Immunolocalization of an anionic peroxidase in differentiating poplar xylem

Peroxidases are the major candidate enzymes involved in dehydrogenative polymerization of monolignols. Peroxidases have the signal sequence at their N-terminus and this suggests that they are transported to extracellular spaces or developing cell walls. In this study, we focused on an anionic peroxidase isozyme encoded by prxA3a, which seems to be related to lignification. To investigate the localization of peroxidase in differentiating xylem cells of poplar (Populus sieboldii × Populus grandidentata), anti-PRX3 antibody was raised against the anionic peroxidase. Western blotting and peroxidase activity inhibition assay showed specificity of the antibody. Labeling by anti-PRX3 antibody was localized in vessels and fibers during the secondary wall formation and was observed along the plasma membrane beside the microtubules. The labeling was not seen in the cell wall, where localization of peroxidases was expected during lignification. The peroxidase isozyme, which is suggested to be involved in monolignol polymerization, is localized on the plasma membrane and its localization might be regulated by microtubules.     

Distribution of lignin interunit bonds in the differentiating xylem of compression and normal woods of Pinus thunbergii

The lignification process and lignin distribution at different stages of cell wall differentiation in the secondary xylem of compression and normal woods of Pinus thunbergii were investigated by thioacidolysis and subsequent desulfuration. We prepared 50-µm-thick, contiguous tangential sections of pine shoots, cut from the cambial zone through to mature xylem. In compression wood, uncondensed guaiacyl (G) and p-hydroxyphenyl (H) lignins were deposited simultaneously from early to late stages of lignification. The various types of G-G, G-H, and H-H dimers were detected in compression wood, and the ratio of G-H and H-H dimers to total dimers increased as lignification proceeded. In contrast, uncondensed and condensed H units were detected in trace amounts in normal wood. Significant differences in the relative distributions of lignin interunit linkages were not observed between compression and normal woods or between differentiating and mature xylems in either compression or normal woods.Part of this report was presented at the 10th International Symposium on Wood and Pulping Chemistry, Yokohama, June, 1999     

Azimuthal variations of sap flux density within Japanese cypress xylem trunks and their effects on tree transpiration estimates

Sap flow techniques are practical tools for estimating tree transpiration. Though many previous studies using sap flow techniques did not consider azimuthal variations of sap flux density (F _d) on xylem trunk to estimate tree transpiration, a few studies reported that ignoring the azimuthal variations in F _d could cause large errors in tree transpiration estimates for some tree species. Therefore, examining azimuthal variations in F _d for major plantation tree species is critical for estimating tree transpiration. Using the thermal dissipation method, we examined azimuthal variations in F _d in six trees of Japanese cypress Chamaecyparis obtusa (Sieb. et Zucc.) Endl., which is one of the most common plantation tree species in Japan. We recorded considerable variations among F _d at four different azimuthal directions. The F _d value for one aspect was more than 100% larger than those for the other aspects. We calculated differences between tree transpiration estimates based on F _d for one to three azimuthal directions and those based on F _d for four aspects. The differences relative to tree transpiration estimates based on F _d for four aspects were typically 30, 20, and 10% in accordance with the F _d for one, two, and three measurement aspects, respectively. This finding indicates that ignoring azimuthal variations could cause large errors in tree transpiration estimates for Japanese cypress.     

Nutrition, xylem cavitation and drought resistance in hybrid poplar

Effects of mineral nutrition on susceptibility to cavitation were examined in four hybrid poplar clones. Two drought-sensitive and two drought-resistant hybrid clones of black cottonwood (Populus trichocarpa Torr. & Gray) and eastern cottonwood (P. deltoides Bartr.) were grown at three concentrations of nitrogen (N) applied factorially with two concentrations of phosphorus (P) in a greenhouse, and subjected to varying degrees of drought stress before measurement of cavitation and of anatomical features that might affect cavitation. Mean vessel pit pore diameters were 0.132 micro m at low P, and 0.074 micro m at high P, but no other significant effects of mineral nutrition on vessel dimensions were observed. Vessel diameter and specific conductivity were greater in the drought-resistant clones than in the drought-susceptible clones. Drought-resistant clones did not reach such low water potentials as drought-sensitive clones during the cavitation induction experiments, suggesting better stomatal and cuticular control of water loss. Scanning electron microscope observations showed less damage to pit membranes, also suggesting greater membrane strength in drought-resistant clones than in drought-sensitive clones. High concentrations of N increased cavitation, whereas high concentrations of P decreased cavitation as measured by both hydraulic flow apparatus and dye perfusion techniques. For one test, cavitation was 48% at high N and low P, but only 28% at high N and high P. We consider that N fertilization may make poplars more susceptible to cavitation on dry sites, but P fertilization may reduce this effect.     

杨树木质素合成酶hct基因的蛋白质序列分析

利用RT-PCR从欧美杨107次生木质部中克隆出一709bp的hct基因片段,其开放阅读框为708bp,编码236个氨基酸,具有HCT共有的DFGWG序列和HXXXQ活性位点,拟表达蛋白含有一个二硫键,属于亲水性稳定型蛋白。克隆片段拟表达蛋白质没有信号肽,也没有跨膜结构域,包含α-螺旋区和少量β-折叠,具有比较多的无规则卷曲。     

Translocation of nitrogen in the xylem of field-grown cherry and poplar trees during remobilization

Studies of small trees growing in pots have established that individual amino acids or amides are translocated in the xylem sap of a range of tree species following bud burst, as a consequence of nitrogen (N) remobilization from storage. This paper reports the first study of N translocation in the xylem of large, deciduous, field-grown trees during N remobilization in the spring. We applied 15N fertilizer to the soil around 10-year-old Prunus avium L. and Populus trichocharpa Torr. & Gray ex Hook var. Hastata (Dode) A. Henry x Populus balsamifera L. var. Michauxii (Dode) Farwell trees before bud burst to label N taken up by the roots. Recovery of unlabeled N in xylem sap and leaves was used to demonstrate that P. avium remobilizes N in both glutamine (Gln) and asparagine (Asn). Sap concentrations of both amides rose sharply after bud burst, peaking 14 days after bud burst for Gln, and remaining high some 45 days for Asn. There was no 15N enrichment of either amide until 21 days after bud burst. In the Populus trees, nearly all the N was translocated in the sap as Gln, the concentration of which peaked and then declined before the amide was enriched with 15N, 40 days after bud burst. Xylem sap of clonal P. avium trees was sampled at different positions in the crown to assess if the amino acid and amide composition of the sap varied within the crown. Sap was sampled during remobilization (when the concentration of Gln was maximal), at the end of remobilization and at the end of the experiment (68 days after bud burst). Although the date of sampling had a highly significant effect on sap composition, the effect of position of sampling was marginal. The results are discussed in relation to N translocation in adult trees and the possibility of measuring N remobilization by calculating the flux of N translocation in the xylem.     

Distribution of lignin in normal and tension wood

Summary The distribution of lignin in normal and tension wood of four hardwood species has been studied by examination in the electron microscope of the lignin skeletons remaining after removal of the polysaccharides with hydrofluoric acid. In normal wood fibers, the S₁ had a higher lignin concentration than the S₂ layer, which was not as highly lignified as in conifer tracheids. Vessels had a high concentration of lignin in both normal and tension wood, while the extent of lignification of the parenchyma was variable.In tension wood fibers, the S₁ and S₂ layers were highly lignified. A thick, unlignified G-layer was often associated with an extremely thin S₂ layer with a high concentration of lignin. In both normal and tension wood, the lignin had the same orientation as the cellulose micro-fibrils in the different cell wall layers. The results confirm the earlier conclusion that, in the species investigated, the same amount of lignin is present in gelatinous as in normal fibers. Evidently, the lignification mechanism operates normally in the non-gelatinous layers of the fibers, as well as in the vessels and in the parenchyma of tension wood.

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Zusammenfassung Die Ligninverteilung im Normalholz und im Druckholz von vier Laubhölzern wurde untersucht. Die Ligningerüste, die nach der Entfernung der Polysaccharide durch Fluorwasser-stoffsäure übrigblieben, wurden im Elektronenmikroskop beobachtet. In den Normalholzfasern hatte die S₁-eine höhere Ligninkonzentration als die S₂-Schicht, die weniger lignifiziert war als in den Koniferentracheiden. Die Gefäße hatten eine hohe Ligninkonzentration in sowohl Normal-als in Zugholz, während der Lignifizierungsgrad der Parenchymzellen variierte.In den Zugholzfasern waren die S₁- und S₂-Schichten völlig lignifiziert. Eine dicke, unlignifizierte G-Schicht war oft mit einer außerordentlich dünnen S₂-Schicht, die eine hohe Ligninkonzentration zeigte, verbunden. Sowohl im Normal- wie auch im Zugholz besaß das Lignin dieselbe Orientierung wie die Cellulosemikrofibrillen in den verschiedenen Zellwandschichten. Die Ergebnisse bestätigen den früheren Schluß, daß in den hier untersuchten Laubhölzern in den gelatinösen und in den normalen Fasern dieselbe Ligninmenge vorliegt. Offenbar läuft der Mechanismus der Lignifizierung in den S₁- und S₂-Schichten der gelatinösen Fasern des Zugholzes normal ab.

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This investigation was supported by the United States Department of Agriculture, Forest Service, through Forest Service Research Grant No. 1, which is hereby gratefully acknowledged.     

Drought-dependent responses of photosynthesis,transpiration and water use efficiency of Japanese cypress and Japanese red pine seedlings

This study was conducted to investigate the potential for modifying drought tolerance of Japanese cypress (Chamaecyparis obtusa Endl.) and Japanese red pine (Pinus densiflora Sieb. et Zucc.). Three-year-old seedlings were controlled for five-months at three different soil water potentials ({ie73-1}). Japanese cypress exposed to high {ie73-2} was able to maintain higher photosynthesis (Ph_n), transpiration (Tr) and stomatal conductance to H₂O (gH₂O) in comparison to low {ie73-3} pretreatments, however, there was no significant difference in Ph_n for Japanese red pine. Soil water potential at the threshold from the maximum to limited Ph_n was higher in high {ie73-4} pretreatments than in low {ie73-5} pretreatments. Net photosynthesis, Tr and gH₂O decreased more rapidly in high {ie73-6} pretreatments than in low {ie73-7} pretreatments. Transpiration decreased more significantly than Ph_n, thus, resulted in increased water use efficiency. All these factors are likely to result in significant improvements in the drought tolerance. Japanese red pine seems more drought-tolerant than Japanese cypress. Japanese cypress is suitable to soil of −0.05 MPa water potential, and Japanese red pine is suitable to −0.16 MPa and even dryer soils.     

Immunohistochemical localization of enzymes related to lignin biosynthesis in the primary xylem of hybrid aspen

We have investigated the spatial regulation of the accumulation of enzymes involved in the biosynthesis of shikimate and lignin during differentiation of primary xylem from the apical meristem via procambium in hybrid aspen (Populus sieboldii x Populus grandidentata). Immuohistochemical staining revealed that, in the top part of shoots, lignification began in a single or just a few adjacent vessel elements and subsequently spread to neighboring cells. The spatial localization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), which is one of the key enzymes in the shikimate pathway, was tightly correlated with the cell-specific deposition of lignin in the primary xylem. We also found that the spatial localization of enzymes in the general phenylpropanoid pathway and in the lignin-specific pathway was closely associated with the cell-specific deposition of lignin and the accumulation of DAHPS. Our data suggest that enzymes that act in the shikimate, general phenylpropanoid, and lignin-specific pathways are initially produced and function coordinately in a single or a few adjacent elements at the start of primary xylem development.     

Tinctoporellus epimiltinus,a causal fungus of butt rot of Japanese cypress

One of the causal fungi of butt rot of Chamaecyparis obtusa in Kyushu Island, Japan, was identified as Tinctoporellus epimiltinus from cultural characters, and by mating and inoculation tests. The cultural characters of the isolates obtained from butt rot of C. obtusa and T. epimiltinus isolated from the basidiocarp tissue and from decayed wood were examined. The morphological characters and chemical reactions of the isolates from butt rot were typical for T. epimiltinus. Di‐mon mating tests were performed between five monospore isolates of T. epimiltinus and five heterokaryotic isolates from the butt rot. All of the monospore isolates examined were heterokaryotized by the isolates from butt rot. Tinctoporellus epimiltinus isolates were inoculated on roots of 24‐year‐old C. obtusa. Butt rot was observed on all inoculated trees 1–2 years after the inoculation, and the inoculated fungus was re‐isolated from the decayed parts of the inoculated trees.     

Effects of root zone trenching on soil nitrogen dynamics in Japanese cedar and cypress plantations

The effects of root exclusion and planted tree species on soil nitrogen (N) dynamics were examined at two plantations, one planted with Japanese cedar and the other with Japanese cypress. We set up ten 1 × 1 × 0.2-m-deep trenched sites and ten untrenched control sites at each plantation. We measured the pool size and leaching of inorganic N at each site for 2 years and the net N mineralization 1 and 2 years after trenching. Despite similar soil conditions, the cedar plantation showed higher net N mineralization than the cypress plantation. Stopped tree uptake of N was expected to cause an increased pool size and leaching of inorganic N at the trenched sites. Nevertheless, we found no significant increase in those variables at both plantations. The trenched cypress sites showed no decrease in the net N mineralization during the 2 years after trenching. However, the net nitrification at the trenched cypress sites increased remarkably at the deeper horizons in comparison with that at the control sites. Enhanced nitrification might result from improved ammonium availability through root exclusion. Net N mineralization at the trenched cedar sites decreased more than 60% compared with that at the control sites 2 years after trenching. Higher nitrification potential at the cedar plantation and enhanced nitrification potential at the trenched cypress sites never resulted in increased leaching of N, due to added fine root litter which acted as an immobilization agent for excess N, thus preventing N loss.     

Incident rainfall partitioning and canopy interception modeling for an abandoned Japanese cypress stand

Interception loss (E _i) in forests has been studied widely. However, E _i parameters and modeling as well as spatial patterns of throughfall (TF) in abandoned Japanese cypress (Chamaecyparis obtusa) plantations remain poorly documented. In this study, gross precipitation (P _G), stemflow (SF), and TF were monitored in an unmanaged 32-year-old Japanese cypress stand throughout the 2011 rainy season. Results indicate that P _G partitioning into TF, SF, and E _i were, respectively, 64.2 ± 3.6, 10.6 ± 0.6, and 25.2 ± 1.1 % of the 880.8 mm cumulative P _G from 29 rainfall events. Direct throughfall proportion (p) and drainage from the canopy contributed about 14 ± 7 and 50 ± 21 % of the total TF for the events, respectively. The mean canopy storage capacity (S) was 2.4 ± 0.7 mm. The coefficient of variability (CV) of TF rate decreased asymptotically with increasing P _G amount, ranging from 16 to 56 % with median 26 %. The CV of TF rate was not significantly correlated with canopy cover (r = 0.152, P = 0.521, n = 20) and distance from the nearest trunk (r = 0.196, P = 0.408, n = 20). Based on the revised Gash analytical model, the total simulated E _i was close to the observed, with a general underestimation magnitude of 5.7 %. The E _i components were quantified, and most of the interception loss (62.9 %) evaporated during rainfall, while 26.8 % evaporated after rainfall ceased. Climatic and forest structural parameters required by the model were identified and analyzed by sensitivity analysis, implying that the revised Gash analytical model is robust and reliable enough for abandoned Japanese cypress plantations in a maritime climate.     

Relation between developmental changes on anatomical structure and on protein pattern in differentiating xylem of tension wood

Tension wood was induced inEucalyptus camaldulensis L. by fixing the stem at an angle. Proteins in the differentiating tissue of tension wood were compared to those of normal wood on sodium dodecyl sulfate polyacrylamide gel electrophoresis. An obvious difference was found in the salt-soluble fraction of 14 days after inclination. At least five bands (19,22,37,41, and 55kDa) were specific in the differentiating tissue of tension wood. These proteins were bound to the cell wall, plasma membrane, or both by their electric charge; they were undetectable until 14 days after inclination. Mature tension wood was observed in the tissue at 14 days. Thus, all differentiating tissue at 14 days was produced after inclination. On the other hand, the differentiating zone at 7 days contained the same tissue, as in tension wood estimated by the vessel number and diameter during the early phase; and the tissue was indistinguishable from normal wood during the late phase. The proteins found here were related to the phenomenon occurring in the late stage of xylem differentiation.     

Evapotranspiration and deep percolation of a small catchment with a mature Japanese cypress plantation

Quantification of hydrological components was conducted in order to clarify the water budget, especially deep percolation (D) into underlying rock, of a small headwater catchment covered by an old Japanese cypress plantation. Precipitation (P), canopy interception (E _i), soil evaporation (E _s) transpiration (E _t), and runoff (R) were observed for 1 year in a 0.41-ha catchment in southern Kyushu, Japan. E _i was calculated from P, throughfall and stemflow measurements, and E _s and E _t were measured using evaporimeters and heat pulse sensors, respectively. From these observations, deep percolation (D) under steady state condition into underlying sedimentary rock was estimated. As for the annual water budget, almost half of P was R and one-third was evapotranspiration (E), which was the sum of the E _i, E _s and E _t. E _i was almost the same magnitude as predicted for an old conifer plantation modeled by Komatsu et al. (J Hydrol 336:361–375, 2007b), but E _t was lower compared to the model. E was not comparable to water loss in the catchment (P minus R). Therefore, D was estimated as a quarter of P and was slightly higher compared to previously reported values for sedimentary rock catchments.     

Chemical kinetics of Japanese cedar,cypress, fir,and spruce and characterization of charcoal

 Thermogravimetric and differential thermal analyses techniques have been applied to investigate the thermal degradation characteristics and chemical kinetics of Japanese cedar, cypress, fir, and spruce. The decomposition of the components could be modeled by an Arrhenius kinetic expression. The kinetic parameters were extracted from the thermogravimetric data using least-squares techniques. The heating rates used for the analyses were 10°, 5°, and 0.33°C/min; and the activation energy and reaction order of the above woods were 7.54, 8.39, 2.87, and 7.88 kJ/mol and 0.71, 0.64, 0.44, and 0.63, respectively. Finally, carbonization was done to produce charcoal from these woods under various operating conditions, and the charcoal was characterized in respect to yield, heating value, electrical conductivity, and X-ray diffraction. The quality of the charcoal from fir was the best among the four types of wood. The charcoal produced is inferior to binchotan (white charcoal) in respect to electrical conductivity and crystalline structure. Received: February 13, 2002 / Accepted: July 12, 2002 Acknowledgment The authors express their gratitude to Professor Yoshida of Applied Chemistry in Tokyo Metropolitan University for performing the TG/DTA in his laboratory and for his valuable suggestions about the analyses.     

Treatment of poplar callus with ferulic and sinapic acids I: incorporation and enhancement of lignin biosynthesis

Ferulic acid (FA), tetradeuteroferulic acid (DFA), sinapic acid (SA), or heptadeuterosinapic acid (DSA) was exogenously supplied to poplar (Populus alba L.) callus. Administration of FA or SA increased the lignin content of the callus to about twice that of the control callus. Gas chromatographic analysis of the alkali hydrolysate of the cell wall residue revealed that only a trace amount of SA was bound to the cell wall, and the amount of FA was less than 2% of the total callus lignin. Thioacidolysis of the DFA-treated callus indicated that DFA is effectively converted to both coniferyl and sinapyl alcohols and then incorporated into the corresponding lignin. Incorporation of DSA into syringyl lignin or guaiacyl lignin was not observed, but yields of syringyl lignin thioacidolysis products were markedly increased by DSA treatment of the callus. These results suggest that SA may not be a precursor of sinapyl alcohol and syringyl lignin per se, but it may induce or enhance the biosynthesis of syringyl lignin in poplar callus.     

Distribution of structure and lignin within growth rings of Norway spruce

A radial core from a Norway spruce (Picea abies (L.) Karst.) estimated to be about 107 years old was cut from a board and was analyzed for density and microfibril angle (MFA). Furthermore, cell geometry, wall thickness and lignin distribution were analyzed on three selected growth rings in detail. Intra-ring differences in the density profiles are also true for cell wall thicknesses as well as radial and tangential lumen diameters. A higher MFA was found for earlywood with a slow decrease toward the latewood region. The lignin was found to remain rather constant throughout the growth rings, which suggests a constant chemical composition of the cell wall material within the growth ring. From the recorded datasets on a cellular level, it can be concluded that the main adaptation regarding structure–property relationships toward the optimization of water transport and mechanical stability is mainly achieved at the cell level.     

Distribution of lignin in normal and compression wood of Pinus taeda L.

Summary The distribution of lignin in normal and compression wood of loblolly pine (Pinus taeda L.) has been studied by the technique of lignin skeletonizing. Hydrolysis of the wood carbohydrates with hydrofluoric acid left normal wood tracheids with a uniform distribution of lignin in the S1 and S2 cell wall layers. However, the S3 region of both earlywood and latewood tracheids consistently retained a dense network of unhydrolyzable material throughout, perhaps lignin.Lignin content in compression wood averaged about 7% more than in normal wood and appears to be concentrated in the outer zone of the S2 layer. The inner S2 region, despite helical checking, is also heavily lignified. The S1 layer, although thicker than normal in compression wood tracheids, contains relatively little lignin.Ray cells, at least in normal wood, appear to be lignified to the same extent, if not more so in certain cases, than the longitudinal tracheids. Other locations where lignin may be concentrated include initial pit border regions and the membranes of bordered pits.This report is a detailed excerpt from the Ph. D. dissertation of R. A. P. Financial support provided by the College of Forestry at Syracuse University and the National Defense Education Act is hereby gratefully acknowledged.