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.     

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.     

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.     

ORIGIN AND EARLY DEVELOPMENT OF NONARTICULATED LATICIFERS IN EMBRYOS OF EUPHORBIA MARGINATA

In E. marginata 12 nonarticulated laticifer initials arise in the cotyledonary node of the young embryo during the early heart stage. The initials arise progressively in the developing embryo, the first laticifers differentiating simultaneously with or shortly before the elements of the pro-cambium. The laticifers occupy a position lateral to the six procambial strands which are formed in the embryo. Upon subsequent growth each laticifer becomes vacuolated and nuclear division unaccompanied by cytokinesis results in the formation of a coenocytic protoplast. The enlarging laticifer produces several branches, one growing into the cotyledon, another growing down along the hypocotyl penetrating toward the root meristem, and one or several growing along intercellular spaces of adjacent cells. No fusion of these branches with one another or adjoining parenchyma cells was observed.     

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.     

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.     

Die Milch macht's – auch bei Pflanzen. Milchröhren und Milchsaft

Laticifers and latex Many different plants have laticifers and produce latex. Latex flows out when the plant is injured and coagulates quickly thus protecting the wound. It is a mixture of vacuoles and cytoplasm and contains various secondary plant products, especially polyterpenes (usually in the form of latex particles) and often toxins. The diversity of the latex corresponds with the diversity of laticifers. Nonarticulated laticifers are long, single cells, articulated ones are derived from cell fusions. Both kinds of laticifers may be branched. Reputed/ill‐reputed is the latex of Papaver somniferum, the source of opium. Technically very important has become the rubber of Hevea brasiliensis, after vulcanization of polyterpenes had been invented.     

Cellulase in latex and its possible significance in cell differentiation

Summary Cellulase was found to be present in the latex of species with articulated laticifers but it could not be detected in the latex of species with nonarticulated laticifers. It is suggested that cellulase is involved in the removal of end walls during the differentiation of articulated laticifers.     

Histochemical study of detailed laticifer structure and rubber biosynthesis-related protein localization in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using spectral confocal laser scanning microscopy

In Hevea brasiliensis, laticifers produce and accumulate rubber particles. Despite observation using histochemical methods, development stage structure and structures with ceasing functions have rarely been described. Spectral confocal laser scanning microscopy with Nile red staining simplifies laticifer structure observation in tangential sections while enhancing the resolution. Laticifer and ray images were extracted from unmixed images and used to monitor changes during growth. A laticifer network structure developed from increased anastomoses between adjoining laticifers outside of the conducting phloem, but because of increased radial division and growth of rays, the network structure ruptured and disintegrated. We also investigated immunohistochemical localization of two rubber particle-associated proteins in the laticifers: small rubber particle protein (SRPP) and rubber elongation factor (REF). Mature bark test results show that SRPP is localized only in the laticifer layers in the conducting phloem; REF is localized in all laticifer layers. Because SRPP plays a positive role in rubber biosynthesis, results show that the rubber biosynthesis capability of laticifers is concentrated where rays and the sieve tube actively transport metabolites.     

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.     

TYPES OF LATICIFERS AND CRYSTALS IN JATROPHA AND THEIR TAXONOMIC IMPLICATIONS

Anatomical examination of 37 species of Jatropha (Euphorbiaceae) has revealed the occurrence of two distinct types of laticifers—articulated, and nonarticulated—in addition to idioblasts that are irregularly shaped individual cells. With the notable exception of J. augustii Pax & K. Hoffm. (section Peltatae (Pax) Dehgan & Webster), these idioblasts occur in the two most advanced sections of the subgenus Curcas (Adans.) Pax, namely Loureira (Cav.) Muell. Arg. ex Pax and Mozinna (Ortega) Pax. The presence of two laticifer types and “idioblastic laticifers” in the genus, in association with the morphological reduction series found with evolutionary advancement are, therefore, significant in the delimitation of sections and subsections. Further evidence is presented by the occurrence of “chambered crystalliferous cells” in subgenus Curcas, but not in subgenus Jatropha (=Adenorhopium Griseb.). Reexamination of laticifers in other genera of the Euphorbiaceae is suggested as a possible means of alleviating the long-standing taxonomic dilemma of this large and morphologically diverse family.     

Laticifers and the classification of Euphorbia: the chemotaxonomy of Euphorbia esula L.

Laticifers and the classification of Euphorbia: the chemotaxonomy of Euphorbia esula L. Articulated and non-articulated laticifer cells represent distinctive cell types of relatively recent origin and occur in only a few families. Both types are of separate phylogenetic origin, reflecting independent evolutionary trends in the Euphorbiaceae. Supra-generic groupings of this family can be segregated into three taxonomic units using the laticifer character; with either articulated laticifers, non-articulated laticifers, or no laticifers. Such units may reflect more natural assemblages than now represented in the classification of this family. Laticifers possess chemical and morphological features of potential application as taxonomic characters to aid in delimiting species and interpreting evolutionary trends. The triterpenoid profile from latex of Euphorbia species has been shown to be diagnostic for a taxon. The qualitative and quantitative composition show a high level of stability under diverse environmental and physiological conditions indicating a genetic basis for triterpenoid synthesis. Triterpenoid profiles of known accessions of European E. esula L. and related presumptive taxa from North America readily separated them into distinctive chemotaxa that include one for E. esula L., whereas morphological features were found inadequate for separating accessions to presumptive taxa. Identification of adventive spurges in North America requires diagnostic analyses of Eurasian leafy spurges for comparison. Laticifer characters used in conjunction with relevant morphological features will provide a broadened insight into phylogenetic relationships with the Euphorbiaceae.     

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。     

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.     

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.     

Laticifers in Euphorbiaceae–a conspectus

Laticifers in Euphorbiaceae–a conspectus. Despite the multitude of references on laticifers in Euphorbiaceae, much of the work concentrated on economically important, and often highly specialized genera, particularly Euphorbia and Hevea, the evolution of the laticifer in the family is poorly understood. In this paper, laticifers, in a representative sample of genera of Euphorbiaceae, are investigated with respect to morphology and distribution. This information is supplemented with a review of existing literature. It seems probable that the articulated and non-articulated types are not as divergent as commonly supposed, since both may undergo intrusive growth, although more ontogenetic studies are needed to clarify the relationship between them.     

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.     

ULTRASTRUCTURE OF DEVELOPING AND MATURE NONARTICULATED LATICIFERS IN THE MILKWEED ASCLEPIAS SYRIACA L. (ASCLEPIADACEAE)

The ultrastructure of developing and mature nonarticulated laticifers in Asclepias syriaca L. (the common milkweed) was studied by conventional fixation and staining techniques and by osmium impregnation techniques. The mature laticifer protoplast in A. syriaca possesses a large central vacuole with an intact vacuolar membrane. Formation of this vacuole apparently results from dilation and subsequent enlargement of endoplasmic reticulum and possibly in part by fusion of smaller vacuoles and limited cellular-lytic autophagy. Widespread digestion or autophagy of cytoplasm within vacuoles is not evident. Nuclei, mitochondria, dictyosomes, and small vesicles are the most prominent components distributed in the peripheral cytoplasm. Plastids appear to degenerate as the laticifer matures. The specialized cellular component, latex, which is the vacuolar content of the laticifer, is interpreted to be produced in the cytoplasm and subsequently incorporated into the large central vacuole. Rubber globules, the most prominent latex component, are surrounded by a membrane that does not have a trilaminate structure. Globules are associated with an electron-dense fibrillar component in the vacuole.     

Histochemical and immunohistochemical identification of laticifer cells in callus cultures derived from anthers of Hevea brasiliensis

Laticifers are highly specialized cells present in over 20 plant families. They are well defined in planta. In vitro development of laticifers was also observed in some plants, but uncertain in the callus cultures of rubber tree, one of the most economically important latex producing plants. In the present study, we provide evidence that laticifer cells present in the callus cultures of rubber tree by histochemical and immunohistochemical studies. They present in the callus mainly as separate non-elongated form, a novel morphology different from the morphology of laticifer cells in planta, excluding their origin from explants. The occurring frequency of laticifer cells in the callus was genotype-dependent and negatively correlated with the somatic embryogenetic ability, suggesting that the presence of laticifer cells in the callus inhibit somatic embryogenesis in tissue culture of rubber tree. The genotypes PR107, RRIM600, Reyan8-79, and Reyan7-33-97 with lower embryogenetic ability compared to Haiken 2 had more laticifer cells, and laticifer clusters were only observed in these genotypes.     

Ultrastructure and Development of the Laticifers of Ficus carica L

Laticifers of Ficus caricaL. are of the branched, non-articulatedtype. They occur in the cortex and pith of the plant axis andpenetrate leaves and inflorescences. Observations were madeon laticifers located in shoot apices. Growing regions of laticifers undergo a sequence of ultrastructuralchanges. These are: (a) a pronounced increase in the vacuolarspace which divides the cytoplasm into separated masses; (b)a development of numerous vesicular structures in the cytoplasm.The vesicular structures are released into the vacuolar space.The whole process is accompanied by disintegration of cytoplasm.Apparently isolated masses of cytoplasm occur in the luminaof laticifer tips in sections taken from dormant apices. Itis assumed that these masses have a role in the initiation ofnew laticifer regions in the next growing season. Ficus caricaL., laticifers, ultrastructure, development differentiation