GRA9, a new Toxoplasma gondii dense granule protein associated with the intravacuolar network of tubular membranes

Important components of the parasitophorous vacuole in which the intracellular protozoan parasite Toxoplasma gondii develops, comprise proteins secreted from apicomplexan specific secretory organelles named the dense granules. Here, we confirm by immunofluorescence and by cryo-electron microscopy that the recently isolated B10 protein (318 amino acids, 41kDa) is a new dense granule protein that should now be referred to as GRA9. Within the vacuolar compartment, GRA9, like GRA2, GRA4 and GRA6, associates with the network of tubular membranes connected to the parasitophorous vacuole delimiting membrane. Like the other GRA proteins, GRA9 is secreted into the vacuole from the anterior end of the parasite. However, unlike GRA2 or GRA6, GRA9 does not transit by the posterior invaginated pocket of the parasite where the network first assembles. Within the dense granules, GRA9 exists in both a soluble and an insoluble state. Like the other GRA proteins, GRA9 is secreted as a soluble form only and like most of the GRA proteins, two forms of GRA9 of the similar molecular weight are detected within the vacuolar space: a soluble form and a membrane associated form. The dual properties of GRA9 are not only ascribed by the presence of amphipathic and hydrophobic alpha-helices but also by the fact that the protein is mainly hydrophilic.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

OCCURRENCE OF THREE NITZSCHIA (BACILLARIOPHYCEAE) TAXA WITHIN COLONIES OF TUBE-FORMING DIATOMS1

In benthic samples from the unchannelized Missouri River, the diatoms Nitzschia dissipata (Kütz.) Grun., N. filiformis (W. Sm.) Schütt and N. pseudofonticola Hust. were observed within the mucilage tubes of four tube-forming diatoms: Cymbella prostrata (Berk.) Cl., C. prostrata var. auerswaldii (Rabh.) Reim., Navicula tripunctata var. schizonemoides (V.H.) Patr., and Nitzschia filiformis. Microscopical observations of live and preserved specimens indicated that “invasion” by Nitzschia occurred primarily in older tubes. Data are presented on the environmental conditions in which the tube-formers and their cohabitants have been found.     

Comparison of tomato root distributions by minirhizotron and destructive sampling

Calibration of minirhizotron data against root length density (RLD) was carried out in a field trial where three drip irrigation depths: surface (R0) and subsurface, 0.20 m (RI) and 0.40 m depth (RII) and two processing tomato cultivars: `Brigade' (CI) and `H3044' (CII) were imposed. For each treatment three minirhizotron tubes were located at 10, 37.5 and 75 cm of the way from one plant row to the next. Roots intersecting the minirizotrons walls were expressed as root length intensity (L _a) and number of roots per unit of minirhizotron wall area (N _ra). Root length density (RLD) was calculated from core samples taken for each minirhizotron tube at two locations: near the top of the minirhizotron (BI) and 15 cm apart from it, facing the minirhizotron wall opposite the plant row (BII). Minirhizotron data were regressed against RLD obtained at BI and BII and with their respective means. The results show that for all the situations studied, better correlations were obtained when RLD was regressed with L _a than with N _ra. Also was evident that the relationship between L _a and RLD was strongly influenced by the location of soil coring. RLD was correlated with L _a trough linear and cubic equations, having the last ones higher determination coefficients. For instance at 10 cm from the plant row when values from the top layer (0–40 cm) were analysed separately, L _a was significantly regressed with RLD measured at BII and described by the equations: RLD = 0.5448 + 0.0071 L _a (R ² = 0.51) and RLD = 0.4823 + 0.0074L _a + 8×10^–5 L _a ² – 5×10^–7 L _a ³ (R ² = 0.61). Under the 40 cm depth the highest coefficients of determination for the linear and cubic equations were respectively 0.47 and 0.88, found when L _a was regressed with RLD measured at BI. For minirhizotrons located at 75 cm from the plant row and for location BI it was possible to analyse jointly data from all depths with coefficients of determination of 0.45 and 0.59 for the linear and cubic equations respectively.     

Electron-Microscopic Evidence of the Vacuolar Nature of Phloem Exudate

We performed electron-microscopic examination of structural diurnal changes in the lumen of sieve tubes and the vacuolar system of corresponding companion cells and changes induced by the experimental blockage of assimilate export from the leaf by its cold-girdling. For these investigations, Cucurbita pepo L. and Helianthus annuus L. plants were used, that is, plant species from groups of symplastic and apoplastic plants, which differ in the type of companion cells and a mode of phloem terminal loading. The examinations showed the complete identity of changes in the electron texture of the sieve-tube lumens and companion-cell vacuoles in both plant species in the course of a day, when the level of assimilates changed, or after export blockage. Similar changes in the structure of the vacuolar labyrinths were stated in the companion cells under normal conditions and after cold-girdling, as related to the rate of sieve-tube loading with the vacuolar exudate. Vacuolar expansion and starch accumulation developing in response to changes in the assimilate level in the evening and after cold blockage of the assimilate export occurred in different types of cells, as dependent on their position in the symplast domains. However, the rate of the process similarly depended on the balance between assimilate synthesis and export. Synchronous changes in the texture of the sieve-tube lumen and companion-cell vacuoles were observed within each complex, but asynchronous changes occurred in different complexes. We suggested this phenomenon for recognizing the particular complexes, when they are grouped in a bundle. We observed no signs of cytoplasm or protein synthetic machinery in the sieve tubes. We concluded that the sieve-tube lumen and vacuoles of companion cells are common in nature. Similar electron texture of the images of the companion-cell vacuolar labyrinth and tube lumens, their connection through the lateral sieve fields, morphological modifications of the companion-cell vacuolar system as dependent on the activity of sieve tube loading—all of these facts imply the continuity of these transport compartments and fluxes in them and the similarity in the composition of the exudates from companion-cell vacuoles and phloem tubes.     

Tobacco pollen tubes – a fast and easy tool for studying lipid droplet association of plant proteins

In recent years, lipid droplets have emerged as dynamic organelles rather than inactive storage sites for triacylglycerol. The number of proteins known to be associated with lipid droplets has increased, but remains small in comparison with those found with other organelles. Also the mechanisms of how lipid droplets are recognized and bound by proteins need deeper investigation. Here, we present a fast, simple and inexpensive approach to assay proteins for their association with lipid droplets in vivo that can help to screen protein candidates or mutated variants of proteins for their association in an efficient manner. For this, a system to transiently transform Nicotiana tabacum pollen grains was used because these naturally contain lipid droplets. We designed vectors for fast cloning of genes as fusions with either mVenus or mCherry. This allowed us to assay colocalization with lipid droplets stained with Nile Red and Bodipy 505/515, respectively. We successfully tested our system not only for proteins from Arabidopsis thaliana, but also for proteins from the moss Physcomitrella patens and the alga Chlamydomonas reinhardtii. The small size of the vector used allows easy exchange of codons by site‐directed mutagenesis. We used this to show that two proline residues in the proline knot of a caleosin are not essential for the binding of lipid droplets. We also demonstrated that peroxisomes are not associated with the lipid droplets in tobacco pollen tubes, which reduces the risk of false interpretation of microscopic data in our system.     

Covalently bound wall proteins of pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination in Lilium longiflorum

Summary A method was worked out using trifluoromethanesulfonic acid (TFMS) as a reagent to split the covalently bound proteins, which are NaCl insoluble, from pollen tube walls of Lilium longiflorum, leaving the peptide bonds essentially intact. After electrophoretic separation, comparisons were made among these proteins from pollen grains and pollen tubes grown in vitro and in styles after self- and cross-pollination. It was found that a) the patterns of covalently bound wall proteins were different between tubes grown in vitro and in vivo; b) fewer bands were found in covalently bound wall proteins than that in noncovalently bound proteins; c) the bands remained almost the same no matter whether the tubes had been cross pollinated or self pollinated, indicating that while the noncovalently bound proteins were involved in incompatibility as shown in the previous paper, the covalently bound proteins may only serve as a structural component, having little to do with incompatibility.     

Among-species differences in pollen quality and quantity limitation: implications for endemics in biodiverse hotspots

Background and Aims

Insufficient pollination is a function of quantity and quality of pollen receipt, and the relative contribution of each to pollen limitation may vary with intrinsic plant traits and extrinsic ecological properties. Community-level studies are essential to evaluate variation across species in quality limitation under common ecological conditions. This study examined whether endemic species are more limited by pollen quantity or quality than non-endemic co-flowering species in three endemic-rich plant communities located in biodiversity hotspots of different continents (Andalusia, California and Yucatan).

Methods

Natural variations in pollen receipt and pollen tube formation were analysed for 20 insect-pollinated plants. Endemic and non-endemic species that co-flowered were paired in order to estimate and compare the quantity and quality components of pre-zygotic pollination success, obtained through piecewise regression analysis of the relationship between pollen grains and pollen tubes of naturally pollinated wilted flowers.

Key Results

Pollen tubes did not frequently exceed the number of ovules per flower. Only the combination of abundant and good quality pollen and a low number of ovules per flower conferred relief from pre-zygotic pollen limitation in the three stochastic pollination environments studied. Quality of pollen receipt was found to be as variable as quantity among study species. The relative pollination success of endemic and non-endemic species, and its quantity and quality components, was community dependent.

Conclusions

Assessing both quality and quantity of pollen receipt is key to determining the ovule fertilization potential of both endemic and widespread plants in biodiverse hotspot regions. Large natural variation among flowers of the same species in the two components and pollen tube formation deserves further analysis in order to estimate the environmental, phenotypic and intraindividual sources of variation that may affect how plants evolve to overcome this limitation in different communities worldwide.