Histological changes during maturation in male and female cones of the cycad Zamia furfuracea and their significance in relation to pollination biology

The cycad Zamia furfuracea L.Fil. Is pollinated by a curculionid beetle, Rhopalotria mollis Sharp which completes its life cycle in male cones of the cycad, and effectively pollinates female coneS. Idioblasts within parenchyma in both male and female cones appear to contain toxic compounds, including at least one neurotoxin, 2-amino-3-(methylamino) propanoic acid (BMAA), and a toxic glycoside, methylazoxymethanol-/!-primeveroside (macrozamin). Idioblasts appear structurally unmodified in male cones throughout the period of pollen maturation, and feeding weevils consume much of the starch-rich microsporophyll parenchyma tissue, including idioblastS. During this activity no appreciable change in morphology or staining reactions of male-cone idioblasts is detectable. Prior to pollen receptivity, female-cone idioblasts resemble those of male cones. Thereafter, many female-cone idioblasts show marked changes in morphology and content not caused by the weevils themselveS. Idioblast changes in female cones are probably associated with the defence of female-cone resources against predation by animals, including pollinating weevils, and may relate to mobilization of toxinS. Absence of similar morphological changes in male-cone idioblasts is correlated with toxin sequestration, enabling the pollinator to breed and feed without intoxication.     

Biosynthesis of L-ascorbic acid and conversion of carbons 1 and 2 of L-ascorbic acid to oxalic acid occurs within individual calcium oxalate crystal idioblasts

L-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various (14)C-labeled compounds and examined by micro-autoradiography for incorporation of (14)C into calcium oxalate crystals. [(14)C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-(14)C]AsA also gave heavy labeling of crystals, whereas [6-(14)C]AsA gave no labeling. Labeled precursors of AsA (L-[1-(14)C]galactose; D-[1-(14)C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, D-[1-(14)C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > L-galactose > D-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via D-mannose and L-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments.     

Two foliar idioblasts of taxonomic significance in Cercidium and Parkinsonia (Leguminosae: Caesalpinioideae)

Leaf anatomy was surveyed for idioblasts in Cercidium (eight to nine species) and Parkinsonia (four to six species), two problematic genera of caesalpinioid legumes variously merged and separated for over a century. Leaflets from 16 accessions (11 dried and five liquid-preserved) of ten species were cleared (protoplasts removed chemically) and stained, supplemented by resin-embedded sections and scanning electron microscopy. Four Parkinsonia species had large, dimorphic, living subepidermal idioblasts (SI)—one form adaxially restricted, the other form abaxial. The fifth species, P. africana, had tiny scalelike leaflets without idioblasts. Idioblast contents are unknown but visibly different from those of adjacent cells. All five Cercidium species lacked SIs, but instead had veinlet idioblasts (VI). These large idioblasts varied in size and abundance, but in C. floridum and C. texanum they were especially large, spheroidal to ovate, thick-walled, and devoid of protoplasm at maturity. VIs occurred mostly as pairs or clusters, with individual cells joined by a large gap resembling a simple perforation plate. Almost all VI units abutted minor veins, but no perforation plate joined the proximal VI to a tracheary element. Cells similar to SIs occur in other Caesalpinieae genera, but VIs are unique to Cercidium. The mutually exclusive distribution of SIs and VIs supports the view that Cercidium and Parkinsonia are different.     

Composition and ultrastructure of the suberized cell wall of isolated crystal idioblasts from Agave americana L. leaves

Styloid-calcium-oxalate-crystal-containing idioblasts possess an interior cell-wall layer which has a lamellar ultrastructure. Idioblasts were isolated by centrifugation of an Agave americana leaf homogenate through 2M sucrose. The aliphatic monomers of the polymeric material from an idioblast fraction were primarily -hydroxy acids (32%) and dicarboxylic acids (35%), with C_18:1 dicarboxylic acid being the most dominant monomer (25%). Nitrobenzene oxidation of the idioblasts yielded syringaldehyde and vanillin in a ratio of 0.46:1. The major class of wax associated with the idioblasts was free fatty acids (34%). A major homologue of both the fatty acid and fatty alcohol fractions of this wax was C₂₂. The hydrocarbon fraction of the wax had a broad chainlength distribution with a large amount of even-numbered (47%) and shorter-chain homologues. The ultrastructure, the composition of the aliphatic and aromatic components of the polymeric material as well as the composition of the wax show that the idioblast cell wall is suberized. The wax and cutin polymer of the epidermis of A. americana leaves were chemically characterized for comparative purposes.Scientific paper No. 6115, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

Leaf anatomy ofDombeya andNesogordonia (Sterculiaceae), emphasizing epidermal and internal idioblasts

We studied leaf anatomy, using clearings, resin sections, and scanning electron microscopy, from mostly herbarium specimens of 123 species ofDombeya and 11 species ofNesogordonia (Sterculiaceae). Species were placed in seven idioblast categories, ranging from those without any to single and bicelled epidermal forms to multicelled nodules and single mesophyll idioblasts. Idioblast contents are possibly mucilaginous, but were not identified. In these two genera the range of foliar idioblast morphology surpasses that known previously for the entire family. Leaves are dorsiventral with mostly abaxial anomocytic stomata and typical palisade and spongy layers; paraveinal mesophyll is lacking. Miniature glandular (clavates, capitates) and nonglandular (mostly stellate) trichomes occur. Prismatic crystals predominate; epidermal prismatics and mesophyll druses are rare.     

Survey of leaf anatomy,especially secretory structures,of tribeCaesalpinieae (Leguminosae,Caesalpinioideae)

We studied leaflet anatomy, emphasizing secretory structures, from herbarium specimens of 128 species of 44 genera of tribeCaesalpinieae, using clearings, resin sections, and scanning electron microscopy. These observations, combined with those from our three earlier papers, provide a survey of 210 species representing all genera. Seventy-three species had secretory structures: 21 had glands or gland-like trichomes, 40 had living mesophyll idioblasts, and nine had cavities (three species each had two different types). Five additional species, all inCercidium (Caesalpinia group), had paired or clustered large spheroidal, thick-walled, empty cells (veinlet idioblasts) interconnected by perforation plate-like gaps. Secretory structures have systematic significance at various taxonomic levels.     

Comparative vegetative anatomy and systematics of Vanilla (Orchidaceae)

Vanilla is a pantropical genus of green-stemmed vines bearing clasping (aerial) and absorbing (terrestrial) roots. Most vanillas bear normal, thick foliage leaves; others produce fugacious bracts. Seventeen species, including both types were studied. Foliage leaves of Vanilla are glabrous, have abaxial, tetracytic stomatal apparatuses, and a homogeneous mesophyll. Species may or may not have a uniseriate hypodermis. Crystals occur in the foliar epidermises of some species, but all species have crystalliferous idioblasts with raphides in the mesophyll. Vascular bundles in leaves are collateral and occur in a single series alternating large and small. Sclerenchyma may or may not be associated with the vascular bundles. Scale leaves may be crescent or C-shaped and usually have abaxial stomatal apparatuses. A hypodermis may or may not be present; the mesophyll contains raphide bundles in idioblasts. Vascular bundles are collateral and occur in a single row sometimes aligned close to the adaxial surface. They may or may not be associated with sclerenchyma. Stems of leafy vanillas show a sclerenchyma band separating cortex from ground tissue; stems of leafless vanillas do not show a sclerenchyma band. Ground tissue of the stem may consist solely of assimilatory cells or mixed assimilatory and water-storage cells. In some species centrally located assimilatory cells are surrounded by layers of water-storage cells. A uniseriate hypodermis is present in all stems. Sclerenchyma may completely surround the scattered collateral vascular bundles, occur only on the phloem side, or be absent. Both aerial and terrestrial roots are notable for their uniseriate velamen the cell walls of which may be unmarked or ornamented with anticlinal strips. Exodermis is uniseriate; the cells vary from barely thickened to strongly thickened. Only the outer and radial walls are thickened. Cortical cells of aerial roots generally have chloroplasts that are lacking from the same tissue of terrestrial roots. Raphide bundles occur in thin-walled cortical idioblasts. Endodermis and pericycle are uniseriate; pericycle cells are all ?-thickened opposite the phloem. Cells of the endodermis are either ?- or ∪-thickened opposite the phloem. Vascular tissue may be embedded in thin- or thick-walled sclerenchyma or in parenchyma. Metaxylem cells are always wider in terrestrial than in aerial roots of the same species. Pith cells are generally parenchymatous but sclerotic in a few species.     

The Occurrence, Type and Location of Calcium Oxalate Crystals in the Leaves of Fourteen Species of Araceae

The occurrence, type and location of calcium oxalate crystalsin the leaves of 14 species belonging to the family Araceaewere studied by light microscopy. The Pizzolato test and theRubeanic acid-silver nitrate test, used to chemically identifyand locate the crystals in cross sections of laminae, showedthe presence of four types of crystals: druses, raphides, prismaticsand crystal sand. Styloids were not observed in any of the species.Crystals identified as calcium oxalate were observed in eachtissue layer of the leaf blade, druses occurring more frequentlyin the palisade mesophyll layers, raphides more often in thespongy mesophyll. Prismatics were sparse, occurring in the mesophyllof only two species. Specialized spindle-shaped crystal idioblasts,located in the spongy mesophyll in all cases, were observedin seven of the 14 aroids. Crystal sand and variations in crystalforms were most frequently observed to be calcium compoundsother than calcium oxalate. Crystals, calcium oxalate, idioblasts, Araceae     

A New Type of Specialized Cell in the Gynoecium of Ornithogalum caudatum (Liliaceae) with Notes on Specialized Cells in Carpels of Other Taxa

Idioblasts differentiate acropetally and centrifugally fromthe base of the ovary and eventually are found throughout thegynoecium Differentiation begins with an increase in cytoplasmicgranularity and culminates with the formation of dense cytoplasmicmasses, arms, and rings which stain for protein, carbohydrateand RNA Idioblastic cell contents eventually are cataboltzedand the cells are emptied of all contents during seed maturationIt is proposed that these cells function as specialized storagecells. Ornithogalum caudatum, carpels, gynoecium, idioblasts, specialized cells     

A chemosystematic study of Veronica: Iridoid glucosides

Caffeoyl-catalpol, isoferuloyl-catalpol, protocatechuoyl-catalpol, benzoyl-catalpol, p-hydroxybenzoyl catalpol (catalposide), vanilloyl-catalpol and cinnamoyl-aucubin have been isolated from several Veronica species (Scrophulariaceae) in a more or less pure state. The first four compounds have never been recorded in plants before. A PC survey of forty-three species of the genus sensu lato has shown a general presence of aucubin and catalpol. They are accompanied in many of the species by a complex mixture of esters, especially esters of catalpol with aromatic acids. In only one species have aucubin esters been found. Loganin has been identified in five species, this being the first time that this compound has been found in the Scrophulariaceae. Loganin is accompanied by unidentified loganin esters in the same five species. From the systematic point of view the complex mixtures of esters of catalpol in Veronica seem to be noteworthy. Species of Globularia, Erinus, Scrophularia Verbascum, Wulfenia, Catalpa and Plantago investigated either totally lacked ester glucosides in leaves or contained mainly different types of esters. The possible systematic meaning of these results is briefly discussed.     

Iridoids from Scrophularia genus

We report here an updated summary about iridoid composition of a series from the genus Scrophularia which have been investigated until now from a phytochemistry point of view. In addition a list is included about iridoids isolated in our laboratory from different plant parts of Scrophularia scorodonia L., which are compared with iridoids from some species of the Scrophularia genus. The present study may serve as a current information to researchers working on phytochemistry and pharmacological aspects from the Scrophularia genus and possibly to serve as a new starting point for future investigations.     

Chemotaxonomy of Veroniceae and its allies in the Plantaginaceae

In a chemosystematic investigation of tribe Veroniceae (Plantaginaceae), representatives of Camptoloma, Sibthorpia, Veronica subg. Pentasepalae and subg. Hebe, Veronicastrum, Wulfenia, and the related Ellisiophyllum and Globularia were examined for non-flavonoid glycosides. From the 14 species studied, 28 different iridoid glucosides and 10 caffeoyl phenylethanoid glucosides (CPGs), as well as salidroside and arbutin were isolated and characterized by NMR; of these, five compounds were previously unknown. It was found that the representatives of Veroniceae, as well as Globularia, were characterized by mannitol, aucubin, catalpol and catalpol esters. Each of the three studied species of Veronica subg. Hebe contained at least one of the 6-O-catalpol esters typical for Veronica s. str. (verminoside), supporting the inclusion of Hebe in Veronica. However, their main constituents were esters of 6-O-rhamnopyranosylcatalpol; a CPG, hebeoside (2'-beta-xylopyranosyl-verbascoside) was isolated from V. (Hebe) salicifolia. The two species of Veronicastrum also contained 6-O-rhamnopyranosylcatalpol esters, including the previously unknown 2',3'- and 3',4'-dicinnamoyl derivatives and, in contrast to the earlier reports, they lacked 6-O-catalpol esters. The main iridoid constituents in the three investigated species of Wulfenia were 10-O-aucubin and 10-O-catalpol esters (isoscrophularioside or globularin) while baldaccioside (10-O-cinnamoyl asystasioside E) was isolated from W. baldaccii. Globularia vulgaris contained 10-O-catalpol esters (e.g., globularin) and, in addition, asperuloside together with its benzoyl analogue named besperuloside. The representatives of Sibthorpia and Ellisiophyllum were almost completely devoid of iridoids; this, however, together with the CPGs present implied a close relationship between the two genera. Camptoloma lyperiiflorum lacked hexitols but contained esters of 6-O-rhamnopyranosylcatalpol different from those found in Veroniceae but known from Buddleja, Scrophularia and Verbascum (Scrophulariaceae s. str.).     

Metabolic differentiations and classification of Verbascum species by NMR-based metabolomics

The genus Verbascum L. (mulleins) comprises of about 360 species of flowering plants in the Scrophulariaceae family. Mulleins have been used in the traditional folk medicine for centuries, for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. Here we report the application of ¹H NMR metabolic fingerprinting in combination with principal component analyses (PCA) in five different Verbascum species. Based on the obtained results mulleins were divided in two groups: group A (Verbascum phlomoides and Verbascum densiflorum) and group B (Verbascum xanthophoeniceum, Verbascum nigrum and Verbascum phoeniceum). Further it was found that the plants in group B accumulate higher amounts of bioactive iridoid and phenylethanoid glycosides. V. xanthophoeniceum and V. nigrum accumulate higher amounts of the pharmaceutically-important harpagoside (∼0.5% on dry weight basis) and verbascoside, forsythoside B and leucosceptoside B (in total 5.6–5.8% on dry weight basis), which underlines the possibility for their application in pharmaceutical industry. To the best of our knowledge this is the first report on the analyses of Verbascum sp. leaf metabolome.     

Fuel treatment effects on soil chemistry and foliar physiology of three coniferous species at the Teakettle Experimental Forest, California, USA

A full factorial design crossing overstory (O) and understory (U) thinning and prescribed burning (B) was started at Teakettle Experimental Forest, California, in 2001 with the aim of achieving shifts in species composition to favor fire-resistant pines over fir. The goal of the present study was to evaluate the use of metabolic changes as early indicators for predicting the effects of these treatments on species-specific growth and long-term productivity of Pinus jeffreyi, Pinus lambertiana, and Abies concolor. Foliar polyamines, amino acids, soluble proteins and chlorophyll were quantified and related with foliar and soil chemistry. Most changes in soil chemistry occurred only with combined treatments (BU or BO). Changes in foliar metabolism were not driven by soil chemistry. In general, the pines were more sensitive to water stress as compared to fir as indicated by proline content. Understory thinning had no effect on any species. Both O and BO had species-specific effects on foliar amino acids, chlorophyll, soluble proteins, and inorganic ions. In addition, both O and BO were the most extreme thinning treatments for pines as they greatly increase the time needed to replenish carbon stocks. With the exception of some amino acids, B by itself had no major effects on foliar inorganic ions, chlorophyll content, and polyamines for any of the three species while BU caused a decrease in chlorophyll in white fir, suggesting a decline in photosynthetic capacity. These data point to BU as the best management option to specifically decrease the growth of fire-sensitive white fir without a major impact on pines.     

Leaf architectural,vascular and photosynthetic acclimation to temperature in two biennials

Acclimation of leaf features to growth temperature was investigated in two biennials (whose life cycle spans summer and winter seasons) using different mechanisms of sugar loading into exporting conduits, Verbascum phoeniceum (employs sugar‐synthesizing enzymes driving symplastic loading through plasmodesmatal wall pores of phloem cells) and Malva neglecta (likely apoplastic loader transporting sugar via membrane transport proteins of phloem cells). In both species, acclimation to lower temperature involved greater maximal photosynthesis rates and vein density per leaf area in close correlation with modification of minor vein cellular features. While the symplastically loading biennial exhibited adjustments in the size of minor leaf vein cells (consistent with adjustment of the level of sugar‐synthesizing enzymes), the putative apoplastic biennial exhibited adjustments in the number of cells (consistent with adjustment of cell membrane area for transporter placement). This upregulation of morphological and anatomical features at lower growth temperature likely contributes to the success of both the species during the winter. Furthermore, while acclimation to low temperature involved greater leaf mass per area in both species, this resulted from greater leaf thickness in V. phoeniceum vs a greater number of mesophyll cells per leaf area in M. neglecta. Both types of adjustments presumably accommodate more chloroplasts per leaf area contributing to photosynthesis. Both biennials exhibited high foliar vein densities (particularly the solar‐tracking M. neglecta), which should aid both sugar export from and delivery of water to the leaves.     

Foliar sclereids in the Magnoliaceae

The foliar sclereids in 136 species representing 11 of the 12 genera of Magnoliaceae were studied and compared. Sclereids occur in four different cell assemblages within the leaf: diffuse idioblasts, mesophyll, dermal system, and the vein sheath including terminal elements. Tropical members tend to have the most highly sclerified leaves. In species of Manglietia the leaves have sclerified spongy mesophyll and either sclerified epidermis or hypodermis. In Talauma , sclerification affects the vein sheath and terminal cells of veinlets, with a specialized thick marginal vein in the Asian taxa but not in the American ones. Liriodendron and the magnolias native to the north temperate zone have only minimal sclerification, which usually is confined to the vein sheath of the midrib and the main lateral veins, and as idioblastic sclereids in the petiole and the midrib near the base of the blade. The two largest genera Magnolia and Michelia are heterogeneous and include species which vary as to their combinations of sclerified elements among the four possible types.     

Diurnal changes in epidermal UV transmittance of plants in naturally high UV environments

Studies were conducted on three herbaceous plant species growing in naturally high solar UV environments in the subalpine of Mauna Kea, Hawaii, USA, to determine if diurnal changes in epidermal UV transmittance (T_UV) occur in these species, and to test whether manipulation of the solar radiation regime could alter these diurnal patterns. Additional field studies were conducted at Logan, Utah, USA, to determine if solar UV was causing diurnal T_UV changes and to evaluate the relationship between diurnal changes in T_UV and UV-absorbing pigments. Under clear skies, T_UV, as measured with a UV-A-pulse amplitude modulation fluorometer for leaves of Verbascum thapsus and Oenothera stricta growing in native soils and Vicia faba growing in pots, was highest at predawn and sunset and lowest at midday. These patterns in T_UV closely tracked diurnal changes in solar radiation and were the result of correlated changes in fluorescence induced by UV-A and blue radiation but not photochemical efficiency (F_v/F_m) or initial fluorescence yield (F_o). The magnitude of the midday reduction in T_UV was greater for young leaves than for older leaves of Verbascum . Imposition of artificial shade eliminated the diurnal changes in T_UV in Verbascum , but reduction in solar UV had no effect on diurnal T_UV changes in Vicia . In Vicia , the diurnal changes in T_UV occurred without detectable changes in the concentration of whole-leaf UV-absorbing compounds. Results suggest that plants actively control diurnal changes in UV shielding, and these changes occur in response to signals other than solar UV; however, the underlying mechanisms responsible for rapid changes in T_UV remain unclear.     

THE TERMINAL IDIOBLASTS IN MAGNOLIACEOUS LEAVES

The idioblasts terminating the foliar veinlets were studied in 152 species of the following magnoliaccous genera: Alcimandra, Aromadendron, Elmerrillia, Kmeria, Liriodendron, Magnolia, Manglietia, Michelia, Paramichelia, and Talauma. In all genera, except Liriodendron, some of the veinlets in mature leaves terminate in enlarged, nonliving cells called tracheoidal elements. Only one wall-facet (rarely 2) in such elements is differentially thickened; this wall lies adjacent to a conventional tracheary element. Ultimate cells of other veinlets in the Magnoliaceae differentiate as thick-walled sclereids, conventional tracheids, clavate tracheids, reticulate-walled dilated tracheids, or secretory cells. The terminal elements differentiate relatively late during leaf enlargement. In the Magnoliaceae, foliar structure is frequently characteristic at the generic level, and in some cases at the species level.     

Differential targeting of the tobacco PR-1 pathogenesis-related proteins to the extracellular space and vacuoles of crystal idioblasts.

Several biochemical and localization studies have shown that the acidic isoforms of the tobacco pathogenesis-related (PR) proteins, PR-1a, -1b and -1c are secreted to the extracellular spaces of leaves in response to pathogen infection or chemical treatment. Here we report the differential accumulation of these proteins within the vacuoles of specialized cells known as crystal idioblasts. In situ hybridization analysis indicated that crystal idioblasts expressed the PR-1 genes at the mRNA level and suggested that PR-1 proteins were synthesized by these cells. Transgenic plants which constitutively express a chimeric gene encoding an acidic PR-1b isoform also accumulated PR-1 protein in the extracellular spaces and within crystal idioblast vacuoles. Analysis of mRNA derived from these transgenic plants indicated that expression of the introduced PR-1b gene was responsible for the accumulation of PR-1 protein in these two distinct locations. The synthesis and accumulation within crystal idioblasts of PR-1 proteins, which are secreted by other cell types, indicates that idioblasts sort these proteins in a unique manner. Moreover, this suggests that protein sorting in higher plants may be modulated in a cell specific manner.     

Antinociceptive and anti-inflammatory effects of saponin and iridoid glycosides from Verbascum pterocalycinum var. mutense Hub.-Mor

The anti-inflammatory and antinociceptive properties of four major compounds from the flowers of Verbascum pterocalycinum var. mutense were investigated. Saponin glycosides called ilwensisaponin A and C and iridoid glycosides known as ajugol and picroside IV were isolated from the methanolic extract. A dose-related anti-inflammatory and antinociceptive response were obtained in this study at doses of 100 and 200 mg/kg. The results of the evaluation of the anti-inflammatory activity induced by carrageenan and PGE1 showed that this species possesses active constituents that could diminish the cyclooxygenase activitiy. No effects were observed in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model. Our results support the anti-inflammatory and analgesic effects of Verbascum pterocalycinum var. mutense. Ilwensisaponins A and C could explain in part the anti-inflammatory and analgesic activities of this species. Although antinociceptive and anti-inflammatory activities of ajugol and picroside IV were found insignificant in the statistical analysis, ilwensisaponin A and C showed notable activity without inducing any apparent acute toxicity as well as gastric damage.