leaf structure and translocation in sugar beet'leaf structure and translocation in sugar...

Plant Physiol. (1969) 44, 45-54

Leaf Structure and Translocation in Sugar Beet'D. R. Geiger and D. A. Cataldo

Department of Biology, University of Dayton, Dayton, Ohio 45409

Received July 17, 1968.

Abstract. Anatomical and ultrastructural details of a translocating 10-cm leaf of sugarbeet (Beta vulgaris L. var. Klein Wanzleben) were correlated with translocation rate datia.The minor veins were found to be 13 times as extensive as the major veins and measure70 cm/cm2 leaf lamina. Measurements disclosed that a 33-,u length of minor vein services29 mesophyll cells with the result that translocate moves an average of 73 ,u or 2.2 celldiameters during transport from mesophyll cells to a minor vein. High-resolution. freeze-dryautoradiography revealed that assimilates accumulate in organelle-rich cells of the minor veinphloem. Correlation of phloem volume and loading rate for minor veins yielded an uptakerate of 735 Amoles of sucrose per g fresh weight of phloem. The arrangement and structuralfeatures of minor veins appeared to be consistent with the concept that vein loading precedestranslocation.

In the search for the mechanism of translocationof organic compounds, various workers have proposedmodels based on active transport of materials byphloem. The proposed mechanisms can be dividedinto those in which the active driving process occursall along the translocation path and those in whichthis process is centered in the phloem of the sourceregions such as the vein endings of the leaf blade(1,8,14,15,22,25,30,34).

Although inhibitor studies offer some hope ofdistinguishing between the alternatives, to date thisapproach has failed to differentiate conclusively be-tween the 2 groups of proposed mechanisms. Diffi-culties include the failure of the effects of chemicalinhibitors to be sufficiently restricted to the pointof application (15) and the production of chillingeffects beyond the inhibition of metabolism (29).

Results of recent localized-chilling experimentssuggest that in the phloem outside of source andsink regions, metabolism serves primarily to main-tain structural organization while in source andpossibly in sink regions it serves to move the trans-locate (10,29). Findings from experiments employ-ing low temperature treatment thus favor mechanismswhich include active vein-loading in the source leaf(1, 3, 8, 25, 30, 33). From a histochemical stand-point, previous studies have suggested a possibleactive loading function for border parenchvma cellsand companion cells in 1 case (30) and for com-panion and transfer cells (Ubergangszellen) in an-other (1). In the present studv, several recentlydevised techniques (6, 7, 18, 19) were used to locatecells which accumulate water-soluble organic com-

1 This research was supported in part by Grant GB-2470 from the National Science Foundation.

pounds in a translocating leaf. In addition, ana-tomical measurements of a translocating leaf werecorrelated with translocation rate data to ascertainthe feasibility of vein loading of minor veins as ameans of driving translocation of organic compouinds.

Materials and Methods

Plan1t Material. Sugar beet plants (Beta viulgarisL. var. Klein Wanzleben) were grown by solutionculture in controlled environment cabinets as de-scribed earlier (11). Studies were performed on10-cm leaves of 5 to 7-week-old plants pruned to asimplified translocation system (11, 12).

Measurement of Venation. To visualize the veinpattern, leaves were allowed to take up a 0.25%(w/v) water solution of acid fuchsin until the dyehad reached the minor veins. Injected leaves werefixed in 80 % ethanol, dehydrated, and then clearedin methvl salicylate. Extent of venation was meas-ured in photographic enlargements of the clearedleaves.

Histological Procedures. Details of vasculartissue were studied in 2-, thick sections of leaf tissuefixed in 3 %o (v/v) glutaraldehyde in pH 7.2 phos-phate buffer or in 10 % acrolein in tap water. Tis-sues were dehydrated in acetone and embedded inhydroxyethvl methacrylate by the method of Ruddell(26). Tissue for ultrastructural studies was pre-pared by fixing 1 mm2 pieces of leaf in 3 % (v/v)glutaraldehyde in pH 7.2 phosphate buffer for 1.5 hrat 4' or in 2 % (w/v) KMnO4 for 15 min at roomtemperature. Glutaraldehyde fixed tissues werewashed for 1.5 to 3 hr in buffer, and postfixed in2 % (w/v) osmium tetroxide in pH 7.4 phosphatebuffer for 2 hr at 4'. Sections were poststained inReynolds' lead acetate.

45

Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

46 PLANT PHYSIOLOGY'

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FIG. 1. (Upper left) Clearedl 10-cm sugar beet leaf, xylem-injected with acid fuchsin, showing major venation.FIG. 2. (Upper right) Detail of small branches of major venation. White rectangle designates positioni of 0.5

111n112 area sholwn in figur-es 3 and 4. Minor veins are not apparent at this magnificationi. X 2.6.FIG. 3. (Lo-wer left) Mesoplhyll cells overlying the minor veins showvn in figure 4. X 125.FIG. 4. (Lox-er right) Pattern of iminor venation in 0.5 mm2 area of 10-cmi sugar beet leaf. X 125. c) connec-

tionis of minor veint net with major veins: cc) presumed companiion cell; ch) chloroplast; m) mesophyll cell; mv)minor vein; o) oxalic acid crystal p) procalnbial cell: pd) plasimiodesimi; pp) phloem parenchyma or companion cell;s) sieve tube: sp) sieve plate; v ) -acuole.


GEIGER AND CATALDO-LEAF STRUCTURE AND TRANSLOCATION

Freeze-dry Autoradiography. Details of steady-state labeling with "4CO2 were described previously( 12). Leaves were sampled after 2 or 4 hr oflabeling at which time tissue activity was 30 to 50

/ic/dm2 leaf. Squares of leaf several millimeters ona side were quickly frozen -vith 8 % (v/v) methyl-cyclohexane in isopentane at -170 to -180( andstored under powdered dry ice prior to dehydrationby a modificationl of the mlethod of Jensen (18).Moisture content of the air streamii was mlaintainedby equilibration with ice at -35o* Dehydration wascarried out at -450 hile the trap was held at -78°.Following 36 to 72 hr of drying. the tissue wasvacuunm infiltrated in 560 paraffin for 30 to 45 min.Sections 6 u in thickness were pressed against a thinfilm of Ilford K-5 emulsion, in total darkness (18).Films of froml 0.2 to 1 u thickness w-ere made byv-arying the speed of w-ithdrawal of the coverglassfrom liquified emlitilsion at 580 ( 19). Autoradio-graphic slides were sampled after periods of 3 to15 days exposure at 40 and developed in KodakMicrodol X at 140 (18).

Resultsand Discussion

As in previous translocation rate studies (10.11,12, 13,29), 10-cni sugar beet leaves were used asexperimental mlaterial. Veiins of the mlidrib andthe first 3 or 4 branches wxere visibly injected withacid fuchsin (figs 1 and 2). Beyond these majorveins, the vascular system is comiposed of a networkof minor veins with relatively few elemlients (figs

3 and 4). A drawing of a representative patternof minor venation in a 0.8 mm2 area of lamina isshown in figure 5A. The minor vein net connectswith a tertiary vein on 1 side and with quaternarybranches on 2 other sides. Examples of paradermaland cross sections of a minor vein have been recon-structed from low magnification electron micrographs(figs 5-7).

To evaluate the relative accessibility of the leafmesophyll to major and minor veins respectively,the extent of each of these classes of venation wasmeasured. A value of 70 + 10 cm of minor veinper cm2 leaf was obtained from a sampling of36 1-mm2 fields. By comparison, the major veinsmeasure 5.5 cm per cm' leaf. Consequently, theminor veins have more than 10 times greater accessi-bilitv to the mesophvll than the major veins. Cal-culations based on measurements from cleared andsectioned leaf material yielded an estimate of 3 X 105mesophyll cells per cm2 leaf lamina. Combiningdata for cell size, vein extent and cell population(table I). it was calculated that a minor vein length,corresponding to the diameter of an average meso-phvll cell, receives translocate from approximately29 mesophvll cells. By contrast, a similar length ofmajor vein is accessible to about 370 cells. In a leaf6.7 cell diameters thick, the cells serviced would bewithin an average lateral distance of 2.2 cell diam-eters or 73 ,u of a minor vein. Wylie (32) foundthe average width of tissue bordering the minor veinsin 66 species of dicotyledons to be 65 K. A samplingof cross sections of 24 minor veins disclosed thatthe average perimeter of contact between the phloemin a minor vein and the adjacent mesophyll is

Table 1. .A1 (lt)I1liCll (hi(d Ph \sioloi(cal Porometcrs for Mintor Veinis in Loz7linina of 10-cmi Leaves of Beta vulgaris

Extent ot minorvenation

No. of mesophyllcells serviced by a

33-, length ofminor veinl

Surface area ofplhloem bundle at

interface withmesophyll cells

in leaf2

Volume of minorvein phloemin a cm2 of

leafs

Rate of exportof sucrose from

leaf4

cells/33-,u length29

cni2/cm2 leaf0.49

mm3/ccm2 leaf0.31

Average diamleter of mesophyll cell is 33 + 5 ,.2 Average perimeter of conitact between phloem of minor vein cross section and mesophyll is 70 + 11 ,u.

Average cross sectional area of minor vein phloem is 440 + 9: ,.4 Data fromi Geiger and Swanson (13).

Table I[. )istrilbittioni of 14C Bet.keein V'ariozns Categories of Comtpounds in the Lainina of a 10-cmi Snlgar Beet LeafAfte VUarious Pcriods of Steady-State Labelintgl

Labeling period80 min 120 min 160 min 240 min

,c % Ac % /c % c %80 % Ethanol insoluble compounds 13.5 48 13.8 49 19.1 63 19.3 62Sucrose 7.7 27 5.6 20 3.7 12 3.9 13Other soluble compounds 7.1 25 9.0 31 7.5 35 8.0 25Total radioactivity in lamina 28.4 100 28.4 100 30.3 100 31.2 100

Details o i method in Geiger and Swanson (13).

cni, 'cni-70 + 10

,Ug/cm2 min1.3 ± 0.4

47


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FIG. 5. (Upper) Diagram of minor venation from leaf area in figure 4. A) pattern of minor veins shovingconnection to branches of major veins; B) paradermal section through minor vein phloem; C) cross section throughminor vein.

FIG. 6. (Lower left) Electron micrograph of cross section through phloem of minor vein similar to figure 5C.X 7500.

FIG 7. (Lower right) Electron micrograph of paradermal section similar to figure jB showing phloemii paren-

chyma and sieve tubes. X 5800. Insert (rectangle) shows details of sieve pore with endoplasmic reticulum tra-versing the pore. X 31,000. See figure 4 for key.

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bright. B) (Lower) Brightfield view of sanme field as A showing pattern ot silver grains at a slightly different

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70 + 11 u giving a combined surface of 0.49 cm2for minor vein phloem per cm2 leaf (table I).

The greater accessibility and the higher surfaceto volume ratio of the minor veins is consistent withthe thesis that they accumulate assimilate as part ofthe translocation process. Several workers (1 30)have proposed that parenchyma cells associated withthe minor veins function in the transfer of assimilatesinto the sieve tubes (1, 8, 14, 30) though they do notagree on the specific cell type involved. To investi-gate the possible transfer role of cells associatedwith leaf veins, we undertook a histoautoradiographicstudy to locate cells capable of accumulating assim-ilates in a translocating leaf. Sugar beet leaves weresupplied with '4CO" under conditionis whiclh resultin a high rate of translocation of labeled sugar (12).Previous studies have shown tllat during the first100 min of steady-state labeling the sucrose in theleaf reaches isotopic saturation (12. 13); thereafter,the spatial distribution of soluble compounds suich assucrose, which have reaclhed tlleir mlaximiunm specificactivity, is accurately reflected in the distribution ofradioactivity throughout the tissue. At the sametinme of sampling, sucrose constittutes 12 to 20 % ofthe label (table II); this proportion decreases withtime as a result of accumulttion of insoluble mlate-rials in the leaf.

The distribution of radioactivity in a paradermlalsection of freeze-dried leaf tissue, as revealed b-high-resolution autoradiography. is shown in figures8 to 10. The grain density in figures 8 to 10 indi-cates a greater amount of labeled material presentitn the phloem than in the cytoplasmn of adjacentmesophyll cells. The sieve tubes are too small tobe located with certainty, but the labeling appearsto be greatest over the cells wvith the dense cyto-plasnm. Control autoradiographs (fig 11) show theabsence of significant numbers of silver grains pro-duced by chenmical actionl or by background radiation.Because a large portion of the label in the leaf isin glycogen (13), protein aind other- insoluble com-potunds, autoradiographs of 80 % ethanol extractedtissue were prepared (fig 12). In contrast to thepattern for non-extracted tissue, there is a highlierconcentration of silver grains over the chloroplastsand cytoplasm of the nmesophyll cells than over theminor veins in the extracted tisstue. Extractioncaused some ioss of resoluition as evidenced by thepresence of grains at the periphery of tiie vacuolesand slightly beyond the edges of the tissue sections.These comparative autoradiographs indicate that alarge portion of the radioactivity in the phloenm is inethanol-soluble compotunds, prestumably, largely insucrose.

This pattern of accumulation b- organelle-riclcells resembles the previously reporlted distributionof energy-related compounds localized in cells postu-lated to function in vein loading during translocation(1, 14, 30). On the basis of phosphatase and tetra-zolium reactions, Bauer implicated companion andtransfer cells of the minor veins of Madia dissitifloira

and Vicia faba in the active loading of tranislocate(figs 10-13 of 1). He also found a lesser conceIn-tration of phosphatase and of formazan in the sievetubes and little activity in the border parenchymaanid mesophyll cells. The recent work of Gunning.Pate and Briartv (14) demonstrates an accumulationof insoluble materials in transfer cells of the minorveins followN-ing xylem injection wvitlh 'H-leucinesolution. These workers also found acid phosplha-tase localized in the cell walls of the transfer cells.

An examination of the tultrastructure of the minorveins revealed specializations which may relate tothe proposed vein loading process. Typicallv. aminor vein is composed of 1 to 3 files of xylemvessels and 4 to 12 files of phloem cells (fig 5C).In the light of vein loading hypotheses. 2 featuresof the phloem appear noteworthy. First is theabundance of mitochondria and other cytoplasmicorganelles in the companion cells and parenchynmacells of the phlo,em (figs 6. 7, 13), wvhich would beexpected in cells involved in active transport. Thesecond feature is the relatively large size of theorganelle-riclh comiipanion cells in the minor v-eins(fig 13) as coml)ared with similar cells found in thepetiole (fig 14). This size relationship. previouslynoted by Morretes (23. 24), may represent an addi-tional adaptationi for vein loading.

A number of calculations were made to correlatethe structural features described above wvith transs-location rate data. During photosxynthesis uildersaturating light initensity, a 10-cm sugar beet leafexports sucrose at the rate of 1.3 pAg/min cm2 or7.8 mg/hr dm2 leaf. On the assumiption that loadingof minor veins is aIn integral part of translocation.the entry of sugar inlto the minor vein phlloemI wX-illoccur at this same rate. The phloem of the miinor-eins occupies a volume of approximately 70 cm X440 p2 (table I) or 3.1 X 10-4 cm23/cm2 leaf; theestimated fresh weight of this phloem is 310 ug/cm2leaf. From this it follows that the sugar uptakerate Nvould be 1.3 pg sucrose/min per 310 pg fr w-tphloem tissue or 4.2 mg sucrose/min g fr wt phloentissue. On a molar basis this is 735 pmolessucrose/hr g fr wt phloenm in the minor veins. Thisrate of vein loading is equal to the rate of uptake ofsugar from a 10 % sucrose solution reported by\Veatherlev for leaf disks of Atropa belladonna (31).In the latter case, nitrogen anaerobiosis reduced theuptake rate to 25 %, suggesting an active transportmechanism is responsible for a major part of theuptake. Another indicariui- of the presence of anactive transport mechanism is the high uptake rateby minor vein phloem of the sugar beet. In thisregard. Bieleski (2) reported that phloem tissueexcised from stemls actively accumulates 9 to 16,umloles sucrose/hr g fr wt of tissue from a 10-1 AI-sucrose solution, a concentration representative ofthe sugar content of photosynthesizing leaf tissue.The rate calculated for nminor veins of the intactsugar beet leaf is 50 to 80 times the rate found b-

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FIG. 9,10. (Upper) Phase contrast view of minor veins in a paradermal section of source leaf; identityc ofindividual cell types difficult to discern. Similar to figure 8A. X 950.

FIG. 11. (Lower left) Freeze-dry autoradiograph of a paradermal section of unlabeled control tissue. X 950.FI(;. 12. (Lower right) Autoradiograph of 80 % (v/v) ethanol-extracted freeze-dried tissue showing labeling

pattern from insoluble materials. X 950. See figure 4 for key.

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FIG. 13. (Upper left) Electron micrograph of cross section of minor vein of leaf showing relationship betweenmesophlyll cell, phloein- parenchyma cell and sieve tube. X 22,000.

FIG. 14. (Uppet right) Ultrastructure of phloeni in cross section of petiole. Note relative sizes ot plhloemii paren-

chylma cells and sieve tubes in minor vein of leaf (figs 6,7,13) and in petiole. X 5800.FI(;. 15. ( Lower left) Plasmodesmiiata between sieve tube andl companion cell in cross sectioni of iminlor vein.

X 20,000.FIG. 16. ((Lower right) Cytoplasmiiic connlectionls between mesolphyll cell, phloeni parenchyiima cell, and(l sieve tube.

X 17,400. See figure 4 for key.

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Bieleski for phloem excised froml the stemii. Thisdifferenice in rates is consistent with the relativelygreater abundance of organelle-rich cells in thephloem of the translocating leaf than in the petiole.In the leaf lamina, the minor vein phloem consistsof 80 to 90 %, by volume, of organelle-rich paren-

chyma cells while in the path or petiole portion ofthe leaf these cells occupy 20 to 30 % of the volume.Consequently, the surface to volume ratio is nmoreconducive to sieve tube loading in the minor veinsthaln in the petiole (figs 14, 15).

Althotugh there is evidence favoring accumulationof assimilates by the phloem parenchyma cells of theminor veinis prior to translocation, many of the de-tails *of the imiodel are tentative and remlain to beexamined. From studies of chloroplasts isolated bynon-aqueous methods, it appears that sucrose is firstproduced in the chloroplasts (4, 28) and quicklymoves into the cytoplasm (28). Evidence for theintercellular miovemient of sucrose in the free space

via the cell walls and intercellular spaces has beenpresented by Hawx-ker (16) and by Kriedemann (20).Several modes of entry of sugar into the phloenihave been suggested as a result of various experi-

enlts. Sotmie -studies give evidence for the hydroly-sis of sucrose prior to its accumulation in cells(5, 27) wvhile others indicate that phosphorylationprecedes entrV- (22) ; still others indicate no hv-drolv-sis prior to uptake (17, 21). The contributionof cell surface structutres in the minor veins is notclear. Cell wall protuberances postullate(d as activein vein loading ( 14), were not observed in sugar

beet. Numierous plalsmodesmiata were observed inthe w-alls of both mieFophyll and phloem cells (fig 16)but their inlportance in vein loading is conjectulral(9).

The l)reseut study denmolnstrates that orgalielle-rich miinlor -eini cells of the translocating leaf accu-

mulate assimilaltes. The structural features of thesecells anid tleier relationship to the mesophvll cells isconsisteint with the concept that parenchyma cells ofthe mn,inor veins actively accumulate sugar lrior toits enltr- inLto the sieve tubes. Correlation of phvsio-logical anid structural mleasuremiielnts reveal nlo major

inconlsisiencies wxi.hI resl)ect to vein-loading theories.

Acknowledgments

authors are grateful to Drs. Mf. Arif Hayat of theIfniversitv of D)avton and Robert M. Giesy of the OhlioState Universitv for their assistance in the electron mi-croscope study alnd to the Ohio State University Collegeof Biology for the generous use of their electron micro-

scrope facilities.WNe alsoz thanik 'Miss Sharon NIcCloskey and fMiss

Mary Anthony for their competent technical assistance.

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53


PLANT l'HYS1OLOGY

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leaf structure and translocation in sugar beet'leaf structure and translocation in sugar...

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