Biochemical and Immunocytological Characterizations of Arabidopsis Pollen Tube Cell Wall

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.Fertilization of flowering plants requires the delivery of the two sperm cells, carried by a fast growing tip-polarized pollen tube, to the egg cell. In plants with dry stigma and solid style such as Arabidopsis (Arabidopsis thaliana), this process begins with the deposition and specific adhesion of the pollen grains on the stigmatic tissue, subsequent hydration of the pollen grains, and germination of pollen tubes (Palanivelu and Preuss, 2000). Pollen tubes invade the papillae cell wall of the stigma, enter the short style, and grow through the apoplast of the specialized transmitting tract (TT) that is filled with a nutrient-rich extracellular matrix (Kandasamy et al., 1994; Lennon et al., 1998). During this invasive growth, pollen tubes are guided to the ovules via signals that need to pass through the cell wall to reach their membrane-associated or intracellular targets (Lord and Russell, 2002; Kim et al., 2003; Boavida et al., 2005; McCormick and Yang, 2005; Johnson and Lord, 2006). In plant species with wet stigma and hollow style such as lily (Lilium longiflorum), adhesion between the pollen tube wall and the TT epidermis extracellular matrix is important for the growth of the pollen tubes toward the ovules (Mollet et al., 2000, 2007; Park et al., 2000; Chae et al., 2007). In addition to being the interface between the tube cells and the surroundings (female sporophyte or culture medium), the pollen tube wall also controls the cell shape, protects the generative cells, and allows resistance against turgor pressure (Geitmann and Steer, 2006; Geitmann, 2010).Most of our knowledge on cell wall polymers of higher plants comes from investigations on vegetative organs in which cells have diffuse growth. The cell wall is mainly composed of polysaccharides (cellulose, hemicellulose, pectin, and occasionally callose, depending on the tissue) and proteoglycans (e.g. extensin and arabinogalactan proteins [AGPs]) forming a complex network with processing enzymes.Pectins are complex wall macromolecules with uncertain supramolecular organization (Vincken et al., 2003) consisting of homogalacturonan (HG) that can be methylesterified and acetylesterified, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (Carpita and McCann, 2000). HG is a polymer of repeated units of (1→4)-α-d-GalUA that can be cross-linked with calcium upon block-wise action of pectin methylesterases (PMEs) on methylesterified HG (Micheli, 2001). RG-II has the same homopolymer backbone as HG but is substituted with four different oligosaccharides composed of unusual sugars, such as apiose, aceric acid, and 3-deoxy-d-manno-2-octulosonic acid, of unknown function (for review, see Caffall and Mohnen, 2009). RG-I consists of the repeating disaccharide (1→4)-α-d-GalUA-(1→2)-α-l-Rha, with a wide variety of side chains attached to the rhamnosyl residues, ranging from monomers to large oligosaccharides such as (1→4)-β-d-galactan, (1→5)-α-l-arabinan, and/or type I arabinogalactan (Caffall and Mohnen, 2009).Xyloglucan (XyG) is the major hemicellulosic polysaccharide of the primary wall of flowering plants. Classic XyG consists of a (1→4)-β-d-glucan backbone substituted with Xyl, Gal-Xyl, or Fuc-Gal-Xyl motifs, which correspond, according to the one-letter code proposed by Fry et al. (1993), to X, L, and F, respectively, G being the unsubstituted glucosyl residue of the glucan backbone. The main XyG fragments released after endo-glucanase treatment of the cell wall from wild-type Arabidopsis vegetative organs are generally XXXG, XXLG/XLXG, XXFG, and XLFG (Zablackis et al., 1995; Lerouxel et al., 2002; Nguema-Ona et al., 2006; Obel et al., 2009). In addition, O-acetylation of XyG can occur, most generally on the galactosyl residues, but its biological function is unknown (Cavalier et al., 2008). In the primary wall, XyG interacts with cellulose microfibrils via hydrogen bonds and participates in the control of cell expansion (Cosgrove, 1999).AGPs and extensin belong to the Hyp-rich glycoproteins superfamily with very high levels of type II arabinogalactan glycosylation (Nothnagel, 1997; Showalter, 2001). These proteoglycans have been implicated in many aspects of plant development, including cell expansion, cell signaling and communication, embryogenesis, wound response, and pollen tube guidance (Wu et al., 1995; Nothnagel, 1997; Seifert and Roberts, 2007; Driouich and Baskin, 2008).Despite the importance of pollen tubes for the delivery of the sperm cells to the egg, little is known about the underlying molecular mechanisms that regulate the mechanical interaction of pollen tubes with female floral tissues. There are very scarce data concerning the different components of the pollen tube cell wall. Past approaches to characterize the pollen tube cell wall are limited to a few plant genera, including Camellia (Nakamura and Suzuki, 1981), Lilium (Jauh and Lord, 1996; Mollet et al., 2002), Nicotiana (Rae et al.,1985; Li et al., 1995; Ferguson et al., 1998; Qin et al., 2007), Pinus (Derksen et al., 1999), and Zea (Rubinstein et al., 1995), and are mostly based on immunocytochemistry. These studies revealed that, depending on the species, the pollen tube cell wall contains epitopes that are found in the polymers described above, including HGs with varying levels of methylesterification, AGPs, extensin-like proteins, and low amounts of cellulose. Unlike most other plant cells, callose, a (1→3)-β-glucan, is predominant and is deposited in the wall back from the tip. Moreover, it is deposited at regular intervals to form callose plugs that maintain the tube cell in the apical expanding region of the tube and separate the viable from the degenerating region of the tube (for review, see Geitmann and Steer, 2006). Only a few reports have investigated the pollen tube of the model plant Arabidopsis. They have focused either on in vivo-grown or on in vitro-grown pollen tubes using monoclonal antibodies (MAbs) directed against a subset of cell wall epitopes present in HG, XyG, and AGPs (Lennon and Lord, 2000; Freshour et al., 2003; Pereira et al., 2006), but quantitative chemical analyses are lacking. This lack of information is most likely due to the fact that substantial amounts of pollen tube material are needed for chemical analysis, and a reproducible and efficient method for liquid culture of Arabidopsis pollen tubes had not been established until recently (Boavida and McCormick, 2007; Bou Daher et al., 2009).Here, we report the composition and localization of different cell wall polymers of in vitro-grown wild-type Arabidopsis pollen tubes based on biochemical analyses coupled to immunocytochemical investigations both at light and transmission electron microscopy (TEM) levels using recently developed MAbs. Our results show distinct patterns of labeling (tip, whole tube, and shank of the tube) depending on the recognized epitope. The most striking observations are (1) the abundance of (1→5)-α-l-arabinan in the tube wall (greater than 40 mol % of Ara), mostly localized, with LM6 and LM13, in the outer wall layer of the tube and (2) an atypical XyG matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profile with over 68% of the oligosaccharide fragments being O-acetylated.     

Global Conservation of Protein Status between Cell Lines and Xenografts

Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.     

Revisiting the Voronoi description of protein-protein interfaces

We developed a model of macromolecular interfaces based on the Voronoi diagram and the related alpha-complex, and we tested its properties on a set of 96 protein-protein complexes taken from the Protein Data Bank. The Voronoi model provides a natural definition of the interfaces, and it yields values of the number of interface atoms and of the interface area that have excellent correlation coefficients with those of the classical model based on solvent accessibility. Nevertheless, some atoms that do not lose solvent accessibility are part of the interface defined by the Voronoi model. The Voronoi model provides robust definitions of the curvature and of the connectivity of the interfaces, and leads to estimates of these features that generally agree with other approaches. Our implementation of the model allows an analysis of protein-water contacts that highlights the role of structural water molecules at protein-protein interfaces.     

Prerequisites for ubiquinone analogs to prevent mitochondrial permeability transition-induced cell death

The permeability transition pore (PTP) is a mitochondrial inner membrane channel involved in cell death. The inhibition of PTP opening has been proved to be an effective strategy to prevent cell death induced by oxidative stress. Several ubiquinone analogs are known to powerfully inhibit PTP opening with an effect depending on the studied cell line. Here, we have studied the effects of ubiquinone 0 (Ub₀), ubiquinone 5 (Ub₅) and ubiquinone 10 (Ub₁₀) on PTP regulation, H₂O₂ production and cell viability in U937 cells. We found that Ub₀ induced both PTP opening and H₂O₂ production. Ub₅ did not regulate PTP opening yet induced H₂O₂ production. Ub₁₀ potently inhibited PTP opening yet induced H₂O₂ production. Both Ub₀ and Ub₅ induced cell death, whereas Ub₁₀ was not toxic. Moreover, Ub₁₀ prevented tert-butyl hydroperoxide-induced PTP opening and subsequent cell death. We conclude that PTP-inhibitor ubiquinone analogs are able to prevent PTP opening-induced cell death only if they are not toxic per se, which is the case when they have no or low pro-oxidant activity.     

Spatial resolution impacts projected plant responses to climate change on topographically complex islands

Aim

Understanding how grain size affects our ability to characterize species responses to ongoing climate change is of crucial importance in the context of an increasing awareness for the substantial difference that exists between coarse spatial resolution macroclimatic data sets and the microclimate actually experienced by organisms. Climate change impacts on biodiversity are expected to peak in mountain areas, wherein the differences between macro and microclimates are precisely the largest. Based on a newly generated fine-scale environmental data for the Canary Islands, we assessed whether data at 100 m resolution is able to provide more accurate predictions than available data at 1 km resolution. We also analysed how future climate suitability predictions of island endemic bryophytes differ depending on the grain size of grids.

Location

Canary Islands.

Time period

Present (1979–2013) and late-century (2071–2100).

Taxa

Bryophytes.

Methods

We compared the accuracy and spatial predictions using ensemble of small models for 14 Macaronesian endemic bryophyte species. We used two climate data sets: CHELSA v1.2 (~1 km) and CanaryClim v1.0 (100 m), a downscaled version of the latter utilizing data from local weather stations. CanaryClim also encompasses future climate data from five individual model intercomparison projects for three warming shared socio-economic pathways.

Results

Species distribution models generated from CHELSA and CanaryClim exhibited a similar accuracy, but CanaryClim predicted buffered warming trends in mid-elevation ridges. CanaryClim consistently returned higher proportions of newly suitable pixels (8%–28%) than CHELSA models (0%–3%). Consequently, the proportion of species predicted to occupy pixels of uncertain suitability was higher with CHELSA (3–8 species) than with CanaryClim (0–2 species).

Main conclusions

The resolution of climate data impacted the predictions rather than the performance of species distribution models. Our results highlight the crucial role that fine-resolution climate data sets can play in predicting the potential distribution of both microrefugia and new suitable range under warming climate.     

alpha-Glucosidase inhibitory constituents from stem bark of Terminalia superba (Combretaceae)

The CH(2)Cl(2)/CH(3)OH (1/1) extract of the dried stem bark of Terminalia superba afforded two compounds, (7S,8R,7'R,8'S)-4'-hydroxy-4-methoxy-7,7'-epoxylignan and meso-(rel 7S,8R,7'R,8'S)-4,4'-dimethoxy-7,7'-epoxylignan along with 11 known compounds. The structures of the compounds were established by analysing the spectroscopic data and also comparing it with the data of previously known analogues. All the isolated compounds were evaluated for their glycosidase inhibition activities. Gallic acid and methyl gallate showed significant alpha-glucosidase inhibition activity.     

Surface‐enhanced Raman scattering‐active photonic crystal fiber probe: Towards next generation liquid biopsy sensor with ultra high sensitivity

The tremendous enhancement factors that surface‐enhanced Raman scattering (SERS) possesses coupled with the flexibility of photonic crystal fibers (PCFs) pave the way to a new generation of ultrasensitive biosensors. Thanks to the unique structure of PCFs, which allows direct incorporation of an analyte into the axially aligned air channels, interaction between the analyte and excitation light could be increased many folds leading to flexible, reliable and sensitive probes that can be used in preclinical or clinical biosensing. SERS‐active PCF probes provide unique opportunity to develop an opto‐fluidic liquid biopsy needle sensor that enables one‐step integrated sample collection and testing for disease diagnosis. Specificity being a key parameter to biosensors, the PCF inside the biopsy needle could be functionalized with targeting moieties to detect specific biomarkers. In this review article, we present some of the most promising recent biosensors based on PCFs including hollow‐core PCFs, suspended‐core PCFs and side‐channel PCFs. We provide a wide range of applications of such platform using Raman spectroscopy, label free SERS or labeled SERS detection and analyze some of the main challenges to be addressed for translating it to a clinically viable next generation sensitive biopsy needle sensing probe.