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.     

Cellular localization of aquaporin mRNA in hybrid poplar stems

? Premise of the study: Aquaporins (AQPs) are channel proteins, and their function is mostly associated with transmembrane water transport. While aquaporin genes are known to be expressed in woody poplar stems, little is known about AQP expression at the cellular level. Localization of AQP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of these genes. ? Methods: Subsets of plants were subjected to 6 wk of high nitrogen fertilization (high N plants) or to a controlled drought. Experimental treatments affected cambial activity and wood anatomy. RNA in situ hybridization was used to characterize spatial expression of three AQP genes in stem cross sections. ? Key results: The strongest labeling consistently occurred in the cambial region and in adjacent xylem and phloem cells. Expression was also detected in rays. Contact cells exhibited high expression, while expression in other ray cells was more variable. High N plants exhibited a broader band of expression in the cambial region than plants receiving only adequate N fertilization (control plants) and plants subjected to drought. ? Conclusions: Water channels in stems were expressed in a manner that allows hydraulic coupling between xylem and other tissues that may serve as water reservoirs, including phloem and pith parenchyma. Expression of AQPs in rays may increase radial flow of water from xylem and phloem to the cambial region where AQPs may help sustain rapid cell division and expansion of developing vessel elements.     

Nitrogen cycling in dense plantings of hybrid poplar and black alder

Summary Nitrogen cycling was studied during the third growing season in pure and mixed plantings (33×33 cm spacing) of hybrid poplar and black alder in southeastern Canada. After 3 years, hybrid poplar growth and N content of living tissues in a plot and of individual hybrid poplar plants increased with the proportion of black alder in a planting. No differences were detected among N contents of individual alder plants regardless of plot treatment. Black alder allocated a larger portion of its N to roots than hybrid poplar. Symbiotic nitrogen fixation was estimated to account for 80% of the nitrogen in aboveground alder tissues in the pure treatment using natural¹⁵N dilution. N return in leaf litter was estimated to be 70kg ha^–1 in the pure alder treatment and decreased to a minimum of 20 kg ha^–1 in the pure hybrid poplar plots. No difference was detected among treatments for throughfall N content. Nitrogen concentration in roots and leaf litterfall of black alder was higher than hybrid poplar. Significant soil N accretion occurred in mixed plantings containing two alders to one poplar and pure black alder plantings. Nitrogen availability (NO₃–N) increased with the amount of black alder in a plot. Results suggest that the early increase in nitrogen accumulation of hybrid poplar in mixed treatments can be attributed to an increase of total soil N availability resulting from the input of large amounts of N from easily mineralizable alder tissue.     

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 x 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 devel...     

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.     

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under‐ or over‐express a xylem‐abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under‐ and over‐expressing aspen lines developed xylem with lower‐specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.     

Variation in embolism occurrence and repair along the stem in drought‐stressed and re‐watered seedlings of a poplar clone

Root pressure and plasma membrane intrinsic protein (PIP) availability in the xylem have been recognized to participate in the refilling of embolized conduits, yet integration of the two mechanisms has not been reported in the same plant. In this study, 4‐month‐old seedlings of a hybrid poplar (Populus alba × Populus glandulosa) clone 84K were subjected to two contrasting soil‐water treatments, with the drought treatment involving withholding of water for 17 days to reduce the soil‐water content to 10% of the saturated field capacity, followed by a re‐watering cycle. The percentage loss of stem hydraulic conductance (PLC) sharply increased, and stomatal conductance and photosynthesis declined in response to drought stress; these processes were gradually restored following the subsequent re‐watering. Embolism was most severe in the middle portions of the stem, followed by the basal and top portions of the stems of seedlings subjected to drought stress and subsequent re‐watering. Although drought stress eliminated root pressure, re‐watering partially restored it in a short period of time. The expression of PIP genes in the xylem was activated by drought stress, and some PIP genes were further stimulated in the top portion after re‐watering. The dynamics of root pressure and differential expression of PIP genes along the stem coincided with changes in PLC, suggesting that root pressure and PIPs work together to refill the embolized vessels. On the basis of the recovery dynamics in PLC and g_smax (maximum stomatal conductance) after re‐watering, the stomatal closure and xylem cavitation exhibited fatigue due to drought stress.     

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.     

Gene expression associated with N-induced shifts in resource allocation in poplar

Surprisingly little is known about molecular mechanisms by which nitrogen (N) availability acts to modulate the growth of forest trees. To address this issue, differential display was used in conjunction with filter‐based arrays to identify 52 partial cDNA clones that were significantly regulated within days in response to limiting or luxuriant levels of NH₄NO₃ fertilization in Populus trichocarpa Torr. & Gray × deltoides Bartr. ex Marsh. A subset of these cDNAs also demonstrated shifts in expression patterns in stem‐girdled trees, a manipulative physiology technique that disrupts phloem transport. Stem girdling also induced changes in glutamine and asparagine pools which were correlated with the observed changes in expression profiles for these genes. The identity of these genes provides insight into biochemical processes that are altered by N availability in poplar. Carbon–nitrogen interactions appear to figure prominently in the N‐response. The gene expression data suggest that N availability modulates the partitioning of C and N resources into metabolic fates that have the potential to alter both wood quality and quantity, including synthesis of vegetative storage proteins, cell wall components, and terpenoids.     

Tracheids in white spruce seedling's long lateral roots in response to nitrogen availability

This study examined how the availability of inorganic nitrogen (N) modified the anatomical characteristics of white spruce (Picea glauca (Moench) Voss) roots related to their hydraulic properties. Seedlings were grown for one growing season in 4 L capacity pots filled with sand under one of three N levels: low (10 ppm), medium (50 ppm) and high (125 ppm). First order lateral roots with intact tips were sampled from dormant seedlings in October. Root segments were collected from 4, 10, and 14 cm distances above the root tip for fixation and sectioning and for maceration. Additional specimens were collected from the 4 and 14 cm distances for maceration and scanning electron microscopy of xylem pits. Root diameter and surface area occupied by the xylem in root cross sections increased basipetally in all treatments but exceptions were found. Higher N-levels significantly increased root diameter and surface area occupied by the xylem. In the two higher N treatments secondary root development was more advanced near the root tip than in the low N treatment. There was a strong positive correlation between root diameter and cross-sectional root area occupied by the xylem (30–50% of the root cross section) but not in portions with little secondary development. Non-conducting space within the xylem occupied 10–13% of its cross-sectional surface. Tracheids of the primary xylem were larger, had larger lumens but thinner cell walls than those of the secondary xylem. Low N treatment seedling tracheids had smaller total cross-sectional area, less lumen, and less cell wall surface area than the two other N treatments. Tracheid diameter means were between 19–20 μm in the high and medium N treatments, and 15.2 μm in the low N treatment. The range was 4.5–51.3 μm. Tracheid length was not significantly affected by N. The average tracheid was about 1000 μm long, and the range was 110–3530 μm. Pit-border diameters ranged between 4.1–20.6 μm (average 10–11 μm) and were not affected by the N treatment. Pit aperture diameters were within 0.62–10.2 μm range (average between 3–4 μm) and were also not significantly affected by the N treatment, although tracheids from the medium N-treatment roots tended to have larger apertures. The pit border diameter equals that of the margo while the aperture size should be similar to that of the torus of the pit membrane. If the capacity for axial water transport in spruce roots is affected by N, it would be by its impact on conduit diameter and, possibly on the pit-membrane pore sizes but not by changes to conduit length and to the size of the pit membrane surface area. This revised version was published online in June 2006 with corrections to the Cover Date.     

Linking irradiance-induced changes in pit membrane ultrastructure with xylem vulnerability to cavitation

The effect of shading on xylem hydraulic traits and xylem anatomy was studied in hybrid poplar (Populus trichocarpa x deltoides, clone H11-11). Hydraulic measurements conducted on stem segments of 3-month-old saplings grown in shaded (SH) or control light (C) conditions indicated that shading resulted in more vulnerable and less efficient xylem. Air is thought to enter vessels through pores in inter-vessel pit membranes, thereby nucleating cavitation. Therefore, we tested if the ultrastructure and/or chemistry of pit membranes differed in SH and C plants. Transmission electron micrographs revealed that pit membranes were thinner in SH, which was paralleled by lower compound middle lamella thickness. Immunolabelling with JIM5 and JIM7 monoclonal antibodies surprisingly indicated that pectic homogalacturonans were not present in the mature pit membrane regardless of the light treatment. Porosity measurements conducted with scanning electron microscopy were significantly affected by the method used for sample dehydration. Drying through a gradual ethanol series seems to be a better alternative to drying directly from a hydrated state for pit membrane observations in poplar. Scanning electron microscopy based estimates of pit membrane porosity probably overestimated real porosity as suggested by the results from the 'rare pit' model.     

The syringaldazine-oxidizing peroxidase PXP 3-4 from poplar xylem: cDNA isolation,characterization and expression

The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases, we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar to those of the corresponding protein from poplar xylem (enzymatic properties, isoelectric point, and migration in a native gel). PXP 3-4 was expressed in the stem and in the root xylem. The data demonstrate that PXP 3-4 (and/or PXP 5) are present in differentiating xylem, supporting a function in secondary cell wall formation.