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: valmg@uenf.br

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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