EMBRYOGENY AND HISTOGENESIS IN NERIUM OLEANDER II. ORIGIN AND DEVELOPMENT OF THE NON-ARTICULATED LATICIFER

Mahlberg , P. G. (U. Pittsburgh, Pittsburgh, Pa.) Embryogeny and histogenesis in Nerium oleander. II. Origin and development of the non-articulated Iaticifer. Amer. Jour. Bot. 48(1): 90–99. Illus. 1961.—Laticifer initials, collectively considered as a laticifer system, are differentiated in the globular embryo from meristematic cells which occupy a position within the potential procambial tissue. A total of usually 28 initials, in Nerium oleander, arise as an irregular ring of cells directly below the embryonic shoot apex, during initiation of the cotyledonary primordia. No anastomoses occur between laticifer initials. During subsequent development of the embryo, the laticifer initials grow in a bi-directional manner and penetrate into the root, cotyledons and toward the shoot apex. Upon enlargement the initials bifurcate repeatedly, many branches penetrate into the cotyledons, others grow into the cortex of the hypocotyl or penetrate between cells of the procambium. Repeated nuclear divisions within each initial result in the formation of a multinucleated protoplast in this cell type. The tips of laticifers occupy intercellular spaces during their growth; they do not penetrate into or through adjacent cells. A plexus of laticifer branches is formed within the cotyledonary node of the mature embryo. No new initials are formed during subsequent growth of the plant, rather certain branches from the cotyledonary nodal plexus penetrate into the enlarging shoot system. The nature of their growth habit and branching suggests that the tips of laticifer initials exhibit an intrusive form of growth.     

MITOSIS IN THE NON-ARTICULATED LATICIFER OF EUPHORBIA MARGINATA

The multinucleate condition in the non-articulated laticifers of embryos of Euphorbia marginata arises as a result of mitosis. Successive stages of mitosis in the nuclei of the laticifer appear in the form of a wave. No sequence of mitotic stages has been noticed in the neighboring longitudinal tiers of cells. This difference in the mitotic pattern in the laticifer and other parenchymatous cells of the embryo suggests that the synthesis of factor(s) responsible for triggering mitosis occurs within the laticifer and does not diffuse to the surrounding cells. The mitotic waves originate distally from the meristems, either in the cotyledonary or hypocotyl portion of the laticifer, and move uni- or bidirectionally along its longitudinal axis. The mitotic stimulus does not start simultaneously in all the laticifers. The variable velocity of the mitotic substance results in aphasic mitotic waves in laticifers of the same embryo. Mitotic aberrations have not been observed in the dividing nuclei of the laticifer. A chromosome estimation made from a polar view of metaphase does not suggest polyploidization in the observed laticifers.     

STRUCTURE AND ONTOGENY OF LATICIFERS IN CICHORIUM INTYBUS (COMPOSITAE)

The branched anastomosed laticifer system in the primary body of Cichorium intybus L. originates in embryos from files of laticiferous members at the boundary between phloic procambium and ground meristem. Upon seed germination, laticiferous members develop perforations in the end walls which become entirely resorbed. Perforations also develop in the longitudinal walls of contiguous laticiferous members and from lateral connections between developing laticifer branches. Additional laticiferous members originate as procambium differentiation proceeds, and their differentiation follows a continuous acropetal sequence in leaf primordia of the plumule. In roots, laticifers closely associated with sieve tubes in the secondary phloem originate from derivatives of fusiform initials in the vascular cambium. These laticifers develop wall perforations and in a mature condition resemble laticifers in the primary body. As the girth of the root increases, laticifers toward the periphery, unlike associated sieve tubes, resist crushing and obliteration. Laticifers vary in width from about 4 to 22 μm; the widest ones occur in involucral bracts and the narrowest ones in florets. There was no evidence that intrusive growth occurs during development of the laticifer system, although such growth may occur during development of occasional branches which extend through ground tissue independent of phloem and terminate in contact with the epidermis. Presence of amorphous callose deposits is related to aging of laticifers and mechanical injury.     

ONTOGENY AND CYTOCHEMISTRY OF ALKALOIDAL VESICLES IN LATICIFERS OF PAPAVER SOMNIFERUM L. (PAPAVERACEAE)

Development of alkaloidal vesicles in laticifers of opium poppy, Papaver somniferum L., was investigated at the ultrastructural level. Laticifer initials possessed abundant endoplasmic reticulum throughout their dense cytoplasm. During differentiation the endoplasmic reticulum organized into long, folded sheets that were parallel to the longitudinal walls along the periphery of the cell. Vesicles appeared to be derived from dilation of endoplasmic reticulum. This relationship was confirmed through cytochemical data obtained with zinc iodide-osmium tetroxide and osmium tetroxide impregnation. Alkaloidal vesicles had electron-dense regions or caps that occurred early in laticifer differentiation, but these caps became less conspicuous in mature cells. Caps appeared to be derived from small particles which condensed along the inner surface of the vesicle membrane and subsequently accumulated at one or two positions along the membrane of the vesicle.     

ULTRASTRUCTURE OF NON-ARTICULATED LATICIFERS IN MATURE EMBRYOS AND SEEDLINGS OF ASCLEPIAS SYRIACA L. (ASCLEPIADACEAE)

The protoplast of the non-articulated branched laticifer in the embryo and seedling of Asclepias syriaca L. was studied at the ultrastructural level and was found to differ from that of adjacent cell types. Embryonal laticifers possess numerous vesicles with electron-dense contents, but lack a large organized central vacuole. Plastids have few lamellae, possess phytoferritin, and accumulate small amounts of starch. Other organelles and membrane systems are similar to those in other cells. After germination, laticifers develop numerous elongated vacuoles by dilation of endoplasmic reticulum. Nuclei in laticifers within the hypocotyl of seedlings are highly lobed and possess dilated perinuclear spaces. Plastids and other organelles are similar to those observed in the protoplast of laticifers in the embryo. The latex or rubber component of the laticifer is not apparent in mature embryos of 72-hr seedlings.     

LATICIFER ULTRASTRUCTURE AND DIFFERENTIATION IN SEEDLINGS OF PAPAVER BRACTEATUM LINDL., POPULATION ARYA II (PAPAVERACEAE)

Laticifers of Papaver bracteatum Lindl., population Arya II, seedlings were examined by electron microscopy. Laticifers were first differentiated in procambium of the radicle associated with phloem about 72 hr after seeds were sown. Proliferation of membrane-bound vesicles of apparent endoplasmic reticulum origin distinguished laticifers from adjacent cells. Vesicles developed electron-dense caps from the internal condensation of small particles. Laticifer initials possessed the usual complement of organelles that became obscured in mature cells by the large, closely packed vesicles. Plastids contained an electron-dense, membrane-bound inclusion, but never developed lamellae or starch grains. Articulation and anastomoses between laticifer elements resulted from gradual removal of wall materials by both cells on opposite sides of the common walls at a perforation site. Differentiation of the laticifer initials and the micromorphology of the protoplast of P. bracteatum is similar to that reported for P. somniferum.     

Investigations of Laticifer Differentiation in Tissue Cultures Derived from Asclepias syriaca L.

Callus cultures of Asclepias syriaca were established from stemexplants and grown in tissue culture. The culture medium onwhich the callus was grown was modified to produce either planfletsof superficial origin on the callus or embryoids which wereanalyzed to determine whether laticifers differentiated in thesestructures. Mature zygotic embryos and adult plants of A. syriacanormally possess a well-developed network of intrusively-growingnon-articulated branched laticifers that arise only once duringplant develop ment from initials differentiated in the youngheart stage embryo. Embryoids were derived from two differentculture media. These embryoids were observed to lack laticifers,although they were similar in their morphology in other respectsto zygotic embryos. Plantlets of superficial origin were formedon each of the media employed in this study. These plantletswere observed to possess laticifers that resemble those in normalshoots. Embryoids and induced shoots represent experimentalsystems in which it may be possible to control for the firsttime the differentiation of the laticifer as a cell type instructures similar to those present in the normal plant.     

FOSSIL LATICIFERS FROM EOCENE BROWN COAL DEPOSITS OF THE GEISELTAL

Thick mats of cellular remains from Eocene brown coal deposits of the Geiseltal near Halle, DDR, were determined to be fossil nonarticulated laticifers. Nuclear magnetic resonance analyses of intact strands showed they consisted of eis-1,4-configuration rubber representing the polymerized isoprenoid contents of individual laticifers. Only remains of laticifers are present; other cells are absent as a result of biodegradation. The long laticifers, often with a surrounding cell wall, retained a tubular shape during their preservation. The isoprenoid content, which filled the entire lumen, possessed a cribriform structural character. The interstices within the rubber represent areas of former protoplasm of the cell. Various configurations in the protoplasm molded by the rubber during the initial phase of fossilization appear as negative images of former nuclei, organelles, and possibly membrane surfaces. The laticifer axes possess branches of several configurations comparable in morphology to those in branched, nonarticulated laticifers in extant plants. Acetone extracts of the rubber contents analyzed by gas-liquid chromatography identified the presence of several hydrocarbons which form a characteristic profile for the laticifer. It is suggested that the distinctive cellular micromorphology, rubber configuration, and hydrocarbon profile of these laticifers can be employed as markers in comparative studies with extant plants to identify the generic or species origin of these laticifers.     

PLASTIDS IN LATICIFERS OF PAPAVER. I. DEVELOPMENT AND CYTOCHEMISTRY OF LATICIFER PLASTIDS IN P. SOMNIFERUM L. (PAPAVERACEAE)

Plastids were observed in all stages of laticifer differentiation in Papaver somniferum L. Plastids in laticifer initials were present as proplastids that later developed electron-dense inclusions, but never possessed the thylakoids or starch grains that characterize chloroplasts in other cells. Electron-dense inclusions in laticifer plastids were membrane-bound and appeared to arise from the accumulation of material within an invagination of the inner plastid membrane. Cytochemical studies of these plastid inclusions indicated that their matrix was not composed of crystalline protein, α-amylose, amylopectin or polysaccharide. The results suggest that the electron-dense, membrane-bound inclusions in laticifer plastids may be composed of lipoprotein.     

Laticifers: An historical perspective

This review describes the development of the laticifer concept, with emphasis upon the nonarticulated type, from early observations of plant exudates and “juices” to the presentation of laticifers by Esau (1953). Classical writers and herbalists described practical applications of these substances. With the advent of the microscope early investigators believed that these substances occurred in structures present in most, if not all, plants and, wrongly, equated these structures to the circulatory system in animals. Introduction of the term, latex, into botany derived from its early use as a term for a blood component by physicians, and not for analogy to milk. However, the origin of the terms, laticifer and laticiferous, remains uncertain. Initial studies of laticifers were marked by the controversy of whether they represented intercellular spaces or elongated cells. Confirmation of their cellular character led to the designation of nonarticulated and articulated laticifers. Nonarticulated laticifers were shown to arise during early embryogeny in some plants. The ontogenetic origin of the articulated laticifer was unclear to early workers, but new laticifers were detected to be formed by cambium activity. Nonarticulated laticifers were described to develop by intrusive growth whereby tips of the cell penetrated between adjacent cells. The coenocytic condition of the nonarticulated laticifer resulted from nuclear divisions along the cell positioned in the growth region of the shoot and the subsequent distribution of the daughter nuclei along the length of the cell.     

Ultrastructure and development of nonarticulated laticifers in seedlings ofEuphorbia maculata L.

The ultrastructure of nonarticulated laticifers in the seedlings ofEuphorbia maculata was studied at various developmental stages. The apical regions of the seedling laticifers growing intrusively contained large nuclei with mainly euchromatin and dense cytoplasm possessing various and many organelles such as rich ribosomes, several small vacuoles, giant mitochondria with dense matrices, rough endoplasmic reticulum, dictyosomes, and proplastids. This result suggested that the apical regions of laticifers were metabolically very active. Laticifers in seedlings at the first-leaf developmental stage did not contain latex particle. In seedlings at second-leaf growth stage, the laticifer cells contained numerous and elongated small vacuoles. These vacuoles appeared to arise by dilation of the endoplasmic reticulum and frequently possessed osmiophilic or electron-dense latex particles. The small vacuoles fused with the large vacuole occupying the central portion of the subapical region of laticifers, and then the latex particles were released into the large central vacuole. The latex particles varied in size and were lightly or darkly stained. Proplastids with a dense matrix and a few osmiophilic plastoglobuli were filled with an elongated starch grain and thus were transformed into amyloplasts. Latex particles were initially produced in the laticifers after seedlings had developed their second young leaves. In seedlings at forth-leaf stage, latex particles with an alveolated rim were found in the laticifers.     

THE STRUCTURE AND DEVELOPMENT OF AN UNUSUAL TYPE OF ARTICULATED LATICIFER IN MAMMILLARIA (CACTACEAE)

Although the laticifers of several species of Mammillaria can technically be classified as being of the articulated type, they differ significantly from all other reported articulated laticifers. They are derived from cells which differentiate only in older tissues, never in meristematic or young regions. The development involves the complete lysis of masses of cells, not just the perforation or resorption of the end walls in a single file of cells. At maturity, the laticifer lumen is lined with a one-to-several layered epithelium which may be quite thick. The laticifers increase in diameter with age, apparently by the lysis of the inner epithelial cells. Laticifers occur in the pith, cortex and tubercles of the vegetative body but were not observed in the roots, flower parts or in seedlings up to eight months old. Seven species were studied, all of which have “milky sap.” and the laticifers of each were virtually identical to the laticifers of the others.     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.     

夹竹桃科2种引种植物分泌结构的解剖学研究

为了解夹竹桃科(Apocynaceae)植物乳汁管的发生发育,对爱之蔓(Ceropegia woodii)和百万心(Dischidia ruscifolia)营养器官中的分泌结构进行了显微观察。结果表明,爱之蔓和百万心营养器官中均有无节分枝乳汁管的分布,茎皮层中的乳汁管大部分具有明显的分枝,叶中乳汁管具明显分枝,分布与走向多与叶脉维管组织平行。另外,爱之蔓营养器官中的分泌结构除乳汁管外,还有分泌腔。这为夹竹桃科植物的系统分类研究提供了解剖学依据。     

CYTOCHEMICAL LOCALIZATION OF CELLULASE ACTIVITY IN ARTICULATED,ANASTOMOSING LATICIFERS OF PAPAVER SOMNIFERUM L. (PAPAVERACEAE)

Cellulase activity was localized at the ultrastructural level in the articulated, anastomosing laticifers of Papaver somniferum. Electron-dense crystalline deposits indicating the presence of cellulase activity were restricted to discrete patches along the laticifer wall in regions of recently formed perforations. This report presents the first direct evidence for the involvement of cellulases in the cell wall perforation process in articulated laticifers.     

DEVELOPMENTAL ANATOMY IN ROOTS OF IPOMOEA PURPUREA. I. RADICLE AND PRIMARY ROOT

The developmental anatomy of the primary root of Ipomoea purpurea was studied at several growth stages, beginning with the radicle. The radicle is generally composed of three superimposed tiers of initials, which produce the vascular cylinder, cortex, and columella; and a peripheral band of lateral rootcap-epidermal initials. The radicular cortex contains 16–19 immature laticifers; none of the tissue regions in the radicle contains mature cells. Following germination and during the first 2–3 cm growth of the primary root the apical meristem and its derivative tissues undergo a series of modifications. Root apical diameter decreases as cells in lateral portions of the rootcap elongate; meanwhile, the columella enlarges vertically. The relationship between cortical and columellar initials changes as fewer mitoses occur in the former while the latter remain active. In longer roots the columellar initials are directly in contact with the vascular initials. Cortical size diminishes during early root growth as cortical laticifers and their associated cells cease to be produced by the outer cortical initials and ground meristem. Early procambium, at the level of vascular pattern initiation, decreases in diameter by cellular reorientation, and the vascular cylinder decreases in overall diameter although the tetrarch pattern remains unchanged.     

Immunolocalization of beta-D-glucans,pectins, and arabinogalactan-proteins during intrusive growth and elongation of nonarticulated laticifers in Asclepias speciosa Torr

Nonarticulated laticifers are latex-containing cells that elongate indefinitely and grow intrusively between the walls of meristematic cells. To identify biochemical mechanisms involved in the growth of nonarticulated laticifers, we have analyzed the distribution of various polysaccharides and proteoglycans in walls of meristematic cells in contact with laticifers, nonadjacent to laticifers, and in laticifer walls. In the shoot apex of Asclepias speciosa, the levels of callose and a (1-->4)-beta-galactan epitope are lower in meristematic walls in contact with laticifers than in nonadjacent walls. In contrast, we did not detect a decline in xyloglucan, homogalacturonan, and arabinogalactan-protein epitopes upon contact of meristematic cells with laticifers. Laticifer elongation is also associated with the development of a homogalacturonan-rich middle lamella between laticifers and their neighboring cells. Furthermore, laticifers lay down walls that differ from those of their surrounding cells. This is particularly evident for epitopes in rhamnogalacturonan I. A (1-->5)-alpha-arabinan epitope in this pectin is more abundant in laticifers than meristematic cells, while the opposite is observed for a (1-->4)-beta-galactan epitope. Also, different cell wall components exhibit distinct distribution patterns within laticifer walls. The (1-->5)-alpha-arabinan epitope is distributed throughout the laticifer walls while certain homogalacturonan and arabinogalactan-protein epitopes are preferentially located in particular regions of laticifer walls. Taken together, our results indicate that laticifer penetration causes changes in the walls of meristematic cells and that there are differences in wall composition within laticifer walls and between laticifers and their surrounding cells.     

Cytochemical localization of pectinase activity in laticifers ofNerium oleander L.

Summary A method is described for the cytochemical localization of pectinase activity at the ultrastructural level. The procedure involves the use of Benedict's reagent to form an electron-dense copper precipitate when reacted with reducing sugars liberated from exogenously supplied pectin. Using this technique, pectinase activity was examined in the nonarticulated, branched laticifers ofNerium oleander. Electron opaque crystalline deposits indicating the presence of pectolytic enzymes were identified in laticifer central vacuoles. Smaller amounts of reaction product were distributed along the middle lamella between laticifers and adjacent cells. This report represents the first direct evidence for the involvement of pectinase in intrusive growth of nonarticulated laticifers.     

FURTHER STUDIES OF THE UNUSUAL TYPE OF LATICIFEROUS CANALS IN MAMMILLARIA (CACTACEAE): STRUCTURE AND DEVELOPMENT OF THE SEMI-MILKY TYPE

The development and structure of the laticifers in several species of the section Subhydrochylus of the genus Mammillaria (Cactaceae) were examined. These laticifers were found to be similar to those of the section Mammillaria in that both types develop from the complete lysis of several rows of parenchyma cells, and both types consist of long, branching, tubular lumens which are lined by epithelia. The laticifers of the section Subhydrochylus differ from those of the section Mammillaria in that those of the former are more irregular in shape, lumen development, and epithelium form. Also, the Subhydrochylus laticifers occur only as a single ring in the outermost cortex and tubercle bases, whereas those of section Mammillaria can be found in pith, medullary rays, cortex and throughout the tubercles. Because the structure of the laticifers in the section Mammillaria is much more regular and orderly, it is postulated that they are the derived type and that the laticifers of the section Subhydrochylus more closely resemble the ancestral condition. Two species, M. elegans and M. tegelbergiana, were found to be intermediate in nature, having characteristics of both types of laticifer systems. Solista pectinata was found to have laticifers similar to those in section Subhydrochylus.