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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC–SHMT enzymatic system, which is therefore required in all plant tissues.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Glycine and serine are two amino acids that are readily interconvertible. Thus their synthesis and catabolism are intimately connected, making difficult the determination of their metabolic fate. The interconversion reaction is catalysed by the serine hydroxymethyltransferase (SHMT), an enzyme requiring tetrahydrofolate (THF) as cofactor (Schirch 1984) and present in the three compartments of the plant cell: cytosol, mitochondria and plastids (Besson et al. 1995). Serine and glycine have important metabolic roles. They are involved in protein biosynthesis and are the precursors of numerous compounds including phospholipids (serine) and purines (glycine). Furthermore, serine is the major source of one-carbon units in many systems (Schirch 1984) and is therefore at the basis of the whole C1 metabolism (Appling 1991). The C1 metabolism is involved in a variety of cellular processes such as methyl transfer reactions, purine, thymidylate and methionine biosynthesis (Cossins & Chen 1997; Rébeillé & Douce 1999a). In plants, two main reactions involving serine and glycine are potential sources of C1 units. The first is the SHMT-catalysed conversion of serine into glycine, which results in the formation of methylenetetrahydrofolate (CH2-THF) (reaction 1).

Glycine + CH2-THF + H2[LEFT RIGHT ARROW] serine + THF (1)

The second is the oxidation of glycine by the glycine decarboxylase complex (GDC), a reaction also leading to the formation of CH2-THF (reaction 2).

Glycine + NAD + THF [RIGHTWARDS ARROW] CO2 + NH3 + NADH + CH2-THF (2)

However, these two activities may vary greatly, depending on the nature of the plant tissue (photosynthetic or non-photosynthetic).

In leaf tissues, glycine and serine are important intermediates of the photorespiratory cycle. This cycle originates from the oxygenase activity of the Rubisco enzyme. The main function of the photorespiratory pathway is to recycle the 2-phosphoglycolate (two-carbon molecule) produced by the oxygenase activity into 3-phosphoglycerate (three-carbon molecule) that must be returned to the Benson–Calvin cycle for ribulose-1,5-bisphosphate regeneration. The conversion of a two-carbon into a three-carbon molecule is carried out in mitochondria by a set of reactions leading to the synthesis of one molecule of serine from two molecules of glycine. Firstly, glycine deriving from phosphoglycolate enters the mitochondria where it is broken down by the GDC with the formation of CO2, NH3 and the concomitant reduction of NAD to NADH (Bourguignon et al. 1988). The remaining carbon, the methylene carbon of glycine, is then transferred to THF to form CH2-THF (reaction 2). The latter compound reacts with a second molecule of glycine to form serine, a reaction catalysed by SHMT (Bourguignon et al. 1988; Rébeilléet al. 1994) (reaction 1). Thus GDC and SHMT activities are intimately coupled in leaf mitochondria. The serine is thereafter converted to phosphoglycerate in a series of reactions involving peroxisomes and chloroplasts. During the greening of leaves, the GDC–SHMT system increases considerably in the matrix space of mitochondria where it represents up to 40% of the soluble proteins (Oliver et al. 1990a; Rébeillé & Douce 1999b). This high level of protein is necessary to sustain the high flux of carbon running through the photorespiratory pathway during the light period (Gerbaud & André 1979; Oliver et al. 1990b). Thus in photosynthetic tissues the mitochondrial GDC–SHMT system and the enzymes of the photorespiratory pathway play an important role in the catabolism of glycine and serine.

In non-photosynthetic tissues, however, the situation is different: there is no photorespiration and GDC is present only in trace amounts, if at all (Bourguignon et al. 1993). The catabolism of glycine and serine in these tissues is only poorly known. Presumably serine could be converted to glycerate which, after phosphorylation, could enter glycolysis (Cheung et al. 1968; for a review see Bourguignon et al. 1999). Serine, after decarboxylation, is also a potential source of ethanolamine (Kinney & Moore 1987). The catabolism of glycine is even less clear. Taking into account that glycine and serine are readily interconvertible, it would be logical to think that a possible route for glycine catabolism involves firstly its conversion into serine. However this reaction, catalysed by SHMT, is not thermodynamically favoured (Besson et al. 1993; Bourguignon et al. 1988), and such a pathway remains hypothetical.

The 13C NMR technique has recently been used to follow C1 metabolism in yeast (Appling et al. 1997; West et al. 1996) and in illuminated Arabidopsis plants (Prabhu et al. 1996; Prabhu et al. 1998). This powerful technique allows the discrimination between the serine originating from glycine through the mitochondrial GDC–SHMT system and the serine originating from glycine through SHMT alone. Indeed, as shown in Fig. 1, labelling at the 2-C position of serine ([2-13C]serine) originates directly from [2-13C]glycine (SHMT), whereas labelling at the 3-C position of serine ([3-13C]serine or [2,3-13C]serine) requires 13CH2-THF, and the cooperation of GDC and SHMT. In the present article, we used the 13C–NMR technique to further determine the catabolism of glycine and serine in non-photosynthetic cell suspension cultures.

image

Figure 1. Representative diagram showing the movement of isotopic label from [2-13C]glycine, added in the culture medium, into serine and CH2-THF.

In these reactions, the isotopic carbon transferred from one compound to another is in bold type and marked with an asterisk. Reaction 1, SHMT; reaction 2, GDC.

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Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Glycine catabolism

Amino acids such as glycine and serine are actively transported across the plasma membrane of plant cells (Bush 1999). As shown in Fig. 2(a), when 1 mm[U-14C]glycine (uniformly labelled) was added in the external medium, this amino acid was rapidly incorporated into the cells where it accumulated (the maximal uptake rate was about 3 μmoles h–1 g–1 FW). Separate control experiments, using an in vivo13C-NMR experiment technique (Aubert et al. 1998), indicated that most of this glycine (70–85%) accumulated in the cytosol (results not shown). Furthermore, Fig. 2(a) also indicates that a significant amount of [14C]CO2 was released during the course of the experiment (the maximal rate of CO2 evolution was about 1 μmole h–1 g–1 FW), suggesting a rapid oxidation of the labelled glycine. Glycine can be oxidized directly through the mitochondrial GDC system or indirectly through the glycolytic pathway after its conversion into serine. Therefore we looked for evidence of the presence of a GDC system in these non-photosynthetic cells and we followed the fate of intracellular glycine using 13C-NMR.

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Figure 2. Glycine catabolism in non-photo- synthetic sycamore cells.

(a) Cells (12 g) were incubated in 100 ml of a culture medium containing 1 mm[U-14C]glycine. [U-14C]glycine uptake, intra- cellular accumulation of 14C compounds and [14C]CO2 efflux were measured as a function of time.

(b) SDS–PAGE analysis of mitochondrial proteins from different origins: lanes 1–2, 2 μg purified P protein (1) or T protein (2) from pea leaves; lanes 3–5, 15 μg mito- chondrial protein from pea leaves (3), potato tubers (4) and sycamore cells (5).

(c) Immunodetection of P protein with an antibody raised against purified P protein from pea leaves: lane 1, 1 μg purified P protein from pea leaves; lane 3, 10 μg pea leaf mitochondrial protein; lanes 4 and 5, 60 μg mitochondrial protein from, respectively, potato tubers and sycamore cells.

(d) Immunodetection of T protein with an antibody raised against purified T protein from pea leaves: lane 2, 1 μg purified T protein from pea leaves; lanes 3–5, conditions as described in (c).

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Generally, mitochondria isolated from non-photosynthetic tissues do not display a significant rate of glycine oxidation (Douce 1985). However, when we used a large amount of mitochondrial proteins (88 mg ml–1), it was possible to observe some glycine-dependent oxygen consumption which was stimulated by the addition of ADP (results not shown). For example, the rate of glycine oxidation by intact mitochondria isolated from potato tubers was about 7 nmoles O2 min–1 mg–1 protein (state 3), a significant but rather low value compared to the rate of glycine oxidation by leaf mitochondria (250 nmoles O2 min–1 mg–1 protein, Neuburger et al. 1986). The presence of a mitochondrial GDC in non-photosynthetic cells was further confirmed by immunodetection with antibodies raised against the P and T proteins of the GDC complex purified from pea leaf mitochondria. The Western blot experiment shown in Fig. 2(c,d) clearly indicates that the antibodies recognized proteins corresponding to the mass of the P and T proteins (98 and 45 kDa, respectively, on SDS–PAGE) in the mitochondria of pea leaf, potato tuber and sycamore cells. Thus GDC is present in non-photosynthetic cells, and this enzymatic system possibly contributed to the glycine oxidation shown in Fig. 2(a).

When [2-13C]glycine (1 mm) was added in the external medium, the labelled molecules detected within the cells by 13C NMR (see Materials and methods) were only glycine and serine, serine representing about 50% of the total 13C pool (Fig. 3). As shown in Fig. 4(a), [2-13C]serine appeared rapidly at first, reflecting the operation of SHMT with [2-13C]glycine and unlabelled CH2-THF (Fig. 1). However, the apparent rate of [2-13C]serine synthesis decreased quickly with time, and the intracellular concentration of this compound reached a near steady-state level after 30 min. In contrast, [2,3-13C]serine increased steadily with time. After 2 h of experiment, its intracellular concentration was three times higher than the level of [2-13C]serine. The formation of one molecule of [2,3-13C]serine requires two molecules of [2-13C]glycine and the coupled operation of GDC plus SHMT (Fig. 1). This indicates that a mitochondrial GDC–SHMT system was present in these-non-photosynthetic cells and played a major role in glycine catabolism, as it is the case during photorespiration in leaves (Oliver et al. 1990b). Figure 4(a) also shows that the rate of [3-13C]serine accumulation was low compared to [2,3-13C]serine. [3-13C]serine is formed from the condensation of an endogenous (unlabelled) glycine and a C1 unit originating from the decarboxylation of a [2-13C]glycine through GDC (see Fig. 1). Presumably, the supply of the endogenous unlabelled glycine was limiting this reaction. The apparent steady-state concentration observed for [2-13C]serine synthesis could be the result of either a rapid turnover of the molecule leading to a near-complete labelling of the pool after 30 min or, on the contrary, a limitation of the SHMT-catalysed reaction by the availability of the endogenous (unlabelled) CH2-THF. In order to discriminate between these two hypotheses, we followed the disappearance of the intracellular [13C]serine pools in a chase experiment. As shown in Fig. 4(b), the pool of [2-13C]glycine decreased rapidly with time, thus confirming the fast catabolism of this amino acid. In contrast, the various serine pools remained approximately constant during the first hours, then decreased in turn when a threshold concentration of [2-13C]glycine was attained. In particular, [2-13C]serine decreased slowly with time, which does not support the rapid turnover hypothesis and suggests therefore that its synthesis was limited by the availability of CH2-THF. From Fig. 4(a,b), the maximal rate of [2,3-13C]serine plus [3-13C]serine synthesis (i.e. the rate of accumulation in Fig. 4a, approximately 0.65 μmole h–1 mg–1 FW, plus the rate of disappearance in Fig. 4b, approximately 0.25 μmole h–1 mg–1 FW) could be roughly estimated to approximately 0.9 μmole h–1 mg–1 FW. This value is representative of the GDC activity because the labelling of one serine at the C3 position required the oxidation of one glycine by the GDC (Fig. 1). This value fits well with the observed maximal rate of glycine-dependent CO2 production shown in Fig. 2(a).

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Figure 3. 13C NMR spectra, in the range 40–65 p.p.m., of perchloric extracts from sycamore cells.

(a) Perchloric extract from a sample of cells with no added 13C compounds in the external medium; the major peaks observed corresponded to natural (1.1%) enrichment of F1, F6 and G6 carbons from sucrose, C2 + C4 from citrate, and C3 from malate.

(b) Perchloric extract from cells incubated for 2 h in presence of [2-13C]glycine. The areas assigned to C2 and C3 of serine are enlarged. For [2,3-13C]serine there are two resonance signals for C2 or C3 because of spin–spin interactions between the two adjacent carbons.

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image

Figure 4. Glycine catabolism in sycamore cells following addition in the external medium of 1 mm[2-13C]glycine.

(a) Time course for the appearance of endogenous [2-13C]glycine and the various 13C pools of serine.

(b) Time course for the disappearance of [2-13C]glycine and the various 13C pools of serine, starting 3 h after the addition of [2-13C]glycine in the external medium (i.e. after all the external [2-13C]glycine has been consumed). The intracellular concentrations were calculated assuming that 1 g FW represents approximately 1 ml.

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Serine catabolism

As shown in Fig. 5(a), when 1 mm[U-14C]serine (uniformly labelled) was added in the external medium, this amino acid was also rapidly incorporated into the cells where it accumulated (the maximal uptake rate was about 4.5 μmoles h–1 g–1 FW). As for glycine, the largest part of this amino acid accumulated in the cytosol (results not shown). The rate of CO2 efflux (approximately 0.3 μmole h–1 g–1 FW) measured in this experiment was significantly lower than that recorded in presence of glycine (1 μmole h–1 mg–1 FW, Fig. 2a). This CO2 production could be the result of either serine catabolism through glycolysis, or serine catabolism through GDC after its conversion into glycine. In order to estimate the catabolism of serine through the glycolytic pathway, we repeated this experiment with [3-14C]serine (Fig. 5b). Indeed, the C3 of serine cannot be released as CO2 by the coupled SHMT plus GDC activities (Fig. 1). In this latter situation, the release of C3 of serine as CO2, which presumably reflected serine oxidation through glycolysis via glycerate formation, was very low, about 0.03 μmole h–1 g–1 FW. To compare this result with that described in Fig. 5(a), we must take into account that serine had only one labelled carbon in this last experiment. Thus, assuming that the three carbons of serine could be released as CO2, it could be postulated that the total amount of CO2 produced from the glycolytic oxidation of serine was, in our experimental conditions, about 0.1 μmole h–1 g–1 FW. Comparison of the estimated rates of CO2 evolution of Fig. 5(a) (0.3 μmole h–1 mg–1 FW) and Fig. 5(b) (0.1 μmole h–1 mg–1 FW) suggests therefore that a large part (possibly two-thirds) of the catabolism of serine involved first its conversion into glycine, then oxidation through the GDC. This was further confirmed by [13C] NMR experiments.

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Figure 5. Serine catabolism in non-photosynthetic sycamore cells.

(a) Cells (12 g) were incubated in 100 ml of a culture medium containing 1 mm[U-14C]serine. [U-14C]serine uptake, intracellular accumulation of 14C compounds and [14C]CO2 efflux were measured as a function of time.

(b) Cells (12 g) were incubated in 100 ml of a culture medium containing 1 mm[3-14C]serine. [3-14C]serine uptake, intra-cellular accumulation of 14C compounds and [14C]CO2 efflux were measured as a function of time. Insert: enlarged area showing the time course of CO2 efflux. The low increase of labelling in the external medium could be the result of either a leakage of [3-14C]serine accumulated within the cells, or excretion of some 14C compounds originating from [3-14C]serine metabolism.

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As shown in Fig. 6, when 1 mm[2-13C]serine was provided to sycamore cells, [2-13C]glycine and the 13C products deriving from glycine catabolism (i.e. [2,3-13C]serine and [3-13C]serine) increased slowly with time. They represented after 2 h less than 4% of the total intracellular 13C pool. Therefore some serine was converted into glycine, which was thereafter catabolized through the GDC–SHMT system. The apparent rate of [2,3-13C]serine plus [3-13C]serine synthesis, representative of the GDC activity, was about 0.18 μmole h–1 g–1 FW, which supports the view that a large part of the CO2 efflux recorded in Fig. 5(a) (0.3 μmole h–1 mg–1 FW) was the result of the GDC activity. Thus serine catabolism firstly required the operation of SHMT to produce glycine. It is very likely that this reaction was not localized in mitochondria where SHMT activity was continuously pushed toward serine formation (the reverse direction) by the operation of GDC. In fact it is most probable that the serine-to-glycine conversion took place in the cytosol, which has a high requirement for C1 units for methionine and S-adenosyl methionine synthesis (Ravanel et al. 1998). However, as shown in Fig. 5(a) and Fig. 6, only a low amount of the incorporated serine was catabolized through glycine, suggesting that the (cytosolic?) SHMT reaction was limited by the availability of THF. In addition, Fig. 6 also indicates that [2-13C]ethanolamine was formed from serine, thus confirming that serine is a potential source of ethanolamine (Kinney & Moore 1987).

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Figure 6. Serine catabolism in sycamore cells following the addition of 1 mm[2-13C]serine.

Insert: enlarged area showing the time-course evolution of the detec13C pools of glycine, serine and ethanolamine. The intracellular concentrations were calculated assuming that 1 g FW represents approximately 1 ml.

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Catabolism of glycine and serine as sources of C1 units

In most cells, serine is recognized as the prime donor of C1 units (Appling 1991). However, two separate reactions (GDC and SHMT) are possible sources of C1 units in sycamore cells, and the above results suggest that glycine oxidation by the GDC reaction is more active than is serine-to-glycine conversion by the SHMT reaction. In order to know which of these two reactions is the prime source of C1 units, we followed the labelling of [13C]methionine as a function of various external 13C substrates. Indeed, methionine is a central compound in C1 metabolism: its synthesis requires the addition of one C1 unit and it is the precursor of S-adenosyl methionine which is, in turn, the donor of methyl groups in numerous reactions (Ravanel et al. 1998). These methylation reactions probably drained the largest part of the pool of C1 units. As shown in Table 1, the concentration of [5-13C]methionine, measured after 15 h of experiment, was much higher with [3-13C]serine than with all the other substrates tested. In fact, the labelled methionine was already detectable after 2 h incubation with [3-13C]serine (not shown). This result confirms that most C1 units derived from the carbon 3 of serine through the SHMT-catalysed reaction. In contrast, glycine was five to six times less efficient than serine for methionine synthesis, and it is possible that [2,3-13C]serine and [3-13C]serine issued from glycine catabolism were, in fact, the sources of C1 units for methionine synthesis. Clearly, the question arises whether [13C]CH2-THF produced by the mitochondrial GDC activity equilibrates with the overall pool. From this point of view, it is noteworthy that [2-13C]serine was not a source of 13C1 units (Table 1), despite a substantial rate of [13C]CH2-THF formation by the GDC activity (equivalent to the GDC-dependent rate of CO2 evolution in Fig. 5(a), approximately 0.2 μmole h–1 g–1 FW). This strongly suggests that the mitochondrial pool of CH2-THF did not equilibrate with the overall intracellular pool.

Table 1.  . Methionine synthesis in sycamore cells after 15 h culture in the presence of three different sources of C1 units (5 mM)
Source of C1 units[5-13C]methionine (mM)
  1. nd, not detected.

[2-13C]glycine0.02
[2-13C]serinend
[3-13C]serine0.14

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Our results indicate that glycine catabolism is coupled to serine synthesis. There are two routes to transform glycine into serine: the first involves SHMT and requires CH2-THF as cofactor, and the second involves the coupling of GDC and SHMT and also requires THF as cofactor (Bourguignon et al. 1999). SHMT is present in the three main cellular compartments (cytosol, mitochondria and chloroplast) (Besson et al. 1995), whereas GDC is present only within mitochondria (Bourguignon et al. 1988; Neuburger et al. 1986). Thus it is tempting to suggest that the formation of [2,3-13C]serine and [3-13C]serine, requiring the GDC–SHMT system, was essentially located within the mitochondria, whereas the synthesis of [2-13C]serine, requiring only SHMT and an endogenous supply of unlabelled CH2-THF, was located mainly in the cytosol or in the plastids. One major observation arguing in favour of a mitochondrial location of [2,3-13C]serine and [3-13C]serine synthesis is that the mitochondrial CH2-THF produced during glycine oxidation apparently did not equilibrate with the cytosolic or plastidial pools. This assumption relies on the results shown in Table 1 indicating that glycine was five to six times less efficient than serine as a source of C1 units (based on methionine synthesis). This is despite the fact that the rate of CH2-THF formation through glycine oxidation (1 μmole h–1 g–1 FW, Fig. 2a) was apparently higher than the rate of CH2-THF formation through the SHMT-dependent serine-to-glycine conversion (about 0.45 μmole h–1 g–1 FW, estimated from Fig. 6). Since [13C]CH2-THF produced by the GDC activity was not a direct source of C1 units, it is likely that the largest part of this compound, if not all, was recycled within the mitochondria for serine formation. Thus the mitochondrial THF was not a limiting factor of the GDC–SHMT system because it was permanently recycled between the two reactions. This suggestion is strongly supported by the fact that the rate of CO2 produced by glycine oxidation (1 μmole h–1 g–1 FW) was very similar to the rate of [2,3-13C]serine plus [3-13C]serine synthesis (0.9 μmole h–1 g–1 FW). The subcellular location of [2-13C]serine synthesis is more difficult to estimate. Taking into account that the largest pool of THF cofactors is probably in the cytosol (Cossins & Chen 1997), it is logical to think that the largest part of [2-13C]serine was synthesized in this compartment. Mitochondria have also a relatively large amount of folate (at least on a protein basis) (Cossins & Chen 1997; Neuburger et al. 1996) and the possibility cannot be excluded that some [2-13C]serine was synthesized from [2-13C]glycine in this compartment, at least at the beginning of the experiment. However, considering the small volume of the mitochondrial fraction and the high turnover of the mitochondrial CH2-THF observed in these experiments, it is likely that the mitochondrial unlabelled CH2-THF would decrease very rapidly.

Our results show that serine is the main donor of C1 units in sycamore cells, as in other cells (Appling 1991). However, the catabolism of serine involved little conversion to glycine, which indicates that only a small number of the incorporated molecules were diverted toward C1 metabolism. Thus, although glycine formation through the SHMT reaction is thermodynamically favoured, this reaction must be tightly regulated. Preliminary experiments involving folate addition in the culture medium suggest that the availability of THF was the prime controller of the reaction. Taken as a whole, these data strongly suggest that catabolism of serine is mainly driven by the need for C1 units. It must be kept in mind that the experimental situations described here may not reflect the usual physiological situation. In particular, the intracellular concentrations of glycine and serine reported in this paper are probably much higher than the naturally occurring ones. In a physiological situation, the rate of serine-to-glycine conversion, which presumably reflects the C1 metabolism activity, is likely to be in balance with the overall cellular metabolism. In such a situation, glycine probably never accumulates because it is oxidized by the mitochondrial GDC–SHMT system as soon as it is produced, leading back to the formation of one serine from two glycines. The final balance for this serine–glycine cycle would be the production of two C1 units, one CO2 and one NH3 for the oxidation of one serine (Fig. 7).

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Figure 7. Hypothetical diagram showing the ‘serine–glycine cycle’ between the cytosol and mitochondria during the catabolism of one serine via SHMT.

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These results raise the question of the role of the GDC in non-photosynthetic tissues such as potato tuber or sycamore cambial cells. Cell metabolism requires a myriad of methylation reactions. Assuming that for each utilization of one C1 unit there is the production of one glycine, it is clear that a relatively fast glycine catabolism is needed. Indeed, accumulation of glycine could rapidly result in a dramatic decrease of C1 unit formation because the SHMT reaction would be pushed toward the formation of serine. Thus the mitochondrial GDC–SHMT coupled reactions are an obligatory route for glycine catabolism in all plant tissues. This system probably plays an important role in tissues with a high protein turnover: rapidly dividing tissues, those with a high protease activity such as senescent tissues, or tissues displaying autophagic processes (Aubert et al. 1996). It will be of interest to know whether the expression of this enzymatic system increases under such conditions, and this question is currently under investigation.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References

Chemicals

[U-14C]glycine (3.85 Gbq mmol–1), [U-14C]serine (5.85 Gbq mmol–1) and [3-14C] serine (2 Gbq mmol–1) were purchased from Amersham Life Science. [2-13C]glycine (99%), [2-13C]serine (99%), [3-13C]serine (99%) and [2-13C]glycolate (99%) were purchased from Leman (Saint Quentin-en-Yvelines, France). To simplify the quantification of intracellular 13C metabolites, the above chemicals were added in the external medium as 100% labelled.

Material

Cambial sycamore (Acer pseudoplatanus L.) cells used in the present study were grown at 20°C as a suspension in liquid nutrient media according to Bligny & Leguay (1987). Under standard conditions, this media did not contain glycine or serine. The cell-suspension cultures were maintained in exponential growth by subculturing every 7 days. In these conditions, the cell-doubling time was approximately 50 h. Thus growth and protein synthesis can be considered as negligible during the time course of most of the experiments described here. For each NMR determination, cells (10 g) were kept in a volume of 0.1 l and stirred continuously at 60 rpm (0.5 g) in the presence of the 13C-labelled compound for the required time. Pea leaf, potato tuber and sycamore cell mitochondria were purified on a Percoll gradient as previously described (Douce et al. 1987).

Determination of the CO2 efflux from glycine and serine catabolism

Cells (approximately 12 g) in a volume of 0.1 l were stirred continuously and incubated in the presence of either 1 mm[U-14C]glycine, 1 mm[U-14C]serine or 1 mm[3-14C]serine (specific activity: 4.6 × 10–6 Gbq μmole–1). The flasks containing the cell suspension culture were aerated with a stream of air. At the exit of the flasks, the stream of air was directed toward a test tube containing 50 ml of a 2 N KOH solution to trap the 14CO2 produced during the time course of the experiment. In separate control experiments it was verified that 85–90% of the CO2 efflux were trapped by this procedure. At each time, aliquots of the KOH solution and of the cell suspension were removed, and the radioactivity present in the KOH solution, the culture medium and the cells was estimated by liquid scintillation counting.

Perchloric acid extract and 13C NMR analysis

At each time point, an aliquot of the culture medium was collected to determine the amount of extracellular 13C-labelled compound that had been incorporated within the cells. Then the cell suspension culture was rapidly rinsed onto a glass fibre filter and washed three times with fresh water to remove all traces of external 13C-labelled compound. The cell wet weight (about 10 g) was rapidly determined, and the cells were quickly frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle with 900 μl of 70% (v/v) perchloric acid. The frozen powder was then placed at 0°C and thawed. The thick suspension thus obtained was centrifuged at 15 000 g for 10 min to remove particulate matter, and the supernatant neutralized with 2 m KHCO3 to pH ≈ 5. The supernatant was then centrifuged at 10 000 g for 10 min to remove KClO4; the resulting supernatant was lyophilized and stored at –80°C. We verified in separate control experiments that this procedure did not lead to a significant loss of 13C metabolites, which indicates that these labelled metabolites were not labile compounds. This freeze-dried material was redissolved in 2.5 ml water containing 10% D2O, neutralized to pH 7.5, and buffered with 50 mm HEPES. Divalent cations (particularly Mn2+ and Mg2+) were chelated by the addition of sufficient amounts of 1,2-cyclohexylenedinitrilotetra-acetic acid ranging from 50 to 100 μmol depending on the samples.

NMR measurements

Spectra of neutralized perchloric acid extracts were recorded on a Bruker NMR spectrometer (AMX 400, WB) equipped with a 10 mm multinuclear probe tuned at 100.6 MHz for 13C NMR studies. The deuterium resonance of D2O was used as a lock signal. The 13C NMR acquisition conditions used were essentially as previously described (Aubert et al. 1998): 90° radio frequency pulses (19 μsec) at 6 sec intervals; spectral width 20 000 Hz; 900 scans; Waltz-16 1H decoupling sequence (with two levels of decoupling: 2.5 W during acquisition time, 0.5 W during delay). Free induction decays were collected as 16 K data points, zero filled to 32 K and processed with a 0.2 Hz exponential line broadening. The 13C NMR spectra are referenced to hexamethyldisiloxane at 2.7 p.p.m.

Identification and quantification of NMR signals

Spectra of standard solutions of known compounds at pH 7.5 were compared with that of a perchloric acid extract of sycamore cells. The definitive assignments were made after running a series of spectra obtained by addition of the authentic compounds to the perchloric acid extracts, according to previous authors (Gout et al. 1993). To determine accurately the total amount of metabolites, we calibrated the peak intensities by the addition of known amounts of the corresponding authentic compounds, and used a 20 sec recycling time to obtain fully relaxed spectra. The yield of recovery was estimated by adding known amounts of authentic compounds to frozen cells before grinding. We have observed that, for all the compounds studied, the overall yield of recovery was ≈80%. The chemical shifts were (in p.p.m.): [2-13C]glycine, 42.4; [2,3-13C]serine, 57.1 and 57.4 or 61 and 61.3; [2-13C]serine, 57.3; [3-13C]serine, 61.1; [2-13C]glycolate, 62.1; [5-13C]methionine, 14.7; [2-13C]ethanolamine, 42.2.

Oxygraphic measurements

Oxygen uptake was measured at 25°C with a Clark-type O2 electrode as previously described (Neuburger et al. 1986). The O2 concentration in air-saturated medium was assumed to be 250 μm. The mitochondria were better than 95% intact as judged by their impermeability to cytochrome c (Douce et al. 1987).

Immunoblotting analysis

Immunoblotting experiments were performed essentially according to previous studies (Macherel et al. 1990; Vauclare et al. 1996) with polyclonal antibodies raised against the P and T proteins of the GDC.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. References
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