Isolation and characterization of paracrystalline structures from transgenic P<Emphasis Type="Italic">ssu-ipt</Emphasis> tobacco

Distinct crystalloids were found in chloroplasts of transgenic Pssu-ipt tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) overproducing endogenous cytokinins. They were present both in rooted (T) and grafted (TC) transgenic plants contrary to control tobacco (C). The fractions enriched by crystalloids were isolated from chloroplasts using a continuous or a discontinuous Percoll gradient. Chlorophyll (Chl) fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHC2) that was not connected to reaction centres of photosystem 2 both in isolated chloroplasts and in the fraction of 80 % Percoll gradient from both types of transgenic tobacco. Further analyses, i.e. pigment contents, polypeptide composition by SDS-PAGE, and immunoblotting support our hypothesis that crystalloids inside chloroplasts of transgenic tobacco are formed by LHC2 aggregates. Treatment with two distinct detergents, chosen with respect to their effects (i.e. β-dodecyl maltoside or Triton X-100), resulted in different degree of disintegration of Chl a/b proteins in transgenic plants compared to the control. Electron microscopic observations and immunogold labelling with specific LHC2 antibodies carried on the resin embedded leaf sections or free suspensions of chloroplasts showed that gold particles were bound preferentially on the outer surface of crystalloids. Three-dimensional reconstruction of chloroplasts and crystalloids proved that paracrystalline structures varied moderately in their size and took up a significant portion of total chloroplast volume.     

Three-dimensional reconstruction of anomalous chloroplasts in transgenic ipt tobacco

Anomalies in the ultrastructure of chloroplasts, from transgenic ipt tobacco, overproducing endogenous cytokinins (CKs) were studied. Detailed analyses of CKs and their metabolites showed that Pssu-ipt tobacco contained enhanced contents of CKs both in leaves and in isolated chloroplasts. The role of CKs in the formation of anomalous structures is suggested. Pssu-ipt chloroplasts frequently formed the distinct peripheral reticulum with a system of caverns that often involved mitochondria and/or peroxisomes. Large crystalloids, which were found in chloroplasts of Pssu-ipt, occupied up to 16% of chloroplast volume. We suggested that the crystalloids were formed by LHC II aggregates. This was supported by analysis of the fluorescence emission spectra at 77°K, chlorophyll a/b ratio, immunogold staining of the structures, and crystallographic unit size analysis.     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.     

Occurrence,composition, and structure of microbody crystalloids in charophycean gametangial sheath cells

Summary An ultrastructural study was made of the cellular sheaths surrounding the sexual organs of five species of algae in the three genera ofCharophyceae: Nitella flexilis, N. mirabilis, Chara brattnii, Tolypella boldii andT. intricata. Microbodies similar in appearance, with crystalline nucleoids, were present in the sheath cells of all five species. The microbodies resembled in size and topographical associations those of other green algae. The hexagonal-shaped crystalloids consisted of parallel arrays of fine tubules of about 15 nm in diameter arranged parallel to the long axis of the crystalloid. In cross sections of the crystalloid, the close packing of the tubules showed hexagonal arrays. The intertubular distance is about 7 nm. At higher magnification there is a suggestion that the walls of these tubules are themselves constructed of smaller tubules. Further electron microscopic observations of diaminobenzidine (DAB)-treated preparations revealed pronounced deposition of reaction product in the microbodies, particularly on the crystalloids. The reaction was completely blocked by the catalase inhibitor, aminotriazole. These results strongly suggest that catalase is involved in this reaction and that catalase is located in the crystalloids.     

The structure,composition and distribution of proteinaceous crystalloids in vegetative cells of the red algaWrangelia plumosa

Summary High concentrations of proteinaceous crystalloids accumulate in vegetative cells of the red algaWrangelia plumosa Harvey but disappear prior to sporogenesis. The distinctly structured crystalloids lack a bounding membrane and appear to autopolymerize within the cytoplasm. Chemical analysis of isolated crystalloids showed the presence of all amino acids except cysteine and cysteic acid. Carbohydrate accounted for 7.5% of the preparation. The crystalloids appear to have a storage function during growth and development.     

Sieve-element plastids ofTriticum andAegilops (Poaceae)

Fourteen taxa of the Triticum-Aegilops group have been investigated for their sieve-element plastids. At maturity they contain dense and thin crystalloid inclusions and are classified into the PIIc' plastid type; onlyAe. comosa var.biaristata lacked the thin crystalloids and thus conforms to the PII c type. The proteinaceous nature of the crystalloids was demonstrated by application of proteolytic enzymes. Ultrastructural evidence suggests that both kinds of crystalloid inclusions are involved in the sealing of sieve-plate pores of injured sieve tubes. Measurements and calculations of the spacings and angles carried out on crystalloid prints permitted the construction of a two- and three-dimensional pattern forT. aestivum thin crystalloids.     

Ultrastructure of sieve-element plastids inCaryophyllales (Centrospermae), evidence for the delimitation and classification of the order

The orderCaryophyllales (Centrospermae) was found to contain specific P-type sieve-element plastids which are characterized by protein inclusions composed of ring-shaped bundles of filaments and of central crystalloids. The sieve-element plastids of 14 families (140 species investigated) fit into this overall characterization, and more specific details are used to delimit the families and arrange them within the order.Phytolaccaceae, the basic family of the order display much diversity: the crystalloids inside their plastids are either globular (most genera) or polygonal (Stegnosperma), starch may also be present (Phytolacca).Nyctaginaceae, with starch inBougainvillea sieve-element plastids, can be derived directly fromPhytolacca. Globular crystalloids are present in most of the families, as inDidiereaceae, Cactaceae, Aizoaceae-Tetragoniaceae, Portulacaceae-Basellaceae-Halophytaceae-Hectorellaceae. Caryophyllaceae andLimeum ofMolluginaceae contain polygonal crystalloids (otherMolluginaceae with globular crystalloids). Crystalloids are entirely absent fromChenopodiaceae (incl.Dysphaniaceae) andAmaranthaceae. The probable relationships between these families are presented diagrammatically in Fig. 13. Bataceae, Gyrostemonaceae, Vivianiaceae, Theligonaceae, Polygonaceae, Plumbaginaceae, Fouquieriaceae, Frankeniaceae, andRhabdodendraceae—all at some time included into theCaryophyllales (Centrospermae) or doubtfully referred to them—develop S-type (or different P-type) sieve-element plastids. Their direct connection to theCaryophyllales therefore is excluded. Finally, evolutionary trends of theCaryophyllales are discussed.Presented in the Symposium Evolution of Centrospermous Families, during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Chemistry and micromorphology of compound epicuticular wax crystalloids (Strelitzia type)

Certain non-liliiflorous taxa within the monocotyledons (e.g.,Strelitzia, Heliconia, Typha) are characterized by compound epicuticular wax rodlets (Strelitzia type). Similar rodlets are also encountered on the surface of the dicotyledonous plantBenincasa hispida (Thunb.)Cogn. Chemical analysis of the surface wax from both sources showed that the rodlets are chemically distinct. The rodlets of the monocotyledons consist exclusively of aliphatic wax lipids, mainly wax esters. In contrast, the rodlets ofBenincasa are cheifly composed of triterpenol acetates and triterpenols. Formation of rodlets is therefore interpreted as ultrastructural convergency. It is concluded that taxonomical studies on wax crystalloids can be misleading when interpreted in terms of micromorphology of crystalloids only.     

Development of P-protein in sieve elements ofMimosa pudica

Summary The P-protein in sieve elements of leaves ofMimosa pudica L. is first discernible as fine fibrous material which forms homogeneous aggregates. Ribosomes, rough endoplasmic reticulum, and dictyosomes with associated vesicles occur in the cytoplasm surrounding the aggregates. The plastids and mitochondria are in a parietal position in the parts of the cell where the nascent P-protein accumulates. In a later stage, the fibrillar material is organized into a three-dimensional system of five- and six-sided elongated compartments. The corners of the compartments appear solid at first, then they become electron lucent in the center and assume tubular form. Aggregates of mature P-protein tubules usually occur near the compartmentalized system. Tubules in pentagonal or hexagonal arrangements may be present in the aggregates and may be partly interconnected. The conclusion was drawn that the P-protein tubules are assembled at the corners of compartments within a continuous orderly system. The fully formed tubules occur first in aggregates, the P-protein bodies. Later the aggregates become loose and partly dispersed. Many of the dispersed tubules assume a loose, extended, helical form characteristic of P-protein in older sieve elements.This work was supported in part by National Science Foundation grant GB-5506. I am also grateful to MissHatsume Kosakai and Mr.Robert H.Gill for technical assistance.     

Crystalloid inclusions in the Sertoli cell of the koala,Phascolarctos cinereus (Marsupialia)

Summary Crystalloid inclusions are a common feature in the basal region of Sertoli cells in the koala, Phascolarctos cinereus. Generally located near the nucleus, they are non membrane-bounded, slender rectangular structures composed of tubules which are orientated at right angles to the long axis of the crystalloid and regularly arranged in rows parallel to this long axis. The tubules in adjacent rows are offset from one another at definite angles and extensively interconnected by filaments. Neither the composition nor function of the crystalloids has been determined, but their association with tonofilaments and the presence of ribosomes in the vicinity suggests that they are most likely proteinaceous.The authors would like to thank Mr. D. Harbrow, Mr. S. Brown and Ms. B. Canty for technical assistance; the Queensland National Parks and Wildlife Service and the Victorian Ministry of Conservation (Fisheries and Wildlife Division) for providing permits to work on this protected species; the staff of the Lone Pine Koala Sanctuary, Brisbane, for their assistance in obtaining animals. This work was supported by a grant from the Australian Research Grants Committee, Number DI-77/15525     

Endoplasmic reticulum derived decorated tubules in the sieve elements ofNymphaea

Summary Bundles of decorated tubules found in the sieve elements ofNymphaea have been studied with the transmission electron microscope. Comparatively straight tubules (100 nm in diameter) arise from the endoplasmic reticulum during early stages of sieveelement development and subsequently associate into bundles of up to 100 tubules that parallel the longitudinal cell axis. From the start of their formation the tubules are structurally distinct from other ER profiles due to their dense decoration with particles. High magnifications reveal an orderly array of the particles (about 24 surround a 100 nm tubule) and suggest a modification of their membrane so that it is no longer dissolvable into a regular three-layered structure. Later during sieve-element ontogeny the decorated tubules get invaginated by smooth ER membranes, thereby squeezing out the intratubular (extracytoplasmic) space. As a result a double mantle is formed that surrounds a plasmatic cylinder. Decorated 100 nm tubules with inner membranes are present in enucleate mature sieve elements ofNymphaea alba andN. tuberosa. Considerably larger tubules (about 200 nm in diameter) were found inN. Candida andN. tetragona and occasionally also inNuphar and Barclaya, two other genera from the same family. The decoration of the tubules and their subsequent invagination by smooth membranes are discussed with respect to the controlled autolysis of sieve elements.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Genetic Control of Development of Malpighian Tubules in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium ofDrosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Effect of cycloheximide on the fine structure of corpus luteum in intact and hypophysectomized rats

Summary In rats, one large intravenous dose of cycloheximide leads to extensive development of two types of membrane-formations in the cells of corpora lutea, within two hours. Both the laminated dense bodies (concentric layers of smooth membranes showing high electron density) and the tubular aggregates (tightly packed smooth tubules with diameter smaller than usual) exhibited obvious connections with endoplasmic reticulum membranes. The reorganization of tubular aggregates gave rise to crystalloids showing hexagonal symmetry. The crystalloids, being obviously unstable, were transformed into smooth fingerprints (concentric arrays of paired agranular membranes showing the same density as endoplasmic reticulum membranes). Hypophysectomy, performed 24 hours previously, moderated but did not totally abolish the development of membranous configurations. The described effect of cycloheximide is considered to represent cellular injury, probably due to membrane-denaturation.This work was supported in part by the Medical Research Council of Canada (Block Term Grant MT-1829), the Ministère des Affaires Sociales, Quebec, and Succession J.A. DeSève. The authors thank the Upjohn Company, Kalamazoo, Michigan, U.S.A., for the cycloheximide used in these experiments.Fellow of the Medical Research Council of Canada.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interaction of Krüppel with other terminal genes. The gap genes tailles and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

The three-dimensional structure of theCyphomandra betacea chloroplast peripheral reticulum

Summary The peripheral reticulum (PR) inCyphomandra betacea chloroplasts originates as vesicles budding from the inner membrane of the chloroplast envelope which elongate to form tubules then aggregate or branch to form discrete PR units. Individual PR units of many coiled tubules may be connected with other units by narrow tubules. Serial sectioning revealed the discrete units to be approximately 650–1,000 nm wide, 400–500 nm high and 500–600 nm deep and to possess a compact morphology. TheCyphomandra PR structure is compared to the morphologies of chloroplast reticula reported for other plant species. A scheme to group PR from different species into 3 distinct morphological categories is outlined and discussed.     

Epicuticular crystals of nonacosan-10-ol: In-vitro reconstitution and factors influencing crystal habits

The primary aerial surfaces of plant species from many families (e.g. Pinaceae, Liliaceae, Ranunculaceae, Papaveraceae) are covered by epicuticular tubules 5–20 μm long and 0.5 μn in diameter. The composition, mechanism of growth and molecular structure of this type of epicuticular aggregates have been studied. Pure nonacosan-10-ol extracted from Picea pungens needle surfaces formed, in vitro, tubular crystals like those occurring in vivo. This crystal habit was obtained irrespective of the type of solvent or substratum, if the solvent was evaporated within minutes. This shows that tubules of nonacosan-10-ol are formed in the kinetic regime of crystallization (limited by the diffusion of molecules from the solution to the crystal surface). Slow evaporation of the solvent or crystallization from the melt resulted in rhombic scales. These planar crystals represent the thermodynamic, stable modification of native nonacosan-10-ol. Homologous impurities in natural nonacosan-10-ol (3–14%) had no effect on the formation of the tubules. However, racemic nonacosan-10-ol invariably crystallized in scales. The phase behaviour of mixtures of natural nonacosan-10-ol and its synthetic racemate as well as synthetic (S)-nonacosan-10-ol provided evidence for the presence of the pure (S)-enantiomer on plant surfaces. The findings are discussed in terms of the mechanisms leading to epicuticular tubules consisting of nonacosan-10-ol and their molecular structure. Crystal structures for the pure enantiomer and the racemate of nonacosan-10-ol are proposed. It is concluded that the principles responsible for the formation of tubules are both the special molecular geometry of the naturally occurring (S)-nonacosan-10-ol and the mobility barrier of the plant cuticle. Further specific biological processes are not necessary for the formation of (S)-nonacosan-10-ol tubules. The alterations of epicuticular structures during ageing or the impact of pollutants are explained as spontaneous transitions between two crystal modifications of (S)-nonacosan-10-ol.     

Vacuolar Reticulum in Oomycete Hyphal Tips: An Additional Component of the CaRegulatory System?

Cultures ofAchlyasp.,Phytophthora cinnamomi, Saprolegnia diclina, S. ferax,andS. parasitica,treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae ofS. ferax.The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca²⁺sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization.     

Purine transport by Malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (p ^p) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.This work was supported by Grant GM22366 from NIH.     

Unusual cell organelles during spermiogenesis in two species of gobies (Gobiidae,Teleostei)

Summary Testes of Lesuerigobius friesii and Gobius bucchichi were studied in adult reproductive fish. During the onset of spermatid development, a peculiar system of alternating rough (RER) and smooth (SER) endoplasmic reticular tubules form rings distally to the cell nucleus. The RER tubules are seen to possess up to 12 ribosomes in cross-section, whereas the SER are strongly electron-dense. Nanotubules connect these stacks of tubules to the developing head and tail of the sperm. With ripening of the sperm these tubules disintegrate within the excessive cytoplasm. It seems likely that these are special forms of Macro-Golgi System that possibly provide protamines for the developing sperm.