companion-cell specific localization of sucrose … physiol. (1 993) 1 01: 899-905 companion-cell...

Plant Physiol. ( 1 993) 1 01: 899-905

Companion-Cell Specific Localization of Sucrose Synthase in Zones of Phloem Loading and Unloading’

Kurt D. Nolte* and Karen E. Koch

Horticultural Sciences Department, 1 151 Fifield Hall, University of Florida, Cainesville, Florida 3261 1

An immunohistochemical approach was used in maize (Zea mays) and citrus (Cifrus paradisi) to address the previously noted association between sucrose synthase and vascular bundles and to determine the localization of the low but detectable levels of sucrose synthase that remain in leaves after the import-export transition. Sucrose synthase protein was immunolocalized at the light microscope leve1 using paraffin sections reacted with rabbit sucrose synthase polyclonal antisera and gold-conjugated goat anti-rabbit immunoglobulin C. lmmunolabel was specifically observed in phloem companion cells of minor and intermediate veins in mature leaves of both species. Similar localization was apparent in the midrib of mature citrus leaves, with additional labeling in selected files of phloem parenchyma cells. A clear companion-cell specificity was evident in the phloem unloading zone of citrus fruit, where high activity of sucrose synthase has been demonstrated in vascular bundles during periods of rapid import. Sucrose synthase protein was not associated with adjacent cells surrounding the vascular strands in this tissue. lhe companion-cell specificity of sucrose synthase in phloem of both importing and exporting structures of these diverse species implies that this may be a widespread association and underscores its potential importance to the physi- ology of vascular bundles.

In their early studies with sugarcane, Hawker and Hatch (1965) reported that a large portion of Suc synthase activity was closely associated with vascular bundles. This observa- tion is consistent with its detection in phloem exudates (Leh- mann, 1973) and more recent evidence demonstrating phloem-specific expression of a maize Suc synthase gene in transgenic tobacco plants (Yang and Russell, 1990). A similar pattern of localization is suggested by work showing elevated levels of activity in the midrib of exporting leaves (Claussen et al., 1985) as well as the vascular bundles of developing citrus fruit (Lowell et al., 1989, Tomlinson et al., 1991). These data suggest that the presence of Suc synthase in or imme- diately adjacent to vascular strands could be a common attribute of these tissues. Therefore, in addition to its suggested functions in sink tissues (provision of precursors for starch accumulation [Choury and Nelson, 19761, cell wall synthesis [Hendrix, 19901, and respiration [Komor et al., 1977]), Suc synthase may also be more directly allied with phloem metabolism and function. Although the close relationship between Suc synthase and carbohydrate utilization/

This research was supported by a grant from the National Science Foundation (Cellular Biochemistry) and by the University of Florida Agricultura1 Experiment Station (joumal series No. R-02939).

* Corresponding author; fax 1-904-392-5653. 899

storage has received substantial attention, the association between the enzyme and transport tissues has yet to be clarified.

Cell-specific immunolocalization of Suc synthase was characterized in vascular bundles to obtain the localization infor- mation needed for initial interpretation of this enzyme’s function in transport tissues. Of particular interest is the possible association between elevated Suc synthase activity (Tomlinson et al., 1991), the high density of mitochondria in companion cells (Warmbrodt et al., 1989), and the metabolic roles both may play in the functioning of phloem. Although the mechanism of phloem loading remains a question of considerable interest, many workers in this area favor an apoplastic step in the selective uptake of solutes into the sieve element/companion-cell complex (Daie, 1989). Such transport is presumably active, involving Suc/H+ symport and action of ATPases concentrated in phloem tissue (Parets- Soler et al., 1990). The H+-cotransport hypothesis, therefore, depends on a continua1 supply of ATP that probably arises from respiration (Komor et al., 1977). Enhanced respiration has long been associated with Suc uptake in algae (Decker and Tanner, 1972) and isolated cells of higher plants (Komor et al., 1977). Thus, Suc synthase, through its ability to cleave SUC, may provide the necessary substrates for respiratory demands of active phloem cells.

Suc cleavage to meet demands for respiratory substrates may be only one component of Suc synthase function in phloem. The enzyme may also have a key role in the synthesis of callose, a (1+3)-/3-~-glucan that is a common structural and/or storage polysaccharide in higher plants (McNairn and Cumer, 1968). Callose has been implicated in the plugging of sieve plates following injury to phloem and in controlling symplastic transport through its deposition in plasmodesmata (Wolf et al., 1991). Previous studies have shown that the preferred substrate for production of callose is UDP-Glc (Morrow and Lucas, 1987). Because callose synthesis is extremely rapid (McNairn and Cumer, 1968), a substantial or a quickly regenerated supply of UDP-Glc is required. Suc synthase is a possible candidate for this metabolic function because of its capacity for UDP-Glc synthesis.

The present study was undertaken to immunolocalize Suc synthase in the transport tissues of maize (Zea mays) and citrus (Citrus parudisi). The presence of Suc synthase in vascular bundles has been well documented (see above); however, the complexity of the conducting tissue itself has made it difficult to determine whether or not Suc synthase was associated with a specific portion of the vascular strand.

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900 Nolle and Koch Plant Physiol. Vol. 101, 1993

The data presented in this report advance previous evidenceof elevated Sue synthase activity in vascular areas by dem-onstrating specific localization in companion cells. Work re-ported here also suggests that the presence of Sue synthasein companion cells of phloem tissue may be widespreadamong species and tissues.

MATERIALS AND METHODS

Preparation of Plant Material

Ten fully expanded, mature leaves were collected randomlyfrom five 10-week-old field-grown maize plants (Zea maysL, W22), and from the most recent spring growth flush offive 8- to 10-year-old containerized 'Marsh* grapefruit (Citrusparadisi Macf.) trees grown in Gainesville, FL. A band 1 cmwide was excised approximately 20 cm from the maize leaftip and from the middle of the citrus leaf. Five grapefruit,each in the expansion phase of growth (stage II, Lowell etal., 1989), were harvested randomly and the region of peelcontaining the major vascular bundles and adjacent segmentepidermis was isolated from the fruit. Portions of both fruitand leaf tissues were cut into 3x3 mm pieces, randomizedand fixed in formalin acetic acid (47.5% [v/v] ethanol, 0.87M glacial acetic acid, 3.7% [v/v] formaldehyde) at 25°C for24 h (Sass, 1940). Fixed tissue was dehydrated at 25°Cthrough a series of increasing concentrations of tertiary butylalcohol and embedded in paraffin at 58°C. Sections 8 /*mthick were dried onto glass slides treated with Haupt's ad-hesive by incubating at 45°C overnight (about 16 h).

Immunohistochemistry

Sue synthase protein was immunolabeled essentially asdescribed earlier (Koch et al., 1992). Deparaffinized, rehy-drated sections were rinsed in PBS with 0.05% Tween 20(PBST) (pH 7.2), then blocked with 5% normal goat serumin PBST for 20 min. Sections were incubated with primaryantibody using a 1:250 dilution of the polyclonal Sue syn-thase antiserum in a humid chamber at room temperaturefor 60 min. Polyclonal antibodies had been raised against acombination of the maize Sue synthases encoded by the Shland Susl genes. The proteins were extracted from wholemaize kernels (W64A x 182E) 22 d after pollination (Koch etal., 1992). Slides were washed with PBST then incubatedwith secondary antibody at room temperature for 60 min.The antisera used was goat anti-rabbit immunoglobulin Gconjugated to 5-nm diameter colloidal gold particles (ZymedLaboratories, Inc., South San Francisco, CA) diluted 1:100 inPBST. The slides were again rinsed in PBST followed by arinse in glass-distilled water. The antigen-antibody complexwas detected by treating the sections with freshly preparedsilver-enhancement reagents (Janseen Biotech N.V., Olen,Belgium) until a black precipitate developed in positivelystained tissue (10-15 min). The tissue was washed in excessglass-distilled water, counterstained with fast green, andpermanently mounted for microscopic evaluation. Bright-field photomicrographs were taken of silver-enhanced im-munogold-stained sections using a light microscope (Opti-phot model, Nikon Inc., Melville, NY) with Kodak T-Max100 film.

Figure 1. Light-level immunogold localizationof Sue synthase present specifically in compan-ion cells of exporting maize leaves. Sections oftissue fixed in formalin acetic acid and em-bedded in paraffin were immunolabeled withSue synthase antisera and anti-rabbit immuno-globulin C conjugated to 5-nm gold particles.Control sections were treated similarly exceptthat preimmune rabbit immunoglobulin C wasused in place of the Sue synthase antisera. Bars= 50 Mm. A, A transverse section of a majorvascular bundle from a fully expanded maizeleaf showing the distribution of Sue synthase inthe companion cells of phloem tissue (box). B,A control section similar to that shown in A buttreated with preimmune serum showing theabsence of immunoreactivity in companioncells (box). C and D, A transverse section of amaize leaf vascular bundle with companion-cell specificity of Sue synthase localizationclearly evident (C) when compared with similarcells in the preimmune control section (D). Eand F, Longitudinal sections of phloem tissuefrom a maize vascular bundle. Note the con-centrated region of gold particles that are lo-calized in a long cylindrical companion cell ofthe antisera-treated sections (E) relative to thepreimmune control treatment (F).



Immunolocalization of Sue Synthase in Companion Cells 901

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B

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Figure 2. Light-level immunogold localizationof Sue synthase in minor and intermediate vas-cular bundles of exporting citrus leaves. Fixa-tion and immunogold labeling are as in Figure1. A, A low-magnification light-micrograph in-dicating regions of companion-cell-specific lo-calization of Sue synthase (arrows) within thephloem tissues of minor vascular bundles. Bar= 250 fim. B, A section treated with preimmunesera (control) with similar minor vascular bun-dle arrangement (arrows) lacking significant im-munoreaction in the transport areas. Bar = 250nm. C and D, An intermediately sized vascularbundle with gold immunolabel. Note the con-centrated areas of gold particles (arrows) dis-tinctly associated with companion cells and theabsence of gold labeling (arrows) in the controlsection (D). Bars = 25 Mm. E and F, Longitudinalsections of immunogold-labeled (E) and preim-mune (F) sections of intermediate vascular bun-dles of citrus leaves. Note the specific distri-bution of immunostaining present in the com-panion cells of phloem tissue (E) that is absentin the control treatment (F). Bars = 250 ̂ m. C,A high-magnification light-level image of a mi-nor vascular bundle from an exporting citrusleaf showing immunogold particles (arrows)clearly localized within the companion cells.Bar = 25 ̂ m. H, A control section of a vascularbundle that is similar to that shown in G buttreated with preimmune antiserum. Note theabsence of gold label in companion cells (ar-rows) relative to the immunochemical reactivityin the companion cells shown in C (arrows).Bar = 25 ̂ m.

RESULTS

Sue synthase in the assimilate loading and transport zonesof exporting maize leaves was localized specifically in thephloem tissue (Fig. 1A). Label was absent from other tissuesof the leaf including epidermis, bundle sheaths, mesophyll,and xylem. Within the phloem, a heavy, black immunogoldlabel appeared to be strongly associated with companioncells. Although optical microscopy does not necessarily pro-vide definitive resolution of cell types, companion cells couldbe distinguished in the present work by their dense cyto-plasm, large, well-differentiated nucleus, minor degree ofvacuolation, characteristic length relative to vascular paren-chyma cells, and close association with sieve elements (Fig.1, C-F). This cellular specificity was observed in all vascularstrands of the maize leaf blade irrespective of bundle size(data not shown). No other phloem cells were immunola-beled. Sue synthase appeared to be restricted to the cytoplasmand did not occur in organelles studied in sections shownhere.

Particular attention was directed toward an inspection ofperipheral cytoplasm in cells with large vacuoles and vascularparenchyma cells with cytoplasmic densities similar to those

of companion cells. The lack of immunolabel in all but thehighly elongated companion cells indicated that results werenot due to cytoplasmic density alone. In addition, Evert et al.(1978) have shown that some of the sieve elements in smalland intermediate bundles of Z. mays are associated withvascular parenchyma instead of companion cells. Althoughsuch vascular parenchyma may replace at least some of thecompanion-cell functions, elevated levels of Sue synthaseprotein were not observed.

Overall, a comparable localization was also evident for Suesynthase in the Sue-loading regions of citrus leaves (Fig. 2, Aand B) and in the midrib (Fig. 3), a tissue associated withsugar export from the leaf. In this dicot, companion-cellspecificity was evident in intermediate-sized vascular bundles(Fig. 2, C-F) as well as in small bundles where the frequencyof companion cells was reduced (Fig. 2, G and H). Althoughthe same close association between Sue synthase and com-panion cells was observed in the midrib of the citrus leaf,immunochemical staining was also apparent in cells of thevascular parenchyma (Fig. 3). These cells, with their conspic-uous vacuoles, thin layer of cytoplasm, and blunt cell ends,occurred in strands or files with varying degrees of Suesynthase immunoreactivity (Fig. 3, E-H). www.plantphysiol.orgon May 29, 2018 - Published by Downloaded from

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902 Nolle and Koch Plant Physiol. Vol. 101, 1993

Figure 3. Transverse and longitudinal sectionsof a portion of the midrib from a fully expandedexporting citrus leaf showing strong light-levelimmunoreactivity in companion cells and pa-renchyma cells within phloem tissue. Fixationand immunogold labeling are as in Figure 1. Aand B, Immunolabel present in the cells asso-ciated with phloem tissue (A, arrows) versuslittle or none found in the sections treated withpreimmune sera (control) (B, arrows). Bars = 50jim. C and D, Transverse section of a midribportion showing the localization of Sue syn-thase in specific cells within the phloem tissue(C, arrows) and its absence in similar cells ofthe control section (D, arrows). Bars = 25 pm.E-H, Longitudinal section of midrib tissue withgold label clearly evident in phloem paren-chyma cells (E and G) and companion cells (G)of anti-Sue synthase-treated tissue and the ab-sence of immunoreactivity in cells of the con-trol sections (F and H). Bars = 50 pm.

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A similar immunostaining pattern was observed in theareas of assimilate unloading in fruit (Fig. 4, A-H). In thevascular bundles of citrus fruit, the Sue synthase signal wasspecifically detected in the companion cells within thephloem tissue.

DISCUSSION

Reports over the past 25 years have increasingly supportedthe possibility that Sue synthase may be associated withvascular bundles (Hawker and Hatch, 1965; Claussen et al.,1985; Tomlinson et al., 1991), and even phloem in particular(Yang and Russell, 1990). However, the physiological role ofthe enzyme in conducting tissues remains unknown. Thepresent study explored the relationship between Sue synthaseand vascular bundles of both photosynthetic and nonphoto-synthetic organs in citrus and maize. At the resolution oflight microscopy, the enzyme was found to be compartment-alized within companion cells of all phloem tissue examined.This includes phloem tissue distinctly linked to Sue loadingand unloading (Figs. 1, 2, and 4) in addition to the midrib, atissue primarily associated with long-distance transport (Fig.3). Thus, the findings presented here could provide a key

insight into the function of Sue synthase in companion cellsthroughout a plant.

The companion cell-specific localization of Sue synthase inmature leaves (Figs. 1-3) offers evidence concerning oneexplanation for how this reversible enzyme, typically cleavingSue in vivo (Avigad, 1982), can be active in organs thatsynthesize and export Sue. Low but detectable levels of Suesynthase activity have been reported in fully expanded leavesof citrus (Schaffer et al., 1987) and maize (Nguyen-Quoc etal., 1990), yet futile cycling could take place if Sue cleavageby Sue synthase and synthesis by Sue phosphate synthaseoccurred within the same cells. These previous observationsmay be reconciled if Sue synthase in mature leaves typicallyexhibits the companion-cell specificity reported here.

Although the exact physiological function of Sue synthasein companion cells remains open for future experimentation,one potential role may lie in the energy demands for Sueloading from the apoplast. Previous physiological and struc-tural investigations suggest that a primary mechanism fortransport of solutes into sieve elements is likely to involveactive accumulation from the apoplast in these systems(Humphreys, 1978; Daie, 1989). A generally favored modelfor apoplastic loading is Suc/H+ cotransport driven by a



Immunolocalization of Sue Synthase in Companion Cells 903

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Figure 4. Portions of dorsal vascular bundles in a phloem unloadingzone of citrus peel tissue showing specific light-level immunogoldlocalization of Sue synthase in companion cells at the light micro-scope level. Tissue fixation and immunogold labeling are as inFigure 1. A and B, Distribution of immunolocalized Sue synthasewithin companion cells of a large vascular bundle (A) and theabsence of reactivity in the sections treated with preimmune sera(B). Bars = 20 Mm. C and D, Localization of Sue synthase incompanion cells showing cell specificity with little or no reactionin any other type of cell. Bars = 20 nm. E-l, Longitudinal sectionslocalizing Sue synthase within companion cells of dorsal vascularbundles. Note the specificity of the gold label in association withcompanion cells (E, C, H, arrows) relative to the control section (F,arrows) and the distribution of Sue synthase-containing cells in thistransport tissue (I). Bars in E-H = 20 nm; bar in I = 100 nm.

proton gradient across the plasmalemma. Formation of sucha gradient is an energy-dependent process, presumably me-diated through activity of a plasmalemma H+-ATPase, suchas that immunolocalized in the phloem tissue of pea leaves(Parets-Soler et al., 1990).

No specific type of cell within the phloem tissue has beenidentified as having an elevated capacity for ATP production,although structural evidence suggests that companion cells

may have such a capacity. First, cytoplasm is more dense andmitochondria more numerous in these versus other cell types.Second, enhanced rates of respiratory activity are indicatedby the extremely condensed conformational state of mito-chondria in companion cells (De Roberts et al., 1975). Thesecontrast sharply with the mitochondria of sieve elements(Esau, 1969), which appear to be internally disorganized tosuch a degree that they have been characterized as modifiedorganelles.

These intriguing ultrastructural observations provide sup-port for the hypothesis suggested by Lehmann (1981), whoused an enzymic approach to propose a specific associationbetween the metabolic capacity in the companion cell andthe availability of ATP for Sue loading. This proposal was anextension of earlier respiratory studies, which suggested thatSue uptake could be mediated through the metabolic energyderived from enhanced respiratory rates as shown in algae(Decker and Tanner, 1972) and higher plants (Komor et al.,1977). The immunolocalization of Sue synthase in companioncells is consistent with these investigations as well as withindications that Sue synthase may be a key enzyme in thepartitioning of carbon through the respiratory pathway(Huber and Akazawa, 1986; Black et al., 1987; Farrar andWilliams, 1990). Elevated activity of Sue synthase in com-panion cells could thus provide the necessary substrates forpotentially high respiratory demands in these cells.

In addition to its association with companion cells, Suesynthase was also localized in specific parenchymatous cellsof the citrus midrib (Fig. 3). This could be particularly signif-icant to the starch storage that occurs here. In an anatomicalstudy of citrus leaf development, Scott et al. (1948) showedthat starch accumulated predominantly in selected paren-chyma cells of midribs. The Sue synthase distribution re-ported here closely resembled that of starch deposition inthese veins. This, together with several investigations show-ing elevated Sue synthase activity in association with starchproduction (Chourey and Nelson, 1976; Doehlert, 1990) im-plies that Sue synthase may supply precursors for starchbiosynthesis in these particular cells of the leaf midrib.

The companion-cell specificity of Sue synthase, describedhere for Sue-loading zones of citrus and maize, was alsoevident in the unloading areas of citrus fruit (Fig. 4). Thismay indicate a similar function for the enzyme in companioncells of both source and sink tissues. Elevated activity of Suesynthase has been shown previously in the functioning trans-port tissues of this assimilate unloading zone during rapidimport (Lowell et al., 1989). Although early work suggestedthat phloem unloading could take place through the activityof an ATPase driven by a Sue-proton antiport (Humphreys,1978), little evidence has been obtained demonstrating thatthis model is directly involved in the unloading process.However, this does not necessarily imply that the overallmechanism of assimilate unloading from phloem is totallyindependent of metabolic energy utilization. Cytochemicallocalization of ATPase activity has demonstrated an associa-tion with the phloem in sink tissues (Oparka, 1986).

One model to explain solute transfer through the phloemand at terminal unloading sites has been the "pump-leak"hypothesis. According to this theory, assimilates, such as Sue,constantly leak from the phloem to the apoplast, where it is



904 Nolte and Koch Plant Physiol. Vol. 101, 1993

either imported by surrounding sink cells or reloaded back into the phloem. It has been suggested that enhanced Suc leakage (unloading) to the apoplast occurs through localized inhibitions of the Suc retrieval mechanism (Patrick, 1990). In such a scenario, the physiological function of Suc synthase within the companion cells could be similar in both importing and exporting organs. The ATP demand in either instance could be supplied by elevated respiration supported in tum by Suc synthase functioning in the cleavage direction.

Suc synthase may have an additional physiological role in companion-cell metabolism besides that of its potential function in the production of precursors for respiration. The transport of assimilates through sieve tubes depends on sev- era1 factors. Among them are the overall area of the sieve plate together with the size and number of the pores within the sieve plate itself. Recent evidence (Wolf et al., 1991) confirms earlier studies showing the potential impact of callose ([ 1+3]-/3-~-glucan) accumulation in the regulation of both cell-to-cell and long-distance transport in plants. Dep- osition of this polysaccharide reduces the size-exclusion limit of the sieve-plate pore in sieve tubes and, hence, constricts the cell-to-cell movement within the strands. Callose accu- mulates extremely rapidly after specific stimuli to phloem (some reports are in the range of seconds [Eschrich, 19751). This, together with its seemingly ubiquitous presence in higher plants, has prompted the view that callose formation may be a standard response to phloem injury. Callose is synthesized by callose synthase, a vectorially arranged enzyme in the plasma membrane (Tighe and Heath, 1982) that is apparently under strict regulation. Evidence suggests that this same enzyme may also be involved in cellulose biosynthesis (Delmer et al., 1985), and that its callose synthase function is normally inactive in healthy cells. The latter appears to be activated by perturbation of plant tissue, however, leading to rapid sealing of the sieve tubes.

Severa1 investigations have shown that callose synthase utilizes UDP-Glc to produce the (1+3)-/3-~-glucan (Morrow and Lucas, 1987) and that this precursor is derived from cytoplasmic metabolism (Kauss, 1985). This is particularly interesting because Suc cleavage by SUC synthase could provide the necessary supply of UDP-Glc for rapid synthesis of callose. Early work in Yucca (Becker et al., 1971) and even Macrosystis, a brown alga (Schmitz and Srivastava, 1974), has shown that the distribution of UDP-Glc in phloem exu- date following a 32P incubation period becomes labeled to a steady state in less than 1 min. The pool of UDP-Glc in sieve tubes is, therefore, either small and/or its metabolic tumover is high. It could be postulated that, in an event where rapid callose synthesis is required, UDP-Glc could be produced through the cleavage of Suc by Suc synthase and diverted from a respiratory role in the companion cell to a synthetic pathway in the sieve element.

The results presented here demonstrate a clear association between Suc synthase and companion cells in the phloem of maize and citrus. Companion-cell specificity was observed in areas where predominant phloem function would presumably have ranged from loading (small leaf bundles) to unloading (fruit dorsal strand) and included areas functioning primarily in long distance transport (midribs). Although the physiological significance of Suc synthase in transport tissues

is unclear, severa1 hypotheses can be proposed. These include maintenance of an equilibrium between phloem Suc and its breakdown products as suggested earlier by Claussen et al. (1985), together with the production of substrates for (a) potentially elevated companion-cell respiration and/or (b) precursors for complex carbohydrates such as callose (and limited instances of very localized starch deposition). The cell-specific function for Suc synthase in phloem tissue thus appears to be distinct from its often more diverse roles in other nontransport cells (Claussen et al., 1985; Huber and Akazawa, 1986; Black et al., 1987).

ACKNOWLEDCMENTS

We thank Dr. Gregory W. Erdos, Department of Microbiology, University of Florida, for his technical assistance with the immunohistochemical techniques, and Dr. Donald R. McCarty and Dr. L. Curtis Hannah, Department of Horticultural Sciences, University of Florida, for their purification of the Suc synthase protein.

Received July 27, 1992; accepted November 20, 1992. Copyright Clearance Center: 0032-0889/93/lOl/0899/07.

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