A Cytochemical Study of Differentiation and Breakdown of Vessel End Walls

A cytochemical approach was used to study the architecture ofthe end wall and its modifications during vessel differentiationin Populus italica and Dianthus caryophyllus. The combinationof cytochemical techniques with selective extractions to removeend wall subunits provides information on the different componentsof the end wall and on the method of perforation. The end wallappears to be formed mainly from pectins and hemicelluloses.No cellulosic components nor lignin were found though the 3,3'-diaminobenzidine assay revealed the presence of peroxidases.That the method of perforation may depend on the species isdiscussed. Dianthus caryophyllus L., carnation, Populus italica, poplar, xylem vessel, cell wall, cytochemistry, vessel differentiation     

Changes in the Distribution of Plasmodesmata in Developing Fibre-tracheid Pit Membranes of Sorbus aucuparia L.

The distribution of plasmodesmata in cambial pit fields anddeveloping fibre-tracheid pit membranes in Sorbus aucupariaL. has been studied using conventional and high voltage transmissionelectron microscopy. In cambial pit fields, plasmodesmata arewidely separated while, during cell enlargement and the stageof secondary wall formation, they occur exclusively in a denselypacked cluster, eccentrically located on the pit membrane ina thickening of primary wall material. It is not clear at themoment whether the cluster arises by aggregation of the cambialpit field plasmodesmata, or by de novo formation in a localizedregion coupled with loss of plasmodesmata outside this region.The significance of these changes is discussed. Sorbus aucuparia L., plasmodesmata, pit membranes, xylem differentiation     

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides × P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofiuorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.     

The Differentiation of Contact Cells and Isolation Cells in the Xylem Ray Parenchyma of Populus maximowiczii

A histochemical analysis was made of the differentiation ofcontact cells and isolation cells in the xylem ray parenchymaof Populus maximowiczii. The contact cells formed secondarywalls at approximately the same time as adjoining vessel elements.The lignification of the cell walls of contact cells and vesselelements began earlier than that of wood fibres and isolationcells. Thus, the formation of the secondary wall, includinglignification, of the contact cells might occur at the sametime as that of the vessel elements to which they are directlyconnected. By contrast, the isolation cells began to form secondarywalls later than the vessel elements and wood fibres in thevicinity of the isolation cells. After the deposition of thesecondary wall, a protective layer was formed in contact cellsbut no isotropic layer was observed in isolation cells. Theresults suggest the importance of vessel elements in the determinationof the differentiation of adjoining ray parenchyma cells.Copyright1999 Annals of Botany Company Contact cell, isolation cell, vessel element, xylem differentiation, Populus maximowiczii Henry.     

Regulation of secondary xylem formation in young hybrid poplars by modifying the expression levels of the <Emphasis Type="Italic">PtaGLIMa</Emphasis> gene

Due to the importance of wood in many industrial applications, a tremendous amount of research has focused on the regulation of secondary xylem formation and wood properties. In this study, we performed functional analysis of PtaGLIM1a, a LIM gene that is predominantly expressed in the differentiation of secondary xylem of the hybrid poplar (Populus tremula × P. alba). With no growth retardation, transgenic poplar plants with increased and reduced expression levels of PtaGlim1a exhibited enhanced and diminished secondary growth, respectively, accompanied by a corresponding change in their lignin abundance. This study demonstrates that the wood-associated PtaGlim1a acts as a positive regulator of secondary xylem formation in poplar trees and could potentially be utilized in modifying the synthesis of plant secondary wall lignin.     

In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata)

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 μM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.     

The Initiation and Development of Secondary Xylem in Axillary Branches of Populus deltoides

The initiation of secondary xylem in elongating axillary branchesof Populus deltoides Bartr. ex Marsh. is independent of thatin the main stem. Although secondary xylem differentiates acropetallyin the main stem, it does not differentiate from the stem intothe axillary branch. Secondary xylem is usually initiated ininternode 4 (occasionally 3) of the axillary branch, and fromthis site it develops both acropetally in the elongating branchand basipetally toward the main stem. Secondary vessel differentiationalways precedes fibre differentiation. Although secondary xylemdifferentiates in internodes that have ceased elongation, itdifferentiates first in traces of the vascular cylinder servingrapidly expanding and maturing foliage leaves. As younger leaveson the branch expand and mature, secondary xylem differentiatesin their traces eventually producing a complete secondary vascularcylinder. Scale leaves do not initiate secondary xylem independentlyin their traces; they are activated by adjacent traces in thevascular cylinder serving foliage leaves. Once established,the primary-secondary vascular transition zone advances acropetallyin a branch just as it does in the main stem. Populus deltoides Bartr. ex Marsh., cottonwood, axillary branches, secondary xylem, plastochron index, post-dormancy development, xylem.     

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Expressing exogenous, apple-derived chalcone synthase in actively lignifying poplar xylem tissue results in less total lignin, improved saccharification rates, and incorporation of naringenin into lignins.     

Ultrastructure of the Cell Wall of Vessel Contact Cells in the Xylem of Tomato Stems

Mature contact cells in the metaxylem of tomato stems are characterizedby a distinctive wall deposit occurring mainly over the areaof the pit membrane that separates the cell from an adjoiningvessel member but also extending as a layer on the secondarywall round the interior of the cell. Cytochemical tests showthe layer contains a low concentration of pectin, no lignin,and a high concentration of polysaccharides. It can be distinguishedfrom the primary and secondary walls by quantitative differencesin the cytochemical staining reactions and by its loose-texturedappearance under the electron microscope. The layer is similarin composition and structure to the ‘protective layer’described in woody plants. The layer has also been found inpea and cotton and may be a characteristic feature of the contactcells in herbaceous plants. Cell wall ultrastructure, contact cells, Lycopersicon esculentum, Mill., protective layer, tomato, xylem     

On Vessel Member Differentiation in the Bean (Phaseolus vulgaris L.)

Certain ultrastructural features of vessel member differentiationwere examined in the primary xylem of petiole of bean (Phaseolusvulgaris L.). The cells used had helical secondary wall thickeningsand simple perforation plates. The primary cell wall increasesin thickness before the helices of secondary wall develop. Ina common wall between two vessel members of different ages,theprimary thickening occurs first in the older cell so thatfor a time the middle lamella is located closer to the youngercell rather than medianly. Apparently the helix is depositedafter the primary wall of a given cell reaches maximum thickness.The perforation of the end wall is preceded by primary thickeningof the part of the wall that is later removed. The marginalregion remains relatively thin and becomes covered with a rimof secondary wall. Vesicles with fibrous content appear nearthe surface within the end wall shortly before the perforationoccurs. A highly vacuolated protoplast with a much enlargednucleus and numerous organdIes is present during cell wall differentiation.After that process is completed, the protoplast disintegratesand the primary wall bearing the helix is hydrolysed where itis exposed to the cell lumen and, under certain conditions,also under the secondary wall.     

Drought‐induced xylem pit membrane damage in aspen and balsam poplar

Drought induces an increase in a tree's vulnerability to a loss of its hydraulic conductivity in many tree species, including two common in western Canada, trembling aspen (Populus tremuloides) and balsam poplar (Populus balsamifera). Termed ‘cavitation fatigue’ or ‘air‐seeding fatigue’, the mechanism of this phenomenon is not well understood, but hypothesized to be a result of damage to xylem pit membranes. To examine the validity of this hypothesis, the effect of drought on the porosity of pit membranes in aspen and balsam poplar was investigated. Controlled drought and bench dehydration treatments were used to induce fatigue and scanning electron microscopy (SEM) was used to image pit membranes for relative porosity evaluations from air‐dried samples after ethanol dehydration. A significant increase in the diameter of the largest pore was found in the drought and dehydration treatments of aspen, while an increase in the percentage of porous pit membranes was found in the dehydration treatments of both species. Additionally, the location of the largest pore per pit membrane was observed to tend toward the periphery of the membrane.     

Fine Structure of Hydrolysed Primary Walls in Tracheary Elements of Petiolar Xylem in Eucalyptus delegatensis

The hydrolysed lateral primary walls of tracheary elements ofthe petiolar xylem of Eucalyptus delegatensis were examinedby electron microscopy. Vessel-vessel and vessel—tracheidhydrolysed walls were strikingly different in appearance fromtracheid—tracheid walls. The difference seemed to be inthe degree to which the primary walls were hydrolysed. The observationssuggest the wall hydrolysis to be an ordered and controlledprocess. Eucalyptus delegatensis, hydrolysed wall, petiolar xylem, tracheary elements     

The proteasome is responsible for caspase-3-like activity during xylem development

Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin β-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.     

Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar

The application of nitrogen-containing fertilisers is one approach used to increase growth rates and productivity of forest tree plantations. However, the effects of nitrogen fertilisation on wood properties have not been systematically assessed. The aim of this work was to document the impacts of nitrogen fertilisation on wood formation and secondary xylem fibre properties. We used three fertilisation treatments in which the level of ammonium nitrate was adjusted to 0, 1 and 10 mM in a complete nutrient solution applied daily over a period of 28 days in standardised greenhouse experiments with clonal material of Populus trichocarpa (Torr and Gray) × deltoides (Bartr. ex Marsh). We showed that there was a short-term and repeatable response in which xylem fibre morphology and secondary cell wall structure adapt to a shift in N availability. Under high-nitrogen exposure, xylem fibres were 17% wider and 18% shorter compared to the adequate nitrogen treatment. A very significant thickening of the fibre cell walls was also observed throughout the stem of trees receiving the high-N treatment. It appeared that cell wall structure was greatly affected by the high-N treatment as fibres developed a modified inner cell wall layer. Histological observations indicated that the internal cell wall layer was enriched in cellulose and chemical determinations showed that wood contained more holocellulose. Together, these results indicate that the response of poplar to nitrogen availability may involve marked effects on secondary xylem formation.     

Empirical Models for Xylogenesis in Populus deltoides

Empirical quantitative models were constructed for Populus deltoidesdescribing temporal and spatial changes in vessel characteristicsof metaxylem, both within individual central leaf traces andwithin all central leaf traces considered as a morphologicalunit at a given transverse level in the stem (the central tracesympodia). Similar models were constructed for secondary vesselcharacteristics. The growth processes of the stem segment throughwhich the vasculature extended were incorporated in these modelsto illustrate how a functional vascular system is maintainedin the stem as a whole. The central trace sympodia representedthe integrals of the temporal and spatial functions for individualcentral leaf traces. Metaxylem vessel production ceased in individualleaf traces two plastochrons before the cessation was reflectedin the central trace sympodia because of the integrative natureof the sympodial complex. A functional continuum of developmentwas apparent between metaxylem vessels of the central tracesympodia and secondary vessels of the stem. The transition betweenmetaxylem and secondary xylem production in the central tracesympodia corresponded with cessation of leaf and internode elongation. Populus deltoides Bartr. ex Marsh., cottonwood, primary xylem, secondary xylem, primary-secondary vascular transition, leaf growth, xylogenesis     

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. Key message We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.     

Plasmodesmata and pit development in secondary xylem elements

Developing pit membranes of secondary xylem elements in Drimys winteri, Fagus sylvatica, Quercus robur, Sorbus aucuparia, Tilia vulgaris and Trochodendron aralioides have been examined by transmission electron microscopy. Absence of plasmodesmata from the membranes of vessel elements and tracheids indicates that their pits develop independently of these structures. On the other hand, plasmodesmata are abundant in pit membranes between fibres, parenchyma cells, and combinations of these cell types in Fagus, Quercus and Tilia. In each case the plasmodesmata pass right through the developing pit membrane. In the case of Sorbus fibres, however, plasmodesmata were absent from the majority of pit membrane profiles seen in sections. Occasionally they were observed in large numbers associated with a swollen region on one side of the pit membrane between fibres and between fibres and parenchyma, radiating from a small area of the middle lamella. In the case of fibre to parenchyma pitting, this swelling was always found on the fibre side of the membrane, while on the other side a small number of plasmodesmata were present completing communication with the parenchyma cytoplasm. These observations are discussed with regard to the role of plasmodesmata in pit formation, and in the differentiation of the various cell types in secondary xylem. The significance their distribution may have for our understanding of xylem evolution is also discussed.