Download Sink to Source Translocation in Soybean

Plant Physiol. (1984) 74, 434-436
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Short Communication
Sink to Source Translocation in Soybean'
Received for publication September 22, 1983
ALAN B. BENNETT*2, BARRY L. SWEGER3, AND ROGER M. SPANSWICK
Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853
ABSTRACE
The possibility that phloem loading may occur in the reproductive sink
tissues of soybeans (Glycine max Meff. cv Chippewa 64) was examined.
When I14Clsucrose was applied to seed coat tissues from which the
developing embryo had been surgically removed, 0.1% to 0.5% of the
radioactivity was translocated to the vegetative plant parts. This sink to
source translocation was largely unaffected by destroying a band of
phloem with steam treatment on the stem above and below the labeled
pod. The same steam treatment, however, completely abolished translocation of I14Cisucrose between mature leaves and developing fruits. These
results indicate that the movement of nutrients from developing seed
coats to the vegetative plant parts occur in the xylem and that phloem
loading does not occur in this sink tissue.
In recent years, several studies have provided evidence suggesting that processes localized in sink tissues are important in
regulating the partitioning of assimilates to storage tissues (4, 7,
13, 15). This realization has led to detailed examinations of the
mechanisms of nutrient uptake by sink tissues (2, 7, 8, 10, 12)
and to the development of techniques to monitor phloem unloading in the sink tissue of developing legume fruits (11, 14).
One model of phloem unloading is that unloading occurs by
passive solute leakage from the phloem wherever active uptake
by the phloem is impaired. This model further proposes that
sinks locally inhibit the phloem loading mechanism and thereby
affect net unloading of the phloem (5). However, evidence has
recently been presented indicating that phloem unloading does
not occur by a passive leakage of solutes but that it occurs by an
energy-dependent and possibly carrier-mediated process (11, 14).
In order to test these two models of phloem unloading, we
have examined directly whether phloem loading could occur in
soybean seedcoat tissue after removal of the developing embryo.
These experiments utilized the techniques developed by Thorne
and Rainbird (11) whereby the metabolic sink tissues (developing
embryo) can be surgically and nondisruptively removed from
the site of phloem unloading (seed coat). This allowed a direct
examination of the potential for phloem loading to occur in the
seed coat when no longer under the influence of the sink storage
tissue.
MATERIALS AND METHODS
Soybeans (Glycine max Merr. cv Chippewa 64) were grown in
a greenhouse with supplemental lighting. Plants were watered
daily and fertilized weekly with liquid fertilizer (Peter's 20-2020). Plants were brought to the laboratory from the greenhouse
when fruits at nodes nine through twelve contained partially
developed seeds. All experiments were carried out in the laboratory.
Access was gained to the seed coat tissue of a central seed in
three-seeded pods by surgically opening the pod and removing
the developing embryo as described by Thorne and Rainbird
(11). This procedure left half of the seed coat intact and still
attached to the pod wall. The seed coat half was filled with a salt
solution (approximately 25 ,l) containing 0.5 mm KCl, 0.5 mM
CaCl2, 0.1 mM MgCl2, and 5 mm Mes adjusted to pH 6.0 with
NaOH, and the pod subsequently was wrapped with parafilm to
prevent dehydration. After 1 h, the salt solution was replaced
and supplemented with 4.2 gCi ['4C]sucrose (New England Nuclear, 3.7 Ci/mol). The salt solution was replenished every hour
and, after 4 h, the plant was severed at the base and the plant
parts lyophilized, oxidized in a Packard Tri-Carb B306 sample
oxidizer, and the radioactivity determined by liquid scintillation
spectroscopy. Counting efficiency was approximately 42%.
Steam girdling of the stem was performed 12 h prior to the
start of labeling and was accomplished with a fine jet of steam
directed at a 0.5-cm band of the main stem for 1 min. A splint
was fashioned to ensure that the structural integrity of the steamgirdled stem was not impaired.
Labeling of a mature leaf with ['4C]sucrose was accomplished
by first abrading the upper leaf surface with a carborundum/
water paste. The ['4C]sucrose (4.2 MCi) was applied to the abraded
surface within a ring of silicon vacuum grease that was subsequently covered with a glass coverslip. Four h after labeling of
the leaf, a single fruit at an intermediate stage of development
was collected from each node and assayed for radioactivity as
described above.
RESULTS
Experiments in which a developing seed coat at node 9 was
labeled with ['4C]sucrose (experiment as diagrammed in Fig. lA)
and the entire plant subsequently monitored for radioactivity
indicated that 0.1 % to 0.5% of the applied 14C was translocated
to the vegetative plant parts (Table I). Approximately 80% of the
translocated radioactivity was recovered in the leaves with lesser
amounts recovered in the internodes, petioles, and developing
fruits (Table I). Of the radioactivity translocated to the leaves,
the largest amounts were found at the 2nd and 4th node above
the labeled pod (data not shown). Since soybean leaves at every
other node share a common vascular system (1) this pattern of
translocation from sink to source suggested that solute movement
was occurring through the vascular system.
Supported by the United States Department of Agriculture Competitive Grants Program grant No. 81-CRCR-1-0758.
2 Present address: Department of Vegetable Crops, University of California, Davis, California 95616.
3 Present address: Department of Biology, Lebanon Valley College,
Annville, Pennsylvania 17003.
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435
SINK TO SOURCE TRANSLOCATION
A
B
C
Table III. Distribution of '4C in Developing Soybean Fruits after 4
Hours ofLabeling of a Mature Leaf at Node 1) with 4.2 MCi of['4C
Sucrose
Plants used were either untreated (control) or were steam girdled on
the main stem above and below node 9. The experiment was performed
as described in "Materials and Methods," and the values shown are the
mean of two experiments.
Fruit at
Node Number
Distribution of Radioactivity
Control
Steam girdled
cpm
FIG. 1. Illustration of a soybean plant and the experimental treatments used in this study. Shaded plant organs indicate where ['4C]sucrose
was applied, and dashed lines (-- -) indicate where bands of phloem
were destroyed by steam girdling. A, The seed coat of a central seed in a
developing fruit at node 9 was labeled with ['4C]sucrose, and the aboveground plant parts were subsequently monitored for radioactivity. B,
The main stem above and below node 10 was steam girdled --- -), the
seed coat of a central seed in a developing fruit at node 10 was labeled
with ['4C]sucrose, and the vegetative leaves were subsequently monitored
for radioactivity. C, The main stem above and below node 9 was steam
girdled (- - -), leaf 11 was labeled with ['4C]sucrose, and the developing
fruits were subsequently monitored for radioactivity.
7
8
9
10
11
12
13
14
15
24,350
296
19,591
135
79,748
341
90
44
31
34
31
44
26
39,772
222
51
13
11
Total
124,626
40,204
In preliminary experiments, sink to source translocation was
not inhibited by the addition of 1 mM NaCN to the seed coat
labeling solution, a treatment expected to inhibit phloem loading.
Additional experiments indicated that treatments which reduced
transpiration (darkness and low temperature) also reduced sink
to source translocation (data not shown). Together these results
suggested that movement of radioactivity from the seed coat
may have occurred by mass flow in the xylem. To test this
possibility, a band of phloem on the main stem above and below
node 10 was destroyed by treatment with steam (steam girdle).
An opened seed coat at node 10 was then labeled with ['4C]
sucrose, and translocation to the vegetative leaves was determined (experiment as diagrammed in Fig. 1B). The extent and
patterns of sink to source translocation were similar in control
and steam-girdled plants (Table II), indicating that this movement of radioactivity did not require functionally intact phloem.
To ensure that the steam treatment of the stem was effective
in disrupting the phloem, the effects of this treatment on transof ['4C]sucrose from a mature leaf to developing fruits
Table II. Distribution of '4C in Leaves after 4 Hours ofLabeling of an location
determined. In these experiments, the stem above and below
Opened Seed Coat in a Developing Soybean Fruit at Node 10 with 4.2 were
node 9 was steam girdled, and ['4C]sucrose was applied to the
MCi of['4C]Sucrose
abraded surface of leaf eleven. After four h, translocation of 14C
Plants used were either untreated (control) or steam girdled on the to the developing fruits was monitored (experiment as diamain stem above and below node 10. The experiments were performed grammed in Fig. 1C). Here it was evident that no 14C was
as described in "Materials and Methods," and the values shown are the translocated beyond the steam girdle (Table III). Since translomean of two (control) or three (steam girdled) experiments.
cation from source to sink is generally regarded as a phloemmediated process, this result demonstrated the effectiveness of
Distribution of Radioactivity
Leaf at
the steam girdle in disrupting the phloem.
Node Number
Control
Steam girdled
cpm
DISCUSSION
6
320
505
The results of the experiments in which the phloem was
7
176
431
disrupted
by steam treatment clearly demonstrated that translo140
202
8
cation
from
the sink did not require intact phloem and suggested
9
196
181
that sink to source translocation occurred predominantly, if not
462
178
10
comletely, by mass flow in the xylem. This result is consistent
11
268
155
with
the findings of Nooden and Murray (9) which indicated
12
336
400
that soybean fruits exerted an influence on leaf senescence via
316
13
261
the xylem. Since mass flow in the xylem is nonselective, this
14
505
163
finding suggests that any seed-derived compounds that are se15
381
132
to the seed apoplast may be translocated to the vegetative
creted
16
235
161
parts of the soybean plant where they may exert an influence on
vegetative growth and development. These results also demonTotal
3335
2739
strate that mass flow of water and solutes from developing seeds
Table I. Distribution of '4C in the Vegetative Plant Parts after 4 Hours
of Labeling of an Opened Seed Coat in a Developing Soybean Fruit at
Node 10 with 4.2 UCi of['4CJSucrose
The experiment was performed as described in "Materials and Methods," and the values shown are the mean of two experiments.
Distribution of Radioactivity
Vegetative Organ
% of total
cpm
5166
80
Leaves
5
Petioles
293
11
737
Intemodes
4
Fruits
248
6444
100
Total
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436
BENNETT ET AL.
can occur in the xylem and may provide the means by which
excess water delivered to the developing seed in the phloem may
be recirculated to the vegetative plant parts. Such a recirculation
of water has been proposed as a mechanism to remove excess
phloem-derived water from developing wheat grains (6).
Quantitatively, source to sink translocation from a leaf to a
fruit two nodes below was 50- to 100-fold greater than translocation in the reverse direction (compare Table II and III) when
measured over a 4-h period. As one would expect, then, this
reverse flow of radioactivity from sink to source is nutritionally
insignificant. However, as discussed above and demonstrated by
Nooden and Murray (9), sink to source translocation may be
physiologically important to sink/source interactions.
With respect to the mechanism of phloem unloading in soybean seed coats, the results presented here indicate that phloem
loading does not occur in this tissue even after removal of the
sink storage tissue. This does not support the model of phloem
unloading which proposes that solutes leave the phloem by
leakage and that sinks locally inhibit the reloading process. By
this model, the activity of the reloading process should have been
apparent after removal of the metabolic sink (i.e., the developing
soybean embryo). Instead, the results presented here suggest that
the phloem in the seed coat does not have the enzymic capacity
for phloem loading and provides circumstantial support for an
alternate model of phloem unloading which involves an active
unloading mechanism.
Acknowledgments-We thank Dr. Francis Hsu for useful suggestions and stimulating discussions.
Plant Physiol. Vol. 74, 1984
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12. THORNE JH 1982 Characterization of the active sucrose transport system of
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13. THORNE JH, HR KOLLER 1974 Influence of assimilate demand on photosynthesis, diffusion resistance, translocation, and carbohydrate levels in soybean
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of Vicia faba L. The effects of several inhibitors on the release of sucrose
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15. WYSE RE, RA SAFTNER 1982 Reduction in sink-mobilizing ability following
periods of high carbon flux. Plant Physiol 69: 226-228
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