Phi thickenings in Brassica oleracea roots are induced by osmotic stress and mechanical effects, both involving jasmonic acid.

Phi thickenings are peculiar secondary cell wall thickenings found in radial walls of cortical cells in plant roots. However, while thickenings are widespread in the plant kingdom, research into their development has been lacking. Here, we describe a simple system for rapid induction of phi thickenings in primary roots of Brassica. Four-day-old seedlings were transferred from control agar plates to new plates containing increased levels of osmotica. Phi thickening development occurred within a narrow region of the differentiation zone proportional to osmolarity, with cellulose deposition and lignification starting after 12h and 15h, respectively. However, osmoprotectants not only failed to induce phi thickenings, but inhibited induction when tested in combination with thickening-inducing osmotica. An independent, biomechanical pathway exists regulating phi thickening induction, with root growth rates and substrate texture being important factors in determining thickening induction. Phi thickening development is also controlled by stress-related plant hormones, most notably jasmonic acid, but also abscisic acid. Our research not only provides the first understanding of the developmental pathways controlling phi thickening induction, but also provides tools with which the functions of these enigmatic structures might be clarified.

Asymmetric wall ingrowth deposition in Arabidopsis phloem parenchyma transfer cells is tightly associated with sieve elements.

In Arabidopsis, polarized deposition of wall ingrowths in phloem parenchyma (PP) transfer cells (TCs) occurs adjacent to cells of the sieve element/companion cell (SE/CC) complex. However, the spatial relationships between these different cell types in minor veins, where phloem loading occurs, are poorly understood. PP TC development and wall ingrowth localization were compared with those of other phloem cells in leaves of Col-0 and the transgenic lines AtSUC2::AtSTP9-GFP (green fluorescent protein) and AtSWEET11::AtSWEET11-GFP that identify CCs and PP cells, respectively. The development of PP TCs in minor veins, indicated by deposition of wall ingrowths, proceeded basipetally in leaves. However, not all PP cells develop wall ingrowths, and higher levels of deposition occur in abaxial- compared with adaxial-positioned PP TCs. Furthermore, the deposition of wall ingrowths was exclusively initiated on and preferentially covered the PP TC/SE interface, rather than the PP TC/CC interface, and only occurred in PP cells that were adjacent to SEs. Collectively, these results demonstrate a tight association between SEs and wall ingrowth deposition in PP TCs and suggest the existence of two subtypes of PP cells in leaf minor veins. Compared with PP cells, PP TCs showed more abundant accumulation of AtSWEET11-GFP, indicating functional differences in phloem loading between PP and PP TCs.

Super-Resolution Fluorescence Imaging of Arabidopsis thaliana Transfer Cell Wall Ingrowths using Pseudo-Schiff Labelling Adapted for the Use of Different Dyes.

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.

Sucrose regulates wall ingrowth deposition in phloem parenchyma transfer cells in Arabidopsis via affecting phloem loading activity.

In Arabidopsis thaliana, phloem parenchyma transfer cells (PPTCs) occur in leaf minor veins and play a pivotal role in phloem loading. Wall ingrowth formation in PPTCs is induced by the phloem loading activity of these cells, which is regulated by sucrose (Suc). The effects of endogenous versus exogenous Suc on wall ingrowth deposition, however, differ. Elevating endogenous Suc levels by increased light enhanced wall ingrowth formation, whereas lowering endogenous Suc levels by dark treatment or genetically in ch-1 resulted in lower levels of deposition. In contrast, exogenously applied Suc, or Suc derived from other organs, repressed wall ingrowth deposition. Analysis of pAtSUC2::GFP plants, used as a marker for phloem loading status, suggested that wall ingrowth formation is correlated with phloem loading activity. Gene expression analysis revealed that exogenous Suc down-regulated expression of AtSWEET11 and 12, whereas endogenous Suc up-regulated AtSWEET11 expression. Analysis of a TREHALOSE 6-PHOSPHATE (T6P) SYNTHASE overexpression line and the hexokinase (HXK)-null mutant, gin2-1, suggested that Suc signalling of wall ingrowth formation is independent of T6P and HXK. Collectively, these results are consistent with the conclusion that Suc regulates wall ingrowth formation via affecting Suc exporting activity in PPTCs.

Phi thickenings in roots: novel secondary wall structures responsive to biotic and abiotic stresses.

Phi thickenings are specialized secondary walls found in root cortical cells. Despite their widespread occurrence throughout the plant kingdom, these specialized thickenings remain poorly understood. First identified by Van Tieghem in 1871, phi thickenings are a lignified and thickened cell wall band that is deposited inside the primary wall, as a ring around the cells' radial walls. Phi thickenings can, however, display structural variations including a fine, reticulate network of wall thickenings extending laterally from the central lignified band. While phi thickenings have been proposed to mechanically strengthen roots, act as a permeability barrier to modulate solute movement, and regulate fungal interactions, these possibilities remain to be experimentally confirmed. Furthermore, since temporal and spatial development of phi thickenings varies widely between species, thickenings may perform diverse roles in different species. Phi thickenings can be induced by abiotic stresses in different species; they can, for example, be induced by heavy metals in the Zn/Cd hyperaccumulator Thlaspi caerulescens, and in a cultivar-specific manner by water stress in Brassica. This latter observation provides an experimental platform to probe phi thickening function, and to identify genetic pathways responsible for their formation. These pathways might be expected to differ from those involved in secondary wall formation in xylem, since phi thickening deposition in not linked to programmed cell death.

Comparative analysis of common regions found in babuviruses and alphasatellite molecules.

Viruses in the genus Babuvirus have multi-component ssDNA genomes and often associate with alphasatellite molecules containing two common motifs, a common-region stem-loop (CR-SL) involved in initiation of rolling-circle replication and a common-region major (CR-M) motif involved in secondary-strand synthesis. We compared known babuvirus genome components and alphasatellite CR-SL and CR-M sequences, defining five divergent CR-SL sequence classes. We identified iterated sequence elements in babuvirus genome components that have particularly conserved sequences and spatial arrangements between known babuviruses.

Induction of anthocyanin in the inner epidermis of red onion leaves by environmental stimuli and transient expression of transcription factors.

KEY MESSAGE: Novel imaging approaches have allowed measurements of the anthocyanin induction in onion epidermal cells that can be induced through water stress or transient expression of exogenous transcription factors. Environmental and genetic mechanisms that allow the normally colourless inner epidermal cells of red onion (Allium cepa) bulbs to accumulate anthocyanin were quantified by both absorbance ratios and fluorescence. We observed that water-stressing excised leaf segments induced anthocyanin formation, and fluorescence indicated that this anthocyanin was spectrally similar to the anthocyanin in the outer epidermal cells. This environmental induction may require a signal emanating from the leaf mesophyll, as induction did not occur in detached epidermal peels. Exogenous transcription factors that successfully drive anthocyanin biosynthesis in other species were also tested through transient gene expression using particle bombardment. Although the petunia R2R3-MYB factor AN2 induced anthocyanin in both excised leaves and epidermal peels, several transcription factors including maize C1 and Lc inhibited normal anthocyanin development in excised leaves. This inhibition may be due to moderate levels of conservation between the exogenous transcription factors and endogenous Allium transcription factors. The over-expressed exogenous transcription factors cannot drive anthocyanin biosynthesis themselves, but bind to the endogenous transcription factors and prevent them from driving anthocyanin biosynthesis.

A temperature-sensitive allele of a putative mRNA splicing helicase down-regulates many cell wall genes and causes radial swelling in Arabidopsis thaliana.

The putative RNA helicase encoded by the Arabidopsis gene At1g32490 is a homolog of the yeast splicing RNA helicases Prp2 and Prp22. We isolated a temperature-sensitive allele (rsw12) of the gene in a screen for root radial swelling mutants. Plants containing this allele grown at the restrictive temperature showed weak radial swelling, were stunted with reduced root elongation, and contained reduced levels of cellulose. The role of the protein was further explored by microarray analysis. By using both fold change cutoffs and a weighted gene coexpression network analysis (WGCNA) to investigate coexpression of genes, we found that the radial swelling phenotype was not linked to genes usually associated with primary cell wall biosynthesis. Instead, the mutation has strong effects on expression of secondary cell wall related genes. Many genes potentially associated with secondary walls were present in the most significant WGCNA module, as were genes coding for arabinogalactans and proteins with GPI anchors. The proportion of up-regulated genes that possess introns in rsw12 was above that expected if splicing was unrelated to the activity of the RNA helicase, suggesting that the helicase does indeed play a role in splicing in Arabidopsis. The phenotype may be due to a change in the expression of one or more genes coding for cell wall proteins.

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.

Identification and in silico characterisation of defective molecules associated with isolates of banana bunchy top virus.

Banana bunchy top virus (BBTV) is a multi-component single-stranded DNA virus. From 267 potentially infected Musa plants, 24 apparently 'defective' BBTV components have been identified. Interestingly, 23/24 of these defective molecules were apparently derived from DNA-R. All of the identified defective molecules had retained at least part of the CR-SL and CR-M but had insertions and/or deletions that in most cases resulted in open reading frame disruptions. Our detection of three monophyletic but diverse (and therefore likely circulating) defective DNA-R lineages suggests that, in many cases, defective DNA-R molecules might remain associated with BBTV genomes for prolonged periods.

The life of phi: the development of phi thickenings in roots of the orchids of the genus Miltoniopsis.

Phi thickenings, bands of secondary wall thickenings that reinforce the primary wall of root cortical cells in a wide range of species, are described for the first time in the epiphytic orchid Miltoniopsis. As with phi thickenings found in other plants, the phi thickenings in Miltoniopsis contain highly aligned cellulose running along the lengths of the thickenings, and are lignified but not suberized. Using a combination of histological and immunocytochemical techniques, thickening development can be categorized into three different stages. Microtubules align lengthwise along the thickening during early and intermediate stages of development, and callose is deposited within the thickening in a pattern similar to the microtubules. These developing thickenings also label with the fluorescently tagged lectin wheat germ agglutinin (WGA). These associations with microtubules and callose, and the WGA labeling, all disappear when the phi thickenings are mature. This pattern of callose and WGA deposition show changes in the thickened cell wall composition and may shed light on the function of phi thickenings in plant roots, a role for which has yet to be established.

Anterior thalamic lesions reduce spine density in both hippocampal CA1 and retrosplenial cortex, but enrichment rescues CA1 spines only.

Injury to the anterior thalamic nuclei (ATN) may affect both hippocampus and retrosplenial cortex thus explaining some parallels between diencephalic and medial temporal lobe amnesias. We found that standard-housed rats with ATN lesions, compared with standard-housed controls, showed reduced spine density in hippocampal CA1 neurons (basal dendrites, -11.2%; apical dendrites, -9.6%) and in retrospenial granular b cortex (Rgb) neurons (apical dendrites, -20.1%) together with spatial memory deficits on cross maze and radial-arm maze tasks. Additional rats with ATN lesions were also shown to display a severe deficit on spatial working memory in the cross-maze, but subsequent enriched housing ameliorated their performance on both this task and the radial-arm maze. These enriched rats with ATN lesions also showed recovery of both basal and apical CA1 spine density to levels comparable to that of the standard-housed controls, but no recovery of Rgb spine density. Inspection of spine types in the CA1 neurons showed that ATN lesions reduced the density of thin spines and mushroom spines, but not stubby spines; while enrichment promoted recovery of thin spines. Comparison with enriched rats that received pseudo-training, which provided comparable task-related experience, but no explicit spatial memory training, suggested that basal CA1 spine density in particular was associated with spatial learning and memory performance. Distal pathology in terms of reduced integrity of hippocampal and retrosplenial microstructure provides clear support for the influence of the ATN lesions on the extended hippocampal system. The reversal by postoperative enrichment of this deficit in the hippocampus but not the retrosplenial cortex may indicate region-specific mechanisms of recovery after ATN injury.

The N-terminal TOG domain of Arabidopsis MOR1 modulates affinity for microtubule polymers.

Microtubule-associated proteins of the highly conserved XMAP215/Dis1 family promote both microtubule growth and shrinkage, and move with the dynamic microtubule ends. The plant homologue, MOR1, is predicted to form a long linear molecule with five N-terminal TOG domains. Within the first (TOG1) domain, the mor1-1 leucine to phenylalanine (L174F) substitution causes temperature-dependent disorganization of microtubule arrays and reduces microtubule growth and shrinkage rates. By expressing the two N-terminal TOG domains (TOG12) of MOR1, both in planta for analysis in living cells and in bacteria for in vitro microtubule-binding and polymerization assays, we determined that the N-terminal domain of MOR1 is crucial for microtubule polymer binding. Tagging TOG12 at the N-terminus interfered with its ability to bind microtubules when stably expressed in Arabidopsis or when transiently overexpressed in leek epidermal cells, and impeded polymerase activity in vitro. In contrast, TOG12 tagged at the C-terminus interacted with microtubules in vivo, rescued the temperature-sensitive mor1-1 phenotype, and promoted microtubule polymerization in vitro. TOG12 constructs containing the L174F mor1-1 point mutation caused microtubule disruption when transiently overexpressed in leek epidermis and increased the affinity of TOG12 for microtubules in vitro. This suggests that the mor1-1 mutant protein makes microtubules less dynamic by binding the microtubule lattice too strongly to support rapid plus-end tracking. We conclude from our results that a balanced microtubule affinity in the N-terminal TOG domain is crucial for the polymerase activity of MOR1.

Molecular Manipulation of the miR160/AUXIN RESPONSE FACTOR Expression Module Impacts Root Development in Arabidopsis thaliana.

In Arabidopsis thaliana (Arabidopsis), microRNA160 (miR160) regulates the expression of AUXIN RESPONSE FACTOR10 (ARF10), ARF16 and ARF17 throughout development, including the development of the root system. We have previously shown that in addition to DOUBLE-STRANDED RNA BINDING1 (DRB1), DRB2 is also involved in controlling the rate of production of specific miRNA cohorts in the tissues where DRB2 is expressed in wild-type Arabidopsis plants. In this study, a miR160 overexpression transgene (MIR160B) and miR160-resistant transgene versions of ARF10 and ARF16 (mARF10 and mARF16) were introduced into wild-type Arabidopsis plants and the drb1 and drb2 single mutants to determine the degree of requirement of DRB2 to regulate the miR160 expression module as part of root development. Via this molecular modification approach, we show that in addition to DRB1, DRB2 is required to regulate the level of miR160 production from its precursor transcripts in Arabidopsis roots. Furthermore, we go on to correlate the altered abundance of miR160 or its ARF10, ARF16 and ARF17 target genes in the generated series of transformant lines with the enhanced development of the root system displayed by these plant lines. More specifically, promotion of primary root elongation likely stemmed from enhancement of miR160-directed ARF17 expression repression, while the promotion of lateral and adventitious root formation was the result of an elevated degree of miR160-directed regulation of ARF17 expression, and to a lesser degree, ARF10 and ARF16 expression. Taken together, the results presented in this study identify the requirement of the functional interplay between DRB1 and DRB2 to tightly control the rate of miR160 production, to in turn ensure the appropriate degree of miR160-directed ARF10, ARF16 and ARF17 gene expression regulation as part of normal root system development in Arabidopsis.

Characterisation of the trans-membrane nucleoporins GP210 and NDC1 in Arabidopsis thaliana.

The nuclear pore is structurally conserved across eukaryotes as are many of the pore's constituent proteins. The transmembrane nuclear pore proteins GP210 and NDC1 span the nuclear envelope holding the nuclear pore in place. Orthologues of GP210 and NDC1 in Arabidopsis were investigated through characterisation of T-DNA insertional mutants. While the T-DNA insert into GP210 reduced expression of the gene, the insert in the NDC1 gene resulted in increased expression in both the ndc1 mutant as well as the ndc1/gp210 double mutant. The ndc1 and gp210 individual mutants showed little phenotypic difference from wild-type plants, but the ndc1/gp210 mutant showed a range of phenotypic effects. As with many plant nuclear pore protein mutants, these effects included non-nuclear phenotypes such as reduced pollen viability, reduced growth and glabrous leaves in mature plants. Importantly, however, ndc1/gp210 exhibited nuclear-specific effects including modifications to nuclear shape in different cell types. We also observed functional changes to nuclear transport in ndc1/gp210 plants, with low levels of cytoplasmic fluorescence observed in cells expressing nuclear-targeted GFP. The lack of phenotypes in individual insertional lines, and the relatively mild phenotype suggests that additional transmembrane nucleoporins, such as the recently-discovered CPR5, likely compensate for their loss.

Uptake and persistence of Mycobacterium avium subsp. paratuberculosis in human monocytes.

Mycobacterium avium subsp. paratuberculosis is a bacterium sometimes found in human blood and tissue samples that may have a role in the etiology of Crohn's disease in humans. To date, however, there have been few studies examining the interactions of these bacteria with human cells. Using the THP-1 human monocytic cell line, this study shows that the uptake and trafficking of M. avium subsp. paratuberculosis in human cells are cholesterol dependent and that these bacteria localize to cholesterol-rich compartments that are slow to acidify. M. avium subsp. paratuberculosis bacteria containing phagosomes stain for the late endosomal marker Rab7, but recruitment of the Rab7-interacting lysosomal protein that regulates the fusion of bacterium-containing phagosomes with lysosomal compartments and facilitates subsequent bacterial clearance is significantly reduced. Disruption of phagosome acidification via this mechanism may contribute to M. avium subsp. paratuberculosis persistence in human cells, but there was no evidence that internalized M. avium subsp. paratuberculosis also affects the survival of bacteria taken up during a secondary phagocytic event.

Cell-to-cell transport via the lumen of the endoplasmic reticulum.

Plasmodesmata are plasma membrane-lined channels through which cytoplasmic molecules move from cell-to-cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3-kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4-kDa recombinant ER-lumenal reporter protein (LRP) from a fragment of the endogenous ER-lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER-GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER-lumenal molecules between cells.

Evidence of multiple introductions of beak and feather disease virus into the Pacific islands of Nouvelle-Caledonie (New Caledonia).

Beak and feather disease virus (BFDV) is a circular ssDNA virus that causes psittacine beak and feather disease and has almost global presence. Here, we report for the first time the presence of in Nouvelle-Calédonie (New Caledonia). One hundred and sixty-eight exotic and 79 endemic birds were sampled in Nouvelle-Calédonie, 26 were found to be positive for BFDV. We characterized the full genomes of 26 isolates and phylogenetic analysis placed nine of the isolates into the BFDV-J strain, with the remaining 17 isolates from Deplanche's Rainbow Lorikeet (Trichoglossus haematodus deplanchii) forming a novel strain, BFDV-P. Of more concern was the discovery of an infected bird from the vulnerable and endemic New Caledonian Parakeet (Cyanoramphus saisseti). Our results reveal that there have been at least two introductions of BFDV into Nouvelle-Calédonie.

Evidence of inter-component recombination, intra-component recombination and reassortment in banana bunchy top virus.

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component, ssDNA virus, which causes widespread banana crop losses throughout tropical Africa and Australasia. We determined the full genome sequences of 12 BBTV isolates from the Kingdom of Tonga and analysed these together with previously determined BBTV sequences to show that reassortment and both inter- and intra-component recombination have all been relatively frequent occurrences during BBTV evolution. We found that whereas DNA-U3 components display evidence of complex inter- and intra-component recombination, all of the South Pacific DNA-R components have a common intra-component recombinant origin spanning the replication-associated protein gene. Altogether, the DNA-U3 and DNA-M components display a greater degree of inter-component recombination than the DNA-R, -S, -C and -M components. The breakpoint distribution of the inter-component recombination events reveals a primary recombination hotspot around the 5' side of the common region major and, in accordance with recombination hotspots detectable in related ssDNA viruses, a secondary recombination hotspot near the origin of virion-strand replication.

Dragonfly cyclovirus, a novel single-stranded DNA virus discovered in dragonflies (Odonata: Anisoptera).

Dragonfly cyclovirus (DfCyV), a new species of ssDNA virus discovered using viral metagenomics in dragonflies (family Libellulidae) from the Kingdom of Tonga. Metagenomic sequences of DfCyV were similar to viruses of the recently proposed genus Cyclovirus within the family Circoviridae. Specific PCRs resulted in the recovery of 21 DfCyV genomes from three dragonfly species (Pantala flavescens, Tholymis tillarga and Diplacodes bipunctata). The 1741 nt DfCyV genomes share >95 % nucleotide identity and are classified into 11 subtypes representing a single strain. The DfCyV genomes share 48-63 % genome-wide nucleotide identity with cycloviruses identified in human faecal samples. Recombination analysis revealed three recombinant DfCyV genomes, suggesting that recombination plays an important role in cyclovirus evolution. To our knowledge, this is the first report of a circular ssDNA virus identified in insects, and the data may help elucidate evolutionary links among novel Circoviridae recently identified in animals and environmental samples.