in several cellular processes, such as the metabolism of one-carbon (C1) units [1], or in signaling mechanisms, where it is one of the three amino acids that are phosphorylated by kinases. Recent major advances in our understanding of specific Ser functions have been achieved in mammals. L-Ser is essential for normal embryonic development, especially for brain morphogenesis [2,3]. It also has a pivotal role in controlling cell proliferation, having been implicated in cancer progression [2,4–9]. L-Ser is an allosteric activator of enzymes like pyruvate kinase M2, which is overexpressed in cancer cells [5]. It also induces metabolic remodeling in cancer cells dependent on the tumor suppressor protein p53, which led researchers to suggest the potential role of L-Ser depletion in the treatment of p53-deficient tumors [7]. Corresponding author: Ros, R. ([email protected]). Keywords: serine biosynthesis; plants; phosphorylated serine biosynthesis pathway. * Current address: Instituto de Biologı´a Molecular y Celular de Plantas (IBMCP), UPVCSIC, 46022 Valencia, Spain 1360-1385/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.06.003
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In plants, deficiencies in molecules derived from L-Ser have drastic consequences. For instance, deficiency in phosphatidylserine, a relatively minor plant cell lipid, leads to alterations in microspore development and to a high embryo abortion rate in Arabidopsis (Arabidopsis thaliana) [10]. Mutants lacking Ser palmitoyltransferase, the enzyme participating in the first committed sphingolipid biosynthesis step by condensation of L-Ser with palmitoyl-CoA, display embryo and male gametophyte lethality [11,12]. Adult plants with reduced sphingolipid content present altered mineral ion homeostasis and are unable to survive [12,13]. L-Ser is also crucial for the regulation of methyl group transfer by providing tetrahydrofolate metabolism with C1 units [14,15]. Folate metabolism has proven essential for embryogenesis, post-embryonic root development, and photorespiration [16,17]. Finally, some evidence suggests that L-Ser is involved in the plant response to biotic and abiotic stresses [18–20]. In mammals and plants, additional non-metabolic functions for Ser have been postulated. L-Ser is the precursor of D-Ser, a well-known neuromodulator [2,21]. In plants, DSer has been assigned a signaling role between the male gametophyte and pistil communication, similar to that observed in animal nervous systems [22]. Arabidopsis knockout mutants for Ser racemase, the enzyme that converts L-Ser into D-Ser, display decreased glutamate receptor-like activity in pistils, affecting Ca2+ influx across the plasma membrane and, in turn, pollen tube growth and morphogenesis [22]. In most organisms, L-Ser is primarily synthesized by the so-called ‘phosphorylated pathway’. In plants, L-Ser biosynthesis proceeds by different pathways (Figure 1), one that is associated with photorespiration, the glycolate pathway [23–27] and two nonphotorespiratory pathways, the phosphorylated and the glycerate pathways [28]. Given its fundamental role in plant metabolism and development, Ser homeostasis is expected to be strictly regulated. However, the coexistence of several L-Ser biosynthetic pathways complicates the understanding of the regulation of this essential process. L-Ser production through the glycolate pathway has been considered the most important [24,26]. No relevant information about the biological significance of the nonphotorespiratory pathways was available until recently [18,29,30], probably because they were considered of minor importance. In this review, we focus on recent findings on the biological function of the phosphorylated pathway of L-Ser biosynthesis (PPSB).
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Plasdial glycolysis Starch Glc-6P Fru-6P Fru-1,6P2 GAP
DHAP
NAD+ GAPCp NADH
NAD+ NADH
Phosphorylated pathway
Glycerate pathway
Glycolate pathway
Calvin cycle
Cytosolic glycolysis
Photorespiraon
1,3-BisPGAP ADP
ADP PGKp ATP
ATP
PGDH
NADH PEP
H2O
PGAP
PSAT 2-oxoglutarate Anapleroc reacons
NADH
THF NAD+
SHMT
GDH
NADH 3-PS
H2O
CO2
Glycerate NAD+
L-glutamate
Isocitrate
GDC
Pi 3-PHP
Pyruvate
OAA
2-Glycine NH3
NAD+
2-PGA
Citrate
3-PGA
3-PGA
3-PGA
Hydroxypyruvate
5,10-CH2THF
Alanine
PSP
Pi
NADH
AH-AT Pyruvate
2-oxoglutarate NADH
Serine pool
Malate NADH Fumarate
FADH2
Suc-CoA Proteins
Succinate
One-carbon metabolism
L-glutamate
TCA cycle
Phospholipids sphingolipids
SAM
Proline Purines Pyrimidines
X Methylaon reacons X-CH3 SAH
Amino acids
Ornine
Glycine Tryptophan Methionine
Auxin
Homocysteine TRENDS in Plant Science
Figure 1. Schematic representation of serine (Ser) biosynthesis in plants and connecting metabolic pathways. The enzymes participating in each pathway are as follows: phosphorylated pathway: PGDH, 3-phosphoglycerate dehydrogenase; PSAT, 3-phosphoserine aminotransferase; PSP, 3-phosphoserine phosphatase. Glycerate pathway: PGAP, 3-phosphoglycerate phosphatase; GDH, glycerate dehydrogenase; AH-AT, alanine-hydroxypyruvate aminotransferase. Photorespiratory pathway (glycolate pathway): GDC, glycine decarboxylase; SHMT, serine hydroxymethyltransferase. Plastidial glycolysis: emphasis is given to the reactions catalyzed by the glyceraldehyde-3phosphate dehydrogenase (GAPCp) and phosphoglycerate kinase (PGKp). Broken lines indicate several enzymatic reactions. Abbreviations: DHAP, dihydroxyacetone phosphate; Fru-1,6P2, fructose-1,6-bisphosphate; Fru-6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; Glc-6P, glucose-6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; SAH, S-adenosylhomocysteine; SAM, S-adenosymethionine; Suc-CoA, succinil Coenzyme A; TCA, tricarboxylic acid; THF, tetrahydrofolate; 1,3BisPGAP, 1,3-bisphosphoglycerate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; 3-PHP, 3-phosphohydroxypyruvate; 3-PS, 3-phosphoserine; 5,10-CH2-THF, 5,10-methylene-tetrahydrofolate.
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The L-Ser biosynthetic pathways in plants: why several pathways? Some of the most important reactions in photorespiration take place within the mitochondria matrix and lead to the biosynthesis of L-Ser through the glycolate pathway (Figure 1). In these reactions, one glycine molecule is decarboxylated and deaminated by the glycine decarboxylase complex (GDC), with the formation of CO2 and NH3 and the concomitant reduction of NAD+ to NADH [24] (Figure 1). The remaining methylene carbon of glycine is transferred to tetrahydrofolate (THF) to form methyleneTHF, which reacts with a second glycine to form L-Ser in a reaction catalyzed by Ser hydroxymethyltransferase (SHMT). The glycerate pathway, which synthesizes L-Ser by dephosphorylation of 3-phosphoglyceric acid (3-PGA) [28], includes the reverse sequence of part of the photorespiratory cycle, from 3-PGA to L-Ser (3-PGA-glycerate-hydroxypyruvate-Ser) (Figure 1). These reactions are catalyzed by enzymes, such as 3-PGA phosphatase (PGAP; catalyzing the conversion of 3-PGA to glycerate), glycerate dehydrogenase (GDH, catalyzing the production of hydroxypyruvate from glycerate), alanine-hydroxypyruvate aminotransferase and glycine hydroxypyruvate aminotransferase (AHAT and GH-AT catalyzing the last conversion of hydroxypyruvate into L-Ser) acting either in the cytosol (i.e., PGAP) or peroxisomes (i.e., GDH, AH-AT, and GH-AT). The activity of the glycerate pathway enzymes in plants has been demonstrated [28] and some of the genes encoding these enzymes have been cloned [31]. However, the functional significance of this pathway remains unknown. PPSB is conserved in animals, plants, and bacteria [19,32]. Plant PPSB synthesizes L-Ser from 3-PGA in the plastids, where it defines a branching point for 3PGA from plastidial glycolysis (Figure 1). It comprises three sequential reactions catalyzed by 3-PGA dehydrogenase (PGDH, EC 1.1.1.95), 3-phosphoserine aminotransferase (PSAT, EC 2.6.1.52), and 3-phosphoserine phosphatase (PSP, EC 3.1.3.3; Figure 1). The precursor
3-PGA is oxidized by PGDH utilizing NAD+ as cofactor to form 3-phosphohydroxypyruvate (3-PHP), which is converted to 3-phosphoserine by PSAT in a transamination reaction using L-glutamate as an amino donor and liberating 2-oxoglutarate. The last step is the conversion of 3phosphoserine into L-Ser in a reaction catalyzed by PSP. Biochemical evidence to support the activity of PPSB enzymes in plants emerged during the 1960–1970s [33– 35]. However, genetic and physiological evidence has been lacking until recently, when the functional characterization of the first and last enzymes of the pathway was achieved [18,29,30]. In the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/), three genes are described to encode putative PGDHs (At1g17745, PGDH; At3g19480, 3-PGDH; and At4g34200, EDA9), two encoding PSAT (At4g35630, PSAT1, and At2g17630, PSAT2) and one encoding PSP (At1g18640). During the 1990s, one PGDH (At1g17745) one PSAT (At4g35630), and the PSP genes were cloned and the encoded proteins demonstrated to have activity in vitro [19,36–38]. Recently, two additional PGDH isoforms (At3g19480 and At4g34200) were also demonstrated to have in vitro enzyme activity [18]. All three PGDH isoforms characterized show a high percentage of identity at the amino acid level, and phylogenetic analysis indicates that they are probably the only members of the PGDH family in Arabidopsis [30]. Therefore, we propose renaming them as PGDH1 (At4g34200, formerly known as EDA9), PGDH2, (At1g17745, formerly known as PGDH), and PGDH3 (At3g19480, formerly known 3-PGDH) (Table 1). In some organisms, PGDH activity is controlled by L-Ser feedback inhibition. In plants, only PGDH1 and PGDH3 are sensitive to L-Ser inhibition, whereas PGDH2 is insensitive. The reason for the different regulation of the PGDH enzymes in plants is not known. However, it might be that, in plants, PPSB functions not only in the biosynthesis of L-Ser, but also in the provision of 2-oxoglutarate, as recently described for human cells [9]. In such a scenario, the PGDH2 enzyme would guarantee a flux through the PPSB pathway even under high Ser concentrations.
Table 1. The PSBP in Arabidopsis thaliana: nomenclature, mutants, and phenotypes Locus
Gene name
At4g34200
Proposed PGDH1
Other EDA9
Mutant name
At1g17745
PGDH2
PGDH
At3g19480
PGDH3
3-PGDH
At4g35630 At2g17630 At1g18640
PSAT1 PSAT2 PSP1
-
Proposed pgdh1-1 pgdh1-2 pgdh1-3 pgdh1-4 pgdh2-1 pgdh2-2 pgdh2-3 pgdh3-1 pgdh3-2 psp1-1 psp1-2
a
Refers to pgdh1-2 mutant.
b
Refers to eda9.1 mutant.
c
Refers to pgdh2-2 mutant.
d
Refers to pgdh2 mutant.
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Other eda9.1 eda9.2 eda9 pgdh2 pgdh1 3-pgdh1 3-pgdh2 psp-1 psp1.1 psp-2
Line SAIL_209G08 GK_155B09 GK_867A04 EDA9 SALK_069543 SALK_149747 SALK_048256 SM_337584 GK_877F12 SALK_062391 SALK_062391 SAIL_658A06
Mutant phenotype
Refs
Embryo lethal Embryo lethal Non-obvious Embryo lethal Non-obvious Non-obvious Non-obvious Non-obvious Non-obvious Embryo lethal Embryo lethal Embryo lethal
[18] [18a,30] b [30] [48] [18] [18c,30] d [30] [30] [30]
[18] [29] [30]
Opinion Given the magnitude of photorespiration (rates of glycine metabolism by isolated leaf mitochondria can exceed 1200 nmol glycine converted to L-Ser per mg of protein per min) [24], L-Ser production through the glycolate pathway has been considered the most important, at least in photosynthetic organs [24,26]. Moreover, L-Ser is easily transported through the phloem [39–41], which would imply that L-Ser synthesized through photorespiration in photosynthetic cells could be supplied to nonphotosynthetic organs. So, why do plants need other L-Ser biosynthetic pathways? One possible explanation is that the activity of the glycolate pathway is restricted to daylight hours, suggesting that alternative L-Ser biosynthetic pathways are required at least during dark periods. A second nonexclusive explanation implies that the L-Ser produced by the glycolate pathway does not reach all plant cell types, either because some cells are devoid of L-Ser transporters or others are not conveniently connected to the vasculature, or indeed a combination of both these hypotheses. PPSB may be important in both photosynthetic and nonphotosynthetic organs PPSB genes are expressed in the shoot and root-apical meristem, vasculature, embryos, anthers, stigma, and pollen grains [18,29,30]. Most of the cells in these tissues lack functional chloroplasts and, therefore, the ability to synthesize L-Ser by the glycolate pathway. According to the hypothesis stated above, this may indicate that PPSB is the only source of L-Ser for these cells. PPSB genes are also expressed in photosynthetic organs, where their expression is regulated by light–dark transitions. For instance, PGDH1 and PGDH3 are induced by 8-h darkness exposure [30]. PGDH1 expression is also upregulated in plants grown at high CO2 concentrations and the L-Ser content is reduced in PGDH1-silenced plants after transfer into a high CO2 atmosphere [18]. These results suggest that, in photosynthetic organs, PPSB is more relevant at night and under conditions when the photorespiratory pathway does not function, than during the daylight hours. However, in nonphotosynthetic organs, such as roots, PGDH family gene expression occurs similarly under light and dark conditions, which would indicate that, in these organs, PPSB is equally important during both day and night. The tight transcriptional control of PGDH family genes at the organ level contrasts with that of PSP1, which is expressed similarly in all organs studied [29,30]. This may be due to the presence of a unique gene compared with the three genes in the PGDH family, but may also suggest that the bottleneck regulating the pathway at transcriptional level is the first reaction of the pathway. Regulating the PGDH activity might be also a checkpoint, because the equilibrium of the reaction catalyzed by the PGDH enzyme lies far on the side of the substrate 3-PGA and represents the rate-limiting step in the PPSB pathway. PPSB is essential for embryo, pollen, and postembryonic root development The first indirect genetic evidence of the biological significance of PPSB in plants came from the phenotypic characterization of mutants of the plastidial glyceraldehyde
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3-phosphate dehydrogenase (GAPCp) from Arabidopsis [42–44]. gapcp1gapcp2 double mutants show a drastic developmental phenotype, including arrested primary root growth and defects in pollen development. It was shown that gapcp1gapcp2 mutant roots displayed a L-Ser deficiency and that L-Ser supplementation to the growing medium recovered the root growth in the mutants [44]. These results suggested that developmental defects observed in gapcp1gapcp2 mutants are caused by the absence of precursor 3-PGA for PPSB supplied by GAPCp activity in plastids (Figure 1). This hypothesis was later confirmed by the molecular and genetic characterization of PPSB genes, indicating that the pathway is essential for embryo, male gametophyte, and postembryonic root development in Arabidopsis [18,29,30]. In psp1 and pgdh1 homozygous mutants, embryo development is arrested at early stages [18,29,30]. Reduction in PSP1 and PGDH1 activity also inhibits postembryonic root growth and leads to an arrest of microspore development at the polarized stage, rendering pollen unviable [18,29,30]. When plants with reduced PGDH activity were grown at high CO2 conditions to prevent photorespiratory L-Ser biosynthesis, leaf initiation was completely inhibited [18], indicating that PPSB is also important for proper shoot apical meristem function under nonphotorespiratory conditions. The root developmental arrest observed in PSP1- and PGDH1-deficient lines may relate to a defect in auxin biosynthesis. The reduced auxin levels found in PGDH1-silenced lines [18] might be achieved through impairment of the biosynthesis of the auxin precursor tryptophan that, in turn, is synthesized by condensation of L-Ser and indole [45,46] (Figure 1). Accordingly, expression of the PPSB genes PGDH1 and PSAT1 is upregulated by MYB51 and MYB34, two transcriptional activators of tryptophan biosynthesis. However, the short root phenotype of PGDH1-silenced lines was not rescued by tryptophan supplementation to the growing medium [18]. PPSB deficiency may also affect C1 metabolism because L-Ser serves as the major intracellular source of C1 tetrahydrofolate adducts (Figure 1). Mutants that are deficient in tetrahydrofolate metabolism display defects in root and embryo development [16,17]. However, neither was the root growth of conditional psp1 mutants rescued by 5formyl-tetrahydrofolate supplementation to the growing medium [47]. Thus, the essential role of L-Ser in root development cannot be ascribed, at least solely, to tetrahydrofolate or auxin metabolism, but might well be due to several, or a combination of several, processes in which LSer is involved. PPSB may connect metabolism, development, and plant responses to environmental stresses Modifications of PPSB expression in Arabidopsis mutants and overexpressing lines significantly altered glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid biosynthesis [29,30], indicating the central role of this pathway in plant primary metabolism. Possemato et al. found that human PPSB, in addition to L-Ser production, may have a key role in anaplerosis of glutamine-derived carbon into the TCA cycle [9]. Specifically, the pathway provides aketoglutarate in the reaction catalyzed by PSAT (Figure 1). 567
Opinion Proliferating cells use TCA-cycle intermediates, such as 2-oxoglutarate, as biosynthetic precursors, and upregulate the anaplerotic reactions that drive glutamine-derived carbon into the TCA cycle to counterbalance biosynthetic efflux. Possemato et al. found that PPSB suppression inhibited cell proliferation even in the presence of externally added L-Ser, which may corroborate the important role of the pathway in supplying TCA intermediaries [9]. A similar mechanism might act in actively dividing plant cells, such as embryos, anthers, or root meristems. If this idea is correct, it opens up the possibility of a conserved signaling mechanism between animals and plants. PPSB has been postulated to have an important role in plants under environmental stresses [19]. L-Ser accumulation in plants grown under low temperature and elevated salinity conditions has been reported ([19] and references therein). Analysis of the promoter region of the Arabidopsis PSP1 and PGDH1 genes showed significant enrichment in sequences present in genes responding to abiotic stresses [29,30]. Hence, overexpression of a PGDH from the cyanobacterium Aphanothece halophytica into Arabidopsis plants enhanced their salt and cold tolerance [20]. By contrast, a function of PPSB in plant–pathogen interactions was recently suggested [18]. The expression of PPSB pathway genes is induced in plants upon infection with necrotrophic pathogens such as Botrytis cinerea. Infection of plants with Botrytis induces the biosynthesis of the phytoalexin camalexin, a tryptophan-derived secondary metabolite. In addition, the content of a major indolic glucosinolate is significantly diminished in roots of plants with reduced PGDH activity. In both cases, the PPSB pathway might be essential to provide L-Ser for the synthesis of tryptophan, the common precursor for the biosynthesis of camalexin and indolic glucosinolate. Concluding remarks and outlook Recent advances in the functional characterization of LSer biosynthetic pathways indicate that L-Ser biosynthesis from the glycolate pathway and subsequent transport through the phloem does not suffice to meet the L-Ser requirements of all cells. PPSB is essential for plant metabolism and development probably because it provides LSer to specific cell types, such as the anther tapetum or meristematic cells. PPSB is a central pathway participating in carbon and nitrogen metabolism and may be one of the missing links connecting metabolism with development in plants, as has been shown in animals. Further research is needed to determine the exact contribution of each L-Ser biosynthetic pathway in different species and metabolisms, for instance, in C4 plants where, due to the low photorespiratory rates, the glycolate pathway can be restricted and the function of the PPSB may be even more important than in C3 plants, or under different environmental conditions. The coordination and connecting links between the different pathways also require further investigation. Specifically, how photorespiration and PPSB interact to regulate L-Ser homeostasis in plants, how the changing environmental conditions will affect these interactions, and the exact consequences for plants, are issues that need answering in the near future. 568
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Acknowledgments This work has been funded by the Spanish Government and the European Union: FEDER/BFU2012-31519, JdlC to J.M-B, and the Valencian Regional Government: PROMETEO/2009/075 and the Deutsche Forschungsgemeinschaft (Grant Kr4245/1-1).
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