The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia

The differentiation of phloem anastomoses linking the longitudinal vascular bundles has been studied in stem internodes of Cucurbita maxima Duchesne, C. pepo L. and Dahlia pinnata Cav. These anastomoses comprise naturally occurring regenerative sieve tubes which redifferentiate from interfascicular parenchyma cells in the young internodes. In all three species, severing a vascular bundle in a young internode resulted in regeneration of xylem to form a curved by-pass immediately around the wound. The numerous phloem anastomoses in these young internodes were not involved in this process, the regenerated vessels originating from interfascicular parenchyma alone. Conversely, in mature internodes of Dahlia, the regenerated vessels originated from initials of the interfascicular cambia, and their phloem anastomoses did not influence the pattern of xylogenesis. On the other hand, in old internodes of Cucurbita, in which an interfascicular cambium was not yet developed, the parenchyma cells between the bundles had lost the ability to redifferentiate into vessel elements, and instead, regenerated vessels were produced in the phloem anastomoses. Thus, the wounded region of the vascular bundle was not bypassed via the shortest, curved pathway, but by more circuitous routes further away from the wound. Some of the regenerated vessels produced in the phloem anastomoses were extremely wide, and presumably efficient conductors of water. It is proposed that the dense network of phloem anastomoses developed during evolution as a mechanism of adaptation to possible damage in mature internodes by providing flexible alternative pathways for efficient xylem regeneration in plants with limited or no interfascicular cambium.This paper is dedicated to the memory of the late Isaac Blachmann (deceased 19 November 1995), father-in-law of the senior author, for encouragement and advice throughout the yearsThis research was supported by an International Scientific Exchange Award to R.A. from the Israel Academy of Sciences and The Royal Society.     

THE TIME COURSE OF SIEVE TUBE AND VESSEL REGENERATION AND THEIR RELATION TO PHLOEM ANASTOMOSES IN MATURE INTERNODES OF COLEUS

Quantitative counts of regenerative sieve tubes and vessels were made in a large number of samples of mature internode #5 of C. blumei, with concomitant study of the fine details of vascular regeneration and the occurrence of the normally developing phloem anastomoses. Such anastomoses were found in many of the plants, but their average number in the small regenerating area was low (viz., 0.9 ± 0.2). With the phloem anastomoses excluded from the counts, the time course of regeneration was clear cut—no strands completed their regeneration around the wound until three days after wounding. More regenerative sieve tubes completed their differentiation under all conditions than did regenerative vessels. The number of sieve tubes and vessels regenerated by four days was closely related to the number of preexisting bundles of that type of vascular cell that had been severed by the transverse wound. The ratio of bundles severed by the wound in the phloem to those in the xylem was 2.14, and the ratio of the regenerative sieve tubes to the regenerative vessels was 2.24. For both tracheary and sieve tube cells the initial regeneration was strongly polar (mostly above the wound), as expected from earlier IAA transport data. The path of tracheary regeneration was obviously related to that of the sieve tubes on the other side of the cambium.     

Histochemical localization of nucleoside triphosphatase activity in assimilate conducting plant tissue

Summary For the histochemical localization of nucleoside triphosphatases at the electron microscopic level, prefixed tissues were incubated with lead nitrate in addition to substrate (GOMORI reaction). While ATP and UTP as substrates gave electron-dense reaction products at the plasmalemma of sieve tubes, companion cells and phloem parenchyma cells, and at plasmodesmata in primary pitfields, AMP gave reaction products only at the tonoplast of parenchyma cells. Since electron-dense deposits also occur in cell walls and vacuoles, energy dispersive X-ray microanalysis was used to distinguish between lead deposits and lead-phosphate deposits. The latter were restricted to the symplast. Among the three plant species used, the leaf bundle phloem ofHordeum distichon showed ATPase activity largely restricted to the phloem cells, except for the thickwalled sieve tubes. Some activity also bordered the chloroplasts of the bundle sheath cells. In the C₄ plantGomphrena globosa, ATPase and UTPase activities appeared to be the greater in phloem parenchyma cells than in sieve tubes. In the phloem of youngMonstera deliciosa roots, ATPase occurred not only at the plasmalemma of sieve tubes, but also around sieve-tube plastids. When compared with AMP as substrate, it appears that nucleoside triphosphates are the natural substrates of the enzyme(s) in the plasmalemma of sieve tubes and phloem parenchyma cells.     

Proliferating sieve elements present in bud phloem anastomoses connect sieve tubes of axillary bud traces to stelar vascular bundles in the aquatic monocotyledonPotamogeton natans L. (Potamogetonaceae)

Summary The stem ofPotamogeton natans is characterized by a central stelar vascular system with reduced xylem and abundant phloem. Wide sieve tubes composed of short sieve-tube members joined by simple sieve plates and associated with companion cells establish an effective conduit for assimilates. At each node the phloem forms a network of parallel sieve elements connecting the stem phloem to leaf and bud traces. InP. natans an axillary bud rarely develops into a side branch, its procambial vascular bundles are each connected to the nodal complex via separate anastomoses. Their most unusual components are the anastomosai sieve elements (ANSE), characterized by thin cell walls pitted all over by tiny callose-lined pores resembling plasmodesmata, which can be detected as bright areas by fluorescence microscopy after staining with aniline blue. Several layers of ANSE make up the centre of an anastomosis and link to both the nodal and bud stelar sieve tubes via mediating (MSE) and connecting sieve elements (CSE). The ultrastructural differentiation of ANSE, MSE, and CSE corresponds to that of normal sieve elements, i.e., in the mature stage they are enucleate, evacuolate, and have lost most of their cytoplasm. Their plastids are of form-P2c, containing many cuneate protein crystals, typical of monocotyledonous sieve elements. Quantitative aspects of the pore areas are discussed in relation to the functional significance of bud anastomoses.Abbreviations ANSE anastomosai sieve elements - CSE connecting sieve elements - FM fluorescence microscopy - LM light microscopy - MSE mediating sieve elements - TEM transmission electron microscopy Dedicated to Professor Dr. Rainer Kollmann on the occasion of his retirement     

Leaf vasculature in Zea mays L.

The vascular system of the Zea mays L. leaf consists of longitudinal strands interconnected by transverse bundles. In any given transverse section the longitudinal strands may be divided into three types of bundle according to size and structure: small, intermediate, large. Virtually all of the longitudinal strands intergrade structurally however, from one bundle type to another as they descend the leaf. For example, all of the strands having large-bundle anatomy appear distally as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. Only the large bundles and the intermediates that arise midway between them extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of longitudinal bundles at the base of the blade, both the total and mean cross-sectional areas of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Das Phloem der Haustorien vonCuscuta

Summary Haustoria ofCuscuta odorata R. & P. andC. grandiflora H.B.K. show continuous traces of sieve elements, connecting the phloem of the host with that of theCuscuta shoot. The continuity of this haustorial phloem is discernible by callose fluorescence after staining with aniline blue. The fine structural criteria for sieve tubes are analyzed electronmicroscopically, with special respect to sieve pores, P-protein, and a distinct wall-standing smooth surfaced ER. Within the central part of the haustorium sieve tubes are elongated, while the elements abutting the phloem of theCuscuta shoot are nearly isodiametric in shape. Both elements are associated with rather large companion cells, derived from an unequal division.

     

Ricerche Sull'infrastruttura del Coleottile di Avena

Abstract

Researches on ultrastructure of Avena coleoptile. 3. The sieve elements. — A study on the ultrastructural organization of the mature sieve elements of Avena coleoptile has been carried out. Data suggest that functional phloem tubes are alive and remain alive until they are working. Judging on morphological basis, the metabolic activity of sieve elements should be of peculiar type and low in comparison to that of the companion cells. In fact the cytoplasm is located in a narrow parietal strand, mitochondria, Golgi apparatus and endoplasmic reticulum are present, but they appear very modified; plastids and nucleus are absent. The cytoplasm is bounded externally by a normal plasmalemma, whilst the vacuole has no visible limits: a tonoplast is, therefore not identifiable.

The strands connecting the superimposed sieve elements with one another through the sieve plate result to be made by a double membrane system very similar to the endoplasmic reticulum, which we believe to realize cytoplasmic continuity between phloem tubes.

The data reported are more favorable to the existence in the sieve tubes of an active mechanism of translocation of organic solutes than a passive mass-flow.

The collaboration of companion cells in the translocation mechanism has been discussed.     

ANATOMICAL FEATURES OF METAPHLOEM IN STEMS OF SABAL,COCOS AND TWO OTHER PALMS

Sieve tubes in metaphloem of palm stems function throughout the life of the plant and merit close investigation. A stem of Sabal palmetto estimated to be 50 years old was sampled extensively. Variation in length of sieve-tube elements throughout this stem was measured and is discussed. In the metaphloem of individual vascular bundles companion cells are not sharply differentiated from other phloem parenchyma cells. Definitive callose deposits and slime are normally absent from mature sieve tubes, even in fixed material. Otherwise no conspicuous structural features which might account for the longevity of sieve tubes can be discerned. Occlusion of phloem strands after leaf fall is initially by callose deposition on sieve plates followed immediately by tylosoid formation. Similar sampling of Cocos nucifera, Washingtonia robusta and to a lesser extent Archontophoenix alexandrae confirmed these results except for quantitative differences.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.     

Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. ex Marsh.

Microautoradiography was used to follow the translocation pathways of ¹⁴C-labeled photosynthate from mature source leaves, through the stem, to immature sink leaves three nodes above. Translocation occurred in specific bundles of the midveins and petioles of both the source and sink leaves and in the interjacent internodes. When each of six major veins in the lamina of an exporting leaf was independently spot-fed ¹⁴CO₂, label was exported through specific bundles in the petiole associated with that vein. When the whole lamina of a mature source leaf was fed ¹⁴CO₂, export occurred through all bundles of the lamina, but acropetal export in the stem was confined to bundles serving certain immature sink leaves. Cross-transfer occurred within the stem via phloem bridges. Leaves approaching maturity translocated photosynthate bidirectionally in adjacent subsidiary bundles of the petiole. That is, petiolar bundles serving the lamina apex were exporting unlabeled photosynthate while those serving the lamina base were simultaneously importing labeled photosynthate. The petioles and midveins of maturing leaves were strong sinks for photosynthate, which was diverted from the export front to differentiating structural tissues. The data support the idea of bidirectional transport in adjacent bundles of the petiole and possibly in adjacent sieve tubes within an individual bundle.Abbreviations C central leaf trace - L left leaf trace - LPI leaf plastochron index - R right leaf trace     

POLARITY OF INDUCTION AND PATTERN OF PRIMARY PHLOEM FIBER DIFFERENTIATION IN COLEUS

The differentiation of primary phloem fibers was studied in Coleus blumei on a quantitative basis. The pattern of fiber differentiation in intact, untreated plants was found to be in the acropetal direction (from a mature internode to a young one). The youngest internodes to differentiate primary phloem fibers were those with cambial activity. In plants grown in the winter, fibers started to differentiate in internodes closer to leaf #2 than in spring-grown plants. A wound changes the pattern of fiber differentiation surrounding it. A wound in which the tissues above and below it were separated with parafilm, prevented fiber differentiation in the tissues directly below the wound, and caused more fiber differentiation in the tissues above and lateral to it. Under wounds with no parafilm separation, few or many fibers differentiated depending on the angle of the wound. The number of fibers under diagonal wounds was five to nine times more than under a horizontal wound. By excision experiments it was found that mature leaves were the source of induction of fiber differentiation. Leaves that produced induction caused fiber differentiation in the internode below them but did not cause fiber differentiation in the internode above. The induction, which can flow through a wound and cause fiber differentiation in at least two internodes below the source, is a polar induction in the basipetal direction (i.e., in the direction from the leaves to the root). Phloem fibers differentiated only in the vascular strands and not from the parenchyma cells between the strands. Therefore, they follow the new regenerative sieve and vessel elements in the pre-existing vascular strands, but do not follow them in their regeneration between the longitudinal strands.     

Complexity untwined: The structure and function of cucumber (Cucumis sativus L.) shoot phloem

Cucurbit phloem is complex, with large sieve tubes on both sides of the xylem (bicollateral phloem), and extrafascicular elements that form an intricate web linking the rest of the vasculature. Little is known of the physical interconnections between these networks or their functional specialization, largely because the extrafascicular phloem strands branch and turn at irregular angles. Here, export in the phloem from specific regions of the lamina of cucumber (Cucumis sativus L.) was mapped using carboxyfluorescein and ¹⁴C as mobile tracers. We also mapped vascular architecture by conventional microscopy and X-ray computed tomography using optimized whole-tissue staining procedures. Differential gene expression in the internal (IP) and external phloem (EP) was analyzed by laser-capture microdissection followed by RNA-sequencing. The vascular bundles of the lamina form a nexus at the petiole junction, emerging in a predictable pattern, each bundle conducting photoassimilate from a specific region of the blade. The vascular bundles of the stem interconnect at the node, facilitating lateral transport around the stem. Elements of the extrafascicular phloem traverse the stem and petiole obliquely, joining the IP and EP of adjacent bundles. Using pairwise comparisons and weighted gene coexpression network analysis, we found differences in gene expression patterns between the petiole and stem and between IP and EP, and we identified hub genes of tissue-specific modules. Genes related to transport were expressed primarily in the EP while those involved in cell differentiation and development as well as amino acid transport and metabolism were expressed mainly in the IP.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

Auxin promotes dormancy callose removal from the phloem ofMagnolia kobus and callose accumulation and earlywood vessel differentiation inQuercus robur

During winter, the phloem of the diffuse-porous tree magnolia (Magnolia kobus DC.) is dormant and is characterized by heavy deposits of dormancy callose. Application of 1-naphthaleneacetic acid (NAA) to either the top or the lower ends of excised dormant branches before bud break resulted in the removal of the dormancy callose from the sieve tubes. In both intact and auxintreated branches, callose degradation occurred first in the recently formed sieve tubes. There was no new vessel differentiation in magnolia before bud break. In contrast, the sieve tubes of the ring-porous oak (Quercus robur L.), which possess massive dormancy callose deposits during winter, were almost callose-free just before bud break. Application of auxin to the top of excised branches before bud break resulted in callose accumulation on the most recently formed sieve tubes. The first earlywood vessels were evident in oak before bud break, and their numbers were increased by auxin application. The early development of phloem and xylem (before bud break) in ring-porous species is an ecological adaptation which prepares the vascular system of these trees to function immediately at the beginning of their growing season which is relatively short.     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.     

The Function of Phloem Connections in Regenerating In Vitro-Grafts*

In order to answer the question whether functioning phloem connections exist between graft partners, phloem transport has been studied in cultured explant-grafts after application of ¹⁴C-sucrose and carboxyfluorescein (CF) to the scion. Autografts of Lycopersicon esculentum and Helianthus annuus were investigated at various regeneration periods. Ungrafted internodes served as controls. A segmental analysis was used to determine the tissue distribution of ¹⁴C-sucrose in a graft. The ¹⁴C-profiles obtained show that sucrose translocation across the graft interface started 4 days after grafting and increased later. The observed translocation appears to occur via wound phloem, since at this time the first complete wound-phloem bridges (shown as files of aniline-blue-positive sieve plates) traverse the graft interface. In 7-d-old autografts, sucrose transport across the graft interface returned to normal again, as indicated by the distribution of the label. In addition, ¹⁴C-profiles reveal accumulation of label in sink tissues. Here the basal callus of the stock, and temporarily the graft union itself, represent the main sinks for labelled sucrose. Translocation of CF was analyzed in hand sections of the grafts. The beginning of translocation into the stock was confirmed with the dye. Moreover, effective phloem translocation across the graft interface, visualized with CF, could undoubtedly be assigned to wound-phloem bridges reconnecting the cut vascular bundles of scion and stock. Thus, the function of phloem connections in regenerated in vitro-grafts is directly shown.     

Leaf vasculature in sugarcane (Saccharum officinarum L.)

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Polarity of IAA Effect on Sieve-Tube and Xylem Regeneration in Coleus and Tomato Stems

A technique is described for the processing of regenerated xylem and sieve tubes from the same wound area for microscopic and quantitative comparison.

Regeneration was examined in internodes of 2 developmental stages in Coleus: internode 2, elongating, characteristic of primary growth; and internode 5, non-elongating, characteristic of secondary growth.

Transport of indoleacetic acid (IAA) in excised number 5 internodes of Coleus is strictly polar, in a basipetal direction, judging by a regeneration bioassay involving both sieve tube strands and xylem cells. Similar results were obtained with tomato.

If isolated number 5 Coleus internodes are not treated with hormone, they regenerate no xylem cells and a small number of sieve tube strands. With increasing concentrations of IAA added apically, the number of regenerated sieve tube strands (and, with higher concentrations, of xylem cells) increases progressively up to 1% IAA, the highest concentration applied.

Internode 2 of Coleus regenerates fewer xylem cells or sieve tube strands than internode 5, whether on the otherwise intact plant or with a given concentration of IAA added apically. The amount of regenerated xylem increases with added apical IAA, except that the highest concentration gives no further increase. The number of xylem cells regenerated in intact plants occurs at the same interpolated IAA concentration as in number 5 internodes. No concentration of IAA tried provided replacement of intact number of sieve tube strands in internode 2.

IAA can exert a regenerative stimulus on both xylem and sieve tubes in the area immediately adjacent to the site of its application.

     

Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata

Rice (Oryza sativa L.) phloem sieve tubes contain RPP13-1, a thioredoxin h protein that moves around the plant via the translocation stream. Such phloem-mobile proteins are thought to be synthesized in the companion cells prior to being transferred, through plasmodesmata, to the enucleate sieve-tube members. In this study, in-situ hybridization experiments confirmed that expression of RPP13-1 is restricted to companion cells within the mature phloem. To test the hypothesis that RPP13-1 enters the sieve tube, via plasmodesmata, recombinant RPP13-1 was expressed in Escherichia coli, extracted, purified and fluorescently labeled with fluorescein isothiocyanate (FITC) for use in microinjection experiments into tobacco (Nicotiana tabacum L.) mesophyll cells. The FITC-RPP13-1 moved from the injected cell into surrounding cells, whereas the E. coli thioredoxin, an evolutionary homolog of RPP13-1, when similarly labeled and injected, failed to move in this same experimental system. In addition, co-injection of RPP13-1 and FITC-dextrans established that RPP13-1 can induce an increase in plasmodesmal size exclusion limit to a value greater than 9.4 but less than 20 kDa. Nine mutant forms of RPP13-1 were constructed and tested for their capacity to move from cell to cell; two such mutants were found to be incapable of movement. Crystal-structure prediction studies were performed on wild-type and mutant RPP13-1 to identify the location of structural motifs required for protein trafficking through plasmodesmata. These studies are discussed with respect to plasmodesmal-mediated transport of macromolecules within the companion cell-sieve tube complex. Received: 6 June 1997 / Accepted 25 June 1997