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Physiological and Molecular Plant Pathology (2000) 56, 227233

doi:10.1006/pmpp.2000.0266, available online at http://www.idealibrary.com on

BSMV infection inhibits chlorophyll biosynthesis in barley plants


A . A L M A S I 1, D . A PAT IN I 3, K. B O K A 2, B . B O D D I 3 and R . G A B O R J A N Y I 1*
1

Plant Protection Institute, Hung. Acad. Sci., 1525 Budapest, P.O. Box 102, Hungary, 2Department of Plant Anatomy, Eotvos
Lorand University, Puskin u. 11/13, Budapest 1088, Hungary and 3Department of Plant Physiology, Eotvos Lorand University,
Muzeum krt 4/a, Budapest 1088, Hungary
(Accepted for publication March 2000)
Protochlorophyllide forms, their phototransformation in the greening process and the ultrastructure of the
etioplasts were studied in BSMV-infected barley plants. The aim of this work was to determine whether
the chlorotic symptoms caused by the infection of this virus result from inhibition of chlorophyll
biosynthesis. 77 K uorescence emission spectra of dark-grown, ash-illuminated and re-darkened leaves
and transmission electron micrographs suggested that the BSMV infection did not cause specic changes
in chlorophyll biosynthesis or in chloroplast development. However, infection accelerated senescence and
c 2000 Academic Press
inhibited or delayed chlorophyll biosynthesis similar to general stress eects. *
Keywords: Barley; Hordeum vulgare L. cv Omega; barley stripe mosaic hordeivirus Russian strain
Atabekov; BSMV; chlorophyll biosynthesis; etioplast ultrastructure; greening; protochlorophyllide; virus
infection.

INTRODUCTION
Virus infection of susceptible plants frequently induces
mosaic, chlorotic or mottling symptoms of systemically
infected leaves. All these pathological changes are closely
related to reduced rates of photosynthesis, and eects on
chloroplast number, ultrastructure and chlorophyll
metabolism [1, 14, 15, 17, 21, 29]. Deterioration of
chloroplast structure, pigment composition and electron
transport can be attributed to the damage caused mostly
in photosystem II (PSII) by the infection stress [24, 20,
24, 30, 38]. Host response to virus infection depends
considerably on the sites of virus replication or on the
accumulation of coat protein or other viral gene products
[23, 25, 26, 37, 42].
Barley stripe mosaic virus (BSMV) induces signicant
chloroplast alterations, such as aberrant swelling, deformation of thylakoid membranes, cytoplasmic invaginations and viral particles attached to the envelope
membrane and small peripheral vesicles [11, 12, 22, 27,
28, 31]. Characteristic modications of 77 K uorescence
emission and excitation spectra were observed in leaves or
isolated plastids of various virus-infected plants, showing
alterations mainly of PSII which may result in decreased
* To whom all correspondence should be addressed.
Abbreviations used in text: BSMV, barley stripe mosaic virus;
Chl, chlorophyll; Chlide, chlorophyllide; Pchlide, protochlorophyllide; PLB, prolamellar body; POR, NADPH : protochlorophyllide oxidoredutase; PSII, photosystem II.

0885-5765/00/060227+07 $35.00/00

light absorbance of PSII [2]. All former experiments were


carried out with fully developed symptomatic leaves, and
showed the nal results of the infection. From these studies
it was not possible to decide whether the deterioration
eect was due to the decomposition of chloroplasts or to a
consequence of inhibited biosynthesis. For example,
chlorosis may reect the disturbance of chlorophyll
biosynthesis. The key regulatory step of this process in
higher plants is the protochlorophyllide (Pchlide)chlorophyllide (Chlide) photoreduction [16] that can be
studied in detail in dark-germinated seedlings. The
NADPH:protochlorophyllide oxidoreductase (POR)
enzyme needs illumination for its activity, therefore
these plants can synthesize protochlorophyllide in darkness. The enzyme POR, the Pchlide and the coenzyme
NADPH form ternary complexes [32], which are integral
units of the tubular network of the etioplast inner
membranes [40], mainly of the prolamellar bodies
(PLB) [6]. These membranes contain aggregates of the
ternary complexes, i.e. several POR-protein subunits
interact among each other to form a molecular environment for interactions among Pchlide molecules [44]. The
result of these interactions is the spectral heterogeneity of
Pchlide in vivo. Complex analyses of the 77 K uorescence
emission spectra of wheat leaves and etioplast inner
membranes showed the presence of main (00) emission
bands at 633, 644, 657 and 670 nm [9, 10]. The 657 nm
emitting band dominates in spectra of 8 day old etiolated
leaves and the complex giving this band has the main role
c 2000 Academic Press
*

228

A. Almasi et al.

in the Chl biosynthesis. Besides this complex, the 644 nm


emitting species is also photoactive. At ash illumination,
these complexes transform into Chlide-containing complexes emitting at 696 and 684 nm, respectively. The
696 nm emitting Chlide complex dominates the spectrum
[8]. A blue shift of the 696 nm band to 680 nm can be
detected which completes within about 30 min after the
ash illumination. This shift corresponds to the
684672 nm blue shift of the absorption bands termed
as Shibata-shift [41]. The 633 nm emitting complex
contains Pchlide and/or protochlorophyll (Pchl) connected to other molecules than the POR-protein [6] and
consequently, this complex cannot be transformed into
Chlide (or Chl) by ash illumination. The role of the
complex emitting at 670 nm, in the Chl biosynthesis is
unknown.
The ratio of the 633, 645 and 657 nm emitting complexes depends on leaf age. In young (23 day old) and
old (older than 1012 days) leaves, the shorter wavelength-emitting complexes are more abundant than the
657 nm band [43].
The ratio of these complexes is also inuenced by the
physiological state of plants, cold stress [5], Cd treatment increases the relative amount of the shortwavelength forms [7].
The aim of this work was to investigate the eects of
BSMV infection on the organization and relative amounts
and/or photoactivity of the Pchlide containing complexes
and this way to assess chlorophyll biosynthesis. Experiments carried out with BSMV-infected barley seedlings
grown in darkness revealed that early virus infection can
interfere with chlorophyll biosynthesis and that damaged
chloroplasts are not the only result of decomposition
processes.

MATERIALS AND METHODS

Plants and viruses


Barley plants (Hordeum vulgare L. cv. Omega) were
germinated in darkness at 258C for 3 days. Then, in a
group of the seedlings, the coleoptyles were inoculated
mechanically with barley stripe mosaic hordeivirus
(BSMV) Russian strain Atabekov. Inoculum was prepared from symptomatic young barley leaves suspended in
0.1 M Sorensen's phosphate buer ( pH 7.2) in a ratio 1 : 5
(g cm 3). Celite was added to the inoculum as abrasive.
The plants were grown on stainless steel wire nets in
hydroponic culture. The plastic boxes containing the
plants were closed with plastic to retain moisture but
permit CO2 and O2 diusion. The plants were grown for
an additional 12 days in the dark at 258C.

77 K spectroscopy
The 77 K uorescence spectra were measured with
Fluoromax-2 spectrouorometer (Jobin YvonSpex)
equipped with a low-temperature accessory. During
measurements, samples were immersed into liquid nitrogen. The excitation wavelength was 440 nm, the optical
slits were 2 nm. An average of three repetitions was
collected in the case of each spectrum. The integration
time was 0.2 s. The spectra were corrected for the
wavelength-dependent sensitivity changes of the instrument and exported in ASCII format for further calculations. Averages of spectra measured from six dierent
plants were calculated, baseline correction and 3-point
linear smoothing was used with the software SPSERV
V3.14 (Copyright: C. Bagyinka, Inst. Biophys. Biol. Res.
Cent. Szeged, Hungary).
Leaf homogenates were prepared in a mixture of 50 %
glycerol and phosphate buer ( pH 6.8) containing 50 %
sucrose. The rst emerging leaves of three dierent plants
were homogenized in cooled mortars, and ltered
through three layers of gauze.
Flash illumination was done with Chinon 900 C
photoash apparatus by varying the distance of illumination to obtain complete phototransformation without
photodestruction of the leaves. For continuous illumination, white light of 100 mmol m 2 s 1 was used. The
chlorophyll content was determined in 80 % acetone.
Absorbance values were measured with a Shimadzu
UVPC spectrophotometer. For concentration calculations, the equation of Porra et al. (1989) was used [34].

Transmission electron microscopy


The rst 0.5 cm pieces of primary leaves were discarded
to avoid dierences in the age of the etioplasts, and the
next 2 cm sections were collected as samples. Tissue
pieces from the leaves were xed in 2 % glutaraldehyde
for 2 h in the dark and postxed in 1 % OsO4 for 3 h.
Fixing solutions were diluted in 70 mM Na-K phosphate
buer ( pH 7.2). After the xing steps, samples were
rinsed in the same buer. Dehydrated samples were
embedded in Durcupan ACM epoxy resin (Fluka Chemie
AG). Ultrathin sections were stained with 5 % uranyl
acetate dissolved in methanol for 5 min and subsequently
treated with Reynolds' lead citrate solution for 5 min
[39]. The sections were investigated by a Hitachi 7100
TEM at 75 kV accelerating voltage.

Serological assay
Virus content of the infected leaves was detected
by ELISA, according to Clark and Adams, 1977 [13]
using anti-BSMV antiserum of Loewe Biochemica Ltd.

BSMV infection inhibits chlorophyll biosynthesis in barley plants


Extinctions were measured at 405 nm wavelength in a
Labsystem Multiscan MS spectrophotometer.

To compare the Pchlide forms in the control and virusinfected leaves, 77 K uorescence spectra were measured.
In spectra of control and virus-infected plants, an emission band at 655 nm dominated. However, the infection
caused an increase of the 633 nm band relative to the
amplitude of the 655 nm band by 12 days after inoculation. This phenomenon varied in dierent plants, among
leaves of dierent ages of the same plants and in sections
along the same leaf. It was the strongest in the old cells at
the tip of the rst (oldest) leaves and less obvious downwards in these leaves or in younger ones. Therefore, the
average spectra of six control and six infected leaf sections
were calculated and compared. These samples were
collected from identical parts of the rst leaves, i.e. the
section between the 2nd and 4th cm (Fig. 1, curves 1 and
2). In several plants the relative increase was more
pronounced (Fig. 1, curve 3). To study the signicance of
the band ratio changes three control and three infected
rst leaves were homogenized in buered sucrose and
glycerol containing solution and the spectra of the leaf
homogenates were compared. This comparison showed
the same phenomenon as the spectra of leaf segments, i.e.
a relative increase of the 633 nm band in the infected
plant material (Fig. 1, inset). No signicant dierences
were found in the spectra of the second leaves of infected
and control plants 12 days after the inoculation. Two
days later, however, similar phenomena were found as in
the case of the rst leaves (data not shown).
Flash illumination caused the phototransformation of
the 655 nm emitting Pchlide complexes in both control
and infected leaves and the 694 nm emitting Chlide form
appeared. In addition to this band, local maximum was
observed at 633 nm and a shoulder at around 677 nm.
The spectra of the control and infected plants were similar
in the Chlide emission region (above 660 nm) (Fig. 2).
The regeneration of the photoactive 655 nm Pchlide
complex and the process of the Shibata-shift were
observed in spectra of plants that were ash illuminated
and then kept in darkness for 6 h. Comparisons of the
spectra of control and infected plants revealed that both
processes were delayed in the infected plants. The
amplitude of the 655 nm band of infected plants was
smaller than that of the control and the Shibata-shift was
not complete. After 6 h the maximum was at 687 nm,
while the control had maximum at 680 nm (Fig. 3). This
phenomenon was expressed only in plants having the
strongest increase of the 633 nm band in their spectra

1.0

0.8

Fluor. emission (rel.)

Fluor. emission (rel.)

77 K spectroscopy

1.0
3

0.6

0.5

0.0
600

640

680

Wavelength (nm)

0.4
0.2
0.0

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620

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660

680

700

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740

Wavelength (nm)

F I G . 1. 77 K uorescence emission spectra of dark-grown,


BSMV-infected and control barley leaves and leaf homogenates in 50 % sucrose (inset). Assignments in the main
panel: curve 1 average spectrum of six infected leaf segments;
curve 2 average spectrum of six control leaf segments;
curve 3 spectrum of an infected leaf with extremely high
extent of infection. Assignments in the inset: curve 1 infected;
curve 2 control. Excitation wavelength 440 nm, slits 2 nm.
1.0

Fluor. emission (rel.)

RESULTS

229

0.8
0.6
2

0.4
0.2
0.0

600

620

640

660

680

700

720

740

Wavelength (nm)

F I G . 2. 77 K uorescence emission spectra of ash illuminated


dark-grown BSMV-infected and control barley leaves. Assignments in the gure: 1 average spectrum of six infected leaf
segments; curve 2 average spectrum of six control leaf
segments. Excitation wavelength 440 nm, slits 2 nm.

before illumination. Other infected plants showed a more


complete Shibata-shift, but the delay was clearly shown
by shoulders of various amplitudes around 685 nm.
The delay of the Chl biosynthesis of infected plants was
observed when dark-grown plants were illuminated with
continuous light for 3 days and their Chl contents were
determined. Chlorotic symptoms appeared in the uppermost parts of the leaves but these were less obvious or did
not appear in the lower segments of the leaves. Thus,

230

A. Almasi et al.

Fluor. emission (rel.)

1.0
2

0.8
0.6
0.4
0.2
0.0

600 620

640

660

680

700

720

740

760

780

Wavelength (nm)

F I G . 3. 77 K uorescence emission spectra of redarkened


BSMV-infected and control barley leaves. The dark-grown
leaves were ash illuminated and then kept in the dark for 6 h.
Assignments in the gure: 1 average spectrum of six infected
leaf segments; curve 2 average spectrum of six control leaf
segments. Excitation wavelength 440 nm, slits 2 nm.

F I G . 4. Electron micrograph of etioplast from the rst leaf of


dark-grown 12 day old control plant. Prolamellar body (PLB) is
well developed and numerous radially positioned prothylakoids
(P) originate from it. Bar 1 mm.

comparisons of Chl contents of identical sections of


control and infected plants were important. The Chl
content of the segments between the 2nd and 4th cm of
virus infected leaves was around 70 % of the controls
(1470 and 2100 mg g 1 fresh weight, respectively). The
plants subsequently survived infection and the etiolated
plants could be greened and developed up to the 6-leaf
stage but the old leaves continuously dried out.

Transmission electron microscopy


In the control plants, most of the etioplasts had typical
structures described in the earlier literature [18, 19, 33,
45]. Both narrow and wide spacing types of prolamellar
bodies (PLBs) were well developed. Prothylakoids connected to PLBs were arranged radially, sometimes in
parallel position or connecting two PLBs incidentally.
Some of them showed several perforations and occasionally paired membranal regions appeared (Fig. 4).
In the BSMV-infected etiolated plants, the etioplasts
showed altered ultrastructure. The size of PLBs was
reduced and their structure was less regular than those of
controls. Wide crystalline-type PLBs and 3-armed tube
elements were common. Early state of transitorial PLB
form was easily observed. Parallel positioned lamellar
prothylakoids occurred frequently while radial prothylakoids were less numerous than in controls, and the
stacking and perforations were abnormal. Numerous
vesicular prothylakoids were also visible in the stroma.
These electrontransparent vesicles were frequent and
some of them were enlarged. Similar, but smaller vesicles

F I G . 5. Electron micrograph of etioplast from the rst leaf of


dark-grown 12 day old BMSV-infected plant. There are some
lamellar prothylakoids arranged parallel (L) connected to the
PLB and several electron-transparent vesicular prothylakoids
(V) are present in the stroma. Bar 1 mm.

were found in some of the etioplasts of the control plants


indicating their ageing (Fig. 5).
Plastoglubuli were near or inside of the PLBs and their
number was similar in the etioplasts of the control and
BSMV-infected plants.

Serological assay
All BSMV-infected plants used in the experiments gave
positive reaction with the anti BSMV antiserum, and all

BSMV infection inhibits chlorophyll biosynthesis in barley plants


control plants proved to be negative. The extinction
values of the positive samples were more than four times
higher than the negative controls.

DISCUSSION
The results of this work proved that 77 K spectroscopy is
an eective tool in identifying eects of BSMV infection
on a molecular level. Pchlide and Chlide molecules
bound in dierent molecular environments (complexes)
have characteristic emission bands the relative amplitudes
of which indicate the distribution of the pigment molecules between dierent complexes. Some of the Pchlide
complexes are photoactive (e.g. the 644 and 655 nm
emitting forms) while others (e.g. the 633 nm emitting
form) are not active under ash illumination. The shifts
of the emission maxima have denite order which is in
correlation with the greening of the leaves.
Symptom appearance in etiolated plants corresponded
to those of green plants and they were more intense in the
old leaves and cells than in the younger tissues. Since
the coleoptyles were infected, the symptoms were due to
the virus replication in the plants. The results indicated
an unequal distribution of the virus in the plants and also
the dierent plants had dierent infection levels. Therefore the measurements were more complicated, the
calculations of average spectra from dierent plants and
the measurement of leaf homogenates were necessary.
It is important to underline that these experiments
needed relatively old etiolated plant material because the
systemic spread of the infection and the symptom appearance required a minimum of 12 days. (This manifestation
was stimulated by the high humidity under the plastic and
the constant 258C temperature.) Thus, at the measurements the plants were 14 days old when certain
decomposition processes started in the etiolated tissues
due to ageing. Therefore, the senescence and the eect of
virus infections developed simultaneously. The etiolated
leaves of the infected plants had symptoms similar to those
of old etiolated tissues, i.e. a relative increase of the
633 nm band. This increase indicates the changes of the
distribution of Pchlide among the complexes of the
etioplast inner membranes. It means the formation of
non-photoactive complexes in which Pchlide is present in
monomeric state and is bound to protein other than POR
[6]. The spectra measured after ash illumination were
very similar because they were normalized at 694 nm, i.e.
at the emission maximum of the Chlide complex. This
Chlide form is produced only from the photoactive Pchlide
complex emitting at 655 nm, therefore the normalization
hides the possible dierences in the amounts of these complexes. Other spectra obtained after ash illumination
followed by a 6 h dark period indicated that the biosynthesis processes are delayed or hindered in the infected

231

tissues because the regeneration of the 655 nm emitting


photoactive complex was inhibited. At the same time, the
Shibata-shift was arrested in a stage when a special form
emitting at 686 nm appeared. This indicates a disturbance in the biosynthesis of the Chl-protein complexes: the
newly synthesised Chl-a molecules cannot be built into
their target proteincomplex molecules if the protein
biosynthesis is inhibited.
The age of etioplasts could also have inuenced their
ultrastructure [35]. Narrow and wide crystalline types,
``hexagonal rings'', 3-armed tubules were described at
dierent stages of etioplast ageing [18, 19, 35]. Etioplast
senescence seems to be a pre-programmed process the
timing of which can be inuenced by starving [36]. Small
vesicle forming prothylakoids resembling those of the
BSMV-infected plant's etioplasts might be interpreted as
ageing and starving stress appearing at the dark-grown
almost 2 week old seedlings.
These results allow us to draw the following general
conclusion: BSMV infection causes non-specic, general
stress responses: the biosynthesis processes of the infected
tissues are delayed and the senescence or ageing phenomena are accelerated. Thus, the mosaic or chlorotic
symptoms can be the result of Chl biosynthesis inhibition,
as well as decomposition of functioning photosynthetic
apparatus formed before infection.
This work was partly supported by a Research Grant of
Hungarian Academy of Sciences (AKP No. 96-192 3,1).

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