Sugar Transport In Conducting Elements of Sugar Beet Leaves`

Planit Plhvsiol. (1060) 44. 717-723
Sugar Transport In Conducting Elements of Sugar Beet Leaves'
P. Trip
Division of Biology, National Research Council of Canada. Ottawa, Canada
Received December 31, 1968.
A bstract. Autoradiography was used to determine the distribution of labeled sugar in
conducting elements of the blade and petiole of sugar beet leaves at intervals ranging from
5 sec to 24 hr. The processes of assimilation by the green cells, collection of sugar in the
minor veins and export in phloem elements were demonstrated visually. It appears that in
minor veins sugar is translocated in companion cells rather than sieve tubes. In major veins
translocation occurs in sieve tubes.
More exact knowledge of the flow of assimilates
from their site of synthesis in green cells into the
conductin,g systenm would be of value in understanding the overall process of translocationi. It is apparent that during this step the composition of the
translocate is determined (6. 8, 11). Sucrose is the
main component in nearly all plan,ts (15). According to Brovchenko (2) sucrose is inverted hefore
entry into phloem cells of sugar beet and is resynthesized in the phloem. Pristupa (9) observed
assimilation by the border parenchvma lining the
vascular bundles of corn and barley leaves. After
a lag period of 15 to 20 mil, label appeared in tlle
vascular bundles.
The anatomy of sugar beet leaves has been
described by Artschwager (1) and bw Esat (3).
For convenience, leaf veins have been given numibers
from I to V in the order of decreasing size (3, 6).
The present study deals particularly Nvith size class
V, the minor veins. While there is general agreement that the phloem of minor veins receives the
assimilates and transmits them to the phloemii of
larger veins for export, there is some confusion as
to which cells are involved. This is partly due to
the lack of physiological evidence aind partly to disagreement on nomenclature and anatomv of minor
veins. The aimn of the present work was to obtain
physiological evidence of the pathway of translocation in the leaf blade usiing autoradiographv of
soluble 14C anid 'II-labeled comiipounids.
Materials and Methods
Plants of Beta vuliaris IL. cultivar Klein \Van1liZ-
leben were germinated in vermiculite and grown in
water culture under conditions described earlier (14).
Glucose-6-3H (specific radioactivity
450 ,uc/
mmole) was introduced to fully expanded leaves of
2-month old plants in aqueous solution via a flap
formed by a cut side vein (13). The leaf blades
were 8 inches long. 14CO0 (44 mc/mmole) was
Issued as N.R.C. No. 10761
assimilated by intact leaves in an assimilation chamber wvith a mercury seal (11). Procedures used in
the assay of radioactivity, lyophilization and autoradiography have been described earlier (12). Material used for anatomical study was fixed in Craf I
(p 18. ref. 10), enmbedded in wax. stained with
safranin land fast green and mounted on glass slides
with nmounting nmediutml. Material for autoradiographv was moulnted on NTB 10 slides a-nd photo-
graphed withotit m)ounting mediumil.
UsS of '4CO(.. A sumnmary of localization of
assiimiilated l4C is given in Figs. 1 to 4. Part of
each of the leaves was pushed throughi a mercury
seal into a chamber containing 14CO.. anld allowed
to assimilate for 30 sec at 2000 ft-c. The feeding
periodl w,as followved by a fltishlilig l)eriod in 12CO.,
of varying lengthl as follows: 0 mi
( Fig. 1),
1 min (Fig. 2), 5 min (Fig. 3). 20 mim *(Fig. 4).
In all 4 cases there was a diffuse pattern of distribution of label throughout the leaf btut in addition, a
concentration of label appeared over the phloem
after ; min and after 20 miii most of the label in the
leaf was associated with the veins. Since all leaves
assimilated 1 4CO., for 30 sec, the amount of radioactivity wzas abouit the same in each leaf. It becomles
appareint that CO., is assimilated througlhout the
thickness of the leaf, that there is a 5 mmin lag period
until a( noticeable quantity of label is associated with
the veins, and that it is not possible fromii '4C autoradiographs to (letermine in which of the phloenii
cells-siev'e tubes, comp)anion cells or parenchvma
cells-the label occurs. In feeding periods of 5, 10,
-and 20 sec results were similar to those pictured in
Fig. 1. The results after flushing periods longer
than 20 min were similar to those pictured in Fig. 4,
i.e. the bulk of the label was in the minor veins.
Use of -H Glutcose. To obtain higher resolution
in the autoradiographs 3H-glucose was flap-fed to
attached leaves. Fifty Iu1 of 0.2 -i 3H-glucose solution was offered for 3 hr. A cross section of the
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FIGS. 1-4. Autoradliograplhs of transverse sectiolns of beet leaves. A period of assimilation
of 14CO., of 30 sec was folloxed by a period of assimilationi of 12CO., of 0 sec, Fig. 1; 1 min,
Fig. 2; 5 min, Fig. 3; anid 20 min. Fig. 4. Tracer moves from the sites of assimilation to the
veins. X215.
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_m EI
X 330.
FIG. 6. Portion of the leaf blade infuse(d with label which appears in irregular patclles. X 330.
FIG. 7. Export of label from the infuse(l area in a minor vein. The label appears over a phloem element.
FIG. 5. Transverse section of flap with label in the xylem.
X 330.
FI(GS. 8, 9. Transverse sections of minor veins. The conmpanioni cells ar-e [unul)ered.
FIG. 10. Autoradiograph of a transverse sectionI of a labeled minior vein.
cells, nlot the sieve tubes (arrows). X 570.
X 1440.
Label appears over the companion
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FIG. 11. Section cut parallel to leaf surface showing miinor vein neti-ork. X 140.
FIG. 12. Parallel section shox-ing a sieve tube between conmpanioni cells (1 andl 2). Companion cells are much
wvider thani sievc tubes. X 570.
FIGS. 13, 14. Autoradiographs of parallel sections showing sinigle anid double streaks of grains overlying minor
veins. Patches nmarked xvith crosses represent tissue, not silver grainis. In Fig. 14 a single sieve tube appears
between 2 labeled coinpanion cells (1 and 2). Fig. 13, X210; Fig. 14, X570.
FIG. 15. Autoradiograph of a longitudinal section of petiolar phloem showinig grains overlying a sieve tube (2).
Cell 1 is a companion cell.
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flap (Fig. .5) shows label confined to the xylem,
mainlv the larger vessels. Fig. 6 shows scattered
patches of label in the mesophvll tissue of the infused
area, and in Fig. /7 a single plhloem elemenit lying
along a xylem vessel contains labeled material. This
material, outside the zone which has been inftused
w ith H-glutcose. w as largely 'H-sucrose as determin,ed by paper chromiiatographv. The tritiated material couild be flushed out of thle miilnor veillns b1
leaving the leaf in I2CO.. for 24 hr anld wvas tlherefore
in tran,sit in the vein.
WN'hiole leaf autoradiograplhs, not slhowni here, hiave
indlicated that the tracer spreads in the area of the
leaf normally supplied with water by the cut vein.
The tracer enters the plhloemii of minior veins in the
infused area either directly from the xylem or -via
the mesoplhyll and is transported otitside the infused
area via the minor vein phloem.
The exported label is present in a cell (Fig. 7)
with a diameter of about 10 ,. The phloem cells
with this diameter are companioni cells. and the
uItiestion arose wh11ethler the comlipanlioIl cells or the
sieve ttubes of minor veins are primarily responsible
for translocationi along the lengtll of the vein. Figs.
8 and 9 represent cross sections of miilnor veins of
size order VJ and IV, respectively. The size relationship between companion cells and sieve tubes is
particularly evident here. In a sample of 21 coImlpanlion cells the dianmeter averaged 9.1 ,x, mleanl
deviation 2 u; the length averaged 45 Ix, mean deviatioin 10 /,E. In a sample of 8 sieve tubes the diameter
averaged 2.4 I. meian deviation 0.6 /U; the lengthl
50 /A, meani deviatioin 14 ,u. Fig. 8 represents the
smallest combination commonly observed in mature
leaves; I sieve tube, 2 companion cells, 1 large xylem
vessel with secondary wall, and 3 small xylem elements. The sieve tubes in the smallest minior veins
(1o not possess slime plugs, sieve plates or callose.
These features are present in sieve tubes of major
veins only. The companiion cells, numnbered in Figs.
8 and 9, all contain dense cytoplasm <and large nuclei.
In safranin-fast green the nucleoli staini a very bright
red and the cytoplasm appears purple. These colors
atre outstanding in tissue preparations anld allowed
(luick identificationi of companion cells in major as
well as minlor veinis.
An autoradiograph of a iiinor veini of size order
IV7 is shlowin in Fig. IC). Grains appear over the
companion cells and nlot over the sieve tubes indicated by arrows. Cell I is a com nl)ilion cell lyiiig
between the sieve tubes and the xylem (as is cell 3
in Fig. 9). A dirt particle overlies 1 of the xylem
cells; it can be distinguished from <a patch of silver
grain,s by its sharper edges.
Fig. 11 gives an indication of the density- of the
netwvork formed by VtJl order minor veins. Companion cells comprise roughly half the total volume
of these veins. The average distance across the
islets of cells enclosed by minor veins was about
100 ix. This is less than half that observed by Esau
(3). A typical arrangement of 2 companion cells,
nunmbered 1 and 2. with a sieve cell in between them
is shown in Fig. 12. The sieve cell abuts another;
both have bulbous ends. Light microscopy failed to
reveal sieve pores in the bulbous end walls.
Ani overall view- of tracer in Vt" order minlor
veins is presenited in Fig. 13. The tracer appears in
shorter or longer tracts about 10 u in diamneter which
-are eitlher single or double. This photograph was
taken from an area in the leaf blade 2 cmn away fronm
the infused zone. At this poinlt there is very little
label in the mlesopllull. Patches marked wvith crosses
represent tissue, not silver grains: thexV appear very
dark because of the air mounlting. In mlore detailed
autoradiograplhs. suichl as Fig,. 14. the tracts appear
over coImlpanlionl cells. In F-ig. 14 the sieve tube
lying in between the 2 coml)anion cells (numbered
1 and 2) is practically devoid of label.
Evidence from the 24 hr flushing experimienits
indicated that the label in minor veins is in transit,
not in storage. If tlle sieve tubes were mainly responsible for transport label xvould be observed in
thenm or at least first in themii. Instead, the companlionl cells were labeled.
In the letiole of the same leaf the label appears
mainly in the sieve tubes as shown in Fig. 15 where
cell 1 is a conmpanion cell, cell 2. a sieve tube. Cross
sections of this petiolar tissue which were alternately
mounted onl emiiulsionl for ra(dioauttography and
mounted onl chrom-alumi for staining and cell idenitification also showed the sieve tubes. not the comilpanlionl cells, to be labeled.
On the basis of anatomical knowledge it has been
assumed (3) that the companion cells of minor veins
serve to accumilulate carbohyldrates from the surrounding tissue and to secrete themi laterally to sieve
tubes. lying alongside the companion cells, for further
transport alonig the minor veins. To describe the
companion cells in physiological rathlel thllan anatomical ternms the! have been nallmed "transfer cells"
by GuInning ct al. (5). These writers have observedl
elaborate wall proturberances, phosphatase activity
in the wvalls, and mitochonldria in transfer cells. but
they were careftul to state that it would be prematlure
to assigni a specific funlctionl to tranisfer cells.
Fischler (4) has carried ouit the milost exhaiistixve
study on miinor Xveins and (ldescrlibe(l the large size
and deiise cytoplasm of the coml)anion cells ( geleitzelleni). Becaiuse the geleitzelleni ( literally: escort
cells) of miinor veinis were intermiediarv in size between comilpanlionl cells of major \veins and leaf
parenchyma cells, he called them "ubergang" or
intermediary cells. He observed that in the largest
v-eins the diameter of the companion cells was
one-eighth that of the sieve tube whereas in the
smallest veins the -diameter of the companion cells
was 8 times that of the sieve tube. This relationship
held for a large numnber of species studied. He
expressed the belief that they temporarily store pro-
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tein (p 248) and tranisfer this protein to the sieve
tubes in which it is exported froml the leaf. This
concept was not used by Esau (3) wrho considered
transition or intermediary (tuiergang) cells to be
parenchyimatic cells which are ontogenetically related
to sieve tuibes. She described their fnilction as
intermediary in the carbohydrate transport anId intvolved in the trainsforimationl of sugars and their
secretion inlto sieve elements. Esau and Fischer also
disagreed on the stricttiure of sieve tuibes in miniilor
veins. Fischer observed sieve tubes miltici snmaller
thain the coml)anion cells, thle absence of sieve plates
and little slimiie and therefor-e conisidlered the sieve
tilbes of minior v-cinis incomplete. Esau has observed
sieve plates and callose hut (loes nlot offer photographs
of these in tile silmallest veinls. Frotim our11 ow-n oabservatioins NN-e agree with Fischer that callose and sieve
plates are not visible ini the smiiallest veins. WVe also
agree with Fischer that sieve tulbe.s in the smallest
veins are miuicih smlaller (diaieter X onle-fouirthi)
thiain the corresponding comlpanion cells. On this
anatomical basis we conclude that the sieve tui-bes of
miitior veins are in(deed incomiiplete and canniot he
considere(l equiivalent to norimial sieve tubes of miajor
veins, i.c. sieve tubes wvith sieve lIlates and lpotential
callose formation. It might be \\-ell to (li.stinguisll
betweeni miniiior veiins adnd ajor xveins on the b)asi.s
of the (diameter of compil)anio i cell): (diameter of
sieve tuibe) rattio rather- thaii on the niumber of cells
ill the vein. In Ilmiolr veinis this ratio
ouild be
greater than 1. in miiajor ones, smaller. The absence
of sieve plates in the incoml)lete sie\ve ttilbes awvaits
confirimiationi .at the electroni microscol)e lexvel, but
atn intermediate type of sieve plate with a few pores
h1as been observed 1b Trip and Colvin; ( 1111published)
aind] been, reported by (;eiger and Cataldo ( inpublished) in imlinor veins of suigar beet leaves. The evid(ence froml autoradiographsl suggests that sieve tubes
of minor veini do not carry sugars as inormiial sieve
tubes do. Thlis function is taken over by the coImlpanioni cells of miinor- veills.
In a radioautographlic study' of the incorporation
of 14C from 14CO.., Pristupa (9) observ ed that the
vascular bundlle sheath of corn leaves became labeled.
Lar-ger bundles became labeled only after 2 hr an(l
lie concltuded that the flow of assimilates to the vein
must require several lhr and that the btunldle sheath
itself must be the miiainl site of aissimilation in order
to observe label associated w\ith minor v,eilns after as
short a time as 2 nlin. Ouir results indicate that in
beet leaves assimilation occurs throughlouit the leaf.
not jtust in, the neighlborhlood of veins and(I that label
accumulates in the leaf blade veins after about 5 min.
Mortimer (7) has observed a 10 mim lag period
between first offering "4CO., and the detection *of
label in the upper part of the petiole. Part of this
lag period (about 5 min, Fig. 3) is due to a time
lag in accumnulation of label in the minor veins. The
other part of the lag is due to the time necessarv for
the tracer to travel along the minor and major veins
into the petiole.
The bulk of the label appeared to be in minlor
veins after 20 min of flushing (Fig. 4). It should
be considered that the proportion of phloem looms
somewhat larger thlia it would if seen 3-dimensionally, (cf. Fig. 11). The bulk of the label may therefore still be outside the veins; this would be in
closer agreement with WVinter and Mortimer's observation (14) that after 30 min only about 20 % of
the assimilated wCwas exported fromii the leaf blade.
In Figs. 1 to 4 there is a broad zoile of grainsi
wvhich extends bevoiid the leaf margiin. Silver gralins
2.5 or closer to the leaf margiin are to be exl)ected
as a resuilt of the !l)resence of 14C withill the Inarginal
leaf cells but graiins 25 to 50 y away from the margin
are thought to be caused by diffusion of labeled
assimilates other than sucrose into the emii'beddinig
Nvax. This zone did Inot occuIr in leaves which NveIe
labeled with radioactive suigars.
The techlnical assistance of -Miss Barbara Sinniiott
anid the photographic \work of 'Mr. R. Whitehea(l are
gratefully acknoNNle(dge(l.
Literature Cited
1. .A\RTSCIIVAGER, E. 1926. Aniatomily of the vegetative organ.s of the suigar beet. l. Agr. Res. 33:
2. BRONVIIENKO, M. 1. 1967. Somiie p)roofs of splitting of sucrose (lurinig its translocationi fromii the
mesophyll to the thin bundlles of sugar- beet leaves.
Fiziol. Rast. 14: 415-24.
3. Es.At-, K. 1967. 'Minor veinis in Bcta. leaves:
Structure relate(d to function. l'roc. Ami1. Phil.
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4. FiSCIIER, A. 1885. Studieni jibe- (lie Siebr6hren
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.s. GUNNING, B. E. S., J. S. PATE, ANI) 1L. CJ. BRIARTv.
1968. Specialized "transfer cells" in min1or veins
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8. N ELSON, C. 1).. H. Ci.As .S I). C. 1MORTI-MER, ANI)
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l)roducts of photosynthesis in soybean. Plant
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Fiziol. Rast. 11: 31-36.
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Physiol. 40: 740-47.
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12. TRIP, P. AND P. R. GORHAM. 1968. Bidirectional
translocation of sugars in sieve tubes of squash
plants. Plant Physiol. 43: 877-82.
13. TRIP, P. AND P. R. GORHAM. 1968. Translocation
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14. WINTER, H. AND D. C. MORTIMER. 1967. Role
of the root in the translocation of the products
of photosynthesis in sugar beet, soybean and pumpkin. Can. J. Botany 45: 1811-22.
15. ZIMMERMANN, M. H. 1957. Translocation of organic substances in trees. I. Nature of the
sugar in the sieve tube exudate in trees. Plant
Physiol. 32: 288-91.
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