An intermediate form of NADP-malic enzyme from the C3 C4 intermediate species Flaveria floridana

Plant Science 147 (1999) 101 – 109 www.elsevier.com/locate/plantsci

An intermediate form of NADP-malic enzyme from the C3 C4 intermediate species Fla6eria floridana Paula Casati, Analia G. Fresco, Carlos S. Andreo *, Marı´a F. Drincovich Centro de Estudios Fotosinte´ticos y Bioquı´micos (CEFOBI), Uni6ersidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina Received 9 March 1999; received in revised form 19 May 1999; accepted 20 May 1999

Abstract NADP-malic enzyme (NADP-ME) characterization and compartmentation between the mesophyll and bundle sheath cells was studied in the C3 –C4 intermediate species of Fla6eria floridana. Although three immunoreactive bands were found in crude leaf extracts, only one isoform was purified to homogeneity, probably due to the low specific activity of the other forms and/or differential susceptibility to proteases’ action. The purified enzyme showed intermediate kinetic features between those from C3 and C4 plants, although the molecular mass was more like that for the enzyme found in C4 plants. The amino terminal sequence of the purified enzyme, despite some changes, was similar to the deduced protein sequence obtained from different Fla6eria NADP-ME cDNA clones although the sequence did not follow the proposed cleavage site of the transient peptide. Localization studies of certain photosynthetic enzymes between the mesophyll and bundle sheath cells indicated that phosphoenolpyruvate carboxylase (PEPC) as well as photosynthetic isoforms of NADP-ME are partially compartmentalized as in C4 plants, while RuBisCO is present in both types of cells. In this way, the intermediate features of the C3 –C4 species F. floridana in relation to C4 plants may be due not only to the lack of compartmentation of RuBisCO, but also to the presence of C4 enzymes with kinetic intermediate features. It is suggested that during the evolution of C4 photosynthesis not only changes in the level of expression and compartmentation of key photosynthetic enzymes have occurred, but also the development of new photosynthetic enzymes with C4-like kinetic features. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: NADP-malic enzyme; Fla6eria floridana; C3 –C4 intermediate; C4 evolution

1. Introduction C3 – C4 intermediate species are thought to represent a stage in the evolutionary transition from the C3 to the C4 photosynthetic mechanism [1–3]. The species reported to be C3 –C4 intermediates belong to different genera, which include both C3 and C4 species, or C3 and C3 –C4 intermediates. All of the intermediate species exhibit some common anatomical and physiological traits, the most important one being the reduced photorespiration rate with CO2 compensation points considerably lower than those observed in C3 species. Two mechanisms are proposed to account for the low * Corresponding author. Tel.: +54-341-4371955; fax: + 54-3414370044. E-mail address: [email protected] (C.S. Andreo)

apparent photorespiration in these intermediate species. In one, which may be common to all intermediates, photorespiratory metabolites generated as a consequence of the ribulose-1,5-bisphosphate carboxylase–oxygenase (RuBisCO) oxygenase reaction in mesophyll cells (MC) are metabolized in bundle sheath cells (BSC) where glycine decarboxylase is located, and the CO2 released by this enzyme is refixed by RuBisCO. In this way, there is a reduced loss of CO2 by photorespiration which occurs without the operation of a C4 cycle. In the other class of intermediates, the operation of a limited C4 cycle between MC and BSC contributes to the further reduction of photorespiration. The genus Fla6eria contains a more or less continuous range of species between those which

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have C3 and C4 metabolism [4]. Among the C3 –C4 intermediate Fla6eria species differences in the level of integration between co-functioning C3 and C4 cycles were identified. On the basis of leaf gas-exchange characteristics [5,6], leaf ultrastructure [7], pulse chase study [6] and carbon-isotope discrimination [8]; Fla6eria ramosissima was identified as having a relatively high degree of integration between the C3 and C4 cycles, Fla6eria linearis as having less well-developed characteristics and Fla6eria floridana, Fla6eria pubescens and Fla6eria anomala as species exhibiting characteristics indicating a level of C3- and C4-cycle integration between these two extremes. In the present report, the intermediate specie F. floridana was used to characterize different isoforms of NADP-malic enzyme (NADP-ME) and to evaluate their expression in different photosynthetic cell types. In previous studies, we detected three isoforms of this enzyme in various Fla6eria intermediate species and suggested that their occurrence is correlated with the C4 pathway evolution [9]. In the past, F. floridana has been one of the better studied C3 –C4 intermediates, in terms of ultrastructure [7], physiology [10,11] and photosynthetic characteristics in its natural habitat [12], which all indicated higher photosynthetic rates relative to C3 plants due to a reduced rate of photorespiration. So, this species possesses both an effective capture of photorespired CO2 and reassimilation by the C3 cycle, demonstrated by evidence that glycine decarboxylase is confined to BSC [13] and by a limited capacity of this species to concentrate CO2 at the active site of RuBisCO, probably through a partial C4 pathway activity [14]. However, this phenomenon has not yet been totally characterized at the biochemical level.

2. Materials and methods

2.1. Plant material F. floridana was propagated vegetatively from shoot cuttings and grown in a compost–sand–perlite mixture (2:1:1 v/v/v). The plants were grown in a greenhouse at a 25/18°C day/night thermoperiod and a 13 – 16 h photoperiod. The third and fourth pairs of leaves from the apex were used for protein preparations and protoplast purification.

2.2. Protein extraction and gel electrophoresis Total protein from the different tissues and cellular fractions was extracted using a buffer containing 100 mM Tris–HCl (pH 7.0), 1 mM EDTA, 10 mM MgCl2, 10 mM b-mercaptoethanol, 10% (v/v) glycerol and 2 mM PMSF. After centrifugation, the extract was used for activity measurements or diluted in 0.25 M Tris– HCl (pH 7.5), 2% (w/v) SDS, 0.5% (v/v) b-mercaptoethanol and boiled for 2 min for SDSPAGE. For the analysis of protein samples by Western blotting, 10% (w/v) polyacrylamide gels containing SDS were used. After electrophoretic separation, the proteins on the gels were electroblotted onto a nitrocellulose membrane for immunoblotting according to Burnette [15]. Affinity purified antimaize 62 kDa NADP-ME IgG (diluted 1:100) was used for detection [16,17]. A serum against RuBisCO spinach large subunit was diluted 1:10 000. Bound antibody were visualized by linking to alkaline phosphatase-conjugated goat anti-rabbit IgG, according to the manufacturer’s instructions (Promega). The molecular masses of the polypeptides were estimated from a plot of the log of molecular mass of marker standards versus migration distance. Protein concentration was determined by the method of Sedmak and Grossberg [18] using bovine serum albumin (BSA) as a standard.

2.3. Purification of NADP-ME from lea6es and measurement of kinetic parameters The protocol used for purification was similar to that described previously for the enzyme from maize [19] with the following modifications: (a) the crude extract was precipitated with 30–70% (NH4)2SO4; (b) the affi-gel blue column was omitted; (c) the enzyme eluted from the hydroxylapatite column was further purified by a Superose 12 HR 12/30 conected to an FPLC system (Pharmacia). The purification process was followed by NADP-ME activity determined at 30°C by monitoring NADPH production at 340 nm with a reaction medium containing 50 mM Tris–HCl (pH 7.5), 0.5 mM NADP, 4 mM L-malate and 10 mM MgCl2.

P. Casati et al. / Plant Science 147 (1999) 101–109

Initial velocity studies were performed by varying the concentration of one of the substrates around its Km, while keeping the other substrates concentrations at saturating levels (0.5 mM NADP, 4 mM L-malate and 10 mM MgCl2). Km values of the substrates were calculated in terms of free concentrations by linear regression in all cases. Different buffer systems were used when analyzing the enzyme activity as a function of pH: 50 mM Mes (pH 5.5–6.5), 50 mM Tricine–MOPS (pH 7.0) and 50 mM Tris–HCl (pH 7.5–8.5). The reaction was started by the addition of L-malate. One international (U.) is defined as the amount that catalyses the formation of 1 mmol NADPH/ min under specified conditions.

2.4. Protein sequencing For the amino-terminal sequence analysis, the purified 62 kDa NADP-ME was run on a 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membrane according to Matsudaira [20]. The sequence was determined using an Applied Biosystems 477A protein sequenator by Dr Laurey Steinke from the Protein Core Structure Facility, Department of Biochemistry, University of Nebraska, Omaha, NE.

2.5. Isolation and purification of leaf protoplasts Protoplasts of three type of cells were previously isolated from leaves of the C4-like specie F. brownii [21,22] and the C3 –C4 intermediate specie F. ramossisima [23]. In the present work, using modifications of these procedures previously described, we also obtained three types of protoplasts from F. floridana leaves called P1, P2 and P3 which differ in cellular density. About 5 g of young leaves (third and fourth pairs) were harvested, sliced into segments (0.5× 10 mm) and digested for 4 h at 25°C in a medium containing 2.5% Cellulase Onozuka R-10, 0.3% Macerozyme R-10 (Yakult Biochemical, Nishinomiya, Japan), 0.5 M mannitol, 10 mM Mes– KOH (pH 5.5), 1 mM CaCl2 and 0.5% BSA. All media used in isolating and purifying protoplasts were the same as in Moore et al. [23], unless otherwise indicated. After filtering through a 230 mm aperture net and washing the leaf segments in 0.5 M manitol, 5 mM Hepes–KOH (pH 7.0), 1 mM CaCl2, 0,2% BSA three times for 20 min, the

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filtrate was centrifuged at 200 ×g for 1 min. The supernatant contained only mesophyll protoplasts (P1), while two types of protoplast fractions were further purified from the pellet, which probably correspond to more dense mesophyll protoplasts (P2) and bundle sheath protoplasts (P3). For purification of P1, the supernatant was diluted with KCl medium, centrifuged at 200×g for 3 min, and the pellet obtained diluted in 2 ml 16% dextran medium and overlayered with 2 ml 3% dextran medium and 2 ml KCl–mannitol medium. Mesophyll protoplasts (P1) were collected from the interface between KCl–mannitol and 3% dextran media. The pellet containing bundle sheath and mesophyll protoplasts was dispersed in 4 ml 16% dextran medium, divided into two tubes and overlayered with 2 ml of 12, 3 and 0.5% dextran media and top layered with KCl–mannitol medium [23]. The bundle sheath protoplasts (P3) floated between the 12 and 3% dextran media and the mesophyll protoplasts (P2) were recovered from the 0.5% dextran and KCl–mannitol media interface. All the fractions were diluted with KCl medium [23] and concentrated by centrifugation at 200×g for 30 s. The three protoplast fractions obtained were examined under a light microscope and distinguished visually by differences in size and in organelle content and distribution. The final pellet of each fraction was resuspended in 100 mM Tris–HCl (pH 7.0), 1 mM EDTA, 10 mM MgCl2, 10 mM b-mercaptoethanol, 20% (v/v) glycerol and 2 mM PMSF and stored in liquid N2. The three protoplast preparations obtained were analyzed for PEPC, NADP-MDH and NADPME activity and Western blotting analysis as previously described. The reaction medium used for activity measurements were: (a) PEPC: 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2, 10 mM KHCO3, 4 mM PEP, 0.15 mM NADH and 10 I.U. MDH; (b) NADP-MDH: (samples were preincubated in 100 mM DTT for 2 h) 50 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.25 mM NADPH and 1 mM OAA; and (c) NADP-ME: 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 0.5 mM NADP and 4 mM L-malate.

2.6. Chemicals NADP, L-malic acid, Tris (Tris (hydroxymethyl) aminoethane), Mes (2-(N-morpholino)ethanosulfonic acid), Tricine (N-tris (hydroxymethyl)

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methylglycine); N-(2-hydroxy-1,1-bis (hydroxymethyl) ethyl) glycine), MOPS (3-(N-morpholino)propanesulfonic acid), Hepes (N-(2hydroxyetghyl)iperazine-N%-2-ethanesulfonic acid), BSA, Dextran (average mol. wt. 39 200) and molecular weight standards for SDS-PAGE were purchased from Sigma (St. Louis, MO, USA). Reagents for Western blotting assays were provided by Promega. PVDF and the nitrocellulose membranes were from BioRad. All other reagents were of analytical grade.

3. Results

3.1. Characterization of purified NADP-ME from F. floridana lea6es Leaf extracts from C3 –C4 intermediate Fla6eria species have higher NADP-ME activity (i.e. F. floridana 0.0776 I.U./mg total protein) than C3 Fla6eria species (i.e. F. pringlei 0.0129 I.U./mg total protein) and lower than C4-like and C4 plants (i.e. F. triner6ia 0.8470 I.U./mg total protein). We have recently shown that this activity is due to different isoforms of NADP-ME that differ in both molecular mass and native isoelectric point [9]. Crude leaf extracts from the C3 –C4 intermediate species F. floridana was found to have three activity bands by isoelectric focusing gel electrophoresis, while Western blotting analysis of SDS-PAGE indicated the presence of three immunoreactive bands of 62, 64 and 72 kDa ([9] and

Fig. 1. (A) Western blot, using maize NADP-ME antibodies, with crude extracts of F. floridana leaves (30 mg) (1) and the purified NADP-ME isoform obtained after the purification protocol (1 mg) (2). (B) Coomassie blue staining of mol. wt. markers (1) and the purified NADP-ME from F. floridana (5 mg) (2). The molecular masses of the markers are indicated in the figure.

Fig. 2. Comparison of amino-terminal sequence from NADPME from different species of Fla6eria. The letters in bold are areas of differences.

Fig. 1A). After studying 13 different species of Fla6eria, we found that the 62 kDa isoform was highly expressed in Fla6eria species expressing C4 photosynthesis, the 72 kDa form was constitutively expressed in all Fla6eria species regardless of the metabolism used for photosynthetic carbon assimilation, and the 64 kDa form was highly expressed in some C3 –C4 and C4-like species [9]. In order to characterize the NADP-ME isoforms in F. floridana leaves a purification protocol (see Section 2) was developed. The purification process was followed by monitoring NADP-ME activity, and the purified protein obtained at the end of the protocol had a molecular mass of 62 kDa, as revealed by Western blotting and Coomasie blue staining (Fig. 1). The enzyme was purified 193-fold, to a final specific activity of 15 I.U./mg protein. The 64 and 72 kDa isoforms could never be obtained although changes in the protocol were performed. In the protocol presented here they were both lost at the saline fractionation step. The inability to purify these proteins may be due to the low specific activity of both proteins, although the intensity of the bands corresponding to these isoforms observed in Western blots on crude extracts using maize NADPME antibodies is stronger than the band for the 62 kDa isoform (Fig. 1A). Another reason for not being able to purify these proteins may be due to their degradation (more susceptible to proteases than the 62 kDa enzyme). The purified malic enzyme from F. floridana leaves was blotted onto a PVDF membrane and the first 10 amino acids of the 62 kDa protein were sequenced. The amino-terminal sequence obtained is indicated in Fig. 2, together with the amino-terminal sequences of NADP-ME from other Fla6eria species. Although some differences were found, this sequence matches NADP-ME from F. triner6ia (C4) [24], F. pringlei (C3) [25] and two

P. Casati et al. / Plant Science 147 (1999) 101–109

isoforms from F. bidentis (C4) [26]; which were all deduced from their corresponding cDNAs (Fig. 2). Borsch and Westhoff [24] postulate, based on the rules of Gavel and von Heijne [27], that the cleavage site of F. triner6ia NADP-ME may be located at residue 61 of the protein. Nevertheless, the F. floridana NADP-ME protein sequence presented here corresponds to residues 72–81 of the deduced primary sequence of the F. triner6ia enzyme. In this way, the processing site and thus, the amino acid composition of the transit peptide could be different in F. floridana than in other NADP-ME. However, some degradation of the protein that we obtained during the purification process can not be ruled out. The kinetic parameters of the purified NADPME from F. floridana were determined. Specific activity of 15 I.U./mg was found for the purified protein, a value that is intermediate between those typically found for C3 and C4 plants (Table 1). At saturating concentrations of the substrates, the dependence of activity on pH revealed a broad maximum of activity centered at pH 7.5, a value similar for the enzymes from C3 and CAM plants [28]. Km values for L-malate and NADP were intermediate between values for C3 and C4 plants (Table 1) [28]. The enzyme was not inhibited by the substrate L-malate at pH 7.0 (not shown), which is different from the behavior observed for NADP-MEs from C4 plants [28] but in a similar way to reports for the enzyme from C3 plants (Ref. [29] and Casati et al. unpublished results). For Mg2 + and Mn2 + , non-linear double reciprocal plots were obtained, suggesting two binding sites for the metal cofactor in the enzyme, as previously described for other NADP-ME isoforms [16,29,30].

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These results indicate that NADP-ME from the C3 –C4 species F. floridana presents unique intermediate kinetic characteristics between those of NADP-ME from C3 and C4 plants, although the molecular mass of the protein is more like that of the enzyme from C4 plants.

3.2. Localization of NADP-ME isoforms in MC and BSC from F. floridana lea6es In order to study the localization of NADP-ME immunoreactive isoforms that differ in abundance in the different photosynthetic types of Fla6eria species [9] we purified mesophyll and bundle sheath protoplasts from F. floridana leaves, where three immunoreactive forms of NADP-ME are expressed (Fig. 1). As in the case of F. ramossisima [23] and F. brownii [21,22], three purified types of cells were obtained in a discontinuous dextran gradient (see Section 2), which were different when examined under a light microscope. Bundle sheath protoplasts had a higher density (with more chloroplasts) than did most of the mesophyll protoplasts, and were visually distinguishable from the two mesophyll preparations (not shown). Although both type of mesophyll protoplasts were less dense, they differed in size; and the denser fraction presented chloroplasts which occupied more surface area. These fractions were called P1, P2 and P3 when ordered by increasing density, corresponding to mesophyll (P1 and P2) and bundle sheath (P3) protoplasts. The activity (I.U./mg protein) of different photosynthetic enzymes implicated in the C4 pathway (NADP-ME, PEPC and NADP-MDH) was measured in each protoplast fraction. The results (Table 2) indicate that P3 and P2 have, respec-

Table 1 Kinetic parameters of NADP-ME purified from the C3–C4 intermediate F. floridana and comparison with NADP-ME from C4 (maize) and C3 (wheat) plantsa Kinetics properties

Maize (photosynthetic tissues)

Wheat

F. floridana

Optimal pH

7.8–8.4 pH 8.0

7.2 pH 7.5

7.5 pH 7.5

Vmax (I.U./mg) Km NADP+ (mM) Km Mg (mM) Km Mn (mM) Km malate (mM) Ki malate (mM)

30.9 8.6 0.23–0.05 1100 0.18 ni (pH 8.0) 4.3 (pH 7.0)

0.98 37 0.20–0.006 0.56–0.066 0.96 ni

15 12 0.16–0.005 0.67–0.03 0.46 ni

a

Maize and wheat NADP-ME data was taken from Ref. [29]. ni, No inhibition was observed.

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Table 2 Activity measurement of NADP-ME, PEPC and NADP-MDH in the three protoplast fractions (P1, P2 and P3) obtained from F. floridana leavesa Fraction

P1 P2 P3

NADP-ME

PEPC

NADP-MDH

I.U./mg

Relative to P1

I.U./mg

Relative to P3

I.U./mg

Relative to P3

0.094 0.325 0.702

1.0 3.5 7.5

0.550 0.160 0.052

10.6 3.1 1.0

0.107 0.096 0.064

1.7 1.5 1.0

a

The purity of each fraction was checked with light microscope observation. Values are means of three preparations, with S.D.s less than 0.01 in all cases.

tively, 7.5 and 3.5 more NADP-ME activity than P1. On the other hand, P1 was enriched in PEPC and NADP-MDH activities, although the compartmentation of PEPC activity was higher than that of NADP-MDH (the ratio of activity between P1:P3 was 10.7 for PEPC and 1.6 for NADPMDH). In order to know which NADP-ME molecular mass form was responsible for NADP-ME activity in each protoplast fraction Western blot analysis was performed with the three samples obtained (Fig. 3A). The results show that the three types of cells express different NADP-ME molecular mass immunoreactive polypeptides, P3 and P2 contain the three isoforms of NADP-ME present in crude extracts of total leaf (72, 64 and 62 kDa), while P1 contains only the 72 kDa isoform. The same protein samples of P1, P2 and P3 were also used to study the cellular localization of RuBisCO, using antibodies against the spinach large subunit (Fig. 3B). The results (Fig. 3B) show that RuBisCO was not restricted to any cell type in this Fla6eria C3 – C4 intermediate species. Moreover, it was present in the three protoplast fractions at almost the same level. In summary, both the activity measurement and Western blotting of the three fractions obtained (P1, P2 and P3) indicate that NADP-ME isozyme expression is cell-specific in this species, with P3 (bundle sheath protoplasts) presenting higher activity and selectively expressing the 62 and 64 kDa forms. The activity measured in P1 (mesophyll protoplasts) apparently corresponds to the 72 kDa form of the enzyme, which is the only form found by Western blotting analysis of this fraction. PEPC activity was also partially differentially distributed among the different cell types, as revealed by activity measurements of the three fractions obtained (Table 2). On the other hand, RuBisCO

has an even distribution as revealed by Western blotting analysis (Fig. 3).

4. Discussion In the present study one isoform of NADP-ME from a Fla6eria C3 –C4 intermediate species was purified to homogeneity. This enzyme showed intermediate kinetic characteristics between those of NADP-ME from C3 and C4 plants, although the molecular mass was very close to that of the enzyme typically found in C4 plants. These results indicate that the purified NADP-ME present in this C3 –C4 intermediate is a unique isoenzyme entity that evolved specifically for the partial C4 pathway present in this species, rather than being derived by enhanced expression of genes already present in the C3 ancestors. On the other hand, the enzyme does not present all the characteristic features of the enzyme found in plants with a complete C4 pathway.

Fig. 3. Western blotting of protein samples (A, 30 mg; B, 5 mg) from total leaf extract (1), P1 (2), P2 (3) and P3 (4) revealed against maize NADP-ME (A) and spinach RuBisCO large subunit (B) antibodies. The calculated molecular mass of the immunoreactive proteins is indicated in the figure.

P. Casati et al. / Plant Science 147 (1999) 101–109

Immunofluorescence techniques previously used to study the localization of RuBisCO and PEPC in F. floridana leaves [31] have shown no strict compartmentation of either enzymes between MC and BSC. Nevertheless, in the present report the study of compartmentation of photosynthetic enzymes in MC and BSC shows that some enzymes are implicated in the C4 cycle, PEPC as well as photosynthetic forms of NADP-ME, are partially compartmentalized as in C4 plants; however, the enzyme RuBisCO is present in both MC and BSC. It is possible that the PEPC protein found in BSC by the immunofluorescent technique may not be functional or may have low specific activity. Relative to the activity of NADP-MDH, we found it to be less compartmentalized than NADP-ME and PEPC in F. floridana. In agreement with our results, Moore et al. [32] reported the presence of NADP-MDH in both mesophyll and bundle sheath chloroplasts; and they suggested that it could be involved in the metabolism of aspartate as a cartbon donor in bundle sheath chloroplasts. With regards to RuBisCO, our results showing the localization of this protein in both MC and BSC are consisting with previous studies which show that F. floridana is capable of incorporating atmospheric CO2 through both C3 and C4 pathways of photosynthesis [6]. Moreover, anatomical and ultrastructural studies of F. floridana leaves revealed that the leaf mesophyll of this plant is typical of that of dicotyledonous C3 plants, but the bundle sheath cells contain granal, starch-containing chloroplasts [7]. The compartmentation of photosynthetic proteins suggests that both photosynthetic pathways are operative within the leaf mesophyll of F. floridana with the C3 system being unconfined and not limited to the BSC as in typical C4 plants. Previous analysis of CO2 compensation points and apparent measures of photorespiration revealed that F. floridana is closely related to C4 plants, although the ultrastructure, leaf anatomy, and the extent of CO2 assimilation into C4 acids and the turnover of part of the C4 acid pool indicates that it is not a true C4 species [7,33]. Particularly, this species has a low efficiency in the transfer of carbon from the C4 to the C3 cycle relative to species with a fully expressed capacity for C4 photosynthesis, low quantum yields and apparent futile C4 pathway cycling of carbon. Although not confirmed, these characteristics were

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attributed to the possibility that this species lacks the typical compartmentation of RuBisCO, PEPC and NADP-ME found in C4 plants [6]. The present report indicate that of these enzymes the only one that is not selectively compartmentalized is RuBisCO, whereas PEPC and NADP-ME have both a high degree of compartmentation. Nevertheless, while the purified isoform of NADP-ME implicated in C4 photosynthesis in this study is practically totally confined to BSC, we demonstrate that its kinetic features are different from the enzyme in C4 plants. This evidence may indicate that not only the compartmentation of an enzyme is necessary for the C4 cycle but also the presence of C4-like kinetic characteristics, among which the most important may be a high specific activity, a low Km for substrates and the typical pH optimum characteristic of the C4 isoform in C4 plants. In the case of PEPC, there is also evidence from some kinetic studies on crude extracts from F. pubescens and F. linearis that this enzyme possesses, at least in these two intermediate species, kinetic properties between those of the enzyme from C3 and C4 plants [34]. Nevertheless, other results suggested that C3 and C3 –C4 intermediate Fla6eria species express PEPC with essentially identical kinetic and regulatory properties [35], and that the electrophoretic/molecular properties of PEPC from F. cronquisti (C3) are very similar, if not identical, to those of F. floridana [36]. Moreover, the light-activation of PEPC found in F. floridana was not attributed to changes in the target enzyme per se, but rather to the level of the protein kinase, protein phosphatase and/or darklight signal(s) [33]. These results indicate that in the case of F. floridana, the evolution of C4 photosynthesis is not necessarily paralleled by a dramatic change in the kinetic or structural properties of PEPC, although our results indicate that such changes have in fact occurred in the enzyme NADP-ME. With regards to the 72 kDa immunoreactive protein, it was previously described as a non-photosynthetic form, as it occurs in non-photosynthetic tissues and in photosynthetic tissues of C3, C3 –C4 and C4 Fla6eria species [9]. The present work reinforces this idea, as this protein is located in both MC and BSC of F. floridana leaves, and is the only form found in MC. With respect to the 64 kDa immunoreactive protein, previous work indicated that it was more abundant in intermediate

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and C4-like species [9]. The results presented here indicate that it is probably a photosynthetic enzyme that may be associated with the C4 cycle. Thus, it is possible that, in Fla6eria, the evolution from the C3 to the C4 isoform of NADP-ME involved the transient presence of a 64 kDa form with cell specific expression. In summary, this study indicates that the intermediate species F. floridana expresses a key photosynthetic enzyme of the C4 cycle with kinetic features between those of C3 and C4 plants. This isoform is also expressed in a cell-specific way, suggesting that the selective intercellular targeting of C4 enzymes such as NADP-ME may be one of the first steps in the evolution to C4 photosynthesis, which may occur even before selective RuBisCO compartmentation. It is also suggested that during the evolution of C4 photosynthesis, not only changes in the level of expression and compartmentation of key photosynthetic enzymes have occurred, but also new photosynthetic enzymes with different structure have been developed that would allow particular C4-like kinetic features.

Acknowledgements The authors thank Dr Ryuzi Kanai for providing Cellulase Onozuka R-10 and Dr Alejandro M. Viale for providing anti-(spinach large subunit RuBisCO) immune serum. This research was supported by grants from the Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET, Argentina) to CSA and MFD. PC is a fellow from CONICET and MFD and CSA are members of the Researcher Career of the same Institution. CSA acknowledges the Rockefeller Foundation for a residency at the Foundation’s Study and Conference Center in Bellagio, Italy.

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