Cellular Aspects of Wood Formation: 20 (Plant Cell Monographs)

By contrast, Rossi et al. However, the physiological regulation of cambial activity in the spring is still not fully understood Funada et al. Evidence for the earlier induction of cambial reactivation by localized heating suggests that increases in the temperature of the stem might be the limiting factor in the onset of cambial reactivation during the quiescent stage of dormancy Barnett and Miller, ; Begum et al.

In addition, the patterns of cambial reactivation and xylem differentiation were almost identical to those in natural systems. Therefore, we postulated that artificial heating might provide a good model system to investigate the cambial biology of trees. Such a model system would allow us to compare detailed cambial activity and xylem differentiation directly over relatively short periods of time Oribe and Kubo, ; Oribe et al.

Present observations showed that an artificial increase in temperature can induce cambial reactivation in a hardwood and conifers. However, the effects of rapid decrease in temperature after cambial reactivation and xylem differentiation remain to be clarified. The main purpose of the present study was to investigate whether decrease in temperature after cambial reactivation can induce any changes in cambial cells and xylem differentiation continue or not in Cryptomeria japonica and Abies firma trees.

Therefore, this paper analyzed the effects of rapid decrease in temperature on the cambium by using heated cambial reactivation system in adult trees of Cryptomeria japonica and in Abies firma seedlings that can be used as a good system for studies of xylogenesis. Cambial reactivation that induced by localized heating was stopped just after cambial reactivation to observe the effects of rapid decrease in temperature on cambial cells.

In addition, presence of nucleus in heated-reactivated cambial cells and non-heated cambial cells were examined to clarify the status of cambial cells whether the cells were alive or not. The possible mechanism of cambial activity in relation with decrease in temperature will be discussed. The Cryptomeria japonica trees were examined from 8 January to 28 February and Abies firma seedlings were examined from 13 January to 3 March In case of adult Cryptomeria japonica trees, electric heating tape Silicone-Rubber Heater; O and M Heater, Nagoya, Japan , 50 cm in length and 30 cm in width, was wrapped at one side of the main stem of each tree at breast height Fig.

In case of Abies firma seedlings, electric heating ribbon Nippon Heater Co. Tokyo, Japan , 6 m in length and 0. No abnormal structures were found by naked eyes in the stems after artificial heating. In adult Cryptomeria japonica trees and Abies firma seedlings, localized heat treatment was started from 8 January and 13 January , respectively.

Continuous heating was applied until cambial reactivation and heating system was stopped on 18 January and 19 January in both species, respectively. After stop of heating, samples were collected from heated and non-heated control portions of the stem until 27 February and 3 March , in both cases, respectively. In case of adult Cryptomeria japonica trees, samples were taken at three to four days intervals from heated stems and non-heated stems under natural conditions throughout the sampling period.

A series of small blocks 2x2x1 cm 3 which contained phloem, cambium and some xylem cells, was removed with a disposable scalpel and chisel with a zigzag fashion to eliminate any effects of wounding from heated stems and stems under natural conditions. Each block was cut into 2 mm thick samples immediately after removal from the tree.

In case of Abies firma , four sample seedlings two from heated stem and two from non-heated control stem were cut in every sampling date at one day interval until 19 January Then sampling was done at one week interval until 3 March For non-heated control sample, we used seedling that was not heated and sample was taken from the same portion of stem at cm above the stem base.

In each sampling date, we tried to collect the seedlings that had almost the same stem height and diameter to avoid any differences among seedlings. The heated and non-heated control portion of stems was cut into 2 mm thick samples immediately after removal from the tree. Preparation of samples for light microscopy: Fixed samples were washed in 0. After washing in phosphate buffer, specimens were dehydrated in a graded ethanol series and embedded in epoxy resin.

Air temperatures during experiments: Daily maximum, average and minimum air temperatures during each experimental period were obtained from the Japan Meteorological Agency that located in Fuchu, Tokyo. Maximum, average and minimum air temperatures from 1 January to 31 March during the first experiment for Cryptomeria japonica and from 1 January to 31 March during the second experiment for Abies firma are shown in Fig.

After stop of heating, in February , the minimum temperature was In February , the minimum temperature was No division of fusiform cambial cells and ray cambial cells was detected in samples of cambium of Cryptomeria japonica and Abies firma that had been collected on 8 January and 13 January , respectively Fig. During dormancy, the cambium consisted of five or six radial layers of radially narrow and compactly arranged cells Fig. Timing of cambial reactivation and xylem differentiation in heated stems: In heated Cryptomeria japonica and Abies firma stems, cambial reactivation occurred after 6 days and 2 days of heating, on 14 January and 15 January , respectively Fig.

After production of radial files of fusiform cambium on 18 January in Cryptomeria japonica and on 19 January in Abies firma , heating system was stopped in both species Fig. Effects of rapid decrease in temperature on cambial cells after stop of heating: One week later of stop of heating, on 25 January and 26 January , the cambial cells became shrunk and cell contents coagulated in phloem and cambial cells in adult Cryptomeria japonica trees and Abies firma seedlings Fig.

The higher magnified image of the same portion of Fig. At that time the structure, shape and size of cambial cells were not at normal condition Fig. Two weeks later of stop of heating, on 1 February and 2 February , the cambial cells were almost at the same condition as well as shrunk cambium observed in both species Fig. In addition, no new cell plates were observed in the cambial zone of Cryptomeria japonica and Abies firma stems indicating that cambial activity was reduced or almost stopped Fig.

Two weeks later of stop of heating, on 1 February and 2 February , in the same sample of Cryptomeria japonica and Abies firma , nucleus was present in ray cambial cells Fig. After one month of stop of heating, on 18 February , the shrunk cambium produced new tracheids with deformed structure of secondary xylem as compared with the normal xylem differentiation in Cryptomeria japonica trees Fig.

Relationship between shrinkage of cambium and temperature data: Due to the stop of heating, temperature decreased rapidly in the heated portions of the stems in both species Fig. When we stopped the heating system in February, the minimum atmospheric temperature was ranged from The temperature profile and microscopic images of cambial cells clearly showed that shrunk cambium with coagulated cell contents produced due to rapid decrease in temperature in adult Cryptomeria japonica trees and Abies firma seedlings in February Fig.

Rapid decrease in temperature on localized-heated stems induced coagulation of cell contents in cambial cells with deformed shape and size of phloem cells in Cryptomeria japonica trees and Abies firma during winter dormancy in February. One week later of stop of heating, shrunk with abnormal structure of longitudinal phloem parenchyma cells were observed in localized-heat-induced differentiating phloem cells. In our previous research, we observed that due to the rapid decrease in temperature, cell wall thickening of phloem fibers started earlier than xylem cells in Cryptomeria japonica trees and Abies firma stems.

In addition, division of phloem cells started prior to cambial reactivation and xylem differentiation in heated stems and under natural conditions at warmer early spring of hybrid poplar Populus sieboldii x Populus grandidentata , indicating that phloem cells were able to make quick response to increase in temperature than cambial cells and xylem cells Begum et al.

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In present study, it was found that coagulation of cell contents occurred in longitudinal phloem parenchyma cells due to stop of heating. This observation suggests that phloem cells might respond to decrease in temperature more rapidly than xylem cells in conifers. It was already proved that temperature was a limiting factor in the onset of cambial reactivation and xylem differentiation during the quiescent dormant state in conifers Savidge and Wareing, ; Barnett and Miller, ; Oribe and Kubo, ; Oribe et al.

Increase in temperature or warmer early spring induced earlier cambial reactivation and xylem differentiation in trees Oribe and Kubo, ; Oribe et al. Similarly, in the present study, earlier cambial reactivation and xylem differentiation was induced by localized heating in Cryptomeria japonica trees and Abies firma stems during winter dormancy in February indicating that temperature is one of the most important trigger for start of cambial reactivation. Under these conditions, no formation of new cell plates occurred in the cambium in March in Cryptomeria japonica trees Begum et al. Thus, low temperatures appear to be very important for maintenance of a quiescent state Begum et al.

In our present study, it was observed that rapid decrease in temperature in heated reactivated cambium induced shrinkage of cambial cells with coagulation of cell contents. In addition, nucleus was observed in those shrunk cambial cells indicating that cambial cells were alive. Therefore, it appears that rapid changes in environmental conditions might induce cellular changes in cambial cells. Certain abnormal environmental conditions can induce the formation of various structures, For example, wider tracheids were produce under water stress condition in plants Landrum, ; Gutierrez et al.

Salinity stress decreased xylem exudation rate and collapsed xylem cells Kabir et al. Drought stress increased vessel wall thickness Mostajeran and Rahimi-Eichi, and showed highest cell wall associated peroxidase activity in leaf cells Hamad et al. Low temperature induced greater degree of shrinkage in cell structure Singh and Pandey, Chilling strees decreased stomatal conductance and increased peroxidase enzymatic activity in plant cells Islam et al.

In general, with increasing methyl jasmonate concentration, chilling injury reduced significantly Zolfagharinasab and Hadian, Deflowering of rachis induced narrower xylem cells which inhibit water conduction to the top of the rachis resulted few or smaller sized pod production Begum et al. Thus, environmental stress plays a significant role on morphology and structure of cells in plant. It was also reported that continuation of cambial activity and xylem differentiation required a constant threshold maximum temperature Begum et al. The results suggest that cambial activity was stopped due to rapid changes of temperature in February.

The present results showed that localized heating during cambial dormancy induced earlier cambial reactivation in conifers and subsequent rapid decrease in temperature just after cambial reactivation induced formation of shrinkage cambium. The results suggest that low temperature might changes endogenous balances that induced shrinkage of cambium with deformed structure of differentiating tracheids which would be helpful to study the mechanism of cambial activity in conifers.

The content of polyphenols is generally higher in the heartwood of old trees than in that of young trees and decreases from the periphery towards the central portion of the stem Gierlinger and Wimmer As the extractives are responsible for natural durability, different grades are achieved depending on species, age and position within the stem Schultz and Nicholas , Taylor et al. The yield of different extractives depends on the extraction method, polarity of the solvent, temperature and time Xu and Chang , Fang et al. For example, pine extractives can be divided into a hydrophilic stilbenes and lignans and a lipophilic part resin acids, long chain fatty acids and triglycerides Figure 1 b.

By sequentially extracting pine heartwood chips with non-polar and polar solvents different extracts can be obtained Lu et al. For example, the extraction with a polar solvent recovers the pinosylvins with high purity Fang et al. Up to now, only a few studies dealing with stilbene impregnation of wood have been conducted. Already in Rennerfelt and Rennerfelt , it was observed that sapwood impregnated with pinosylvins became more stable against fungal degradation.

Later on, Celimene et al. Recently, the textile industry has become interested in polyphenol impregnation to inhibit the bacterial growth Sanchez-Sanchez et al. Furthermore, novel food packaging e. Heartwood extractives have been mainly investigated by wet-chemical and chromatographic methods Ekeberg et al. To investigate extractives in context with microstructure, TOF-SIMS imaging has been applied in Cryptomeria japonica trees and showed that the extractives tend to accumulate near the radial rays Saito et al.

Recently Belt et al. On the micro-level, conglomerating phenolics were reported in the lumen as well as a higher amount of phenolics in the compound middle lamella CML and cell corner CC. Confocal Raman Microscopy relies on the inelastic scattering of incident monochromatic light hv 0 , wherein the energy of the scattered light changes upon interaction with molecular vibrations e.

Important new insights on the molecular structure of wood polymers, especially cellulose and lignin, could be gained Agarwal , , , Agarwal et al.

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The main objective of this study was to monitor heartwood formation with CRM exactly in the transition zone of native pine samples Pinus sylvestris and compare it with an in-vitro impregnation. The experimental design focuses on chemical changes occurring in tracheid cell walls and radial rays with the aim to understand the transport pathways of natural and in-vitro extractives impregnation and wood functionalization.

Never-dried wood samples were obtained from pine P. Rectangular pieces were cut out from sapwood and heartwood in both north and south cardinal directions of the disk see Table S1 available as Supplementary Data at Tree Physiology Online to record the most important wood characteristics e. The dried wood was conditioned to room temperature in a desiccator and the initial dry weight was recorded.

Crude extractives were obtained using a Soxhlet extractor ml with 23 g dry pine heartwood chips. Each block was placed in a vial with 6 ml extract solution under ambient pressure for 20 h see Figure S2a and b available as Supplementary Data at Tree Physiology Online. Thereafter the blocks were repeatedly impregnated in the vacuum mbar. One cycle took 10 min at room temperature and at least three cycles were carried out. Before sectioning, the blocks were immersed with Deuterium D 2 O under vacuum to reduce the overlap of the OH and CH stretching bands during the Raman measurements.

During the cutting process, only D 2 O was used to avoid drying of the specimen and maintaining the native water content. The thin sections were placed on a standard glass slide with a drop of D 2 O, covered with a glass coverslip 0. The sapwood—heartwood boundary was marked on the bottom of the slide and measured immediately or kept frozen until CRM measurement. The orientation of the sample with respect to the laser polarization the radial direction within the y -axis of the table was kept constant during all measurements.

All Raman scans were taken with a lateral resolution of 0. The control Four WITec acquisition software was used to set experimental parameters for hyperspectral image acquisition. For further information, these reference spectra can be also found on the webpage www.

The extracted spectra were analyzed with Opus 7. Before the Raman images were generated based on integration of specific bands, a cosmic ray removal filter spike half-width 2 was applied. Based on the integrated images, average spectra of distinct areas of the samples e. Raman mappings were acquired in-situ with a lateral resolution of nm along five annual rings to reveal the nature and distribution of the extractives in pine P.

The cellulose Raman images Figure 2 b depict the cell wall and show a rather constant intensity along sapwood and heartwood small changes come from slight changes in focal plane or uneven sample surface. On the contrary, the intensity related to the aromatic ring stretching vibration from the phenolic compounds increases clearly towards the heartwood Figure 2 c. Reference spectra from characteristic pine heartwood extractives indeed clearly show that also pinosylvins contribute to the intensity of this band details in Figure S3 available as Supplementary Data at Tree Physiology Online.

In-situ Raman mapping of pine tracheids during heartwood formation. Raman measurement areas are marked by the numbered rectangles 1—9. Within the heartwood, the highest intensity dark red is found in the CC, CML, S3 layer, within the pit membrane and in various deposits in the lumen Figure 2 d. The higher accumulation in the CML becomes again clear in the intensity profile.

Moreover, a slightly higher content in the S3 layer compared with the S2 layer, described by a bow-shaped intensity profile with three main peaks Figure 2 e, image 8 is visible: Both layers CML, S3 are tiny layers and close to the limit of the spatial resolution of the instrument, but still, a higher content can be detected Figure 2 e, images 8 and 9. To examine the chemical changes and composition during heartwood formation within the secondary cell wall and the CC selectively, average spectra were extracted.

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How heartwood differs—spectral view of heartwood formation. However, the question is whether this tiny wavenumber difference is enough to discriminate between both substances in wood. These bands arise from vibration 12 of the benzene ring Varsanyi , Colthup et al. Pinosylvins show these two bands because both rings will exhibit this vibrational mode. However, there will be a difference between the two rings due to the different nature of the substituents OH, OMe.

Changing the environment of the rings hydrogen-bonding etc. Nevertheless, the latter one is in general weaker and not completely free from lignin contribution as it is already present in the sapwood Figure 3 c and d. Summarized, the average spectra extracted from CC and cell wall in sapwood, transition zone and heartwood indicate that the main difference was the increase of phenolic components, identified as pinosylvins and mainly PSMME Figure 3. The spatial distribution is similar to that for lignin as higher amounts of pinosylvins were deposited in the CCs than in the cell wall.

Beside in the CC and cell wall pinosylvins have been detected in deposits in the lumen Figure 2 d. The deposits in the lumen are always stuck to the S3 layer and are large at the border between sapwood and transition zone, and become very small and even more tightly attached to the S3 in the heartwood Figure 4 a. The extracted average spectra of the visualized droplets in each annual ring show clearly the change in composition from sapwood to heartwood Figure 4 b.

The spectra of the droplets in the sapwood are dominated by bands of lipids: Comparison with linoleic and oleic acid see Figure S3 available as Supplementary Data at Tree Physiology Online confirms that the sapwood deposit spectrum resembles the fatty acid spectra with all bands clearly present.

The highest intensity of phenolics is observed in the heartwood deposit in the annual ring 18th, which highly resembled the pinosylvins reference spectra see Figure S3 available as Supplementary Data at Tree Physiology Online. Learning from similarities and differences of various deposits.

Although in former studies no details on the chemistry of observed droplets could be given, Magel et al.

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These droplets stick to the pits and further to the lumen of neighbor tracheids after the disintegration of the protoplast. Fromm concluded that the cell walls in heartwood are infiltrated with these droplets. Also, Schneider et al. A recent study by Belt et al. In our study, we were able to show the transition of the droplets from a lipidic to an aromatic character. On the other hand, the deposits found in heartwood have still minor lipidic traces, confirming the co-existence of lipids and aromatics once again.

It is known that the pinosylvins in the transition zone are synthesized from triglycerides besides from sucrose Lim et al. So the fatty acids and triglycerides are involved in the synthesis, but could also play a role in transportation. By sequential extraction, these lipophilic and hydrophilic components of pine wood were further investigated. The loadings give the wavenumbers that are important for this group separation Figure 5 b.

Within the lipophilic side, abietic acid and the deposit in the transitions zone Pi TZ are the ones closest to the hydrophilic part. This is due to the higher amount of stilbenes in the deposit see spectrum Figure S3 available as Supplementary Data at Tree Physiology Online and the rather low CH-stretching of the abietic acid compared with the fatty acids Figure 5 a; see Figure S3 available as Supplementary Data at Tree Physiology Online.

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The chemical structure of abietic acid is different as it belongs to the resin acids. The authors showed also that the linoleic and oleic acid are the most common fatty acids in Scots pine sapwood. Based on spectral signature of sapwood deposits, located in the score plot very near linoleic acid Figure 5 c , it is revealed that these lipophilics are mainly found within the lumen of sapwood. Based on this band it is possible to conclude that the native deposits in the sapwood contain triglycerides.

These lipids are, in our study, mainly found in the deposits in the lumen Figure 4 a and in rays Figure 6. Multivariate comparison of spectra from pine references, extractives and native deposits. Note that these are an abstract representation of the spectral profiles with negative values. PC-2 shows mostly the hydrophilic part. Pinosylvins transported through radial rays. The intensity scale is from 0 to 5 for all images a—f , except image d 0— Native spruce sapwood lacks pinosylvins Figure 6 c as reported in the literature Hovelstad et al.

Impregnating the pinosylvin-free spruce sapwood with hydrophilic pine extract results in a similar pinosylvins distribution pattern like in pine heartwood Figure 6 e; see Figure S4a available as Supplementary Data at Tree Physiology Online. Within the ray, the highest pinosylvins amount was detected and associated with lipids Figure 6 f. The average spectra from the deposits of native pine and impregnated spruce Figure 6 g coincide very well, pointing to the same chemical composition.

Although the EtOH extract used for impregnation contains only a small amount of lipids, the deposits found after impregnation show strong lipid bands. Therefore, these native lipids seem to be involved in the transport of pinosylvins also in the artificial impregnation of spruce. We thus hypothesize that the hydrophilic pinosylvins are associated with the lipids for transportation as summarized in Figure 6 h in a schematic drawing.