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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
1
2
3
4
5
>200 kDa
AA
AA
77 kDa
58 kDa
50 kDa
A
A
B
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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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