Sandra L. Jackson

Fluorescence Microscopy Study of Protein Adsorption at Modified Glassy Carbon Surfaces

Glassy carbon (GC) surfaces were modified with thin films by means of electrochemically assisted reduction of aryl diazonium salts and oxidation of primary amines. GC plates with modified and unmodified areas were exposed to solutions of fluorescently labelled bovine serum albumin (BSA-FITC). Fluorescence microscopy was found to be a simple and direct method for comparing adsorption of BSA-FITC between the different areas. Modification with methylbenzene, hexylbenzene, poly(ethylene glycol) benzene, and tetraethylene glycol diamine groups increases protein adsorption relative to unmodified GC. Hexylamine and the poly(ethylene glycol) diamine ED-2003 reduce protein adsorption. The results give insight into some factors controlling protein adsorption at these surface films.

The Distribution of un-esterified and Methyl-Esterified Pectic Polysaccharides in Pinus Radiata

A cationic dye which binds acidic polymers such as pectin and monoclonal antibodies, directed against un-esterified and methyl-esterified (JIM5) and only methyl-esterified (JIM7) pectin epitopes, were used, in conjunction with light microscopy, confocal microscopy and immunogold electron microscopy, to study the spatial distribution of pectin in the xylem tissue of Pinus radiata D. Don. Histochemistry demonstrated that pectin was located in the compound middle lamella (CML) of the maturing tracheid cell wall, in addition to the pit membranes and the CML of the ray cell walls. Immunogold labeling showed differential distribution of the pectin epitopes within the CML of the maturing cell walls. Moreover, in the xylem, the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues. Neither epitope occurred in the secondary walls of the xylem cells. These patterns of epitope expression were not maintained in the mature cell. These results represent the first demonstration of restricted spatial patterns of distribution of these epitopes in the xylem tissue of radiata pine and are consistent with results from other coniferous gymnosperms.

Intra-ring checking in Pinus radiata D. Don: the occurrence of cell wall fracture, cell collapse, and lignin distribution

Ultrastructural and cytochemical features of tracheid cell walls were examined in oven-dried Pinus radiata D. Don disks that demonstrated a range in severity of the wood quality flaw referred to as “intra-ring checking,” from severe to none. Observations of the tracheid cell wall at the ultrastructural level included the localization of the origin of tears between adjacent cells, and the occurrence of tracheid collapse. Cytochemical analysis focused on determination of the spatial distribution of lignin within the cell wall layers. Tracheid lignin content was further quantified using the Klason and acetyl bromide methods. We found considerable homogeneity in the point of failure in the wood demonstrating intra-ring checking, with 80% of the tears occurring at the compound middle lamella (CML)/S1 cell wall interface. In these samples, tracheid collapse was observed adjacent to the tear as well as between tears, and the cell walls appeared to have altered lignin distribution, particularly in the S1 wall layer. We suggest that alterations in the CML/S1 layers create a weak point in the cell wall, making it prone to the observed tears. The mechanisms that may be involved in the occurrence of intra-ring checking are discussed at the morphological level.

RGD-containing peptides and cyclic AMP have antagonistic roles in the morphology of Mucor rouxii

Summary.The tripeptide Arg-Gly-Asp (RGD) (1 mM) as well as the polymer ProNectin F (20 nM) added to culture medium of the fungus Mucor rouxii (defined medium) produced a delay in the switch from isodiametric growth to tip growth; at the time of germination the mother cell had a 4.6 times larger volume with 3.6 times more germ tubes per cell than control germinating sporangiospores. Disruption of the actin network with 2 µg of cytochalasin A per ml blocked the switch to tip growth; the effect was analogous to the one of 150 µM dibutyryl–cyclic AMP (cAMP), which we previously described to promote isodiametric growth via protein kinase A. 150 µM dibutyryl-cAMP antagonises partially the effect of 1 mM RGD; the cells still emit several germ tubes per mother cell but their number is smaller and the volume of the cell at germ tube emission is larger than with RGD alone. At higher concentrations the dibutyryl-cAMP overrides completely the effect of RGD. Our results suggest that M. rouxii has an RGD recognition system and demonstrate that RGD-containing peptides have a profound effect on the isotropic stage of growth and on the establishment of cell polarity and that cAMP analogues can override this effect.

Are Rays and Resin Canals Causal Sites for Intra-Ring Checking in the Wood of Pinus Radiata?

Pinus radiata D. Don (radiata pine) wood can develop a wood quality defect called ‘intra-ring checking’ (checks) during kiln drying. A study was conducted to examine if rays and resin canals were the initiation sites of checks, and if the presence of the rays and resin canals increased the susceptibility of radiata pine wood to checking. The structural features associated with checking were observed in images of thirteen oven-dried radiata pine disks. Six of the sixty checks observed were associated with rays and resin canals. It is clear from the observations that rays and resin canals could not be the primary sites for check development. A comparative study showed some differences between the checked and non-checked wood with respect to rays and resin canals. Checked wood showed a higher amount of tissue area occupied by rays than the nonchecked wood. Hence, it is possible that rays can influence the tendency of wood to check. Such a relationship was not seen with respect to resin canals. However, a difference in the arrangement of resin canals was observed between checked and non-checked wood. Checked wood showed a scattered arrangement of resin canals, while the non-checked wood showed a linear arrangement.

An investigation of the effects of Ca2+ channel inhibitors on branching and chemotropism in the oomycete Achlya bisexualis: Support for a role for Ca2+ in apical dominance

In an attempt to better understand branching and chemotropism, we describe the effects of Ca²+ channel inhibitors on these processes in Achlya bisexualis, using a branch induction technique and whole plate assays. Branching appears to be a two step process with the initial formation of a bump from which a branch emerges. Verapamil increased numbers of branches in whole plate assays and decreased the distance from the first branch to the tip. In induction assays verapamil increased the number of bumps formed, although in some hyphae it inhibited the transition from an initial bump to a branch. When a branch formed it did not affect the time taken to branch. It had no effect on chemotropism. Lanthanum (La³+) and gadolinium (Gd³+) also increased branching in whole plate assays but their effect was much less marked and they had no effect on bump/branch number in induction assays. Gd³+ decreased the time taken to branch. Both La³+ and Gd³+ increased chemotropism. These data suggest firstly that the respective inhibitors may affect different parts of the branching process and secondly that Ca²+ influx through channels may not be a requirement for branching, indeed such movements may suppress branching. This would fit with elevated Ca²+ at the tip playing a role in apical dominance.

Mucor rouxii ultrastructure: cyclic AMP and actin cytoskeleton

Summary.A comparative analysis of the effect of two compounds, dibutyryl–cyclic-AMP (dbcAMP) and latrunculin B, on the morphology and ultrastructure of the dimorphic fungus Mucor rouxii under aerobic growth conditions is presented. dbcAMP acts through the sustained activation of protein kinase A, and latrunculin B through the disruption of the actin cytoskeleton. Upon addition of these compounds to the growth medium at any stage of the germination process, cells lost polarised growth and switched to isodiametric growth. The effect was reversible. The morphologies, visualised by light microscopy or scanning electron microscopy (SEM), were alike. A switch from a rough to a smooth surface was observed by SEM when cells were repolarised by removal of the added compound. Ultrastructural changes under both conditions, as observed by transmission electron microscopy, were similar, the main feature being the enlargement of the cell wall, with irregular depositions, and detachment from the cell membrane. dbcAMP-treated cells showed a decrease in the number of glycogen granules compared with control and latrunculin B-treated cells. F-actin staining with fluorescein isothiocyanate–phalloidin showed that both dbcAMP- and latrunculin B-treated cells displayed a much lower fluorescence than control cells, with only a few pale plaques. The results suggest that the sustained activation of protein kinase A, which impairs polarised growth, might exert its effect through a modification of actin cytoskeleton organisation, very probably also involving an integrinlike pathway, as judged by the cell wall detachment and loss of cell adhesiveness of the dbcAMP-treated isodiametric cells.

Compression Wood does not Form in the Roots of Pinus Radiata

We investigated the potential for the roots of Pinus radiata D. Don to form compression wood. Compression wood was not observed in either the tap or any lateral roots further than 300 mm from the base of the stem. This suggests that either the roots do not experience the stresses required to induce compression wood formation, or that they lack the ability to form it. Roots artificially subjected to mechanical stress also failed to develop compression wood. It is therefore unlikely that an absence of a compressive load on buried roots can account for the lack of compression wood. Application of auxin to the cambia of lateral roots was similarly ineffective at inducing the formation of compression wood. These observations suggest that the buried roots of radiata pine lack the ability to develop compression wood. We also report the formation of an atypical S3 wall layer in the mechanically-stressed and auxin-treated tracheids and suggest that a reaction wood that is different to compression wood may well form in roots.