Nigel Chaffey

Red fluorescent protein

One of the many useful cell biological techniques of the past few years has been the development of green fluorescent protein (GFP) as a reporter for gene activity, in which the gene of interest and the GFP gene are expressed together. Thus, the appearance of GFP fluorescence can be used to visualize both the expression of the other gene and its cellular location. Although GFP variants exist with different emission wavelengths, more variety is needed to permit multi-fluorescent imaging, or to overcome problems of inherent tissue fluorescence. The recent publication of the atomic resolution structure of red fluorescent protein (RFP) by Mark Wall and colleagues [Nat. Struct. Biol. (2000) 7, 1133–1138] should permit engineering of its fluorescent properties so that FPs can be even more useful as gene-reporters. NC

Cambium: old challenges – new opportunities

Abstract Trees represent a, probably the, major component of the biosphere and have a unique place in the history of Mankind. One of their most fascinating features is the process of secondary growth which is effected principally by the secondary vascular system, the developmental continuum of secondary phloem, vascular cambium, and secondary xylem. However, for too long assumptions about the developmental biology of trees have had to be based upon studies of primary growth systems within annual, herbaceous species because study of the secondary vascular system had been largely ignored. Even when attempts are made to understand some of the most fundamental features of the secondary vascular system, such as xylogenesis, the current model system, isolated Zinnia mesophyll cells, is not entirely appropriate to the situation in the intact tree. Some deficiencies of the Zinnia system are discussed, and the advantages of the genus Populus as a model for study of the hardwood secondary vascular system are considered. Some of the new approaches which are poised to lead to significant advances in our knowledge of the cell bio-logy of the secondary vascular system of trees – spe-cifically of the cell wall, the plasmalemma, and the cytoskeleton – are discussed. The value of one of these new techniques – immunocytochemistry – is demonstrated by a consideration of recent work on the role of the cytoskeleton in the hardwood secondary vascular system.

Wood formation in trees: cell and molecular biology techniques.

Introduction. An Introduction to the Problems of Working with Trees. Wood Microscopical Techniques. Conventional (Chemical-fixation) Transmission Electron Microscopy and Cytochemistry of Angiosperm Trees. Chemical and Cryo-fixation for Transmission Electron Microscopy of Gymnosperm Cambial Cells. Deep-etching Electron Microscopy and 3-Dimensional Cell Wall Architecture. Secondary Ion Mass Spectometry Microscopy: Application to the Analysis of Woody Samples. Immunolocalization of the Cytoskeleton in the Secondary Vascular System of Angiosperm Trees and its Visualisation Using Epifluorescence Microscopy. Immunolocalization and Visualization of the Cytoskeleton in Gymnosperms using Confocal Laser Scanning Microscopy. Cell Walls of Woody Plants: Autoradiography and Ultraviolet Microscopy. Cell Walls of Woody Tissues: Cytochemical, Biochemical and Molecular Analysis of Pectins and Pectin Methylesterases. Immunolocalization of Enzymes of Lignification. Sampling of Cambial Region Tissues for High Resolution Analysis. Biochemistry and Quantitative Histochemistry of Wood. Protein Analysis in Perennial Tissues. The Use of GUS Histochemistry to Visualize Lignification Gene Expression In Situ during Wood Formation. In Situ Hybridization. Random Amplification of Polymorphic DNA and Reverse transcription Chain Reaction of RNA in Studies of Sapwood and Heartwood.

A cytoskeletal basis for wood formation in angiosperm trees: the involvement of cortical microtubules

Abstract. Rearrangements of cortical microtubules (CMTs) during the differentiation of axial secondary xylem elements within taproots and shoots of Aesculus hippocastanum L. (horse-chestnut) are described. A correlative approach was employed using indirect immunofluorescence microscopy of α-tubulin in 6- to 10-μm sections and transmission electron microscopy of ultrathin sections. All cell types – fibres, vessel elements and axial parenchyma – derive from fusiform cambial cells which contain randomly oriented CMTs. At the early stages of development, fibres and axial parenchyma cells possess helically arranged CMTs, which increase in number as secondary wall thickening proceeds and simple pits develop. In contrast, incipient vessel elements are distinguished by the marking out of sites of bordered pits; these sites first appear as microtubule-free regions within the reticulum of randomly oriented CMTs that characterises their precursor fusiform cambial cells. Subsequently, the ring of CMTs which develops at the periphery of the microtubule-free region decreases in diameter as the over-arching pit border is formed. Like bordered pits, large-diameter, non-bordered pits (contact pits) which develop between vessel elements and adjacent contact ray cells originate as microtubule-free regions and are also associated with development of a ring of CMTs at the periphery. In the case of contact pits, however, there is no reduction in the diameter of the CMT ring during pit development. Tertiary cell wall thickenings are also a feature of vessel elements and appear to form at sites where bands of laterally associated, transversely oriented CMTs, separated from each other by microtubule-free zones, are found. Later, these bands of CMTs become narrower, and separate into pairs of microtubule bundles located on each side of the developing wall thickening. Development of perforations between vessel elements is also associated with the presence of a ring of CMTs at their periphery.

The cytoskeleton facilitates a three-dimensional symplasmic continuum in the long-lived ray and axial parenchyma cells of angiosperm trees

The microtubule (MT), microfilament (MF) and myosin components of the cytoskeleton were studied in the long-lived ray and axial parenchyma cells of the secondary xylem (wood) and secondary phloem of two angiosperm trees, Aesculus hippocastanum L. (horse-chestnut) and Populus tremula L. x P. tremuloides Michx. (hybrid aspen), using indirect immunofluorescence localisation and transmission electron microscopy. MTs and MFs were bundled and oriented axially (parallel to the cells long axis) within all parenchyma cell types after they had fully differentiated. Additionally, actin and myosin were immunolocalised at the thin-walled membranes of the pits, which linked cells in neighbouring files of both ray and axial parenchyma, and at the pits between axial and ray parenchyma cells themselves. Anti-callose antibody immunolocated the plasmodesmata at the pit membranes, and in the same pattern as that of anti-myosin. Ray cells are important symplasmic pathways between the xylem and the phloem throughout the life of trees. We hypothesise that the MT and MF components of the cytoskeleton in the ray and axial parenchyma cells are involved in the transport of materials within those cells, and, in association with the acto-myosin of plasmodesmata at pit fields, are also important in intercellular transport. Thus, the symplasmic coupling between ray cells, between axial parenchyma cells, and between axial parenchyma and ray cells represents an extensive three-dimensional communication pathway permeating the tree from the phloem through the cambium into the wood. We suggest that this cytoskeletal pathway has an important role in delivery of photosynthate, and mobilised reserves, to the actively dividing cambium, and in the movement of materials to sites of reserve deposition, principally within the wood. This pathway could also have an important role in co-ordinating developmental processes throughout the tree.

Cortical microtubule involvement in bordered pit formation in secondary xylem vessel elements of Aesculus hippocastanum L. (Hippocastanaceae) : a correlative study using electron microscopy and indirect immunofluorescence microscopy

SummaryA correlative study, using indirect immunofluorescence microscopy (IIF) of anti-α-tubulin stained sections and transmission electron microscopy (TEM), gave details of the involvement of cortical microtubules (CMTs) in the development of bordered pits in secondary xylem vessel elements ofAesculus hippocastanum L. In addition, aspects of wall cytochemistry were studied during this process using the Thiéry (PATAg) test, immunolocalization with the monoclonal antibodies JIM5 and JIM7, and a range of other cytochemical procedures. IIF showed that the alternately-arranged pits are pre-figured as perforations within a reticulum of randomly-oriented CMTs before any secondary wall thickening is obvious. Each incipient pit border is subsequently delimited by a circle of CMTs whose diameter decreases as deposition of secondary wall takes place around the perforation. These IIF observations are corroborated by a parallel TEM study. During the period of bordered pit formation, the secondary walls of the cell are lignifying. At maturity, however, the pit membrane is unlignified and continues to stain strongly with the monoclonal antibody JIM5, a marker of primary, “juvenile” wall. The results are discussed in terms of the relationship of the CMT cytoskeleton with development of bordered pits.

Visualization of the cytoskeleton within the secondary vascular system of hardwood species

A single fixation technique has been devised to demonstrate localization of α‐tubulin (for microtubules) and F‐actin (for microfilaments) within the secondary vascular system of hardwood trees by indirect immunofluorescence microscopy using butyl‐methylmethacrylate‐embedded material. Application of this technique to problems of cytomorphogenesis during secondary growth and its versatility are demonstrated with the hardwood species Aesculus hippocastanum L., Salix viminalis L., S. burjatica Nazarov × S. viminalis L., Hedera helix L., Acer platanoides L., Platanus sp., Quercus ilex L. and Liriodendron tulipifera L., and in the softwood Pinus pinea L. The methods employed have considerable scope for advancing knowledge of the role of the cytoskeleton in differentiation within the secondary vascular system of woody species.

Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not

Abstract The plant cytoskeleton has been implicated in a variety of morphogenetic events in higher plants. Most of this work, however, has concentrated on epidermal cells or primary tissues. We have investigated the cortical microtubular (CMT) and microfilament (MF) components of the cytoskeleton in a secondary tissue  –  active vascular cambium of Aesculus hippocastanum L. (horse-chestnut)  –  and followed the changes in these components during the early stages of differentiation of fusiform cambial derivatives to axial elements of the secondary vascular system. A correlative approach was used employing indirect immunofluorescence microscopy of α-tubulin on 6 μm sections, and transmission electron microscopy of 60 nm sections. The study has demonstrated a rearrangement of the CMT cytoskeleton, from random to helical, as fusiform vascular cambial cells begin to differentiate as secondary phloem vascular tissue. A similar CMT rearrangement is seen as fusiform cambial cells begin to differentiate as secondary xylem fibres. This rearrangement is interpreted as evidence of determination of cambial derivatives towards vascular development. Axially-oriented MF bundles are present in fusiform cambial cells and their axial orientation is retained in the vascular derivatives at early stages of their development even though the CMTs have become rearranged.

Microfibril orientation in wood cells: new angles on an old topic

Although it is essential to obtain experimental evidence about any role for microtubules in microfibril orientation, the difficulty of accessing the developing wood cells in trees has to date required the use of excised, fixed material. A system in which cell-biological events can be followed, and experimentally manipulated, in vivo is clearly desirable and will permit a much better understanding of all aspects of wood-cell differentiation. Arabidopsis, which also undergoes substantial wood-formation10xWood formation in forest trees: from Arabidopsis to Zinnia. Chaffey, N.J. Trends Plant Sci. 1999; 4: 203–204Abstract | Full Text | Full Text PDF | PubMedSee all References10, might have a role to play here, in tandem with poplar, the model hardwood tree, of course!

Plasmatubules in transfer cells of pea (Pisum sativum L.)

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461–465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.