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1.  Computing Competition for Light in the GREENLAB Model of Plant Growth: A Contribution to the Study of the Effects of Density on Resource Acquisition and Architectural Development 
Annals of Botany  2007;101(8):1207-1219.
Background and Aims
The dynamical system of plant growth GREENLAB was originally developed for individual plants, without explicitly taking into account interplant competition for light. Inspired by the competition models developed in the context of forest science for mono-specific stands, we propose to adapt the method of crown projection onto the x–y plane to GREENLAB, in order to study the effects of density on resource acquisition and on architectural development.
The empirical production equation of GREENLAB is extrapolated to stands by computing the exposed photosynthetic foliage area of each plant. The computation is based on the combination of Poisson models of leaf distribution for all the neighbouring plants whose crown projection surfaces overlap. To study the effects of density on architectural development, we link the proposed competition model to the model of interaction between functional growth and structural development introduced by Mathieu (2006, PhD Thesis, Ecole Centrale de Paris, France).
Key Results and Conclusions
The model is applied to mono-specific field crops and forest stands. For high-density crops at full cover, the model is shown to be equivalent to the classical equation of field crop production ( Howell and Musick, 1985, in Les besoins en eau des cultures; Paris: INRA Editions). However, our method is more accurate at the early stages of growth (before cover) or in the case of intermediate densities. It may potentially account for local effects, such as uneven spacing, variation in the time of plant emergence or variation in seed biomass. The application of the model to trees illustrates the expression of plant plasticity in response to competition for light. Density strongly impacts on tree architectural development through interactions with the source–sink balances during growth. The effects of density on tree height and radial growth that are commonly observed in real stands appear as emerging properties of the model.
PMCID: PMC2710279  PMID: 18037666
Functional–structural plant models; GREENLAB; competition for light; Beer–Lambert Law; plant plasticity; dynamical system
2.  Models for Forest Ecosystem Management: A European Perspective 
Annals of Botany  2007;101(8):1065-1087.
Forest management in Europe is committed to sustainability. In the face of climate change and accompanying risks, however, planning in order to achieve this aim becomes increasingly challenging, underlining the need for new and innovative methods. Models potentially integrate a wide range of system knowledge and present scenarios of variables important for any management decision. In the past, however, model development has mainly focused on specific purposes whereas today we are increasingly aware of the need for the whole range of information that can be provided by models. It is therefore assumed helpful to review the various approaches that are available for specific tasks and to discuss how they can be used for future management strategies.
Here we develop a concept for the role of models in forest ecosystem management based on historical analyses. Five paradigms of forest management are identified: (1) multiple uses, (2) dominant use, (3) environmentally sensitive multiple uses, (4) full ecosystem approach and (5) eco-regional perspective. An overview of model approaches is given that is dedicated to this purpose and to developments of different kinds of approaches. It is discussed how these models can contribute to goal setting, decision support and development of guidelines for forestry operations. Furthermore, it is shown how scenario analysis, including stand and landscape visualization, can be used to depict alternatives, make long-term consequences of different options transparent, and ease participation of different stakeholder groups and education.
In our opinion, the current challenge of forest ecosystem management in Europe is to integrate system knowledge from different temporal and spatial scales and from various disciplines. For this purpose, using a set of models with different focus that can be selected from a kind of toolbox according to particular needs is more promising than developing one overarching model, covering ecological, production and landscape issues equally well.
PMCID: PMC2710278  PMID: 17954471
Ecosystem management; management paradigms; decision support in Europe; sustainability; models; spatial and temporal scales; scaling; scenario generation; visualization
3.  Understanding the Impact of Root Morphology on Overturning Mechanisms: A Modelling Approach 
Annals of Botany  2007;101(8):1267-1280.
Background and Aims
The Finite Element Method (FEM) has been used in recent years to simulate overturning processes in trees. This study aimed at using FEM to determine the role of individual roots in tree anchorage with regard to different rooting patterns, and to estimate stress distribution in the soil and roots during overturning.
The FEM was used to carry out 2-D simulations of tree uprooting in saturated soft clay and loamy sand-like soil. The anchorage model consisted of a root system embedded in a soil block. Two root patterns were used and individual roots removed to determine their contribution to anchorage.
Key Results
In clay-like soil the size of the root–soil plate formed during overturning was defined by the longest roots. Consequently, all other roots localized within this plate had no influence on anchorage strength. In sand-like soil, removing individual root elements altered anchorage resistance. This result was due to a modification of the shape and size of the root–soil plate, as well as the location of the rotation axis. The tap root and deeper roots had more influence on overturning resistance in sand-like soil compared with clay-like soil. Mechanical stresses were higher in the most superficial roots and also in leeward roots in sand-like soil. The relative difference in stresses between the upper and lower sides of lateral roots was sensitive to root insertion angle. Assuming that root eccentricity is a response to mechanical stresses, these results explain why eccentricity differs depending on root architecture.
A simple 2-D Finite Element model was developed to better understand the mechanisms involved during tree overturning. It has been shown how root system morphology and soil mechanical properties can modify the shape of the root plate slip surface as well as the position of the rotation axis, which are major components of tree anchorage.
PMCID: PMC2710277  PMID: 17942593
Acclimative growth; anchorage; biomechanics; tree uprooting; rotation axis; root architecture; root eccentricity; secondary growth; von Mises stresses
4.  A System for Modelling Cell–Cell Interactions during Plant Morphogenesis 
Annals of Botany  2007;101(8):1255-1265.
Background and aims
During the development of multicellular organisms, cells are capable of interacting with each other through a range of biological and physical mechanisms. A description of these networks of cell–cell interactions is essential for an understanding of how cellular activity is co-ordinated in regionalized functional entities such as tissues or organs. The difficulty of experimenting on living tissues has been a major limitation to describing such systems, and computer modelling appears particularly helpful to characterize the behaviour of multicellular systems. The experimental difficulties inherent to the multitude of parallel interactions that underlie cellular morphogenesis have led to the need for computer models.
A new generic model of plant cellular morphogenesis is described that expresses interactions amongst cellular entities explicitly: the plant is described as a multi-scale structure, and interactions between distinct entities is established through a topological neighbourhood. Tissues are represented as 2D biphasic systems where the cell wall responds to turgor pressure through a viscous yielding of the cell wall.
Key Results
This principle was used in the development of the CellModeller software, a generic tool dedicated to the analysis and modelling of plant morphogenesis. The system was applied to three contrasting study cases illustrating genetic, hormonal and mechanical factors involved in plant morphogenesis.
Plant morphogenesis is fundamentally a cellular process and the CellModeller software, through its underlying generic model, provides an advanced research tool to analyse coupled physical and biological morphogenetic mechanisms.
PMCID: PMC2710276  PMID: 17921524
Pattern formation; dynamic model; CellModeller; multicellular systems; morphogenesis; cell–cell interaction
5.  Parameter Optimization and Field Validation of the Functional–Structural Model GREENLAB for Maize at Different Population Densities 
Annals of Botany  2007;101(8):1185-1194.
Background and Aims
Plant population density (PPD) influences plant growth greatly. Functional–structural plant models such as GREENLAB can be used to simulate plant development and growth and PPD effects on plant functioning and architectural behaviour can be investigated. This study aims to evaluate the ability of GREENLAB to predict maize growth and development at different PPDs.
Two field experiments were conducted on irrigated fields in the North China Plain with a block design of four replications. Each experiment included three PPDs: 2·8, 5·6 and 11·1 plants m−2. Detailed observations were made on the dimensions and fresh biomass of above-ground plant organs for each phytomer throughout the seasons. Growth stage-specific target files (a description of plant organ weight and dimension according to plant topological structure) were established from the measured data required for GREENLAB parameterization. Parameter optimization was conducted using a generalized least square method for the entire growth cycles for all PPDs and years. Data from in situ plant digitization were used to establish geometrical symbol files for organs that were then applied to translate model output directly into 3-D representation for each time step of the model execution.
Key Results
The analysis indicated that the parameter values of organ sink variation function, and the values of most of the relative sink strength parameters varied little among years and PPDs, but the biomass production parameter, computed plant projection surface and internode relative sink strength varied with PPD. Simulations of maize plant growth based on the fitted parameters were reasonably good as indicated by the linearity and slopes similar to unity for the comparison of simulated and observed values. Based on the parameter values fitted from different PPDs, shoot (including vegetative and reproductive parts of the plant) and cob fresh biomass for other PPDs were simulated. Three-dimensional representation of individual plant and plant stand from the model output with two contrasting PPDs were presented with which the PPD effect on plant growth can be easily recognized.
This study showed that GREENLAB model has the ability to capture plant plasticity induced by PPD. The relatively stable parameter values strengthened the hypothesis that one set of equations can govern dynamic organ growth. With further validation, this model can be used for agronomic applications such as yield optimization.
PMCID: PMC2710275  PMID: 17921525
Functional–structural plant model; GREENLAB; plant architecture; source–sink relationship; plant population density; maize (Zea mays); model parameterization
6.  The Derivation of Sink Functions of Wheat Organs using the GREENLAB Model 
Annals of Botany  2007;101(8):1099-1108.
Background and Aims
In traditional crop growth models assimilate production and partitioning are described with empirical equations. In the GREENLAB functional–structural model, however, allocation of carbon to different kinds of organs depends on the number and relative sink strengths of growing organs present in the crop architecture. The aim of this study is to generate sink functions of wheat (Triticum aestivum) organs by calibrating the GREENLAB model using a dedicated data set, consisting of time series on the mass of individual organs (the ‘target data’).
An experiment was conducted on spring wheat (Triticum aestivum, ‘Minaret’), in a growth chamber from, 2004 to, 2005. Four harvests were made of six plants each to determine the size and mass of individual organs, including the root system, leaf blades, sheaths, internodes and ears of the main stem and different tillers. Leaf status (appearance, expansion, maturity and death) of these 24 plants was recorded. With the structures and mass of organs of four individual sample plants, the GREENLAB model was calibrated using a non-linear least-square-root fitting method, the aim of which was to minimize the difference in mass of the organs between measured data and model output, and to provide the parameter values of the model (the sink strengths of organs of each type, age and tiller order, and two empirical parameters linked to biomass production).
Key Results and Conclusions
The masses of all measured organs from one plant from each harvest were fitted simultaneously. With estimated parameters for sink and source functions, the model predicted the mass and size of individual organs at each position of the wheat structure in a mechanistic way. In addition, there was close agreement between experimentally observed and simulated values of leaf area index.
PMCID: PMC2710274  PMID: 18045794
Wheat; Triticum aestivum ‘Minaret’; tiller; GREENLAB; organ mass; functional–structural model; model calibration; multi-fitting; source–sink
7.  Using Three-dimensional Plant Root Architecture in Models of Shallow-slope Stability 
Annals of Botany  2007;101(8):1281-1293.
The contribution of vegetation to shallow-slope stability is of major importance in landslide-prone regions. However, existing slope stability models use only limited plant root architectural parameters. This study aims to provide a chain of tools useful for determining the contribution of tree roots to soil reinforcement.
Three-dimensional digitizing in situ was used to obtain accurate root system architecture data for mature Quercus alba in two forest stands. These data were used as input to tools developed, which analyse the spatial position of roots, topology and geometry. The contribution of roots to soil reinforcement was determined by calculating additional soil cohesion using the limit equilibrium model, and the factor of safety (FOS) using an existing slope stability model, Slip4Ex.
Key Results
Existing models may incorrectly estimate the additional soil cohesion provided by roots, as the spatial position of roots crossing the potential slip surface is usually not taken into account. However, most soil reinforcement by roots occurs close to the tree stem and is negligible at a distance >1·0 m from the tree, and therefore global values of FOS for a slope do not take into account local slippage along the slope.
Within a forest stand on a landslide-prone slope, soil fixation by roots can be minimal between uniform rows of trees, leading to local soil slippage. Therefore, staggered rows of trees would improve overall slope stability, as trees would arrest the downward movement of soil. The chain of tools consisting of both software (free for non-commercial use) and functions available from the first author will enable a more accurate description and use of root architectural parameters in standard slope stability analyses.
PMCID: PMC2710273  PMID: 17766845
Landslide; root area ratio; slope stability; 3D digitizing; Quercus alba; soil cohesion; soil internal friction angle
8.  AmapSim: A Structural Whole-plant Simulator Based on Botanical Knowledge and Designed to Host External Functional Models 
Annals of Botany  2007;101(8):1125-1138.
Background and Aims
AmapSim is a tool that implements a structural plant growth model based on a botanical theory and simulates plant morphogenesis to produce accurate, complex and detailed plant architectures. This software is the result of more than a decade of research and development devoted to plant architecture. New advances in the software development have yielded plug-in external functions that open up the simulator to functional processes.
The simulation of plant topology is based on the growth of a set of virtual buds whose activity is modelled using stochastic processes. The geometry of the resulting axes is modelled by simple descriptive functions. The potential growth of each bud is represented by means of a numerical value called physiological age, which controls the value for each parameter in the model. The set of possible values for physiological ages is called the reference axis. In order to mimic morphological and architectural metamorphosis, the value allocated for the physiological age of buds evolves along this reference axis according to an oriented finite state automaton whose occupation and transition law follows a semi-Markovian function.
Key Results
Simulations were performed on tomato plants to demostrate how the AmapSim simulator can interface external modules, e.g. a GREENLAB growth model and a radiosity model.
The algorithmic ability provided by AmapSim, e.g. the reference axis, enables unified control to be exercised over plant development parameter values, depending on the biological process target: how to affect the local pertinent process, i.e. the pertinent parameter(s), while keeping the rest unchanged. This opening up to external functions also offers a broadened field of applications and thus allows feedback between plant growth and the physical environment.
PMCID: PMC2710271  PMID: 17766310
Simulation software; physiological age; reference axis; FSPM; plant growth modelling; plant architecture
9.  Exploring Ecological Significance of Tree Crown Plasticity through Three-dimensional Modelling 
Annals of Botany  2007;101(8):1221-1231.
Background and Aims
Morphogenetic plasticity may be as important as physiological plasticity in determining plant adaptability to changing environmental conditions. This study examines the importance of crown plasticity of trees in stands.
A three-dimensional forest simulator is used to explore the impact of crown shape plasticity on tree growth. Crown deformation is mediated through the local response to light and overall allometric constraints governing tree dimensions. By altering shape response parameters of Hevea brasiliensis the impact of increased or decreased plasticity is explored in a variety of competitive environments defined by various combinations of tree density and relative frequency of different strategies. The possible interactions between plasticity and growth rate and plasticity and below-ground competition are also explored.
Key Results
Crown plasticity confers competitive superiority in all cases studied. Interactions with other processes may downplay or enhance this competitive advantage.
Simulation results strongly suggest that crown plasticity does have a significant impact on tree performance in nature and that commonly observed crown shape deformation response of trees is of adaptive value.
PMCID: PMC2710270  PMID: 17720977
Crown plasticity; 3D simulation; individual-based model; competition
10.  A Rule-based Model of Barley Morphogenesis, with Special Respect to Shading and Gibberellic Acid Signal Transduction 
Annals of Botany  2007;101(8):1109-1123.
Background and Aims
Functional–structural plant models (FSPM) constitute a paradigm in plant modelling that combines 3D structural and graphical modelling with the simulation of plant processes. While structural aspects of plant development could so far be represented using rule-based formalisms such as Lindenmayer systems, process models were traditionally written using a procedural code. The faithful representation of structures interacting with functions across scales, however, requires a new modelling formalism. Therefore relational growth grammars (RGG) were developed on the basis of Lindenmayer systems.
In order to implement and test RGG, a new modelling language, the eXtended L-system language (XL) was created. Models using XL are interpreted by the interactive, Java-based modelling platform GroIMP. Three models, a semi-quantitative gibberellic acid (GA) signal transduction model, and a phytochrome-based shade detection and object avoidance model, both coupled to an existing morphogenetic structural model of barley (Hordeum vulgare L.), serve as examples to demonstrate the versatility and suitability of RGG and XL to represent the interaction of diverse biological processes across hierarchical scales.
Key Results
The dynamics of the concentrations in the signal transduction network could be modelled qualitatively and the phenotypes of GA-response mutants faithfully reproduced. The light model used here was simple to use yet effective enough to carry out local measurement of red:far-red ratios. Suppression of tillering at low red:far-red ratios could be simulated.
The RGG formalism is suitable for implementation of multi-scaled FSPM of plants interacting with their environment via hormonal control. However, their ensuing complexity requires careful design. On the positive side, such an FSPM displays knowledge gaps better thereby guiding future experimental design.
PMCID: PMC2710269  PMID: 17766311
Barley; Hordeum vulgare L.; functional-structural plant model (FSPM); extended L-System language; relational growth grammars; morphogenesis; gibberellic acid; plant hormone; signal transduction; shade detection; object avoidance; computer graphics
11.  Rhythms and Alternating Patterns in Plants as Emergent Properties of a Model of Interaction between Development and Functioning 
Annals of Botany  2007;101(8):1233-1242.
Background and Aims
To model plasticity of plants in their environment, a new version of the functional–structural model GREENLAB has been developed with full interactions between architecture and functioning. Emergent properties of this model were revealed by simulations, in particular the automatic generation of rhythms in plant development. Such behaviour can be observed in natural phenomena such as the appearance of fruit (cucumber or capsicum plants, for example) or branch formation in trees.
In the model, a single variable, the source–sink ratio controls different events in plant architecture. In particular, the number of fruits and branch formation are determined as increasing functions of this ratio. For some sets of well-chosen parameters of the model, the dynamical evolution of the ratio during plant growth generates rhythms.
Key Results and Conclusions
Cyclic patterns in branch formation or fruit appearance emerge without being forced by the model. The model is based on the theory of discrete dynamical systems. The mathematical formalism helps us to explain rhythm generation and to control the behaviour of the system. Rhythms can appear during both the exponential and stabilized phases of growth, but the causes are different as shown by an analytical study of the system. Simulated plant behaviours are very close to those observed on real plants. With a small number of parameters, the model gives very interesting results from a qualitative point of view. It will soon be subjected to experimental data to estimate the model parameters.
PMCID: PMC2710268  PMID: 17715304
Rhythms; plasticity; plant growth model; GREENLAB; interactions; branching system; fructification; emergent properties
12.  A Dynamical Model of Environmental Effects on Allocation to Carbon-based Secondary Compounds in Juvenile Trees 
Annals of Botany  2007;101(8):1089-1098.
Background and Aims
Patterns and variations in concentration of carbon-based secondary compounds in plant tissues have been explained by means of different complementary and, in some cases, contradictory plant defence hypotheses for more than 20 years. These hypotheses are conceptual models which consider environmental impacts on plant internal demands. In the present study, a mathematical model is presented, which converts and integrates the concepts of the ‘Growth–Differentiation Balance’ hypothesis and the ‘Protein Competition’ model into a dynamic plant growth model, that was tested with concentration data of polyphenols in leaves of juvenile apple, beech and spruce trees. The modelling approach is part of the plant growth model PLATHO that considers simultaneously different environmental impacts on the most important physiological processes of plants.
The modelling approach for plant internal resource allocation is based on a priority scheme assuming that growth processes have priority over allocation to secondary compounds and that growth-related metabolism is more strongly affected by nitrogen deficiency than defence-related secondary metabolism.
Key Results
It is shown that the model can reproduce the effect of nitrogen fertilization on allocation patterns in apple trees and the effects of elevated CO2 and competition in juvenile beech and spruce trees. The analysis of model behaviour reveals that large fluctuations in plant internal availability of carbon and nitrogen are possible within a single vegetation period. Furthermore, the model displays a non-linear allocation behaviour to carbon-based secondary compounds.
The simulation results corroborate the underlying assumptions of the presented modelling approach for resource partitioning between growth-related primary metabolism and defence-related secondary metabolism. Thus, the dynamical modelling approach, which considers variable source and sink strengths of plant internal resources within different phenological growth stages, presents a successful translation of existing concepts into a dynamic mathematical model.
PMCID: PMC2710266  PMID: 17693454
Plant growth; carbon-based secondary compounds; plant defence hypotheses; simulation model; phenolic allocation; nitrogen; carbon dioxide; Malus domestica; Fagus sylvatica; Picea abies
13.  Quantitative Genetics and Functional–Structural Plant Growth Models: Simulation of Quantitative Trait Loci Detection for Model Parameters and Application to Potential Yield Optimization 
Annals of Botany  2007;101(8):1243-1254.
Background and Aims
Prediction of phenotypic traits from new genotypes under untested environmental conditions is crucial to build simulations of breeding strategies to improve target traits. Although the plant response to environmental stresses is characterized by both architectural and functional plasticity, recent attempts to integrate biological knowledge into genetics models have mainly concerned specific physiological processes or crop models without architecture, and thus may prove limited when studying genotype × environment interactions. Consequently, this paper presents a simulation study introducing genetics into a functional–structural growth model, which gives access to more fundamental traits for quantitative trait loci (QTL) detection and thus to promising tools for yield optimization.
The GREENLAB model was selected as a reasonable choice to link growth model parameters to QTL. Virtual genes and virtual chromosomes were defined to build a simple genetic model that drove the settings of the species-specific parameters of the model. The QTL Cartographer software was used to study QTL detection of simulated plant traits. A genetic algorithm was implemented to define the ideotype for yield maximization based on the model parameters and the associated allelic combination.
Key Results and Conclusions
By keeping the environmental factors constant and using a virtual population with a large number of individuals generated by a Mendelian genetic model, results for an ideal case could be simulated. Virtual QTL detection was compared in the case of phenotypic traits – such as cob weight – and when traits were model parameters, and was found to be more accurate in the latter case. The practical interest of this approach is illustrated by calculating the parameters (and the corresponding genotype) associated with yield optimization of a GREENLAB maize model. The paper discusses the potentials of GREENLAB to represent environment × genotype interactions, in particular through its main state variable, the ratio of biomass supply over demand.
PMCID: PMC2710265  PMID: 17766844
Plant growth model; GREENLAB; genetics; QTL; breeding; yield optimization; genetic algorithm; Zea mays
14.  A Century of B Chromosomes in Plants: So What? 
Annals of Botany  2007;101(6):767-775.
Supernumerary B chromosomes (Bs) are a major source of intraspecific variation in nuclear DNA amounts in numerous species of plants. They favour large genomes, and create polymorphisms for DNA variation in natural populations. By studying Bs we can gain useful knowledge about the organization, function and evolution of genomes. There are also significant biological questions concerning the origin and structural organization of Bs, and the way in which these selfish elements can establish themselves by exploiting the replicative machinery of their host genome nucleus.
It is a sine qua non that Bs originate from the A chromosomes, in a variety of ways. We can study their modes of drive and ask how it is that chromosomes which apparently lack genes can have control over their own drive process which leads to their survival in natural populations. Molecular cytogenetic studies are opening up new avenues of investigation. Population equilibria for B frequencies are determined by a balance between accumulation and harmful effects. Bs are also subject to meiotic loss due to polysomy and to elimination at meiosis as univalents. These balancing forces can be seen in the context of host/parasite interaction, based on a dissection of the genetic elements in both As and Bs (in maize) which interact to bring about a stable equilibrium, at least for a snapshot in time.
Aside from their intrinsic enigmatic properties, B chromosomes make useful experimental tools to study genome organization. Thus far they have not been exploited for their applications, other than through the use of A-B translocations used for gene mapping in maize; but there are opportunities to use them to modulate the frequency and distribution of recombination, to diploidize allopolyploids, to study centromeres and to be developed as plant artificial chromosomes; given that they can be structurally modified and their inheritance stabilized.
PMCID: PMC2710218  PMID: 17704238
B chromosomes; DNA polymorphisms; host/parasite interaction; mitotic/meiotic drive; applications; genome organization/evolution; centromeres
15.  Anthocyanin Inhibits Propidium Iodide DNA Fluorescence in Euphorbia pulcherrima: Implications for Genome Size Variation and Flow Cytometry 
Annals of Botany  2007;101(6):777-790.
Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics.
DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia).
Key Results
There were large differences in PI staining (35–70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2·8–6·9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1·69–1·76 pg) than green leaves (1·81 pg). Chopping pea or poinsettia tissue in buffer with 0–200 µm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin.
Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or correlated with environmental factors known to induce variation in pigmentation.
PMCID: PMC2710214  PMID: 18158306
Anthocyanin; cyanidin-3-rutinoside; DNA staining inhibitors; Euphorbia pulcherrima; flow cytometry; genome size artefacts; nuclear DNA amount; genome plasticity
16.  Biosystematics and Conservation: A Case Study with Two Enigmatic and Uncommon Species of Crassula from New Zealand 
Annals of Botany  2007;101(6):881-899.
Background and Aims
Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their distinctiveness is assessed so that their taxonomic and conservation status can be clarified.
Populations of these two species were analysed using morphological, chromosomal and DNA sequence data.
Key Results
It proved impossible to differentiate between these two species using 12 key morphological characters. Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n = 42 to 2n = 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showed that differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from the same population.
The results suggest that these plants are a species complex that has evolved through interspecific hybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problem for their conservation.
PMCID: PMC2710211  PMID: 18055560
Chromosome variation; Crassula; Crassula hunua; Crassula ruamahanga; Crassulaceae, conservation; phylogenetics; taxonomy; New Zealand flora
17.  Dissecting Meiosis of Rye using Translational Proteomics 
Annals of Botany  2007;101(6):873-880.
Background and Aims
Much of our understanding of the genetic control of meiosis has come from recent studies of model organisms, which have given us valuable insights into processes such as recombination and the synapsis of chromosomes. The challenge now is to determine to what extent these models are representative of other groups of organisms, and to what extent generalisations can be made as to how meiosis works. Through a comparative proteomic approach with Arabidopsis thaliana, this study describes the spatial and temporal expression of key structural and recombinogenic proteins of cereal rye (Secale cereale).
Antibodies to two synaptonemal complex-associated proteins (Asy1 and Zyp1) and two recombination-related proteins (Spo11 and Rad51) of A. thaliana were bound to meiocytes throughout meiotic prophase of rye, and visualized using conventional fluorescence microscopy and confocal laser scanning microscopy. Western analysis was performed on proteins extracted from pooled prophase I anthers, as a prelude to more advanced proteomic investigations.
Key Results
The four antibodies of A. thaliana reliably detected their epitopes in rye. The expression profile of Rad51 is consistent with its role in recombination. Asy1 protein is shown for the first time to cap the ends of bivalents. Western analysis reveals structural variants of the transverse filament protein Zyp1.
Asy1 cores are assembled by elongation of early foci. The persistence of foci of Spo11 to late prophase does not fit the current model of molecular recombination. The putative structural variants of Zyp1 may indicate modification of the protein as bivalents are assembled.
PMCID: PMC2710209  PMID: 17766846
Rye; Secale cereale; meiosis; proteins; immunocytology; western analysis
18.  Cytochemistry and C-values: The Less-well-known World of Nuclear DNA Amounts 
Annals of Botany  2007;101(6):791-804.
In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect.
The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern.
In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.
PMCID: PMC2710206  PMID: 17951594
C-values; Feulgen densitometry; Drosera rotundifolia; flow cytometry; genome size; Pisum sativum; polyphenols; staining inhibitors; standardization; tannins
19.  Cytosolic Nitrate Ion Homeostasis: Could it Have a Role in Sensing Nitrogen Status? 
Annals of Botany  2007;101(4):485-489.
Background and Aims
There is ongoing debate regarding homeostasis of cytosolic nutrient ion concentrations. This deliberation centres on the question of whether homeostasis occurs for some nutrients and, if so, what are the consequences for how plants sense their nutrient status. Particularly for nitrate, this controversy has focused on the methods used and the cellular pools which they measure. Cytoplasm and cytosol have been distinguished and it has been suggested that two ranges of nitrate values can be separated depending on whether the method separates the pools found in organelles.
The present study defines homeostasis of nutrient ions and discusses how whole organ averaging techniques can hide important cellular differences that can help to explain some of the discrepancies between results reported by various methods. These results are considered in relation to a possible role in signalling nutrient status, and have relevance to other averaging techniques such as the use of ‘omics’ technologies.
PMCID: PMC2710192  PMID: 18089584
Hordeum vulgare; homeostasis; cytosolic nitrate; compartmentation; ion-selective microelectrodes
20.  In vivo Visualization of the Water-refilling Process in Xylem Vessels Using X-ray Micro-imaging 
Annals of Botany  2007;101(4):595-602.
Background and Aims
Xylem vessels containing gases (embolized) must be refilled with water if they are to resume transport of water through the plant, so refilling is of great importance for the maintenance of water balance in plants. However, the refilling process is poorly understood because of inadequate examination methods. Simultaneous measurements of plant anatomy and vessel refilling are essential to elucidate the mechanisms involved. In the present work, a new technique based on phase-contrast X-ray imaging is presented that visualizes, in vivo and in real time, both xylem anatomy and refilling of embolized vessels.
With the synchrotron X-ray micro-imaging technique, the refilling of xylem vessels of leaves and a stem of Phyllostachys bambusoides with water is demonstrated under different conditions. The technique employs phase contrast imaging of X-ray beams, which are transformed into visible light and are photographed by a charge coupled device camera. X-ray images were captured consecutively at every 0·5 s with an exposure time of 10 ms.
Key Results
The interface (meniscus) between the water and gas phases in refilling the xylem vessels is displayed. During refilling, the rising menisci in embolized vessels showed repetitive flow, i.e. they temporarily stopped at the end walls of the vessel elements while gas bubbles were removed. The meniscus then passed through the end wall at a faster rate than the speed of flow in the main vessels. In the light, the speed of refilling in a specific vessel was slower than that in the dark, but this rate increased again after repeated periods in darkness.
Real-time, non-destructive X-ray micro-imaging is an important, useful and novel technique to study the relationship between xylem structure and the refilling of embolized vessels in intact plants. It provides new insight into understanding the mechanisms of water transport and the refilling of embolized vessels, which are not understood well.
PMCID: PMC2710191  PMID: 18077466
Micro-imaging; Phyllostachys bambusoides; water refilling; X-ray; xylem vessel
21.  Does Germination Success Differ with Respect to Seed Mass and Germination Season? Experimental Testing of Plant Functional Trait Responses to Grassland Management 
Annals of Botany  2007;101(4):541-548.
Background and Aims
Plant functional trait responses to processes such as grassland management have been analysed frequently; however, the scaling-up from individual traits to the outcomes of vegetation dynamics has seldom been tested. In this experiment, germination success was studied with respect to the relationships between grassland management (mowing and grazing), as well as abandonment, and two traits that are relevant for seedling recruitment: seed mass and germination season. On the basis of discussions in the literature and indirect trait analyses in our previous studies, the following hypotheses are proposed: (1) with respect to seed mass, mowing and grazing favour the germination of small seeds, whereas after abandonment the germination success of larger seeds is higher; and (2) with respect to germination season, mowing and grazing favour autumn-germinating seeds, whereas succession promotes spring-germinating seeds.
The germination experiment took place in a semi-natural, dry grassland in north-east Germany. Seeds of eight herbaceous species that differ with respect to seed mass and germination season were sown in mown, grazed and abandoned plots. Germination success was documented during the following year.
Key Results and Conclusions
Contrary to the hypothesis, germination of small seeds was not promoted by mowing or grazing and they germinated relatively more often than expected in the abandoned plots. A relationship between abandonment and gaps of bare soil below the vegetation cover that favour germination of small seeds was likely, but could not be proved statistically. It is possible that the small seeds suffered less from predation. Mowing favoured autumn germination, which could be explained by the removal of biomass in late summer. Contrary to our expectation, there was relatively more spring germination after grazing than after mowing, yet vegetation height was smallest in spring. Generally, germination season was found to be related to the temporal occurrence of favourable light conditions.
PMCID: PMC2710190  PMID: 18083716
Field experiment; functional analysis; germination success; seed characteristics; semi-natural grassland; assembly rules
22.  Surface Hydrophobicity Causes SO2 Tolerance in Lichens 
Annals of Botany  2007;101(4):531-539.
Background and Aims
The superhydrophobicity of the thallus surface in one of the most SO2-tolerant lichen species, Lecanora conizaeoides, suggests that surface hydrophobicity could be a general feature of lichen symbioses controlling their tolerance to SO2. The study described here tests this hypothesis.
Water droplets of the size of a raindrop were placed on the surface of air-dry thalli in 50 lichen species of known SO2 tolerance and contact angles were measured to quantify hydrophobicity.
Key Results
The wettability of lichen thalli ranges from strongly hydrophobic to strongly hydrophilic. SO2 tolerance of the studied lichen species increased with increasing hydrophobicity of the thallus surface. Extraction of extracellular lichen secondary metabolites with acetone reduced, but did not abolish the hydrophobicity of lichen thalli.
Surface hydrophobicity is the main factor controlling SO2 tolerance in lichens. It presumably originally evolved as an adaptation to wet habitats preventing the depression of net photosynthesis due to supersaturation of the thallus with water. Hydrophilicity of lichen thalli is an adaptation to dry or humid, but not directly rain-exposed habitats. The crucial role of surface hydrophobicity in SO2 also explains why many markedly SO2-tolerant species are additionally tolerant to other (chemically unrelated) toxic substances including heavy metals.
PMCID: PMC2710189  PMID: 18077467
Contact angle; hydrophilicity; hydrophobicity; lotus effect; cortex; sulphur dioxide; air pollution; water uptake; lichens
23.  Pattern Selection in Plants: Coupling Chemical Dynamics to Surface Growth in Three Dimensions 
Annals of Botany  2007;101(3):361-374.
Background and Aims
A study is made by computation of the interplay between the pattern formation of growth catalysts on a plant surface and the expansion of the surface to generate organismal shape. Consideration is made of the localization of morphogenetically active regions, and the occurrence within them of symmetry-breaking processes such as branching from an initially dome-shaped tip or meristem. Representation of a changing and growing three-dimensional shape is necessary, as two-dimensional work cannot distinguish, for example, formation of an annulus from dichotomous branching.
For the formation of patterns of chemical concentrations, the Brusselator reaction-diffusion model is used, applied on a hemispherical shell and generating patterns that initiate as surface spherical harmonics. The initial shape is hemispherical, represented as a mesh of triangles. These are combined into finite elements, each made up of all the triangles surrounding each node. Chemical pattern is converted into shape change by moving nodes outwards according to the concentration of growth catalyst at each, to relieve misfits caused by area increase of the finite element. New triangles are added to restore the refinement of the mesh in rapidly growing regions.
Key Results
The postulated mechanism successfully generates: tip growth (or stalk extension by an apical meristem) to ten times original hemisphere height; tip flattening and resumption of apical advance; and dichotomous branching and higher-order branching to make whorled structures. Control of the branching plane in successive dichotomous branchings is tackled with partial success and clarification of the issues.
The representation of a growing plant surface in computations by an expanding mesh that has no artefacts constraining changes of shape and symmetry has been achieved. It is shown that one type of pattern-forming mechanism, Turing-type reaction-diffusion, acting within a surface to pattern a growth catalyst, can generate some of the most important types of morphogenesis in plant development.
PMCID: PMC2701828  PMID: 18045793
Morphogenesis; pattern formation; surface expansion; symmetry breaking; finite element modelling; reaction-diffusion; tip growth; dichotomous branching; whorl formation; surface spherical harmonics; Micrasterias
24.  Reproductive Modes in Leiothrix (Eriocaulaceae) in South-eastern Brazil: The Role of Microenvironmental Heterogeneity 
Annals of Botany  2007;101(3):353-360.
Background and Aims
The genus Leiothrix (Eriocaulaceae) is restricted to South America and contains 37 taxa. The genus is most species-rich in the mountains of Minas Gerais, where 25 species occur, 19 of them in the Serra do Cipó. Leiothrix taxa that inhabit different microhabitats exhibit a number of reproductive modes. Rhizomatous taxa produce seeds plentifully; therefore, this group were defined as rhizomatous seed-producing. The pseudoviviparous reproductive mode was divided into canopy-forming and rooted. In the first, ramets remain attached to a parental rosette suspended by scapes, whereas in the second, ramets take root and may or may not remain attached to a parental rosette. In this study, it is proposed that microenvironmental heterogeneity is an important factor generating and maintaining reproductive modes in Leiothrix.
Soil analyses and vegetation cover estimates were performed for five Leiothrix taxa occurring in 19 areas along the Serra do Cipó. In these 19 points of the Serra do Cipó, soil data were collected from 27 populations of each species, and vegetation cover data were collected from 20 populations, due to fire that occurred in the region and destroyed most of the vegetation. For each population, three replicates were made. A discriminant function analysis was performed, in an attempt to test the effect of microhabitat features in the differentiation of the reproductive modes.
Key Results
Discriminant function analyses separated the three groups of reproductive modes based mainly on percentage vegetation cover. The pseudoviviparous canopy-forming group occurs under densely crowded conditions, while the pseudoviviparous rooted and rhizomatous seed-producing groups occur in areas with sparse vegetation cover. However, the group pseudoviviparous rooted occurs in soils constituted of exposed sand, while the rhizomatous seed-producing group occurs, frequently, on mat-forming mosses.
Microenvironmental heterogeneity, specifically heterogeneity in percentage cover of vegetation, appears to have influenced the generation and maintenance of reproductive modes in Leiothrix. Reproductive variation within Leiothrix taxa occupying different microenvironments results from a response to fine-scale habitat variation. Therefore, it is proposed that ecological speciation is an important process in adaptive radiation in this genus.
PMCID: PMC2701826  PMID: 17998571
Clonal growth; environmental heterogeneity; Leiothrix; life history; morphological variation; pseudovivipary; reproductive modes; rupestrian grasslands
25.  Genome Size and GC Content Evolution of Festuca: Ancestral Expansion and Subsequent Reduction 
Annals of Botany  2007;101(3):421-433.
Background and Aims
Plant evolution is well known to be frequently associated with remarkable changes in genome size and composition; however, the knowledge of long-term evolutionary dynamics of these processes still remains very limited. Here a study is made of the fine dynamics of quantitative genome evolution in Festuca (fescue), the largest genus in Poaceae (grasses).
Using flow cytometry (PI, DAPI), measurements were made of DNA content (2C-value), monoploid genome size (Cx-value), average chromosome size (C/n-value) and cytosine + guanine (GC) content of 101 Festuca taxa and 14 of their close relatives. The results were compared with the existing phylogeny based on ITS and trnL-F sequences.
Key Results
The divergence of the fescue lineage from related Poeae was predated by about a 2-fold monoploid genome and chromosome size enlargement, and apparent GC content enrichment. The backward reduction of these parameters, running parallel in both main evolutionary lineages of fine-leaved and broad-leaved fescues, appears to diverge among the existing species groups. The most dramatic reductions are associated with the most recently and rapidly evolving groups which, in combination with recent intraspecific genome size variability, indicate that the reduction process is probably ongoing and evolutionarily young. This dynamics may be a consequence of GC-rich retrotransposon proliferation and removal. Polyploids derived from parents with a large genome size and high GC content (mostly allopolyploids) had smaller Cx- and C/n-values and only slightly deviated from parental GC content, whereas polyploids derived from parents with small genome and low GC content (mostly autopolyploids) generally had a markedly increased GC content and slightly higher Cx- and C/n-values.
The present study indicates the high potential of general quantitative characters of the genome for understanding the long-term processes of genome evolution, testing evolutionary hypotheses and their usefulness for large-scale genomic projects. Taken together, the results suggest that there is an evolutionary advantage for small genomes in Festuca.
PMCID: PMC2701825  PMID: 18158307
Festuca; fescue; grasses; genome size evolution; chromosome size; base composition; GC content; polyploidy; phylogeny; retrotransposon dynamics; flow cytometry

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