EVOLUTION OF THE LATICIFER IN EUPHORBIA AS INTERPRETED FROM STARCH GRAIN MORPHOLOGY

The morphology of plastid starch grains from several succulent and nonsucculent species of Euphorbia was examined in parenchyma and in non-articulated laticifiers. Several classes of grains were identified: small oblong-round, rod, somewhat osteoid, osteoid, lobed osteoid, discoid, and round grains. Parenchyma possessed only small oblong-round grains, whereas grains of different morphology were present in laticifers. Different species of this genus can be characterized by the morphology of the large, or mature, starch grains present in the laticifer. The rod-shaped grain, which was somewhat wider at the midregion than at the ends, was present in several nonsucculent forms, E. terracina, E. pulcherrima and E. heterophylla. The somewhat osteoid grain was represented by the succulent species E. viguieri, E. milii, and E. mauritanica, where the mature grains developed somewhat enlarged ends. Grains with much enlarged ends were represented in the succulent species of E. abyssinica, E. pseudocactus, and E. tirucalli. Alteration of the pattern of starch deposition in which several lobes were formed at the ends of the grain has given rise to a lobed osteoid class (E. inconstantia). Lobes also may be formed with greater frequency along the midregion of the grain in this than in other species. Euphorbia lactea had the most complex grain in which lobing was frequent at the ends as well as along the midregion, resulting in a large discoid grain. Subclasses in which grains differed significantly in length between species were evident in all classes containing several species. The average length of grains in any subclass was similar for subclasses between the classes. The study suggests that the elongated grain of the laticifer was derived from the round or oblong grain present in the more primitive parenchymatous cell. Progressive changes in the pattern of starch deposition have given rise to osteoid and discoid grains of increasing morphological complexity which is interpreted to represent trends in laticifer evolution between different species of Euphorbia as reflected by subtle changes for starch deposition within the plastids of this cell.     

A COMPARISON OF ALPHA-AMYLASES FROM THE LATEX OF THREE SELECTED SPECIES OF EUPHORBIA (EUPHORBIACEAE)

A technique for the partial purification of α-amylases from latex of Euphorbia heterophylla, E. marginata, and E. tirucalli is described. The enzymes were found to be similar to other higher plant amylases using the criteria of molecular weight, pH characteristics, kinetics, number of isozymes, and blue value-reducing value patterns. Carbohydrases other than α-amylases were not detectable in latex. The amylases were employed to examine their capacity to digest latex starch grains which are common components of the laticiferous cell in this genus. Laticifer starch grains are not susceptible to in vitro amylolysis. Removal of the starch grain membrane with Triton X-100, damaging the grain, or treating the grains with α-amylases from diverse biological sources had little effect upon hydrolysis. Grains incubated with pullulanase followed by α-amylase caused a slight but significant increase in hydrolysis of raw laticifer starch grains. These studies indicate that the nonarticulated laticifer in Euphorbia is a cul de sac for certain primary and secondary metabolic products and that the indigestible and morphologically complex starch grains in the latex have evolved to function in a secondary role within the laticifer.     

CHARACTERIZATION OF STARCH GRAINS IN THE NONARTICULATED LATICIFER OF EUPHORBIA PULCHERRIMA (POINSETTIA)

Starch grain morphology in laticifer amyloplasts of Euphorbia pulcherrima Willd. (poinsettia) was examined for evidence of starch metabolism in vegetative and flowering plants. Laticifer starch grains in vegetative plants were rod shaped with lengths ranging from 3 to 60 μm. Average grain size was significantly larger in stems than leaves, and in older than younger tissues. Starch grain length frequency was unimodal and approximated a normal probability distribution in stems, but was skewed positively toward smaller grains in leaves. Frequency distributions were shifted toward larger grains in older tissues. Under short-day photoperiod (flowering) conditions, round starch grains formed in latex of stems, and the average length of rod-shaped grains decreased in latex of stems and leaves. Round grains did not occur in laticifers of leaves or bracts. Round starch grains often occurred in aggregates of two or more subunits. Changes in size and shape of latex starch grains indicate that amyloplasts in fully differentiated laticifers metabolize starch. Identification of metabolically active amyloplasts in differentiated laticifers suggests that the function of these organelles may involve starch mobilization under certain physiological conditions.     

LATEX AND LATICIFER STARCH CONTENT OF DEVELOPING LEAVES OF EUPHORBIA PULCHERRIMA

Laticifer starch accumulation was compared to laticifer growth for developing leaves of Euphorbia pulcherrima Willd. (poinsettia). Measurements of the laticifer-specific triterpenol, cycloartenol, in latex and in whole leaf extracts were used to calculate the total latex volume in leaves of different developmental stages. Latex volume and starch concentration in the latex were used to determine total laticifer starch and to compare laticifer growth and starch synthesis. Young leaves contained the highest latex and laticifer starch contents on dry wt and leaf area bases. In older expanding leaves, laticifer growth produced an increase in total latex volume accompanied by an increase in total laticifer starch. Laticifer growth and starch accumulation stopped upon cessation of leaf expansion. Starch concentration was similar in latex from all leaves, but differed between plant organs. Thus, laticifer starch accumulation correlated with laticifer growth, but mobilization of the starch out of the laticifer was not observed in old or senescent leaves. This is evidence that laticifer starch grains function within the laticifer independently of degradation or export to other cell types.     

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.     

Differentiation of Non-articulated Laticifers in Poinsettia (Euphorbia pulcherrima Willd.)

Differentiation of non-articulated laticifers in poinsettia(Euphorbia pulcherrima Willd.) was studied ultra-structurally.Growing laticifers show: (1) a multinucleate apical region containingabundant ribosomes but few other differentiated organelles and(2) a sub-apical zone where the cytoplasm is dominated by vacuolesof diverse morphology with latex particles. These particlesappear first within narrow tubular vacuoles developed especiallyin the peripheral cytoplasm. During vacuolation of the laticifer,portions of cytoplasm, including some of the nuclei, becomeisolated by the enlarging and fusing vacuoles; eventually thesebecome lysed, except the latex particles which remain in thecentral vacuole. During differentiation of a laticifer branch,the cytoplasm contains the usual organelles, including a fewmicrobodies and coated vesicles. The plastids that lie withinthe peripheral cytoplasm differentiate into amyloplasts witha single elongated starch grain. Towards the end of differentiationthe cytoplasm becomes restricted to a thin parietal layer, withthe remaining organelles reduced or degenerate, surroundinga central vacuole filled with latex particles. Euphorbia pulcherrima Willd, poinsettia, ultrastructure, differentiation, laticifers     

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.     

Subcellular fractionation of triterpenoid biosynthesis in Euphorbia lathyris latex

Latex isolated from laticifer cells of Euphorbia lathyris maintained its ability to synthesize triterpenols (and their esters) from acetate. When the latex was centrifugated at 5 000 g for 15 min. this biosynthetic activity could be subdivided into two separate fractions; the acetate to β-hydroxymethyl glutaryl-coenzyme A activity remained in the supernatant, while the β-hydroxymethyl glutaryl-coenzyme A to triterpenol activity was pelleted. Further purification of the pellet by isopycnic centrifugation on Percoll gradients yielded at least three particles: latex particles, starch grains, and a single membrane-bound organelle. Electron micrographs were made of all of these latex particles. The single membrane-bound organelle was only observed in the region of the density gradient that exhibited the ability to incorporate mevalonic acid into the triterpenoids. In addition, the enzyme β-hydroxymethyl glutaryl-coenzyme A reductase (EC 1.1.1.34) was found in the 5000 g pellet, while β-hydroxymethyl glutaryl-coenzyme A lyase (EC 4.1.3.4) remained in the supernatant.     

A palynotaxonomic study of the genus Filipendula (Rosaceae)

Pollen grains from 15 species (18 taxa) of the genus Filipendula were examined with light and scan-ning electron microscopy. It was revealed that the pollen grains are isopolar, tricolporate, with scabrate or scabrate-microechinate surface. The pollen morphology was compared with the conventional classification sys-tems of the genus by different authors, and supported Shimizu's system (1961), in which the genus was divided into three subgenera. The monotypic subgen. Hypogyna is characterized by pollen lacking fastigium and thickened costae colpi. The other monotypic subgen. Filipendula differs from others by pollen having larger grain, larger pore size, longitudinally elliptic fastigium and thickened costae colpi. The largest subgen. Ulmaria is distinguished by pollen having rounded or latitudinally elliptic fastigium and thickened costae colpi. Sectional classification was not supported by the pollen morphology due to insufficient variability.     

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.     

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.     

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.     

No Latex Starch Utilization in Euphorbia esula L

Utilization of leaf, stem, root, and latex starch was monitored in Euphorbia esula L. plants. Leaf, stem, and root starch decreased rapidly during a 52 day light starvation period while latex starch did not. Scanning electron and light microscope studies provided additional evidence that no changes in latex starch granules had occurred. Amylase activity (6.6 units per milligram protein) could be isolated from latex. However, latex starch granules were extremely resistant to enzymic hydrolysis by latex amylases, Bacillus subtilis α-amylase, and by amyloglucosidase from Aspergillus niger. Results indicate that latex starch grains do not function as utilizable carbohydrate in this species under these conditions.     

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.     

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.     

Distribution and Organization of Non-articulated Laticifers in Mature Tissues of Poinsettia (Euphorbia pulcherrima Willd.)

The distribution and cytological organization of non-articulatedbranched laticifers in the mature root, stem, and leaf tissuesof poinsettia. Euphorbia pulcherrima Willd., were studied bythe use of optical and electron microscopy. The laticifers occurin all parts of the plant body, being well represented in certainparenchymatous tissues and the phloem. The mature region ofthe laticifer has a living protoplast showing a thin parietalcytoplasm, bounded by plasmalemma and a tonoplast, which enclosesa large continuous central vacuole containing the milky latexfluid. The protoplast is multinucleate and possesses large amyloplasts,each enclosing a single elongated starch grain. Sparseness andpoor differentiation of the other components of the protoplast,mostly mitochondria, ribosomes and endoplasmic reticulum, suggestlow metabolism in the mature region of the laticifer. The latexof the central vacuole is dominated by spherical particles,0.3–1 µm in diameter, each with a dense matrix andan eccentric core of lighter staining material. In some laticifersthe latex particles fuse into coagulated masses. Euphorbia pulcherrima Willd, poinsettia, laticifers, ultrastructure, cytology     

Accumulation of non-utilizable starch in laticifers of Euphorbia heterophylla and E. myrsinites

Starch biosynthesis and degradation was studied in seedlings and mature plants of Euphorbia heterophylla L. and E. myrsinites L. Mature embryos, which lack starch grains in the non-articulated laticifers, develop into seedlings that accumulate starch rapidly when grown either in the light or the dark. Starch accumulation in laticifers of dark-grown seedlings was ca. 47 and 43% of total starch in light-grown controls in E. heterophylla and E. myrsinites, respectively. In light-grown seedlings, starch was present in laticifers as well as parenchyma of stems and leaves, whereas in dark-grown seedlings starch synthesis was almost exclusively limited to laticifers. In 7-month-old plants placed into total darkness, the starch in chyma was depleted within 6 d, whereas starch in laticifers was not mobilized. The starch content of latex in plants during development of floral primordia, flowering, and subsequent fruit formation remained rather constant. The results indicate that laticifers in seedlings divert embryonal storage reserves to synthesize starch even under stress conditions (darkness) in contrast to other cells, and that starch accumulated in laticifers does not serve as a metabolic reserve. The laticifer in Euphorbia functions in the accumulation and storage of secondary metabolites yet retains the capacity to produce, but not utilize starch, a primary metabolite.     

PECTINASE IN ASCLEPIAS LATEX AND ITS POSSIBLE ROLE IN LATICIFER GROWTH AND DEVELOPMENT

A pectinase with a pH optimum of 5.2 is present in the latex of the common milkweed, Asclepias syriaca L. The enzyme was partially purified from the serum fraction of fresh latex by dialysis and ammonium sulfate fractionation. Enzyme activity was detected by a viscometry assay and by the dinitrosalicylic acid assay for reducing sugars. Pectin and polygalacturonic acid could serve as substrates for the enzyme. Pectolytic activity in latex presents a basis for describing the development of the non-articulated branched laticifer system. Enzyme activity may facilitate intrusive tip growth of the laticifer among other cells by solubilizing pectic substances of the middle lamella and also may be important for loosening wall material of the laticifer itself to facilitate extension growth.     

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.