Department of Field Crops, Vegetables, and Genetics and the Otto
Warburg Center for Agricultural Biotechnology, Faculty of Agricultural,
Food, and Environmental Quality Sciences, The Hebrew University of
Jerusalem, Rehovot 76100, Israel
Viral infection often affects carbon assimilation and metabolism in
host plants. To better understand the effect of cucumber mosaic virus
(CMV) infection on sugar transport, carbohydrate levels and the amounts
of the various sugars in the phloem sap were determined in infected
melon (Cucumis melo L.) plants. Source leaves infected
with CMV were characterized by high concentrations of reducing sugars
and relatively low starch levels. The altered level of carbohydrates
was accompanied by increased respiration and decreased net
photosynthetic rates in the infected leaves. Although stachyose was the
predominant sugar in phloem sap collected from petioles of control
leaves, sucrose (Suc) was a major sugar in the phloem sap of infected
leaves. Moreover, analyses of the newly fixed
14CO2 revealed a high proportion of radioactive
Suc in the phloem sap of infected leaves 60 min post-labeling. The
alteration in phloem sap sugar composition was found in source, but not
old, leaves. Moreover, elevations in Suc concentration were also
evident in source leaves that did not exhibit symptoms or contain
detectable amounts of virus particles. The mode by which CMV infection
may cause alterations in sugar transport is discussed in terms of the
mechanism by which sugars are loaded into the phloem of cucurbit plants.
 |
INTRODUCTION |
One of the key factors controlling
carbohydrate allocation to the various plant organs is the process of
phloem loading. Photosynthetic carbon assimilation and Suc synthesis
take place within mesophyll cells of mature source leaves. The flow of
Suc is governed by cell-to-cell transport through mesophyll cells and
vascular tissues, after which Suc is loaded into the companion
cell-sieve element (CC-SE) complex (van Bel, 1993
). Phloem loading is
defined by Geiger (1975) as "the process by which the major
translocated substances are selectively and actively delivered to the
sieve tubes in the source region prior to translocation." Structural studies have suggested two possible mechanisms for phloem loading. In
most agronomically important crops, the transport of Suc to the loading
region is via the symplasmic pathway, followed by an apoplasmic step to
transfer Suc into the CC-SE complex (van Bel, 1993; Turgeon, 1996).
Cucurbits, however, are characterized by the presence of numerous
plasmodesmata (PD) connecting the minor veins with the surrounding
bundle sheath (BS), suggesting a symplasmic pathway along the entire
route from the mesophyll to the CC-SE complex. Based on these
structural observations and the fact that the main translocated sugar
in cucurbits is stachyose, an alternative model for phloem loading was
developed (Turgeon, 1991; Grusak et al., 1996). According to this
model, Suc is synthesized in the mesophyll and then diffuses through
the BS into the intermediary cells (IC, a specific type of CC in minor
veins of cucurbit plants) through the abundant PD that connect the two
cell types. Raffinose and stachyose are synthesized from Suc in the IC
(to maintain the diffusion gradient for Suc). It is thought, however,
that these sugar molecules are not able to diffuse back into the
mesophyll because they are larger than the size exclusion limit (SEL)
of the connecting PD and, as a result, the concentration of raffinose and stachyose rises in the phloem. This hypothesis is based on the
assumption that PD between the BS and IC must be somewhat smaller
(400-500 D) than is common for PD at other interfaces (800-1,000 D)
in order to trap the oligosaccharides in the IC.
Numerous reports have indicated that carbohydrate metabolism in
the source leaf is influenced by viral infection (Tecsi et al., 1994a,
1994b, 1996). Infected source leaves are usually characterized by
reduced photosynthetic rate, a decrease in the concentration of soluble
sugars, and often starch accumulation (Goodman et al., 1986; Fraser,
1987).
Detailed analysis of the host response to cucumber mosaic virus (CMV)
infection was performed in cotyledons of marrow (Cucurbita pepo L.) plants (Tecsi et al., 1994a, 1994b). An increase in
starch and a decrease in Suc contents were observed during the first few days after infection. However, later the infected cotyledons incorporated less of the recently fixed carbon into structural carbohydrates and more into soluble sugars (Tecsi et al., 1994b). Fine-mapping of the lesions formed by CMV in cotyledons of marrow plants was used to define the relationships between viral accumulation, starch content, and photosynthetic activity (Tecsi et al., 1994a). This
study indicates that viral replication within specific cells initiates
a series of metabolic events in the neighboring cells over several
days, following infection. Interestingly, the alteration in
photosynthetic activity and starch accumulation was not found to be a
direct consequence of viral replication.
The effect of viral movement proteins (MPs) on PD function is well
documented (Carrington et al., 1996; Gilbertson and Lucas, 1996). It is
therefore logical to assume that alteration of plasmodesmal function in
virally infected plants will affect the process of phloem loading
particularly in plants that are determined to be symplasmic loaders. In
this paper, we examined the effect of CMV infection on carbon
assimilation and transport in melon (Cucumis melo L.)
plants. We show that viral infection causes a significant increase in
the proportion of Suc within the phloem. Moreover, this effect was also
evident in the phloem sap of leaves in which the virus was absent,
indicating that the influence over the loading of sugars into the
phloem cannot be attributed to a simple interference of virus particles
with this process.
| |
RESULTS |
CMV Infection Alters Assimilation Rate and Carbohydrate Metabolism
in Source Leaves
Typical CMV symptoms were observed 8 to 10 d post-inoculation
(DPI). First symptoms were evident in the uppermost leaves, with
symptoms observed in older leaves a few days later. It is important to
note that in all experiments, the first (and usually even the second)
leaf above the infected one did not exhibit any symptoms, even 30 DPI.
Daytime changes in carbohydrate levels were analyzed in 5-week-old
plants, 2 weeks after inoculation with CMV. Although discs were taken
from the leaf area between the minor veins, it was impossible to
completely eliminate the presence of minor ribs in the samples. As
indicated in Figure 1, levels of primary
sugars (Glc and Fru) were significantly higher in CMV-infected plants than in controls. Similar results were obtained in the morning and
afternoon hours. Starch levels were significantly lower in the infected
plants at both sampling hours (Fig.
2).
View larger version (33K):
[in this window]
[in a new window]
|
Figure 1.
Effect of CMV infection on sugar content in source
leaves (leaf 5) of melon plants. Leaf discs were taken at the beginning
of the light period (A) and in the afternoon (B). White columns,
Noninfected plants; dotted columns, CMV-infected plants. Data are the
means of five measurements. Columns followed by different letters are
statistically different at the 5% level.
|
|
View larger version (23K):
[in this window]
[in a new window]
|
Figure 2.
Effect of CMV infection on starch content (as Glc
equivalents) in source leaves (leaf 5) of melon plants. Leaf discs were
taken at the beginning of the light period (AM) and in the afternoon
(PM). White columns, Noninfected plants; dotted columns, CMV-infected
plants. Data are the means of five measurements. Columns followed by
different letters are statistically different at the 5% level.
|
|
Analysis of photosynthetic activity in leaf 5 (exhibiting typical
mosaic symptoms) indicated no differences in gross photosynthesis between infected and control plants (Table
I). However, the values of respiration
rate for the infected leaves were almost twice than those for the
control plants, resulting in a significant CMV-imposed reduction in net
photosynthesis with values close to zero in the infected leaves (Table
I).
View this table:
[in this window]
[in a new window]
|
Table I.
Photosynthesis (Pn) and respiration rates of control
and CMV-infected melon plants
Plants were grown in a temperature-controlled growth chamber with an
average photon flux density of 250 µmol m 2
s1 at the level of the measured leaf (no. 5).
Measurements were performed 2 weeks post-inoculation. Values followed
by the same letter within a column are not statistically different at
the 5% level.
|
|
Effect of CMV Infection on Phloem Sap Sugar Composition
Phloem sap was collected from the petiole of leaf 5 before the
beginning of the light period and after 8 h of the photoperiod. Stachyose was found to be the major sugar present in the phloem sap of
healthy plants (Fig. 3). Only low levels
of Suc were detected in these petioles, about 20% to 25% of the
stachyose levels. A significant increase in the concentration of Suc in
the phloem sap was evident in petioles of CMV-infected plants. Both at
the beginning of the light period and in the afternoon, Suc
concentration was about 6-fold that of stachyose. The Suc to stachyose
ratio in the sap collected from infected plants was about 20 times
higher than that of controls.
View larger version (23K):
[in this window]
[in a new window]
|
Figure 3.
Effect of CMV infection on sugar content of phloem
sap collected from petioles of melon source leaves. Sap was collected
at the beginning of the light period (AM) and in the afternoon (PM).
White columns, Noninfected plants; dotted columns, CMV-infected plants.
Data are the means of five measurements (±SE).
|
|
To further explore the effect of CMV infection on phloem sap
composition, a specific experiment was designed in which cotyledons were infected with CMV just after expansion (to ensure infection of all
the true leaves). In this particular experiment, phloem sap was
collected from young-mature and old leaves 5 weeks after inoculation
when all plants had nine to 10 leaves. The selected old leaves were the
oldest in each plant (leaf 1) and the young-mature ones were leaves 7 and 8. No significant differences were observed in stachyose or
raffinose levels of phloem sap collected from young versus old petioles
(Table II). All sugars were more
concentrated (though not statistically significant) in the phloem sap
of infected versus control leaves. However again, the most pronounced
effect of CMV infection was on the level of Suc, which was over 10 times higher in sap collected from young infected petioles, relative to
their respective controls. Interestingly, the rise in Suc concentration due to CMV infection was relatively small in petioles of old leaves, with no significant differences between the infected and control plants.
View this table:
[in this window]
[in a new window]
|
Table II.
Sugar content in the early afternoon (2 PM) in the phloem sap of control and CMV-infected melon
plants
Plants were grown in a growth chamber (25°C/18°C day/night) with a
12-h photoperiod. Sap was collected from petioles of young-mature (nos.
7 and 8) and old (no. 1) leaves 5 weeks after the inoculation of
cotyledons. Five plants were used for each treatment. Values followed
by the same letter within a column are not statistically different at
the 5% level.
|
|
A pulse-labeling experiment was performed as an alternative approach to
verifying the effect of CMV infection on phloem-sugars composition.
This experiment was conducted in a temperature-controlled greenhouse
under natural irradiation regimes. Net photosynthetic rate under these
conditions was 14.8 µmol CO2
m2 s1 for the control
plants, significantly higher than the value obtained for the
CMV-infected plants (7.7 µmol CO2
m2 s1). Phloem sap was
collected from petioles of the labeled leaves 30 and 60 min after
labeling and the radioactivity of each sugar was determined. As
indicated in Figure 4 radioactivity level
of phloem sugars nearly doubled from 30 to 60 min post-labeling. In
control plants this increase was solely due to the increase in the
radioactivity of stachyose and raffinose, while the radioactivity of
Suc remained constant at a low level. However, in CMV-infected leaves,
the elevated radioactivity of the phloem sugars was made up of an
increase in the radioactivity of stachyose plus raffinose and a
significant increase (more than double) in the radioactivity of Suc.
These results indicated alterations in the process of newly fixed
carbon metabolism and transport in CMV-infected leaves.
View larger version (32K):
[in this window]
[in a new window]
|
Figure 4.
14C sugars fractionated from
phloem sap of melon source leaves (leaf 5). Sap was collected 30 and 60 min after labeling the leaf with
14CO2. White columns,
Noninfected plants; dotted columns, CMV-infected plants. Data are the
means of three measurements (±SE).
|
|
The Effect of CMV Infection on Phloem Sap Sugar Composition Is
Independent of the Presence of Virus Particles
Long-distance movement of virus particles is known to occur via
the phloem, following the stream of sugar transport (Maule, 1991;
Gilbertson and Lucas, 1996). The nonsignificant effect of CMV on phloem
Suc concentration in old leaves (Table II) could have been due to the
fact that these specific leaves were too old to be sources. However, it
is also possible that they did not contain high levels of virus particles.
Further study was aimed at exploring the relationship between
progression of the systemic infection and alteration in Suc levels in
the phloem sap of different leaves. Phloem sap was collected from
different leaves at different times post-infection. First symptoms were
observed in leaves 5 and 6 at 10 DPI, at which stage the plants had six
true leaves. As indicated in Figure 5A,
Suc concentration in the phloem sap collected from petioles of leaf 5 was 15 times higher than values determined in control leaves. However,
a significant elevation in Suc concentration was also evident in leaf
3, which did not show any typical mosaic symptoms. Protein, as well as
RNA analyses, were employed to verify the presence of virus particles
in the latter leaves and according to both analyses, no virus particles
were detected. A similar phenomenon was observed when leaves were
sampled 16 DPI (Fig. 5B). On this date, the plants had eight true
leaves and symptoms were observed in all leaves except for leaves 2 and
3 (located above the infected leaf). Again, a significant increase in
Suc concentration was found in petioles of leaf 3, although virus particles could not be detected by either western blot or reverse transcriptase-PCR.
View larger version (48K):
[in this window]
[in a new window]
|
Figure 5.
Presence or absence of CMV particles in different
leaves of CMV-infected melon plants. Presence of CMV in the various
leaves was determined using western blot of leaf extracts probed with
antiserum raised against purified CMV-CP, or by analysis of CMV-MP RNA
using reverse transcriptase-PCR. Leaves 3 and 5 from two representative
plants (1 and 2) were sampled 10 DPI (A) and leaves 3 and 4 from other
representative plants (3-6) were sampled 16 DPI (B). Values indicate
the concentration of Suc (mg mL1) in the phloem
sap of petioles collected from the respective leaves. Data in
parentheses indicate the Suc concentration (mg
mL1) in phloem sap of respective control
petioles collected from noninfected plants. ud, Undetected.
|
|
To further investigate whether CMV infection causes other functional
alterations in the noninfected leaf (no. 2), photosynthetic rate and
leaf sugar content were determined in an additional experiment. As
indicated in Table III, similar
photosynthetic and respiration rates were obtained for leaves of
infected and noninfected plants. It is important to note that the
differences in the rate of net photosynthesis and respiration in leaf 5 (exhibiting typical symptoms) from the same set of plants were similar
to those presented in Table II. Moreover, the levels of soluble sugars
extracted from infected and control leaves (no. 2) were almost
identical (Table IV). Nevertheless, once
again, phloem collected from petioles of leaves from the infected
plants contained significantly higher levels of Suc (Fig.
6). The Suc to stachyose ratio in the sap collected from the infected plant (leaf 2) was about 40 times higher
than in its respective control.
View this table:
[in this window]
[in a new window]
|
Table III.
Photosynthesis (Pn) and respiration rates of
control and CMV-infected melon plants
Plants were grown in a temperature-controlled growth chamber with an
average photon flux density of 200 µmol m2
s1 at the level of the measured (no. 2) leaf. Measured
leaves from both control and CMV-infected plants did not exhibit any
symptoms. Measurements were performed 2 weeks post-inoculation. Values
followed by the same letter within a column are not statistically
different at the 5% level.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV.
Sugar content in leaves of different ages from
CMV- infected and control melon plants
Samples were collected in the early afternoon (2 PM) from
young fully expanded (no. 5) and fully mature (no. 2) leaves 2 weeks
after inoculation. Plants were grown in a temperature-controlled growth
chamber (25°C/18°C day/night) with a 12-h photoperiod. Five plants
were used for each treatment. Values followed by the same letter within
a column are not statistically different at the 5% level.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Figure 6.
Effect of CMV infection on sugar content of phloem
sap collected from petioles of melon leaves. Sap was collected 2 weeks
after inoculation from leaf 5 displaying typical symptoms and leaf 2, which was symptomless. White columns, Noninfected plants; dotted
columns, CMV-infected plants. Data are the means of five measurements
(±SE).
|
|
| |
DISCUSSION |
Systemic viral infection often causes mosaic symptoms and/or
necrotic lesions, indicative of structural changes in the chloroplasts, altered carbon metabolism, and the accumulation of starch grains (Goodman et al., 1986; Fraser, 1987). A gradual decline in net photosynthesis has been observed in cotyledons of marrow plants during
the first week after infection with CMV; however, the most pronounced
effect observed in the infected cotyledons is a sharp increase in
respiration rate (Tecsi et al., 1994b). A significant increase in
respiration rate after CMV infection was observed in our study as well
(Table I). It is interesting that the gross photosynthetic rate, 2 weeks post infection, was similar for control and infected source
leaves. Significantly higher net photosynthetic rate in control as
compared with CMV-infected leaves was observed under natural sunlight
in greenhouse grown plants. Nevertheless, the differences in gross
photosynthetic rate were only about 20%. These results indicate that,
despite the severe mosaic symptoms observed at this stage of plant
development, the potential photosynthetic activity of CMV-infected
leaves is relatively high.
CMV infection significantly altered carbohydrate metabolism. A sharp
increase in the concentrations of Fru and particularly Glc was observed
in the infected leaves. These changes are associated with a decrease in
leaf starch content (Figs. 1 and 2). An earlier study indicated an
increase in reducing sugars and a reduction in starch content due to
CMV-induced higher starch hydrolase and lower ADP-Glc pyrophosphorylase
activities (Tecsi et al., 1994b). The inhibition of starch accumulation
and/or starch degradation is probably due to the increased demand for
soluble sugars (mainly Glc) required to maintain the high respiration rate.
Perhaps the most important effect of CMV infection observed in the
present study was the striking alteration in sugar composition within
the phloem sap. Similar to data presented in previous reports (Madore,
1991; Mitchell et al., 1992), stachyose was the predominant sugar in
the phloem sap of control plants (Fig. 3). As indicated in Figures 3
and 6, CMV infection caused only small changes in the phloem sap
stachyose concentration; in contrast, Suc concentration increased
dramatically. The outcome of these changes was a 15- to 40-fold
increase in the Suc to stachyose ratio. Note that the concentration of
sugars in the phloem sap collected from cut petioles may not be
identical to their concentration in vivo. Dilution of the collected sap
by solutes and water from the cut surface and xylem elements could
reduce the resultant values. None of our samples contained even traces
of monosaccharides, which suggests that the collected solute did not
contain detectable contamination from neighboring cells. Nevertheless,
water delivered from the xylem elements may have produced lower
concentration values. However, even if the collected samples were
diluted with water, it is important to note that stachyose
concentrations were similar for CMV-infected and control leaves,
emphasizing the dramatic changes in Suc concentration.
The increase in Suc level might be explained by the initiation of
stachyose hydrolysis following CMV infection. If this were the case,
however, there would be a parallel increase in Gal concentration in the
phloem sap. The fact that the alteration in Suc concentration was not
accompanied by detectable changes in other sugars concentration indicates that CMV infection affects the localization of Suc rather than its metabolism. An analysis of sugar levels in minor veins using a
microdissection procedure has indicated that IC may contain considerable amounts of Suc; however, Haritatos et al. (1996) suggested
that this Suc is localized in the vacuoles. One could argue that CMV
infection may have caused a perturbation in the sugars' localization
within the phloem cells. As Suc can easily diffuse from the IC into the
SE, an increase in IC cytosolic Suc concentration would result in a
high concentration of Suc within the phloem sap as well. Assuming that
in melon plants, transport of Suc from the mesophyll to the IC-SE
complex is via diffusion through PD, such an increase in IC Suc
concentration would likely result in Suc leakage back toward the
mesophyll, resulting in the inhibition (or even cessation) of phloem
loading. The level of total radioactivity in source leaves appeared to
decline more rapidly in CMV-infected versus control plants (data not
shown). This result, together with the fact that the increased
radioactivity in phloem total sugar was similar (about 2-fold) in
infected and control plants (Fig. 4), indicated that the export rate of
newly fixed carbon is not inhibited in CMV-infected plants.
An alternative explanation for the CMV-induced alteration in phloem sap
Suc concentration may relate to the functioning of the CMV-MP. A
significant increase in plasmodesmal SEL has been observed in
transgenic tobacco plants expressing the CMV 3a protein (Vaquero et
al., 1994; Ding et al., 1995), indicating that this is the CMV-MP.
Based on the model for phloem loading of sugars in cucurbit plants
(Turgeon, 1991; Grusak et al., 1996), an alteration in the SEL of PD at
the BS-IC interface could result in diffusion of stachyose from the IC
to the BS, thereby impairing the process of phloem loading and sugar
transport. However, even if CMV-MP or another viral protein altered
plasmodesmal functioning in infected melon plants, the observed
increase in Suc concentration within the phloem sap cannot be simply
attributed to such changes. On the contrary, an increase in the
plasmodesmal SEL would destroy the Suc trap in the IC, as described by
Grusak et al. (1996), and inhibit Suc traffic into the minor veins.
Hence, the basis for the change in Suc concentration may lie in an
alteration of the cellular controls that regulate phloem loading via
the symplasmic and apoplasmic routes. Recall that, in symplasmic
loaders, apoplasmic and symplasmic loading mechanisms may function
concurrently in the same plant (van Bel, 1993; Grusak et al., 1996).
This hypothesis is founded on structural considerations; e.g. the
abaxial phloem of the smallest minor vein is comprised of only IC and
SE, whereas in the larger minor veins, ordinary CCs are also present.
The ICs have been suggested to load Suc symplasmically and convert it
to raffinose sugars, while the ordinary CCs load Suc via an apoplasmic
step (Schmitz et al., 1987; Turgeon et al., 1993). In addition, a
transcript highly homologous to a Suc transporter (SUT1) was recently
identified in the phloem sap of Cucurbita maxima
(Ruiz-Medrano et al., 1999). Clearly, manipulation of plasmodesmal function by viral infection in the source leaves of plants that are
defined as symplasmic loaders could have a dramatic effect on plant
development
even a lethal one. It is logical to assume that in such a
case, the plant response would include an activation of defense
mechanisms to inhibit this effect, and such a mechanism could include a
shift from symplasmic to apoplasmic loading. The outcome of such a
shift would be increased Suc level in the phloem. The rapid elevation
of radioactive Suc within the phloem of CMV-infected leaves (Fig. 4)
further supports the assumption that a high Suc level in the phloem of
these plants is associated with an alteration in the process of phloem
loading. In this respect it is interesting to note that other stress
responses, such as low temperature and abscisic acid treatment, also
induced an increase in melon phloem sap Suc concentration (Mitchell and
Madore, 1992). Moreover, the activation of defense mechanisms includes
a process mediated by information perceived by distant tissues. In such
a case the information could also be perceived by leaves in which virus
particles are still absent, as seems to have occurred here with the
observed Suc level in the phloem sap of noninfected leaves (Figs. 5 and 6). Further studies are aimed at elucidating the basis for the CMV-induced change in sugar levels in the phloem of infected melon plants.
| |
MATERIALS AND METHODS |
Plant Material
Melon plants (Cucumis melo L. cv Hale's Best
Jumbo) were grown in a coconut mixture in 15-cm-diameter plastic pots.
Two-week-old seedlings were transferred to a temperature-controlled
chamber (model F15, Conviron, Asheville, NC) with a 12-h photoperiod
and 25°C/18°C day/night temperature. Photon flux density was 200 to 300 µmol m2 s1 at canopy level. Auxiliary
buds were removed as they appeared, such that all plants had one main
stem. In some experiments, the seedlings were transferred to an
insect-free, temperature controlled greenhouse (approximately
25°C/18°C day/night temperatures, respectively), with a natural
sunlight (midday average photon flux density of 1,200-1,500 µmol
m1 s1).
CMV Inoculation
When the plants were 3 weeks old and had four developed true
leaves above laminar length of 5 cm, the oldest leaf (the first above
the cotyledons) was inoculated with CMV (strain Fny) using carborundum
as an abrasive and 500 µL of inoculum containing 40 µg of virus.
Control plants were subjected to a similar inoculation procedure with water.
To verify the presence of virus particles in specific leaves, leaf
discs were collected for either protein or RNA analyses. Proteins were
extracted from 0.5 g fresh weight of tissue using denaturing
buffer (75 mM Tris
[Tris(hydroxymethyl)-aminomethane], pH 6.8, 9 M urea,
4.5% [w/v] SDS, and 7.5% [v/v] mercaptoethanol) at a 1:2
ratio. Proteins were then separated by 12% (w/v) SDS-PAGE and blotted
onto nitrocellulose paper (Towbin et al., 1979). To detect the CMV-coat
protein, we used rabbit polyclonal antibodies (kindly provided by Dr.
Garcia-Luque, CIB Consejo Superior de Investigaciones
Científicas, Madrid) diluted 1:2,000. Goat anti-rabbit horseradish peroxidase conjugate was used as a second antibody, and
detection was performed with an ECL detection kit (Amersham, Buckinghamshire, UK).
Viral RNA was detected by reverse transcriptase-PCR analysis. Total RNA
was extracted from leaf samples (200 mg) with Tri-Reagent (Molecular
Research, Cincinnati) following the manufacturer's instructions. cDNA
was prepared with specific antisense primer based on the CMV-MP 3'
primer (5'-GACCGTTAACCACCTGCG-3') followed by PCR analysis with the
addition of a CMV-MP 5' primer (5'-CCCGAGGCATGGCTTTCC-3'). The PCR
product was detected on a 1.2% (w/v) agarose gel stained with ethidium bromide.
Gas-Exchange Measurements
Net photosynthesis (measured as CO2 uptake) was
determined using a closed, portable, infrared gas-exchange system (LI
6200, LI-COR, Lincoln, NE) as described by Olesinski et al. (1996). All
analyses (unless otherwise indicated) were carried out 2 weeks post-inoculation at the stage when plants had eight true leaves (Fig.
7). Leaf 1 (the infected leaf) was
defined as the oldest one, and leaf 8 the youngest, i.e. the last to
reach a length of 5 cm. Measurements were carried out on the youngest
fully expanded leaf (no. 5) at noon with an initial CO2
concentration in the chamber of 350 ± 10 µL L1; a
30-s measurement was begun immediately after a reduction in CO2 concentration was detected. For respiration analyses,
the chamber was covered with black cloth for 2 min and measurements were begun immediately after an increase in CO2
concentration was detected. Gross photosynthesis was calculated as the
sum of the net photosynthesis and respiration rates.
View larger version (55K):
[in this window]
[in a new window]
|
Figure 7.
Representation of a melon plant in which
photosynthetic rate, carbohydrate levels, and export of
photoassimilates were studied. Five-week-old plants (2 weeks
post-inoculation) were used in most experiments. The inoculated leaf
was the oldest one (leaf 1) and the youngest fully expanded leaf, 5, was sampled in most experiments. Leaf 2 (and usually also 3) did not
exhibit any symptoms.
|
|
Starch and Sugar Determination
Leaf carbohydrate content was determined as described earlier
(Olesinski et al., 1996). Briefly, four leaf discs (1 cm2
each) were sampled between the small ribs of each leaf. Soluble sugars
were extracted in 80% (w/v) ethanol, and after evaporating the
supernatant, sugars were redissolved in water and filtered through a
0.45-µm membrane HPLC filter (Whatman, Maidstone, UK). Sugars were
separated in an analytical HPLC system (Pump System 320, Kontron,
Switzerland) fitted with a Sugar-Pak I column (6.5 × 300 mm,
Waters, Milford, MA) using an refractive-index detector (LDC
Analytical, Riviera Beach, FL). Starch content was determined in the
ethanol-water-extracted leaf discs following starch conversion by
amyloglucosidase (Sigma Chemical, St. Louis). Starch content as Glc
equivalents was determined using the Sigma (HK) quantitative Glc
determination kit.
Phloem exudate (sap) was collected from cut petioles using
microcapillary pipettes calibrated to 20 µL. The first drops were blotted for several seconds onto filter paper and the phloem sap exudate thereafter (5-10 µL) was immediately transferred to an Eppendorf tube containing 300 µL of ice-cold 50 µg
mL1 EDTA in HPLC-grade water. The solution was than
centrifuged at 4°C, 10,000 rpm for 5 min and stored at 
20°C.
Sugars in the sap were determined using the above described HPLC system.
Radioactive Labeling with 14CO2
Pulse-chase experiments were conducted according to the
procedure described by Olesinski et al. (1995). In short, an attached leaf was sealed into a 4-L Plexiglas chamber where it was held between
two layers of nylon monofilament. 14CO2 was
released into the chamber to give an initial specific activity of about
2 × 105 Bq mg1 carbon. After 15 chase min,
the leaf was released from the chamber and used for analysis of
14C-photosynthate export. Total radioactivity of each
14C-labeled leaf was monitored for 120 min using a portable
Geiger-Muller tube (RAM-DA, model GM-10, Rotem, Beer-Sheva, Israel)
that was placed on the adaxial surface of the leaf.
Partitioning of newly fixed carbon within phloem sap sugars was
determined 30 and 60 min post-labeling. Sugars were identified and
fractionated by HPLC and radioactivity of each fraction was measured in
a liquid scintillation counter (Tri-Carb, model 1600 TR,
Packard, Groningen, The Netherlands).
Statistical Analysis
The data are presented as the means of five replications
(plants), except for the comparison between the 14C
fractionated from phloem sap of various leaves (Fig. 4). The Student's
t test was used to detect differences between infected and non-infected plants. All statistically significant differences were
tested at the P 
5% level.
Received November 22, 1999; accepted February 7, 2000.
TOP
ABSTRACT
INTRODUCTION