Background and Aims
Cambial reactivation in trees occurs from late winter to early spring when photosynthesis is minimal or almost non-existent. Reserve materials might be important for wood formation in trees. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules in cambium and phloem were examined from cambial dormancy to the start of xylem differentiation in locally heated stems of Cryptomeria japonica trees in winter.
Electric heating tape was wrapped on one side of the stem of Cryptomeria japonica trees at breast height in winter. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules were determined by image analysis of optical digital images obtained by confocal laser scanning microscopy.
Localized heating induced earlier cambial reactivation and xylem differentiation in stems of Cryptomeria japonica, as compared with non-heated stems. There were clear changes in the respective localizations and levels of starch and lipids (as droplets) determined in terms of relative areas on images, from cambial dormancy to the start of xylem differentiation in heated stems. In heated stems, the levels and number of starch granules fell from cambial reactivation to the start of xylem differentiation. There was a significant decrease in the relative area occupied by lipid droplets in the cambium from cambial reactivation to the start of xylem differentiation in heated stems.
The results showed clearly that the levels and number of storage starch granules in cambium and phloem cells and levels of lipids (as droplets) in the cambium decreased from cambial reactivation to the start of xylem differentiation in heated stems during the winter. The observations suggest that starch and lipid droplets might be needed as sources of energy for the initiation of cambial cell division and the differentiation of xylem in Cryptomeria japonica.
Cambial reactivation; confocal laser scanning microscopy; Cryptomeria japonica; lipid; starch; xylem differentiation
• Background and Aims The effect of heating and cooling on cambial activity and cell differentiation in part of the stem of Norway spruce (Picea abies) was investigated.
• Methods A heating experiment (23–25 °C) was carried out in spring, before normal reactivation of the cambium, and cooling (9–11 °C) at the height of cambial activity in summer. The cambium, xylem and phloem were investigated by means of light- and transmission electron microscopy and UV-microspectrophotometry in tissues sampled from living trees.
• Key Results Localized heating for 10 d initiated cambial divisions on the phloem side and after 20 d also on the xylem side. In a control tree, regular cambial activity started after 30 d. In the heat-treated sample, up to 15 earlywood cells undergoing differentiation were found to be present. The response of the cambium to stem cooling was less pronounced, and no anatomical differences were detected between the control and cool-treated samples after 10 or 20 d. After 30 d, latewood started to form in the sample exposed to cooling. In addition, almost no radially expanding tracheids were observed and the cambium consisted of only five layers of cells. Low temperatures reduced cambial activity, as indicated by the decreased proportion of latewood. On the phloem side, no alterations were observed among cool-treated and non-treated samples.
• Conclusions Heating and cooling can influence cambial activity and cell differentiation in Norway spruce. However, at the ultrastructural and topochemical levels, no changes were observed in the pattern of secondary cell-wall formation and lignification or in lignin structure, respectively.
Norway spruce; Picea abies; cambium; xylem; phloem; cell differentiation; heating; cooling; light microscopy; transmission electron microscopy; UV-microspectrophotometry
Background and Aims
Cambium reactivation after dormancy and budbreak in deciduous trees requires a supply of mobilized reserve materials. The pathway and mode of transfer of these materials are poorly understood.
Transport of reserve materials during cambium reactivation in Populus nigra was investigated by conventional and immunocytochemical TEM analyses, SDS–PAGE, western blotting and intracellular microinjection of fluorescent dyes.
Proteinaceous compounds stored in vacuoles and protein bodies of vascular cells and ray cells disappeared within 3 weeks after cambial reactivation and budbreak. Some of these proteins (32 kDa, 30 kDa and 15 kDa) were labelled by lectin antibodies in SDS–PAGE. The same antibodies were localized to plasmodesmata (PDs) between phloem parenchyma, ray cells and fusiform cambial cells. In addition, proteinaceous particles were localized inside the cytoplasmic sleeves of these PDs during budbreak. During this period, the functional diameter of PDs was about 2·2 nm which corresponds approximately to the Stokes' radius of the detected 15-kDa protein.
Lectin-like reserve proteins or their degradation products seem to be transferred through PDs of phloem parenchyma and rays during cambial reactivation and budbreak. PD transfer of storage proteins is a novelty which supports the concept of symplasmic nutrient supply to the cambial region.
Cambial region; lectins; plasmodesmal trafficking; Populus nigra ‘italica’; size exclusion limit; storage proteins; vascular tissues
Background and Aims
Latewood formation in conifers occurs during the later part of the growing season, when the cell division activity of the cambium declines. Changes in temperature might be important for wood formation in trees. Therefore, the effects of a rapid decrease in temperature on cellular morphology of tracheids were investigated in localized heating-induced cambial reactivation in Cryptomeria japonica trees and in Abies firma seedlings.
Electric heating tape and heating ribbon were wrapped on the stems of C. japonica trees and A. firma seedlings. Heating was discontinued when 11 or 12 and eight or nine radial files of differentiating and differentiated tracheids had been produced in C. japonica and A. firma stems, respectively. Tracheid diameter, cell wall thickness, percentage of cell wall area and percentage of lumen area were determined by image analysis of transverse sections and scanning electron microscopy.
Localized heating induced earlier cambial reactivation and xylem differentiation in stems of C. japonica and A. firma as compared with non-heated stems. One week after cessation of heating, there were no obvious changes in the dimensions of the differentiating tracheids in the samples from adult C. japonica. In contrast, tracheids with a smaller diameter were observed in A. firma seedlings after 1 week of cessation of heating. Two or three weeks after cessation of heating, tracheids with reduced diameters and thickened cell walls were found. The results showed that the rapid decrease in temperature produced slender tracheids with obvious thickening of cell walls that resembled latewood cells.
The results suggest that a localized decrease in temperature of stems induces changes in the diameter and cell wall thickness of differentiating tracheids, indicating that cambium and its derivatives can respond directly to changes in temperature.
Cambial activity; conifers; latewood formation; morphology of tracheids; rapid decrease in temperature
Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula×alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 nl l−1). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.
Cellulose; lignin; ozone; poplar; tension wood
Background and Aims
Teak forms xylem rings that potentially carry records of carbon sequestration and climate in the tropics. These records are only useful when the structural variations of tree rings and their periodicity of formation are known.
The seasonality of ring formation in mature teak trees was examined via correlative analysis of cambial activity, xylem and phloem formation, and climate throughout 1·5 years. Xylem and phloem differentiation were visualized by light microscopy and scanning electron microscopy.
A 3 month dry season resulted in semi-deciduousness, cambial dormancy and formation of annual xylem growth rings (AXGRs). Intra-annual xylem and phloem growth was characterized by variable intensity. Morphometric features of cambium such as cambium thickness and differentiating xylem layers were positively correlated. Cambium thickness was strongly correlated with monthly rainfall (R2 = 0·7535). In all sampled trees, xylem growth zones (XGZs) were formed within the AXGRs during the seasonal development of new foliage. When trees achieved full leaf, the xylem in the new XGZs appeared completely differentiated and functional for water transport. Two phloem growth rings were formed in one growing season.
The seasonal formation pattern and microstructure of teak xylem suggest that AXGRs and XGZs can be used as proxies for analyses of the tree history and climate at annual and intra-annual resolution.
Growth rings; teak; Tectona grandis; vascular cambium; xylem and phloem formation
Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (3H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.
Background and Aims
During their lifetime, tree stems take a series of successive nested shapes. Individual tree growth models traditionally focus on apical growth and architecture. However, cambial growth, which is distributed over a surface layer wrapping the whole organism, equally contributes to plant form and function. This study aims at providing a framework to simulate how organism shape evolves as a result of a secondary growth process that occurs at the cellular scale.
The development of the vascular cambium is modelled as an expanding surface using the level set method. The surface consists of multiple compartments following distinct expansion rules. Growth behaviour can be formulated as a mathematical function of surface state variables and independent variables to describe biological processes.
The model was coupled to an architectural model and to a forest stand model to simulate cambium dynamics and wood formation at the scale of the organism. The model is able to simulate competition between cambia, surface irregularities and local features. Predicting the shapes associated with arbitrarily complex growth functions does not add complexity to the numerical method itself.
Despite their slenderness, it is sometimes useful to conceive of trees as expanding surfaces. The proposed mathematical framework provides a way to integrate through time and space the biological and physical mechanisms underlying cambium activity. It can be used either to test growth hypotheses or to generate detailed maps of wood internal structure.
Dynamic model; level sets; surface growth; vascular cambium; wood formation
The genus Populus includes poplars, aspens and cottonwoods, which will be collectively referred to as poplars hereafter unless otherwise specified. Poplars are the dominant tree species in many forest ecosystems in the Northern Hemisphere and are of substantial economic value in plantation forestry. Poplar has been established as a model system for genomics studies of growth, development, and adaptation of woody perennial plants including secondary xylem formation, dormancy, adaptation to local environments, and biotic interactions.
As part of the poplar genome sequencing project and the development of genomic resources for poplar, we have generated a full-length (FL)-cDNA collection using the biotinylated CAP trapper method. We constructed four FLcDNA libraries using RNA from xylem, phloem and cambium, and green shoot tips and leaves from the P. trichocarpa Nisqually-1 genotype, as well as insect-attacked leaves of the P. trichocarpa × P. deltoides hybrid. Following careful selection of candidate cDNA clones, we used a combined strategy of paired end reads and primer walking to generate a set of 4,664 high-accuracy, sequence-verified FLcDNAs, which clustered into 3,990 putative unique genes. Mapping FLcDNAs to the poplar genome sequence combined with BLAST comparisons to previously predicted protein coding sequences in the poplar genome identified 39 FLcDNAs that likely localize to gaps in the current genome sequence assembly. Another 173 FLcDNAs mapped to the genome sequence but were not included among the previously predicted genes in the poplar genome. Comparative sequence analysis against Arabidopsis thaliana and other species in the non-redundant database of GenBank revealed that 11.5% of the poplar FLcDNAs display no significant sequence similarity to other plant proteins. By mapping the poplar FLcDNAs against transcriptome data previously obtained with a 15.5 K cDNA microarray, we identified 153 FLcDNA clones for genes that were differentially expressed in poplar leaves attacked by forest tent caterpillars.
This study has generated a high-quality FLcDNA resource for poplar and the third largest FLcDNA collection published to date for any plant species. We successfully used the FLcDNA sequences to reassess gene prediction in the poplar genome sequence, perform comparative sequence annotation, and identify differentially expressed transcripts associated with defense against insects. The FLcDNA sequences will be essential to the ongoing curation and annotation of the poplar genome, in particular for targeting gaps in the current genome assembly and further improvement of gene predictions. The physical FLcDNA clones will serve as useful reagents for functional genomics research in areas such as analysis of gene functions in defense against insects and perennial growth. Sequences from this study have been deposited in NCBI GenBank under the accession numbers EF144175 to EF148838.
The raffinose family of oligosaccharides (RFOs) serve as transport carbohydrates in the phloem, storage compounds in sink tissues, and putative biological agents to combat both abiotic and biotic stress in several plant species. To investigate further the functional roles of this class of compounds in trees, two cDNAs encoding galactinol synthase (GolS, EC 126.96.36.199), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus alba×grandidentata). Phylogenetic analyses of the Populus GolS isoforms with other known GolS proteins suggested a putative role for these enzymes during biotic or abiotic stress in hybrid poplar. The predicted protein sequences of both isoforms (Pa×gGolSI and Pa×gGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII resulted in Km values for UPD-galactose of 0.80 and 0.65 mM and Vmax values of 657.5 and 1245 nM min−1, respectively. Pa×gGolSI inherently possessed a broader pH and temperature range when compared with Pa×gGolSII. Interestingly, spatial and temporal expression analyses revealed that Pa×gGolSII transcript levels varied seasonally, while Pa×gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.
galactinol synthase; GolS; raffinose family of oligosaccharides; hybrid poplar; abiotic stress; biotic stress
Two thaumatin-like proteins (TLPs) were previously identified in phloem exudate of hybrid poplar (Populus trichocarpa × P. deltoides) using proteomics methods, and their sieve element localization confirmed by immunofluorescence. In the current study, we analyzed different tissues to further understand TLP expression and localization in poplar, and used immunogold labelling to determine intracellular localization.
Immunofluorescence using a TLP antiserum confirmed the presence of TLP in punctate, organelle-like structures within sieve elements. On western blots, the antiserum labeled two constitutively expressed proteins with distinct expression patterns. Immunogold labelling suggested that TLPs are associated with starch granules and starch-containing plastids in sieve elements and phloem parenchyma cells. In addition, the antiserum recognized TLPs in the inner cell wall and sieve plate region of sieve elements.
TLP localization in poplar cells and tissues is complex. TLP1 is expressed predominantly in tissues with a prominent vascular system such as midveins, petioles and stems, whereas the second TLP is primarily expressed in starch-storing plastids found in young leaves and the shoot apex.
The diameter of vascular conduits increases towards the stem base. It has been suggested that this profile is an efficient anatomical feature for reducing the hydraulic resistance when trees grow taller. However, the mechanism that controls the cell diameter along the plant is not fully understood. The timing of cell differentiation along the stem was investigated. Cambial activity and cell differentiation were investigated in a Picea abies tree (11.5 m in height) collecting microsamples at nine different heights (from 1 to 9 m) along the stem with a 4 d time interval. Wood sections (8–12 μm thick) were stained and observed under a light microscope with polarized light to differentiate the developing xylem cells. Cell wall lignification was detected using cresyl violet acetate. The first enlarging cells appeared almost simultaneously along the tree axis indicating that cambium activation is not height-dependent. A significant increase in the duration of the cell expansion phase was observed towards the tree base: at 9 m from the ground, xylem cells expanded for 7 d, at 6 m for 14 d, and at 3 m for 19 d. The duration of the expansion phase is positively correlated with the lumen area of the tracheids (r2=0.68, P < 0.01) at the same height. By contrast, thickness of the cell wall of the earlywood did not show any trend with height. The lumen area of the conduits down the stem appeared linearly dependent on time during which differentiating cells remained in the expansion phase. However, the inductive signal of such long-distance patterned differentiation remains to be identified.
Auxin; cambium; cell differentiation; conduit tapering; Picea abies polar pattern growth
Background and Aims
Studies on xylogenesis focus essentially on the stem, whereas there is basically no information about the intra-annual growth of other parts of the tree. As roots strongly influence carbon allocation and tree development, knowledge of the dynamics of xylem production and maturation in roots at a short time scale is required for a better understanding of the phenomenon of tree growth. This study compared cambial activity and xylem formation in stem and roots in two conifers of the boreal forest in Canada.
Wood microcores were collected weekly in stem and roots of ten Abies balsamea and ten Picea mariana during the 2004–2006 growing seasons. Cross-sections were cut using a rotary microtome, stained with cresyl violet acetate and observed under visible and polarized light. The number of cells in the cambial zone and in differentiation, plus the number of mature cells, was counted along the developing xylem.
Xylem formation lasted from the end of May to the end of September, with no difference between stem and roots in 2004–2005. On the contrary, in 2006 a 1-week earlier beginning of cell differentiation was observed in the stem, with cell wall thickening and lignification in roots ending up to 22 d later than in the stem. Cell production in the stem was concentrated early in the season, in June, while most cell divisions in roots occurred 1 month later.
The intra-annual dynamics of growth observed in stem and roots could be related to the different amount of cells produced by the cambium and the patterns of air and soil temperature occurring in spring.
Abies balsamea; boreal forest; cambium; cell differentiation; cell wall thickening; lignification; Picea mariana; root; stem; xylem
Wood formation requires a continuous supply of carbohydrates for structural growth and metabolism. In the montane belt of the central Austrian Alps we monitored the temporal dynamics of xylem growth and non-structural carbohydrates (NSC) in stem sapwood of Pinus sylvestris L. during the growing season 2009, which was characterized by exceptional soil dryness within the study area. Soil water content dropped below 10 % at the time of maximum xylem growth end of May. Histological analyses have been used to describe cambial activity and xylem growth. Determination of NSC was performed using specific enzymatic assays revealing that total NSC ranged from 0.8 to 1.7 % dry matter throughout the year. Significant variations (P < 0.05) of the size of the NSC pool were observed during the growing season. Starch showed persistent abundance throughout the year reaching a maximum shortly before onset of late wood formation in mid-July. Seasonal dynamics of NSC and xylem growth suggest that (i) high sink activity occurred at start of the growing season in spring and during late wood formation in summer and (ii) there was no particular shortage in NSC, which caused P. sylvestris to draw upon stem reserves more heavily during drought in 2009.
Background and Aims
Our knowledge about the influences of environmental factors on tree growth is principally based on the study of dominant trees. However, tree social status may influence intra-annual dynamics of growth, leading to differential responses to environmental conditions. The aim was to determine whether within-stand differences in stem diameters of trees belonging to different crown classes resulted from variations in the length of the growing period or in the rate of cell production.
Cambial activity was monitored weekly in 2006 for three crown classes in a 40-year-old silver-fir (Abies alba) plantation near Nancy (France). Timings, duration and rate of tracheid production were assessed from anatomical observations of the developing xylem.
Cambial activity started earlier, stopped later and lasted longer in dominant trees than in intermediate and suppressed ones. The onset of cambial activity was estimated to have taken 3 weeks to spread to 90 % of the trees in the stand, while the cessation needed 6 weeks. Cambial activity was more intense in dominant trees than in intermediate and suppressed ones. It was estimated that about 75 % of tree-ring width variability was attributable to the rate of cell production and only 25 % to its duration. Moreover, growth duration was correlated to tree height, while growth rate was better correlated to crown area.
These results show that, in a closed conifer forest, stem diameter variations resulted principally from differences in the rate of xylem cell production rather than in its duration. Tree size interacts with environmental factors to control the timings, duration and rate of cambial activity through functional processes involving source–sink relationships principally, but also hormonal controls.
Cambial activity; forest-stand structure; silver fir (Abies alba); tree-ring formation; tree-to-tree competition; social status; wood anatomy; xylem cell differentiation
Trees will have to cope with increasing levels of CO2 and ozone in the atmosphere. The purpose of this work was to assess whether the lignification process could be altered in the wood of poplars under elevated CO2 and/or ozone. Young poplars were exposed either to charcoal-filtered air (control), to elevated CO2 (800 μl l−1), to ozone (200 nl l−1) or to a combination of elevated CO2 and ozone in controlled chambers. Lignification was analysed at different levels: biosynthesis pathway activities (enzyme and transcript), lignin content, and capacity to incorporate new assimilates by using 13C labelling. Elevated CO2 and ozone had opposite effects on many parameters (growth, biomass, cambial activity, wood cell wall thickness) except on lignin content which was increased by elevated CO2 and/or ozone. However, this increased lignification was due to different response mechanisms. Under elevated CO2, carbon supply to the stem and effective lignin synthesis were enhanced, leading to increased lignin content, although there was a reduction in the level of some enzyme and transcript involved in the lignin pathway. Ozone treatment induced a reduction in carbon supply and effective lignin synthesis as well as transcripts from all steps of the lignin pathway and some corresponding enzyme activities. However, lignin content was increased under ozone probably due to variations in other major components of the cell wall. Both mechanisms seemed to coexist under combined treatment and resulted in a high increase in lignin content.
13C labelling; elevated CO2; lignin; ozone; poplar; wood
We investigate cambial growth periodicity in Brachystegia spiciformis, a dominant tree species in the seasonally dry miombo woodland of southern Africa. To better understand how the brevi-deciduous (experiencing a short, drought-induced leaf fall period) leaf phenology of this species can be linked to a distinct period of cambial activity, we applied a bi-weekly pinning to six trees in western Zambia over the course of one year. Our results show that the onset and end of cambial growth was synchronous between trees, but was not concurrent with the onset and end of the rainy season. The relatively short (three to four months maximum) cambial growth season corresponded to the core of the rainy season, when 75% of the annual precipitation fell, and to the period when the trees were at full photosynthetic capacity. Tree-ring studies of this species have found a significant relationship between annual tree growth and precipitation, but we did not observe such a correlation at intra-annual resolution in this study. Furthermore, a substantial rainfall event occurring after the end of the cambial growth season did not induce xylem initiation or false ring formation. Low sample replication should be taken into account when interpreting the results of this study, but our findings can be used to refine the carbon allocation component of process-based terrestrial ecosystem models and can thus contribute to a more detailed estimation of the role of the miombo woodland in the terrestrial carbon cycle. Furthermore, we provide a physiological foundation for the use of Brachystegia spiciformis tree-ring records in paleoclimate research.
The fine structure of ash cambium was studied after glutaraldehyde-osmium tetroxide fixation. The fusiform and ray initials are essentially alike, and both have the basic complement of organelles and membranes typical of parenchyma cells. The varied behavior of the two types of initials and the role of cambium in oriented production of the xylem and phloem are still unexplained phenomena. Actively growing cambial cells are highly vacuolate. They are rich in endoplasmic reticulum of the rough cisternal form, ribosomes, dictyosomes, and coated vesicles. Microtubules are present in the peripheral cytoplasm. The plasmalemma appears to be continuous with the endoplasmic reticulum and produces coated vesicles as well as micropinocytotic vesicles with smooth surfaces. The plastids have varying amounts of an intralamellar inclusion which may be a lipoprotein. The quiescent cambium is deficient in rough ER and coated vesicles and has certain structures which may be condensed proteins.
Background and Aims
Alterations of plasmodesma (PD) connectivity are likely to be very important for plant development. Here, the repetitive division pattern of cambial initials in Populus nigra ‘italica’ was studied to follow the development of the PD network during maturation. Furthermore, seasonal changes were investigated in order to trace indications for developmental and functional adaptations.
Cambium samples of P. nigra twigs, collected in summer, autumn and spring, were chemically fixed for transmission electron microscopy. The parameters, PD density (number of PDs per square micrometre cell-wall area) and PD frequency (total number of PDs per average cell-wall area), were determined for radial and tangential cell interfaces deposited in chronological order.
Data sets, presented in plasmodesmograms, show a strong variability in the PD network throughout the year. In summer, high PD numbers occur at the division wall which, after PD doubling by longitudinal fission, decline with further development both at the xylem and the phloem side. In autumn, the number of PDs at the division wall is low as they are in subsequent tangential interfaces. In spring, the first cell division coincides with a massive increase in PD numbers, in particular at the division wall. Only the radial walls between initials maintain their PD equipment throughout the year. This feature can be exploited for identification of the initial layer.
PD networks in the cambium go through a strict developmental programme depending on the season, which is associated with changing functional requirements. For instance, PD numbers correlate with proliferative activity and potential pathways for intercellular signalling. Increases in PD numbers are ascribed to longitudinal fission as a major mechanism, whereas the decline in older derivatives is ascribed to PD degradation.
Cambium; meristem initials; plasmodesmata; Populus nigra ‘italica’; seasonal conditions; ultrastructure
Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups.
Full-length cDNA sequences were isolated from poplar and white spruce. Phylogenetic reconstruction indicated that some of the gymnosperm sequences were derived from lineages that diverged earlier than angiosperm sequences, and seem to have been lost in angiosperm lineages. Transcript accumulation profiles were assessed by RT-qPCR on tissue panels from both species and in poplar trees in response to an inhibitor of polar auxin transport. The overall transcript profiles HD-Zip III complexes in white spruce and poplar exhibited substantial differences, reflecting their evolutionary history. Furthermore, two poplar sequences homologous to HD-Zip III genes involved in xylem development in Arabidopsis and Zinnia were over-expressed in poplar plants. PtaHB1 over-expression produced noticeable effects on petiole and primary shoot fibre development, suggesting that PtaHB1 is involved in primary xylem development. We also obtained evidence indicating that expression of PtaHB1 affected the transcriptome by altering the accumulation of 48 distinct transcripts, many of which are predicted to be involved in growth and cell wall synthesis. Most of them were down-regulated, as was the case for several of the poplar HD-Zip III sequences. No visible physiological effect of over-expression was observed on PtaHB7 transgenic trees, suggesting that PtaHB1 and PtaHB7 likely have distinct roles in tree development, which is in agreement with the functions that have been assigned to close homologs in herbaceous plants.
This study provides an overview of HD-zip III genes related to woody plant development and identifies sequences putatively involved in secondary vascular growth in angiosperms and in gymnosperms. These gene sequences are candidate regulators of wood formation and could be a source of molecular markers for tree breeding related to wood properties.
The progression of events in the development of pine wilt disease following the invasion by Bursaphelenchus xylophilus is reviewed from early migration through pine tissues until symptom development on foliage. Disease resistance in pines, especially the hypersensitive reaction that is successful in controlling many potential pests and pathogens, is explored. Pathologies resulting from the activities of pinewood nematode include cortical trails and cavities; formation of cambial gaps and traumatic resin cysts; browning and death of cortex, phloem, cambium, and ray tissues; granulation and shrinkage of cell cytoplasm in rays; and destruction of resin canal epithelial and ray parenchyma cells. Death of parenchyma, production of toxins, and leakage of oleoresins and other material into tracheids are typical of the hypersensitive reaction occurring in pines following migration of small numbers of pinewood nematodes. The hypothesis presented is that a spreading hypersensitive reaction results in some of the observed pathologies and symptoms and eventually causes pine death. The growth-differentiation balance hypothesis is used to help explain predisposition, oleoresin production and toxicity, susceptibility and resistance, and the effects of variation in climate on host pines as related to pinewilt disease.
Bursaphelenchus xylophilus; pathogenesis; pathology; pine wilt disease; pinewood nematode
Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.
Percentage and rate of mortality in 2-4-year-old conifers depended upon the numbers of pinewood nematodes Bursaphelenchus xylophilus inoculated into their stems. In addition, percentage of conifer mortality was greater for spring inoculations when cambial activity was greater than for late summer and fall inoculations. Gross and histological examination of stems revealed destruction of the cambial layer, including fusiform and ray intitials and their derivatives. These data suggest that cambial and ray destruction causes tree death through blockage of tracheids by gas, oleoresin, or metabolites from dying ray tissues.
histopathology; Bursaphelenchus xylophilus; pinewood nematode; pinewi!t disease; conifer death; mortality; Pinus spp.
The relationship between stem CO2 efflux (ES), cambial activity and xylem production in Pinus cembra was determined at the timberline (1950 m a.s.l.) of the Central Austrian Alps, throughout one year. ES was measured continuously from June 2006 to August 2007 using an infrared gas-analysis system. Cambial activity and xylem production was determined by repeated microcore sampling of the developing tree ring and radial increment was monitored using automated point dendrometers. Aside of temperature, the number of living tracheids and cambial cells was predominantly responsible for ES: ES normalized to 10°C (ES10) was significantly correlated to number of living cells throughout the year (r2 = 0,574; p < 0,001). However, elevated ES and missing correlation between ES10 and xylem production was detected during cambial reactivation in April and during transition from active phase to rest, which occurred in August and lasted until early September. Results of this study indicate that (i) during seasonal variations in cambial activity non-linearity between ES and xylem production occurs and (ii) elevated metabolic activity during transition stages in the cambial activity-dormancy cycle influence the carbon budget of Pinus cembra. Daily radial stem increment was primarily influenced by the number of enlarging cells and was not correlated to ES.
cambial reactivation; dormancy; Pinus cembra; radial stem growth; sap flow; stem CO2 efflux; stem respiration; xylem production
We determined the temporal dynamic of cambial activity and xylem development of stone pine (Pinus cembra L.) throughout the treeline ecotone. Repeated micro-sampling of the developing tree ring was carried out during the growing seasons 2006 and 2007 at the timberline (1950 m a.s.l.), treeline (2110 m a.s.l.) and within the krummholz belt (2180 m a.s.l.) and the influence of climate variables on intra-annual wood formation was determined.
At the beginning of both growing seasons, highest numbers of cambial and enlarging cells were observed at the treeline. Soil temperatures at time of initiation of cambial activity were c. 1.5 °C higher at treeline (open canopy) compared to timberline (closed canopy), suggesting that a threshold root-zone temperature is involved in triggering onset of above ground stem growth.
The rate of xylem cell production determined in two weekly intervals during June through August 2006-2007 was significantly correlated with air temperature (temperature sums expressed as degree-days and mean daily maximum temperature) at the timberline only. Lack of significant relationships between tracheid production and temperature variables at the treeline and within the krummholz belt support past dendroclimatological studies that more extreme environmental conditions (e.g., wind exposure, frost desiccation, late frost) increasingly control tree growth above timberline.
Results of this study revealed that spatial and temporal (i.e. year-to-year) variability in timing and dynamic of wood formation of Pinus cembra is strongly influenced by local site factors within the treeline ecotone and the dynamics of seasonal temperature variation, respectively.
Cambium; intra-annual growth; Pinus cembra; temperature; tracheid production