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Immunolocalization of maize transglutaminase and its substrates in plant cells and in Escherichia coli transformed cells M. Santos*,1, E.Villalobos1, P.Carvajal-Vallejos1, E. Barberá3, A. Campos2 and J.M.Torné1 1 2 3

Department of Molecular Genetics, Laboratori de Genetica Molecular Vegetal, Consorci CSIC-IRTA. IBMB. Jordi Girona 18-26. 08034 Barcelona, Spain Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av da Republica, Apt. 127, 2781-901Oeiras, Portugal Grup de Quimica Biològica I Biotecnologia, Institut Quimic de Sarrià. Universitat Ramon Llull. 08017 Barcrelona. Spain

Plant transglutaminases (TGases) are poorly characterized in contrast to mammalian members of the super family. Two cDNAs encoding active maize chloroplast TGase, TGZ15 and TGZ21 were cloned for the first time in plants by our group (Patent WWO03102128). The plant TGase was shown to be preferentially present in the grana-appressed thylakoids of mesophyll light-exposed chloroplasts by subcellular localisation, with the abundance depending on the degree of grana development, and the enzyme activity being light dependent. Using antibodies obtained from the plant protein, the enzyme has been immunolocalized at different stages of chloroplast development, in dark-grown maize embryogenic callus, etiolated and light-grown plants as well as co-immunolocalized with its LHCII antenna protein substrates. Variants of maize chloroplast transglutaminase have also been sub-cloned into a pET28 vector and overexpressed in Escherichia coli cells, with the recombinant protein immunodetected preferentially in bacterial-formed inclusion bodies. Keywords chloroplast; grana; maize, thylakoids; transglutaminase; vesicles

1. Introduction Transglutaminases (TGases, EC 2.3.2.13) are intracellular and extracellular enzymes that catalyse calcium-dependent post-translational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. These enzymes were detected for the first time in animals, where they modify structural proteins [1], and are widely distributed in bacteria, animals, and plants. The primary structure of TGase was first established in 1986 for coagulation factor XIIIA. Animal transglutaminases have a catalytic triad of cysteine, histidine, and aspartate (asparagine), with the reaction proceeding via an intermediate linked to the cysteine. In recent years, many studies on TGase have been performed in tissues from humans, lower vertebrates, bacteria, algae and yeasts. In general, their sequence homology is moderate and, in some cases, there is no structural or functional similarity. Cloning of TGs has allowed a better understanding of their implication in cellular differentiation processes, tissue stabilization and apoptosis [2] [3]. Of all the reactions that are catalysed by TGases, protein crosslinking has probably attracted the greatest interest, although the significance of TGase mediated post-translational modifications of proteins by deamidation and amine incorporation is also well recognized. Research on plant TGases is less developed than in mammalian systems. TGase-like activity in plants was first observed in pea seedlings [4] and in sprout apices of Helianthus tuberosus [5]. Del Duca et al. [6], using animal polyclonal antibodies, identified apoproteins of the antenna complex (such as LHCPII) as endogenous substrates of TGase in leaf chloroplasts of Helianthus tuberosus. Our group detected a unique 58 kDa band in maize (Zea mays L.) meristematic calli and *

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chloroplasts that was light sensitive, and showed a daily rhythm [7]. Subsequently, a polyclonal antibody, obtained from purified chloroplast TGase, was used in a comparative study of the subcellular localization of this TGase in various maize cell types [8]. We also cloned two maize cDNAs (TGZ15 and TGZ21), expressing active, light-dependent chloroplast TG, for the first time in plants [9] [10]. In addition to Zea mays, sequence analysis of DNA/protein databases of Arabidopsis thaliana and Oryza sativa also show the presence of related enzymes, with low homology to known animal TG sequences [11]. A complete description of the TG immunolocalization study, using antibodies obtained from the plant protein and commercial antibodies against its preferential chloroplast substrates (LHCII antenna proteins), at different stages of chloroplast development are described here. TGase immunolocalization was carried out on tissues from dark-grown maize embryogenic callus, etiolated plants and different stages of maize light-grown plants. In order to produce pure and active TG for functional and structural studies, as well as for further applications, variants of maize chloroplast transglutaminase were subcloned into a pET28 vector and over-expressed in Escherichia coli BL21 (DE3) cells [12]. The immunolocalization of the over-expressed protein in bacterial inclusion bodies is also presented. This work forms part of a more complete study of the implication of the enzyme in photosynthesis-related processes such as photoprotection of the antenna proteins and its possible role in the regulation of thylakoid stacking.

2. Immunolocalization of a transglutaminase related to grana development in maize 2.1

Western blot immunodetection

To confirm the presence of transglutaminase in maize protein extracts, a polyclonal antibody (AbH) obtained from chicken, using a chloroplast purified TGase of Helianthus tuberosus leaves as antigen, was used to immunolocalize the enzyme [8]. The specificity of the antibody used was tested in western blots and in an enzymatic TGase assay, using maize mature leaf protein extracts. The immunological probes recognised a band of 58 kDa, corresponding to TGase (Fig. 1A), at a dilution of 1:10000 (antibody - blocking solution). In the TGase enzyme assay, the addition of 1 mg antibody per ml of maize leaf extract in the reaction mixture gave 68% inhibition of TGase activity with respect to control extracts. Figure 1 shows the results from western blots of maize protein extracts of different organs and embryogenic callus cell types. Fig. 1A shows the influence of light treatment on 20-day old maize leaf extracts. In dark-grown plantlets (0), bands of 150, 77 and 58 kDa were obtained, with the 58 kDa band giving the strongest signal. After two hours of illumination (2) only the 77 and 58 kDa bands were obtained, with the concentration of the 58 kDa band increasing until 8 hours of light exposure, supporting the results from the enzymatic assay. Figure 1B shows the different isoforms obtained in maize embryogenic callus, at various stages of differentiation as a result of being exposed to different light intensities. As with the leaf extracts, a 77 kDa band appeared in dark conditions, its intensity decreasing with light intensity increase. In the same conditions, 58 and 50 kDa bands appeared.

kD

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2

3

4

5 >200 kDa

AA AA 77 kDa 58 kDa 50 kDa

A A

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Fig 1- Western blot analysis of proteins from different maize tissues separated by SDS-PAGE (Laemmli 1970) using 1:10000 AbH dilution and a peroxidase-conjugated donkey anti-chicken IgY as secondary antibody. A- Maize leaves from young plants grown from 0 to 48 h in continuous light (Villalobos et al., Gene 2004). B- Embryogenic callus grown in different light intensities. 1, dark, 2, 30 days low light; 3 and 4, 200 days low light , 5, high light.

2.2 TGase activity 2.2.1 Embryogenic callus In order to analyse the differences observed in the western blot analyses, the TGase activity expressed as putrescine incorporated to proteins, in undifferentiated embryogenic callus, after different periods of culture under strong or weak light conditions, as well as the effect of hormone deprivation and plant differentiation was assayed (Table I). After 6 hours of culture under light conditions, all the samples had significantly higher TGase activity with respect to the 0 hour control samples, independent of light intensity. However, after 9 hours of exposure to light, significant differences in enzymatic activity between samples cultured under strong light and those cultured under weak light were obtained. Table I. TGase activity expressed as putrescine incorporation (pmol/ mg protein h-1) in embryogenic callus after different periods of culture with different light and hormone conditions. (Villalobos et al., Protoplasma 2001).

Time (h)/conditions l + 9 µM 2,4-D L + 9 µM 2, 4-D L + 0 µM 2,4-D

0 16 ± 1.7 16 ± 1.7 16 ± 1.7

6 60.2 ± 10.2 (b) * 41.1 ± 9.5 (a) 34.9 ± 10.5 (a)

9 81.7 ± 8.3 (b) 132.4 ± 5.7 (a) 119.5 ± 4.6 (a)

L= strong light culture; l= weak light culture. * Different letters in the same column indicate significant differences at 0.01> n ≥ 0.001 level. 2,4-D= 2,4 dichlorophenoxyacetic acid.

2.2.2

Membrane-chloroplast

As in the case of differentiating embryogenic callus, the TGase activity in thylakoids and grana protein extracts from 20-day old greenhouse-grown maize plants was significantly higher when the assay was performed in the light (Fig 2a). Similar results were obtained when the enzymatic assays were done with dark-grown plants, assayed in light or dark conditions. [10].

Bound PU (pm ol/h·m g prot)

GH

Fig 2- Effect of light on TGase activity (pmol/ mg protein h-1) of thylakoid (a) and grana (b) leaf extracts. GH= greenhouse grown plants (From: Villalobos et al. Gene 2004).

400 300 200 100 0 a

b Assay conditions

In view of the previous results, we cloned TGase DNA in maize (TGZ) using the chloroplast TGase antibody, detailed earlier, and a maize-leaf cDNA library. Two cDNAs coding for this enzyme, TGZ15 and TGZ21, which have been shown to code for active TGs and possess the catalytic Cys-His-Asp triad, were isolated [9] [10].

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2.3 Ultrastructural localization of maize TGase 2.3.1 Immature and adult maize leaves In the first immunolocalization study using the AbH antibody non-appressed thylakoid structures were present in chloroplasts of the 7-day germinated plantlet (Fig. 3A), and gold labelling was observed only in a few cases (Fig. 3B), and with the highest antibody concentration (1:4000). All the MET observations were using a Hitachi H 600 operated at 80 kV (for further information on methodology see [8].

Fig 3- Electron microscopy of AbH immunolabelled sections of young maize plant leaves. A, entire chloroplast without grana appressed thylakoids. B, randomly immunolabelled thylakoids of the same chloroplast. chl, chloroplast, cy, cytosol. Bars: A, 0.5µ; B, 50 nm. (Villalobos et al. Protoplasma 2001)

In adult leaves (Fig 4), the enzyme was preferentially present in the grana-appressed thylakoids of lightexposed mesophyll chloroplasts and also dispersed in bundle-sheath cell chloroplasts. The abundance depended on the degree of grana development, and the enzyme activity was light dependent. The enzyme was not found in other organelles such as mitochondria.

E

Fig 4- MET of AbH immunolabeled sections of adult maize plant leaves. C, general aspect of mesophyll and bundle-sheath chloroplasts (chl). D, scarce immunolabelling of thylakoids in a bunddlesheath chl. E, absence of gold labelling in a mesophyll chl with pre-immune serum. F, granaconcentrated immunolabelling in a mesophyll chl. BSC, bundle-sheath cell; MC, mesophyll cell; e, stroma; s, starch grain; g, grana. Bars: C, 1µm; D and F, 50 nm; E, 100 nm. (Villalobos et al. Protoplasma, 2001).

2.3.2 Maize embryogenic callus In ultra thin sections of embryogenic callus grown in maintenance medium and dark conditions only a few proplastids were present, with no gold immunolabelling (Fig 5A). However, in the same cell types,

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significant gold labelling was detected in dense spherical vesicles, with a diameter between 200 and 700 nm, which were dispersed in the cytoplasm (Fig 5A, a). The gold particles were deposited in the electron-dense core, surrounded by an electron-translucent layer. The same type of vesicles were present in abundance near chloroplasts from weak-light cultured callus (figure not shown), but were not detected in the other cell types under examination. At the end of the subculture period of embryogenic callus in proliferation medium, under weak light conditions (Fig. 5b), many immature chloroplasts without normal grana were observed (Fig. 5B). In these chloroplasts, gold labelling was detected in the thylakoid-forming grana, distributed randomly. In those chloroplasts with normal grana structures, gold labelling was specifically concentrated in thylakoid-appressed grana (Fig. 5C). After 12 hours of embryo culture in plant differentiation medium under strong light conditions, chloroplasts developed mature grana and an abundance of starch grains, as expected for differentiating embryos. In the grana-appressed thylakoids of these chloroplasts there was a high level of labelling (using 1:4000 and 1:8000 antibody dilutions) (Fig. 5E).

A

B

a

E

2,0 mm

e D C

b

Fig 5- AbH immunlabelled sections of maize embryogenic callus cultured on different media and under different light intensities. A, Proplastid and vesicle (inset a: bar, 100 nm). B, general aspect of an immature chloroplast (chl) from cells of embryogenic-callus cultured in proliferation medium under weak light. C, grana-concentrated labelling in a chl of an embryoid cell cultured under strong light in differentiation medium. D, cell as in C, but showing absence of gold labelling after pre-treatment using pre-immune serum. Bars: A and B, 200 nm; C-E, 100 nm. (Villalobos et al. Protoplasma, 2001). a, thin sections of embryogenic callus cultured in proliferation medium showing dividing cells. b, general aspect of embryogenic callus cultured in differentiation medium, e, embryoid.

Figure 6 shows the differences in the number of gold particles from the different chloroplast cell types described in 2.3.1 and 2.3.2. The predominance of TGZ in mesophyll chloroplasts, mature leaves and, in particular, in differentiated embryoids is evident.

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70 gold particles

60 50 40 30 20 10 0 yl

mlm

mlb

gc

ecn

ecd1

ecd2

cell type

Fig 6- Number of gold particles per unit area (1µ2) in appressed (black bars) and non-appressed (white bars) thylakoids of the different chloroplast (chl) cell types. All the samp les were treated with 1:4000 antibody AbH concentration. Data ± SE are the mean of five replicates. yl= young leaf chl; mlm= mature leaf mesophyll chl; mlb= mature leaf bundle-sheath chl; gc= green callus chl; ecn= embryogenic callus non-differentiated chl under weak light; ecd1= embryogenic callus differentiated chl 12h under strong light; ecd2= embryogenic callus differentiated chl 8 days under strong light. (From: Villalobos et al. Protoplasma, 2001).

The increase of TGase with chloroplast differentiation and its specific detection in the thylakoid appressed grana indicated that this enzyme might be related to the LHCII proteins of the antenna complex, which are localized in the same grana structures [12]. The 58 kDa band present in the lightexposed cell types suggests that this may be the active TGase form, present in the ultra-structural observations of the differentiated chloroplasts. In a study of the location of the thylakoid membrane system during structural differentiation of green plant chloroplasts, Murakami and Toda [12] found no immuno-gold labelling of the LHCPII protein on the etioplasts and etiochloroplasts in the early stage of greening of various plants, including barley and spinach. This is in agreement with our observations on cells of dark-grown maize tissues immunolabelled with the plant TGase antibody. After 4 hours of illumination, small amounts of gold particles were found randomly in the thylakoid membrane system, where the differentiation of grana and stroma thylakoids is initiated. This also coincides with our observations on the immature chloroplasts of maize embryogenic callus, cultured under weak light intensity, and young maize leaves (Figs. 2, 3 and 4). As in LHCII immunolabelled spinach chloroplasts, with the progress of grana formation in maize cells (differentiated embryos and adult leaves), the number of gold particles on the thylakoids increased significantly and were concentrated in the grana. Moreover, the increase in TGase with chloroplast differentiation, together with significant levels being present in mesophyll chloroplasts of the maize C4-photosynthesis cell system, supports the relation between this TGase and the presence of grana, leading to the hypothesis that the presence of chloroplast TGase is concomitant with that of PSII in the greening process and grana formation. With respect to the dense spherical vesicles observed only in the non-differentiated embryogenic callus, their size and structure indicate that they may be similar to the transport vesicles observed in storage cells of various species, such as wheat [13]. In these cases, transport is Golgi-mediated and the vesicles are derived from the ER, containing the precursor form of a storage protein which is transported in these vesicles to protein-storage vacuoles, where the mature form is generated by proteolysis via a vacuolar processing enzyme [14]. Further studies are needed on the presence of TGase (probably in its inactive form) in these vesicles, which are observed only in callus cells without differentiated chloroplasts, in order to relate this to the chloroplast differentiation process.

3- Co-immunolocalization of transglutaminase and its substrates, the LHCII antenna proteins, in different maize tissues In view of these results on the possible co-localization of chloroplast TGase with its photosynthetic substrates (LHCII antenna proteins) during the different stages of chloroplast differentiation, a

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complementary study using the AbH antibody, and two new, specific maize TGase antibodies (AbQ and AbTGZ4) together with commercial LHCII antibodies, was carried out, using embryogenic maize callus and maize plants grown under different light conditions. 3.1 Etiolated, young and adult maize plant leaves The presence of TGase in parallel with its protein substrates, the LHCII antenna proteins, during the different stages of chloroplast development, related to the presence or absence of light, was studied. This was done using immunodetection of the proteins in 9-day etiolated dark-grown tissues, and different periods of light exposure in greenhouse grown plants with three anti-maize TGase antibodies and three anti-LHCII antibodies. 3.1.1 Western blot analyses Figure 7 shows a western blot analysis of 20-day old maize thylakoid protein extracts using an antibody against the LHCbII protein (Agrisera). It can be seen that, using TE buffer (lane a), a 77 kDa band was detected, which coincides with one of the TGase bands detected with AbH, as well as a 34 kDa band. When B4 buffer is used (lane b) the 77 kDa band is not detected, but the 34 kDa band (the monomer form of LHCbII) is clearly detected. These results indicate that the 77 kda band could be an enzyme/substrate complex. kDa 97 77 kDa

66

45

30 kDa

Fig. 7- Western blot analysis against LHCbII ftrom thylakoid extracts of 20 days old maize plants. a, TE buffer (Tris-HCl, NaCl and SDS; pH 8); b, B4 buffer (NaCl, MgCl2, HEPES and sucrose, pH 8).

29

a

b

As may be seen in Fig. 8, the treatment with both antibodies against the enzyme and the substrate seems to confirm the presence of an enzyme/substrate complex.

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A 1

2

B 3

1

2

3

210 kD

77 kD 58 kD

Fig. 8- Western blot of the same extracts as in Fig. 7, treated with AbH (anti TGase) (A) and AbLCHbII (B)

29-34 kD

3.1.2 Co-immunolocalization with the LHCII proteins Subcellular co-immunolocalization of both TGase and LHCII proteins, in different stages of chloroplast development, was then carried out. The results in etioplasts from plants cultured for 9 days in dark conditions are shown in Fig. 9. The thylakoids in the bundle sheath etioplasts were normal (A), but prolamellar bodies (plb), the result of the abnormal accumulation of tubes in a quasi-crystalline lattice that is only present under these conditions, were formed in the mesophyll etioplasts (B), In these abnormal structures, TGase and LHCII signals were present mainly in the cytoplasm (A) and near the cell wall (E). There was no protein signal in the bundle-sheath etioplasts (A) and very little signal in the mesophyll plb (C and D).

A

B me be

C

D

plb

pl

E

pd

pl Fig. 9- Co-immunolocalization of TGase and LHCII proteins in the etioplasts of 9 day-old maize plants grown in dark conditions. Bars: A and B, 1 µm; C, D and E, 0.2 µm. be, bundle-sheath etioplasts; me, mesophyll etioplast; plb, prolamellar bodies; pd, plasmodesmata.

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When plants were grown for 1 hour in light conditions, more of both proteins was detected in the mesophyll (Fig 10, A) than in the bundle-sheath thylakoids (Fig 10, B). After 3 days under light conditions, TGase and LHCII were mainly concentrated in the grana of the mesophyll chloroplasts (Fig 10, D and E) and barely detected in the bundle-sheath thylakoids (Fig 10, C), as we had observed previously in mature plants. These observations confirm the results obtained with western blot analyses, and agree with biochemical analyses which demonstrate that LHCII antenna proteins have the same cellular localisation as that of chloroplast TGase during the greening process from etioplast to chloroplast. These results also give more experimental data on the relationship between these two types of proteins in the photosynthesis related processes, especially in thylakoid appression to form the grana apparatus, where Photosystem II is located.

A

B

C

D g

E

g Fig. 10- Co-immunolocalization of TGase and LHCbII proteins in 1-h (A and B) and 3-day old (C, D and E) maize plants grown in light conditions. Bars. A and B,0.2 µm; C, 0.2 µm; D and E, 0.5 µm. Dark arrows indicate LHCII. Light arrows indicate TGase. G, grana.

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3.2 Dark-grown maize embryogenic callus As observed in embryogenic callus immunolabelled with anti-TGase antibodies, the double labelling with the anti-LHCII antibodies demonstrated the presence of electro-dense vesicles, never detected in the leaf of intact plants, that contain the TGase enzyme and its substrate, the antenna apoproteins LHCII (Fig. 11D). These protein vesicles are always present in dark-grown embryogenic cells and also during the first stages of chloroplast differentiation under light conditions. After this period, when differentiation from the embryo structures advances, these structures were no longer present in the cells and the TGase-LHCII signal was in the chloroplast appressed thylakoids (grana). To our knowledge, this is the first time this has been reported. LHCII proteins are present in protein transport vesicles in an in vitro differentiating system such as embryogenic callus (Fig. 11A). These vesicles are presumably generated directly from the endoplasmic reticulum (Fig. 11A and B) and transport the LHCII protein to its target organelle, the chloroplast (Fig. 11C), where it is imported when the light conditions permit chloroplast development. The same route may be detected for the enzyme TGase in this system.

A

B

C

v

C

v v prb v

p

D

Fig. 11- Co-immunolocalization of LHCII apoproteins and TGase in maize embryogenic callus cells grown in dark conditions. A, general aspect of an electro-dense vesicle enveloped by polyribosomes (prb) of the ER in the cytoplasm; B, three protein-containing vesicles; C, vesicle near a differentiating proplastid of a callus cell grown in the light for 3 days. D, detail of a vesicle showing the two-protein immunolocalization.. v, vesicle, c, cytoplasm; p, proplastid. Bar: 0.2 µm. Dark arrows indicate the presence of LHCII. Light arrows indicate the presence of TGase.

3.3 Escherichia coli cells harbouring the TGZ gene After the transformation of BL21 E. coli cells with a pET28 expression vector containing the maize TGZ4 gene [15], the over-expression of the protein was inducted with IPTG. The presence of the protein in the bacterial cell was demonstrated by western blotting and TEM microscopy with the T7 commercial antibody (pET system), the anti-TGZ peptide antibody PAB/18QA, designed against a part of the Cterminal region (active site) of the deduced protein sequence, and with the specific AbTGZ4, obtained from the purified, heterologously over-expressed TGZ4 protein.

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Western blot analysis of the over-expressed protein extract (Fig. 12A) demonstrated that TGZ was present in the total cell extract, in the soluble fraction and, preferentially, in the insoluble fraction. After purifying the protein and producing the anti-TGZ antibody, western blot analysis of maize thylakoid protein extracts under different growth conditions demonstrated that this antibody also detected the plant protein (Fig 12B).

T

SF

IF

A

B Fig. 12- A, Western blot analysis of E. coli overexpressed TGZ4 using the T7 antibody. T, total bacterial protein extracts; SF, soluble fraction; IF,insoluble protein fraction. B, TGZ immunolocalization using the AbTGZ4 in maize thylakoid extracts from plants grown in different light conditions. 1, etiolated plant; 2 and 3, plants grown under light conditions for 3 and 20 days respectively. M = Molecular marker. Arrows show different forms of maize chloroplast TG. (Carvajal et al. Biotech. Lett. 2007).

In the sub cellular immunolocalization study, using induced TGZ-transformed E. coli cells (Fig. 13), maize TGase was predominantly accumulated and abundantly detected in an inclusion body that is formed in the bacterial cell after induction, which was not present in the non-transformed cells (Fig 13, D). As may be seen, although the protein may be localized with the T7 (Fig 13, A) and the PAB/18QA antibodies (Fig 13, B), the most abundant localization was that obtained with the AbTGZ4, probably due to its more elevated antigens recognition capacity against the complete protein sequence.

A

C

B

D

Fig. 13- TEM subcellular immunolocalization of TGZ4p in inclusion bodies of E.coli transformed and induced cells. Localization with: A, the T7-Tag antibody (1:2000), B, the PAB/181QA antibody (1:200), C, the TGZ4 antibody (1:3000). C1= E.coli transformed and induced cell non-immunotreated with secondary Ab . D= Non-transformed E.coli cell. (C and D, from: Carvajal et al. Biotech. Lett. 2007).

Conclusions The results presented in this review demonstrate the relationship between an enzyme recently cloned in plants and its specific substrates, from the point of view of its sub cellular localization. The novelty of the observations and the obtaining of new antibodies that shows a clear specificity against the original protein, in its natural system and in a heterologous system give more added data about the possible functionality of the enzyme in plants. This function is probably related to the thylakoid appresssing

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process during chloroplast development and/or with photoprotection related phenomena in which the regulation of the grana size becomes important for plant survival. Acknowledgements The support by Nuria Cortadellas from the Servei de Microscopia de la Universitat de Barcelona is gratefully acknowledged. This work has been financed by the Spanish PN, (MCYT BFI2003-003318), MEC (BFU2006-15115-CO2-01), and IQS-CEUB grant (2001SGR 00327), it includes results from the theses of Villalobos and Carvajal. Figures 3,4,5,6 were published in Protoplasma, 216, Villalobos, E, Torné, JM, Rigau, J, Ollés, I, Claparols, I and Santos, M , Immunogold localization of a transglutaminase related to grana development in different maize cell types, 155-163, Copyright Springer (2001) Figures 1A, 2 were published in Gene, 336, Villalobos E, Santos M, Talavera D, Rodríguez-Falcón M, Torné JM, Molecular cloning and characterization of a maize transglutaminase complementary DNA, 93-104, Copyright Springer (2004) Figures 12, 13 were published in Biotech. Lett. DOI: 10.1007/s10529-007-9377-7, Carvajal-Vallejos,P.K., Campos, A., Fuentes-Prior, P., Villalobos, E., Almeida,A.M., Barberà, E., Torné, J. M. and Santos, M., Purification and in vitro refolding of maize chloroplast transglutaminase over expressed in Escherichia coli/ (In Press), Copyright Springer (2007)

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