Journal of Experimental Botany, Vol. 54, No. 387, pp. 1523±1535, June 2003 DOI: 10.1093/jxb/erg171

REVIEW ARTICLE Genetic manipulation of glycine decarboxylation

Hermann Bauwe1 and UÈ ner Kolukisaoglu Abteilung P¯anzenphysiologie der UniversitaÈt Rostock, Albert-Einstein-Strasse 3, D-18051 Rostock, Germany

Received 2 October 2002; Accepted 11 March 2003 Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021

Abstract complex that occurs in all organisms, prokaryotes and eukaryotes. GDC, together with serine hydroxymethyl- The glycine±serine interconversion, catalysed by gly- transferase (SHMT), is responsible for the inter-conversion cine decarboxylase and serine hydroxymethyltransfer- of glycine and serine, an essential and ubiquitous step of ase, is an important reaction of primary metabolism in primary metabolism. In Escherichia coli, 15% of all all organisms including plants, by providing one-car- carbon atoms assimilated from glucose are estimated to bon units for many biosynthetic reactions. In plants, pass through the glycine±serine pathway (Wilson et al., in addition, it is an integral part of the photorespira- 1993). In eukaryotes, GDC is present exclusively in the tory metabolic pathway and produces large amounts mitochondria, whereas isoforms of SHMT also occur in the of photorespiratory CO within mitochondria. 2 cytosol and, in plants, in plastids. The term `glycine±serine Although controversial, there is signi®cant evidence that this process, by the relocation of glycine decar- interconversion' might suggest that the central importance boxylase within the leaves from the mesophyll to the of this pathway is just the synthesis of serine from glycine and vice versa. However, in both directions of the bundle-sheath, contributed to the evolution of C4 photosynthesis. In this review, some aspects of cur- concerted reaction of GDC and SHMT, tetrahydrofolate 5 10 rent knowledge about glycine decarboxylase and ser- (THF) becomes N ,N -methylenated making these reac- ine hydroxymethyltransferase and the role of these tions the most important source of active one-carbon-units enzymes in metabolism, about the corresponding for a number of biosynthetic processes such as the genes and their expression as well as about mutants biosynthesis of methionine, pyrimidines, and purines and anti-sense plants related to these genes or pro- (Fig. 1). Glycine and serine itself are precursors for cesses will be summarized and discussed. From a chlorophyll, glutathione, tryptophan, phosphatidylcholine comparison of the available information about the and related phospholipids, and ethanolamine. The role of number and organization of GDC and SHMT genes in GDC in all organisms is to interconnect the metabolism of the genomes of Arabidopsis thaliana and Oryza sativa one-, two-, and three-carbon compounds (reviewed by it appears that these and, possibly, other genes Kikuchi, 1973; Oliver, 1994; Cossins, 2000; Hanson and related to photorespiration, are similarly organized Roje, 2001; Douce et al., 2001). It is therefore not even in only very distantly related angiosperms. surprising, that a malfunction of GDC results in serious metabolic consequences. Humans, for example, can suffer from non-ketotic hyperglycinemia, an inherited and Key words: Arabidopsis, genetic engineering, glycine incurable disease with devastating and often lethal symp- decarboxylase, mutant analysis, one-carbon metabolism, photo- toms (Kure et al., 1997). Plants are not able to perform respiration, photosynthesis, serine hydroxymethyltransferase, oxygenic photosynthesis without GDC or SHMT and, with reduced activities of these enzymes, will usually show severe growth retardation (Somerville, 2001; Wingler et al., 1997; Heineke et al., 2001). Introduction Compared with other organisms, the photorespiratory Glycine decarboxylase (GDC, also named glycine-cleav- pathway of plants provides a novel role for both GDC and age-system or glycine dehydrogenase) is a multi-protein SHMT. In plants, GDC and SHMT are integral

1 To whom correspondence should be addressed. Fax: +49 381 498 6112. E-mail: [email protected] 5 10 Abbreviations: CH2-THF, N ,N -methylene tetrahydrofolate; GDC, glycine decarboxylase; LPD, dihydrolipoamide dehydrogenase; SHMT, serine hydroxymethyltransferase.

Journal of Experimental Botany, Vol. 54, No. 387, ã Society for Experimental Biology 2003; all rights reserved 1524 Bauwe and Kolukisaoglu understood. Secondly, the glycine±serine interconversion, by providing one-carbon units, is directly related to many biosynthetic processes outside the photorespiratory path-

way. Finally, in photosynthesizing organs of C3 plants, GDC is the major source of internally generated CO2 and, as will be discussed in more detail later, may in¯uence

CO2 concentration gradients within leaves. Several excellent recent reviews cover different aspects of the biochemistry and enzymology of glycine decarbox- ylation and its relation to plant metabolism (for example Douce et al., 2001; Mouillon et al., 1999; Hanson and

Roje, 2001). In this review, these aspects will only be Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 Fig. 1. Schematic presentation of the glycine±serine interconversion and its connection to one-carbon metabolism in different subcellular discussed brie¯y, instead the focus will be on the compartments. Circles P, T, H, and L represent the four protein underlying genetics and on the results obtained with components of glycine decarboxylase and circle S represents serine mutants and transgenic plants. As stated above, GDC hydroxymethyltransferase (Cossins, 2000; Ravanel et al., 2001). closely co-operates with SHMT both during the photo- respiratory decarboxylation of glycine and the supply of components of primary metabolism not only in the context one-carbon units for other biosynthetic processes. of `house-keeping' glycine±serine interconversion as dis- Therefore, both GDC and SHMT will be covered in this cussed above. Their additional function in plants is the survey. breakdown of glycine that originates, after several enzymatic reactions, from the oxygenase reaction of Rubisco (Bowes et al., 1971; Tolbert, 1973). By this side Protein components and reactions of the glycine±serine interconversion reaction of oxygenic photosynthesis, 2-phosphoglycolate is produced and, by the action of ten different enzymes The general course of the individual reactions is well including GDC and SHMT, is subsequently recycled as 3- known from the work of several groups over many years phosphoglycerate to the Calvin cycle. The contributing (Kikuchi, 1973; Oliver, 1994; Bourguignon et al., 1988; enzymes are localized in three different organelles, Walker and Oliver, 1986a). More details of the involved chloroplasts, peroxisomes, and mitochondria. In C3 plants, catalytic mechanisms can be expected from crystallo- if grown under illumination in ambient air, glycine graphic data in the near future. Strongly simpli®ed, the synthesis occurs at very high rates and requires a high course of the reactions in the context of the photorespira- capacity for mitochondrial glycine oxidation. In fact, tory pathway can be described by the following equations: glycine is the preferred substrate of mitochondria and becomes very rapidly oxidized (Day et al., 1985; KroÈmer GDC: + and Heldt, 1991) leading to relatively low glycine Glycine + NAD + THF ® Methylene-THF + CO2 + concentrations in leaves (Leidreiter et al., 1995). NH3 + NADH GDC, under unstressed conditions, represents the sole SHMT: source of photorespiratory CO2 and NH3 and functions as Glycine + Methylene-THF + H2O ® Serine + THF an important link between photorespiration and other GDC/SHMT: + metabolic pathways such as nitrate and ammonia assimi- 2 Glycine + NAD ® Serine + CO2 +NH3 + NADH lation. Much of the earlier work on photorespiration was directed towards attempts to reduce the massive net CO GDC comprises four protein components (Fig. 1). All 2 four individual proteins, which have been designated P, T, losses that occur in C3 plants especially in warm environ- ments. From research conducted over the past 20 years, it H, and L protein, are nuclear encoded and targeted into the is now clear that attempts to abolish or even reduce mitochondrial matrix. photorespiration by reducing the activity of individual enzymes of the photorespiratory pathway, except ribulose- P protein (EC 1.4.4.2) 1,5-bisphosphate oxygenase, will not lead to improved P protein, a pyridoxal-5-phosphate containing homodimer plant performance. of about 200 kDa, is the actual glycine decarboxylating What then can be the purpose of continuing attempts to subunit. P protein has also been identi®ed as the binding manipulate glycine decarboxylation genetically? Firstly, it protein of a host-speci®c toxin, victorin (Wolpert et al., appears that regulatory interactions exist between photo- 1994). The product of the P protein-catalysed decarbox- respiration and photosynthesis triggered by metabolite ylation of glycine is CO2 and not bicarbonate (Sarojini and levels. The nature of these interactions is not well Oliver, 1983). The remaining amino methylene moiety is Manipulation of glycine decarboxylation 1525 transferred to the distal sulphur atom of the oxidized dehydrogenase complex (Luethy et al., 1996). By contrast lipoamide arm of H protein (Douce et al., 2001). with pea, where it was reported that mitochondrial L protein is encoded by a single gene and shared between a- H protein ketoacid dehydrogenase complexes and GDC (Turner and H protein, a 14 kDa lipoamide (5[3-(1,2) dithiolanyl] Ireland, 1992; Bourguignon et al., 1992, 1996), two genes pentanoic acid) containing non-enzyme protein, interacts encoding mitochondrial L protein (mtLPD1 and mtLPD2) as a co-substrate with all three enzyme proteins of the have been reported for Arabidopsis thaliana. mtLPD1, complex. The three-dimensional structures of all forms of seems to provide L protein for GDC whereas the mtLPD2 H protein have been resolved (Pares et al., 1994, 1995; gene product mainly interacts with a-ketoacid dehydro- Cohen-Addad et al., 1995; Macherel et al., 1996; Faure genases (Lutziger and Oliver, 2001). However, from the et al., 2000; reviewed in Douce et al., 2001). Lipoylation high sequence identity of 92%, the authors conclude that

of H protein is catalysed by a lipoate±protein ligase (Wada both L proteins can work in either multienzyme complex. Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 et al., 2001a) and occurs after import of the apoprotein into In a more recent analysis of the mRNA and subunit protein the mitochondria (Fujiwara et al., 1990) where lipoic acid levels of the pea leaf mitochondrial pyruvate dehydrogen- is synthesized from fatty acid precursors (Wada et al., ase complex it was shown that, in sharp contrast to all other 1997). Once aminomethylated, the lipoate arm becomes subunits, the activity of the E3 subunit (L protein) was locked within a cleft at the surface of the H protein and highest in mature, fully expanded leaves, re¯ecting its role released only by interaction with T protein which induces a as a component of GDC (Luethy et al., 2001). Pea change in the overall conformation of the H protein (Douce chloroplasts contain a lipoamide dehydrogenase that is and Neuburger, 1999). In some plants, tissue-speci®c different from the mitochondrial isoenzyme (Conner et al., alternative splicing results in two H proteins with or 1996). Similarly, two plastidic LPD genes were identi®ed without an N-terminal extension of two amino acids. The in Arabidopsis thaliana that are only 33% identical to their possible effects of this extension onto the H protein's mitochondrial counterparts (Lutziger and Oliver, 2000). properties are not yet known (Kopriva et al., 1995a, Apparently, the plastidic LPD is part of the plastidic 1996a). pyruvate dehydrogenase. There is experimental evidence T protein (E.C. 2.1.2.10) that LPD is present in soybean nodules, too, and that this LPD is identical to ferric leghaemoglobin reductase-2 T protein, a 45 kDa monomeric aminomethyl transferase, (Moran et al., 2002). needs THF and H protein as co-substrates. One of the conserved domains of T protein shows signi®cant similar- Molecular interactions between GDC components ity to a domain of formyltetrahydrofolate synthetase from both prokaryotes and eukaryotes suggesting that T protein In green leaves, GDC can be present in concentrations of ±1 is not as unique as generally thought (Kopriva et al., up to 200 mg ml (Oliver, 1994; Douce et al., 1994). The 1995b). T protein takes over the aminomethylene group for ratio of the protein subunits has been roughly estimated as further processing. The methylene group becomes trans- 4P:27H:9T:2L (Oliver et al., 1990). It is not yet well ferred to tetrahydrofolate resulting in the synthesis of understood how the GDC subunits interact with one 5 10 another. They are probably able spontaneously to assemble N ,N -methylene tetrahydrofolate (CH2-THF) and NH3 is released. During these reactions, the lipoamide arm of H within the mitochondrial matrix as can be concluded from protein becomes full reduced and, to be ready for the next their behaviour in vitro at protein concentrations above cycle, needs to be re-oxidized. 0.25 mg ml±1 with the H protein possibly building a kind of central core (Oliver et al., 1990; Oliver, 1994) or the L protein (EC 1.8.1.4) `structural and mechanistic heart' of the complex (Douce This reoxidation is achieved by the L protein (dihydroli- et al., 2001). Structure±function relationships of and poamide dehydrogenase, LPD). L protein is present as a between the individual subunits are now becoming clearer homodimer of about 100 kDa containing FAD as a co- from crystallographic data for some of the respective enzyme. During the oxidation of reduced H protein, FAD proteins and the analysis of their interaction by nuclear is reduced to FADH2 which, in turn, becomes immediately magnetic resonance studies (Faure et al., 2000; Neuburger reoxidized by NAD+ resulting in the synthesis of one et al., 2000; Pares et al., 1995; Douce et al., 2001). Several NADH per decarboxylated glycine. The three-dimensional lines of evidence strongly suggest that, except the catalytic structure of L protein has been resolved (Faure et al., interaction with the lipoyl arm, there is no apparent 2000). molecular recognition and interaction between L protein L protein is a component not only of GDC but, as the so- and the reduced H protein. It is assumed that the main role called E3 subunit, also of a-ketoacid dehydrogenase of H protein could be to maintain the hydrophobic lipoate complexes, namely pyruvate dehydrogenase, a-ketogluta- in a state that is freely accessible to the catalytic site of the rate dehydrogenase and the branched chain a-ketoacid L protein (Faure et al., 2000; Neuburger et al., 2000). As 1526 Bauwe and Kolukisaoglu far as is known, no crystallographic data are available for units for biosynthetic reactions within the cell including the T protein and for the P protein. chloroplasts and cytosol (Appling, 1991; Mouillon et al., Corresponding cDNAs and genes have been cloned and 1999). CH2-THF itself can be converted to methyl-, analysed over the last ten years by several groups and from methenyl- and formyl-THF thus providing one-carbon different plant sources. More recently, sequences of GDC units for a number of different biosynthetic reactions, such genes became available from genome and full-length as the biosynthesis of methionine, purines, pyrimidines, cDNA sequencing projects for a vast number of organisms. and lipids, not only in plants but in all organisms (Cossins Some of the genes and their expression behaviour have and Chen, 1997; Hanson et al., 2000; Hanson and Roje, been analysed in more detail (Macherel et al., 1992; 2001). Srinivasan and Oliver, 1995; Kopriva et al., 1995a; Bauwe et al., 1995; Vauclare et al., 1998). For several genes Possible contributions of glycine decarboxylase

encoding GDC subunits, induction by light has been Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 for the evolution of C plants observed (Walker and Oliver, 1986b; Kim et al., 1991; 4 Macherel et al., 1990; Turner et al., 1992b; Vauclare et al., The majority of C4 plants evolved about six to eight 1998; Ma et al., 2001). In the case of H protein and SHMT, million years ago under conditions of relatively low negative effects of methyljasmonate on the transcript atmospheric CO2 concentrations that, by favouring ener- levels were reported (Schenk et al., 2000). getically wasteful photorespiratory processes, increase the so-called Rubisco penalty (Edwards et al., 2001). C4 SHMT (EC 2.1.2.1) photosynthesis evolved polyphyletically and differs from SHMT (also named glycine hydroxymethyltransferase), a the ancestral C3 photosynthesis in a number of features. tetramer of pyridoxal-5-phosphate containing 53 kDa The major achievement of C4 plants relative to C3 plants subunits, catalyses the reversible conversion of serine is the presence of a highly ef®cient CO2 concentrating 5 10 and THF to glycine and N ,N -methylene THF (Schirch, mechanism, the C4 cycle, leading to CO2 levels within the 1982; Mouillon et al., 1999). In photosynthetic cells, by bundle-sheath of C4 plant leaves in excess of 20 times their high photorespiratory production of glycine, the atmospheric concentrations (Hatch, 1987; Kellog, 1999). mitochondrial SHMT reaction ¯ows in the reverse direc- Besides other effects, this results in a suppression of tion, i.e. towards the synthesis of serine. primary photorespiration (rates of internal CO2 generation During the photorespiratory decarboxylation of glycine by decarboxylation of glycine) by greatly reduced synthe- in plants, a high mitochondrial activity of SHMT is needed sis of phosphoglycolate, the initial substrate of the not only to synthesize serine but also permanently to photorespiratory carbon oxidation cycle. Usually, C4 recycle the methylenated THF to THF for its reuse in the plants show a specialized leaf anatomy, `Kranz' anatomy GDC reaction. It was shown that CH2-THF is not perfectly (Haberlandt, 1914), with two distinctive and co-operating channelled between T protein and SHMT and that high types of photosynthetic cells, namely mesophyll and CH2-THF/THF rates prevail during steady-state glycine bundle-sheath cells. Very much like the enzymes of the oxidation in mitochondrial matrix extracts (Rebeille et al., photosynthetic carbon reduction cycle and the decarbox- 1994). ylating enzymes of the C4 cycle, GDC is present in the By contrast with animal cells, which need an external bundle-sheath but not in the mesophyll of C4 plant leaves supply of folate (Appling, 1991), plant cells are able to (Ohnishi and Kanai, 1983). Photorespiratory CO2 is synthesize folate in their mitochondria. Plant mitochondria therefore released only within the bundle-sheath and contain 100±150-fold more THF than chloroplasts becomes ef®ciently recaptured. Collectively, these related (Neuburger et al., 1996; Ravanel et al., 2001). The biochemical and cell-biological aspects of C4 photosyn- cytosolic concentrations have not yet been estimated. thesis result in the high CO2 assimilation rates of C4 plants, The mitochondrial CH2-THF/THF pool does not equili- even under conditions of low stomatal conductance (for a brate with the cytosolic or plastidic pools (Bourguignon recent comprehensive treatise see Sage and Monson, et al., 1988; Mouillon et al., 1999). Therefore, it is not 1999). regarded as a direct major source of one-carbon units for Several recent reports provide evidence that C4 photo- biosynthetic reactions outside the mitochondria (Mouillon synthesis does not necessarily require Kranz anatomy. This et al., 1999). has been shown for two species of the Chenopodiaceae SHMT is present not only in mitochondria but in at least family, namely Borszczowia aralocaspica and Bienertia two other intracellular compartments, the cytosol and the cycloptera (Voznesenskaya et al., 2001b, 2002; reviewed chloroplasts (Turner et al., 1992a; Besson et al., 1995). in Sage, 2002). In these two succulent halophytic plants, The photorespiratory cycle is thus able, via export of C4 photosynthesis is accomplished by the separation of serine, to provide one-carbon units for use in biosynthetic two types of chloroplasts and other organelles between the pathways outside of the mitochondria. It is assumed that two opposite ends (B. aralocaspica) or between two cytosolic SHMT represents the major source of one-carbon concentric cytoplasmic layers (Bi. cycloptera) of the Manipulation of glycine decarboxylation 1527 individual chlorenchymatic cells. Chloroplasts in the distal Interestingly, at least one C3±C4 intermediate plant, (B. aralocaspica) or outer (Bi. cycloptera) cytosolic layer, Salsola arbusculiformis, has been identi®ed within the respectively, in contrast to the more proximally or Chenopodiaceae (Voznesenskaya et al., 2001a). The leaf centrally located chloroplasts, for example, lack grana anatomy of this plant, in contrast to the Salsoloid Kranz and do not accumulate starch but contain most of the leaf anatomy that is typical for Salsola C4 species, was pyruvate orthophosphate dikinase. Moreover, mitochon- described as being Kranz-like. The authors did not dria were found exclusively within the central cytoplasmic speci®cally examine the intercellular distribution of GDC layer of Bi. cycloptera. It is not yet clear whether this type in this species but found that a very high fraction of bundle-sheath cell volume is occupied by mitochondria of C4 photosynthesis is related to the evolution of the (50% relative to the respective chloroplast volume). This `classical' Kranz-type of C4 photosynthesis in the Chenopodiaceae or, alternatively, represents a separate suggests the possibility of a similar role for GDC in S.

arbusculiformis as in C ±C intermediate plants from other Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 `non-classical' type of C4 photosynthesis. It is important to 3 4 families. note that this single-cell type of C4 photosynthesis is not Except GDC, there is no other enzyme which is typical for the large number of C4 plants present in this family and has not yet been found within other plant speci®cally con®ned to the bundle-sheath of C3±C4 plants. As already mentioned, GDC is the major source of CO2 families. Hence, single-cell C4 photosynthesis as found in B. aralocaspica and Bi. cycloptera could possibly be a internally generated from photorespiratory processes. relatively rare adaptation to salty habitats. Estimates concerning the rate of photorespiratory CO2 release in C3 plants vary, depending on the method used Apparently, C4 photosynthesis must have evolved step- by-step by the successive modi®cation of many genes. In for determination, from about 25% to about 100% of net genera of several families, species have been identi®ed that photosynthesis (Zelitch, 1979; Peterson, 1983; PaÈrnik and possess no or no fully developed C cycle (Rawsthorne and Keerberg, 1995). Despite these uncertainties it can be 4 stated that, as a general rule, rates of photosynthesis and Bauwe, 1998). Probably not all these C3±C4 intermediate plants can be regarded as derived from extinct predeces- photorespiration are of the same order of magnitude in C3 plants. In the mesophyll of C plant leaves, photorespira- sors of C plants. However at least in Flaveria, a genus that 3 4 tion moves freshly assimilated carbon from the chlor- includes a relatively broad range of species with varying oplasts into the mitochondria where it is released as degrees of C /C photosynthesis, phylogenetic studies 3 4 photorespiratory CO . Due to corresponding high carbon strongly suggest that C ±C intermediate representatives 2 3 4 ¯uxes this process can be compared with a carbon can be regarded as being derived from the extinct concentrating mechanism that is futile because the inlet evolutionary links between C and C Flaveria species 3 4 (chloroplasts) and the outlet (mitochondria) are present (Kopriva et al., 1996b). within the same cell. One of the most characteristic features of C3±C4 The situation is different in C3±C4 intermediate plants intermediate plants, relative to C3 plants, are high where photorespiratory glycine is produced with high rates reassimilation rates for photorespiratory CO2 leading to both in the mesophyll and in the bundle-sheath, but can be greatly reduced rates of apparent photorespiration decarboxylated only by the mitochondria of the bundle- (Holbrook et al., 1985; Bauwe et al., 1987). On a sheath. It is therefore tempting to speculate that these biochemical level, leaves of C3±C4 intermediate plants combined features may result in elevated CO2 concentra- contain relatively high concentrations of glycine (Holaday tions within the bundle-sheath. This hypothesis has been and Chollet, 1984). Signi®cant progress has been made in 14 tested by quantitative CO2 labelling experiments with the explanation of the underlying molecular and cell- leaves of several Flaveria species designed to permit the biological events, but they are still far from being fully determination of in vivo carboxylation/oxygenation ratios understood (Rawsthorne, 1992; Rawsthorne and Bauwe, of ribulose-1,5-bisphosphate. These data indicate that the 1998). According to current knowledge, both the meso- C3±C4 intermediate species Flaveria anomala has about a phyll and the bundle-sheath of C3±C4 intermediate plant 2-fold increased carboxylation/oxygenation ratio of ribu- leaves contain functionally complete carbon reduction lose-1,5-bisphosphate ratio relative to the C3 plant cycles. In contrast to the bundle-sheath cells, however, that Flaveria cronquistii. Because there are no signi®cant contain the full enzyme set of the photorespiratory cycle differences between these two species in their in vitro the mesophyll mitochondria of C3±C4 intermediate plants af®nity of Rubisco to CO2 and O2 (Bauwe, 1984) it was lack at least one of the GDC subunits rendering the enzyme concluded that Rubisco operates under an approximately inactive (Hylton et al., 1988; Morgan et al., 1993). It was doubled mean CO2 concentration in leaves of the C3±C4 concluded that most of the photorespiratory glycine intermediate plant (BassuÈner, 1985; U Bauwe and O produced in the mesophyll of C3±C4 intermediate plants Keerberg, unpublished data). moves to the bundle sheath, where it can be decarboxy- From all the ®ndings discussed above it is most likely lated. that the photorespiratory cycle of C3±C4 intermediate 1528 Bauwe and Kolukisaoglu plants, by the exclusive presence of GDC in the bundle- biosynthesis of a number of photosynthetic and photo- sheath and by using glycine as the vehicle, is capable of respiratory enzymes in C4 plant leaves is more complex transporting large amounts of freshly assimilated carbon than has been thought previously (Bailey et al., 2000). In from mesophyll chloroplasts (the `pump's' inlet) to addition, there were some initial reports on the use of bundle-sheath mitochondria (the `pump's' outlet) where mutagenized tobacco callus cultures (Berlyn, 1978; Zelitch it is released as photorespiratory CO2 leading to elevated and Berlyn, 1982) but these studies apparently have not CO2 concentrations within the bundle-sheath. This gly- been continued. More recently, antisense plants with cine-to-serine conversion possibly provided one of the reduced contents of GDC subunits and SHMT were biochemical starting points for the evolution of C4 plants. studied (Heineke et al., 2001; Winzer et al., 2001; There are many other open questions related to the Bauwe et al., 1999). evolution of C4 from C3 via C3±C4 intermediate plants. For Barley mutants obtained by chemical mutagenesis example, some characteristics of C4-photosynthesis have Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 been reported for the cells surrounding the vascular Two mutants of barley, LaPr 85/55 and LaPr 87/30, were bundles in stems and petioles of C3 plants like tobacco isolated that grow well in 0.7% CO2 but accumulate or celery (Hibberd and Quick, 2002). It must also be glycine 5±10-fold relative to wild-type levels and show mentioned that the possible effects of a relocation of GDC reduced levels of glutamate and alanine in combination for the evolution of C4 plants are controversial (Monson, with rapid senescence when exposed to air (Blackwell 1999; Edwards et al., 2001). It cannot be ruled out that, in et al., 1990). These mutants behaved differently insofar different families, quite different evolutionary scenarios that LaPr 85/55 was able to metabolize much more 14C- led to the evolution of C4 plants. glycine into sugars than LaPr 87/30 after 2 h (70% and 4%, In the authors' opinion, the detailed analysis of the respectively). SHMT activity was not affected, however, physiological and adaptive implications caused by the the data corresponded well with reduced GDC activities presence of a primary CO2 concentrating mechanism (measured via the glycine±bicarbonate exchange reaction driven by GDC in the context of the photorespiratory cycle that needs H and P protein, but no T or L protein) with 70% is an important key for a better understanding of the wild-type activity with LaPr 85/55 and only 14% with evolution of C4 photosynthesis. To test this hypothesis LaPr 87/30, respectively. Protein blotting showed severely further it appears as an intriguing task to attempt a reduced levels of P and H protein (10% remaining) and a relocation of GDC in a C3 plant. Such experiments require slight reduction in T protein (50%). Supply of 40 mM at least two prerequisites. Firstly, a mutant that does not serine through the xylem stream was able to at least 14 contain endogenous GDC and, secondly, genes encoding partially (70%) restore wild-type CO2 ®xation rates for GDC subunits under the control of bundle-sheath speci®c both mutants. A mutation in a glycine transporter was promoters to supplement the mutant with a functional suggested for LaPr 85/55 (Blackwell et al., 1990) and a photorespiratory cycle. Appropriate GDC genes have been reduction in H protein down to 1% relative to wild-type has cloned and characterized from C3±C4 intermediate and C4 been shown for homozygous LaPr 87/30 plants (Wingler Flaveria species (Chu, 1996; Chu et al., 1998; Nan et al., et al., 1997). 1998; Nan and Bauwe, 1998; Cossu, 1997; Cossu and From a more detailed analysis of LaPr 87/30, including Bauwe, 1998). The current situation with respect to heterozygote lines, it was concluded that the biosynthesis available GDC defective mutants will be discussed below. and activity of GDC biosynthesis in vivo is determined by the biosynthesis of H protein. More speci®cally, P protein content in LaPr 87/30 heterozygous lines was reduced by Genetic manipulation of glycine decarboxylation 25% but GDC activity increased linearly with increasing H Historically, three programmes for the identi®cation of protein content. The authors also suggested that photo- photorespiratory mutants in chemically mutagenized seed respiratory carbon ¯ux is not restricted by GDC activity sets were performed. The ®rst mutant screen was devized (Wingler et al., 1997, 2000). for Arabidopsis thaliana. The analysis of corresponding Studies with LaPr 87/30 on a cellular level revealed mutants was very fruitful for a short time, but has not signi®cant changes in the redox status of the cells such as received very much attention during the last decade over-reduction and over-energization of chloroplasts (Somerville and Ogren, 1982a; Somerville, 1984, 2001). (Igamberdiev et al., 2001a). Surprisingly, these studies A second mutant screen was performed with barley also revealed a rate of glycine oxidation both in leaf (Kendall et al., 1983; Blackwell et al., 1988). The analysis cuttings and in intact mitochondria of 30±40% relative to of these mutants has continued over the years (Wingler the wild type. However, the authors could not exclude that et al., 2000). The third programme was directed towards this effect was due to growth of the plants under low light the C4 plant Amaranthus edulis (Dever et al., 1995; which might result in lower GDC levels in wild-type Wingler et al., 1999). Notably, immunocytochemical plants. In addition, the level of alternative oxidase was studies with this plant indicate that the cell-speci®c reduced. It was also shown that 13C/12C isotope fractiona- Manipulation of glycine decarboxylation 1529 Table 1. Summary of genes encoding GDC subunits or SHMT in A. thaliana (The Arabidopsis Genome Initiative, 2000) Designation of SHM1±SHM5 corresponds to the proposal by McClung et al. (2000). Direct experimental evidence for the predicted subcellular localization is not available.

Genes encoding Symbol MIPS-No. cDNA Encoded amino acids Subcellular localization References H protein AtGDH1 At2g35370 M82921 165 Mitochondrial (Srinivasan and Oliver, 1992) AtGDH2 At2g35120 AY056106 156 Mitochondrial AtGDH3 At1g32470 AF385740 166 L protein AtmLPD1 At3g17240 AF228639 507 Mitochondrial (Lutziger and Oliver, 2001) AtmLPD2 At1g48030 AF228640 507 Mitochondrial (Lutziger and Oliver, 2001) P protein AtGDP1 At4g33010 AY065004 1037 Mitochondrial AtGDP2 At2g26080 1044 Mitochondrial T protein AtGDT1 At1g11860 AY125509 408 Mitochondrial SHMT AtSHM1 At4g37930 AJ271726 517 Mitochondrial (McClung et al., 2000) AtSHM2 At5g26780 AY035117 517 Mitochondrial Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 AtSHM3 At4g32520 AF375450 529 Chloroplastic AtSHM4 At4g13930 AF361589 471 Cytosolic AtSHM5 At4g13890 470 Cytosolic AtSHM6 At1g22020 AY125514 599 Nuclear AtSHM7 At1g36370 AY084945 598 Nuclear

tion is higher in LaPr 87/30 relative to the wild type P protein is encoded by two genes, AtGDP1 and (Igamberdiev et al., 2001b). AtGDP2. The derived proteins are 90% identical to each The TIGR Barley Gene Index (http://www.tigr.org/tdb/ other. Two loci, gld1 (originally named glyD) and gld2, hvgi/index.html), at the time of this writing, includes two have been identi®ed by chemical mutagenesis (Somerville entries for tentative consensus sequences (TC) corres- and Ogren, 1982b; Artus et al., 1994). The major charac- ponding to H protein genes, TC8419 (green leaf prefer- teristics of gld1 were high accumulation of glycine under ence) and TC8850 (root and caryopsis preference). The normal air, no decline in glycine concentrations during a strong metabolic effects, as described above, suggest that following dark period, reduced rate of photosynthesis, no the gene corresponding to TC8419 is affected in LaPr 87/ glycine oxidation by isolated mitochondria, and no 30. It also indicates that the second gene is not able to take glycine±bicarbonate carbon exchange. By the osmotic- over the tasks, most likely because of its preferential swelling technique, no indication could be found for an expression in non-photosynthetic organs. impaired glycine transport into mitochondria (Somerville and Ogren, 1982b). The affected locus was mapped to Arabidopsis thaliana mutants obtained by chemical chromosome 2 about 40 cM from the er-py region (Artus and insertional mutagenesis et al., 1994). This chromosome harbours one gene About 20 years ago, the use of A. thaliana in a genetic encoding P protein, AtGDP2, and two genes encoding H approach to resolve controversial ideas about the mech- protein, AtGDH1 and AtGDH2, however, the mapped anism of photorespiration led to the identi®cation of a position of gld1 does not correspond with any of these loci number of mutants with defects in enzymes of the (Fig. 2). photorespiratory cycle (for a historical view compare Very similar to gld1, mutation of gld2 reduced glycine± Somerville, 2001). Unfortunately, as mentioned above, bicarbonate exchange rates by 70±80% and glycine following their initial characterization (Somerville and oxidation by isolated mitochondria by more than 90%. Ogren, 1982b, 1981), not much effort has been put into a The affected locus was mapped to chromosome 5 at a more detailed analysis of mutants where genes encoding distance of about 21 cM from tt3 (Artus et al., 1994). From GDC subunits or SHMT were affected. their biochemical data and under the assumption that gld1 The availability of the complete genome nucleotide represents a GDP locus, the authors hypothesized that the sequence of A. thaliana (The Arabidopsis Genome gld2 mutation most likely represents a defect in the H or T Initiative, 2000) revealed the existence of small multi- protein or in glycine transport into the mitochondria (Artus gene families for all GDC components, except T protein et al., 1994). However, the nucleotide sequence of which is encoded by a single gene, and SHMT (Table 1). chromosome 5 does not contain a gene for a functional This knowledge opens new opportunities for a closer GDC subunit. These data support the idea that glycine investigation of the genetics and transcriptional regulation transport instead of GDC biosynthesis could be affected as of corresponding genes, for example, during the ontoge- it has already been suggested as a possible alternative by netic development of A. thaliana. Artus et al. (1994). Unfortunately, knowledge about 1530 Bauwe and Kolukisaoglu 60% identical to the homologue protein encoded by AtGDH2. In promoter studies and other experiments with AtGDH1, transcriptional activation by light was shown (Srinivasan and Oliver, 1992). T protein is the only GDC subunit that is encoded by a single-copy gene in A. thaliana. This singular occurrence could indicate a central role of T protein in the regulation of GDC biosynthesis and might explain the, as yet unsuccessful, search for insertional mutants for this gene in this laboratory (UÈ Kolukisaoglu and H Bauwe, unpub- lished data).

An inspection of the A. thaliana genome sequence Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 reveals the presence of seven SHM genes in A. thaliana, AtSHM1 to AtSHM7 (Table 1; Fig. 2). For reasons of Fig. 2. Approximate positions of genes encoding GDC protein conformity, the designation of genes encoding SHMT as components and SHMT on Arabidopsis thaliana chromosomes 1 to 5. SHM will be adopted (instead of STM) as suggested by McClung et al. (2000). glycine transport into the mitochondria is very limited. Recent studies have shown that AtSHM1 expression is Although 20 years ago it was suggested that glycine/serine high in leaves with light inducibility, suggesting that antiporters might reside in the inner mitochondrial mem- SHM1 encodes a photorespiratory SHMT, and circadian brane (Walker et al., 1982) such transporters have not yet oscillations in transcript abundance. Similar to AtSHM1, been identi®ed (Oliver, 1994; Laloi, 1999). the expression of AtSHM2 is strongly induced by light in These data suggest that, most likely, neither gld1 nor leaves, but not in roots. AtSHM4 is expressed with low gld2 represent genes encoding GDC components. At least abundance only in roots and in ¯owers. This gene does not theoretically, similar metabolic effects as observed with show a light response but, like AtSHM1, shows circadian gld1 and gld2 could be induced, for example, by mutation oscillations as well (McClung et al., 2000; Ho et al., 1999). of the lipoate±protein ligase that is required for the Using a positional cloning approach, an A. thaliana SHM1 lipoylation of H protein at the e-amino group of a lysine mutant has been identi®ed (Renne et al., 2001). This residue. In A. thaliana, both a mitochondrial (LIP2, mutant is unable to grow under ambient conditions, but can At1g04640, Wada et al., 2001a) and a plastidic form be recovered under 1500 ppm CO2. Biochemical data are (LIP2p, At4g31050, Wada et al., 2001b) have been cloned not yet available. and characterized. However, due to their chromosomal Meanwhile, the complete cDNA sequence of AtSHM3 is location, these genes are clearly no candidates for the loci available from the RAFL project (Seki et al., 2002). From de®ned by mutations gld1 and gld2. these new data and the correction of the deduced N- L protein is encoded by four genes in A. thaliana, two of terminus, a plastidic targeting appears as more likely than each encoding plastidic (Lutziger and Oliver, 2000) and the formerly assumed cytosolic localization (McClung mitochondrial lipoamide dehydrogenases (Lutziger and et al., 2000). The proteins encoded by AtSHM6 and Oliver, 2001). Although the genes encoding mitochondrial AtSHM7 differ from those encoded by AtSHM1-AtSHM5 proteins are expressed in all organs, the isologue genes by long N-terminal extensions of more than 100 amino show distinctly different expression patterns both with acids. According to PSORT (http://psort.nibb.ac.jp) and respect to their organ preference and their response to TargetP (http://genome.cbs.dtu.dk/services/TargetP/) light. An insertional knockout mutant for AtLPD2 did not these proteins are candidates for becoming targeted to show any apparent morphological phenotypic change. By the nucleus. contrast to the unchanged CO2 release from pyruvate, a Three allelic A. thaliana putative SHM mutants have 14 14 25% reduction in CO2 release from [1- C]glycine was been isolated following chemical mutagenesis in Ogren's observed. It was suggested that the two proteins, once in laboratory (Somerville and Ogren, 1981). They showed the mitochondrial matrix, are interchangeable among the severe growth retardation under ambient air conditions different multienzyme complexes of GDC and a-ketoacid and, like the other photorespiratory mutants, plants had to dehydrogenases (Lutziger and Oliver, 2001). be grown under an elevated CO2 concentration. Total Knowledge about H and T proteins in Arabidopsis is SHMT activity in leaves was about 15% relative to the much more limited. With three members, H-protein is the wild-type total and the mutants were shown to be de®cient only GDC subunit that is encoded by a multigene family. both in glycine decarboxylation and in the conversion of In addition, a pseudogene exists on chromosome 5 glycine to serine. The authors concluded that these mutants (F26C17). Notably, the AtGDH1 and AtGDH3 encoded do not possess any mitochondrial SHMT at all. However, proteins are 92% identical to each other but only about this conclusion could not be supported by more recent data Manipulation of glycine decarboxylation 1531 which indicate that the level of SHM1 transcripts is locus is known. By contrast, antisense or RNAi-based unaltered in the stm mutant (Beckmann et al., 1997). The approaches allow the evaluation of more general effects. locus affected in this mutant (Nottingham Stock Centre Such an approach is useful if no mutants are available, for N8010) has been mapped to chromosome 5 (A Weber, example, due to their lethality, or if the intended studies personal communication). More recent ®ne mapping data concern multigene families. support this result but, surprisingly, indicate that the stm Transgenic potato plants with about 60±70% less P locus is probably not related to SHM2 or to any other of the protein relative to wild-type potato plants and a corres- seven SHM genes in A. thaliana (Schilling et al., 2001). ponding decrease in the ability of leaf mitochondria to This supports the view that the stm mutation might affect a decarboxylate glycine were indistinguishable from wild- locus that is required for SHMT activity, but is distinct type plants when grown under 800 ppm CO2 (Heineke from SHM loci encoding SHMT protein (McClung et al., et al., 2001; Winzer et al., 2001). When grown under

2000). ambient CO2 and moderate light, there were no clear Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 Taken together, the mutant data discussed above, phenotypic changes, except the early senescence of older especially those related to P and L protein of GDC and leaves. Photosynthetic and growth rates were reduced, but those related to SHMT in A. thaliana, raise several the plants were viable under ambient air and produced questions. First of all, the two loci gld and smt are probably tubers. Glycine concentrations, especially in fully ex- only indirectly related to the proper function of GDC and panded leaves, were elevated by up to about 100-fold SHMT. Nevertheless, the quite massive effects of the during illumination. Notably, nearly all of the glycine respective mutations indicate important, but as yet accumulated during the day in leaves of the antisense unknown, functions in glycine±serine metabolism. potato plants was metabolized during the following night. Secondly, P protein, mitochondrial L protein and mito- This was accompanied by distinctly increased levels of chondrial SHMT are all encoded by two genes in A. serine at the end of the night. thaliana. It is not known, whether the respective genes are Similarly, leaves of transgenic potato plants with equally important or, alternatively, whether they serve severely reduced amounts of SHMT contained up to 100- different functions in different organs or developmental fold elevated levels of glycine relative to the wild type. contexts. Photosynthesis rates were reduced and the degree of this The recent publication of a draft sequence of the rice reduction was correlated with glycine levels, i.e. with the genome (Yu et al., 2002; Goff et al., 2002) opened the reduction in SHMT activity. These negative effects on opportunity to compare the number and structure of GDC growth were greatly elevated by higher light intensity. and SHMT encoding genes, respectively, in a monocoty- Two lines were unable to grow in ambient air even under ledonous plant. Searches for GDC and SHMT encoding moderate light intensity but could be recovered in 2000 ml ±1 sequences in the genome of rice and comparisons to their l CO2 (Bauwe et al., 1999). orthologues in A. thaliana revealed two important insights. Collectively, the data obtained with transgenic plants First, the genomic structure of these genes and the deduced suggest that the photosynthetic±photorespiratory metabol- protein structures are very similar in both species. Second, ism of potato plants responds ¯exibly to limited changes in and perhaps more important, the number of rice homo- the capacity of leaves to decarboxylate glycine. GDC logues to the A. thaliana GDC and SHMT genes seems to seemingly operates far below substrate saturation in wild- be roughly equal. For instance, only one orthologue of type plants held under `normal' conditions. This provides AtGDT, a single copy gene in A. thaliana, was found in the the opportunity to respond rapidly to enhanced rates of rice genome. There are also seven rice OsSHM genes with photorespiration as they occur during increased tempera- exon±intron boundaries identical to those found in the tures or under conditions of stomatal closure during different AtSHM genes (data not shown). Due to the periods of insuf®cient water supply. Under such circum- preliminary character of the rice genome sequence these stances, perhaps much like transgenic plants with moder- data just represent estimations. However, it can be already ately reduced GDC activity, GDC operates under higher concluded that the information about content and organ- saturation with glycine, thus achieving a similar steady- ization of GDC and SHMT genes extracted from the A. state throughput as during normal photosynthesis. At least thaliana genome is transferable to a cereal. On the basis of in potato, GDC exerts high control over the level of this knowledge from two distantly related model plants it is glycine, but only low control over the ¯ux rates through the likely that a similar organization of photorespiratory genes interconnected cycles of photosynthesis and photorespira- exists in other angiosperms, too. tion (Heineke et al., 2001).

Plants with reduced content of GDC subunits or SHMT by antisense approaches Conclusions Mutational approaches provide the possibility to study the The glycine±serine interconversion, catalysed by GDC and function of individual genes as soon as the mutagenized SHMT, is an important reaction of primary metabolism in 1532 Bauwe and Kolukisaoglu all organisms including plants. Quite generally, this two P-isoproteins of the glycine cleavage system from Flaveria reaction provides one-carbon units for many biosynthetic pringlei. European Journal of Biochemistry 234, 116±124. Bauwe H, Keerberg O, BassuÈner R, PaÈrnik T, BassuÈner B. 1987. reactions. In plants, in addition to this general role in Reassimilation of carbon dioxide by Flaveria (Asteraceae) metabolism, it is an integral part of the photorespiratory species representing different types of photosynthesis. Planta metabolic pathway in which glycine is produced with high 172, 214±218. rates from Calvin cycle intermediates and converted into Bauwe U, Danek F, Driscoll S, Bauwe H. 1999. Effects of reduced serine within the mitochondria. Large amounts of photo- SHMT activities on photosynthesis and photorespiration in potato leaves. Proceedings of the XVI International Botanical Congress, respiratory CO2 are produced by this plant-speci®c path- Saint Louis, USA. Abstract 3300. way. Several lines of evidence suggest that this latter Beckmann K, Dzuibany C, Biehler K, Fock H, Hell R, Migge A, process, by relocation of GDC from one leaf-cell type (the Becker TW. 1997. Photosynthesis and ¯uorescence quenching, mesophyll) to another (the bundle-sheath) contributed to and the mRNA levels of plastidic glutamine synthetase or of mitochondrial serine hydroxymethyltransferase (SHMT) in the

the evolution of C plants. Although this hypothesis is Downloaded from https://academic.oup.com/jxb/article/54/387/1523/540368 by guest on 26 September 2021 4 leaves of the wild-type and of the SHMT-de®cient stm mutant of controversial (compare Edwards et al., 2001), it is Arabidopsis thaliana in relation to the rate of photorespiration. regarded as most likely that changes in the intercellular Planta 202, 379±386. distribution of GDC are capable of signi®cantly in¯uen- Berlyn MB. 1978. A mutational approach to the study of photorespiration. In: Siegelman HW, Hind G, eds. cing the concentration of CO2 within the respective cells. If this is true, they will unavoidably modify the relative rates Photosynthetic carbon assimilation. London, New York: Plenum Publishing Corporation, 153±164. of carbon ¯ux into the photosynthetic carbon reduction Besson V, Neuburger M, Rebeille F, Douce R. 1995. Evidence for cycle and the photorespiratory carbon oxidation cycle thus three serine hydroxymethyltransferases in green leaf cells: in¯uencing the ef®ciency of photosynthesis. Is this in¯u- puri®cation and characterization of the mitochondrial and ence on the overall ef®ciency of photosynthesis very minor chloroplastic isoforms. Plant Physiology and Biochemistry 33, or is it perhaps of greater signi®cance? In light of the 665±673. Blackwell RD, Murray AJS, Lea PJ. 1990. Photorespiratory progress made with the analysis of genes and mutants mutants of the mitochondrial conversion of glycine to serine. related to photorespiratory processes and the cloning of Plant Physiology 94, 1316±1322. GDC genes from C3±C4 intermediate plants this question Blackwell RD, Murray AJS, Lea PJ, Kendall A, Hall NP, now can be targeted. Turner JC, Wallsgrove RM. 1988. The value of mutants unable to carry out photorespiration. 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