Cell corner middle lamella in hydroids of dendroid moss Hypnodendron menziesii gametophyte is prominently thickened: a proposed role in the mechanical support function.

MAIN CONCLUSION: Significantly thickened corner middle lamella of the hydroid cell wall in the stipe of dendroid moss Hypnodendron menziesii has a mechanical support function. The hydroid cell walls of the erect stipe of Hypnodendron menziesii were investigated using light microscopy (LM), transmission electron microscopy (TEM), and TEM-immunogold labeling in support of the proposed biomechanical function for the highly thickened cell corner middle lamellae. The statistical analyses of dimensions of hydroid cell and wall parameters revealed a strong positive correlation between the area of hydroid cell and (i) the hydroid cell walls adhering to thick corner middle lamella, (ii) the area of the thick cell wall at hydroid corners, and (iii) the maximum thickness of cell wall at hydroid corners. The total area of the thick cell wall at the hydroid corners concomitantly increased with the area of the hydroid cell wall adhering to the middle lamella, and with the increased number of hydroids surrounding a reference hydroid. The results suggest that markedly thickened middle lamellae of the hydroid cell wall in Hypnodendron likely function by preventing hydroid cells from collapsing under the tensile forces generated from the transpirational pull on the water column. The specific localization of (1→4)- ß-D-galactan and (1,5)-α-L-arabinan in the interface region of the hydroid cell wall and the thick middle lamella is consistent with these cell wall components being involved in the mechanical strengthening of the interface through firm adhesion as well as elasticity, ensuring the structural stability of this cell wall region, which may be prone to delamination/fracturing from the various internal and external pressures imposed. The copious presence of homogalacturonan in the thick middle lamella may further enhance the strength and flexibility of hydroid cell walls.

Heteromannans are the predominant hemicelluloses in the gametophytic stem of the umbrella moss Hypnodendron menziesii and occur in the walls of all cell types.

MAIN CONCLUSION: Heteromannans are the predominant hemicelluloses in the gametophytic stem of the moss Hypnodendron menziesii and occur in the walls of all cell types Little is known about the cell-wall polysaccharides of mosses. Monosaccharide analysis of cell walls isolated from the stem of the umbrella moss Hypnodendron menziesii was consistent with heteromannans, probably galactoglucomannans, being the predominant hemicellulosic polysaccharides in the walls. Immunofluorescence and immunogold microscopy with the monoclonal antibody LM21, specific for heteromannans, showed that these polysaccharides were present in the walls of all stem cell types. These cell types, except the hydroids, have secondary walls. Experiments in which sections were pre-treated with 0.1 M sodium carbonate and with the enzyme pectate lyase indicated that the heteromannans have O-acetyl groups that limit LM21 binding and the cell walls contain pectic homogalacturonan that masks detection of heteromannans using LM21. Therefore, to fully detect heteromannans in the cell walls, it was essential to use these pre-treatments to remove the O-acetyl groups from the heteromannans and pectic homogalacturonan from the cell walls. Fluorescence microscopy experiments with a second monoclonal antibody, LM22, also specific for heteromannans, showed similar results, but the binding was considerably weaker than with LM21, possibly as a result of subtle structural differences in the epitopes of the two antibodies. Although heteromannans occur abundantly in the cell walls of many species in basal lineages of tracheophytes, prior to the present study, research on the distribution of these polysaccharides in the walls of different cell types in mosses was confined to the model species Physcomitrium patens.

Dimensional Changes of Tracheids during Drying of Radiata Pine (Pinus radiata D. Don) Compression Woods: A Study Using Variable-Pressure Scanning Electron Microscopy (VP-SEM).

Variable-pressure scanning electron microscopy was used to investigate the dimensional changes in longitudinal, tangential and radial directions, on wetting and drying, of tracheids of opposite wood (OW) and three grades of compression woods (CWs), including severe CW (SCW) and two grades of mild compression wood (MCW) (MCW1 and MCW2) in corewood of radiata pine (Pinus radiata) saplings. The CW was formed on the underside and OW on the upper side of slightly tilted stems. In the longitudinal direction, the shrinkage of SCW tracheids was ~300% greater than that of OW tracheids, with the shrinkage of the MCW1 and MCW2 tracheids being intermediate. Longitudinal swelling was also investigated and hysteresis was demonstrated for the tracheids of all corewood types, with the extent of hysteresis increasing with CW severity. A statistical association was found between longitudinal shrinkage and the content of lignin and galactosyl residues in the cell-wall matrix. The galactosyl residues are present mostly as (1→4)-ß-galactans, which are known to have a high capacity for binding water and swell on hydration. The small proportions of (1→3)-ß-glucans in the CWs have similar properties. These polysaccharides may play a functional role in the longitudinal shrinking and swelling of CW tracheids. Tangential shrinkage of tracheids was greater than radial shrinkage but both were greatest for OW and least for SCW, with the MCW1 and MCW2 being intermediate.

Location and characterization of lignin in tracheid cell walls of radiata pine (Pinus radiata D. Don) compression woods.

Tilted stems of softwoods form compression wood (CW) and opposite wood (OW) on their lower and upper sides, respectively. More is known about the most severe form of CW, severe CW (SCW), but mild CWs (MCWs) also occur widely. Two grades of MCWs, MCW1 and MCW2, as well as SCW and OW were identified in the stems of radiata pine (Pinus radiata) that had been slightly tilted. The four wood types were identified by the distribution of lignin in the tracheid walls determined by fluorescence microscopy. A solution of the fluorescent dye acridine orange (AO) (0.02% at pH 6 or 7) was shown to metachromatically stain the tracheid walls and can also be used to determine lignin distribution. The lignified walls fluoresced orange to yellow depending on the lignin concentration. Microscopically well-characterized discs (0.5 mm diameter) of the wood types were used to determine lignin concentrations and lignin monomer compositions using the acetyl bromide method and thioacidolysis, respectively. Lignin concentration and the proportion of p-hydroxyphenyl units (H-units) relative to guaiacyl (G-units) increased with CW severity, with <1% H-units in OW and up to 14% in SCW. Lignin H-units can be used as a marker for CW and CW severity. Similar discs were also examined by Raman and FTIR micro-spectroscopies coupled with principal component analysis (PCA) to determine if these techniques can be used to differentiate the four different wood types. Both techniques were able to do this, particularly Raman micro-spectroscopy.

Tracheid cell-wall structures and locations of (1 → 4)-ß-D-galactans and (1 → 3)-ß-D-glucans in compression woods of radiata pine (Pinus radiata D. Don).

BACKGROUND: Compression wood (CW) forms on the underside of tilted stems of coniferous gymnosperms and opposite wood (OW) on the upperside. The tracheid walls of these wood types differ structurally and chemically. Although much is known about the most severe form of CW, severe CW (SCW), mild CWs (MCWs), also occur, but less is known about them. In this study, tracheid wall structures and compositions of two grades of MCWs (1 and 2) and SCW were investigated and compared with OW in slightly tilted radiata pine (Pinus radiata) stems. RESULTS: The four wood types were identified by the distribution of lignin in their tracheid walls. Only the tracheid walls of OW and MCW1 had a S3 layer and this was thin in MCW1. The tracheid walls of only SCW had a S2 layer with helical cavities in the inner region (S2i). Using immunomicroscopy, (1 → 4)-ß-D-galactans and (1 → 3)-ß-D-glucans were detected in the tracheid walls of all CWs, but in only trace amounts in OW. The (1 → 4)-ß-D-galactans were located in the outer region of the S2 layer, whereas the (1 → 3)-ß-D-glucans were in the inner S2i region. The areas and intensities of labelling increased with CW severity. The antibody for (1 → 4)-ß-D-galactans was also used to identify the locations and relative amounts of these galactans in whole stem cross sections based on the formation of an insoluble dye. Areas containing the four wood types were clearly differentiated depending on colour intensity. The neutral monosaccharide compositions of the non-cellulosic polysaccharides of these wood types were determined on small, well defined discs, and showed the proportion of galactose was higher for CWs and increased with severity. CONCLUSION: The presence of an S3 wall layer is a marker for very MCW and the presence of helical cavities in the S2 wall layer for SCW. The occurrence and proportions of (1 → 4)-ß-D-galactans and (1 → 3)-ß-D-glucans can be used as markers for CW and its severity. The proportions of galactose were consistent with the labelling results for (1 → 4)-ß-D-galactans.

Molecular characterisation of a novel recombinant Ribgrass mosaic virus strain FSHS.

BACKGROUND: The genus Tobamovirus (Virgaviridae) comprises 33 accepted species with the recent addition of eight new viruses and is divided in to three subgroups based on the origin of assembly of the virion and host range. Within the subgroup 1 tobamoviruses the orchid-associated tobamovirus was hypothesized to be a chimeric derivative of recombinations between genome fragments from subgroup 3 and 1. Recombination events involving RdRp, movement and coat protein genes are recorded within subgroup 1 and 2. However natural recombinations have not previously been reported between subgroup 3 tobamoviruses. FINDINGS: The organization and phylogenetic analyses of the complete genome and the different ORFs placed the new isolate within the Ribgrass mosaic virus clade of subgroup 3 tobamoviruses. Recombination detection analyses indicated that the isolate was a chimeric genome with fragments of high similarity to Ribgrass mosaic virus (RMV) strains NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1) infecting herbaceous Plantago sp. and woody Actinidia spp., respectively. The recombinant differed across the whole genome by 3-8 % from other published RMV genomes. CONCLUSION: In this investigation we report an intra-specific recombination between RMV strains NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1), in the replicase component between viral-methyltransferase and viral-helicase regions, resulting in a novel RMV strain FSHS (JQ319720.1) that represents the first described natural recombinant within the RMV cluster of subgroup 3 tobamoviruses.

Quantification of (1→4)-ß-d-Galactans in Compression Wood Using an Immuno-Dot Assay.

Compression wood is a type of reaction wood formed on the underside of softwood stems when they are tilted from the vertical and on the underside of branches. Its quantification is still a matter of some scientific debate. We developed a new technique that has the potential to do this based on the higher proportions of (1→4)-ß-d-galactans that occur in tracheid cell walls of compression wood. Wood was milled, partially delignified, and the non-cellulosic polysaccharides, including the (1→4)-ß-d-galactans, extracted with 6 M sodium hydroxide. After neutralizing, the solution was serially diluted, and the (1→4)-ß-d-galactans determined by an immuno-dot assay using the monoclonal antibody LM5, which specifically recognizes this polysaccharide. Spots were quantified using a dilution series of a commercially available (1→4)-ß-d-galactan from lupin seeds. Using this method, compression and opposite woods from radiata pine (Pinus radiata) were easily distinguished based on the amounts of (1→4)-ß-d-galactans extracted. The non-cellulosic polysaccharides in the milled wood samples were also hydrolysed using 2 M trifluoroacetic acid followed by the separation and quantification of the released neutral monosaccharides by high performance anion exchange chromatography. This confirmed that the compression woods contained higher proportions of galactose-containing polysaccharides than the opposite woods.

Characterization of the complete genome of a novel citrivirus infecting Actinidia chinensis.

A ssRNA virus from kiwifruit (Actinidia spp.) was identified as a member of the family Betaflexiviridae. It was mechanically transmitted to the herbaceous indicators Nicotiana benthamiana, N. clevelandii, N. glutinosa and N. occidentalis. The complete genome was comprised of three ORFs and a 3'poly (A) tail. Phylogenetic analysis of the entire genome indicated it was a novel member of the genus Citrivirus (family Betaflexiviridae). The complete nucleotide sequence differed from that of citrus leaf blotch virus (CLBV) by ~ 26 %. The movement protein (ORF2) and coat protein (ORF3) shared 95-96 % and 90-92 % amino acid sequence identity, respectively, with CLBV. The replicase polyprotein (ORF1) was distinctly different from published CLBV sequences, with 78-79 % amino acid sequence identity, while the 5' UTR and 3' UTR differed from CLBV by 28 % and 29 %, respectively. The sequence differences indicate that the citrivirus from Actinidia is either a divergent strain of CLBV or a member of a new citrivirus species.

Characterization of the complete genome of ribgrass mosaic virus isolated from Plantago major L. from New Zealand and Actinidia spp. from China.

The complete genomes of tobamovirus isolates from Plantago major L. from New Zealand (NZ-439), Plantago sp. from Germany (Kons 1105), Actinidia chinensis (Actinidia-AC) and A. deliciosa (Actinidia-AD) from China were sequenced and compared to previously published tobamovirus genomes. Their genome organization and phylogenetic analysis of the putative replicase component, replicase readthrough component, movement protein, coat protein and complete genome placed all four isolates in subgroup 3 of the tobamoviruses. The complete genomes differed from each other by <8.5% and from published sequences of turnip vein clearing virus and youcai mosaic virus by about 12-13% and 19-20%, respectively. The aa sequences of the individual ORFs of the Plantago and Actinidia isolates differed from each other by <4% and were most similar to published (partial) sequences of ribgrass mosaic virus (RMV). We propose that these sequences constitute the first complete published sequences for RMV.

Detection and characterisation of two novel vitiviruses infecting Actinidia.

Two co-infecting novel vitiviruses from Actinidia chinensis were identified from mechanically inoculated Nicotiana occidentalis. Both virus genomes were sequenced and share 64% nucleotide identity. Their overall structure is typical of vitiviruses, with five open reading frames (ORFs) and a polyadenylated 3' end. Open reading frame 4 (ORF4) encodes the coat protein, the most conserved gene of the vitiviruses, in which they share 75% amino acid identity, 61-68% with grapevine virus B, 55-59% with grapevine virus A, and 37-42% with grapevine virus E. Based on the molecular criteria for species demarcation in the family Betaflexiviridae, these are two novel viruses, tentatively named Actinidia virus A and Actinidia virus B.

A generic method to identify plant viruses by high-resolution tandem mass spectrometry of their coat proteins.

Although a number of protocols have been developed for detection of viruses at the genus or family level, universal approaches to detect and identify unknown viruses are still required. High-resolution tandem mass spectrometry was used to identify accurately peptide masses and their constituent sequences from partially purified plant virus preparations. Analysis of the peptide fragment masses against a virus database using pattern-matching algorithms identified sequences with homology to known virus peptides and also predicted peptides using de novo sequence analysis. This method provided sufficient information to confirm the identity of two known viruses that were included as controls (Cucumber mosaic virus and Tomato spotted wilt virus) and to identify unknown viruses in six viral isolates. The unknown viruses have been identified as four common viruses (Alfalfa mosaic virus, Tobacco streak virus, Citrus leaf blotch virus and Ribgrass mosaic virus), and two novel viruses (a potexvirus and a vitivirus). The identification of viruses from five distinct families by the tandem mass spectrometric determination of their coat protein demonstrates that this is a useful method for initial virus identification. This method, complemented with molecular or immunological procedures, provides a rapid and convenient way to identify both known and novel plant viruses.

Production and characterisation of two wheat-bran fractions: an aleurone-rich and a pericarp-rich fraction.

Wheat bran is a good source of dietary fibre in the form of cell walls, but contains a number of different cell types. We describe a large-scale procedure for the production of an aleurone-rich and a pericarp-rich fraction from hard, Australian wheat. The fractions were characterised by field-emission scanning electron microscopy, by using a range of bright-field stains, colour reagents, and fluorochromes, and by chemical analysis of the walls. The aleurone fraction included the seed coat with its cuticle. Only the pericarp walls showed a histochemical reaction for lignin. The concentrations of ester-linked ferulic acid and (1-->3),(1-->4)-beta-glucans were greater in the aleurone-rich fraction than in the pericarp-rich fraction. The results are consistent with the arabinoxylans in the walls of the pericarp-rich fraction being more highly substituted with arabinose than those in the walls of the aleurone-rich fraction. When the fractions were fed as a dietary supplement to rats and walls were isolated from the faeces, it was found that the pericarp walls were not degraded, but the aleurone walls were partially degraded.