Plant Physiol. (1988) 87, 179-182
0032-0889/88/87/0179/04/$Ol .00/0
Different Patterns of Vein Loading of Exogenous [14C]Sucrose in
Leaves of Pisum sativum and Coleus blumeil
Received for publication October 5, 1987 and in revised form January 25, 1988
ROBERT TURGEON* AND LARRY E. WIMMERS2
Section of Plant Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853
The implicit assumption in this interpretation is that autoradiographic images reveal the tissue types which accumulate suVein loading of exogenous [14Cjsucrose was studied using short uptake crose from the medium, i.e. that [14C]sucrose enters the veins
and wash periods to distinguish between direct loading into veins and directly and that movement of label from the mesophyll to the
loading via mesophyll tissue. Mature leaf tissue of Pisum sativum L. cv veins is negligible during the course of the experiment. However,
Little Marvel, or Cokus blumei Benth. cv Candidum, was abraded and this assumption may not be valid in uptake experiments lasting
leaf discs were floated on ['4C]sucrose solution for I or 2 minutes. Discs more than a few minutes, since photoassimilate labeled by the
were then washed for 1 to 30 min either at room temperature or in the exposure of a leaf to '4CO is transported from the mesophyll to
cold and were frozen, lyophilized, and autoradiographed. In P. sativum, the phloem within this time frame (12, 19). Indeed, indirect
veins were clearly labeled after 1 minute uptake and 1 minute wash pe- evidence for such transfer has been obtained by the analysis of
riods. Autoradiographic images did not change appreciably with longer sucrose uptake into enzymically isolated veins and mesophyll
times of uptake or wash. Vein loading was inhibited by p-chloromercuri- cells (1, 17). In this paper we provide direct evidence for transbenzenesulfonic acid. These results indicate that uptake of exogenous port of exogenously applied ['4C]sucrose from mesophyll to misucrose occurs directly into the veins in this species. When C. blumei leaf nor veins in Coleus blumei leaf tissue and demonstrate that, in
discs were floated on [14C]sucrose for 2 minutes and washed in the cold, Pisum sativum, labeled sucrose is accumulated directly by the
the mesophyll was labeled but little, if any, minor vein loading occurred. veins.
ABSTRACT
When discs were labeled for 2 minutes and washed at room temperature,
label was transferred from the mesophyll to the veins within minutes.
These results indicate that there may be different patterns of phloem
loading of photosynthetically derived sucrose in these two species.
There is increasing evidence that phloem loading is a regulatory step in the partitioning of photoassimilate (6). The manner
in which loading is controlled must, in turn, be dependent on
the pathway which photoassimilates take as they are transported
from the chloroplasts of mesophyll cells to the SE-CC3 complex.
The consensus is that phloem loading involves an apoplastic step,
i.e. photoassimilate at some point along the transport pathway
enters the extracellular space and is then loaded into the SE-CC
complex against a concentration gradient (6). However, this view
has not gone unchallenged; although compelling evidence is lacking it has been suggested that, in some species, photoassimilate
follows a symplastic route, passing from cell to cell through plasmodesmata from mesophyll to phloem (8, 10, 11).
The hypothesis of apoplastic phloem loading is supported by
a number of observations (6). One of the most crucial of these
is that exogenous radiolabeled sucrose is taken up into the veins
of mature (exporting) but not immature (importing) leaf tissue
(2). Although some of the label is accumulated by mesophyll
cells (1, 17, 20), enough is loaded into the veins to provide a
clear image of the vascular network. This preferential uptake of
exogenous sucrose into veins is thought to simulate the in vivo
loading of apoplastic photoassimilate.
' Supported by National Science Foundation grant number DMB8510090
and by Hatch project 185401.
2 Present address: Department of Vegetable Crops, University of California, Davis, CA 95616.
3 Abbreviation: SE-CC, sieve element-companion cell.
MATERIALS AND METHODS
Plant Material. Pea plants (Pisum sativum L., cv Little Marvel)
were grown in a controlled-environment room. Plants were illuminated for 16 h per 24 h period with two vertically mounted
1000 W metal halide lamps (M1000/c/v metalarc: Sylvania, Danvers, MA) and eight 40 W fluorescent lamps. Light intensity was
1000 ,tmol photons m- s ' at leaf level. Day and night temperatures were 200 + 20C and 140 + 20C, respectively. Seeds
were obtained from Agway (Syracuse, NY).
Coleus plants (Coleus blumei Benth., cv Candidum) with variegated leaves were grown in a greenhouse under conditions
described previously (15). Seeds were obtained from Geo. W.
Park Seed Co. (Greenwood, SC).
Uptake Studies. Mature leaves were abraded with carborundum, and 5.6-mm diameter discs were removed with a cork borer
under the surface of 2(N-morpholino)ethanesulfonic acid (MES)
buffer (20 mM MES + 20 mm CaCl2 adjusted to pH 5.5 with
KOH). Discs were randomized and transferred, abraded side
down, to the surface of fresh buffer in small (3.5 cm diameter)
plastic Petri dishes. The buffer was removed and replaced with
2.5 ml of a solution consisting of the same buffer and [U-'4C]sucrose
(1 mm at 108 Bq mmol -) at room temperature for periods described in the text. At the end of the uptake period, the ['4C]sucrose
solution was quickly removed, and the discs were washed with
room temperature or cold (1-20C) buffer. The lengths of the
wash periods varied, as described in the text. Changes of wash
buffer were made on a schedule of 1, 2, 3, 5, and 10 min and at
5 min intervals thereafter, although in short-term experiments
this schedule was interrupted. During cold washes, Petri dishes
were placed in crushed ice. During the uptake and wash periods,
the discs were continuously agitated on a reciprocal or rotary
shaker and exposed to room light.
Autoradiography. At the end of the wash period, the final
buffer change was removed and the Petri dish was filled with
powdered dry ice. Discs were lyophilized at - 300C, compressed
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180
TURGEON AND WIMMERS
Plant
Physiol. Vol. 87,
1988
between polished steel plates in a vice, attached to thin cardboard
sheets with double stick tape, and autoradiographed with Kodak
XAR-5 film (Eastman Kodak, Rochester, NY) as described (14).
Exposure times were 3 to 11 d.
RESULTS
Vein Loading in Pea Leaf Discs. Leaf discs from abraded pea
leaves were floated on [14C]sucrose solution for 1 min and then
washed in buffer for 1 min prior to freezing and autoradiography.
The half-time of exchange of the free space in pea leaf discs is
1 to 2 min, as determined by compartmentation efflux analysis
(data not shown). Therefore, these time periods were sufficient
to allow uptake of a limited amount of labeled sugar and then
to remove almost half the unincorporated label from the free
space. The minor vein network was clearly visible in autoradiographs (Fig. 1A). A considerable amount of label was usually
seen at the rim of the discs indicating that label entered preferentially, but not exclusively, through the cut edge. The image
of the minor veins was usually more clear when the uptake period
was extended to 2 min (Fig. 1B). To determine the effect on
uptake of the sulfhydryl-modifying compound p-chloromercuribenzenesulfonic acid (PCMBS), discs were floated on [14C]sucrose
with or without PCMBS (2 mM) for 1 min and washed for 10
min in the cold. PCMBS clearly inhibited vein loading (Fig. 1,
C and D).
Vein Loading in Coleus Leaf Discs. Discs from abraded, green,
C. blumei leaf tissue were floated on [14C]sucrose solution for 2
min and washed in cold buffer for 10 min. Autoradiographs of
these discs revealed almost uniform uptake, although larger veins
were often labeled (Fig. 2A). The disc illustrated in Figure 2A
is representative of the majority of samples, but there was some
FIG. 2. Autoradiographs of C. blumei leaf tissue floated on ['4C]sucrose
solution at room temperature for 2 min and washed in buffer at room
temperature or in the cold (1-2°C) prior to freezing and lyophilization.
A, 10 min wash in cold; B, 2 min wash at room temperature followed
by 8 min wash in cold; C, 5 min wash at room temperature followed by
5 min wash in cold; D, 10 min wash at room temperature; E, 30 min
wash at room temperature; F, smaller piece of tissue treated as in E.
Autoradiographic and photographic exposure times were the same in all
cases. Scale, 1.5 mm.
FIG. 1. Autoradiographs of P. saivum leaf discs floated on ['4C]sucrose
solution for 1 or 2 min at room temperature and washed in buffer at
room temperature or in the cold (1-2°C) prior to freezing and lyophilization. A, 1 min uptake, 1 min wash at room temperature; B, 2 min
uptake, 1 min wash at room temperature; C, 1 min uptake, 10 min wash
in cold; D, 1 min uptake in the presence of PCMBS (2 mM), 10 min
wash in cold. Autoradiographs were used as negatives; therefore, white
regions indicate the presence of 14C. Autoradiographic and photographic
exposure times were the same in all cases. Scale, 1.5 mm.
variation in the appearance of autoradiographs; in the occasional
disc no loading could be distinguished in any of the vein classes,
whereas in others some of the minor veins were apparent. In the
latter case, the contrast between silver grain density over minor
veins and surrounding tissue was minimal. A few minor veins
are visible in Figure 2A. Approximately the same range of images
was obtained when the uptake period was reduced to 1 min
(autoradiographs not shown). Since the half-time of exchange of
the free space in C. blumei leaf discs is 1.8 min in the cold (data
not shown), approximately 98% of the label in the free space
was removed in the 10 min wash. Therefore, the absence of minor
vein loading in autoradiographs cannot be attributed to masking
of the veins by high levels of extracellular label.
To allow time for movement of label from the mesophyll to
the veins, discs which had been exposed to [14C]sucrose for 2
min were washed in room temperature buffer for varying periods.
This room temperature incubation was followed by a cold buffer
wash to extend the wash period to a total of 10 min. After 2 min
wash at room temperature, followed by 8 min cold wash, more
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VEIN LOADING PA1TERNS IN PEA AND COLEUS
of the minor veins were visible in autoradiographs contrasted
against a slightly lighter background of mesophyll labeling (Fig.
2B). After 5 min at room temperature, followed by 5 min of cold
wash, the veins were more distinct (Fig. 2C). After 10 min of
wash at room temperature the minor veins were very clear, and
the amount of label in the mesophyll was obviously reduced (Fig.
2D). The vein pattern was even more distinct when the 2 min
uptake period was followed by a 30 min wash in room temperature buffer (Fig. 2E). These results indicate that exogenous
[14C]sucrose is taken up primarily by the mesophyll in mature
C. blumei green leaf tissue and is subsequently transported to
the minor veins in a period of minutes. This transport is inhibited
by cold.
Since label was visible in some of the larger veins in leaf discs
after 2 min of uptake, we considered the possibility that minor
vein labeling during the subsequent incubation at room temperature could be due to movement of label from the larger to the
smaller veins in the phloem rather than movement from the
mesophyll to the minor veins. To test this possibility, small pieces
of tissue (approximately 1-5 mm2) were excised from an abraded
leaf with a scalpel taking care to exclude large veins. These tissue
pieces were exposed to [14C]sucrose for 2 min and were then
washed in room temperature buffer for 30 min; minor veins were
clearly evident (Fig. 2F).
We also considered the possibility that the minimal labeling
of minor veins after short uptake periods was due to wounding
of the tissue. Experiments with excised leaf and storage tissues
indicate that transport may be transiently reduced by wounding
caused by excision (9, 18). Abraded C. blumei leaf discs were
floated, at room temperature, on a solution containing 25 mm
MES buffer (pH 5.5), 25 mM CaCl2, 10 mm sucrose, and 25 ,ug/
ml ampicillin for 12 h, with agitation. At the end of this period,
discs were washed in three changes of the same buffer (plus
CaCl2) for a total of 30 min to remove exogenous sucrose. The
discs were then floated on [14C]sucrose solution for 2 min at
room temperature and washed for 10 min in either room temperature or cold buffer as described above. Results were essentially identical to those obtained with 'unaged' discs; minor vein
labeling was apparent after incubation in room temperature buffer,
but not in cold buffer (autoradiographs not shown).
DISCUSSION
The results reported here demonstrate that patterns of vein
loading of exogenous sucrose in P. sativum and C. blumei differ
significantly. Sucrose is translocated in both pea (13) and Coleus
(4, 5). In pea leaves, labeled sucrose enters the veins directly
and can be detected in uptake experiments lasting only 1 or 2
min. Given longer periods of uptake and wash, the vein image
in autoradiographs does not change significantly. Based on these
results, we cannot exclude the possibility that some label which
is taken up by the mesophyll tissue is subsequently transported
to, and enters, the veins. However, if this transfer from mesophyll to minor veins occurs it cannot be detected visually against
the much larger background of direct uptake into the veins. The
autoradiographic image after 2 min of uptake is more clear than
that obtained after 1 min of uptake when the wash periods are
the same. This difference could be due to transfer of label from
mesophyll to veins during the second minute of uptake but we
believe it is more likely due to different penetration times of
label into the mesophyll and veins. Since the bundle sheath encloses the vein, it is to be expected that exogenous sucrose will
take longer to reach the minor vein phloem than it will to reach
mesophyll cells. Given only 1 min of uptake, a period within the
half time of washout of the tissue, the mesophyll cells may have
had significantly more time than the phloem to accumulate label.
Pea leaf minor vein phloem contains transfer cells, i.e. cells
with elaborate wall ingrowths that increase plasmalemma surface
181
area (7). Our results support the widely held view that phloem
transfer cells are specialized for the uptake of solute from the
apoplast.
In contrast to the veins of pea, those of C. blumei do not
appear to accumulate much more exogenous ['4C]sucrose than
does the mesophyll. In some autoradiographs, the veins are visible after short labeling periods, but the difference in density
between veins and mesophyll is slight. The clear labeling pattern
and obvious difference in density between veins and mesophyll
seen in autoradiographs after long uptake and chase periods is
due to movement of label from the mesophyll to the veins.
These results may reflect a difference in the mechanism of
phloem loading of photosynthetically derived sucrose in C. blumei and P. sativum. It is interesting to note that, in contrast to
pea minor vein companion (transfer) cells that are specialized
for uptake from the apoplast, some of the companion cells of C.
blumei minor veins are 'intermediary' cells (3), i.e. they are
especially large, contain dense cytoplasm, and are connected to
surrounding cells by numerous plasmodesmata (16). These plasmodesmata may provide sufficient intercellular continuity to allow symplastic phloem loading (11, 16). A symplastic pathway
from mesophyll to minor veins would explain the observation
that sucrose is transferred from one tissue to the other without
being washed away during continuous agitation of the discs in
buffer. However, while our results are consistent with a symplastic pathway of phloem loading in C. blumei, they do not
prove it. In fact, one possible interpretation of our results is that
exogenously supplied sucrose is not transferred from the mesophyll to the sieve element-companion (intermediary) cell complex of the minor veins at all but enters other cell types such as
the phloem or xylem parenchyma. The cellular location of radiolabeled sucrose in the veins can only be resolved by microautoradiography.
While the results reported here do not provide compelling
proof for symplastic phloem loading in C. blumei, they demonstrate that sucrose is not accumulated directly by the minor
vein phloem in this species as would be expected if the mechanism
of loading of photosynthetically derived sucrose involves an apoplastic step and uptake from the free-space by sucrose-proton
co-transport (6). The results also indicate that pathways of photoassimilate transport may not be the same in all species and that
more attention must be devoted to analyzing patterns of loading
in short-term experiments. Caution should be used in the interpretation of autoradiographic and kinetic data until the temporal
pattern of sucrose uptake and transfer between different cell
types is established.
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