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. Results 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 717- Published by www.plantphysiol.org Downloaded from on June 18, 2017 Copyright © 1969 American Society of Plant Biologists. All rights reserved. 718R PLANT PHYSIOLOGY A 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. Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1969 American Society of Plant Biologists. All rights reserved. TRIP _W m - _m EI 719 SUGAR TRANSPORT IN 'MINOR V'EINS e! -w --m - 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 Insert Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1969 American Society of Plant Biologists. All rights reserved. 721 TRIP - SUGAR TRANSPORT IN MINOR VEINS gA"tP NO 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. X 570. from on June 18, 2017 - Published by www.plantphysiol.org Downloaded Insert Copyright © 1969 American Society of Plant Biologists. All rights reserved. TRIP - SUGAR TRANSPORT IN 'MINOR V'EINS 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, 723 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. Discussion 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- Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1969 American Society of Plant Biologists. All rights reserved. 74PLAN'T PHYSIOLOGY 724 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. Acknowledgment 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: 143-76. 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. Soc. III, 4: 219-33. 4. FiSCIIER, A. 1885. Studieni jibe- (lie Siebr6hren der Dikotylenblitter. Sachsisclhe Akad. Wiss. Ber. 37: 245-90. .s. GUNNING, B. E. S., J. S. PATE, ANI) 1L. CJ. BRIARTv. 1968. Specialized "transfer cells" in min1or veins of leaves and their possible significanice in phloei translocation. .l. Cell Biol. 37: C7-C12. 6. KIURSANOV, A. L., M. 1. BROVCTIENKO, AND A. N. P,AsRISKAYA. 1959. Flow of assimilates to the con(lucting tissuie in rhubarb ( Rheum r-haponticni, L.) leav-es. Fiziol. Rast. 6: 527-36. 7. ]MORTIMER, D. C. 1965. Tranislocatiotn of tlle products of photosynithesis in sugar- beet petioles. Can. J. Botany 43: 269-80. 8. N ELSON, C. 1).. H. Ci.As .S I). C. 1MORTI-MER, ANI) P. R. GORIIHAM. 1961. Selective translocation of l)roducts of photosynthesis in soybean. Plant Physiol. 36: 581-88. 9. PRISTPA. N. A. 1964. Redistributioin of radioactive assimilates in the leaf tissues of cereals. Fiziol. Rast. 11: 31-36. 10. SASS, J. E. 1958. Botanical Microtechnique. Iow%a State College Press. Amnes, Iowa. 11. TRIP, P., C. D. NELSON, AND G. KROTKOV. 1965. Selective and preferential translocation of 14C_ labeled sugars in white ash and lilac. Plant Physiol. 40: 740-47. Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1969 American Society of Plant Biologists. All rights reserved. TRIP SUGAR TRANSPORT IN MINOR VEINS 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 of radioactive sugars in vascular tissues of soybean plants. Can. J. Botany 46: 1129-33. 14. WINTER, H. AND D. C. MORTIMER. 1967. Role 725 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. Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1969 American Society of Plant Biologists. All rights reserved.
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