Additional file 4: Table S4. of Transcriptomic analysis reveals the key immune-related signalling pathways of Sebastiscus marmoratus in response to infection with the parasitic ciliate Cryptocaryon irritans

Immune systems with differentially expressed genes between C. irritans-infected (B, C, D, E) and non-infected (A) S. marmoratus. (DOCX 133 kb

A domain-reduction approach to bridging-scale simulation of one-dimensional nanostructures

We present a domain-reduction approach for the simulation of one-dimensional nanocrystalline structures. In this approach, the domain of interest is partitioned into coarse and fine scale regions and the coupling between the two is implemented through a bridging-scale interfacial boundary condition. The atomistic simulation is used in the fine scale region, while the discrete Fourier transform is applied to the coarse scale region to yield a compact Green‟s function formulation that represents the effects of the coarse scale domain upon the fine/coarse scale interface. This approach facilitates the simulations for the fine scale, without the requirement to simulate the entire coarse scale domain. After the illustration in a simple 1D problem and comparison with analytical solutions, the proposed method is then implemented for carbon nanotube structures. The robustness of the proposed multiscale method is demonstrated after comparison and verification of our results with benchmark results from fully atomistic simulations

<i>Ann5</i> overexpression did not influence pollen germination and tube growth in response to LatB.

<p>(A) Representative images of pollen grains and tubes from the Lat52-GFP, Lat52-Ann5-GFP 1 and 3 and Ann5Pro-Ann5 1 homozygous lines growing on germination medium containing 1 or 2 nM LatB for 3 h. Bar  = 50 µm. (B) Pollen germination ratios of the Lat52-GFP, Lat52-Ann5-GFP 1 and 3 and Ann5Pro-Ann5 1 homozygous lines after the pollen had germinated for 3 h in the presence of 1 or 2 nM LatB. Values represent the means ± _SD. Eight hundred pollen grains and tubes were counted. (C) Pollen tube lengths of the Lat52-GFP, Lat52-Ann5-GFP 1 and 3 and Ann5Pro-Ann5 1 homozygous lines after the pollen germinated normally for 2 h and then for 2 h in the presence of 2.5 or 5 nM LatB. Values represent the means ± _SD. Five hundred pollen tubes were measured.</p

Ann5 binds to negatively charged phospholipids and this association is stimulated by Ca²⁺.

<p>(A) Multiple sequence alignment of the deduced amino acid sequences of repeats I and IV from <i>Arabidopsis thaliana</i> AnnAt1 to 8. Ca²⁺-binding sites of type II GXGTD-(37 residues)-E/D are indicated by blue and red shadows. The residues with red asterisks indicate the locations of PCR-based site-directed mutagenesis. The mutants of repeats I and IV were named Ann5G26EG28E and Ann5G257EG259E, respectively, and the conserved glycine residues were replaced by glutamic acid residues. (B) Predicted three-dimensional structure of the Ann5 protein. The annexin core domain was composed of four homologous repeats that are colored in red (repeat I), green (repeat II), yellow (repeat III) and blue (repeat IV) and shaped as a slightly curved disc. The convex surface on which calcium ions bind (magenta spheres) participates in peripheral membrane binding. The type II Ca²⁺-binding sites are labeled with sticks, and the ribbon illustrates the highly α-helical structure. (C) Phospholipid-binding properties of the recombinant His6-Ann5, His6-Ann5G26EG28E, His6-Ann5G257EG259E and His6-Ann5G26EG28EG257EG259E proteins. The individual protein (50 µg) was incubated with liposomes (1∶1 PC/PS) in the presence of increasing Ca²⁺ concentrations. (-) denotes that the reaction mixtures contained neither liposomes nor Ca²⁺. (D) Comparison of the Ca²⁺-dependent phospholipid binding abilities of Ann5 and its mutants using densitometry analysis of the signal intensities of blots as described in (C). The phospholipid binding amount of Ann5 in 200 µM Ca²⁺ is normalized as 1 (control). The relative intensities are displayed as fold-binding over the control. Values represent mean ± _SD (n = 6).</p

Identification of the constructs concerning Ann5.

<p>(A) Schematic representations of the constructs concerning Ann5. (B) <i>Ann5</i>-specific primers were designed to identify the level of overexpression of the <i>Ann5</i> transcript by RT-PCR performed for 25 cycles. Total RNA was extracted from open flowers of the wild-type (WT), Lat52-GFP, Lat52-Ann5-GFP 1, 2 and 3 and Ann5Pro-Ann5 1, 2 and 3 homozygous lines. <i>EF4A</i> was used as the control. (C) Pollen grains from the WT, Lat52-GFP and Lat52-Ann5-GFP 1, 2 and 3 lines were observed by epi-fluorescence microscopy with a GFP filter. Homo, homozygous lines; Hetero, heterozygous lines. Half of the pollen grains in the heterozygous lines expressed Ann5-GFP, and the other half did not express Ann5-GFP. Bar  = 20 µm. (D) Densitometry analysis of the results presented in (C). Fluorescence intensities (arbitrary units) across the whole pollen grain were calculated. More than 50 pollen grains for each line were quantified. Values represent the means ± _SD. *P<0.05 and **P<0.01 by Student's <i>t</i> test.</p

Analysis of the cytoplasmic streaming velocity in pollen tubes of the overexpression lines.

<p>(A) The velocity of cytoplasmic streaming in pollen tubes from the overexpression lines Lat52-GFP, Lat52-Ann5-GFP 1 and 3, Lat52-G26-GFP 1 and Lat52-G257-GFP 1 under normal conditions. Pollen grains were cultured on germination medium for 4 h. Cytosolic particles exhibiting continuous movement were selected at random for velocity analysis using the Image J software. Values represent the means ± _SD (n = 25). (B) The relative velocity of cytoplasmic streaming in pollen tubes from the Lat52-GFP, Lat52-Ann5-GFP 1 and 3, Lat52-G26-GFP 1 and Lat52-G257-GFP 1 overexpression lines in response to 0.6 µM BFA. The pollen had germinated normally for 2 h, followed by 2 h in the presence of 0.6 µM BFA. The velocity of the particles of each individual line in normal conditions was normalized to 1. The relative velocity was displayed as the proportion over the control. Values represent the means ± _SD. *P<0.05 and **P<0.01 (n = 25) by Student's <i>t</i> test.</p

Comparison of pollen germination rates and pollen tube lengths between the Lat52-GFP, Lat52-Ann5-GFP 1, Lat52-G26-GFP 1 and 2 and Lat52-G257-GFP 1 and 2 homozygous lines.

<p>For pollen germination rate, the pollen germinated for 3 h in the presence of 0.6 µM BFA. Six hundred pollen grains and tubes were counted. For pollen tube length, the pollen germinated normally for 2 h and then for 2 h in the presence of 0.6 µM BFA. Three hundred pollen tubes were measured. Values represent the means ± _SD. *P<0.05 and **P<0.01 by Student's <i>t</i> test.</p

<i>Ann5G26EG28E</i> or <i>Ann5G257EG259E</i> overexpression decreased the resistance of pollen to BFA compared with <i>Ann5</i> overexpression.

<p>Pollen grains and tubes from the Lat52-GFP, Lat52-Ann5-GFP 1, Lat52-G26-GFP 1 and 2 and Lat52-G257-GFP 1 and 2 homozygous lines growing on germination medium containing 0.6 µM BFA in vitro. Medium containing 50 nM DMSO was used as the control. The images were captured after culturing for 3 h. Bar  = 50 µm.</p

Overexpression of <i>Ann5</i> increased the resistance of pollen germination and tube growth to BFA.

<p>Representative micrographs of pollen grains and tubes from the <i>Ann5</i>-overexpressing lines growing on <i>Arabidopsis</i> pollen germination medium containing 0.3 µM or 0.6 µM BFA for 3 h in vitro. Medium containing 50 nM DMSO was used as the control. Bar  = 50 µm.</p

Annexin5 Plays a Vital Role in <i>Arabidopsis</i> Pollen Development via Ca²⁺-Dependent Membrane Trafficking

<div><p>The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative “linker” between Ca²⁺ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of <i>Arabidopsis</i> annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca²⁺ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of <i>Ann5</i>-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of <i>Ann5</i> promoted Ca²⁺-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of <i>Ann5</i> did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca²⁺-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.</p></div