Pathway of phloem loading in the C3 tropical orchid hybrid Oncidium Goldiana

Abstract

The apoplast of mature leaves of the tropical orchid Oncidium Goldiana was perfused with 0.5 mM p-chloromercuribenzenesulphonic acid (PCMBS) via the transpiration stream in order to test the mode of phloem loading. The
efficacy of introducing PCMBS by perfusion was shown by saffranin O dye movement in the veins and leaf apoplast in control
experiments. Photoassimilate export as the result of phloem loading was measured by collection of 14CO2-derived photoassimilates from the basal cut-ends of intact leaves.

Phloem loading and translocation of photoassimilates was inhibited by 89% in leaves perfused with PCMBS for 1 h. The effect
of PCMBS on leaf photosynthesis was minimal. The amount of radiocarbon fixed by PCMBS-treated leaves averaged 89% of control
leaves perfused with distilled water. A negative correlation between the total amount of photoassimilate exuded and the calculated
concentration of PCMBS in the leaf apoplast was also observed. The results indicate that phloem loading in Oncidium Goldiana occurs via the apoplastic pathway.

Full text

Journal
of
Experimental
Botany,
Vol.
47,
No.
305,
pp.
1935-1939,
December 1996
Journal
of
Experimental
Botany
Pathway
of
phloem loading
in the
C
3
tropical orchid
hybrid Oncidium Goldiana
Carl Khee Yew Ng and Choy Sin Hew
1
School
of
Biological Sciences, The National University
of
Singapore, Lower Kent Ridge Road,
Singapore 119260, Republic
of
Singapore
Received
21
March 1996; Accepted 22 July 1996
Abstract
The apoplast
of
mature leaves
of the
tropical orchid
Oncidium Goldiana
was
perfused with
0.5
niM
p-
chloromercuribenzenesulphonic acid (PCMBS)
via the
transpiration stream
in
order
to
test
the
mode
of
phloem loading. The efficacy
of
introducing PCMBS by
perfusion
was
shown
by
saffranin
0 dye
movement
in
the
veins and leaf apoplast
in
control experiments.
Photoassimilate export
as the
result
of
phloem load-
ing
was
measured
by
collection
of
14
C0
2
-derived
photoassimilates from
the
basal cut-ends
of
intact
leaves.
Phloem loading
and
translocation
of
photoassim-
ilates
was
inhibited
by 89% in
leaves perfused with
PCMBS for
1
h. The effect
of
PCMBS on leaf photosyn-
thesis was minimal.
The
amount
of
radiocarbon fixed
by PCMBS-treated leaves averaged
89% of
control
leaves perfused with distilled water.
A
negative
cor-
relation between
the
total amount
of
photoassimilate
exuded and the calculated concentration
of
PCMBS
in
the leaf apoplast was also observed. The results
indi-
cate that phloem loading
in
Oncidium
Goldiana occurs
via
the
apoplastic pathway.
Key words: Phloem loading, apoplast, PCMBS, tropical
orchid.
Introduction
Phloem transport
of
photoassimilates
is an
important
determinant
of the
growth
and
development
of
plants
and
a
decisive factor
in
crop productivity (Wardlaw,
1990).
Phloem loading involves
a
series
of
discrete
and
well-defined transport steps from source cells
to
sieve
tubes.
The loading process is highly dynamic and flexible,
involving both physiological
and
structural factors
(van
Bel, 1992a,
b,
1993).
The
apoplast concept
of
phloem
loading has dominated phloem transport physiology since
the seventies (Giaquinta, 1983). Numerous studies have
concentrated
on
finding
a
unifying concept
on
phloem
loading,
but it has
become increasingly clear that
the
mode
of
phloem loading may differ between plant species
(Flora
and
Madore, 1996; Gamalei, 1989, 1991; Madore
and Lucas, 1987; Turgeon and Beebe,
1991;
Turgeon
and
Gowan, 1990; Turgeon
and
Webb, 1976; Turgeon
and
Wimmers, 1988; van Bel et
al,
1992, 1994).
It
is even
not
excluded that different modes
of
phloem loading
can
occur within
a
leaf (van Bel, 1992a).
Extensive efforts have been directed towards elucidating
the pathways of phloem loading in dicotyledonous plants.
Comparatively few studies of phloem loading in monoco-
tyledonous plants have been conducted, exceptions being
those
on
maize, sugarcane
and
barley (Evert
et al, 1996;
Fritz
et
al., 1989; Robinson-Beers
and
Evert, 1991a,
b).
It
was
observed that
the
pathway
of
phloem loading
adopted
by
dicotyledonous plants
is
strongly correlated
with
the
minor-vein anatomy (Flora
and
Madore,
1996;
Turgeon
and
Beebe, 1991; Turgeon
and
Gowan,
1990;
van
Bel et al., 1992,
1994).
The
correlation between
minor-vein anatomy and carbohydrate transport in mono-
cotyledonous plant species remains
to be
determined.
Evert
and
co-workers (1996) observed that vein ultra-
structure
of
monocotyledons
is
very different from that
in dicotyledons.
It is
doubtful, therefore, whether
the
minor-vein classification adopted
for
dicotyledons can
be
used likewise
for
monocotyledons (Evert
et
al., 1996).
The objective
of
the present study was
to
elucidate
the
pathway
of
phloem loading
in the
tropical epiphytic
orchid hybrid
Oncidium
Goldiana through administration
of radiolabelled carbon dioxide
to
intact leaves subjected
to PCMBS perfusion.
1
To whom correspondence should be addressed. Fax:
+65
779
5671.
E-mail: sbshewcs@leonis.nus.sg
© Oxford University Press 1996
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1936 Ng and Hew
Materials and methods
Plant material
Experiments were performed with the sympodial thin-leaved
orchid hybrid Oncidium Goldiana (O. sphacelatum x
O. flexuosum), a C
3
shade plant (Hew and Yong, 1994) at the
flowering stage (Fig. 1). Plants were darkened 4d prior to use.
Mature leaves (L
3
) subtending the inflorescence were used for
all experiments.
Infiltration of PCMBS
Mature leaves were detached under water by cutting the leaf
sheath close to the base of the pseudobulb (Fig. 1). The leaf
was then immediately passed through a slit in a Parafilm cover
into a 10 ml cup containing 3 ml of solution (either distilled
water or distilled water with 0.5 mM PCMBS) so that the leaf
sheath base was below the surface of the fluid (van Bel el al.,
1994).
Perfusion of distilled water or distilled water with
0.5 mM PCMBS was by subjecting the leaves to vacuum (50 cm
Hg) for
1
h. After 1 h, the leaves were removed and the cups
were weighed. The difference in weight before and after
perfusion enabled calculation of the amount of PCMBS present
in the treated leaves.
After a few millimetres had been cut off from the base of the
leaf,
the leaves were transferred to 10 ml cups containing 3 ml
of distilled water with 10 mM Na-EDTA to prevent sealing of
the sieve tubes by callose formation.
™CO
2
assimilation and quantification
The leaves were illuminated at an irradiance of
200
fi
molrrr
2
s~
1
at leaf height and exposed to
14
CO
2
.
14
CO
2
was generated from Na
2
14
CO
3
(1.96 GBq mmol"
1
; Amersham
International, England) in a small glass vial (lml; lcm in
diameter) as described by Clifford et al. (1992) using an excess
of 0.1 M H
2
SO
4
for a 10
M
l droplet of Na
2
14
CO
3
(2 KBq). The
glass vial was attached to the abaxial surface of the leaf lamina
using an adhesive (Blu-tack, Bostick, Australia) that lined the
circumference of the open end of the vial, forming a gas-tight
chamber. To liberate
14
CO
2
, H
2
SO
4
was injected into the vial
through a hole on its side. After injection, the hole was
immediately sealed with adhesive (Blu-tack, Bostick, Australia).
L2
Pseudobulb
Inflorescence
Stem \J
Roots
FTg. 1. Diagrammatic representation of Oncidium Goldiana at the
flowering stage. Arrow indicates the point of detachment of mature leaf
L
3
from the base of the pseudobulb.
After 30 min of feeding
14
CO
2
, the glass assimilation vials
were removed. The amount of
14
C fixed by the leaf was
determined in discs (3 mm in diameter) punched out of the area
exposed to
14
CO
2
. Each disc was subjected to a wet combustion
method for extraction of total radiocarbon (Clifford et al.,
1973).
The tissue was oxidized in 4.0 ml chromic acid, and
14
CO
2
released from the oxidation process was trapped in
6.0 ml of 0.25 N NaOH. An aliquot (0.5 ml) of the NaOH was
mixed with 4.5 ml of Ecoscint scintillation cocktail (National
Diagnostics, USA) for liquid scintillation counting with a
Beckman LS 6000LL scintillation counter. High counting
efficiency was maintained (93% and 96%) and counts in absolute
disintegrations were corrected for background.
Collection and quantification of phloem exudate
The leaves were placed in cups containing 1.5 ml of distilled
water with 10 mM Na-EDTA solution to collect phloem
exudate. To prevent EDTA uptake via the xylem, transpiration
was retarded during exudation in darkness by maintaining a
high relative humidity in the atmosphere. This was achieved by
enclosing the leaves in a glass chamber, the bottom of which
was filled with water. The cups were replaced every 2 h (for
24 h) after the start of collection of phloem exudate. A 0.5 ml
sample of each solution was shaken with 4.5 ml of Ecoscint
scintillation cocktail (National Diagnostics, USA) and assayed
by scintillation spectrometry for the presence of
14
C-exudate as
described above. The statistical significance of the differences
between the phloem exudates of PCMBS-treated and control
leaves was determined using the Student's Mest.
Identification of labelled translocates
Identification of labelled compounds was conducted essentially
as described by King and Zeevaart (1974). The exudates (1 ml)
were evaporated to dryness at ambient temperature (25 °C) and
redissolved in
1
ml of 80% ethanol. Aliquots (2/il) were then
applied as small spots to commercially coated silica gel 60 thin-
layer chromatography plates (0.25 mm; 20 x 20 cm; Merck,
Germany). TLC plates were first chromatographed in phenol-
water (72:28, w/w) for 3 h in one direction, and in rt-butanol-
propionic acid-water (10:5:7, by vol.) for 3 h in the second
direction. Radioactive compounds were located by exposing the
chromatograms to Fuji Medical X-ray films for 6 weeks.
Radioactive spots were eluted and re-chromatographed with
standard sugars.
Results
Effect of PCMBS in the apoplast on "CO
2
assimilation and
the loading of
u
C-photoassimilates
Penetration of PCMBS into the apoplast of detached
leaves via the transpiration stream was achieved by perfu-
sion under vacuum at a pressure of 50 cm Hg for
1
h.
Microscopic observations of whole leaves indicated that
1%
saffranin O dye introduced in the same manner readily
penetrated the vein network (data not shown). The con-
centration of PCMBS in the apoplast was calculated from
the concentration of the uptake solution (0.5 mM), the
amount of solution infiltrated (obtained by difference in
weight of solution before and after the experiment), the
water content of the leaf
(91%
of fresh weight), and the
assumption that apoplast water constitutes 15% of total
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Phloem loading in Oncidium 1937
leaf water content (Turgeon
and
Gowan, 1990).
The
mean apoplast concentration
of
PCMBS delivered
by
infiltration averaged 0.25 +
0.07
mM
in
leaves
of O.
Goldiana.
The effect
of
PCMBS
on
photosynthesis was minimal.
The amount
of
radiocarbon fixed by PCMBS-treated and
control leaves
was
110.35+19.50 (dpm mm"
2
min"
1
)
and 124.35±9.35 (dpm mm"
2
min"
1
), respectively.
The
amount
of
radiocarbon fixed
by
PCMBS-treated leaves
averaged 89%
of
control leaves.
Collection of phloem exudate
Experiments with PCMBS-treated and control leaves were
always conducted simultaneously. The EDTA method
of
phloem exudate collection
was
regarded
as
convenient
for
orchid leaves (Neo, 1993). The rate
of
phloem exuda-
tion
of
14
C was low
during
the
first
10
h of
exudation,
but
the
release
of
14
C-exudate increased substantially
from
12
h of
exudation onwards.
At the end of the
exudation period (24 h), control leaves had exuded about
0.55%
of the
14
C
fixed.
In
comparison, PCMBS-treated
leaves exuded only about 0.06%
of the
14
C
fixed.
It is
evident that phloem loading
in
PCMBS-treated leaves
was inhibited
by
89% (Fig. 2).
The
rate
of
leakage
of
14
C-photoassimilates after 24
h
was taken
as a
reference
point
to
record
the
effect
of
PCMBS
on
phloem loading
and export (Fig. 2). The difference
in
the amount
of
14
C-
exudate between PCMBS-treated leaves
and
control
leaves was statistically significant (P<0.05).
A significant negative correlation
was
also observed
between
the
calculated concentration
of
PCMBS
in
treated leaves
and the
total amount
of
14
C-exuded after
24
h
(Fig. 3). There
was a
decrease
in the
exudation
of
I
a
s
o
•a
8
5
2000
1600-
1200-
12
16 20 24
Time (hours)
Fig.
2.
Cumulative time-course
of
14
C-photoassimilate
in the
phloem
exudate collected
at the cut end of
PCMBS-treated (circles)
and
control
(squares) leaves
of
Oncidium Goldiana.
The
leakage
is
expressed
as the
absolute amount
of
radioactivity (mean±SE, n
=
5).
y= -550.526x +296.711
r
2
= 0.891
0
0.1 0.2 0.3 0.4 0.5
PCMBS Concentration (mM)
Fig. 3. Relationship between
the
total amount
of
14
C
leakage after
24 h
and
the
calculated concentration
of
PCMBS
in the
apoplast
of
treated
leaves
of
Oncidium Goldiana.
14
C-photoassimilates from
the cut end of
treated leaves
with increasing concentrations
of
PCMBS
in the
leaf
apoplast.
Identification of translocated compounds
Autoradiography
of the
exudate after
14
CO
2
-feeding
showed only one radioactive spot which chromatographed
with standard
14
C-sucrose (data
not
shown).
Discussion
The data reported here indicated that the PCMBS concen-
tration
in the
apoplast
of O.
Goldiana
is
sufficient
to
block carrier-mediated uptake
of
photoassimilates from
the apoplast. The PCMBS effect must be attributed solely
to
an
interference with
the
phloem loading machinery,
since photosynthesis was hardly inhibited
by
PCMBS.
It
is noteworthy that
the
uptake system seems extremely
sensitive
to
PCMBS. Studies involving dicotyledons have
demonstrated effective concentrations
of
PCMBS which
is 10-20 times the present estimate (van Bel
et ai,
1994).
In
Vicia
faba, phloem loading
of
sucrose
was
inhibited
by
45%
with 0.05 mM
of
PCMBS (M'Batchi
and
Delrot,
1984).
They also observed that inhibition
of
phloem
loading by PCMBS was concentration dependent. Phloem
loading was inhibited
by
60,
67 and
72% with 0.10,
0.50
and
1
mM
PCMBS, respectively (M'Batchi
and
Delrot,
1984).
The extreme sensitivity
to
PCMBS
in the
present
study
is
interesting.
The
underlying mechanism
of
this
sensitivity
is
unknown
and it
remains
to be
established
whether there
is
widespread occurrence
of
this phenom-
enon among
the
orchids
and
monocotyledons.
One feature that
is
evident from
the
present study
is
the slow rate
of
phloem loading
and
translocation.
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1938 Ng and Hew
Translocation and export of
14
C-photoassimilates was
very slow in the first 10 h after
14
CO
2
-dosing, increasing
linearly only from 12 h onwards. This was slower than
the observed rates of assimilate translocation in vivo
involving whole plants. In whole plants, 4% of the total
14
C-photoassimilate was exported after 6h (Yong and
Hew, 1995). In attached leaves of
Pisum,
50% of the total
14
C-photoassimilate was exported after 2 h (Jahnke et al.,
1989).
The retardation could be attributed to the absence
of sink organs and the lack of utilization of photoassim-
ilates in the in
vitro
system used in this study, which could
also account for the extremely long period (24 h) of time
required for collection of phloem exudate. In the majority
of plants studied, both symplastic phloem loaders
(Philadelphus,
Epilobium, Lythrum,
Origanum)
and apo-
plastic phloem loaders
{Ranunculus,
Bellis, Centranthus,
Impatiens),
the rates of
14
C-leakage from sieve tubes was
low only in the first 2 h after
14
C0
2
-dosing, increasing
substantially and linearly from 2 h onwards (van Bel
et
al.,
1994). In comparison, the situation in O. Goldiana
is rather peculiar. The underlying mechanisms contribut-
ing to the slow rates of translocation in O. Goldiana
remains to be established. It is likely that the slow rate
of phloem loading and translocation is responsible for
the slow growth of orchids in general (Arditti, 1992).
It is well documented that sucrose is not the only
transport sugar (Zimmermann and Ziegler, 1975). The
sugar composition of phloem exudate collected from
petioles and stems (Zimmerman and Ziegler, 1975) is a
highly diverse mixture of mono- (glucose, fructose), and
oligosaccharides (raffinose, stachyose, verbacose), sugar
alcohols (sorbitol, mannitol), and other components. It
is noteworthy that families which exhibit symplastic
phloem loading translocate 20-80% of the sugars in the
form of raffinose-related compounds (Gamalei, 1989; van
Bel et al., 1994). In contrast, families which load photo-
assimilates via the apoplast translocate almost exclusively
sucrose (Gamalei, 1989; van Bel et al., 1994). The trans-
located sugar in O. Goldiana was found to be sucrose
only. The nature of the translocated sugar further sup-
ports the conclusion from PCMBS studies that
O. Goldiana executes apoplastic phloem loading.
It has been postulated that light, temperature and water
stress are important driving forces in the evolution of
phloem loading strategies (van Bel and Gamalei, 1991,
1992).
The light-limited environment of the forest floor
may have increased the selective advantage for evolution
of apoplastic phloem loading. In addition, temperature
may also have been important in driving the evolution of
an apoplastic loading strategy. This is due to the need of
a more efficient loading system under temperate condi-
tions (van Bel and Gamalei, 1992). While these appear
to be logical assumptions supported by numerous studies,
it can not fully explain the strategy of phloem loading in
O. Goldiana.
The apoplastic phloem loading strategy adopted by
O. Goldiana is unlikely to be driven by low light condi-
tions as a selective pressure. This is because O. Goldiana,
being epiphytic, is unlikely to encounter excessive light
limitations as compared to understorey herbaceous plant
species. However, it is also possible that evolution of
epiphytism occurred at a later period after the adoption
of an apoplastic loading strategy. Its occurrence in the
tropics also effectively rules out temperature as a causal
factor in the evolution of an apoplastic mode of phloem
loading. The most plausible factor is water stress. The
epiphytic habitat is characterized by extremes of environ-
mental stress, both water and nutrient (Benzing et al.,
1983;
Dressier, 1990).
Therefore, water stress as a driving force for the
evolution of an apoplastic route of phloem loading can
not be ruled out. It has been proposed that water shortage
in leaves results in greater demands for higher osmotic
potentials which is required for maintenance of cellular
integrity and the phloem loading process itself (van Bel
and Gamalei, 1992). In addition, plasmodesmata appear
to close with increasing turgor differences (Cote et al.,
1987;
Zawadzki and Fensom, 1986). This would make
symplastic phloem loading extremely drought-sensitive.
This is further supported by the predominance of apo-
plastic phloem loading in families well-adapted to water
stress (Gamalei, 1989). Epiphytic orchids, including
O. Goldiana are well-adapted to water stress, implying
that water stress may have been a decisive factor in the
evolution of apoplastic phloem loading in O. Goldiana.
Conclusions
Phloem loading in O. Goldiana does not support the
hypothesis that the apoplastic mode of phloem loading is
a more efficient system for translocation of photoassim-
ilates in dicotyledons (van Bel and Gamalei, 1992). When
compared to herbaceous dicotyledons, both symplastic
and apoplastic phloem loaders (van Bel et al., 1994), the
rate of phloem loading and translocation of photoassim-
ilates in orchids is extremely slow in both whole plant
systems (Yong and Hew, 1995) and in detached leaves.
If the slow growth rate of trees and shrubs is associated
with a symplastic mode of phloem loading, the correlation
does not hold for O. Goldiana.
The occurrence of this slow rate of translocation and
the extreme sensitivity to PCMBS in other orchid species
of contrasting habitat remains to be established. More
systematic investigations of the strategies of phloem load-
ing adopted by monocotyledons of differing growth forms
and habitat is necessary for a better understanding of the
physiology of phloem loading in this group of plants.
Acknowledgements
We thank Mr TK Ong for his technical assistance. We are also
grateful to the reviewers for their comments. CKY Ng is
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grateful to the National University of Singapore for providing
a scholarship during the course of this project. This project is
supported by a grant from The National University of Singapore
(RP950351).
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