A new putative cellulose-synthesizing complex ofColeochaete scutata

Summary Cells of the charophycean alga,Coleochaete scutata active in cell wall formation were freeze fractured in the search for cellulose synthesizing complexes (TCs) since this alga is considered to be among the most advanced and a progenitor to land plant evolution. We have found a new TC which consists of two geometrically distinctive particle complexes complementary to one another in the plasma membrane and occasionally associated with microfibril impressions. In the E-fracture face is found a cluster of 8–50 closely packed particles, each with a diameter of 5–17 nm. Most of these particles are confined within an 80 nm circle. In the P-fracture face is found an 8-fold symmetrical arrangement of 10 nm particles circumferentially arranged around a 28 nm central particle. The TCs ofC. scutata are quite distinctive from the rosette/globule TCs of land plants. The 5.5×3.1 nm microfibril inC. scutata is also distinctive from the 3.5×3.5 nm microfibril typical of land plants. The phylogenetic implications of this unique TC in land plant evolution are discussed.     

Ultrastructure of freeze-substituted pollen tubes ofLilium longiflorum

Summary In view of the importance of the lily pollen tube as an experimental model and the improvements in ultrastructural detail that can now be attained by the use of rapid freeze fixation and freeze substitution (RF-FS), we have reexamined the ultrastructure of these cells in material prepared by RF-FS. Several previously unreported details have been revealed: (1) the cytoplasm is organized into axial slow and fast lanes, each with a distinct structure; (2) long, straight microtubule (MT) and microfilament (MF) bundles occur in the cytoplasm of the fast lanes and are coaligned with every organelle present; (3) the cortical cytoplasm contains complexes of coaligned MTs, MFs, and endoplasmic reticulum (ER); (4) the cortical ER is arranged in a tight hexagonal pattern and individual elements are closely appressed to the plasma membrane with no space between; (5) mitochondria and ER extend into the extreme apex along the flanks of the pollen tube, and vesicles and ER are packed into an inverted cone-shaped area at the center of the apex; (6) MF bundles in the tip region are fewer, finer, and in random orientation in comparison to those of the fast lanes; (7) the generative cell (GC) cell wall complex contains patches of plasmodesmata; (8) The GC cytoplasm contains groups of spiny vesicles that are closely associated with and seem to be fusing with or pinching off from mitochondria, and (9) the vegetative nucleus (VN) contains internal MT-like structures as well as numerous cytoplasmic MTs associated with its membrane and also located between the VN and GC.Abbrevations CF chemical fixation - ER endoplasmic reticulum - GC generative cell - MF microfilament - MT microtubule - PD plasmodesmata - PM plasma membrane - RF-FS rapid freeze fixation-freeze substitution - VN vegetative nucleus     

Effects of 2,6-dichlorobenzonitrile and Tinopal LPW on the structure of the cellulose synthesizing complexes ofVaucheria hamata

Summary The effects of 2,6-dichlorobenzonitrile (DCB, a known inhibitor of cellulose synthesis) and Tinopal LPW (TPL, an agent which disrupts glucan crystallization) on the structure of cellulose synthesizing complexes (terminal complexes, TCs) in the xanthophycean algaVaucheria hamata were investigated. DCB (10 M) inhibits nascent fibril formation from the TC subunit (based on the absence of impressions) although it does not alter the overall shape of the rectangular TC during the short treatment of 20 min. With a prolonged treatment (60 min), the arrangement of TC subunits becomes disordered, and particles generally exhibited as doublets of subunits are released from each other. DCB also interferes with the formation of the overall shape of the TC although it does not disturb the conversion into TC rows of the subunits (the zymogenic precursor of the TC) packed in the globules. A 15 min treatment with TPL (1 mM) destroys the TC integrity by reducing the subunits into small fragments or particulate aggregates. The particulate rows of the TC are interrupted at many points, and fragments and particulate aggregates are dispersed by prolonged treatment (45 min) with TPL. Unlike DCB, TPL inhibits the conversion of globule subunits into TC rows. New insights on the structural characteristics necessary for cellulose microfibril assembly and possible mechanisms for the biogenesis of theVaucheria TC come from these data.Abbreviations DCB 2,6-dichlorobenzonitrile - TPL Tinopal LPW - TC terminal complex     

Relaxation process after the cooling jump across the pretransition of dipalmitoylphosphatidylcholine bilayers

The relaxation processes involved in the pretransition of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles were investigated by the measurements of transmitted light intensity under crossed polars and freeze fracture electron microscopy. Temperature jump experiments from the ripple (P_β′) phase into the gel (L_β′) phase suggest that there must be at least three relaxation stages, one of which has a relaxation time much shorter than those described previously. Freeze fracture electron microscopy observations of the surface structure during the relaxation process reveals that the defects in the ripple pattern, i.e. the open terminal ends of each ripple ridge line, may be the primary origin of the growth of the L_β′ phase, and that the phase conversion may take place exclusively at the pointed end of a ripple ridge line. Thus the L_β′ phase grows almost one-dimensionally between ripple structures.     

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca²⁺ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca²⁺ gradient.Abbreviations ER endoplasmic reticulum - MES 2-[N-morpholino] ethanesulfonic acid - MFs microfilaments     

Second messenger and cAMP-dependent protein kinase responses to dehydration and anoxia stresses in frogs

The effects of whole body dehydration (up to 40% of total body water lost) or anoxia exposure (up to 2 days under N₂ gas) at 5 °C on tissue levels of adenosine 3′–5′ cyclic monophosphate (cAMP) and the percentage of cAMP-dependent protein kinase present as the free catalytic subunit (PKAc), as well as the levels of the protein kinase C (PKC) second messenger, inositol 1,4,5-trisphosphate (IP₃), were assessed in two anurans, the freeze-tolerant wood frog, Rana sylvatica, and the freeze-intolerant leopard frog, Rana pipiens. Dehydration of wood frogs resulted in a rapid elevation of liver cAMP and PKAc; cAMP was 3.4-fold greater than control values in animals that had lost 5% of total body water, whereas PKAc was elevated threefold in 20% dehydrated frogs. These results indicate protein kinase A mediation of the liver glycogenolysis and hyperglycemia that is induced by dehydration in this species. Skeletal muscle PKAc content also rose with dehydration but neither cAMP nor PKAc was affected by dehydration in leopard frog tissues. Anoxia exposure had different effects on signal transduction systems. PKAc was elevated after 1 h anoxia in R. sylvatica brain and was sustained over time but the enzyme was unaffected in other organs; by contrast, R. pipiens showed variable responses by PKAc to anoxia in three organs. Both species showed rapid (within 30 min) and large (3 to 7.8-fold) increases in IP₃ in liver of anoxic frogs that decreased slowly with continued anoxia. IP₃ also increased quickly in heart of anoxia-exposed wood frogs. This suggests that PKC may mediate various metabolic adjustments that promote hypoxia/anoxia resistance such as coordinating metabolic rate depression. A progressive rise in liver IP₃ during dehydration in wood frogs (reaching fourfold higher than controls in 40% dehydrated animals) may also mediate similar hypoxia resistance adaptations under this stress since anurans experience progressive hypoxia due to increased blood viscosity when water loss reaches high values. The patterns of second messenger and PKAc changes in wood frog liver during dehydration closely parallel the changes seen in these same parameters during natural freezing suggesting that the freeze tolerance of selected terrestrially hibernating anurans may have evolved out of various anuran mechanisms of dehydration resistance. Accepted: 2 January 1997     

Maintenance of the supercooled state in the gut fluid of overwintering pyrochroid beetle larvae, Dendroides canadensis : role of ice nucleators and antifreeze proteins

  The effect of gut fluid ice nucleators and antifreeze proteins on maintenance of supercooling was explored in fire-colored beetle larvae, Dendroides canadensis, via seasonal monitoring of supercooling points, antifreeze protein activity and ice nucleator activity of gut fluid and/or larvae. During cold hardening in the field, freeze-avoiding larvae evacuated their guts and depressed larval supercooling points. Analysis of gut fluid indicated supercooling points and ice nucleator activity decreased, whereas antifreeze protein activity increased as winter approached. Suspensions of bacteria isolated from guts of feeding larvae collected in spring/summer had higher supercooling points than those from midwinter-collected non-feeding larvae, suggesting bacterial ice nucleators are removed from midwinter gut fluid. The ice nucleation active bacterium Pseudomonas fluorescens was isolated from gut fluid of feeding larvae but was absent in winter. When mixed with purified D.␣canadensis hemolymph antifreeze proteins (structurally similar and/or identical to those in gut fluid), the cumulative ice nucleus spectra of P. fluorescens suspensions were shifted to lower temperatures indicating an inhibitory effect on the bacteria's ice-nucleating phenotype. By extending larval supercooling capacity, both gut clearing and masking of bacterial ice nucleators by antifreeze proteins may contribute to overwintering survival in supercooled insects. Accepted: 8 August 1996     

Impact of winter climate change on nitrogen biogeochemistry in forest ecosystems: A synthesis from Japanese case studies

Nitrogen (N) is a critical ecological and environmental indicator under changing environments. The impact of winter climate change on N biogeochemical processes in forest ecosystems has gained increasing recognition. Decreasing snowfall has caused a decrease in the heat insulation properties of the snowpack, resulting in an increase in the frequency and magnitude of freezing and thawing cycles in surface soil, where biological processes are most active. Here I synthesize recent research findings from integrated field observations and experiments conducted in northern Japan and compare these results with previous research outcomes from other regions to identify current research gaps and develop the next research agenda to further advance our understanding of this complex problem. Japanese case studies indicated that net ammonium production (ammonification) was mostly dominant in terms of available soil N fertility in cold environments and was sensitive to the increase in soil freezing and thawing cycles because of the decreased snowpack. On the other hands, nitrate dynamics were more stable or conservative than those of ammonium. The soil characteristics (i.e., N pool and microbial activities) were significant explanatory factors of the responses of soil N dynamics and N leakage among different soils to increased freezing–thawing cycles at watershed and national scale. This synthesis indicates that winter climate change had significant impacts on soil N biogeochemistry (such as soil N pool size and microbial N transformation) during the winter and snowmelt season and also during the following growing season. Several research gaps and possible research topics (path dependency and soil microbial community composition) are also presented by synthesizing the current research findings. Further field experiments and observations quantifying the pools and fluxes of inorganic N with modeling analysis under freeze–thaw environments would contribute to increase the understandings of N transformation processes under winter climate change.     

A method for rapid freeze fixation of plant cells

Summary We describe here an apparatus that permits rapid freeze fixation of whole cells, which are then prepared by freeze substitution and resin embedment for examination in the EM. The freezing device utilizes a rotary solenoid that rapidly plunges the specimen holder, a formvar-film-covered thin wire loop, into a well of stirred liquid propane at –180C. The rotary solenoid allows for an adjustable, repeatable immersion rate. Substitution takes place at –80 C in acetone with 2% OsO₄ and is followed by en bloc staining in either hafnium tetrachloride or uranyl acetate. We have utilized these techniques on plant cells, for which there has been relatively little published work when compared to other organisms. The results show that, with the versatile specimen holder and rapid, repeatable immersion rates, different cell types, including pollen, stamen hairs, and germinating moss spores, can be rapidly frozen with repeatable success. The improved preservation achieved with rapid freeze fixation over conventional chemical fixation reveals itself particularly in the structure of the plasmamembrane, the cytoskeleton, chromatin, and certain endomembrane systems.     

Supramolecular organization of chlorosomes (chlorobium vesicles) and of their membrane attachment sites in Chlorobium Limicola

The photosynthetic green bacterium Chlorobium limicola 6230 has been examined by freeze-fracture electron microscopy to investigate the size, form, distribution and supramolecular architecture of its chlorosomes (chlorobium vesicles) as well as the chlorosome attachment sites on the cytoplasmic membrane. The oblong chlorosomes that underlie the cytoplasmic membrane show a considerable variation in size from about 40 × 70 nm to 100 × 260 nm and exhibit no particular orientation. The chlorosome core, which appears to be hydrophobic in nature, contains between 10 and 30 rod-shaped elements (approx. 10 nm in diameter) surrounded by an unetchable matrix. The rod elements are closely packed and extend the full length of the chlorosome. Separating the chlorosome core from the cytoplasm is a approx. 3 nm thick lipid-like envelope layer, which exhibits no substructure. A 5–6 nm thick, crystalline baseplate connects the chlorosome to the cytoplasmic membrane. The ridges of the baseplate lattice make an angle of between 40° and 60° with the longitudinal axis of the chlorosome and have a repeating distance of approx. 6 nm. In addition, each ridge exhibits a granular substructure with a periodicity of approx. 3.3 nm. The cytoplasmic membrane regions adjacent to the baseplates are enriched in large (greater than 9 nm) intramembrane particles, most of which belong to approx. 10 nm and approx. 12.5 nm particle size categories. Each chlorosome attachment site contains between 20 and 30 very large (greater than 12.0 nm diameter) intramembrane particles.The following interpretive model of a chlorosome is discussed in terms of biophysical, biochemical and structural information reported by others: it is proposed that the bacteriochlorophyll c (BChl c; chlorobium chlorophyll) is located in the rod elements of the core and that it is complexed with specific proteins. The cytoplasm-associated envelope layer is depicted as consisting of a monolayer of galactosyl diacylglycerol molecules. BChl a-protein complexes in a planar lattice configuration most likely make up the crystalline baseplate. The greater than 12-nm particles in the chlorosome attachment sites of the cytoplasmic membrane, finally, may correspond to complexes containing a reaction center and non-crystalline light-harvesting BChl a. The crystalline nature of the baseplate is consistent with the notion that it serves two functions: besides transferring excitation energy to the reaction centers it could also function as a distributor of this energy amongst the reaction centers.