Chromoplasts of Palisota barteri,and the molecular structure of chromoplast tubules

Ripe, deep-red fruits of Palisota barteri contain tubulous chromoplasts which develop from unpigmented leucoplasts. These plastids contain, besides large spherical inclusion bodies, numerous osmiophilic globules which, in the course of ripening, frequently show transition states to tubular structures. The tubules contain, in all stages of their development, acylated -citraurin, which is also the main pigment of Citrus fruits. The tubular structures have been isolated, fragmented by French-pressure treatment, and separated into three fractions on sucrose gradients. The lightest fraction (1.044 g·cm^-3) contained thick fragments (saccules) with diameters of 50–60 nm, whereas the heaviest (1.083 g·cm^-3) consisted of tubules 20–25 nm in diameter. The relative amounts of polar lipids, proteins, and carotenoids of the different fractions are consistent with a molecular structure model of tubules and saccules, according to which a wick of longitudinally oriented carotenoid molecules of variable thickness is coated by a monolayer of polar lipids and proteins. High-resolution negative-stainings showed the surface of the tubules to be covered with globular particles of about 6 nm diameter. The main protein of all fractions is a 30-kDa polypeptide; it is assumed that the particles are oligomers of this specific protein.Abbreviations IB inclusion body - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Chromoplasts of Tropaeolum majus L.: Lipid synthesis in whole organelles and subfractions

Isolation of tubulous chromoplasts from Tropaeolum majus L. petals was achieved in pure form. Their main substructures-lipid bodies, tubules, and envelope membranes-have been enriched. Whole chromoplasts as well as substructures have been tested for their activities in lipid synthesis. The following activities were found: fatty acid synthesis from acetate, glycosyl transfer reactions from UDP-galactose and UDP-glucose to galactolipids and sterols, acyltransferase reactions from palmitoyl-CoA, and a very active acyl-CoA hydrolase (EC 3.1.2.2.). Fatty acid synthesis was restricted to whole chromoplasts. Glycosyl- and acyltransferases were essentially confined to envelope membranes, whereas acyl-CoA hydrolase was found in all fractions. The chemical composition of chromoplast subfractions was determined. The lipid bodies consisted mainly of galactolipids and carotenoid esters in a 1:1 ratio, together with small amounts of protein.     

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis)

Summary. The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of -carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as membranous chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.     

Chromoplasts of Tropaeolum majus L.: Structure and development

Summary The fine structure of chromoplasts in epidermal cells of flower petals of Tropaeolum has been investigated by light, polarizing, and electron microscopy at different stages of development. The pale greenish-yellow petals still enclosed in the bud contain barely differentiated chloroplasts with few, irregular grana. The chromoplasts of unfolding petals show differently oriented bundles of tubules with variable diameters (mean: 17 nm). Thylakoid membranes become reduced more and more. The tubular bundles are intermingled with numerous isodiametric bodies of ca. 50 nm diameter; these bodies are better discernible at later stages when the chromoplasts possess a less dense matrix. The chromoplasts of open flowers are in a state of disorganization at a time when the cytoplasm still appears normal. A comparison is made between chromoplast tubules and tubular structures described from other kinds of plastids. The observations are discussed in view of chromoplast typology and with regard to possible processes underlying chromoplast differentiation in flowers.Abbreviations in Figures Chr chromoplast - CT chromoplast tubules - Cy cytoplasm - D dictyosome - IB isodiametric body - M mitochondrion - MT microtubule - oG osmiophilic globule - S S-body - St starch grain - V vacuole All micrographs from glutaraldehyde-OsO₄-fixed material, unless otherwise specified. The bar designates 1 m (multiples or fractions of it indicated).     

Early metabolic changes in regenerating microfragments of the liverwort Riella helicophylla (Bory et Mont.) Mont.: Protein and RNA synthesis,free α-amino concentration

Microfragments of constant size were isolated from the thallus of Riella by a rapid punching procedure. Thus it was possible to study various metabolic parameters especially during the first hours after fragment isolation. Protein synthesis increases rapidly after a slight decrease at 30 min. The earliest significant increase in RNA synthesis occurs at 8 h, indicating a different activation pattern. The concentration of -amino compounds drops at 30 min and then increases continuously, thus exhibiting a close relationship with the measured alterations in protein synthesis. Another indication of metabolic conversions during regeneration is provided by the changing level of free radioactive leucine, which shows a marked turning point at 24 h after fragmentation. Analysis of free -amino concentrations in small regions of larger fragments also indicates the establishment of new intercellular correlations only a few hours after isolation of the cells from the meristem. Up to the 8th h after fragmentation, the contents of both the adaxial and peripheral fragment regions increase. Thereafter only the adaxial (regenerating) cells continue accumulating; the peripheral (nonregenerating) ones remain at the same level.     

Tubule bundle-inclusions in vacuoles of Tamarix aphylla L. mesophyll cells

Summary Vacuoles of differentiating mesophyll cells of Tamarix aphylla contain an amorphous electron-dense material in which stacks of parallel aligned striations are embedded. Cross-sections of the striations disclosed that they represent profiles of longitudinally sectioned bundles of tubules (tubule outer diameter 9.0 nm, tubule wall thickness 1.8 nm). In advanced mesophyll cell development, the amorphous vacuolar material disappears, whereas the bundles of tubules turn into bundles of double helices (double helix diameter 14.5 nm). Cytochemical treatment of mesophyll cells with the enzymes pepsin and trypsin has revealed that both the bundles of tubules/double helices and the embedding material are constituted of protein. The possible functional role of the vacuolar inclusions is discussed.     

Floral ontogeny in<Emphasis Type="Italic"> Lespedeza thunbergii</Emphasis> (Leguminosae: Papilionoideae: Desmodieae): variations from the unidirectional mode of organ formation

Floral ontogeny of Lespedeza thunbergii was studied with the use of scanning electron microscopy (SEM). The ontogeny varies in all whorls from the undirectional mode, which has been long held to be the rule in Leguminosae. In the sepal whorl, the lateral and the adaxial sepals are formed simultaneously, which is interpreted as a tendency towards whorled organ formation. Whorled organ formation is shown in the petal whorl. The antesepalous stamen whorl varies least from the unidirectional mode. Here, the adaxial stamens are formed successively. This is seen as a remnant of an original helical organ formation in Papilionoideae. Within the antepetalous stamen whorl, the two abaxial stamens and the adaxial stamen are formed first, followed by the two lateral stamens. This is a rarely found phenomenon, which is hard to interpret at the present state of knowledge. Concerning the mature flower, it is shown that nectar stomata are found in a distinct area on the adaxial side of the flower. The presented new characteristics should be an initial step toward further work on taxa of the tribe Desmodieae. These studies will broaden the data set and enable a detailed phylogenetic analysis.     

Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer

Chimeric -glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BrdUrd bromodeoxyuridine - GUS -glucuronidase - Km^R kanamycin resistant - T-DNA transferred DNA     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Imaging of chlorophyll-a-fluorescence in leaves: Topography of photosynthetic oscillations in leaves of Glechoma hederacea

Images of chlorophyll-a-fluorescence oscillations were recorded using a camera-based fluorescence imaging system. Oscillations with frequencies around 1 per min were initiated by a transient decrease in light intensity during assimilation at an elevated CO₂-concentration. The oscillation was inhomogenously distributed over the leaf. In cells adjacent to minor veins, frequency and damping rate was high, if there was any oscillation. In contrast, the amplitude was highest in cells most distant from phloem elements (maximal distance about 300 m). The appearance of minor veins in oscillation images is explained by a gradient in the metabolic control in the mesophyll between minor veins and by transport of sugar from distant cells to phloem elements. The potential of fluorescence imaging to visualize microscopic source-sink interactions and metabolic domains in the mesophyll is discussed.Abbreviations P_i inorganic phosphate - Fru2,6BP fructose-2,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - SPS sucrose-phosphate synthetase - HP hexosephosphate     

Nuclear-magnetic-resonance imaging of leaves ofMesembryanthemum crystallinum L. plants grown at high salinity

Differences in water binding were measured in the leaf cells ofMesembryanthemum crystallinum L. plants grown under high-salinity conditions by using nuclear-magnetic-resonance (NMR) imaging. The 7-Tesla proton NMR imaging system yielded a spatial resolution of 20·20·100 m³. Images recorded with different spin-echo times (4.4 ms to 18 ms) showed that the water concentrations in the bladder cells (located on the upper and lower leaf surface), in the mesophyll cells and in the water-conducting vessels were nearly identical. All of the water in the bladder cells and in the water-conducting vessels was found to be mobile, whilst part of the water in the mesophyll cells was bound. Patches of mesophyll cells could be identified which bound water more strongly than the surrounding mesophyll cells. Optical investigations of leaf cross-sections revealed two types of mesophyll cells of different sizes and chloroplast contents. It is therefore likely that in the small-sized mesophyll cells water is strongly bound. A long-term asymmetric water exchange between the mesophyll cells and the bladder cells during Crassulacean acid metabolism has been described in the literature. The high density of these mesophyll cells in the lower epidermis is a possible cause of this asymmetry.Abbreviations CAM Crassulacean acid metabolism - NMR nuclear magnetic resonance - T_E spin-echo time     

Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

Ultrastructural localization of photosynthetic activity in thylakoids during chloroplast development in maize

The localization of photosynthetic activity in developing maize (Zea mays L.) chloroplasts was studied in situ by two electron-microscopic-cytochemical methods. The activity of photosystem I was detected by photooxidation of 3,3-diaminobenzidine (DAB) and the activity of the photosystem II by photoreduction of thiocarbamyl nitrotetrazolium blue (TCNBT). During the transformation of proplastids into chloroplasts, at the base of the leaf blade the DAB reaction appeared before the TCNBT reaction. A positive DAB reaction was observed in the single thylakoids of plastids in cells located only about 0.5 mm above the base. Dark, osmiophilic DAB polymers accumulated in the lumina of the thylakoids. Plastid envelopes and tubules of the prolamellar bodies in immature chloroplasts were DAB-negative. In fully differentiated leaf tissue the DAB reaction was intense in the thylakoids of bundle-sheath chloroplasts, as well as in the stroma thylakoids and the peripheral grana thylakoids of mesophyll chloroplats. The photoreduction of TCNBT started in leaf tissue about 1 mm above the base. Dark granular material of reduced TCNBT appeared mostly in the partitions of grana, i.e. interthylakoidally, but some granules were also attached to the stroma thylakoids. The membranes of plastid envelopes and the tubules of prolamellar bodies showed a negative TCNBT reaction. Young bundle-sheath chloroplasts contained some reduced TCNBT in their grana; these deposits largely disappeared in the course of further differentiation. In mature leaf tissue the photoreduction of TCNBT was conspicuous in the grana of mesophyll chloroplasts, but very weak in the single thylakoids and in the granal rudiments of bundle-sheath chloroplasts.Abbreviations DAB 3,3-diaminobenzidine·4 HCl - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS(I,II) photosystem (I,II) - TCNBT thiocarbamyl nitrotetrazolium blue chloride     

The vacuolar-tubular continuum in living trichomes of chickpea (Cicer arietinum) provides a rapid means of solute delivery from base to tip

Summary A vacuolar continuum exists from base to tip in the secretory trichomes of chickpea (Cicer arietinum). This continuum is seen in living trichomes which have been labeled with Lucifer yellow CH and examined with confocal microscopy. It encompasses the large vacuole of the lower stalk cell, the vacuoles and tubules of the central stalk cell, the thin tubules of the upper stalk cell, and the tubules and vacuoles of the secretory head cells. The vacuolar-tubular system is structurally distinct within each cell, forming a gradient of large vacuoles in the lower stalk cell, thick tubules in the central stalk cell, and thin anastamozing tubules in the upper stalk cell. This membrane system appears to be continuous between trichome cells, as thin tubules emanate from plasmodesmata between stalk cells and between the upper stalk and lower head cell. In the upper stalk cell, the thin tubules of this continuum are streaming up and down the long axis of the cell at 0.67 m/s. The larger vacuolar-tubular system in the central and lower stalk cells is also slowly moving, with apparent peristalsis occurring in the central cell. The vacuolar-tubular system of the secretory head cells is completely labeled with Lucifer yellow when the dye has only partly diffused up the long walls of the trichome, indicating that the streaming tubular system delivers solute through the stalk cells to the secretory head cells faster than diffusion through the trichome walls. In the lower head cells, tubules emanate from the plasmodesmata connecting to the upper stalk cell, and these tubules are continuous with the head cell vacuoles. In addition, another layer of thin tubules forms along the edges of the secretory head cells, at the site of exocytotic secretion. We propose that the continuous vacuolar-tubular system in these trichomes functions to rapidly deliver solute from the base of the trichome to the secretory head cells. This system provides a pathway for the transport of secretory material.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Water relations of individual leaf cells ofMesembryanthemum crystallinum plants grown at low and high salinity

Summary The effects of saline conditions on the water relations of cells in intact leaf tissue of the facultative CAM plantMesembryanthemum crystallinum were studied using the pressure probe technique. During a 12-hr light/dark regime a maximum in turgor pressure was recorded for the mesophyll cells of salttreated (CAM) plants at the beginning of the light period followed 6 hr later by a pressure maximum in the bladder cells of the upper epidermis. In contrast, the turgor pressure in the bladder cells of the lower epidermis remained constant during light/dark regime. Turgor pressure maxima were not observed in untreated (C₃) plants.This finding strongly supports the assumption that water movement during malate accumulation and degradation in salttreated plants occurs predominantly between the mesophyll cells and the bladder cells of the upper epidermis. The necessary calculations take differences in the compartment volumes and in the elastic moduli of the cell walls () of the bladder cells of the lower and upper epidermis into account.Measurements of the kinetics of water transport showed that the half-time of water exchange for the two sorts of bladder cells were nearly identical in CAM plants and in C₃ plants. The absolute values of the half-times increased by about 45% in salttreated plants (about 113 sec) compared to the control plants (78 sec). Simultaneously, the half-time of water exchange of the mesophyll cells increased by about 60% from 14 sec (untreated plants) to 22 sec (salt-exposed plants). The leaves of this plant are apparently able to closely maintain the time of propagation of short-term osmotic pressure changes over a large salinity range.A cumulative plot of the data measured on both C₃ and CAM plants showed that the differences between the values of the elastic moduli of bladder cells from the lower and from the upper epidermis are due to differences in volume and suggested that the intrinsic elastic properties of the differently located bladder cells of C₃ and CAM plants were identical.A cumulative plot of the hydraulic conductivity of the membrane obtained both on mesophyll and on bladder cells of salttreated and of untreated plantsvs. the individual turgor pressure yielded a relationship well-known from giant algal cells and some higher plant cells: The hydraulic conductivity increased at very low pressure, indicating that the water permeability properties of the membrane of the various cell types of C₃ and CAM plants are pressure dependent, but otherwise identical.The results suggest that a few fundamental physical relationships control the adaptation of the tissue cells to salinity.     

TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium

To study the effect of auxin on direct somatic embryogenesis from leaf cultures ofOncidium `Gower Ramsey', 1-cm-long explants have been cultured in vitro testing IAA, 2,4-, quercetin, TIBA and PCIB. On a modified MS medium devoid of plant growth regulators, leaf cells of three regions (leaf tips, adaxial sides and cut ends) formed somatic embryos. After 8 weeks in culture, the frequencies of embryo-forming explants were 55, 52.5 and 30 % on leaf tips, adaxial sides and cut ends, respectively, and the numbers of embryos per dish was 89.3. Except for TIBA, other growth regulators (IAA, 2,4-, quercetin, PCIB) and their combinations tested, all retarded direct embryo formation. In the presence of 0.1 and 0.5 M TIBA, leaf tip, adaxial sides and cuts end of explants gave almost the same embryogenic response as the control. However, 10 and 27.5 % of explants were induced to form embryos from abaxial sides, and these explants did not form embryos on cut ends. In addition, after 8weeks in culture, TIBA at 0.5M highly promoted the mean numbers of embryos per dish to 134.2.     

Ultrastructural indications for coexistence of symplastic and apoplastic phloem loading in Commelina benghalensis leaves

The ultrastructural ontogeny of Commelina benghalensis minor-vein elements was followed. The mature minor vein has a restricted number of elements: a sheath of six to eight mestome cells encloses one xylem vessel, three to five vascular parenchyma cells, a companion cell, a thin-walled protophloem sieve-tube member and a thick-walled metaphloem sieve-tube member. The protophloem sieve-tube member (diameter 4–5 m; wall thickness 0.12 m) and the companion cell originated from a common mother cell. The metaphloem sieve-tube member (diameter 3 m; wall thickness 0.2 m) developed from the same precursor cell as the phloem parenchyma cells. Counting the plasmodesmatal frequencies demonstrated a symplastic continuum from mesophyll to the minor-vein phloem. The metaphloem sievetube member and the phloem parenchyma cells are the termini of this symplast. The protophloem sieve-tube member and companion cell constitute an insulated symplastic domain. The symplastic route, mesophyll to metaphloem sieve tube, appears to offer a path for symplastic loading; the protophloem sieve tube may be capable of accumulation from the apoplast. A similar two-way system of loading may exist in a number of plant families. Plasmodesmograms (a novel way to depict cell elements, plasmodesmatal frequencies and vein architecture) of some other species also displayed the anatomical requirements for two routes from mesophyll to sieve tube and indicate the potential coexistence of symplastic and apoplastic loading.     

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