Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana

The functional symplastic connections between primary and developinglateral roots of Arabidopsis were studied non-invasively usingconfocal laser scanning microscopy (CLSM), following ester-loadingof the phloem with carboxyfluorescein (CF). Prior to the formationof lateral primordia in the pericycle, the phloem of the primaryroot behaved as an isolated conducting domain. However, thedifferentiation of phloem connector elements within the dividingpericycle allowed the rapid establishment of intercellular communicationbetween the phloem and the cells of the lateral primordium.This communication was often established prior to the completeemergence of the lateral root from the parent root. Shortlyafter its emergence, functional conducting phloem became differentiatedwithin the developing lateral root. A progressive isolationbetween the phloem and surrounding cells at the base of thelateral root was observed as the lateral continued to grow;the new phloem conducting CF to the elongation zone where itwas unloaded symplastically from the protophloem into surroundingcells of the cortex and stele, a feature mirroring the patternfound near the apex of growing primary roots. Anomalous patternsof intercellular communication were found which indicated thatpreviously functional symplastic pathways may have become sealedoff following the emergence of some of the lateral roots. Key words: Arabidopsis, carboxyfluorescein, confocal laser scanning microscopy (CLSM), intercellular transport, lateral roots, phloem (unloading), symplast     

Real-time imaging of phloem unloading in the root tip of Arabidopsis

Confocal laser scanning microscopy (CLSM) has been used to image phloem transport and unloading in the root tip of Arabidopsis. The fluorescent probe 5(6) carboxyfluorescein (CF) was ester loaded into a single cotyledon and the entire seedling placed within an observation chamber under the microscope. Translocation of CF to the root tip was rapid, followed by unloading into discrete concentric files of cells. The position of the prominent unloading ‘zone’ corresponded precisely with that of the two protophloem files of sieve elements, demonstrating a functional role of these cells in symplastic sieve-element unloading. Symplastic transport following unloading was confined to the elongating zone of the root with little basipetal transport to more mature cells. Following photobleaching of the unloading zone, phloem transport was restored immediately into the protophloem sieve elements, followed rapidly by lateral, symplastic sieve-element unloading. The results demonstrate that phloem transport processes can now be imaged in real time, and non-invasively, within an intact plant system.     

The fluorescent probe HPTS as a phloem-mobile, symplastic tracer: an evaluation using confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) has been used to evaluatethe use of the fluorescent probe HPTS (8-hydroxypyrene-1,3,6-trisulphonicacid) as a symplastic tracer. HPTS-acetate was used to loadHPTS into the phloem and its movement was followed in threesystems where symplastic unloading has been proposed. In Arabidopsisroot tips and Abutilon nectaries the intercellular distributionof HPTS differed markedly from that observed with 5-(and 6)-carboxyfluorescein(CF)- HPTS was observed in the nuclei and cytoplasm whilst CFwas rapidly transferred into the vacuoles. In contrast, bothHPTS and CF accumulated in the vacuoles of the vascular parenchymaand nucellus cells following unloading from the phloem of thedeveloping barley caryopsis. The results indicate that HPTShas a number of advantages as a symplastic probe compared withCF. The findings are discussed in relation to the influenceof vacuolar sequestration on dye distribution. Key words: Confocal laser scanning microscopy (CLSM), HPTS, intercellular transport, phloem (unloading)     

Phloem mobility of fluorescent xenobiotics in Arabidopsis in relation to their physicochemical properties

The transport of a range of fluorescent probes within the rootphloem of Arabidopsis thaliana has been imaged using the confocallaser scanning microscope. The phloem mobility of these probesand their subsequent subcellular distribution in the cells ofthe ‘unloading zone’, close to the root tip, arediscussed in relation to a structure-activity relations (SAR)model. This is a generalized model describing the interactionof low molecular weight xenobiotics with living cells, basedon the physicochemical properties of the former. The work demonstratesthat the model can be used to predict the phloem mobility ofxenobiotics, but only partly predicts the subcellular distributionof phloem-mobile probes following unloading. The potential useof phloem-mobile fluorescent probes as physiological indicatorsis discussed. Key words: Arabidopsis, confocal laser scanning microscopy (CLSM), fluorescent probes, phloem transport, xenobiotic transport     

STUDIES ON THE ROOT OF HORDEUM VULGARE L.– ULTRASTRUCTURE OF THE SEMINAL ROOT WITH SPECIAL REFERENCE TO THE PHLOEM

Seminal root tissue of Hordeum vulgare L. var. Barsoy was fixed in glutaraldehyde and osmium tetroxide and studied with the light and electron microscopes. The roots consist of an epidermis, 6–7 layers of cortical cells, a uniseriate endodermis and a central vascular cylinder. Cytologically, the cortical and endodermal cells are similar except for the presence of tubular-like invaginations of the plasmalemma, especially near the plasmodesmata, in the former. The vascular cylinder consists of a uniseriate pericycle surrounding 6–9 phloem strands occurring on alternating radii with an equal number of xylem bundles. The center of the root contains a single, late maturing metaxylem vessel element. Each phloem strand consists of one protophloem sieve element, two companion cells and 1–3 metaphloem sieve elements. The protophloem element and companion cells are contiguous with the pericycle. Metaphloem sieve elements are contiguous with companion cells and are separated from tracheary elements by xylem parenchyma cells. The protoplasts of contiguous cells of the root are joined by various numbers of cytoplasmic connections. With the exception of the pore-plasmodesmata connections between sieve-tube members and parenchymatic elements, the plasmodesmata between various cell types are similar in structure. The distribution of plasmodesmata supports a symplastic pathway for organic solute unloading and transport from the phloem to the cortex. Based on the arrangement of cell types and plasmodesmatal frequencies between various cell types of the root, the major symplastic pathway from sieve elements to cortex appears to be via the companion and xylem parenchyma cells.     

Living sieve cells of conifers as visualized by confocal,laser-scanning fluorescence microscopy

Summary Confocal laser scanning microscopy (CLSM) and fluorochromes were used to visualize the assimilate-conducting sieve cells of conifers in vivo. When still nucleate, the cytoplasm of these cells shows streaming and occupies the cell periphery including the pitlike, thin wall regions where sieve areas would develop. During differentiation the nuclear fluorescence and the central vacuoles disappear. At maturity and after ER-specific staining the sieve areas are the most conspicuous character of sieve cells. Those linking two sieve cells are covered on either side with prominent amounts of ER, while those leading to a Strasburger (=albuminous) cell show fluorescence on the sieve-cell side only. Within the sieve-area wall fluorescence appears also in the common median cavity which is part of the symplastic path between sieve cells. Electron microscopy (EM) depicts the ER as complexes of densely convoluted tubules of smooth ER, equally on either side of a sieve area, provided that the fixation of this sensitive tissue is appropriate. Purposeful wounding causes a swelling and vesiculation of the ER-tubules which is visible in both CLSM and EM. Electron micrographs of ER-complexes at sieve areas -in this paper demonstrated in vivo -have often been argued to be artefacts, since they should raise flow resistance considerably and are not consistent with the Münch hypothesis on phloem transport. The implications of this location for phloem transport are discussed.Abbreviations CLSM confocal laser scanning microscopy - DiOC 3,3-dioxacarbocyanine iodide - EM electron microscopy - ER endoplasmic reticulum - FDA fluorescein diacetate     

Root Cell Ultrastructure in Developing Aerenchyma Tissue of Three Wetland Species

Patterns of root cortex cell development and ultrastructurewere analysed in Sagittaria lancifolia L., Thalia geniculataL. and Pontederia cordata L. using scanning and transmissionelectron microscopy (SEM, TEM). In all three species, cortexcells were arranged in radial columns extending from the endodermisto the hypodermis/epidermis. During gas space formation, thecortex cells elongated parallel to the root radius and shrankin the plane perpendicular to the radius leaving long and thinrows of cortex cells extending from the endodermis to the epidermis.Although the cortex cells appeared collapsed in tissue withwell-developed gas spaces, TEM revealed that the cortical cellsas well as the epidermal cells maintained intact membranes andmany normal organelles. Formation of root cortex tissue withwell-developed gas spaces does not require cell death in thesespecies. Living cortex cells in root tissue with mature gasspaces could provide a symplastic pathway for transport betweenthe root stele and the living epidermal cells. Copyright 2000Annals of Botany Company Sagittaria lancifolia, Thalia geniculata, Pontederia cordata, aerenchyma, root, wetland, development     

Gas and Liquids in Intercellular Spaces of Maize Roots

Oils are spontaneously absorbed by gas-filled intercellularspaces (IS) in maize root cortex. The network of these spacesin living root sections was imaged by confocal laser scanningmicroscopy using a fluorescent solution of Nile red in oil.The gas volume fraction (GVF) of root segments was quantifiedby the increase in weight (differentiated zones) or tissue density(2–3 mm root tips) due to complete vacuum infiltration.Cooling to 6 °C or inhibition of oxidative phosphorylationdiminished the GVF of root tips but did not significantly affectthe GVF of differentiated root zones. The threshold pressuredifference for measurable infiltration of isolated root segmentsis lower (10 to 15  kPa) than the threshold for infiltrationof comparable zones of attached roots or of detached roots withthe cut surface sealed (>60 kPa). In the absence of an opencut, pressure-driven infiltration of the root cortex is acceleratedby microscopic fissures within the epidermal/hypodermal barrier.The GVF of the root cortex was reduced after transferring rootsfrom sugar solutions (0.1 to 0.3M ) to water. This points toefficient water transport from the medium to sugar-containingcortical cell walls through epidermal and hypodermal protoplasts.When 2-cm-long primary roots were vacuum infiltrated in situand then allowed to grow on aerated mineral medium for a further5 d, cortical IS of the originally infiltrated root bases remainedfilled with liquid but the subsequently grown apical root zoneshad a normal GVF. Copyright 1999 Annals of Botany Company Apoplastic and protoplasmic route, maize, infiltration, intercellular spaces, oil absorption, confocal laser scanning microscope, water transport, Zea mays L.     

Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules

Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway.     

Symplastic continuity in Agrobacterium tumefaciens-induced tumours

The Agrobacterium tumefaciens-induced plant tumour is regarded as a strong sink, containing a well-developed vascular system that guarantees an efficient supply of water and nutrients from the host plant into the tumour. The phloem transport and unloading of the fluorescent dye carboxyfluorescein (CF) was studied to examine the potential pathways for unloading of a low-molecular-mass solute, and was compared with the symplastic movement of potato virus X expressing a green fluorescent protein-coat protein fusion (PVX.GFP-CP). The distribution of both CF and PVX.GFP-CP in the host plant, Nicotiana benthamiana, demonstrated a clear symplastic pathway between the phloem of the host stem and the cells of the tumour, and also a considerable capacity for subsequent cell-to-cell transport between tumour cells. This same pattern of CF transport was also demonstrated independently for the host species Cucurbita maxima and Ricinus communis. In addition to entering the tumour, CF and PVX both moved through the vascular rays of the host stem towards the stele. The results confirm that host and tumour tissues in the Agrobacterium gall are in direct symplastic continuity and emphasize an important symplastic pathway for radial solute transport in stems.Key words: Agrobacterium tumefaciens, carboxyfluorescein, GFP, symplastic phloem unloading, plant tumour, vascular rays      

The Behaviour of Two Cell Populations in the Pericycle of Allium cepa, Pisum sativum, and Daucus carota During Early Lateral Root Development

The length of cells of the pericycle, endodermis and middlecortex not actively involved in lateral root primordia (LRP)development was measured in primary roots of Allium cepa, Pisumsativum and Daucus carota. The presence of two cell populationsin the pericycle was demonstrated in all three species. In Alliumcepa and Pisum sativum, pericyclic cells located opposite xylempoles were significantly shorter than cells lying opposite phloempoles. In both species, LRP originated opposite xylem poles.Our results, furthermore, strongly suggest that in regions ofthe root far from the apical meristem, numerous pericyclic cellsundergo transverse division both previous to and during LRPinitiation, decreasing in mean length throughout this period.In Daucus carota, LRP begin to form in pericyclic cells locatednext to the phloem poles, such cells were significantly shorterthan those opposite xylem poles, even in areas of the primaryroot located close to the root tip. Cells also appear to dividetransversely in regions far from the root tip in this species,leading to a conspicuous drop in the mean length of those cellslocated in portions of the pericycle destined to give rise toLRP. Two different cell populations can also be distinguishedin the endodermis of Allium cepa and Pisum sativum, althoughobservations were less conclusive in Daucus carota. In all threespecies, length of cortical cells was unaffected by their positionopposite xylem or phloem poles Allium cepa, carrot, cell division, cell length, Daucus carota, endodermis, lateral root development, onion, pea, pericycle, Pisum sativum     

Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. I. Extent of transport through the coat symplast

The extent of post-phloem solute transport through the coatsymplasts of developing seeds of Vicia faba L. and Phaseolusvulgaris L. was evaluated. For Vicia seed coats, the membrane-impermeantfluorochrome, CF, moved radially from the chalazal vein to reachthe chlorenchyma and thin-walled parenchyma transfer cell layers.Thereafter, the fluorochrome moved laterally in these two celllayers around the entire circumference of the seed coat. Transferof CF from the chalazal vein was inhibited by plasmolysis ofattached ‘empty’ seed coats. In contrast, the spreadof phloem imported CF was restricted to the ground parenchymaof Phaseolus seed coats. Fluorochrome loaded into the outermostground parenchyma cell layer was rendered immobile followingplasmolysis of excised seed-coat halves. Phloem-imported [¹⁴C]sucroseand the slowly membrane permeable sugar, L-[¹⁴C]glucose, werepartitioned identically between the vascular and non-vascularregions of intact Vicia seed coats. For ¹⁴C-photosynthates,these partitioning patterns in attached ‘empty’Vicia seed coats were unaffected by PCMBS, but inhibited byplasmolysis. Tissue autoradiographs of intact Phaseolus seedcoats demonstrated that a pulse of ¹⁴C-photosynthate moved fromthe veins to the grounds tissues. In excised Vicia seed coats,preloaded with ¹⁴C-photosynthates, the cellular distributionof residual ¹⁴C-label was unaffected by PCMBS. In contrast,PCMBS caused the ¹⁴C-photosynthate levels to be elevated inthe veins and ground parenchyma relative to the branch parenchymaof Phaseolusseed coat halves. Based on the above findings, itis concluded that the phloem of Vicia seed coats is interconnectedto two major symplastic domains; one comprises the chlorenchyma,the other the thin-walled parenchyma plus thin-walled parenchymatransfer cells. For Phaseolusseed coats, the phloem forms amajor symplastic domain with the ground parenchyma. Key words: Phaseolus vulgaris L, phloem unloading, photosynthate transport, seed coat, symplast, Vicia faba L     

Root structure and cellular chloride,sodium and potassium distribution in salinized grapevines

X-ray microanalysis was used to study the patterns of K+, Na+ and Cl- accumulation in salinized (25 mm NaCl) and non-salinized grapevine (Vitis) roots. The aim was to determine whether NaCl affects patterns of Cl- accumulation differentially in the roots of a Cl--excluding genotype and a non-excluding genotype. Two regions of fibrous roots were analysed: (1) a region 2-3 mm basipetal to the root tip; and (2) a region of the root 10-12 mm basipetal to the root tip where the outermost layer is the hypodermis. The ion contents of the hypodermis, cortex, endodermis and pericycle vacuoles were analysed. Data were also collected from the cytoplasm of the endodermal and pericycle cells. The analyses showed that the ion profiles of the hypodermis and the endodermis were significantly different from those of the cortex and pericycle. The hypodermis and endodermis had higher K+ and lower Na+ and Cl- than surrounding cells. Some changes due to salinity such as increased K+ concentrations in the hypodermis were also noted. Chloride concentrations did not differ between the genotypes in the hypodermis, across the cortex or in the endodermis, but were higher in the pericycle of the excluder in comparison with the non-excluding genotype. However, K+/Na+ ratios of the cortex and endodermis were higher in the excluder. The pericycle cells exhibited the greatest ability to sequester Na+ and Cl- in vacuoles. Overall the data show cell-type-specific ion accumulation patterns and small but significant differences were found between genotypes. The possibility that these accumulation patterns arise from differences in uptake properties of cell types and/or result from the spatial distribution of the cell types along the competing symplastic and apoplastic ion transport pathways across the root is discussed.     

Frequencies of plasmodesmata in Allium cepa L. roots: implications for solute transport pathways.

Plasmodesmatal frequencies (PFs) were analysed in Allium cepa L. roots with a mature exodermis (100 mm from the tip). For all interfaces within the root, the numbers of plasmodesmata (PD) microm(-2) wall surface (Fw) were calculated from measurements of 60 walls on ultrathin sections. For tissues ranging from the epidermis up to the stelar parenchyma, the frequencies were also expressed as total PD numbers mm(-1) root length (Fn), which is most instructive for considering the radial transport of ions and photosynthates (because the tissues were arranged in concentric cylinders). The Fn values were constantly high at the interfaces of exodermis-central cortex, central cortex-endodermis and endodermis-pericycle (4.05x10(5), 5.13x10(5), and 5.64x10(5), respectively). If the plasmodesmata are functional, a considerable symplastic transport pathway exists between the exodermis and pericycle. Two interfaces had especially low PFs: epidermis-exodermis (Fn=8.96x10(4)) and pericycle-stelar parenchyma (Fn=6.44x10(4)). This suggests that there is significant membrane transport across the interface of epidermis-exodermis (through short cells) and direct transfer of ions from pericycle to protoxylem vessels. In the phloem, the highest PF was detected at the metaphloem sieve element-companion cell interface (Fw=0.42), and all other interfaces had much lower PFs (around 0.10). In the pericycle, the radial walls had a high PF (Fw=0.75), a feature that could permit lateral circulation of solutes, thus facilitating ion (inward) and photosynthate (outward) delivery.     

DISTRIBUTION AND RELATIONSHIP OF CELL DIVISION AND MATURATION EVENTS IN PISUM SATIVUM (FABACEAE) SEEDLING ROOTS

Pea roots have open apical organization, where discrete initial cells do not exist. Differentiation of all tissues occurs in cylinders and vascular sectors that blend gradually with each other. This study reports the distribution of dividing cells and their relationship to maturation events in the 2 mm root tip, and in the 8–10 and 18–20 mm segments. Up to 200 μm from the root body/cap junction, cell division is uniformly distributed throughout all meristem regions. By 350 to 500 μ, xylem tracheary elements and cells of the pith parenchyma and middle cortex have stopped dividing. At this level cell division is almost entirely restricted to two cylinders, one composed of the inner root cap, the epidermis, and the outer cortex (outer cortex cylinder) and another composed of cells of the inner cortex, the pericycle and vascular tissue (inner cortex cylinder). When the protophloem matures, all cells in the phloem sector of the inner cortex cylinder, including the 1 layered pericycle, the endodermis and the phloem parenchyma, stop dividing. The 3–4 layered pericycle in the xylem sectors continues dividing until about 10 mm from the body/cap junction following the maturation of the protoxylem tracheary elements.     

Studies on the longitudinal and lateral transport of IAA in the shoots of etiolated corn seedlings

The auxin, indole-3-acetic acid, and the symplastic probe, carboxyfluorescein diacetate, were applied to the cut mesocotyl base or coleoptile apex of etiolated Zea mays seedlings and their transport measured and tissue distribution determined. The longitudinal transport of indole-3-acetate was strongly basipolar, while that of carboxyfluorescein was essentially apolar. The longitudinal transport of IAA, like carboxyfluorescein, was mainly in the stele. IAA exhibited a much higher lateral mobility from stele to cortex than did carboxyfluorescein. Based on the calculation of moles probe/kg fw, IAA is 4 times more concentrated in the stele than in the cortex while CF is 24 times higher in concentration in the stele than in the cortex. The structure of the node and the mesocotyl regions just below the node, regions of maximum growth, were examined and plasmodesmatal structure and frequency in these regions determined. The plasmodesmatal frequency, about 3 per micrometer2, between all cell types of the mesocotyl was found to be about 5-8 fold higher than that found for the root. Hypotheses of lateral auxin transport are discussed.     

Phloem Loading and Transport of Endogenously or Exogenously Labelled Photo-Assimilate in Bean, Beet, Maize and Cucurbit

Transport of carbon-11 labelled photo-assimilate was monitoredin Phaseolus vulgaris. Beta vulgaris, Zea mays, and Cucwbitopepo. The region of leaf to be labelled was first abraded anda solution passed over it to gain access to its apoplast andmonitor changes in label therein. With PCMBS in the bathingsolution the rate of washout of label into the bathing solutionincreased, but the effect of phloem loading was very variablefor each species: on some occasions transport was hardly affected,on others it was halted. This was true even for Cucurbito pepo,where a symplastic pathway of loading has been widely acceptedand suggests that PCMBS affects symplastic transport or thatthere is an apoplastic step in Cucurbito pepo. Apoplastic pHhad little effect on transport or label washout unless a veryacid (pH 40) buffer was introduced, contrary to notions of hydrogenion co-transport for sugar uptake. Anoxia caused phloem loadingto decrease immediately and label washout to increase in allspecies. It is suggested that both symplastic and apoplasticpathways can operate in all species but that their proportionvaries according to species and ambient and/or growth conditions. Key words: Phloem loading, photo-assimilate transport     

巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因(编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001。将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP)标记的巴西固氮螺菌Yu62菌株。用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8d、12d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙。用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内。     

Induction of phloem unloading in Arabidopsis thaliana roots by the parasitic nematode Heterodera schachtii.

Phloem unloading of both the fluorescent probe carboxyfluorescein (CF) and 14C-labeled solutes was induced in Arabidopsis thaliana L. roots by the parasitic nematode Heterodera schachtii Schmidt. Confocal laser scanning microscopy demonstrated that anomalous unloading of CF from the sieve element companion cell complexes occurred specifically into the syncytium, the nematode-induced feeding structure located within the stele of the root. From this syncytial complex of modified root cells, both fluorescent and radioactive labels were withdrawn by feeding nematodes. Movement of CF was unidirectional from the phloem to the syncytium. A range of low-molecular-weight fluorescent probes (including CF) microinjected into the syncytium stayed in this structure, demonstrating that it is symplastically isolated from the surrounding root tissue. The mechanism of unloading in this host-pathogen relationship therefore appears to be apoplastic. Our results provide unequivocal evidence that sedentary cyst-forming nematodes have direct access to phloem-derived solutes.     

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H⁺-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H⁺-ATPase antibody clones, raised against the PM H⁺-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H⁺-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K⁺ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes. Received: 3 July 2000 / Accepted: 12 October 2000