Peeking into Pit Fields: A Multiple Twinning Model of Secondary Plasmodesmata Formation in Tobacco[W]

In higher plants, plasmodesmata (PD) are major conduits for cell–cell communication. Primary PD are laid down at cytokinesis, while secondary PD arise during wall extension. During leaf development, the basal cell walls of trichomes extend radially without division, providing a convenient system for studying the origin of secondary PD. We devised a simple freeze-fracture protocol for examining large numbers of PD in surface view. In the postcytokinetic wall, simple PD were distributed randomly. As the wall extended, PD became twinned at the cell periphery. Additional secondary pores were inserted at right angles to these, giving rise to pit fields composed of several paired PD. During wall extension, the number of PD increased fivefold due to the insertion of secondary PD. Our data are consistent with a model in which a subset of the original primary PD pores function as templates for the insertion of new secondary PD, spatially fixing the position of future pit fields. Many of the new PD shared the same wall collar as the original PD pore, suggesting that new PD pores may arise by fissions of existing PD progenitors. Different models of secondary PD formation are discussed. Our data are supported by a computational model, Plasmodesmap, which accurately simulates the formation of radial pit fields during cell wall extension based on the occurrence of multiple PD twinning events in the cell wall. The model predicts PD distributions with striking resemblance to those seen on fractured wall faces

The Psychology of Removing Group Members and Recruiting New Ones

The removal of group members and the recruiting of new ones are central processes in the maintenance of a group, yet they receive little study. Bases for determining who will be expelled and who initiated are stated in the form of propositions. A number of hypotheses are then offered concerning conditions that may cause events to run counter to these propositions

The Phytocalpain Defective Kernel 1 Is a Novel Arabidopsis Growth Regulator Whose Activity Is Regulated by Proteolytic Processing[W]

The role of the unique plant calpain Defective Kernel 1 (DEK1) in development has remained unclear due to the severity of mutant phenotypes. Here, we used complementation studies of the embryo-lethal mutant to dissect DEK1 protein behavior and to show that DEK1 plays a key role in growth regulation in Arabidopsis thaliana. We show that although full-length DEK1 protein localizes to membranes, it undergoes intramolecular autolytic cleavage events that release the calpain domain into the cytoplasm. The active calpain domain alone is not only necessary for DEK1 function but is sufficient for full complementation of dek1 mutants. A novel set of phenotypes, including leaf ruffling, increased leaf thickness, and abnormalities of epidermal cell interdigitation, was caused by expression of the constitutively active calpain domain. This analysis of the novel phenotypes produced by DEK1 under- and overexpression, as well as DEK1 subcellular localization and protein processing, has revealed a fundamental role for DEK1-mediated signaling in growth regulation

Colony morphology of MAP kinase mutants.

<p>All MAP kinase mutants showed macroscopic colony phenotypes clearly distinct from the wild type and between the three MAP kinase pathways, but highly conserved within each cascade. (<b>A</b>) CWI-MAP kinase mutants (Δ<i>mik-1</i>, Δ<i>mek-1</i> and Δ<i>mak-1</i>) typically showed increased autolysis resulting in rosette-like colony growth, and slow colony extension even on nutrient rich media. (<b>B</b>) MAP kinase mutants of the PR pathway (Δ<i>nrc-1</i>, Δ<i>mek-2</i> and Δ<i>mak-2</i>) were characterized by short aerial hyphae and conidiation starting from the colony center. (<b>C</b>) Colony phenotypes of OS-MAP kinase mutants (<i>Δos-4</i>, <i>Δos-5</i> and <i>Δos-2</i>) comprised reduced aerial hyphae in the colony center, elevated carotenoid biosynthesis and intense production of ‘sticky’ aerial hyphae and macroconidiophores were foremost at the plate edge. (<b>D</b>) Wild type controls, and the ‘old’ Δ<i>os-2</i> strain FGSC11436, which displayed a colony phenotype different to that of the genuine <i>os</i> mutants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone.0042565.s004" target="_blank">Figure S4</a> for a more detailed genotypic and phenotypic comparison between the two <i>Δos-2</i> mutants FGSC11436 and FGSC17933).</p

Protoperithecial development in MAP kinase gene-deletion mutants.

<p>In comparison to the wild type, which formed regular, subspherical protoperithecia 40–80 µm in diameter, only mutants of the PR- and CWI-MAP kinase cascades formed protoperithecial-like structures of similar appearance. These however, did vary in size, shape and degree of pigmentation and were not clearly discernable as protoperithecia even to an experienced microscopist using the stereomicroscopy technique shown here. It was these observations that warranted investigations using more powerful microscopic techniques, as used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone-0042565-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone-0042565-g006" target="_blank">6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone-0042565-g008" target="_blank">8</a>. Protoperithecial-like structures could not be observed in any of the newly generated OS-MAP kinase mutants. In contrast to the other <i>os</i> mutants, Δ<i>os-2</i> FGSC11436 showed disorganized mycelial architecture, typical of hyphal fusion defects. The Δ<i>nrc-1</i> strains generated from FGSC18162 by vegetative homokaryon selection (HS) showed no phenotypic differences compared to Δ<i>nrc-1</i> FGSC11466. In order to calibrate the results, all strains were inoculated onto cellophane over LSA medium (and SCM for comparison), and incubated for 5–7 days at 25°C dependent on the rate of developmental of the mutant strain. By cutting out cellophane squares carrying mycelium the same samples as shown here were subsequently prepared for LTSEM. Finally, these female cultures were fertilized with opposite mating type conidia of the wild type to confirm female sterility. All scale bars, 50 µm.</p

Optical sectioning of developing protoperithecia.

<p>Montages of selected optical sections through developing protoperithecia of the rescued Δ<i>mak-2</i> strain expressing MAK-2-GFP (NCAL043). (<b>A</b>) The small dimensions of a late stage ascogonial coil are fully accessible to optical sectioning when labelled with CFW and MAK-2-GFP. Scale bar, 5 µm. (<b>B</b>) With increasing size, CFW dye is unable to penetrate the interior of the developing fruitbody, and consequently cannot be used to optically section the interior of the ascogonium. Fluorescently labelled MAK-2, however, allows visualization of the whole protoperithecium. Scale bar, 10 µm. (<b>C</b>) Optical sectioning of a mature protoperithecium reveals the complex and tightly wound hyphal network comprising this structure. Scale bar, 20 µm. (<b>D</b>) Middle section of a protoperithecium expressing MAK-2-GFP. The corresponding surface plot shows that fluorescence intensity peaks in the central core region, suggesting that MAK-2-GFP accumulates in the ascogonial coil tissue. (<b>E</b>) MAK-1-GFP fluorescence in the rescued <i>Δmak-1</i> strain (NCAL010) also peaked in the central ascogonium region, however, was not as pronounced as in the case of MAK-2. Scale bar, 10 µm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone.0042565.s010" target="_blank">Movies S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone.0042565.s011" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone.0042565.s012" target="_blank">S7</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#pone.0042565.s013" target="_blank">S8</a> show full z-stacks of optically sectioned protoperithecia.</p

Protoperithecial morphogenesis of <i>N. crassa</i> wild type.

<p>LTSEM of the main stages of protoperithecial development. (<b>A</b>) Two ascogonial coils differentiated from the vegetative mycelium of a two day-old culture. These two coils have formed on branches (<i>bh</i>) off the main arterial trunk hyphae (<i>th</i>). Some of the surrounding branches have fused with each other, they are therefore considered to be fusion hyphae (<i>fh</i>). Vegetative hyphal fusion is instrumental in the establishment of a fully co-operative interconnected mycelium. Scale bar, 50 µm. (<b>B</b>) Higher magnification of the ascogonial coil boxed in (A). On careful inspection a septum can be seen on the lower part of the coil (aligned with arrowheads). Scale bar, 5 µm. (<b>C</b>) A slightly expanded ascogonial coil again formed on a side branch of a trunk hypha, the coil is being wrapped around by enveloping hyphae. Scale bar, 20 µm. (<b>D</b>) A slightly later stage where enveloping hyphae (arrowheads) originating from the ascogonium have wrapped around the central ascogonial coil (<i>ac</i>). These enveloping hyphae exhibit septation and branching. The ‘parent hypha’ (<i>ph</i>) of the ascogonial coil can be clearly defined, and is separated from the developing fruitbody by a basal septum (<i>bs</i>). (<b>E</b>) The subspherical shape of the protoperithecium becomes evident after additional enveloping hyphae have formed a protective casing around the ascogonium. Trunk hyphae (<i>th</i>), their branches (<i>bh</i>) and fusion hyphae (<i>fh</i>) can be clearly distinguished. Scale bar, 5 µm. (<b>F</b>) Mature protoperithecium, with visible ECM secretion ‘gluing’ enveloping hyphae together, and a trichogyne (arrowhead) emerging from its center. Scale bar, 20 µm. (<b>G</b>) Enlarged view of the boxed area in (D) showing ECM strands between hyphae (arrowhead). Scale bar, 2 µm. (<b>H</b>) Enlarged view of the boxed area in (B) showing ECM strands (arrowhead) between the tightly attaching revolutions of the ascogonial coil. Scale bar, 1 µm. (<b>I</b>) Enlarged view of the boxed area in (F) showing the surface hyphae of the protoperithecium evenly covered in ECM. Scale bar, 5 µm.</p

ECM and hyphal adhesion seem essential for the organized assembly of enveloping hyphae into protoperithecia.

<p><b>(OS)</b> Despite several attempts, ascogonial coils, let alone protoperithecial-like structures, could not be identified in mycelia of the three OS-MAP kinase mutants. Large areas of the mycelium were collapsed, indicating extensive lysis of vegetative hyphae. Hyphal loops (<i>a.k.a.</i> hyphal coils or lassoes), as shown here in Δ<i>os-2</i> (arrowhead in E) were occasionally observed in all three mutants. These structures are frequently found in the wild type, and although their function is unknown, a connection to sexual development seems unlikely (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042565#s4" target="_blank">discussion</a>). Scale bars: (A) 100 µm; (B, C, E) 50 µm; (D, F) 25 µm. <b>(CWI)</b> Δ<i>mik-1</i>, Δ<i>mek-1</i> and Δ<i>mak-1</i> strains initiated ascogonial coils and differentiated enveloping hyphae. The assembled multicellular structures, however, remained loose hyphal aggregations and ECM was absent, suggesting that hyphal adhesion was not sufficient to form subspherical protoperithecia. Scale bars: (G) 10 µm; (H, I, K, L) 25 µm; (J) 50 µm. <b>(PR)</b> Δ<i>nrc-1</i>, Δ<i>mek-2</i> and Δ<i>mak-2</i> strains produced ECM, and hyphal aggregations resembled better-organized and more spherical ‘early-stage’ protoperithecia. Nevertheless, trichogynes have not been observed in these strains, and sexual development did not progress beyond this stage. Scale bars: (M–R) 25 µm.</p