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.     

Analysis of early processes in wound-induced vascular regeneration using TED3 and ZeHB3 as molecular markers

Interruption of the vascular bundles of Zinnia internodes induced transdifferentiation of cells into tracheary elements (TEs) or sieve elements (SEs) within 4 d of wounding. The early stage of the regeneration processes was analyzed using two molecular marker genes, TED3 and ZeHB3, which are expressed specifically in TE precursor cells and immature phloem cells, respectively. An increase in the numbers of TED3 and ZeHB3 mRNA-expressing cells always preceded an increase in the numbers of TEs and SEs formed. The earliest sign of vascular differentiation was the appearance 24 h after wounding of a layer(s) of TED3 mRNA-expressing cells in the inter- and intrafascicular cambial-like regions along the severed vascular bundles. In contrast, the number of ZeHB3 mRNA-expressing cells decreased dramatically along the severed bundles 24 h after wounding, and increased again 36 h after wounding. These results clearly indicate that xylem and phloem differentiation are not synchronized during vascular regeneration. Treatment with 10(-3) M colchicine abolished the expression of ZeHB3 mRNA in pith parenchyma, but not TED3 mRNA; this suggests that cell division is a prerequisite for the transdifferentiation of pith parenchymal cells into immature phloem cells expressing ZeHB3. In contrast, transdifferentiation of pith parenchymal cells to TE precursor cells does not require preceding cell division. However, the inhibition of cell division prevented the formation of both radial files of TEs and the cambial-like layer(s) of TED3 mRNA-expressing cells, and, ultimately, vascular regeneration altogether. These results imply that wound-induced cambial-like activity in and between severed vascular bundles is essential for vascular regeneration.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): QUANTITATIVE ASPECTS,AND SOLUTE CONCENTRATIONS OF THE SIEVE-TUBE MEMBERS

The vascular system of the leaf of Populus deltoides Bartr. ex Marsh, was examined quantitatively, and plasmolytic studies were carried out to determine the solute concentrations of sieve-tube members at various locations in the leaf. Both the total number and total crosssectional area of each cell type decreases with decreasing vein size. Although the proportion of phloem occupied by sieve tubes varies considerably from location to location, a linear relationship exists between cross-sectional area of the vascular bundles and both total and mean cross-sectional area of sieve tubes. Collectively, the cross-sectional area of all tertiary and minor veins feeding into a secondary exceeds the total cross-sectional area of sieve tubes at the base of that secondary. Moreover, the total volume of sieve tubes in the “catchment area” of a secondary vein is much greater than the total sieve tube volume of the secondary itself. Both tracheary elements and sieve-tube members undergo a reduction in both total and mean crosssectional area in the constricted zone at the base of the leaf. The plasmolytic studies revealed the presence of positive concentration gradients in sieve tubes of the lamina from the minor veins and tips of the secondaries to the bases of the secondaries and their associated subjacent midvein bundles and from the upper to lower portions of the median bundle of the midvein.     

The functional significance of phloem anastomoses in stems ofDahlia pinnata Cav.

The role of phloem anastomoses in translocation was studied experimentally in intact and wounded internodes ofDahlia pinnata Cav. Translocation was visualized with fluorescein, a fluorescent dye capable of movement in the phloem. Translocation was analyzed in large areas of living phloem tissue which were peeled from the xylem at the cambium region. Under normal conditions, fluorescein was observed in sieve tubes of the longitudinal phloem strands but very rarely in the sieve tubes of the anastomoses. However, when a few longitudinal strands were severed, fluorescein was translocated through the anastomoses located around the wound within 24 h. It is suggested that the phloem anastomoses in mature internodes ofDahlia serve mainly as an emergency system which enable a fast response to damage by providing alternative pathways for assimilates around the stem. A possible regulatory mechanism based on differences in resistance to flow in longitudinal versus lateral sieve tubes is discussed. This study was supported by an International Scientific Exchange award and an operating grant to C.A.P. from the Natural Sciences and Engineering Research Council of Canada.     

A THREE-DIMENSIONAL RECONSTRUCTION OF THE VASCULAR SYSTEM TO THE LODICULES,ANDROECIUM, AND GYNOECIUM OF A FERTILE FLORET OF PANICUM DICHOTOMIFLORUM (GRAMINEAE)

A three-dimensional reconstruction of a fertile floret stele of Panicum dichotomiflorum approaching anthesis was made by a new technique using superimposition of tracings of 80, 1-μm thick serial sections, cleared tracing film, and mounting adhesive. From a collateral bundle, which also served as the median trace to the fertile lemma, most of the vascular tissue branched adaxially and horizontally to become the sole vascular supply to the two lodicules, three stamens, and pistil. The xylem branched at a low level to form a broad and long vessel plexus. The phloem branched at a higher level to overlay the vessel plexus on the right and left with an arc of horizontal sieve tubes in a phloem plexus. Those sieve tubes and vessels which rose after branching from the horizontal plexi assumed a more vertical course in the floret stele. Traces to the right and left lodicules arose from the lower abaxial portions of the flanks of the floret stele. Vessels ascended vertically from the xylem plexus and passed through the phloem plexi and joined with the sieve tubes there to exit at the same level and become the right and left lodicule traces. The vascular tissues to the three filament traces arose from different higher levels of the stele. The sieve tubes for the median filament trace arose vertically from the abaxial side between but above the lodicule traces. At higher levels the sieve tubes for the lateral filaments rose from the horizontal arcs of the flanks of the stele and departed it tangentially. The vessels destined to the filament traces arose in the center of the floret stele from adaxial portions of the horizontal plexus, ascended between the arcs of phloem, exited the stele simultaneously above the phloem of the traces, and followed the courses of their respective sieve tubes. The adaxially displaced apex of the floret stele then contained the vascular tissue related to the pistil. All the sieve tubes and vessels of the floret stele were embedded in a matrix of intermediary cells. The peripheral intermediary cells associated with the vessel plexus were xylem transfer cells with pronounced wall ingrowths. At higher levels in the floret stele, intermediary cells in scattered locations near sieve tubes or vessels had less conspicuous wall ingrowths. No preferred orientation of transfer cells with any particular trace was noted.     

Wound-induced and naturally occurring regenerative differentiation of xylem in Zea mays L.

The regenerative differentiation of xylem, both around a wound in the stem and at the root junction was studied in seedlings of maize. The regeneration of vessels around a wound was very small, being limited to the very young internodes and sharply declining basipetally. There were more regenerative vessel elements and they differentiated faster above the wound than below it. The regenerative vessel elements around the wound were characterized by helical or annular pattern of secondary wall thickenings. Wounding also resulted in the development of additional vascular anastomoses in the leaf immediately above the wound, and in differentiation of discontinuous vessels in adjacent bundles. Regenerative vessel elements were very common where the adventitious roots connected with the stem internodes, and exhibited pitted or reticulated secondary wall thickenings.     

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.     

Ultrastructure of and plasmodesmatal frequency in mature leaves of sugarcane

Vascular bundles and contiguous tissues of leaf blades of sugarcane (Saccharum interspecific hybrid L62–96) were examined with light and transmission electron microscopes to determine their cellular composition and the frequency of plasmodesmata between the various cell combinations. The large vascular bundles typically are surrounded by two bundle sheaths, an outer chlorenchymatous bundle sheath and an inner mestome sheath. In addition to a chlorenchymatous bundle sheath, a partial mestome sheath borders the phloem of the intermediate vascular bundles, and at least some mestome-sheath cells border the phloem of the small vascular bundles. Both the walls of the chlorenchymatous bundlesheath cells and of the mestome-sheath cells possess suberin lamellae. The phloem of all small and intermediate vascular bundles contains both thick- and thin-walled sieve tubes. Only the thin-walled sieve tubes have companion cells, with which they are united symplastically by pore-plasmodesmata connections. Plasmodesmata are abundant at the Kranz mesophyll-cell-bundlesheath-cell interface associated with all sized bundles. Plasmodesmata are also abundant at the bundle-sheathcell-vascular-parenchyma-cell, vascular-parenchyma-cellvascular-parenchyma-cell, and mestome-sheath-cell-vascular-parenchyma-cell interfaces in small and intermediate bundles. The thin-walled sieve tubes and companion cells of the large vascular bundles are symplastically isolated from all other cell types of the leaf. The same condition is essentially present in the sieve-tube-companion-cell complexes of the small and intermediate vascular bundles. Although few plasmodesmata connect either the thin-walled sieve tubes or their companion cells to the mestome sheath of small and intermediate bundles, plasmodesmata are somewhat more numerous between the companion cells and vascular-parenchyma cells. The thick-walled sieve tubes are united with vascular-parenchyma cells by pore-plasmodesmata connections. The vascular-parenchyma cells, in turn, have numerous plasmodesmatal connections with the bundle-sheath cells.This study was supported by National Science Foundation grants DCB 87-01116 and DCB 90-01759 to R.F.E. and a University of Wisconsin-Madison Dean's Fellowship to K. R.-B. We also thank Claudia Lipke and Kandis Elliot for photographic and artistic assistance, respectively.     

The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 μg/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 μg/mL and 20 μg/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 μg/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.     

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.     

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.     

大麦浆片结构及其在开花过程中的变化

浆片由表皮、基本组织和维管束三部分组成。表皮上不具气孔,细胞外壁角质化。维管束为有限外韧型,呈散生状分布。浆片中管束数与其所含导管数因品种（系）而异,可育系多于不育系。大维管束由数个导管、筛管及伴胞和维管束薄壁细胞组成,且其维管束薄壁细胞壁厚、核大、质浓,线粒体丰富,中、小维管束一般不含导管。     

LEAF VASCULATURE IN BARLEY,HORDEUM VULGARE (POACEAE)

The vascular system of the Hordeum vulgare L. leaf consists of multiple longitudinal strands interconnected by transverse bundles. In any transverse section, the longitudinal strands can be categorized into three bundle types: small, intermediate, and large. Individual longitudinal strands intergrade structurally from one bundle type into another as they descend the leaf. At their distal ends, they have the anatomy of a small bundle. As they descend the leaf, most intergrade into intermediate bundle and then into large bundle types. All strands with large bundle anatomy extend basipetally into the stem. Typically, the other longitudinal strands, which do not intergrade structurally into large bundles, do not enter the sheath, but fuse with other longitudinal strands above the junction of the blade with the sheath. Despite the decrease in number of longitudinal bundles entering the sheath, an increase takes place in the total crosssectional area of sieve tubes and tracheary elements. A linear relationship exists between leaf width and total bundle number in the blade but not in the sheath. Moreover, a linear relationship exists between cross-sectional area of vascular bundles and both total and mean cross-sectional area of tracheary elements and thin-walled sieve tubes.     

VASCULAR SYSTEM OF LODICULES OF DACTYLIS GLOMERATA L.

The vascular system for the two lodicules in a floret of Dactylis glomerata L. was studied in serial sections. The floret stele contained a few modified tracheary elements and xylem transfer cells enveloped by a phloem of squat sieve-tube members and intermediary cells. A single sieve tube and associated phloem parenchyma exited the right and left sides of the stele and upon nearing the base of each lodicule branched and formed the minor veins of the lodicule. The minor veins underwent limited branching and anastomosing to form a small three-dimensional system which described an arc during its ascent in the adaxial portion of each lodicule. The sieve tubes in the minor veins extended halfway up the lodicule and contained short sieve-tube members with transverse, slightly oblique, or lateral simple sieve plates. The associated phloem parenchyma cells were intermediary cells, companion cells, and less intimate parenchyma cells. Intermediary cells terminated the minor veins and touched the distal ends of the terminal sieve-tube members, which lacked distal sieve plates. Although the transverse area of the sieve-tube members remained constant up the lodicule, the transverse area of the associated phloem parenchyma fluctuated.     

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.     

The development of the tuber in seedlings of five species of Dioscorea from Nigeria

Tubers in all five species develop from the hypocotyl region of the seedlingS. A perivascular cambium arises cutting off mainly starch-storing parenchyma and collateral vascular bundles to the inside. A phellogen gives rise to cork on the outside. Between the two cambial layers there may or may not be layers of parenchyma, not storing starch but containing raphides. The vascular bundles consist of xylem with vessels, scalariform tracheids and parenchyma; and phloem, with sieve tubes and parenchyma.     

Structurally,phloem unloading in the maize leaf cannot be symplastic

Developing longitudinal vascular bundles of the leaf blades of maize (Zea mays L. cv. W273) were examined with the transmission electron microscope to determine the frequency of plasmodesmata between the sieve tubes and their neighboring cells. Of particular interest were the protophloem sieve tubes, the first sieve tubes to mature in importing (all large and some intermediate) bundles. The protophloem sieve tubes, most of which lack companion cells, intergrade structurally with the thin-walled metaphloem sieve tubes. Both the protophloem sieve tubes and the thin-walled metaphloem sieve tubes and their companion cells (the sieve tube-companion cell complexes) are virtually isolated symplastically from the rest of the leaf, precluding a symplastic mechanism of phloem unloading in the leaf blade of maize.     

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     

浙贝母鳞片衰退和物质撤退的超微结构观察

本文以浙贝母衰退鳞片为材料,观察了细胞内含物降解和运输的过程。细胞内含物降解时,内膜系统产生许多囊泡,这些囊泡有降解和运输两方面的作用。降解产物在细胞中表现为颗粒和丝状物形式,它们在细胞间转移通过共质体和质外体两条途径,转移方式多种多样．降解产物经过细胞间转移,最终汇集到维管束,再通过维管束运往新生器官。转移细胞在物质运往筛分子的过程中起着重要作用。韧皮部是降解产物运输的主要通道,导管的一部分可能也参与了这种运输．     

The three-dimensional structure of vascular tissues in Agrobacterium tumefaciens-induced crown galls and in the host stems of Ricinus communis L.

The three-dimensional pattern of phloem and xylem in 10-d-to two-month-old tumors induced by Agrobacterium tumefaciens (C58) and in adjacent Ricinus communis L. stem tissues was studied in thick sections by clearing with lactic acid and by staining with lacmoid. The crown galls contained two types of vascular strands: treelike branched bundles, which developed towards the tumor surface in fast-growing regions, and globular bundles in the slowly developing parts. Both types of vascular bundles contained xylem and phloem and were continuous with the vascular system of the host plant. The tumor bundles were interconnected by a dense net of phloem anastomoses, consisting of sieve tubes but no vessels. These vascular patterns reflect the apparent synthesis sites, concentration gradients and flow pathways of the plant hormones additionally produced in the tumors upon expression of the T-DNA-encoded genes. The A. tumefaciens-induced crown gall affected vascular differentiation in the host stem. In the basipetal direction, the tumor induced more xylem differentiation directly below it, where the crown-gall bundles joined the vascular system of the host. In the centripetal direction, the crown gall caused the development of pathologic xylem characterized by narrow vessels, giant rays and absence of fibers. On the other hand, most probably as a consequence of its gibberellic acid content, the host plant stimulated a local differentiation of regenerative phloem and xylem fibers with unique ramifications, only at the base of the tumor. However, fibers were absent from the main body of the crown gall. The study shows that A. tumefaciens-induced crown galls are characterized by a sophisticated network of vascular tissues in the tumor and are accompanied by a perturbated vessel system in the host. The hormonal mechanisms controlling vascular differentiation in the tumor and neighboring host tissues are discussed. In addition, the gall constriction hypothesis is proposed for explaining the mechanism which gives priority in water supply to the growing gall over the host shoot.We thank Dr. Zs. Koncz (Max-Planck-Institut für Züchtungsforschung, Köln, Germany) for Agrobacterium strains and the Deutsche Forschungsgemeinschaft (SFB 199) for financial support to C.I.U.