Enio Egon

Sosinski Júnior
email: esosinski@gmail.com

Pascal Carrère

Frédérique Louault

Jean-François Soussana

Development of a functional approach in a grassland vegetation Development of a functional approach in a grassland vegetation Development of a functional approach in a grassland vegetation Development of a functional approach in a grassland vegetation sub sub sub sub----model model model model

Keywords: extensification, grassland vegetation, models, simulation, functional attributes, vegetation communities extensificação

To improve our understanding of grassland dynamics under different levels of utilization, a functional description of the vegetation was introduced in a deterministic model. The selected traits and their parameterization were based on the results of a longterm experiment in which temperate grasslands were managed for 12 years with three levels of herbage use: high, medium and low. The integration of functional attributes of the community species within the model's parameters can be seen as a new step in the study of the grassland ecosystem. With this tool, it is possible to decrease the number of interconnections in the system and consequently decrease the complexity. In this work a, functional definition of three different grassland communities was introduced into the subvegetation model. This was done by interchanging the model's parameters with the functional attributes of the communities. From the conceptual point of view, the subvegetation model works adequately and it seems suitable to simulate the dynamic of grassland vegetation described by functional traits. The model fits experimental data well for high levels of utilization, but was poorly adjusted at medium and low levels of herbage use. We believe this is due to a better simulation of green biomass fluxes than for senescence or reproductive fluxes. Some possible improvements of the model are discussed.

Introduction Introduction Introduction Introduction

To understand the relationship between the vegetation response to the intensity and frequency of grazing herbivores impact in relation to environmental conditions, simulation models have been developed. These are to provide a simplified framework to better understand grazing systems and to be able to simulate their complexity [START_REF] Marriott | Structure and dynamics of grazed vegetation[END_REF]. The Institut National de la Recherche Agronomique (INRA) has developed a simulation model with the aim of building a spatial multi-agent simulator capable of conducting a Acta Sci. Anim. Sci. Maringá, v. 30, n. 2, p. 145-153, 2008 realistic representation of the management of permanent grassland vegetation under grazing regimes. It was conceived as a research tool to explore animal behavior in large heterogeneous spaces. Broad functional responses based on well-demonstrated biological laws and long developed theoretical frameworks were to be used to reach the target without creating an overly complicated model [START_REF] Pérochon | Design of a spatial multi-agent model of a perennial grassland ecosystem grazed by a herd of ruminants[END_REF][START_REF] Baumont | Développement d'un modèle multi-agents spatialisé d'un troupeau de ruminants pâturant une prairie hétérogène[END_REF][START_REF] Carrère | Design of a spatial model of a perennial grassland grazed by a herd of ruminants: the vegetation sub-model[END_REF]. The role of such a model could be to explore a large number of hypotheses in the spatiotemporal heterogeneity in plant-animal interactions [START_REF] Marriott | Structure and dynamics of grazed vegetation[END_REF][START_REF] Laca | Modeling spatial aspects of plant-animal interactions[END_REF].

Recent researches in natural and semi-natural European grassland vegetation are focusing on the reduction of the intensity of grassland ecosystem utilization [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF]. The main concerns of these works have been with those areas that have recently changed to a less intensive agricultural management. Some works concern low fertilizer application, others are pointing to the impact on decreasing the stocking rate. The principal question is to identify and quantify the process that drives plant communities in a system where the relationship between vegetation composition and grazing herbivores is dynamic in time and space (Taiton et al., 1996). Grazing herbivores actions affect the structure and composition of the vegetation by changing the vertical and horizontal structure and quality of the vegetation, which in turn will affect the animal choices [START_REF] Marriott | Structure and dynamics of grazed vegetation[END_REF].

A grazed plant community consists of many populations coexisting in a meta-equilibrium between abiotic and biotic conditions [START_REF] Bullock | Plant competition and population dynamics[END_REF]. It underlies dynamic processes where the number of individual plants in a population varies in time and space because some new individuals thrive and others die as a consequence of environmental variables or animal actions (Taiton et al., 1996). The community composition depends on how these factors affect the performances and competitive abilities of each individual within the population. Many species can coexist in nature because they have some attributes that interact with the environment to affect the demographic performances and competitive abilities. Not all mechanisms which regulate the plant abundance in a grazed plant community ecosystem are understood [START_REF] Bullock | Plant competition and population dynamics[END_REF] and a full comprehension will expend a lot of effort, time and money: a complete understanding of population establishment and competition in a community would require a detailed study of each species, the interaction within and between them, the environmental condition involved and the patterns of distribution in time and space of the species (Gordon, 2000).

One possibility to solve the problem has been the use of non-phylogenetic classification of plants responding in similar ways to a given factor. This kind of approach serves to complement the species-based classification with a more functional approach, facilitating the association of some plants characteristics with particular response to grazing, without the need to identify all the species [START_REF] Landsberg | Grazing response groups among understorey plants in arid rangelands[END_REF]. Also it can provide a picture that could be applied to different regional floras. Several studies have attempted to identify functional groups of species with similar ecological roles. These groups are called functional types and can be defined as groupings of plants with similar functions and sharing common biologic characteristics [START_REF] Díaz | Can grazing response of herbaceous plants be predicted from simple vegetative traits?[END_REF], i.e. groups of populations in which the responses may not necessarily be plastic, but rather as substitutions of certain populations by other populations with different traits, more adapted to the new conditions or with the same functional effects in the ecosystem. Recent studies are trying to identify these linkages between the relation of the species contribution to the ecosystem process and the species response to changes in environment [START_REF] Lavorel | Predicting changes in community composition and ecosystem functioning from plant traits: revisiting the Holy Grail[END_REF]. It means a new grouping of plants that will compose the vegetation community defining a mosaic in the landscape. The functional classification could be seen as a new scale of description of vegetation, used to simplify and improve the perception of the process by using the plants themselves as indicators [START_REF] Pillar | Character-based community analysis: theory and application program[END_REF].

The vegetation patterns in grassland plots in an experimental area after 12 years of contrasting management was described by [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF], indicating marked differences in species composition among treatments. Three major plant functional types of response in relation to the level of herbage use (cutting and grazing) characterized the differences when the treatments were described by traits aggregated at the community scale. Two types co-dominated the high use plots and one type described the other treatments. In the high use treatment, one type is competitive (fast-growing), grazing tolerant and avoiding moderate grazing, while the other was assumed by the authors to be conservative and only avoiding moderate grazing. For the medium and low use treatments, the characteristic type is competitive to light but with little grazing avoidance and no grazing tolerance.

The aim of this work is to integrate the functional description at the community level in a Acta Sci. Anim. Sci. Maringá, v. 30, n. 2, p. 145-153, 2008 determinist model that simulates vegetation dynamic. A functional approach assumes that the plant species that develop in response to a given factor (e.g. herbage use efficiency, nutrient availability) display some converging traits, which in turn condition their performance in the community. The basic hypothesis is that the model can simulate different patterns of grassland vegetation response described by a functional approach. The work focuses particularly on the simulation of perennial grassland vegetation communities managed under three different levels of exploitation, described in [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF]. We discuss how to integrate the traits description of each community in the model setup and its implications. We define the parameters in the grazing-vegetation model that can be used as links between the functional community description (faciès) and the vegetation community described by traits and determine the level of agreement of the simulation outputs with the experimental data when using a functional approach.

Model description Model description Model description Model description

The prototype has been implemented with a Linux system and GNU C++. The vegetation submodel was built using object-oriented analysis. At field scale, the spatial distribution of the vegetation is managed by an input file, which gives the position of different vegetation types (i.e. plant communities formed by a given mix of species) within the plot. Each vegetation type is associated with a cell. A simulation is driven by broad parameters determining the plot size, the simulation duration and climatic conditions. More specific parameters define a faciès. The climatic conditions are given by an environmental input file that allows the model to manage time using a thermal calendar based on the sum of average temperatures, which turns on or off the growth, flowering and senescence functions. These processes follow continuous functions, which are discrete in the sub-model using a daily iteration [START_REF] Carrère | Design of a spatial model of a perennial grassland grazed by a herd of ruminants: the vegetation sub-model[END_REF]. The simulator was conceived as a spatial multi-agent model to be able to represent the different aspects of animal behavior and the heterogeneity of the vegetation. The simulator is composed of three sub-models: vegetation, animal, and spatial (herd displacement) [START_REF] Baumont | Développement d'un modèle multi-agents spatialisé d'un troupeau de ruminants pâturant une prairie hétérogène[END_REF].

Vegetation Sub Vegetation Sub Vegetation Sub Vegetation Sub----model model model model

According to [START_REF] Pérochon | Design of a spatial multi-agent model of a perennial grassland ecosystem grazed by a herd of ruminants[END_REF] and [START_REF] Carrère | Design of a spatial model of a perennial grassland grazed by a herd of ruminants: the vegetation sub-model[END_REF], the vegetation sub-model simulates dynamics of biomass flows in the vegetation in relation with the defoliation performed by animals. The grassland plot is divided into elementary hexagonal cells of 0.1 m 2 , which correspond to the size of a feeding station for sheep. The defoliation process is considered homogeneous within the cell. The size of a cell is a trade-off between biological and modeling constraints, i.e. it has to be small enough to keep the homogeneity, but not too small to allow the modeling of a large area (e.g. up to ten hectares) avoiding an excessive number of cells. The use of a hexagonal cell facilitates the modeling of animal movements because the center of each cell is at equal distance of the center of the six adjacent cells.

Inside the cell, the vegetation is described by the following compartments: green leaves and shoots (GV), dry leaves and shoots (DV), green stems and ears (GR) and dry stems and ears (DR). Each of the four compartments is defined by state variables (biomass and height), two qualitative variables (fiber content and digestibility) and two allometric functions representing the relationships between state variables (vertical distribution of the biomass, and specific leaf area, which is the leaf area produced for each gram of biomass). Interactions between the four compartments are simulated by six functions which calculate the flow of biomass: one growth function, one reproductive function, two senescence functions, and two functions simulating abscission of dead tissues and their disappearance in the litter. Many equations are involved in those functions where time is managed using a thermal calendar, which determines activation or deactivation of these functions and therefore the vegetation development.

Only the above ground vegetation is simulated in the cell; soil activity is taken into account through a nutrition index representing soil fertility. The biomass produced in the GV compartment is then reallocated towards the three other compartments using reproductive (GV through GR) or senescence (GV through DV; GR through DR) functions. The biomass may be defoliated by an animal, by linking with the animal sub-module or by abscission of dead tissues, through a litter function not yet implemented. The spatial partition of the vegetation in the plot among the cells corresponds to the partition of the different plant community types called faciès. Each faciès is associated with a cell and described by a set of parameter values used by the cell functions. A set of parameters describing the vegetation and summarizing its properties defines a faciès. The parameters will complete the six functions of the vegetation sub-model and will allow the model to simulate different plant community dynamics. The growth of each cell is calculated in the GV compartment using three functions: the light dependent on radiation intensity function, Acta Sci. Anim. Sci. Maringá, v. 30, n. 2, p. 145-153, 2008 temperature function and fertility function. The biomass produced in the GV is then distributed between GV, GR or DV compartments, using flowering or senescence functions. The flowering function is driven by temperature and depends on soil fertility. The senescence flux varies with the average daily temperature and also considers biomass losses through respiration and through the abscission of shoot litter. Outputs of the vegetation sub-model consist in the state and qualitative variables of each compartment and weighted averages of the compartments for each cell. These output data for the vegetation sub-model are inputs data for the animal sub-model.

Material and Material and Material and Material and methods methods methods methods

The experimental data to be used in the process of parameterization came from an experiment established in 1989 on permanent grassland located at Theix, (Auvergne, France). Six individual plots were subjected to three levels of herbage use in order to simulate a process of de-intensification of use. The three management regimes go from the mort (high H and medium M) to the least intensive (low L); all were first grazed in mid-April, and only H was cut for hay at mid-June. The H and M plots were also grazed three times, in late July, late September and late November. At each grazing period, eight ewes per plot performed grazing until the grassland height in the plots H reached 5.5 to 6.5 cm. There was no fertilization supply.

Since 1989, vegetation surveys were performed to observe the development of vegetation in the growing season. The proposal was to measure the biomass position and translocation year-round. This work was done in all herbage uses at the same time before and after each treatment was conducted. We chose the data from 1998 because it was closest to the year when the traits were measured (2000) and because the biomass partition sort data base was most complete for this year. Sampling was obtained within a 3.5 by 0.1 m sampling unit replicated three times in each herbage use and before each grazing or cutting treatment. The collected samples were sorted in green or dry leaves, ears, stems etc., and were dried and weighted. The experimental area has been described by [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF] as speciespoor vegetation which was fully exploited by grazing and cutting with regular organic and inorganic fertilizer supply, before the experiment start in 1989. After 12 years (2000) of application of differential management, a vegetation survey and species traits measurements were performed in each of the six plots, one month after the early spring grazing. Plant traits were measured on the most abundant species (i.e., those accounting for at least 85% of the vegetation biomass). Seventeen traits, including leaf traits, plant digestibility, plant height and phenology were evaluated with a method adapted from [START_REF] Weiher | Challenging theophrastus: a common core list of plant traits for functional ecology[END_REF]. The mean value for each trait was calculated at the community level for the three levels of herbage use, and significant differences between treatments were found for most of the traits [START_REF] Louault | Long term effect of a reduce herbage use in a semi-natural grassland[END_REF]. The results [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF] suggest the possibility of describing the three different degrees of herbage use according to the following functional community approach:

H -high level of herbage use led the plant community to an increase in the abundance of short and early flowering plants with medium size lamina of low dry matter content.

L -low level of herbage use favored tall plants with medium date flowering and plants with medium size lamina of higher dry matter content when compared with H.

M -medium level of herbage use favored a mixed plant community composed of the two types already described above and a third type which is characterized by low height with early flowering an very small lamina of low dry matter content.

The environmental data corresponds to daily temperature and global radiation observations that were collected for the last 17 years by the meteorological station in St. Genest Champanelle. The global radiation data was previous transformed from Joule cm 2 to Megajoule cm 2 (PARi) using the formula ((X/100)*0.47). After that, mean values for 17 years for both variables were calculated.

Functional parameters Functional parameters Functional parameters Functional parameters

Among the 43 parameters of the vegetation submodel, some are predominantly functional and were considered as traits of the plant community, and some describe vegetation structure. Functional parameters are those that can be considered plant traits because they are related to some physiological or phenological aspect of the vegetation. In this first approach, parameters linked to the plasticity of the plant and their phenology and life cycles were considered. These parameters probably should best represent the adaptive responses of the communities to different disturbances or changes in aboveground biomass. Therefore, the parameters chosen are known to have an impact in the competitive abilities of the plants to the amount of light energy intercepted. The interception of light depends of the amount of dry material and its distribution in space, which determines the quantity of leaves by unit of Acta Sci. Anim. Sci. Maringá, v. 30, n. 2, p. 145-153, 2008 surface and its capacity of light interception. The light utilization efficiency is regulated for light conversion efficiency of the leaves and is controlled by the environment being dependent on radiation intensity, daily temperature and nutrition index.

Based on this information, the first parameters are related to the life cycle of the plant community (earliness and duration of flowering) and to growth ability (SLA), and the second parameters inform us about vegetation state at the beginning of the simulation (beginning of the year) and concern the quantity, quality and vertical distribution of the biomass. For 17 parameters, values are given at the community level for the three levels of herbage use (Table 1), values of the remaining 26 staying the same for all three plant communities.

Simula Simula Simula Simulation tion tion tion

Simulations were performed to describe the dynamics of the total biomass and of its four compartments for a one-year period, starting from the first of January. Input parameters describe plant communities under the three levels of herbage use. The environmental file corresponds to climatic (temperature and light) data of the year 1998 and the average of 17 years. A set of simulations was conducted including disturbance, i.e. biomass removal at each cutting or grazing event. It is important to stress that the spatial aspect was not considered in the simulations. All the work with the prototype was done in a single cell. The use of more cells is reasonable when the animal is present; otherwise it does not matter, since there is no interaction between cells.

The first simulations were done in order to comprehend the prototype functioning and to verify the basic set of data correction, i.e. to analyze whether the model works properly with the basic data prepared. The second set of simulations used the same files as above, including the 1998 environmental file during a period of a year starting from January 1 st until the end of December. They did not consider the management of the treatments because the idea in this simulation was to observe the profile of each faciès along the year, comparing them in terms of the differences in biomass production, without any impact of different managements. In each sequence, the starting biomass for all compartments and the number of days simulated were based on the experimental data. The profiles were observed to identify similarities between the simulation and the experimental data. The estimated data based on the simulations performed above were compared with the empirical data to observe the level of adjustment. Graphics with both of these data sets were elaborated in order to examine the fitness of simulated data compared to the experimental data.

Results

Results Results

Results and and and and discussion discussion discussion discussion

Table 1 shows the parameters for the three faciès descriptions included in the input file. They correspond to the three levels of utilization in the experiment data, and the state variables total biomass (T) and biomass of each compartment were chosen as model outputs for further analysis.

Table 1. Values of parameters of the growth model for the three plant communities under the three levels of herbage use (High, Medium and Low).

Tabela 1. Valores dos parâmetros do modelo de crescimento para as três comunidades vegetais submetidas a três níveis de intensidades de uso (Alta, Média e Baixa).

Parameters (units)

Parâmetros (nidades)

High

Alta

Medium

Média

Low

Baixa

Plant traits

Atributos das plantas

Specific leaf area (m 2 kg -1 DM) Área específica da folha (m 2 kg -1 MS) Maringá, v. 30, n. 2, p. 145-153, 2008 The first simulations of the prototype used as environmental variables the mean average of 17 years worth of data. This simulation was performed to observe whether the adjustment of the experimental data described by the three experimental faciès had been coherent with the standard. The results profile (Figure 1) shows a reasonable behavior for all faciès along the year despite the larger amount of total biomass (Wcell) produced (around 900 g m -2 ) by the three experimental faciès. With the same basic faciès data, we tried to change the environmental data to see whether the results would keep coherence with the ones found using the 1998 environment data set. The idea was to compare the simulations results and choose one of then. The results (Figure 2) show that the maximum Wcell produced by the four faciès in 1998 based data was lower than the other. Also the total biomass of the cell (Wcell) started to grow earlier compared to the average of 17 years' data based simulation. We decided to work with the 1998 environmental data file. After that, the focus was to observe the model output performance along the year looking for the dynamic of total biomass without management. The profiles of simulation (Figure 2) show that the Wcell for each faciès started at different levels (point 1) and decreased during the ending winter time until the beginning of the growing season. It could be observed that the faciès high start to grow first (45 days) followed by the others faciès (point 2). From this point, the three curves showed a larger rate of biomass accumulation until they reach almost the same high-level plateau (point 3). For the last phase, the same pattern of biomass decreases could be observed in all faciès (point 4).

In Figure 3, experimental data is being compared with estimated data in terms of total biomass of the cell (Wcell). It can be observed that at high and medium levels of exploitation, the model agreed well with experimental data, but not at low level of herbage use. At the low level of exploitation, the simulated data estimate higher values (400 to 800 g m -2 ) in comparison with experimental data (400 to 600 g m -2 ). For each treatment alone, it is possible to observe that the simulation estimated better for the lower values of the most intensive management than for the higher ones. This fact could be linked to the fact that the model had problems to simulate the compartments Wdv and Wr (Figure 4). In these compartments, the model output showed a bad agreement with the experimental data, especially for the lower level of exploitation. However, in the green vegetative compartment (Wgv), the simulation data fit well with the experimental data at high and medium levels of exploitation, and performed not too badly in the most extensive treatment. The simulations performed with the community description based on the functional traits showed that the sub-vegetation model correctly estimated the dynamics of the vegetation. The model showed reasonable responses to the environmental variables, climate and fertility, performing a coherent profile for all faciès during the year. In the profile of the simulation without management, it can be observed that the vegetation followed the classical profile described in [START_REF] Lemaire | Tissues flows in grazed plant communities[END_REF]. The biomass started from a lower level (point 1), in winter, and at the start of the warm season (point 2). The biomass grows until reaching an adequate plateau of total biomass production (point 3). After that, it begins to decrease according to the senescent rules (point 4). It can be observed that at the beginning of the simulation, the higher amount of biomass in the faciès L and M when compared to H is due to the accumulation of the dry and reproductive compartments, in accordance with the experimental data and the observations in [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF]. Nearly 55% and 36% of the Wcell of faciès L is given by the Wdv and Wr compartments, respectively, and the same proportion is found for the M faciès. From [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF] the experimental treatments of low herbage utilization lead to an increase in plant height with a significant correlation to later maturity. Nevertheless, the opposite was described for the faciès H where 66% of the total biomass was given by the Wgv compartment.

However, at the start of the growing season (point 2) it can be observed that the faciès H response was faster in terms of total biomass production than that of the others. The small proportion of senescent material from the last year in the well used treatments allowed for a net accumulation of herbage equals to the net assimilation rate of the canopy [START_REF] Lemaire | Tissues flows in grazed plant communities[END_REF]. At this early stage of regrowth, there is little death of leaf material in H treatments, in contrast to the M and L treatments, where a high increase of death tissue can be observed. Yet, according to the same authors, the regrowth in highly used plots increases the leaf area index (LAI) by increasing leaf size and Acta Sci. Anim. Sci. Maringá, v. 30, n. 2, p. 145-153, 2008 appearance, which improves the light interception and gross photosynthesis. This can be translated into the model by faciès parameters linked to the biomass production (SLA) or the canopy structure of the community. In fact, these characteristics are attributes of the species interacting with the environmental to affect the demographic performances and competitive abilities of the plants [START_REF] Bullock | Plant competition and population dynamics[END_REF].

Referring to what has been discussed above, the net herbage accumulation rate will be at its maximum until the rate of senescent material equalizes it or the reproduction phase starts [START_REF] Lemaire | Tissues flows in grazed plant communities[END_REF]. At this part of the simulation, other important traits related to the phenology and life cycle of the plants will affect the competitive abilities of the faciès. The reproductive activation attribute is a response to the start of the translocation of biomass produced in the Wr compartment, changing the pattern of the net accumulation rate and the reproductive activation deactivation for the duration of this period. It is later and longer in the L Faciès because maturity is reached later in tall plants than in short plants [START_REF] Louault | Plant traits and functional types in response to reduced disturbance in a semi-natural grassland[END_REF].

In contrast with this first phase of the process, all the faciès showed the same dynamics of biomass production in the second part of the year. After a period of strong net accumulation of in the spring, the faciès likely equated in their rate of production and percentage of dead tissues (point 3). These results suggest a good adjustment of the model to the limit of production given by the environmental variables (climate and fertility).

Therefore, it can be observed in the simulation results that the dynamics of each faciès are consequence of a different structure of the community, determined by its level of exploitation i.e. by spatial and temporal heterogeneity of grazing [START_REF] Laca | Modeling spatial aspects of plant-animal interactions[END_REF]. However, grazing or any kind of heterogeneous disturbance is not part of the model yet. While the temporal component should be further developed in the model, the spatial heterogeneity of the grassland vegetation is summarized in each faciès which represent a "functional" community description. The faciès reduce the spatial heterogeneity of the communities by means of the averages of the functional attributes of the plant community in homogeneous cells.

Conclusion Conclusion Conclusion Conclusion

The traits of the plants in a community interact with the factors, altering the individual plants ability to compete. This ability is altered by the presence of the animal increasing the number of different species by increasing the complexity of the system. Models are used to represent this complexity in a simpler manner. The sub-vegetation model was used to simulate grassland vegetation dynamic under three different levels of herbage use by grazers. These three communities were described by aggregates of traits involved in the response of the community to the level of use. These descriptions were introduced into the model concept by the faciès definition. Each faciès was related to a different community by defining their parameters according to the respective values of the community's traits. A correct simulation of the grassland vegetation dynamics described by functional traits was achieved by the sub-vegetation model. The model simulates well the dynamics of the different faciès, but performed better in the simulation of the growing flux than in phenology or the senescent flux. The structural aspect of the vegetation still is not being simulated well, especially in plots under low utilization and showing high spatial heterogeneity. The model needs to be improved to account for the complexity of the grazing system.

We suggest that the lack of agreement between observed and simulated results is due to a poor estimation of senescence and abscission fluxes. The largest discrepancies were shown for the winter period under high herbage use treatment. Under medium and low treatments, the large amounts of biomass were for long regrowth periods without disturbance, during which the tissues become old and start their senescence. Under high level of herbage use, the structure of the sward is more homogeneous than under low level of exploitation [START_REF] Parsons | Dynamics of heterogeneity in a grazed sward[END_REF]. We suggest that the discrepancies registered in our study partly arose because the errors of prediction were lower for a homogeneous structure than for a heterogeneous structure. One way to improve the model would be to use a stochastic and spatial approach, specifically combining plant and animal models. According to the changes of trait values observed in the vegetation at different levels of herbage use, the modification of the values of few parameters of the growth model (designated as traits), enable us to simulate contrasted biomass dynamics. Increases in the prediction performance of the model could be achieved through a better description of senescence mechanisms, and also by integrating a spatial approach.

Figure 1 .

 1 Figure 1. Profiles of W cell compartment for all faciès using the average of 17 years environmental data file.

Figura 1 .Figure 2 .

 12 Figura 1. Perfis do compartimento W das células para todos os faciès utilizando arquivo de dados ambientais com a média de 17 anos.

Figura 2 .

 2 Figura 2. Perfis do compartimento W das células para todos os faciès utilizando arquivo de dados ambientais do ano 1998. Os números indicam aspectos importantes dos perfis, e são descritos no texto .

Figure 3 .

 3 Figure 3. The experimental versus the simulated Wcell (g m -2 ) for the three treatments (H, high; M, medium; L, low) considering the management. The diagonal line indicates the point where the estimate is equal to the experimental data.

Figura 3 .)ExFigure 4 .

 34 Figura 3. Valores experimentais versus simulados de Wcell (g m -2 ) para os três tratamentos (H, alta; M, média; L, baixa intensidade). A linha diagonal indica os pontos onde os dados estimados são iguais aos experimentais.

Figura 4 .

 4 Figura 4. Valores experimentais versus simulados para os compartimentos Wgv, Wdv, Wr (de cima para baixo) nos três tratamentos (esquerda para direita) segundo a intensidade do manejo. A linha diagonal indica os pontos onde os dados estimados são iguais aos experimentais.

Received on July 24, 2007. Accepted onMay 19, 2008.

Acknowledgements Acknowledgements Acknowledgements Acknowledgements

Enio Sosinski received a Capes (Brazil) PhD grant through a cooperation between Capes (Brazil) and Cofecub (France). This work was supported by "INRA-AIP pâturage", FNADT.


Vincent Baillard

Cécile Sulmon

Anne-Kristel Bittebière

Cendrine Mony

Simon Devin

Elise Billoir

Can SSD models help assessing interspecific

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Interspecific competition impact on organism responses to chemical stress : an SSD-based approach.

Materials & methods

Isoproturon tolerance Low

Medium High

Competitiveness Low

Poa trivialis Poa pratensis Trisetum flavescens

High

Arrhenatherum elatius Lolium multiflorum Dactylis glomerata

Experimental design

Modelling process (see also comm' WE474 and WE368)

Grass strips are vegetated areas that act as buffer strips against agricultural pollutants flows towards streams. They present a community of plant species that are simultaneously exposed to various pollutants  interactions between competition and chemical stress responses is an important topic. The objective of this study is to :

-assess whether interspecific competition modifies tolerance of herbaceous plants to the herbicide isoproturon -evaluate SSD, a tool widely used in risk assessment that permits to integer data from monospecific tests to produce a theoretical sensitivity distribution of the community.

Examples of fit SSD examples (IC10 values)

In every case, HC 5 values derived with BMDsd, IC 10 or 50 and EC 10 or 50 , used in every different SSD types used are 1,04 to 7,21 times lower for organisms in presence of the competitor.

-6 herbaceous grass species (3 isoproturon tolerance levels * 2 competitiveness levels), -Bromus erectus as model competitor -25-days exposure to 6 isoproturon concentrations, from 0 to 1.75 µM, in presence and absence of Bromus erectus (8 replicates).

-measurement of 12 endpoints expected to respond to isoproturon and/or competition stress (linked to biomass, photosynthesis, resource allocation…).

S1 S2 S3

The choice of toxicity value used and the handling of multiple toxicity values for a same species can result in important changes in final HC 5 values and their differences between situations with and without interspecific competition.

: HC 5

Toxicity effect Competition effect

Both

Interspecific competition  decrease in tolerance to isoproturon  impact on obtained SSD models.

Depending on species and endpoints, fitted curves present competition effect (differences at intercept) and/or toxicity effect (differences in toxicity values). It seems important to assess in the future whether this ecological interaction, and others, should be further highlighted in risk assessment. Other experiments are actually in progress to define if these conclusions can be applied to metabolomic data and to complex assemblages of tested species.

Effect of toxic

Effect of competition

  Cécile SULMON 2 , Anne-Kristel BITTEBIERE 3 , Cendrine MONY 2 , Simon DEVIN 1 , Elise BILLOIR 1 1 : LIEC, CNRS UMR 7360, Université de Lorraine, France 2 : ECOBIO, CNRS UMR 6553, Université de Rennes 1, France 3 : LEHNA, CNRS UMR 5023, Université de Lyon 1, France Contact : vincent.baillard@univ-lorraine.fr for each species (quadratic model) Model fitting (linear, exponential, Hill, Gaussian and log-Gaussian) Best-fit selected according to AIC values IC 10 and 50 (x% change compared to the control) BMD sd (You don't know that one? Ask me for explanations!)

  Toxic value choice & processing of multiple toxic value for each species  changes in HC 5 values and their modification by competition.


Interspecific competition impact on organism responses to chemical stress : an SSD-based approach.

Materials & methods

Isoproturon tolerance Low

Medium High

Competitiveness Low

Poa trivialis Poa pratensis Trisetum flavescens

High

Arrhenatherum elatius Lolium multiflorum Dactylis glomerata

Experimental design

Modelling process (see also comm' WE474 and WE368)

Grass strips are vegetated areas that act as buffer strips against agricultural pollutants flows towards streams. They present a community of plant species that are simultaneously exposed to various pollutants  interactions between competition and chemical stress responses is an important topic. The objective of this study is to :

-assess whether interspecific competition modifies tolerance of herbaceous plants to the herbicide isoproturon -evaluate SSD, a tool widely used in risk assessment that permits to integer data from monospecific tests to produce a theoretical sensitivity distribution of the community.

Examples of fit SSD examples (IC10 values)

In every case, HC 5 values derived with BMDsd, IC 10 or 50 and EC 10 or 50 , used in every different SSD types used are 1,04 to 7,21 times lower for organisms in presence of the competitor.

-6 herbaceous grass species (3 isoproturon tolerance levels * 2 competitiveness levels), -Bromus erectus as model competitor -25-days exposure to 6 isoproturon concentrations, from 0 to 1.75 µM, in presence and absence of Bromus erectus (8 replicates).

-measurement of 12 endpoints expected to respond to isoproturon and/or competition stress (linked to biomass, photosynthesis, resource allocation…).

S1 S2 S3

The choice of toxicity value used and the handling of multiple toxicity values for a same species can result in important changes in final HC 5 values and their differences between situations with and without interspecific competition.

: HC 5

Toxicity effect Competition effect

Both

Interspecific competition  decrease in tolerance to isoproturon  impact on obtained SSD models.

Depending on species and endpoints, fitted curves present competition effect (differences at intercept) and/or toxicity effect (differences in toxicity values). It seems important to assess in the future whether this ecological interaction, and others, should be further highlighted in risk assessment. Other experiments are actually in progress to define if these conclusions can be applied to metabolomic data and to complex assemblages of tested species.

Effect of toxic

Effect of competition

  Cécile SULMON 2 , Anne-Kristel BITTEBIERE 3 , Cendrine MONY 2 , Simon DEVIN 1 , Elise BILLOIR 1 1 : LIEC, CNRS UMR 7360, Université de Lorraine, France 2 : ECOBIO, CNRS UMR 6553, Université de Rennes 1, France 3 : LEHNA, CNRS UMR 5023, Université de Lyon 1, France Contact : vincent.baillard@univ-lorraine.fr for each species (quadratic model) Model fitting (linear, exponential, Hill, Gaussian and log-Gaussian) Best-fit selected according to AIC values IC 10 and 50 (x% change compared to the control) BMD sd (You don't know that one? Ask me for explanations!)

  Toxic value choice & processing of multiple toxic value for each species  changes in HC 5 values and their modification by competition.


Damien Guillaumot

Marie-Anne Lelu-Walter

Agnès Germot

Fanny Meytraud

Louis Noël Gastinel

Catherine Riou-Khamlichi
email: catherine.riou@unilim.fr

Damien Guillaumota

Marie-Anne Lelu-Walterb

Agnès Germotc

Fanny Meytrauda

Louis Gastinela

Catherine Riou-Khamlichia

Expression patterns of

Keywords: APETALA2, Hybrid Larch, Somatic embryogenesis aa, amino acid, AP2, APETALA2, EM, embryonal mass, Ll, Larix x marschlinsii, Pa, Picea abies, Pt, Pinus thunbergii, RACE, rapid amplification of cDNA ends, RT-PCR, reverse transcriptase polymerase chain reaction, week, wk

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Introduction

In gymnosperms, embryogenesis is divided into three distinct phases : proembryogeny, all stages before the elongation of the suspensor, early embryogeny before root and shoot meristem initiation and late embryogeny with primary meristem formation [START_REF] Von Aderkas | Woody Plant Biotechnology[END_REF][START_REF] Von Arnold | Plant Cell Tiss[END_REF]. For gymnosperm embryogenesis, few molecular data are available due to the high complexity of this process especially during the two first phases, the absence of molecular databases and the lack of a conifer model such as Arabidopsis and poplar. Nevertheless, somatic embryogenesis which mimics zygotic embryogenesis is now well described for some gymnosperms such as spruce (Picea glauca (Moench) Voss), Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and hybrid larch (Larix x marschlinsii Coaz) [START_REF] Von Aderkas | Woody Plant Biotechnology[END_REF]Lelu and Label, 1994;Gutmann et al., 1996;[START_REF] Von Arnold | Plant Cell Tiss[END_REF]. Of these three conifer models, somatic embryogenesis is fastest in hybrid larch, with embryos maturing in four weeks, by which time they have acquired the physiological capability to withstand desiccation and to initiate germination [START_REF] Von Aderkas | [END_REF], Mathieu et al., 2006).

To gain insight molecular markers of hybrid larch somatic embryogenesis, we focused our study on the APETALA2/ERF super family proteins, the two-AP2-domain proteins. These proteins are transcription factors involved in several developmental events from flowering to vegetative development in Arabidopsis, petunia and maize (Jofuku et al., 1994;[START_REF] Chuck | Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii)[END_REF]Riechmann and Meyerowitz, 1998;Maes et al., 1999). APETALA2 could also influence embryo development by controlling size and cell number (Jofuku et al., 1994(Jofuku et al., , 2005;;Ohto et al., 2005). APETALA2-Like genes were also characterized in Norway spruce (Picea abies (L.) Karst) and pine (Pinus thunbergii Parl.) (Vahala et al., 2001;Shigyo and Ito, 2004;Nilsson et al., 2007). We hypothesized that these proteins could also be characterized in hybrid larch, where they may play a role in somatic embryogenesis. We characterized two APETALA2-like cDNA from hybrid larch named LmAP2L1 and LmAP2L2. Using RT-PCR analyses, transcript expression patterns during somatic embryogenesis and germination of hybrid larch were examinated.

Material and Methods

Experiments were conducted with a highly embryogenic line of hybrid larch, Larix x marschlinsii (L. kaempferi (Lamb.) x L. decidua (Miller)) designated 69-18 obtained by secondary embryogenesis as previously described (Lelu et al., 1994 a,b). Embryonal mass (EM) was subcultured every 2 wks in darkness onto fresh proliferation medium which consisted of basal MSG medium supplemented with 0.01 M glutamine, 9 μM 2,4-D, 2.3 μM BA. Maturation was a step-wise process in which EM was first transferred for 1 wk to plant growth regulator-free MSG medium with 1% activated charcoal after which they were transferred for at least 4 wks onto MSG medium supplemented with ABA 60 μM as previously described [START_REF] Von Aderkas | [END_REF]. Four week-old cotyledonary somatic embryos were transferred onto plant growth regulator-free MSG medium for germination and plantlet development.

Total RNAs were extracted with a 70 °C pre-heated CTAB extraction buffer as described by Chang et al. (1993). RNAs were quantified at 260 and 280 nm by spectrophotometry. Two μg of total RNAs extracted from EM and 4 week-old embryos after 14 or 25 days of germination were used to synthesize complementary DNA. The cDNA template obtained was amplified by PCR using AP2-domain-specific primers designed from Picea abies and Pinus thunbergii PaAP2L1 and L2 and PtAP2L1 and L2 nucleic acid sequences respectively (Vahala et al., 2001;Shigyo and Ito, 2004) Two μg of total RNAs extracted from the different tissues described below and from the different somatic embryogenesis stages were used to synthesize complementary DNA as detailed earlier.

Primers were Lmcdc2sens : 5'GAGCTAGCAAAGGATCCTCGTCTAATCA 3' and Lmcdc2 reverse: 5' CAGCTAATATACAGGTCAGGGTACAAAACC 3'; these primers amplified a fragment of 587 pb. Accession number for Lmcdc2 is DQ988343. For L1 and L2, we used a set of two primers CAT6 sens 5' CGTGGCGTGACATTCTACAG and CAT7 reverse 5' TTGGGGTGAGAATCCTGATG which allowed to visualize two fragments: a short fragment (800 pb) corresponding to LmAP2L1 and a large fragment (1000 pb) corresponding to LmAP2L2 in the same PCR reaction (25 cycles and hybridization temperature 52°C). Gel images were treated by the Image Quant TL (Amersham, UK) and the ratio: volume of control band (Lmcdc2) on volume of band corresponding to LmAP2L1 or LmAP2L2 was calculated. A ratio closed to 1 corresponds to a strong expression of transcripts.

The deduced amino acid sequences of two-AP2 genes were obtained from NCBI DNA databases. Sequences were aligned with the program CLUSTALW (1.83). Positions including gaps due to the alignment were removed. So, for phylogenetic analysis, 31 sequences and 133 positions were retained comprising the two AP2 domains and the linker. Maximum likelihood tree was obtained using the method implemented in the PROTML version 2.3 software (Adachi and Hasegawa, 1996). It was built by the quick-add Operational Taxonomic Units (OTUs) search, the Jones-Taylor-Thornton (JTT)-f model of amino acid substitution and retaining the 50-top ranking trees. Bootstrap proportions were calculated by the resampling estimated log-likelihood (RELL) method upon all top-ranking trees (Kishino et al., 1990). The best tree has a lnL of -1869.8.

Results and discussion

Primers for cloning both forms of larch two-AP2-domain genes were designed from the sequences of Norway spruce and black pine (Vahala et al., 2001;Shigyo and Ito, 2004). The cDNA encoding LmAP2L1 was entirely characterized and encoded a putative protein of 529 aa containing the two-AP2 domain and a potential miR172 binding site (Figure 1A and1C). Furthermore, LlAP2L1 sequence contained a conserved domain downstream the AP2 domain which function remains unknown (Figure 1B). This motif named C domain by Vahala et al. (2001) could be considered as a signature of Pinacea L1 forms. Indeed, the 10 aa motif (DPDLDLSLGNS) showed an aspartic acid in the third position which was only found in the gymnosperm L1 forms and monocotyledon plants like sweet potato, rice and hyacinthus whereas all the other plant sequences had an asparagine. In fact, LmAP2L1 putative amino acid sequence showed 86.59 % identity with PtAP2L1 and PaAP2L1. In contrast, LmAP2L1 presented only 30.77 % identity with N terminal truncated putative sequence encoded by LmAP2L2. Similar divergence was also observed for the L2 and L1 forms of pine and spruce species. Nevertheless, LmAP2L2 amino acid partial sequence (404 aa) showed a greater 2). A 21 nucleotide micro-RNA, miRNA172, was completely complementary to Arabidopsis AP2 form which suggests that AP2L1 was its target (Chen, 2004). This was also shown for the regulation of glossy15 encoded a two-AP2 domain protein from maize (Lauter et al., 2005;Nilsson et al., 2007). L1 forms from Norway spruce, pine and hybrid larch had this site totally complementary and located at 3' end of cDNA. This motif was also described in primitive spermaphytes such as Ginkgo biloba, Cycas revoluta (Kim et al., 2006). Thus, a translation regulation by miRNA172 could down-modulate LmAP2L1 but further investigations should be necessary to show it and greatly interesting to enlarge the role of RNA interference in spermaphyte kingdom. By contrast, this site was really more divergent for the three AP2L2 forms suggesting that this protein was probably not regulated by translational regulation as we suppose it is the case for L1 forms.

Due to the high degree of amino acid conservation for AP2 domains and the fact that AP2 is a multigenic family, numerous nodes in the phylogenetic tree received low bootstrap supports weakening in some cases the evolutionary interpretation. Taking together, the 7 Pinaceae double AP2-domain proteins showed over the alignment portion more than 81% of identity. However, the three L1 forms were clearly orthologs, with a clade supported by bootstrap proportion (BP) of 100%. The L2 forms looked more divergent (Figure 1 and Figure 2). Indeed, they did not form a monophyletic group in the tree. Only LmAP2L2 and PaAP2L2 could be recognized as orthologs with BP of 100%. PtAP2L2 did not cluster with its counterparts. Two explanations could be proposed (i), the sequence of L2 form in pine is divergent due to a change in the selective pressure acting on the gene, or (ii) the sequence corresponds to a paralogous gene and the real ortholog of L2 form in pine had not been identified yet. A third form was recently described in Picea abies (Nilsson et al., 2007). PaAP2L3 is undoubtedly the sister group of Gnetum parvifolium AP2, with a bootstrap proportion of 98%. Considering the phylogenetic link between gnetophytes and conifers (Hajibabaei et al., 2006) and the apparent single AP2 form present in G. parvifolium, Cycas revoluta and Gingko biloba (Shigyo et al., 2006), we hypothesized that L1 and L2 forms in Pinaceae could have arisen by lineage-specific duplications of an ancestral AP2L3-like gene.

We studied hybrid larch early and late somatic embryogenesis. Early somatic embryogenesis essentially corresponded to EM on proliferation medium, and late somatic embryogenesis corresponds to somatic embryo differentiation on activated charcoal and ABA media [START_REF] Von Aderkas | Woody Plant Biotechnology[END_REF]. EM was a very proliferative tissue which contains a suspensor with very long cells and embryonal mass with actively dividing cells (Figure 3A). Detection of LmAP2L1 and LmAP2L2 transcripts was performed by RT-PCR analysis on 69-18 EM. LmAP2L2 was highly expressed in EM (Figure 3B). Strong expression of PaAP2L2 was also observed in Norway spruce EM which is consistent with the fact the two forms are orthologous (Vahala et al., 2001). Moreover LmAP2L1 and PaAP2L1 transcripts were never detected in EM (Figure 3B; Vahala et al., 2001).

To gain insight into both patterns of gene expression, we induced somatic embryo differentiation from EM (Figure 3A). While LmAP2L2 expression decreased when EM was placed under activated charcoal condition, it rapidly increased after one week on ABA which corresponds to the beginning of the formation of globular shape somatic embryos (Figure 3A-B). As described for PaAP2L2, LmAP2L2 expression remained high and relatively constant when differentiation occurred in 2 to 3 week-old embryos, which, by this time, had cotyledons and meristems (Figure 3A). As LmAP2L2 was then constitutively expressed, it was not a molecular marker of hybrid larch somatic embryogenesis. In contrast, LmAP2L1 expression started to be detected in 2 week-old somatic embryos which were not able to . This result suggests that LmAP2L1 could be a molecular marker of hybrid larch late somatic embryogenesis which did not seem to be the case for its ortholog PaAP2L1 (Vahala et al., 2001). LmAP2L1 expression corresponded to the moment when embryos started to store lipids and proteins and increase in mass (Lelu-Walter, personal communication). Indeed after 3 wks on ABA, mature embryos were able to germinate because they accumulated storage useful for further germination (Lelu andLabel 1994, Gutmann et al., 1996;Label and Lelu, 2000). Our hypothesis is that, during late somatic embryogenesis, LmAP2L1 expression was linked to the capacity of embryos to germinate which was also a role suggested for Arabidopsis two-AP2 domain protein (Ohto et al., 2005). Nevertheless, even when embryos matured after 6 wks on ABA, both transcript forms were detected although LmAP2L2 expression level was always higher than LmAP2L1 (Figure 3B). As LmAP2L1 remained expressed in very old embryos (12 week-old), it might have further roles that we should investigate.

Early vegetative growth was monitored on 4 week-old somatic embryos after 14 and 25 days of germination. After 25 days of germination, young conifers had green needles and well developed roots. Whereas LmAP2L1 and LmAP2L2 transcripts were strongly detected in somatic embryos germinating for 14 days (ratios closed to 1), both mRNA expression levels decreased after 25 days of germination. This was particularly observed for LmAP2L2 whose expression remained difficult to detect (Figure 3C). As AP2 proteins were known to be involved in organ formation, this result suggested that L1 and L2 forms could also act in further mechanisms. This was also consistent with previous work published by Jofuku et al. (2005) where APETALA2 was also detected in Arabidopsis leaves and stem.

Conclusions

So far, the two hybrid larch cDNAs encoding two-AP2 domain proteins constitute the first set of molecular data for this species. We showed that LmAP2L1 is probably a good molecular marker of somatic late embryogenesis while LmAP2L2 was constitutively expressed during this fundamental process of plant cell life. Nevertheless, LmAP2L2 expression seemed to disappear during post-embryogenic development while LmAP2L1 remained quite highly expressed. Expression of LmAP2L1 was clearly detected when embryos accumulate lipids and proteins and thus able to properly germinate. Furthermore, LmAP2L1 with its orthologs PaAP2L1 and PtAP2L1 constitute a gymnosperm specific sub-group. LmAP2L2 was also orthologous to PaAP2L2. As both forms were not regulated in the same manner at the transcriptional level, they are probably not redundant in function. Nevertheless, they could also be regulated at the translational level via microRNA172 especially LmAP2L1. It should be interesting to gain insight the role(s) of both forms by RNAinterference and transgenesis (Gleeson et al., 2005;Mathieu et al., 2006). Further investigations should be necessary to understand the roles of these two forms during embryogenesis and vegetative development. In bold the new sequences, LmAP2L1 and LmAP2AL2, described in this paper. R1 (yellow box) and R2 (blue box) correspond to sub-domains of the AP2 double domain (Kim et al., 2006), minus 2 amino acids at N-terminal and 6 at C-terminal, respectively. A 25 amino acid region also called linker region is located between R1 and R2 sub-domains. Stars point conserved residues and arrow heads, pertinent positions to discriminate L1, L2 and L3 forms. Are also shown for a given position, in green similar residues and in red non similar ones. B) A conserved domain is just found downstream to the AP2 double domain. Its consensus sequence is The majority of these sequences except Amborella and Dendrobium were also described in previous works (Kim et al., 2006;Shigyo et al., 2006). Bootstrap values are indicated for branches supported by at least 50 %. The scale bar corresponds to 0.1 amino acid substitution per position. LmAP2L1 and LmAP2L2 of larch are in bold. LmAP2L2 and the lower band to LmAP2L1. Lanes 1-2 correspond to 4 week-old somatic embryos after 25 and 14 days of germination, respectively. Lane 3 corresponds to 4 week-old somatic embryos non germinated. Normal condition of germination takes place under 16 h photoperiod. Numbers under pictures correspond to calculated ratios of LmAP2L2 (first line) and LmAP2L1 gene expression levels compared to hybrid larch Lmcdc2 expression level, respectively. nd : not determined. Gene expression pattern was checked three times using three independent sets of plant material.

D P/Q D/N L D L S L G N/I S/
Comment citer ce document : Guillaumot, D., Lelu

Figure 1 :

 1 Figure1: Alignment of conserved regions of the two-AP2 domain proteins in conifer species, Larix x marschlinsii (Lm), Picea abies (Pa) and Pinus thunbergii (Pt). A) Portion of the alignment used for the phylogenetic reconstruction. In bold the new sequences, LmAP2L1 and LmAP2AL2, described in this paper. R1 (yellow box) and R2 (blue box) correspond to sub-domains of the AP2 double domain(Kim et al., 2006), minus 2 amino acids at N-terminal and 6 at C-terminal, respectively. A 25 amino acid region also called linker region is located between R1 and R2 sub-domains. Stars point conserved residues and arrow heads, pertinent positions to discriminate L1, L2 and L3 forms. Are also shown for a given position, in green similar residues and in red non similar ones. B) A conserved domain is just found downstream to the AP2 double domain. Its consensus sequence is D P/Q D/N L D L S L G N/I S/G with PD and QN couples specific to L1 group and L2 group respectively. C) Putative binding site of miRNA172 in the cDNA of the 7 two-AP2 forms from Pinaceae. The nucleic acids that are identical to the complementary DNA of the miR172 are in bold. GenBank accession numbers for the different forms of AP2 genes are reported in material and methods section.

Figure 3 :

 3 Figure 3: LmAP2L1 and LmAP2L2 gene expression patterns during somatic embryogenesis and germination of hybrid larch. RT-PCR was performed with primers allowing amplification of L1 and L2 forms in the same reaction. Upper band corresponds to LmAP2L2 (1000 bp) and the lower band to LmAP2L1 (800 bp). A) Pictures of EM and developing somatic embryos. Upper panel, from left to right: white EM (bar: 1 mm), EM placed on activated charcoal for 1 wk (AC, bar: 0.3 mm), embryos on ABA for 1 to 2 wks (bar: 0.5 mm), for 3 wks (bar: 0.5 mm) and for 4 to 12 wks (bar: 0.75 mm). Pictures were taken under binocular microscope under visible light. Lower panel: 7 day-old EM observed

  -Walter, M.-A., Germot, A., Meytraud, F., Gastinel, L., Riou-Khamlichi, C. (2008). Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii). Journal of Plant Physiology, 165 (9), 1003-1010. DOI : 10.1016/j.jplph.2007.08.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Comment citer ce document : Guillaumot, D., Lelu-Walter, M.-A., Germot, A., Meytraud, F., Gastinel, L., Riou-Khamlichi, C. (2008). Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii). Journal of Plant Physiology, 165 (9), 1003-1010. DOI : 10.1016/j.jplph.2007.08.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Comment citer ce document : Guillaumot, D., Lelu-Walter, M.-A., Germot, A., Meytraud, F., Gastinel, L., Riou-Khamlichi, C. (2008). Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii). Journal of Plant Physiology, 165 (9), 1003-1010. DOI : 10.1016/j.jplph.2007.08.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript

  Author manuscript percentage of identity with the gymnosperm L2 forms (around 44 %) especially with PaAP2L2. This result linked to the phylogenetic analysis suggested that LmAP2L2 and PaAP2L2 are orthologs (Figure

Comment citer ce document : Guillaumot, D., Lelu-Walter, M.-A., Germot, A., Meytraud, F., Gastinel, L., Riou-Khamlichi, C. (2008). Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii). Journal of Plant Physiology, 165 (9), 1003-1010. DOI : 10.1016/j.jplph.2007.08.009 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur /

  G with PD and QN couples specific to L1 group and L2 group respectively. C) Putative binding site of miRNA172 in the cDNA of the 7 two-AP2 forms from Pinaceae. The nucleic acids that are identical to the complementary DNA of the miR172 are in bold. GenBank accession numbers for the different forms of AP2 genes are reported in material and methods section.

	Figure 2: Maximum likelihood tree of the two-AP2 domain subfamily. Phylogenetic
	reconstruction was based on amino acid comparison of 31 double AP2-domain-containing
	genes selected from representative gymnosperms and angiosperms. It is rooted on the
	Aintegumenta (ANT) group, considering the existence of two monophyletic groups, AP2 and
	ANT, in the AP2 subfamily (Shigyo and Ito, 2004). GenBank accession numbers for the AP2
	group are: CV006845 for Amborella trichopoda, AP2 NP_195410 and TOE1 NP_565674 for
	Arabidopsis thaliana, LIPLESS1 AAO52746 and LIPLESS2 AAO52747 for Antirrhinum
	majus, CrAP2L1 BAE48512 for Cycas revoluta, AP2 AAZ95247 for Dendrobium
	crumenatum, GbAP2L1 BAE48514 for Gingko biloba, GpAP2L1 BAE48516 for Gnetum
	parvifolium, HvAP2L1 AAL50205 for Hordeum vulgare, HAP2 AAD22495 for Hyacinthus
	orientalis, InAP2B BAD36744 for Ipomoea nil, LmAP2L1 DQ988340 and LmAP2L2
	DQ988341 for Larix x marschlinsii, AHAP2 AAL57045 for Malus x domestica, AP2D23
	AAW78371 for Oryza sativa, PhAP2B AAD39440 for Petunia x hybrida, PaAP2L1
	AAG32658, PaAP2L2 AAG32659 and PaAP2L3 AAY79346 for Picea abies, PtAP2L1
	BAD16603 and PtAP2L2 BAD16604 for Pinus thunbergii. Genbank accession numbers for
	the ANT group are: AINTEGUMENTA AAA91040, BABY BOOM AAM33803 and
	PLETHORA1 AAS86335 for Arabidopsis thaliana, BABY BOOM1 AAM33802 for
	Brassica napus, CrANTL1 BAE48513 for Cycas revoluta, GbANTL1 BAE48515 for Gingko
	biloba, (1) NP_915190 and (2) XP_474162 for Oryza sativa, PtANTL1 BAD16602 for Pinus
	thunbergii.

  fluorescence microscopy. DAPI staining was performed for observation of nuclei from EM and its suspensor. Bar: 100 μm. B) Expression of LmAP2L1 and LmAP2L2 gene transcripts during somatic embryogenesis of hybrid larch embryogenic line. EM; AC : EM placed on activated charcoal for one week; Lanes 1 to 7 correspond to embryos placed for 1 to 7 wks on ABA medium. Lane 8 corresponds to 12 wk-old embryos. C) Gene expression patterns of LmAP2L1 and LmAP2L2 during germination. Upper band corresponds to

	under visible and
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript

). Journal of Plant Physiology, 165 (9), 1003-1010. DOI : 10.1016/j.jplph.2007.08.009

Acknowledgements

We are grateful to Pr A. Oulmouden and L. Forestier for their help for cDNA sequencing. This work was supported by a Grant from the region Limousin (France).