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1.  Induction of Cambial Reactivation by Localized Heating in a Deciduous Hardwood Hybrid Poplar (Populus sieboldii × P. grandidentata) 
Annals of Botany  2007;100(3):439-447.
Background and Aims
The timing of cambial reactivation plays an important role in the control of both the quantity and the quality of wood. The effect of localized heating on cambial reactivation in the main stem of a deciduous hardwood hybrid poplar (Populus sieboldii × P. grandidentata) was investigated.
Methods
Electric heating tape (20–22 °C) was wrapped at one side of the main stem of cloned hybrid poplar trees at breast height in winter. Small blocks were collected from both heated and non-heated control portions of the stem for sequential observations of cambial activity and for studies of the localization of storage starch around the cambium from dormancy to reactivation by light microscopy.
Key Results
Cell division in phloem began earlier than cambial reactivation in locally heated portions of stems. Moreover, the cambial reactivation induced by localized heating occurred earlier than natural cambial reactivation. In heated stems, well-developed secondary xylem was produced that had almost the same structure as the natural xylem. When cambial reactivation was induced by heating, the buds of trees had not yet burst, indicating that there was no close temporal relationship between bud burst and cambial reactivation. In heated stems, the amount of storage starch decreased near the cambium upon reactivation of the cambium. After cambial reactivation, storage starch disappeared completely. Storage starch appeared again, near the cambium, during xylem differentiation in heated stems.
Conclusions
The results suggest that, in deciduous diffuse-porous hardwood poplar growing in a temperate zone, the temperature in the stem is a limiting factor for reactivation of phloem and cambium. An increase in temperature might induce the conversion of storage starch to sucrose for the activation of cambial cell division and secondary xylem. Localized heating in poplar stems provides a useful experimental system for studies of cambial biology.
doi:10.1093/aob/mcm130
PMCID: PMC2533603  PMID: 17621596
Populus sieboldii × Populus grandidentata; localized heating, cambial reactivation; model system; storage starch; xylem differentiation
2.  Changes in the localization and levels of starch and lipids in cambium and phloem during cambial reactivation by artificial heating of main stems of Cryptomeria japonica trees 
Annals of Botany  2010;106(6):885-895.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcq185
PMCID: PMC2990657  PMID: 21037242
Cambial reactivation; confocal laser scanning microscopy; Cryptomeria japonica; lipid; starch; xylem differentiation
3.  Characterization of Transcriptome Remodeling during Cambium Formation Identifies MOL1 and RUL1 As Opposing Regulators of Secondary Growth 
PLoS Genetics  2011;7(2):e1001312.
Cell-to-cell communication is crucial for the development of multicellular organisms, especially during the generation of new tissues and organs. Secondary growth—the lateral expansion of plant growth axes—is a highly dynamic process that depends on the activity of the cambium. The cambium is a stem cell–like tissue whose activity is responsible for wood production and, thus, for the establishment of extended shoot and root systems. Attempts to study cambium regulation at the molecular level have been hampered by the limitations of performing genetic analyses in trees and by the difficulty of accessing this tissue in model systems such as Arabidopsis thaliana. Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity. Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments. By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation. In summary, we provide a means for investigating cambium regulation in unprecedented depth and present two signaling components that control a process responsible for the accumulation of a large proportion of terrestrial biomass.
Author Summary
In contrast to animals, plants have the capacity to grow and form new organs throughout their entire life cycle, thereby building up some of the largest organisms on earth. This remarkable capacity is based on the activity of stem cell–like tissues—the meristems—located at shoot and root apices and, in a large repertoire of species, in lateral positions at the flanks of growth axes. In comparison to apical meristems, our knowledge of the molecular mechanisms controlling the activity of lateral meristems like the cambium is very limited. This is despite the fact that lateral growth is responsible for wood formation, and thus for the accumulation of large amounts of terrestrial biomass, and for fixation of atmospheric CO2. Here, we introduce an in vitro system by which cambium initiation can be stimulated under controlled conditions in stems of the reference plant Arabidopsis thaliana. By revealing genome-wide and tissue-specific alterations in transcript accumulation during cambium initiation, we identify two novel receptor-like kinases, namely MOL1 and RUL1, as opposing cambium regulators. These findings demonstrate that our in vitro system represents a valuable tool for studying cambium regulation and open up possibilities to dissect lateral growth in plants from novel perspectives.
doi:10.1371/journal.pgen.1001312
PMCID: PMC3040665  PMID: 21379334
4.  A meta-analysis of cambium phenology and growth: linear and non-linear patterns in conifers of the northern hemisphere 
Annals of Botany  2013;112(9):1911-1920.
Background and Aims
Ongoing global warming has been implicated in shifting phenological patterns such as the timing and duration of the growing season across a wide variety of ecosystems. Linear models are routinely used to extrapolate these observed shifts in phenology into the future and to estimate changes in associated ecosystem properties such as net primary productivity. Yet, in nature, linear relationships may be special cases. Biological processes frequently follow more complex, non-linear patterns according to limiting factors that generate shifts and discontinuities, or contain thresholds beyond which responses change abruptly. This study investigates to what extent cambium phenology is associated with xylem growth and differentiation across conifer species of the northern hemisphere.
Methods
Xylem cell production is compared with the periods of cambial activity and cell differentiation assessed on a weekly time scale on histological sections of cambium and wood tissue collected from the stems of nine species in Canada and Europe over 1–9 years per site from 1998 to 2011.
Key Results
The dynamics of xylogenesis were surprisingly homogeneous among conifer species, although dispersions from the average were obviously observed. Within the range analysed, the relationships between the phenological timings were linear, with several slopes showing values close to or not statistically different from 1. The relationships between the phenological timings and cell production were distinctly non-linear, and involved an exponential pattern
Conclusions
The trees adjust their phenological timings according to linear patterns. Thus, shifts of one phenological phase are associated with synchronous and comparable shifts of the successive phases. However, small increases in the duration of xylogenesis could correspond to a substantial increase in cell production. The findings suggest that the length of the growing season and the resulting amount of growth could respond differently to changes in environmental conditions.
doi:10.1093/aob/mct243
PMCID: PMC3838565  PMID: 24201138
Cambium; cell differentiation; cell production; climate change; conifers; growth; meristem; phenology; productivity; secondary wall formation; xylogenesis
5.  Fluctuations of cambial activity in relation to precipitation result in annual rings and intra-annual growth zones of xylem and phloem in teak (Tectona grandis) in Ivory Coast 
Annals of Botany  2012;110(4):861-873.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcs145
PMCID: PMC3423803  PMID: 22805529
Growth rings; teak; Tectona grandis; vascular cambium; xylem and phloem formation
6.  A rapid decrease in temperature induces latewood formation in artificially reactivated cambium of conifer stems 
Annals of Botany  2012;110(4):875-885.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcs149
PMCID: PMC3423807  PMID: 22843340
Cambial activity; conifers; latewood formation; morphology of tracheids; rapid decrease in temperature
7.  A Patchy Growth via Successive and Simultaneous Cambia: Key to Success of the Most Widespread Mangrove Species Avicennia marina? 
Annals of Botany  2007;101(1):49-58.
Background and Aims
Secondary growth via successive cambia has been intriguing researchers for decades. Insight into the mechanism of growth layer formation is, however, limited to the cellular level. The present study aims to clarify secondary growth via successive cambia in the mangrove species Avicennia marina on a macroscopic level, addressing the formation of the growth layer network as a whole. In addition, previously suggested effects of salinity on growth layer formation were reconsidered.
Methods
A 1-year cambial marking experiment was performed on 80 trees from eight sites in two mangrove forests in Kenya. Environmental (soil water salinity and nutrients, soil texture, inundation frequency) and tree characteristics (diameter, height, leaf area index) were recorded for each site. Both groups of variables were analysed in relation to annual number of growth layers, annual radial increment and average growth layer width of stem discs.
Key Results
Between trees of the same site, the number of growth layers formed during the 1-year study period varied from only part of a growth layer up to four growth layers, and was highly correlated to the corresponding radial increment (0–5 mm year–1), even along the different sides of asymmetric stem discs. The radial increment was unrelated to salinity, but the growth layer width decreased with increasing salinity and decreasing tree height.
Conclusions
A patchy growth mechanism was proposed, with an optimal growth at distinct moments in time at different positions around the stem circumference. This strategy creates the opportunity to form several growth layers simultaneously, as observed in 14 % of the studied trees, which may optimize tree growth under favourable conditions. Strong evidence was provided for a mainly endogenous trigger controlling cambium differentiation, with an additional influence of current environmental conditions in a trade-off between hydraulic efficiency and mechanical stability.
doi:10.1093/aob/mcm280
PMCID: PMC2701843  PMID: 18006508
Avicenia marina; cambial marking; mangrove; phloem; salinity; secondary growth; successive cambia; xylem
8.  Effect of Local Heating and Cooling on Cambial Activity and Cell Differentiation in the Stem of Norway Spruce (Picea abies) 
Annals of Botany  2006;97(6):943-951.
• 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.
doi:10.1093/aob/mcl050
PMCID: PMC2803384  PMID: 16613904
Norway spruce; Picea abies; cambium; xylem; phloem; cell differentiation; heating; cooling; light microscopy; transmission electron microscopy; UV-microspectrophotometry
9.  Cambial Activity and Intra-annual Xylem Formation in Roots and Stems of Abies balsamea and Picea mariana 
Annals of Botany  2008;102(5):667-674.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcn146
PMCID: PMC2712372  PMID: 18708643
Abies balsamea; boreal forest; cambium; cell differentiation; cell wall thickening; lignification; Picea mariana; root; stem; xylem
10.  A Dynamic, Architectural Plant Model Simulating Resource‐dependent Growth 
Annals of Botany  2004;93(5):591-602.
• Background and Aims Physiological and architectural plant models have originally been developed for different purposes and therefore have little in common, thus making combined applications difficult. There is, however, an increasing demand for crop models that simulate the genetic and resource‐dependent variability of plant geometry and architecture, because man is increasingly able to transform plant production systems through combined genetic and environmental engineering.
• Model GREENLAB is presented, a mathematical plant model that simulates interactions between plant structure and function. Dual‐scale automaton is used to simulate plant organogenesis from germination to maturity on the basis of organogenetic growth cycles that have constant thermal time. Plant fresh biomass production is computed from transpiration, assuming transpiration efficiency to be constant and atmospheric demand to be the driving force, under non‐limiting water supply. The fresh biomass is then distributed among expanding organs according to their relative demand. Demand for organ growth is estimated from allometric relationships (e.g. leaf surface to weight ratios) and kinetics of potential growth rate for each organ type. These are obtained through parameter optimization against empirical, morphological data sets by running the model in inverted mode. Potential growth rates are then used as estimates of relative sink strength in the model. These and other ‘hidden’ plant parameters are calibrated using the non‐linear, least‐square method.
• Key Results and Conclusions The model reproduced accurately the dynamics of plant growth, architecture and geometry of various annual and woody plants, enabling 3D visualization. It was also able to simulate the variability of leaf size on the plant and compensatory growth following pruning, as a result of internal competition for resources. The potential of the model’s underlying concepts to predict the plant’s phenotypic plasticity is discussed.
doi:10.1093/aob/mch078
PMCID: PMC4242319  PMID: 15056562
Plant architecture; phenotypic plasticity; demand functions; competition among sinks; source–sink relationships; structural‐functional models
11.  Successive Cambia: A Developmental Oddity or an Adaptive Structure? 
PLoS ONE  2011;6(1):e16558.
Background
Secondary growth by successive cambia is a rare phenomenon in woody plant species. Only few plant species, within different phylogenetic clades, have secondary growth by more than one vascular cambium. Often, these successive cambia are organised concentrically. In the mangrove genus Avicennia however, the successive cambia seem to have a more complex organisation. This study aimed (i) at understanding the development of successive cambia by giving a three-dimensional description of the hydraulic architecture of Avicennia and (ii) at unveiling the possible adaptive nature of growth by successive cambia through a study of the ecological distribution of plant species with concentric internal phloem.
Results
Avicennia had a complex network of non-cylindrical wood patches, the complexity of which increased with more stressful ecological conditions. As internal phloem has been suggested to play a role in water storage and embolism repair, the spatial organisation of Avicennia wood could provide advantages in the ecologically stressful conditions species of this mangrove genus are growing in. Furthermore, we could observe that 84.9% of the woody shrub and tree species with concentric internal phloem occurred in either dry or saline environments strengthening the hypothesis that successive cambia provide the necessary advantages for survival in harsh environmental conditions.
Conclusions
Successive cambia are an ecologically important characteristic, which seems strongly related with water-limited environments.
doi:10.1371/journal.pone.0016558
PMCID: PMC3031581  PMID: 21304983
12.  A stochastic model of tree architecture and biomass partitioning: application to Mongolian Scots pines 
Annals of Botany  2010;107(5):781-792.
Background and Aims
Mongolian Scots pine (Pinus sylvestris var. mongolica) is one of the principal species used for windbreak and sand stabilization in arid and semi-arid areas in northern China. A model-assisted analysis of its canopy architectural development and functions is valuable for better understanding its behaviour and roles in fragile ecosystems. However, due to the intrinsic complexity and variability of trees, the parametric identification of such models is currently a major obstacle to their evaluation and their validation with respect to real data. The aim of this paper was to present the mathematical framework of a stochastic functional–structural model (GL2) and its parameterization for Mongolian Scots pines, taking into account inter-plant variability in terms of topological development and biomass partitioning.
Methods
In GL2, plant organogenesis is determined by the realization of random variables representing the behaviour of axillary or apical buds. The associated probabilities are calibrated for Mongolian Scots pines using experimental data including means and variances of the numbers of organs per plant in each order-based class. The functional part of the model relies on the principles of source–sink regulation and is parameterized by direct observations of living trees and the inversion method using measured data for organ mass and dimensions.
Key Results
The final calibration accuracy satisfies both organogenetic and morphogenetic processes. Our hypothesis for the number of organs following a binomial distribution is found to be consistent with the real data. Based on the calibrated parameters, stochastic simulations of the growth of Mongolian Scots pines in plantations are generated by the Monte Carlo method, allowing analysis of the inter-individual variability of the number of organs and biomass partitioning. Three-dimensional (3D) architectures of young Mongolian Scots pines were simulated for 4-, 6- and 8-year-old trees.
Conclusions
This work provides a new method for characterizing tree structures and biomass allocation that can be used to build a 3D virtual Mongolian Scots pine forest. The work paves the way for bridging the gap between a single-plant model and a stand model.
doi:10.1093/aob/mcq218
PMCID: PMC3077980  PMID: 21062760
Pinus sylvestris var. mongolica; functional–structural plant model; canopy architecture; three-dimensional; forest canopy; virtual plant; GreenLab, parameterization
13.  Immunolocalization indicates plasmodesmal trafficking of storage proteins during cambial reactivation in Populus nigra 
Annals of Botany  2010;106(3):385-394.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcq130
PMCID: PMC2924828  PMID: 20584737
Cambial region; lectins; plasmodesmal trafficking; Populus nigra ‘italica’; size exclusion limit; storage proteins; vascular tissues
14.  Transcriptome Characteristics and Six Alternative Expressed Genes Positively Correlated with the Phase Transition of Annual Cambial Activities in Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook) 
PLoS ONE  2013;8(8):e71562.
Background
The molecular mechanisms that govern cambial activity in angiosperms are well established, but little is known about these molecular mechanisms in gymnosperms. Chinese fir (Cunninghamia lanceolata (Lamb.) Hook), a diploid (2n  = 2x  = 22) gymnosperm, is one of the most important industrial and commercial timber species in China. Here, we performed transcriptome sequencing to identify the repertoire of genes expressed in cambium tissue of Chinese fir.
Methodology/Principal Findings
Based on previous studies, the four stage-specific cambial tissues of Chinese fir were defined using transmission electron microscopy (TEM). In total, 20 million sequencing reads (3.6 Gb) were obtained using Illumina sequencing from Chinese fir cambium tissue collected at active growth stage, with a mean length of 131 bp and a N50 of 90 bp. SOAPdenovo software was used to assemble 62,895 unigenes. These unigenes were further functionally annotated by comparing their sequences to public protein databases. Expression analysis revealed that the altered expression of six homologous genes (ClWOX1, ClWOX4, ClCLV1-like, ClCLV-like, ClCLE12, and ClPIN1-like) correlated positively with changes in cambial activities; moreover, these six genes might be directly involved in cambial function in Chinese fir. Further, the full-length cDNAs and DNAs for ClWOX1 and ClWOX4 were cloned and analyzed.
Conclusions
In this study, a large number of tissue/stage-specific unigene sequences were generated from the active growth stage of Chinese fir cambium. Transcriptome sequencing of Chinese fir not only provides extensive genetic resources for understanding the molecular mechanisms underlying cambial activities in Chinese fir, but also is expected to be an important foundation for future genetic studies of Chinese fir. This study indicates that ClWOX1 and ClWOX4 could be possible reverse genetic target genes for revealing the molecular mechanisms of cambial activities in Chinese fir.
doi:10.1371/journal.pone.0071562
PMCID: PMC3741379  PMID: 23951189
15.  Functional Multi-Locus QTL Mapping of Temporal Trends in Scots Pine Wood Traits 
G3: Genes|Genomes|Genetics  2014;4(12):2365-2379.
Quantitative trait loci (QTL) mapping of wood properties in conifer species has focused on single time point measurements or on trait means based on heterogeneous wood samples (e.g., increment cores), thus ignoring systematic within-tree trends. In this study, functional QTL mapping was performed for a set of important wood properties in increment cores from a 17-yr-old Scots pine (Pinus sylvestris L.) full-sib family with the aim of detecting wood trait QTL for general intercepts (means) and for linear slopes by increasing cambial age. Two multi-locus functional QTL analysis approaches were proposed and their performances were compared on trait datasets comprising 2 to 9 time points, 91 to 455 individual tree measurements and genotype datasets of amplified length polymorphisms (AFLP), and single nucleotide polymorphism (SNP) markers. The first method was a multilevel LASSO analysis whereby trend parameter estimation and QTL mapping were conducted consecutively; the second method was our Bayesian linear mixed model whereby trends and underlying genetic effects were estimated simultaneously. We also compared several different hypothesis testing methods under either the LASSO or the Bayesian framework to perform QTL inference. In total, five and four significant QTL were observed for the intercepts and slopes, respectively, across wood traits such as earlywood percentage, wood density, radial fiberwidth, and spiral grain angle. Four of these QTL were represented by candidate gene SNPs, thus providing promising targets for future research in QTL mapping and molecular function. Bayesian and LASSO methods both detected similar sets of QTL given datasets that comprised large numbers of individuals.
doi:10.1534/g3.114.014068
PMCID: PMC4267932  PMID: 25305041
functional QTL mapping; wood quality traits; Scots pine; multi-locus model; shrinkage estimation
16.  Dynamic changes in transcripts during regeneration of the secondary vascular system in Populus tomentosa Carr. revealed by cDNA microarrays 
BMC Genomics  2009;10:215.
Background
Wood is the end product of secondary vascular system development, which begins from the cambium. The wood formation process includes four major stages: cell expansion, secondary wall biosynthesis, lignification, and programmed cell death. Transcriptional profiling is a rapid way to screen for genes involved in these stages and their transitions, providing the basis for understanding the molecular mechanisms that control this process.
Results
In this study, cDNA microarrays were prepared from a subtracted cDNA library (cambium zone versus leaf) of Chinese white poplar (Populus tomentosa Carr.) and employed to analyze the transcriptional profiles during the regeneration of the secondary vascular system, a platform established in our previous study. Two hundred and seven genes showed transcript-level differences at the different regeneration stages. Dramatic transcriptional changes were observed at cambium initiation, cambium formation and differentiation, and xylem development, suggesting that these up- or downregulated genes play important roles in these stage transitions. Transcription factors such as AUX/IAA and PINHEAD, which were previously shown to be involved in meristem and vascular tissue differentiation, were strongly transcribed at the stages when cambial cells were initiated and underwent differentiation, whereas genes encoding MYB proteins and several small heat shock proteins were strongly transcribed at the stage when xylem development begins.
Conclusion
Employing this method, we observed dynamic changes in gene transcript levels at the key stages, including cambium initiation, cambium formation and differentiation, and xylem development, suggesting that these up- or downregulated genes are strongly involved in these stage transitions. Further studies of these genes could help elucidate their roles in wood formation.
doi:10.1186/1471-2164-10-215
PMCID: PMC2685409  PMID: 19426563
17.  Cambial activity related to tree size in a mature silver-fir plantation 
Annals of Botany  2011;108(3):429-438.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcr168
PMCID: PMC3158687  PMID: 21816842
Cambial activity; forest-stand structure; silver fir (Abies alba); tree-ring formation; tree-to-tree competition; social status; wood anatomy; xylem cell differentiation
18.  Deep sequencing on a genome-wide scale reveals diverse stage-specific microRNAs in cambium during dormancy-release induced by chilling in poplar 
BMC Plant Biology  2014;14(1):267.
Background
Trees in temperate zones show periodicity by alternating active and dormant states to adapt to environmental conditions. Although phytohormones and transcriptional regulation were found to be involved in growth cessation and dormancy transition, little is known about the mechanisms of the dormancy-active growth transition, especially dormancy maintenance and release. Small RNAs are a group of short non-coding RNAs regulating gene expressions at the post-transcriptional level during plant development and the responses to environmental stress. No report on the expression profiling of small RNAs in the cambial meristem during the dormancy-active growth transition has been reported to date.
Results
Three small RNA libraries from the cambium of poplar, representing endodormancy induced by short day conditions, ecodormancy induced by chilling and active growth induced by long day conditions, respectively, were generated and sequenced by Illumina high-throughput sequencing technology. This yielded 123 known microRNAs (miRNAs) with significant expression changes, which included developmental-, phytohormone- and stress-related miRNAs. Interestingly, miR156 and miR172 showed opposite expression patterns in the cambial dormancy-active growth transition. Additionally, miR160, which is involved in the auxin signaling pathway, was expressed specifically during endodormancy release by chilling. Furthermore, 275 novel miRNAs expressed in the cambial zone were identified, and 34 of them had high detection frequencies and unique expression patterns. Finally, the target genes of these novel miRNAs were predicted and some were validated experimentally by 5′RACE.
Conclusions
Our results provided a comprehensive analysis of small RNAs in the cambial meristem during dormancy-release at the genome-wide level and novel evidence of miRNAs involved in the regulation of this biological process.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0267-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0267-6
PMCID: PMC4189724  PMID: 25269469
Cambium; Chilling; Ecodormancy; Endodormancy; MiRNAs; Poplar
19.  THE SIGNIFICANCE OF THE CAMBIUM IN THE STUDY OF CERTAIN PHYSIOLOGICAL PROBLEMS 
1. The adjacent, undifferentiated, uninucleated cells of the lateral meristem or cambium are of two distinct shapes and sizes: (1) small, more or less isodiametric initials which are of the same general order of magnitude as the cells of the terminal meristem and embryo; and (2) large, elongated initials which in certain cases may attain a length of more than 10,000 micra and a volume of 10,000,000 cubic micra. The large initials may be induced to divide to form small initials, and the latter to regenerate elongated cells of normal dimensions. Thus, the cambium affords an unusually favorable medium for the study of a number of fundamental physiological and cytological problems. 2. A study of the cambium reveals the fact that there is a very-much greater variability in the size of meristematic cells in plants than was suspected by Sachs or Strasburger, and that the working sphere of the nucleus is by no means so restricted as assumed by these investigators. 3. Although the larger cambial initials of Pinus strobus tend to have larger nuclei, the nucleocytoplasmic-relation varies within wide limits and the diploid number of chromosomes is constant. The conditions in the cambium do not support Winkler's view that there is a close correlation between chromosomal number (chromosomal mass) and cell size in the somatic tissue of plants, and that giant cells are hyperchromatic. 4. The process of cell plate formation in the cambium is a remarkable phenomenon, and one which is significant in discussing the relative merits of various theories concerning the dynamics of karyokinesis and cytokinesis. 5. The newly formed partition membranes in the cambial initials frequently intersect the side walls at angles of varying degrees of acuteness, which is in contradiction to Errera's (Plateau's) Law of Minimal Area.
PMCID: PMC2140393  PMID: 19871827
20.  Season-associated modifications in symplasmic organization of the cambium in Populus nigra 
Annals of Botany  2010;105(3):375-387.
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.
Methods
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.
Key Results
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.
Conclusions
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.
doi:10.1093/aob/mcp300
PMCID: PMC2826250  PMID: 20045870
Cambium; meristem initials; plasmodesmata; Populus nigra ‘italica’; seasonal conditions; ultrastructure
21.  A functional–structural model for radiata pine (Pinus radiata) focusing on tree architecture and wood quality 
Annals of Botany  2011;108(6):1155-1178.
Backgrounds and Aims
Functional–structural models are interesting tools to relate environmental and management conditions with forest growth. Their three-dimensional images can reveal important characteristics of wood used for industrial products. Like virtual laboratories, they can be used to evaluate relationships among species, sites and management, and to support silvicultural design and decision processes. Our aim was to develop a functional–structural model for radiata pine (Pinus radiata) given its economic importance in many countries.
Methods
The plant model uses the L-system language. The structure of the model is based on operational units, which obey particular rules, and execute photosynthesis, respiration and morphogenesis, according to their particular characteristics. Plant allometry is adhered to so that harmonic growth and plant development are achieved. Environmental signals for morphogenesis are used. Dynamic turnover guides the normal evolution of the tree. Monthly steps allow for detailed information of wood characteristics. The model is independent of traditional forest inventory relationships and is conceived as a mechanistic model. For model parameterization, three databases which generated new information relating to P. radiata were analysed and incorporated.
Key Results
Simulations under different and contrasting environmental and management conditions were run and statistically tested. The model was validated against forest inventory data for the same sites and times and against true crown architectural data. The performance of the model for 6-year-old trees was encouraging. Total height, diameter and lengths of growth units were adequately estimated. Branch diameters were slightly overestimated. Wood density values were not satisfactory, but the cyclical pattern and increase of growth rings were reasonably well modelled.
Conclusions
The model was able to reproduce the development and growth of the species based on mechanistic formulations. It may be valuable in assessing stand behaviour under different environmental and management conditions, assisting in decision-making with regard to management, and as a research tool to formulate hypothesis regarding forest tree growth and development.
doi:10.1093/aob/mcr156
PMCID: PMC3189843  PMID: 21987452
Functional–structural plant model; wood quality; internodes; knots; wood density; growth ring; photosynthesis; respiration; allometry; plant architecture; carbon allocation; Pinus radiata
22.  Integrated Analysis of Tropical Trees Growth: A Multivariate Approach 
Annals of Botany  2006;98(3):637-645.
• Background and Aims One of the problems analysing cause–effect relationships of growth and environmental factors is that a single factor could be correlated with other ones directly influencing growth. One attempt to understand tropical trees' growth cause–effect relationships is integrating research about anatomical, physiological and environmental factors that influence growth in order to develop mathematical models. The relevance is to understand the nature of the process of growth and to model this as a function of the environment.
• Methods The relationships of Aphananthe monoica, Pleuranthodendron lindenii and Psychotria costivenia radial growth and phenology with environmental factors (local climate, vertical strata microclimate and physical and chemical soil variables) were evaluated from April 2000 to September 2001. The association among these groups of variables was determined by generalized canonical correlation analysis (GCCA), which considers the probable associations of three or more data groups and the selection of the most important variables for each data group.
• Key Results The GCCA allowed determination of a general model of relationships among tree phenology and radial growth with climate, microclimate and soil factors. A strong influence of climate in phenology and radial growth existed. Leaf initiation and cambial activity periods were associated with maximum temperature and day length, and vascular tissue differentiation with soil moisture and rainfall. The analyses of individual species detected different relationships for the three species.
• Conclusions The analyses of the individual species suggest that each one takes advantage in a different way of the environment in which they are growing, allowing them to coexist.
doi:10.1093/aob/mcl142
PMCID: PMC2803565  PMID: 16822807
Phenology; radial growth; climatic periodicity; microclimate; tropical trees; multivariate analysis; Aphananthe monoica; Pleuranthodendron lindenii; Psychotria costivenia
23.  Genome-wide transcriptome analysis of the transition from primary to secondary stem development in Populus trichocarpa 
BMC Genomics  2010;11:150.
Background
With its genome sequence and other experimental attributes, Populus trichocarpa has become the model species for genomic studies of wood development. Wood is derived from secondary growth of tree stems, and begins with the development of a ring of vascular cambium in the young developing stem. The terminal region of the developing shoot provides a steep developmental gradient from primary to secondary growth that facilitates identification of genes that play specialized functions during each of these phases of growth.
Results
Using a genomic microarray representing the majority of the transcriptome, we profiled gene expression in stem segments that spanned primary to secondary growth. We found 3,016 genes that were differentially expressed during stem development (Q-value ≤ 0.05; >2-fold expression variation), and 15% of these genes encode proteins with no significant identities to known genes. We identified all gene family members putatively involved in secondary growth for carbohydrate active enzymes, tubulins, actins, actin depolymerizing factors, fasciclin-like AGPs, and vascular development-associated transcription factors. Almost 70% of expressed transcription factors were upregulated during the transition to secondary growth. The primary shoot elongation region of the stem contained specific carbohydrate active enzyme and expansin family members that are likely to function in primary cell wall synthesis and modification. Genes involved in plant defense and protective functions were also dominant in the primary growth region.
Conclusion
Our results describe the global patterns of gene expression that occur during the transition from primary to secondary stem growth. We were able to identify three major patterns of gene expression and over-represented gene ontology categories during stem development. The new regulatory factors and cell wall biogenesis genes that we identified provide candidate genes for further functional characterization, as well as new tools for molecular breeding and biotechnology aimed at improvement of tree growth rate, crown form, and wood quality.
doi:10.1186/1471-2164-11-150
PMCID: PMC2846914  PMID: 20199690
24.  A Comparative Computer Simulation of Dendritic Morphology 
PLoS Computational Biology  2008;4(6):e1000089.
Computational modeling of neuronal morphology is a powerful tool for understanding developmental processes and structure-function relationships. We present a multifaceted approach based on stochastic sampling of morphological measures from digital reconstructions of real cells. We examined how dendritic elongation, branching, and taper are controlled by three morphometric determinants: Branch Order, Radius, and Path Distance from the soma. Virtual dendrites were simulated starting from 3,715 neuronal trees reconstructed in 16 different laboratories, including morphological classes as diverse as spinal motoneurons and dentate granule cells. Several emergent morphometrics were used to compare real and virtual trees. Relating model parameters to Branch Order best constrained the number of terminations for most morphological classes, except pyramidal cell apical trees, which were better described by a dependence on Path Distance. In contrast, bifurcation asymmetry was best constrained by Radius for apical, but Path Distance for basal trees. All determinants showed similar performance in capturing total surface area, while surface area asymmetry was best determined by Path Distance. Grouping by other characteristics, such as size, asymmetry, arborizations, or animal species, showed smaller differences than observed between apical and basal, pointing to the biological importance of this separation. Hybrid models using combinations of the determinants confirmed these trends and allowed a detailed characterization of morphological relations. The differential findings between morphological groups suggest different underlying developmental mechanisms. By comparing the effects of several morphometric determinants on the simulation of different neuronal classes, this approach sheds light on possible growth mechanism variations responsible for the observed neuronal diversity.
Author Summary
Neurons in the brain have a variety of complex arbor shapes that help determine both their interconnectivity and functional roles. Molecular biology is beginning to uncover important details on the development of these tree-like structures, but how and why vastly different shapes arise is still largely unknown. We developed a novel set of computer models of branching in which measurements of real nerve cell structures digitally traced from microscopic imaging are resampled to create virtual trees. The different rules that the models use to create the most similar virtual trees to the real data support specific hypotheses regarding development. Surprisingly, the arborizations that differed most in the optimal rules were found on opposite sides of the same type of neuron, namely apical and basal trees of pyramidal cells. The details of the rules suggest that pyramidal cell trees may respond in unique and complex ways to their external environment. By better understanding how these trees are formed in the brain, we can learn more about their normal function and why they are often malformed in neurological diseases.
doi:10.1371/journal.pcbi.1000089
PMCID: PMC2376061  PMID: 18483611
25.  Chemical combination effects predict connectivity in biological systems 
Chemical synergies can be novel probes of biological systems.Simulated response shapes depend on target connectivity in a pathway.Experiments with yeast and cancer cells confirm simulated effects.Profiles across many combinations yield target location information.
Living organisms are built of interacting components, whose function and dysfunction can be described through dynamic network models (Davidson et al, 2002). Systems Biology involves the iterative construction of such models (Ideker et al, 2001), and may eventually improve the understanding of diseases using in silico simulations. Such simulations may eventually permit drugs to be prioritized for clinical trials, reducing potential risks and increasing the likelihood of successful outcomes. Given the complexity of biological systems, constructing realistic models will require large and diverse sets of connectivity data.
Chemical combinations provide a new window into biological connectivity. Information gleaned from targeted combinations, such as paired mutations (Tong et al, 2004), has proven to be especially useful for revealing functional interactions between components. We have been screening chemical combinations for therapeutic synergies (Borisy et al, 2003; Zimmermann et al, 2007), collecting full-dose matrices where combinations are tested in all possible pairings of serially diluted single agent doses (Figure 1). Such screens yield a variety of response surfaces with distinct shapes for combinations that work through different known mechanisms, suggesting that combination effects may contain information on the nature of functional connections between drug targets.
Simulations of biological pathways predict synergistic responses to inhibitors that depend on target connectivity. We explored theoretical predictions by simulating a metabolic pathway with pairs of inhibitors aimed at different targets with varying doses. We found that the shape of each combination response depended on how the inhibitor pair's targets were connected in the pathway (Figure 2). The predicted response shapes were robust to plausible variations in the simulated pathway that did not affect the network topology (e.g., kinetic assumptions, parameter values, and nonlinear response functions), but were very sensitive to topological alterations in the modelled network (e.g., feedback regulation or changing the type of junction at a branch point). These findings suggest that connectivity of the inhibitor targets has a major influence on combination response morphology.
The predicted shapes were experimentally confirmed in yeast combination experiments. The proliferation experiment used drugs focused on the sterol biosynthesis pathway, which is mostly linear between the targets covered in this study, and is known to be regulated by negative feedback (Gardner et al, 2001). The combinations between sterol inhibitors confirmed expectations from our simulations, showing dose-additive responses for pairs targeting the same enzyme and strong synergies across enzymes of the shape predicted in our simulations for linear pathways under negative feedback. Combinations across pathways showed much more variable responses with a trend towards less synergy on average.
Further experimental support was obtained from human cells. A combination screen of 90 annotated drugs in a human tumour cell line (HCT116) proliferation assay produced strong synergies for combinations within pathways and more variable effects between targeted functions. Synergy profiles (sets of all synergy scores involving each drug) also showed a greater degree of similarity for pairs of drugs with related targets. Finally, the most extreme outliers were dominated by inhibitors of kinases that are especially critical for HCT116 proliferation (Awwad et al, 2003), with effects that are consistent across mechanistic replicates, showing that chemical combinations can highlight biologically relevant cellular processes.
This study demonstrates the potential of chemical combinations for exploring functional connectivity in biological systems. This information complements genetic studies by providing more details through variable dosing, by directly targeting single domains of multi-domain proteins, and by probing cell types that are not amenable to mutagenesis. Responses from large chemical combination screens can be used to identify molecular targets through chemical–genetic profiling (Macdonald et al, 2006), or to directly constrain network models by means of a prediction-validation procedure (Ideker et al, 2001). This initial exploration can be extended to cover a wider range of response shapes and network topologies, as well as to combinations of three or more chemical agents. Moreover, this approach may even be applicable to non-biological systems where responses to targeted perturbations can be measured.
Efforts to construct therapeutically useful models of biological systems require large and diverse sets of data on functional connections between their components. Here we show that cellular responses to combinations of chemicals reveal how their biological targets are connected. Simulations of pathways with pairs of inhibitors at varying doses predict distinct response surface shapes that are reproduced in a yeast experiment, with further support from a larger screen using human tumour cells. The response morphology yields detailed connectivity constraints between nearby targets, and synergy profiles across many combinations show relatedness between targets in the whole network. Constraints from chemical combinations complement genetic studies, because they probe different cellular components and can be applied to disease models that are not amenable to mutagenesis. Chemical probes also offer increased flexibility, as they can be continuously dosed, temporally controlled, and readily combined. After extending this initial study to cover a wider range of combination effects and pathway topologies, chemical combinations may be used to refine network models or to identify novel targets. This response surface methodology may even apply to non-biological systems where responses to targeted perturbations can be measured.
doi:10.1038/msb4100116
PMCID: PMC1828746  PMID: 17332758
chemical genetics; combinations and synergy; metabolic and regulatory networks; simulation and data analysis

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