SHORT COMMUNICATION
Ultrastructural changes during early infection of Vignaunguiculata and Phaseolus vulgaris leaves by Xanthomonasaxonopodis pv. phaseoli and an unexpected associationbetween chloroplast and mitochondrion
Andre de O. Carvalho • Maura Da Cunha • Rosana Rodrigues • Claudia P. Sudre •
Izabela S. Santos • Katia V. S. Fernandes • Guilherme R. Rabelo •
Valdirene M. Gomes
Received: 4 May 2010 / Revised: 29 November 2010 / Accepted: 31 January 2011 / Published online: 16 February 2011
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2011
Abstract Common bacterial blight, caused by Xantho-
monas axonopodis pv. phaseoli, is an important disease
affecting beans causing considerable yield losses in many
producing areas. Our aim was to investigate Xap–beans
interactions in the first 48 h after infection analyzing
structurally relevant changes by electron microscopy.
Scanning electron microscopy analysis demonstrated
morphological changes on leaf surfaces of both species.
Transmission electron microscopy showed different types
of chloroplast structural disorganization in both paren-
chyma and bundle sheath cells of Vigna unguiculata which
were only observed in parenchyma cells of Phaseolus
vulgaris. Additionally, we report an unexpected physical
association between the chloroplasts and mitochondria
observed only to P. vulgaris-infected plants.
Keywords Mitochondria–chloroplasts association �Xanthomonas axonopodis pv. phaseoli �Vigna unguiculata � Phaseolus vulgaris
Introduction
The Gram-negative bacterium, Xanthomonas axonopodis
pv. phaseoli (Xap), is the causal agent of common bacterial
blight in beans. Its principal host is Phaseolus vulgaris L.,
although other legume species are known to be hosts,
among them V. unguiculata (L.) Walp. (Swings and
Civerolo 1993). Infected seeds are one of the most efficient
forms of disease dispersal between cultivated areas,
through long-distance human transportation. The disease
has a causal connection with crop yield reduction and, in
part, is caused by factors associated with facilities in the
transmission of the bacteria, such as seed contamination,
bacterial persistence in the field, deficiency in chemical
control and lack of availability of resistant cultivars
(Swings and Civerolo 1993; Agrios 1997).
The cowpea (Vigna unguiculata L. Walp) is a tropical
legume originating from Africa and cultivated in most
tropical regions of the world. In Brazil, it is mostly culti-
vated in the Northeastern region and is the main protein
source of daily diets for most part of the poor population
(Phillips et al. 2003). Diseases caused by pathogens,
including fungi, bacteria, viruses, and nematodes, consti-
tute one of the most important constraints on the produc-
tion in all cultivating areas (Fatokun et al. 2002). The use
of resistant crop varieties assisted with proper culture
Communicated by B. Barna.
A. de O. Carvalho � I. S. Santos � V. M. Gomes (&)
Laboratorio de Fisiologia e Bioquımica de Microrganismos,
Centro de Biocienicas e Biotecnologia, Universidade Estadual
do Norte Fluminense-Darcy Ribeiro, CEP 28013-600 Campos
dos Goytacazes, RJ, Brazil
e-mail: [email protected]
M. Da Cunha � G. R. Rabelo
Laboratorio de Biologia Celular e Tecidual, Centro de
Biocienicas e Biotecnologia, Universidade Estadual do Norte
Fluminense-Darcy Ribeiro, CEP 28013-600 Campos dos
Goytacazes, RJ, Brazil
R. Rodrigues � C. P. Sudre
Laboratorio de Melhoramento Genetico Vegetal, Centro de
Ciencias e Tecnologia Agropecuaria, Universidade Estadual do
Norte Fluminense-Darcy Ribeiro, CEP 28013-600 Campos dos
Goytacazes, RJ, Brazil
K. V. S. Fernandes
Laboratorio de Quımica e Funcao de Proteınas e Peptıdeos,
Centro de Biocienicas e Biotecnologia, Universidade Estadual
do Norte Fluminense-Darcy Ribeiro, CEP 28013-600 Campos
dos Goytacazes, RJ, Brazil
123
Acta Physiol Plant (2011) 33:2025–2033
DOI 10.1007/s11738-011-0726-8
practices and the use of healthy seeds are some of the best
ways of avoiding losses, especially in regard to bacterial
diseases (Agrios 1997). Because of the social importance
and economic potential attributed to this culture, studies to
improve agronomic and nutritional traits of cowpea are of
major relevance (Fatokun et al. 2002).
The common bean (Phaseolus vulgaris L.) (bean) was
originated in the Central and South America (Toro et al.
1990) and their seeds have a great importance because they
represent the main legume grain used as human food in the
world. Brazil is the largest producer of common bean
where the culture is socially important because it is farmed
as a subsistence culture and is the main income of small
farmers. The culture lacks technology and the use of pes-
ticides and fertilizers is rare (Santos et al. 2003). Because
of the social and economic implications attributed to bean
cultivars, the use of resistant cultivars represents a prom-
ising, simple, and economically effective method of dis-
ease control, especially when combined with the use of
healthy seeds and adequate crop practices.
We have planned to study the interaction of bean plants
and Xap by electron microscopy during the first 48 h after
Xap infection. We have found morphological changes in
cowpea and bean tissues since first hours after infection.
However, we found an unexpected physical association
between chloroplast and mitochondrion of bean-infected
leaves. For the best we know this is the first report of
association of chloroplast and mitochondrion related to a
pathological state.
Materials and methods
Cowpea (Vigna unguiculata L. (Walp.)) seeds (cultivar
EPACE-10) were supplied by the ‘‘Centro de Ciencias
Agrarias’’, ‘‘Universidade Federal do Ceara’’, Fortaleza,
Brazil. Common bean (Phaseolus vulgaris L.) seeds (BAC-
6 line) which are resistant to the bacterial plant pathogen
Xap (Rodrigues et al. 1999) were supplied by ‘‘Centro de
Ciencias e Tecnologias Agropecuarias’’, ‘‘Universidade
Estadual do Norte Fluminense’’, Campos dos Goytacazes,
Brazil. Seeds of both plants were separately sowed onto
five-liter plastic plots, six seeds per pot, and 3 days after
emergence, the plants were thinned out to two per pot. The
pots were filled with a sand-soil-organic material mixture
(1:1:1, w/w/w). Fertilizer containing nitrogen was added to
the soil 10 days after plant emergence. Plants were kept in
the greenhouse on continuous illumination, exposed to
natural daylight for the entire experimental period.
Xanthomonas axonopodis pv. phaseoli (Xap), isolate
CNF 15, was provided by the ‘‘Centro Nacional de Pes-
quisa Arroz-Feijao’’—Embrapa/CNPAF. The bacteria were
cultivated and a freshly harvested cell suspension was used
as the inoculum (108 cells mL-1), as described by Rodri-
gues Neto et al. (1986).
Bean plants at 25 days after germination were inocu-
lated by spraying a fine mist of the bacterial suspension on
both sides of one leaf. For control purposes, bean plants
were mock-inoculated with a saline solution and submitted
to the same experimental conditions as the test plants. Test
and control plants were collected at 6, 12, 24, and 48 h
after inoculation. Disease leaf reaction was previously
assessed using the following scale, according to Rodrigues
et al. (1999): 1.0 = no symptoms; 2.0 = 1–5%;
3.0 = 6–25%; 4.0 = 26–50%; 5.0 = [50% of the inocu-
lated area presenting symptoms. The final score was the
arithmetic mean of eight independent assessments. Plants
rated with grades \1.0 were considered highly resistant;
plants with means rating (x) varying from 1.1 \ x \ 2.0
were considered resistant; plants with grades 2.1 \ x \ 3.0
were classified as moderately resistant; plants with grades
varying from 3.1 \ x \ 4.0 were classified as susceptible
while plants graded from 4.1 \ x \ 5.0 were highly sus-
ceptible. Inoculated leaves and non-inoculated leaves were
detached and immediately fixed as described in electron
microscopy.
For transmission electron microscopy (TEM) analysis
fragments of infected and control leaves of both plants
(three infected plants and two control plants) were col-
lected and immediately fixed for 2 h at room temperature
in an aqueous solution containing 2.5% glutaraldehyde
(v/v) and 4% paraformaldehyde (v/v) in 50 mM cacodylate
buffer (pH 7.2). After fixation the materials were washed,
post-fixed in 1% (w/v) osmium tetroxide in corresponding
buffer for 1 h at room temperature. The samples were
dehydrated in a graded acetone series (30, 50, 70, 90 and
100% (v/v)) and embedded in Epox resin (Polybed).
Ultrathin sections (0.1 lm) were laid on copper grids,
stained with uranyl acetate for 10 min followed by lead
citrate for 5 min and then were observed with a Zeiss 900
transmission electron microscope (Zeiss company,
Germany) operating at 80 kV (Agizzio et al. 2006).
For scanning electron microscopy (SEM) analysis, after
the fixation described above, the leaf fragments were
dehydrated in a graded acetone series and submitted to
critical point on a Critical Point Dryer (Bal-Tec CPC 030),
using liquid carbon dioxide as a substituting liquid. The
dried materials were covered with a layer of 20 nm gold in
a Sputter Coaster (Bal-Tec SCD 050) and then observed in
a DSEM 962 Zeiss scanning electron microscope (Zeiss
Company, Germany) (Agizzio et al. 2006).
The number of stomata which present morphological
changes, as describe in the ‘‘Results and discussion’’ sec-
tion, were counted, 20 fields for cowpea and 10 fields for
bean, 0.04 mm2 each, for the abaxial leaf blade for both
species for all studied time. The numbers of opened and
2026 Acta Physiol Plant (2011) 33:2025–2033
123
Fig. 1 Scanning electron
microscopy of cowpea (a–f) and
bean (g–j) leaves at different
times after Xap inoculation.
This figure set shows the
morphological changes found in
glandular trichome (a, b) and in
stomata (d, e) during the period
of 6–48 h. a, b Glandular
trichome of the infected leaves.
c Glandular trichome of a
control leaf. Rupture of the
external cap after 12 h (a) and
structure collapse after 48 h
(b) may be observed. d Stomata
of the infected leaf after 48 h.
e Stoma where bacteria may be
observed inside the stomatal
pore after 12 h and f Stomata of
a control leaf. g Stomata of a
control leaf. h Stomata of an
infected leaf showing
distribution of Xap after 24 h.
Note some shrunken cells.
i Glandular trichome of a
infected leaf. j Non-glandular
trichome of infected leaf. Bar,
7.1 lm in (b), 2.4 lm in (c),
2.8 lm in (a, d, e), 1 lm in (f),5.5 lm in (g), 4.3 lm in (h),
5.1 lm in (i), 8.3 lm in (j)
Acta Physiol Plant (2011) 33:2025–2033 2027
123
closed stomata of the abaxial leaf blade (10 fields of
0.04 mm2 each) were also counted for control and for the
time of 6 h after inoculation for the both species. These
data were treated statistically with ANOVA test and t test
for morphological changes and opened and closed stomata,
respectively.
Results and discussion
Control plants, either bean or cowpea, presented no disease
symptoms and were rated with grade 1.0. For inoculated
plants, bean mean grade was 1.3 while cowpea ranged 2.6,
twice as much than observed for bean. The effects of Xap
on the leaf surfaces of cowpea and bean at different times
can be observed in Fig. 1a–j. Cowpea glandular trichomes
present a release of the external cap (Fig. 1a) and, in more
severe cases, a destruction of the entire structure (Fig. 1b),
which was not observed in the control leaves (Fig. 1c). In
leaves of cowpea at 48 h, the distribution of Xap near the
stomata (Fig. 1d) could be observed. In the stomata, the
guard and subsidiary cells were seen to be shrunken, with
slight differences between 12 (Fig. 1e) and 48 h (Fig. 1d).
In Fig. 1e was observed an enlargement of the stomatal
pore and the presence of bacteria inside it. In control leaves
these effects were not observed (Fig. 1f). The stomata of
bean plants, (Fig. 1g, h), the morphological changes were
very similar to those found in cowpea, although the effect
in those cells is more accentuated showing them to be
withered and shrunken. In contrast, no changes were
observed in the glandular trichomes (Fig. 1i) and also in
non-glandular trichomes (Fig. 1j). The number of stomata
presenting morphological changes for both plant species is
present in Table 1.
Xap was found inside the stomatal pore of both cowpea
and bean guard cells (Fig. 1e, h), that was directly related
to the changes observed on these structures and could be
associated with the way the bacterium enters the plant
(Agrios 1997). We can see from the pictures of the leaf
surface of cowpea and bean that Xap is associated with cell
structures that are believed to be microenvironment of
nutrient release such as stomata and trichomes (Melotto
et al. 2008). For plant foliar pathogenic bacteria, associa-
tion with stomata give them the opportunity to enter in the
leaf tissues to obtain more nutrients and consequently to
infect the plant. It has been demonstrated that plants sense
bacteria and respond to it closing the stomata. On the other
hand bacteria whose developed a strict relationship with
stomata can induce the reopening of stomatal pore by the
releasing of virulent factors. For the model foliar pathogen
Pseudomonas syringae coronatine was identified as
the substance that induces the stomatal pore reaperture
(Melotto et al. 2008). We have counted the opened and
closed stomata to the first time after inoculation for both
plant species and we have found that the stomal pores
associated with Xap are opened indicating that a similar
mechanism may be present in this pathosystem (Table 2).
The structural changes present on glandular trichomes of
cowpea, at the initial phase, might be related to glandular
secretion. It has been demonstrated for other plant species
that when secretion is released from the glandular tri-
chome, rupture of the cuticle or tearing of the outer cap can
occur (Serrato-Valenti et al. 1997; Sacchetti et al. 1999).
The composition of the secretion, characterized in other
plant species, showed the presence of different chemical
substances and some of them possess antimicrobial activity
and contribute to the plant defense against pathogens and
pests (Carter et al. 1989; Ascensao and Pais 1998; Dixon
2001). The observed changes in glandular trichomes could
be a defense response of cowpea to Xap inoculation. As
these changes were not observed on control plants we
presume that they are somehow connected with the process
of Xap infection.
The infected plants responded macroscopically to Xap
inoculation, presenting the release of the external cap of the
trichome (Fig. 1a), the shrunkage of guard and subsidiary
cells (Fig. 1d, e, h) and distribution of Xap near stomata
(Fig. 1d, h). These effects were reflected internally on the
ultrastructure of plant cells. Analysis of ultrastructural
aspects revealed that inoculation with Xap has affected the
cellular organization level, particularly the chloroplasts. In
both infected species changes were found, however, in
cowpea they were more severe (Fig. 2a–j) and were
Table 1 Means of stomata presenting morphological changes in the
abaxial leaf blade
Vigna unguiculata Phaseolus vulgaris
Without With Without With
Control 25.45 ± 4.11a 1.90 ± 1.17a 7.70 ± 1.49ad 2.40 ± 0.52a
6 h 7.20 ± 2.75b 4.55 ± 1.61b 5.10 ± 1.85a 2.40 ± 1.17a
12 h 2.50 ± 1.05c 1.50 ± 1.19a 12.60 ± 2.12b 3.00 ± 1.33a
24 h 5.20 ± 1.77b 3.50 ± 1.24b 7.30 ± 3.77ab 1.80 ± 1.14a
48 h 0.1 ± 0.31d 9.55 ± 2.26c 8.40 ± 2.17cd 2.80 ± 1.32a
Means with the same letter are not significantly different (ANOVA,
P \ 0.05)
Table 2 Means of open and closed stomata in the abaxial leaf blade
after 6 h after inoculation
Vigna unguiculata Phaseolus vulgaris
Opened Closed Opened Closed
Control 5.00 ± 2.12a 13.67 ± 4.39a 3.40 ± 0.97a 6.70 ± 1.16a
6 h 14.00 ± 3.00b 0.89 ± 1.62b 5.50 ± 1.18a 2.00 ± 1.94a
Means with the same letter are not significantly different (t test,
P \ 0.05)
2028 Acta Physiol Plant (2011) 33:2025–2033
123
observed in bundle sheath cells (Fig. 2h–j) and stomata
(Fig. 2e) as compared to the control (Fig. 2a–c). In infected
cowpea plants, the mesophyll chloroplasts present changes
in shape, becoming more spherical. In some cases, a light
disorganized membrane system, accumulation of starch
grains and plastoglobuli were observed (Fig. 2d). The
chloroplasts of bundle sheath cells showed a higher signif-
icant loss of structure and, in some cases, plasmolysis was
observed (Fig. 2i, j). In the mesophyll chloroplasts of bean
cells, the ultrastructural changes were very similar to those
found in cowpea (Fig. 3a–i). The chloroplasts of bundle
sheath cells of bean seemed to be slightly affected. In bean
epidermal cells, some degraded plastids were observed in an
advanced state (Fig. 3i). Some multivesicular bodies were
observed at cowpea and bean cells (Fig. 3g, h). Mitochon-
dria and nuclei presented minor changes in the Xap-beans
interaction. In general, the mitochondria demonstrated
slightly swollen cristae and no great changes were observed
in the nuclei apart from some chromatin condensation at the
periphery of nuclear membrane.
Other works have also demonstrated alteration very
similar to that we have found in chloroplast ultrastructure.
Plants of Nicotiana benthamiana infected with pepper mild
mottle tobamovirus showed chloroplasts deformed, swol-
len, containing large and irregular starch grains, disorga-
nized lamellar structures and presence of plastoglobuli
Fig. 2 Transmission electron microscopy of cowpea leaves at
different times after Xap inoculation. a Cellular structure of a typical
spongy parenchyma cell of control plants. b Vascular bundle and
bundle sheath cells of control plants. c Chloroplast of a bundle sheath
cells of control plants. d Chloroplast of a spongy mesophyll cell at
12 h after Xap inoculation. e Guard cell after 12 h Xap interaction, the
chloroplast is degraded and some membranous structures are
observed in the cytoplasm. f A nucleus from a mesophyll cell at
12 h. g Xap adhered to the surface of cowpea leaf. h Bundle sheath of
a leaf at 12 h. i, j Bundle sheath cell chloroplasts after 12 h. Esextracellular space, CW cell wall, N nuclei, Mt mitochondria,
C chloroplasts, V vacuoles, Sg starch grain, asterisks bundle sheath
and filled triangle bundle sheath cells, arrow heads plastoglobuli.
Bar, 1.4 lm in (a), 3.5 lm in (b), 0.5 lm in (c), 0.75 lm in (d–f),2.4 lm in (h), 0.21 lm in (g), 0.3 lm in (i, j)
Acta Physiol Plant (2011) 33:2025–2033 2029
123
(Perez-Bueno et al. 2006). Other types of stresses, such as
drought, can provoke similar chloroplast ultrastructural
disorganization as described above as demonstrated in
needles of Norway spruce (Zellning et al. 2010). Normal
development can provoke similar changes. Chloroplasts in
senescence leaves of a super-high-yield Oryza sativa
variety, at 47 days after full expansion, showed spherical
shape, accumulation of large starch grains, increased
presence of plastoglobuli and swollen thylakoid mem-
branes (Zhang et al. 2010).
In interaction of Gram-negative bacteria and plant cells,
the effector molecules of pathogenicity are delivered directly
inside the plant cell cytoplasm and thus require the attach-
ment of bacteria to the plant cell wall (Bonas and van den
Ackerveken 1997). Our results from TEM (Figs. 2, 3) were
unable to show the presence of Xap in the vascular sheath of
both plants. The presence of Xap was detected in the stomatal
pore of both plants and additionally its presence was only
detected in the intercellular space of bean parenchyma cells
(Fig. 3f). The infection caused relevant structural changes on
Fig. 3 Transmission electron microscopy of bean leaves after
inoculation with Xap. a Spongy parenchyma cell of control plant.
b Mesophyll cells after 48 h of Xap interaction. c Chloroplasts from a
parenchyma cell after 48 h showing the disorganization of grana and
thylakoids. d Spongy parenchyma cell after 12 h of interaction. e Xapadhered to the leaf surface after 48 h of interaction. f Xap in
intercellular space. g Multivesicular body found in guard cell at 48 h
of interaction. h Guard cells after 48 h Xap interaction where is
possible to observe a bacterium inside the stomatal pore. In general,
the same structural changes were observed as those of cowpea, such
as the degradation of chloroplasts and the presence of some
membranous structures in the cytoplasm. i Plastid of an epidermal
cell in advanced stage of degradation after 48 h. Es extracellular
space, CW cell wall, N nuclei, Mt mitochondria, C chloroplasts,
V vacuoles, Sg starch grain, asterisks bundle sheath and filled trianglebundle sheath cells, arrow heads plastoglobuli, arrow position of
multivesicular bodies on cytoplasm of guard cells. Bar, 0.5 lm in
(a, i), 2 lm in (b), 0.75 lm in (c, d), 0.3 lm in (e, f), 0.19 lm in (g),
1.3 lm in (h)
2030 Acta Physiol Plant (2011) 33:2025–2033
123
both species. In cowpea, these changes were shown to occur
in the chloroplasts with two distinct intensities. Those of the
parenchyma cells presented less structural disorganization,
while those of the vascular system presented a strong struc-
tural disorganization. For bean, these changes were only
observed in chloroplasts of parenchyma cells. Due to the
rapidity in which the changes happened we suggest that some
diffusible factor released by the bacteria could be involved in
the observed changes. The xanthomonads produce
extracellular polysaccharides (EPS) called xanthan gum. In
regard to pathogenicity, xanthan is involved with pathogen
virulence in some systems. Its infiltration on host plants
produces symptoms such as yellowing, browning, and water-
soaking lesions (Walkes and O’Garro 1996; Dharmapuri and
Sonti 1999). The aforementioned observations reflect that
Xap have infected the bean plants.
While we were looking for the ultrastructural changes
we observed an unexpected association between
Fig. 4 Association between
chloroplasts and mitochondria
in leaves of bean infected with
Xap. The chloroplasts presented
the same structural changes
observed in Fig. 3, such as
disorganization of grana and
thylakoids, accumulation of
starch grains and plastoglobuli.
a Chloroplasts after 12 h
showing a projection.
b Chloroplast after 48 h of Xapinteraction with a projection
near to a mitochondria.
c Chloroplast after 12 h of Xapinteraction with a mitochondria
in its interior and also a portion
of the cytoplasm, the
mitochondria present normal
ultrastructure and d Chloroplast
12 h after Xap infection with a
portion of the cytoplasm.
e, f Chloroplast and a
mitochondrion of control cells,
respectively. N nuclei, Mtmitochondria, C chloroplast, Sgstarch grain, arrow headsplastoglobuli, arrows indicate
mitochondria in the proximity
of a projection b or in intimate
association with a chloroplast
c. Bar, 0.35 lm in (a, c),
0.5 lm in (b), 0.75 lm in (d),
0.15 (e), and 0.21 (f)
Acta Physiol Plant (2011) 33:2025–2033 2031
123
chloroplasts and mitochondria of bean plants (Fig. 4a–f). It
was found cytoplasmic projections in the chloroplasts.
Some projections were also observed in the vicinity of
mitochondrion (Fig. 4b) and chloroplast with an intimate
association with mitochondrion (Fig. 4c). In the Fig. 4d
seems that only a cytoplasmatic portion was associated or
possibly engulfed. Neither of these results was found to the
cowpea-infected plants. The effects of the Xap infection, as
have been demonstrated to bean plants (Fig. 3a–i), are
present in these chloroplasts as the light disorganized
membrane system, accumulation of starch grains, and
plastoglobuli.
Other studies also related the close association of
mitochondria and projections of the chloroplast. A similar
phenomenon has been reported in plants of Z. mays and
Hyptis suaveolens grown in conditions of low light inten-
sities (Montes and Bradbeer 1976), which that we had found
describing a similar phenomenon. They have also showed
that the mitochondria were localized in deep invaginations
of chloroplasts as demonstrated by serial sections. Differ-
ently from the chloroplasts of Z. mays and H. suaveolens
associated to mitochondria which did not present starch
grains, the chloroplasts of bean-infected plants presented
starch grains and they were great and deformed. They also
presented plastoglobuli and a light disorganized membrane
system. These effects were related to the Xap infection as
can be observed in Fig. 4. Montes and Bradbeer (1976)
have showed that the mitochondria associated with chlo-
roplasts of Z. mays and H. suaveolens presented an apparent
normal ultrastructure. We have also found normal ultra-
structure for the associated mitochondria.
Other stress types have been shown to induce such
alteration. In transgenic tobacco plants (Pssu-ipt) over-
producing cytokinin (a senescence inhibiting hormone) the
chloroplasts also present irregular shape and protrusions of
the chloroplast envelop into the cytoplasm (Synkova et al.
2003). High-alpine plants such as Ranunclus glacialis,
Geum reptans, Oxyria digyna, and Poa alpine var. vivipara
form plastid protrusion and close association with mito-
chondrion (Lutz and Engel 2007).
Montes and Bradbeer (1976) speculated that the asso-
ciation of the chloroplasts and mitochondria in leaves of
Z. mays and H. suaveolens grown in low light intensities
could be related to an energetic coupling between the two
organelles. Synkova et al. (2003) also credit the phenom-
enon of association of plant organelles to way plants to
cope with stress conditions. Lutz and Engel (2007) also
speculated that the observed protrusion of plastid in high-
alpine plants could facilitate or speed up the exchange of
metabolites between cytoplasm and plastid as a way to
high-alpine plants to adapt to extreme environment of the
alpes. Kohler et al. (1997) had demonstrated that chloro-
plast projections are able to exchange proteins and
speculated that exchange of macromolecules could be the
functions of these projections. In our case bean plants were
grown in normal day light conditions which have incited us
to relate this phenomenon to the Xap infection. Any
reported information related to such phenomenon and the
pathogenic state could not be found in the literature.
Additionally, in general, in pathogen-infected plants is
observed a reduction of the photosynthesis rate caused by
the perturbation of the normal plant physiological condi-
tions upon the pathogen stablishment or replication in plant
tissues (Akhkha et al. 2003; Synkova et al. 2006). For these
reasons we can not discharge the possibility of the asso-
ciation found in this work be a way into bean plant with-
stand Xap infection. Additional studies will be necessary to
unravel the physiological role of such association in the
pathological state.
Acknowledgments This study forms part of the DS degree thesis of
A. O. Carvalho, carried out at the Universidade Estadual do Norte
Fluminense-Darcy Ribeiro (UENF). We acknowledge the financial
support of the Brazilian agencies Conselho Nacional de Desen-
volvimento Cientıfico e Tecnologico (CNPq) and Fundacao Carlos
Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro
(FAPERJ), the UENF and the International Foundation for Science
(IFS), Stockholm, Sweden (grant C/2806–3F). We are grateful to B.
R. Ferreira for the preparation of samples for microscopy, M. A. da
Silva Carvalho for photography and to M. T. Gobo and L. C. D. Souza
for technical assistance.
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