Nazzareno Diodato

Luca Brocca

Gianni Bellocchi
email: giannibellocchi@yahoo.com

Francesco Fiorillo

Francesco Maria Guadagno

Libera Esposito

Luisa Vernacchia

Complexity-reduction modelling for assessing the macro-scale patterns of historical soil moisture in the Euro-Mediterranean region

Keywords: Campania Plain (Italy), Nitrate, Probability Kriging

  L'archive ouverte pluridisciplinaire

Introduction

Rainfall is the main source for replenishment of groundwater resources that is the water taken into the ground after having saturated the soil [START_REF] Buchan | Pollution of Ground Water in Europe[END_REF]. Soil profile evolution follows land-use changes, with implications for groundwater quantity and quality [START_REF] Huang | Soil Profile Evolution Following Land-Use Change: Implications for Groundwater Quantity and Quality[END_REF]. Effective rainfall moves into the soil associated with point-source contaminants such as chloride and nitrogen compounds. This has occurred at high rates since the 1950s, with intensification of agriculture and urbanization [START_REF] Visser | Comparison of Methods for the Detection and Extrapolation of Trends in Groundwater Quality[END_REF]. In agricultural areas, in particular, an excessive use of fertilizers has directly or indirectly affected the groundwater quality [START_REF] Böhlke | Groundwater Recharge and Agricultural Contamination[END_REF]. The main cause of sub-surface water pollution in Europe is the input of nutrients to agricultural land (inorganic fertilisers and manure), and point-sources of pollution (organic and industrial waste) exceeding the output of nutrients from agricultural land (mainly harvested crop yield) and biological depuration, respectively [START_REF] De Wit | The Contribution of Agriculture to Nutrient Pollution in Three European Rivers, with Reference to the European Nitrates Directive[END_REF][START_REF] Kraft | Nitrate Impacts on Groundwater from Irrigated-Vegetable Systems in a Humid North-Central US Sand Plain[END_REF][START_REF] Nas | Groundwater Contamination by Nitrates in the City of Konya (Turkey): A GIS Perspective[END_REF].

Natural reactions of atmospheric forms of nitrogen with rainwater result in the formation of nitrate ( 3 NO  ) and ammonium ( 4 NH  ) ions [START_REF] Schröder | The Effects of Nutrient Losses from Agriculture on Ground and Surface Water Quality: The Position of Sci-ence in Developing Indicators for Regulation[END_REF]. Nitrate is a common compound, naturally generated from the nitrogen cycle. However, anthropogenic sources have greatly increased the 3 NO  concentration, particularly in groundwater [START_REF] Chand | Nitrate Pollution: A Menace to Human, Soil, Water and Plant[END_REF][START_REF] Vomocil | Fertilizers: Best Management Practices to Control Nutrients[END_REF]. The largest anthropogenic sources are septic tanks, nitrogen-rich fertilizers applied to turf grass, and agricultural processes. Nitrogen fertilizers are extensively applied in agriculture to increase crop production, but excess nitrogen supplies can cause air, soil, and water pollution. Arguably, one of the most widespread and damaging impacts of agricultural over-application of nitrogen fertilizers is the degradation of groundwater quality and contamination of drinking water supplies, which can pose immediate risks to human health [START_REF] Hallberg | Nitrate[END_REF][START_REF] Lord | Agricultural Nitrogen Balance and Water Quality in the UK[END_REF][START_REF] Rail | Groundwater Contamination: Sources, Control, and Preventive Measures[END_REF]. When nitrate-nitrogen concentrations reach excessive levels there can be harmful biological consequences for the organisms in the groundwater habitat. Human interest is of primary concern when setting guidelines for acceptable ni-trate levels and proper agricultural practices. However, though climate change has resulted in discharge depletion during the last decades, the advancements occurred in agricultural technology has accelerated the total flux of nitrogen in the soil across European western regions (Figure 1(a)).

Vulnerable lands for high nitrate concentration are in turn recorded in Italy (red colour in Figure 1(b)).

Although studies have been performed attempting to link nitrate consumption to human illnesses, only for methemoglobinemia (also infant cyanosis or blue-baby syndrome) ingestion of water containing high nitrate concentrations proved to be a significant cause. Since 1945, there have been over 2000 cases of infant methemoglobinemia reported in Europe and North America with 7% -8% of the afflicted infants dying [START_REF] Johnson | Fatal Outcome of Methemoglobinemia in an Infant[END_REF]. However, problems can be severe as shown in a 1950 report, documenting of 144 cases of infant methemoglobinemia with 14 deaths in a 30-day period in Minnesota, USA [START_REF] Johnson | Fatal Outcome of Methemoglobinemia in an Infant[END_REF]. Even if this case was isolated, it points to the fact that groundwater can be used only when the quantity and quality is properly assessed [START_REF] Kharad | GIS Based Groundwater Assessment Model[END_REF]. For this, quality control becomes an important issue for the assessment of aquifers, especially those subject to both point and nonpoint pollution sources. Geographic Information Systems (GIS) have been used in the map classification of groundwater quality, based principally on modelling approaches and vulnerability methods [START_REF] Butler | Ground-Water Quality Classification Using GIS Contouring Methods for Cedar Valley, Iron County, Utah[END_REF][START_REF] Asadi | Remote Sensing and GIS Techniques for Evaluation of Groundwater Quality in Municipal Corporation of Hyderabad (Zone-V), India[END_REF][START_REF] Balakrishnan | Groundwater Quality Mapping Using Geographic Information System (GIS): A Case Study of Gulbarga City, Karnataka[END_REF]. There are, however, situations with large uncertainty, or in which a particular pollutant can be diluted in a zone and accumulated in an-other, also depending on the delay time-or migration time-that can operates for this purpose. Uncertainty, migration times and spatial patterns, interconnected with both the recharge of the acquifer and the modality of monitoring, could however be not discernible by standard GIS or parametric approaches in hydro-geostatistics, such as ordinary kriging (OK). Aware of this, some hydrogeological scientists have proposed softer approaches based on simulation or indicator kriging (IK) to attenuate spatial uncertainty in groundwater quality mapping [START_REF] Carlson | Geostatistical Analysis and Simulation of Nonpoint Source Groundwater Nitrate Contamination: A Case Study[END_REF][START_REF] Liu | Evaluation of Arsenic Contamination Potential Using Indicator Kriging in the Yun-Lin Aquifer (Taiwan)[END_REF][START_REF] Diodato | Computational Uncertainty Analysis of Groundwater Recharge in Catchment[END_REF][START_REF] Piccini | Application of Indicator Kriging to Evaluate the Probability of Exceeding Nitrate Contamination Thresholds[END_REF].

Considering the above aspects of groundwater contamination and use of non-parametric hydro-geostatistical approaches in groundwater quality mapping, the present study was undertaken to map the groundwater quality in Campania Plain (Southern Italy) using Box-Cox probability kriging (PK). The main objective of this work is to make groundwater quality assessment based on the available physical-chemical data from 158 locations. The purposes of this assessment are (1) to provide an overview of present groundwater quality, and (2) to determine spatial distribution of groundwater quality parameters such as nitrate (NO 3 ) to generate groundwater quality probability map for a target zone (the Campania Plain, southern Italy).

Materials and Methods

Climatological and Geological Setting

The study area (approximately 500 km 2 ) is the southern Climate is very variable in spring and autumn and more stable in winter and summer. Dry season, from May to September, not enable to recharge the soil before of November-December, in order to inter-annual variability. A typical seasonal trend of different terms of water balance is illustrated in Figure 3, where the climogram for the Campania Plain describes the humid period (October-March) followed by the dry-summer season (June-August). Transitional periods (April-May and September) are the growing seasons where vegetation is green.

The graben system of the area began to form during the Pliocene, between Mesozoic carbonate sequence outcropping to the east and south of the plain. During the Pleistocene this structural depression was filled with pyroclastic deposits (ashes, pumice, scoriae and tuffs) of Neapolitan volcanoes (Phlegraean Fields and Somma-Vesuvius) and alluvial (mainly sandy and partly clayey) and marine (mainly silt) sediments to a thickness of some thousands of metres. The first few hundred metres beneath the soil of the circumvesuvian plain, the zone of the most active groundwater circulation, are made up of the pyroclastic deposits (ashes, pumice and scoriae) and alluvial deposits interposed with marine sediments marshy layers and paleosols. The tuff aquitards divide the flow into two overlapping levels (Figure 4).

Travertine, debris and conglomerates lie at the base of the limestone mountains, while approaching the Somma-Vesuvius volcano lava flows prevail, interbedded with pyroclastic deposits.

The carbonate aquifers are the most important aquifers located at the boundaries of the plain. The Somma-Vesuvius volcano has a radial water table. Groundwater flows towards the sea and also feeds the aquifer of the surrounding plain. The aquifer of the Campania Plain is characteristically extremely heterogeneous due to granulometric variation in the unconsolidated sediments, the degree of fissuring of rock and complex stratification of deposits.

The map (Figure 5(a)) shows that the aquifer of the Campania Plain flows from NE to SW and highlights the presence of one axe of groundwater drainage to the East of Naples. There are two groundwater divides: the first is between San Giuseppe Vesuviano and Palma Campania (in SE of the map) and the second is between Cancello and Caivano (in NW of the map).

The hydraulic gradient varies from a few units per thousand to a few units per hundred. In particular, the highest values of the piezometric gradient found at the foot of Lattari mountains, may be related to high transmissivity from carbonate aquifers (10 -1 -10 -2 m 2 •s -1 ) compared with values in other areas the plain (10 -2 -10 -4 m 2 •s -1 ).

The main contributions to aquifer recharge are provided by direct infiltration and groundwater inflow from the nearby volcanic and carbonate aquifers. The mean annual effective infiltration is of 59.6 × 10 6 m 3 •yr -1 [START_REF] Esposito | Groundwater Flow Evolution in the Circum-Vesuvian Plain (Italy)[END_REF]. In the Figure 5(b) is shown the map of land-use, with the urban and agricultural activity covering most of the study area. The urban areas are developed, however, in the absence of infrastructure networks. Therefore, we assume a deep percolation of organic waste in the aquifer.

Nitrate Sampling

Groundwater pollution sampling was initiated since 1992 . in Campania Plain and continued in 2001 and 2006 years [START_REF] Celico | Limiti di Applicabilità Delle Carte Della Vulnerabilità All'Inquinamento Degli Acquiferi Nella Previsione Dello Stato di Contaminazione Antropica Delle Acque Sotterranee[END_REF][START_REF] Corniello | Origine Dell'Inquinamento da Nitrati Nelle Falde Dell'Area di Acerra (Piana Campana)[END_REF]. However, only in 1992 a wide and resolute sampling was conducted covering a surface of about 500 km 2 .

In 158 wells used for the reconstruction of the paper in the aquifer isopiezometric curves were also collected water samples for the determination of nitrate concentrations. The analysis refers to the period of low water of the aquifer. Nitrate in water samples was colorimetrically measured [START_REF] Fishman | Methods for Determination of Inorganic Substances in Water and Fluvial Sediments[END_REF][START_REF] Fishman | Methods of Analysis by the US Geological Survey National Water Quality Laboratory-Determination of Inorganic and Organic Constituents in Water and Fluvial Sediments[END_REF]. The wells were managed by one of the leading institutions of central-southern aqueduct (ARIN, Naples Water Resources Company, http://www.arin.na.it).

Probability Kriging

Kriging and its derivatives have been recognized as the main spatial interpolation techniques from 1970s. Kriging is a method for making optimal, unbiased estimates of regionalized variables at unsampled points. It is possible to have a good estimation of the selected variable using the values collected in the surrounding stations and a structural analysis [START_REF] Journel | Mining Geostatistics[END_REF]. Probability kriging (PK) is a specific method belonging to the family of kriging, introduced by as a non-linear and non-parametric method using indicator variables [START_REF] Sullivan | Conditional Recovery Estimation through Probability Kriging-Theory and Practice[END_REF]. PK represents an effort for calculating estimates that are less sensitive than indicator kriging to the choice and number of cut-offs (thresholds) and estimates better reflecting local variability [START_REF] Hohn | Petroleum and Geostatistics[END_REF]. PK is a composite kriging of the indicator data using the rank-order transform as additional variable [START_REF] Goovaerts | Geostatistics for Natural Resources Evaluation[END_REF]. Replacing the values of the primary variable in PK by indicator data and using the rank-order transform data, the indica-

tor code,  , α k  I z u
, is assigned as the pollutant variable [START_REF] Deutsch | GSLIB Geostatistical Software Library and User's Guide[END_REF][START_REF] Johnston | Using ArcGis Geostatistical Analyst[END_REF]. A vector-based GIS software package ArcGIS 9.2 was used to map, query, and analyze the data in this study.

Decision-Making in the Presence of Uncertainty

In the present paper, environmental risk assessments related to nitrate pollution are derived from the probability that a pollutant leaching rate can migrate from sub-surface to groundwater. The selected thresholds or the action level often determine the introduction or not of certain measures. The concentration in natural water is less than 10 mg•L -1 . Water containing more than 100 mg•L -1 is bitter to taste and causes physiological distress. Water in shallow wells containing more than 50 mg•L -1 causes methemoglobinemia, the so-called blue baby syndrome in humans [START_REF] Balakrishnan | Groundwater Quality Mapping Using Geographic Information System (GIS): A Case Study of Gulbarga City, Karnataka, India[END_REF]. Availability of the regionalization between indicator pollutant at nitrate threshold concentrations for each location s α within the study area would allow a grid layer α(s α ) referring to "probable hydrogeological effectiveness" of the nitrate pollutant on the basis of the estimate

  ; α k PK when actually I z    s
groundwater quality, and the second, z k2 = 50 mg•L -1 , which represents the maximum values for the drinking groundwater quality.

Results and Discussion

The ability to identify the true spatial variability of a dataset depends to a great extent, on ancillary knowledge of the underlying measured phenomenon. This is why exploratory data analysis is often the first step in hydrogeostatistical studies. Postplot, initial contour maps and basic statistics are used as a preliminary description of the dataset in a spatial context and to develop a strategy for future evaluation [START_REF] Carlson | Geostatistical Analysis and Simulation of Nonpoint Source Groundwater Nitrate Contamination: A Case Study[END_REF].

Distribution of the Data

The presence of nitrates is considered an indicator of pollution in the Campania Plain because of the intensive agricultural land use, urbanization and industrial practices throughout the area. In several wells, nitrate concentrations exceed the maximum allowable concentration (50 mg•L -1 ) as set by the Italian law. The statistics of nitrate data collected in October 1993 are summarized with mean and median values equal to 57 mg•L -1 , and 43 mg•L -1 , respectively. The maximum value is 239 mg•L -1 . Figure 6(a) shows a strong skewed distribution. This is why a Box-Cox transformation (with 0.4 transformation parameter) was adopted. The transformation results in a quasinormal distribution (Figure 6(b)).

Spatial Structural Modelling

A two-stage model of regionalization was fitted using an iterative procedure [START_REF] Goovaerts | Geostatistics for Natural Resources Evaluation[END_REF]. Stage 1 assumes an isotropic model and executes a first run of the experimental spatial structures on the scaled data,  

    1 s s z z z       
, where z(s α ) is used to denote the j th measurement of a variable at the α th spatial locations s α , and σ is the sample standard deviation. With stage 2, numbers such as lag (assumed equal 7), lag size h (assumed equal to 2000 m), and range (a) are set to represent the limit of spatial dependence, while nugget is set to represent the unexplained spatial variability, calibrated interactively. Also at, this stage it has been assumed an isotropic semivariogram model for the pollutant indicator spatial pattern. In this way, Figure 7 shows the experimental semivariogram (dots) computed from 158 nitrate data, with spherical permissible models fitted (blue curves).

Semivariogram values increase with the separation distance, reflecting the assumption that nearby nitrate-pollutant data tend to be more similar than data that are farther apart. The modelled variograms have been tested by the cross-validation method. The semivariograms reach 2000 to 10,000 m before dipping around a sill value.

    k Z z   s
. The European Commission suggested two limits for nitrates: a first threshold, z k1 = 25 mg•L -1 , which represents the minimum concentration needed to overcome the drinking

                  h h h h h
The three spatial structures for high nitrate concentration lead us to believe that there are different sources of pollution resulting from the landscape overlying the aquifer. At the local level the presence of nitrates may result from direct leakage into the groundwater of organic waste. At larger range, instead, nitrate may have derived from non-point source as agricultural manures.

The symbolic term in Equations ( 3) and ( 4),

  Sph , a     h , is the spherical correlation function equal to  (3) 3 3 1 2 2 h h a a                        .       2
                    h h h h h a  h (4)
It represents a dimensional semivariance of unit sill with ranges given by the circle with a 1 = 10000, z k1 = 25 mg•L -1 , and a 2 = 3000 a 3 = 10000 meters, for z k2 . Ideally, the value of the semivariogram should reach a minimum value when the separation vector h is zero. In the case study, this is not true firstly because the measurement error exists in nitrate sampling data, and secondly because the distribution and number of wells could be poor. The range relative to the large threshold, z k2 = 50 mg•L -1 , has a minor nugget than with z k1 = 25 mg•L -1 but the nugget/total sill ratio is roughly the same (0.46 -0.47), which indicates moderate spatial correlation for both the thresholds.

Spatial Pattern of Estimation and Hazard Classification

Figures 8(a) and (b) show probability-kriged maps, based on 500 m by 500 m grid across Campania Plain. In kriged model pattern area of Figure 8(a), it is possible to observe a distinct separation between East (minor) and West (major) zone of nitrate map, which is consistent with the pattern of groundwater flow outlined in par. 2.1. The areas having probability of 50% that the nitrate concentration exceeded 25 and 50 mg•L -1 are narrow, intermediate bands (in light blue). This indicates that nitrate concentration is characterized by high gradient when its concentration is near the threshold values.

Especially in the map of Figure 8(a), it appears a distinct separation between little free zone at East (blue) and a large polluted area at West (red-orange-yellow) including Naples metropolitan area. This map accounts for areas exceeding 25 mg•L -1 for about 70% of the total surface.

In Figure 8(b), it is mapped the probability that nitrate concentration exceeded the 50 mg•L -1 , with high probability that remains at West, although not all areas are strongly polluted. In this case, areas with very high probability are aligned along a diagonal line crossing Maddaloni, Acerra, Lufrano and Naples municipalities.

As already outlined [START_REF] Celico | Limiti di Applicabilità Delle Carte Della Vulnerabilità All'Inquinamento Degli Acquiferi Nella Previsione Dello Stato di Contaminazione Antropica Delle Acque Sotterranee[END_REF], this trend takes into account the groundwater inflow that is carried out in correspondence of the foothills (cf. par. 2.1), but also the concentration of the pollutant that originates from the surface and effective infiltration. Due to the source of surface contamination, the pollutant load in the vicinity of the foothills is not particularly high, when compared to that of more central areas of the plain. This is due both to the limited extension of the foothills, on which the effective infiltration of rain takes place, and to a major capacity of denitrification of the plain subject to agricultural manure. In the most central part of the plain, there has been a general increase in the spread of nitrates due to the flow of water and potential pollution. Comparing land-use chart and nitrate probability maps, we can see an increase in hazard only where intensive agriculture mixes with more urbanized areas.

At the time of sampling, the only areas remaining free from high concentrations can be individuated between the surrounding of Palma Campania, Nola, Marigliano and Roccarainola municipalities. Local patterns with high nitrate concentration were also founded in Somma Vesuviana and Palma Campania, especially in PK [>50 mg•L -1 ].

However, no spatial pattern variability can be detected below 2000 m, due to nugget effect. This represents unexplained or random variance, which is either caused variability of data that cannot be detected at the scale of sampling, or measurement errors within Campania Plain. Although the monitoring phase refers to the period of lean ground when less effective infiltration is present, the maps presented here suggest a dramatic pattern with still high probability of exceeding the above limits. Therefore, it is suspected that nitrate concentrations in well water can be high during any month of the year and the issue of nitrate contamination and health effects should not be ignored.

Cross-Validation Results and Spatial Error

Qualitative Assessment

The error involved on the expansion of the information from point to landscapes through probability kriging es- If we consider OK experiment, measurements versus the predicted values of nitrate are in disagreement (see the scatter diagram of Figure 9(c)). Also, mean error is greater than 0.01, although the other errors are comparable with PK approach. However, a large bias remains in the relative scatter plot, indicating that OK is not suitable for interpreting spatial pattern in Campania Plain. This study has attempted to predict the spatial distribution and uncertainty of groundwater nitrate concentration across some municipalities of the Campania Plain (Southern Italy). Probability kriging, a type of nonparametric geostatistical techniques, was applied to the groundwater nitrate hazard data for two distribution maps related to the threshold values of 25 and 50 mg•L -1 , respectively. Geostatistics can provide tools to describe spatial and hazard behavior of hydrochemical parameters. Groundwater nitrate concentrations were log-normally distributed. The spherical model was found to be the best model representing the spatial variability of groundwater probability nitrate maps. The average value of the variograms for the spatial analysis was in a range of 2000 -10,000 m in the spherical model. Nitrate pollution in the groundwater occurred most in the urban and periurban centre of the municipalities because of nitrate excess from biological, agricultural and, secondarily, industrial production. Although the modelling results indicate that the probability kriged groundwater nitrate maps satisfactorily matched the observed groundwater nitrate distribution, a newer and more continuos sampling is needed for reaching the areas with more hazard.

Discussion and Conclusions

Figure 1 .

 1 Figure 1. (a) World total nitrogen flux per grid cell (50 × 50 km) (data from http://wwdrii.sr.unh.edu/download.html), and (b) itrate vulnerable area with concentration above 50 mg•L -1 (red) in Italy [37]. n

Figure 3 .

 3 Figure 3. Climogram with representations of the seasonal regime of deficit (yellow band) and surplus water (blue bands) from precipitation for Campania Plain (arranged by New-LocClim FAO software, http://www.fao.org/nr/climpag/pub/en0201_en.asp).

Figure 4 .

 4 Figure 4. Geological profile of Campania Plain (tv = vegetal soil; pz = pozzolane, tf = tuffs; IC = Ignimbrite Campana; s = sand; l = silt).

Figure 5 .

 5 Figure 5. (a) Map of isopiezometric of groundwater level and (b) land-use cover across the Campania Plain.

Figure 6 .

 6 Figure 6. (a) Statistical distribution of nitrate concentration for the original series and (b) Box-Cox transformed series (nitrate classes data along X-axis are in mg•L -1 ).

SemivarianceFigure 7 .

 7 Figure 7. (a) Experimental semivariance (dots) and related spherical models (curves) for z k > 25 mg•L -1 ), and (b) for z k > 50 mg•L -1 ). The semivariogram in (b) is composed by nugget effect plus two spherical structures. In particular, while unidirectional semivariograms in Figure 7(a) was modelled as a combination of two distinct spatial structures-nugget variance and a spherical structure for z k1 -the semivariogram in Figure 7(b) was composed by nugget plus two spherical structures for z k2 (Figure 6(b)) (see also Equations (3) and (4)). 

Figure 8 .

 8 Figure 8. Spatial patterns of kriged-probability maps exceeding the nitrate threshold-value of (a) 25 mg•L -1 , and (b) 50 g•L -1 across Campania Plain for October 1992. m

Figure 10

 10 Figure 10 shows that nitrate concentrations have increased compared to the first sampling in 1993. Analyzing both the graphs of Figure 10(a), it appears that nitrate concentration has increased both in time and in space. Therefore, a strong temporal persistence and enrichment of nitrate concentrations seems to be present in the aquifer. However the few sampling points of 2006 (black dots in Figure 10(b)) does not allow us to compare the relative map with the one developed in this work.This study has attempted to predict the spatial distribution and uncertainty of groundwater nitrate concentration across some municipalities of the Campania Plain (Southern Italy). Probability kriging, a type of nonparametric geostatistical techniques, was applied to the groundwater nitrate hazard data for two distribution maps related to

Figure 9 .

 9 Figure 9. Scatterplot of cross-validation between nitrate-measured and indicator probability kriging for the nitrate-thresholds of (a) 25 mg•L -1 , and (b) 50 mg•L -1 , and (c) for ordinary kriging. Vertical lines in both graphs (a) and (b) represent the respective thresholds. In graph (c), a large bias exists for ordinary kriging application.

Figure 10 .

 10 Figure 10. (a) Trend of nitrate concentration in Somma Vesuviana from 1991 to 2004 [38], and (b) iso-nitrate pattern upon ampania Plain in 2006 (from ENEA, http://eboals.bologna.enea.it/ambtd/regi-lagni/volume-2/3-vol2-ac_sot_all6.html). C

  ).

	 				 
	2.75				2.75
	2.2				2.2
	1.65				1.65
	1.1				1.1
	0.55				0.55
	0	0.18 0.36 0.54 0.72 0.9		1.08 1.26 1.4	0	0.18 0.36 0.54 0.72 0.9	1.08 1.26 1.4
		D i stance, h 10	-4	(meters)		D i stance, h 10	-4	(meters)


B Dedieu
email: dedieu@clermont.inra.fr

G Serviere

social and environmental sustainability in sheep and goat production systems

Keywords:

In France, several work models, which underlie research and tools for "work advice", coexist in approaches to analyse how livestock farming systems (LFS) function. We describe three models: "resource work", "organised work", "work and subjectivity" and specify the way in which LFS research has mobilised social science disciplines to construct them. We illustrate how they complement each other to examine the transformations in livestock farming systems, the ways in which the farming activity is carried out, the conceptions of work, as well as the workforce and technical systems.

I -Introduction

Integrating the work dimension into the analysis of how livestock farming systems function goes back twenty years or more in France. It has two notable characteristics: the first is that it questioned the livestock farming system concept itself, and the second is that it was based on a virtually permanent to and fro between research and development. The first characteristic arose from an explicit request from farmers who had indicated the partial and insufficient nature of representations of livestock farming systems that focused on the interactions between a decisional sub-model and a sub-model elaborating technical performances as proposed by [START_REF] Landais | Recherche sur les systèmes d'élevage. Questions et perspectives[END_REF]. These farmers in the Massif Central (Center France) closely related the specificity of certain livestock management choices (whole year lambings, light lambs sales) and the fragility of their mixed systems, associating dairy cows and ewes, with the problems of work [START_REF] Dedieu | L'étude du travail dans les exploitations d'élevage : proposition de méthode et premiers résultats sur les systèmes mixtes vaches laitières et brebis en Margeride[END_REF]. In fact the livestock farming models of the time had hardly any viewpoint on work, except as a structural detail, linked to economic approaches, in the form of quantification of the workforce present [UTA (annual work unit), UTH, (human worker unit) UMO (manpower unit), etc.]. The second characteristic is that of a dynamic closely associating research and extension and particularly the INRA and the Institut de l'Elevage. This partnership is described by [START_REF] Kling | The "Work on livestock farms" network (WLFN): Building a new domain of learning and intervention[END_REF]. We underline here two important features: (i) a common questioning as to the necessity of renewing together not only the research models but also the methods and tools of guidance for advisers; and (ii) a strong interest shown by the farmers interviewed (and there will have been several thousand of them) in tackling their farming system by shedding light on their work, independently of the evolution of subjects that have mobilised R&E for the past 20 years (extensification, quality, multifunctionality, livestock and environment etc.).

During this period, research into livestock farming systems (LFS) radically renewed its ways of characterising and evaluating its central subject (herd management and resources that are allocated to it, its determinants and its consequences) as well as its relations with social sciences [START_REF] Gibon | Livestock farming systems research in Europe and its potential contribution for managing towards sustainability in livestock farming[END_REF]. In France, the theme of work was one of the fundamental aspects of this renewal. We describe in this article the major models of work with livestock which coexist at the present time in the community of researchers and extension agents who are interested in livestock in the farm enterprise. Our work base is threefold: the scientific publications which have marked different stages of evolution of the work representation framework; a wide knowledge of the methods and tools constructed and used in the framework of advice (Collectif, 2009); our expertise as actors in the research and extension partnership. When talking of "models", the term is used here not in the mathematical or reference system sense, but as a "stylised framework of thought", which generates questionings, distinct issues of collaborations, and which proposes a different look at herd management and more generally the management of transformations which affect this sector. We do not claim to present an exhaustive range of viewpoints, or to take account of all the disciplinary progress on the theme of work. We have extracted here a few polarities in debates on how to tackle "work in livestock farming" in France.

II -Three "livestock management-work" models

The "systemic agronomy" was a precursor of approaches associating technical vision and formalised questions about work (Sebillotte, 1986). The OTELO model, a strong mix of agronomy and management sciences [START_REF] Attonaty | La simulation de l'organisation du travail comme outil de gestion de l'exploitation agricole. Application à la grande culture[END_REF], was a very accomplished attempt to analyse and model the interactions between technical choices, days available and work (manpower, mechanisation and worksite output) at peak periods in the cropping calendar. OTELO is a simulator that is still used by agronomists to estimate the room for manoeuvre available to farmers to change one practice or another. In LFS approaches, the objective was to reveal the consistencies in decisions, livestock farming practices and work but at the scale of a farm year, and not just at periods of strong competition between tasks. Moreover, livestock farming is marked by considerable activity on a daily basis which cannot be put off [START_REF] Madelrieux | Qualification and assessment of work organisation in livestock farms[END_REF] which is not found in main crops. To understand and reason the competition between types of tasks according to their rhythm and their capacity for being put off is one of the issues of the study of work in livestock farming. LFS scientists have therefore chosen other conceptual ways and have mobilised other frameworks of social sciences.

We distinguish three models of work in livestock farming that we call: "work as a resource", "work as an organisation", "work and subjectivity". Their order of presentation has only very partially a chronological aspect and the three models coexist today as the foundations of a wide variety of methods and tools for advice (Collectif, 2009 ;[START_REF] Cournut | Transformation des systèmes d'élevage et du travail des éleveurs, Numéro thématique[END_REF].

The "work as a resource" model

When viewing the farm as an enterprise, and the livestock farming system as the "man -herdresources" triptych [START_REF] Dedieu | Système d'élevage, un concept pour raisonner les transformations de l'élevage[END_REF], work is a resource which must be optimised in the framework of a farmer project aimed at economic excellence. This approach is of course very much inspired by economics, for which work is an essential production factor whose effectiveness can be followed and evaluated with combined performance indicators, its variation factors identified and its improvement sought.

For economists, the efficiency and/or productivity of work is most often measured by dimension ratios (Livestock Unit, Agricultural Area) brought to times of work. One way of quantifying work duration is to take as the base the "full time equivalent" Annual Work Unit. Given the very crude and prescriptive nature of the value of the denominator, these types of ratios do not give a precise evaluation of efficiency. In the late 1980s, economists (in particular [START_REF] Jean | Durée et intensité du travail des agriculteurs dans la crise économique[END_REF][START_REF] Brangeon | La durée annuelle du travail professionnel agricole en élevage porcin et laitier[END_REF] studied the durations and intensity of farming work, paying particular attention to the livestock farm (herbivore, pigs) with the aim of comparing them with other professional categories. The time budget and work budget were then the reference methods. [START_REF] Lacroix | Mesurer le travail agricole: de l'enregistrement à la reconstitution analytique[END_REF] made a comparative analysis of declarative approaches, by timing and recording farmers (budgets) on criteria of accuracy and reliability, and also on their selectivity. They proposed a method of "analytical reconstitution" for the estimation of working times. Its pragmatic character (by declaration) and its accuracy (on the basis of temporal markers making it possible to limit risks of inaccurate memory) greatly inspired the Work Assessment method [START_REF] Dedieu | Bilan travail pour l'étude du fonctionnement des exploitations d'élevage[END_REF] which quantifies working times devoted to implementing livestock and land management practices.

So the approaches developed in the resource work model sought for greater accuracy in the estimation of work duration and the appreciation of work-livestock effectiveness by ratios such as: "work duration over the year/production unit" for example in hours per sow [START_REF] Salaün | Le travail en production porcine -Synthèse bibliographique[END_REF]; in hours of routine work per dairy cow or per ewe; in number of days of seasonal work per hectare in herbivores, where non differable (routine) and differable (seasonal) ryhtms of tasks have always been distinguished. The analysis of the indicator value dispersion (Fig. 1) and the identification of its causes constitute the first line of progress. These approaches have two major characteristics. On the one hand, the different elementary tasks which make up the routine work (RW) or the seasonal work in herbivores, and the total work in monogastric farms as well as the various workers (permanent or occasional, paid or voluntary) are considered as being able to be added together. On the other hand, the herd management appears:

(i) either as the expression of a production system (breeder vs breeder-fattener in pigs or beef cattle for example) whose definition will make it possible to differentiate the ad hoc system of reference for the comparative analysis and the establishment of margins for progress;

(ii) or as one of the variation factors of annual working times (Fig. 2). The essential part of the margins for improvement resides in the categorisation and study of specific work posts whose variability is established at grass roots level (milking, the lambing period) and the gains in productivity that are potentially easier to obtain. The analyses then focus on the implementation of practices in their environment (milking parlour, calving barn, etc.).

J a n v 1 F é v 2 A v r i l 1 M a i 2 J u i l l e t 1 A o û t 2 O c t 1 N o v 2
Hours / day

Work as an organisation: A complex system of activities over time

This work model takes account of the farmer's project in his economic dimension but also his search for "quality of life". It comes within the association, promoted by the professional Livestock Institutes Networks, of the terms "viable" and "liveable", the latter of the two now often amounting to the search for time kept for something other than the farming activity. Work is seen as an organisation and formalised as a system of interactions between herd managements, equipment and buildings, the workforce and the other activities (including private activities which may be the pivot of the organisation) [START_REF] Madelrieux | Patterns of work organisation in livestock farms: the ATELAGE approach[END_REF]. This system of interactions is pressurized by the dimensioning of activities, of the composition -and expectations -of the organising kernel of the work and the issues of technical changes.

U-shaped profile

Long summer period: 7 to 8 months Very marked break of RW duration with the winter period which is quite short (4 to 5 months)

This farm in the Limousin is characterised by calvings grouped in the autumnearly winter with a farmer receiving good help from his volunteer retired father in the winter

Flat profile

No significant modification of the RW over the year

This farm in the Pyrénées-Atlantiques is characterised by calving spread out over the whole year with the farmer working alone

This model is largely derived from studies by ergonomists. We can quote the studies by [START_REF] Cellier | Eléments d'analyse temporelle du travail agricole. Diversité et fractionnement de l'activité des éleveurs ovin-lait[END_REF] and by [START_REF] Bages | Conditions de travail et mode de vie des éleveurs de brebis laitières du Sud-Aveyron (région de Roquefort)[END_REF] in dairy ewe farms in the Roquefort area, which were a strong source of inspiration because these authors had identified the problem of structuring activities which have very varied temporal characteristics (rhythms, possibility of putting off an activity), which will become the routine work (daily, not able to be put off or concentrated) and the seasonal work in a first stage, then a more detailed elaborated categorisation (activities with daily and non daily rhythms), according to [START_REF] Madelrieux | ATELAGE : un modèle pour qualifier l'organisation du travail dans les exploitations d'élevage[END_REF] (Table 1). Fixed position in the day → activity with fixed daily routine (e.g. : milking) Linked to the possible deferability of a task whose positioning depends on the farmer → free daily routine activity (e.g.: taking water to the animals in the enclosure)

Activity with a daily rhythm Position not "fixed" in the day Linked to the unpredictable nature of the positioning of the task, a task which cannot be put off, the activity is under conditions → activity of the "fire fighter" (e.g.: selling direct from the farm, variable according to the presence and number of clients) Not able to be put off → non daily routine activity (e.g.: work on station 5 days a week, selling on the market 1 day a week)

Repeated

Can be put off → activity of the "repeated manipulation" (e.g.: visit to the animals at summer pasture once a week)

Of an occasional type

Single → activity of the "manipulation" type (e.g.: prophylaxis) Deadlines at start and end and during this interval: accomplishment of the task to its end; not able to be put off → activity of the "worksite" (e.g.: manure spreading) Deadline defined from which accomplishment of the task to its end; cannot be put off → activity of the "harvest" (e.g.: hay-making, harvest) Accomplishment of the task until its end before a defined end deadline ; cannot be put off → activity of the "preparation" (e.g.: preparation of paddocks, equipment…)

Activity with a non daily rhythm (ANQ)

Of the interval type with deadlines Deadline of start and end and, during this interval, the farmer accomplishes what he can of the task; can be put off → activity of the "maintenance" (e.g.: clearing scrub)

In this work model, the livestock farm management can be adjusted and adapted in an explicit way, like the other levers (workforce, equipment, etc.). The adoption of particular techniques (once-a-day milking, for example) or even the reconfiguration of the livestock system (for example the consequences on reproduction, replacement and feed practices of concentrating calvings to close the milking parlour) [START_REF] Cournut | Simplification des conduites d'élevage en bovins laitiers[END_REF][START_REF] Hostiou | Relations entre conduites d'élevage et organisation du travail : une étude dans des élevages bovin laitier en France[END_REF], aim at changing the work organisation. Tasks and workers can no longer be added together, the tasks are considered according to their temporal characteristics, the workers according to their commitment in the farming activity (the base unit), the way in which their work is recompensed (gift, work, money in the Work Assessment) and by their central role or not (the organising kernel) in the choices of work organisation. In these approaches, the task-worker associations (the work activities of the ergonomists) become the elementary bricks of the "work" system still reasoned at year scale. The year becomes a chain of periods with different and evolving issues and characteristics of organisation (Fig. 3). The methods that operationalize this type of model propose quantitative or qualitative indicators (for example the proportion of mutual help in carrying out the seasonal work, description of typical days with and without the temporary employee) to take account of the organisation and appreciate the tensions and margins for manoeuvre.

Work and subjectivity: the subjective rationalities of the work

This model is centred on the man, his subjectivity, what he brings into play in the work and what the work enables him to be and become. This comes within all the viewpoints of sociologists and psycho-sociologists about work [START_REF] Dufour | Le travail comme rapport social et comme activité de travail[END_REF]. What interests us here is the original way of questioning the content of the actual work, linked to herd management choices.

Inspired by work by [START_REF] Dejours | L'évaluation du travail à l'épreuve du réel -Critique des fondements de l'évaluation[END_REF], the approach formalises the subjective reasons for "working", in reference to economic, relational and identity rationalities.

It is in reference to these rationalities that certain livestock farming practices (castration, bottle feeding, herding…) and the consistencies of certain production systems qualified for example as "industrial", "organic", "multiple job holding" [START_REF] Porcher | L'élevage, un partage de sens entre hommes et animaux : intersubjectivité des relations entre éleveurs et animaux dans le travail[END_REF]2008) are looked at again, as to the satisfaction they give or how they harm deep-seated reasons for "being a livestock farmer". [START_REF] Fiorelli | Comprendre les compromis faits par les éleveurs pour organiser leur travail: Proposition d'un cadre d'analyse à partir du cas d'éleveurs pluriactifs[END_REF] place in relationship the rationalities of the work and its organisation via a qualification grid of the rationalities and the identification of what, for the people, constitutes the pivot or lever in this organisation. It takes into account the need for money, the taste for technology, the search for a professional identity, the way in which the body itself is called upon, and finally the relations with others, and with the animals (Table 2). Work organisation is envisaged at the scale of the year via the characteristics and relations between farming activity and other activities, management of working and non working time, dimensioning of activities, management of the workforce, livestock farming technical choices. The pivot characteristic means that these components are not adjustable unlike the "lever" characteristic.

III -Consequences on the study of livestock farming systems

This very summary presentation of the models emphasises three ways of developing the interactions between the notions of work and livestock farming systems whose differences are focused on numerous points. The first concerns the contour of the system studied, the second the human base of the livestock system and the third the way of considering livestock management itself.

The contour of the system studied

The "work as a resource" model is in fact very much focused on the livestock activity and very strongly on its competitiveness. The "work as an organisation" model places the farming activity in relation with all the others (economic and private) which influence this organisation (even including football on Sunday afternoon, which can play a role in the abandonment of Sunday evening milking). The "work and subjectivity" model can take an interest in other activities (agricultural or not) (see above) or ignore their existence [START_REF] Porcher | L'élevage, un partage de sens entre hommes et animaux : intersubjectivité des relations entre éleveurs et animaux dans le travail[END_REF].

The human base of the livestock system

The differences focus on:

(i) The definition of the objectives assigned to the livestock activity: to produce more efficiently; a system that is viable, liveable and reproducible; to find fulfilment in work.

(ii) Qualification markers, indicators to appreciate the room for manoeuvre and regulations of the organisation, indicators of work efficiency, etc.

(iii) What defines "the livestock farmer". He is a technical-economic manager, a work organiser and a sensitive worker by turns in each of the three models. In the first case, he represents "the family" in a simplified form of the family-farm of [START_REF] Osty | L'exploitation agricole vue comme un système. Diffusion de l'innovation et contribution au développement[END_REF]. In the second case, "the livestock farmer" is a collection of people whose combination of activities and expectations for non-work define the organisation framework of the farming work. In the third case, each person is unique, whatever his professional status (farmer, spouse, farm worker, etc.).

Livestock farm management

The expression of farm management varies according to the different models: a production system; a set of practices in the annual calendar, generating tasks with a daily and non-daily rhythm, that can be more or less put off; emblematic practices given to people by working with livestock.

In the majority of applications of the "resource" model, management participates in the prescribed work; it is defined in comparison with economic, regulatory and sector imperatives. The debate about effectiveness then centres on automation and rationalisation of the use of manpower. In the model of organised work, livestock management is one of the elements of the work system, one of the levers of work organisation. It can therefore be adapted according to the evolution of the challenges of this organisation and according to the possibilities or the desire of the farmers to manipulate the other levers. The simplification of livestock managements is often the case of farms with moderate capacities for investment in equipment and buildings or of taking on staff [START_REF] Cournut | Quelles adaptations des systèmes laitiers pour réduire la contrainte "travail[END_REF] or for whom low indebtedness and autonomy in work are primordial.

With regard to technical innovation, work can be a limiting resource, "a brake" on the adoption of innovative technical systems (for example [START_REF] Mak | Continued innovation in a Cambodian rice-based farming system: farmer testing and recombination of new elements[END_REF]. For the organised work model [START_REF] Madelrieux | Qualification and assessment of work organisation in livestock farms[END_REF] the technical system is at the crossroads between the performance development scheme and the work organisation. The processes designing innovative systems must therefore be able to integrate this essential component of plasticity (Beguin, 2007 ;[START_REF] Hostiou | Diversity of forage system work and adoption of intensive techniques in dairy cattle farms of Amazonia[END_REF] : is there a dedicated work organisation or does the technical system authorise a wide range of work organisations? For the "work and subjectivity" model, the innovations can be analysed as to how they limit or increase suffering at work for certain people, because they materialize the animal, unlike what is at issue in relational rationalities, or because they modify the body itself at work.

IV -Linking these models…

The models even make their mark on methods and operational tools. However, they are often very widely inspired by a single model. For example, "EPI work" (Chauvat and Serviere, 2009) intended to accompany installation projects is very close to the ATELAGE model which is emblematic of the approach of work as an organisation [START_REF] Madelrieux | ATELAGE : un modèle pour qualifier l'organisation du travail dans les exploitations d'élevage[END_REF]. The ACTEL approach (improvement of working conditions in a dairy farm) (Collectif, 2009) is based on the work-resource developments of the Work Assessment method.

In a context of mutations of work collectives, of continual enlargement of the dimensions of livestock farms, of evolution of relationships at work, the future of study and research on work in livestock farming rests on our capacities to integrate the three dimensions: economic competitiveness, controlled organisation to make livestock coexist with other activities (weekends and holidays have become a new standard of "working well") and finally, the sense as to what is still a life-time commitment and a very specific way of relating to the animal. The Work Assessment enriched the work as a resource and as an organisation models [START_REF] Dedieu | Caractériser et évaluer l'organisation du travail en élevage. La méthode "Bilan Travail[END_REF]. These bridges are today reinforced by new methodological proposals (Hostiou and Dedieu 2010). New approaches are attempting to build bridges between the organised work and work and subjectivity models [START_REF] Fiorelli | Comprendre les compromis faits par les éleveurs pour organiser leur travail: Proposition d'un cadre d'analyse à partir du cas d'éleveurs pluriactifs[END_REF].

Sociologists emphasise the diversity of conceptions of work (difficult, organised, impassioned) [START_REF] Dufour | Le travail en élevage laitier : Des conceptions, des noyaux organisateurs et des profils d'organisation variés[END_REF], of conceptions of one's job (businessmen/artisans/herdsmen) [START_REF] Commandeur | Styles of pig farming and family labour in the Netherlands[END_REF] and indicate new ways in the livestock world of looking at the workforce and work statutes in agriculture (association, salaried staff). The contribution of sociology is essential for understanding the dynamics of norms of work, the job and the organisational choices of livestock farmers. Thus for instance, the permanent paid worker often comes within a dynamic and in representations that are "entrepreneurial", corresponding to the resource-work model. But the analysis of the brakes on recourse to this manpower makes it necessary to identify how paid workers fit into an annual organisation of work (weekends, holidays, work entrusted and work reserved) and what personal satisfaction and what recognition are to be found in the occupations of paid farm workers today.

Another line of thought is the joint application of these models in the framework of two archetypes of the conception of livestock systems [START_REF] Meynard | Recherches sur la conception de systèmes agricoles innovants[END_REF]:

(i) Improving the existing situation (stable conception) with expectations of greater efficiency of work, less hard work, more room for manoeuvre or the elimination of practices that people judge negatively, (ii) Changing the system (innovative conception) with the search for new internal consistencies between livestock management, work collectives, combinations of activities and paying attention to the subjectivity and values of the workers [START_REF] Beguin | Réflexions sur les enjeux d'une prise en compte du travail agricole dans la conception d'une agriculture durable[END_REF].

Fig. 1 .

 1 Fig. 1. Herd size and efficiency of the total beef cattle routine work in a sample of large herds (Cournut et al., 2009).

Fig. 2 .

 2 Fig. 2. Two evolution profiles of annual routine work, in relation with reproduction management (Cournut et al., 2009).

Fig. 3 .

 3 Fig. 3. Profile of chain of organisational sequences over a year: evolution of the degree of involvement of the organising kernel in the non-daily activities (example of a farmer in the North Alps -Madelrieux et al., 2009).

Table 1 . Activity typology according to the temporal characteristic of their task (Madelrieux et al., 2006)

 1

Table 2 . The five subjective rationalities in work. Example of a multiple job holding farmer (sheep- farming and earth-moving work) (Fiorelli et al., 2007) ( ≠ : in opposition with earth-moving activity ; = as earth-moving activity)

 2

	Subjective	Farmer's appreciations
	Rationalities
	Economical	An uncertain, unpredictable income, difficult to obtain ≠ earth-moving
		An income to repay property loans, not for the family
	Identity	The pride of heading such a large property after setting out with nothing
		The heady feeling of always undertaking more = earth-moving
	Technical	Acquiring equipment to spend the least time possible on work : as it doesn't
		pay too much, better not spend too long on it
	Relational	Working alone = earth-moving
	Body commitment	Hard work ≠ earth-moving


Dominique Arnaud

Annabelle Déjardin

Jean-Charles Leplé

Marie-Claude Lesage-Descauses

Nathalie Boizot

Marc Villar

Hélène Bénédetti

Gilles Pilate
email: pilate@orleans.inra.fr

Expression analysis of LIM gene family in poplar, toward an updated phylogenetic classification

Background: Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied. Findings: In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues.

Conclusions:

The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins.

Background

Plant LIM domain proteins are related to animal Cystein Rich Proteins (CRP), and contain two LIM domains characterized by the consensus sequence [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF][START_REF] Eliasson | Molecular and expression analysis of a LIM protein gene family from flowering plants[END_REF]. In animals, the CRP proteins mostly expressed in muscle tissues are involved in muscle differentiation, transcriptional regulation and actin organization [START_REF] Kadrmas | The LIM domain: from the cytoskeleton to the nucleus[END_REF][START_REF] Weiskirchen | The CRP/MLP/TLP family of LIM domain proteins: acting by connecting[END_REF]. In plant, the tobacco NtWLIM1 protein functions in the nucleus as a transcription factor regulating the expression of genes involved in lignin biosynthesis [START_REF] Kawaoka | Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis[END_REF], and in the cytoplasm as an actin binding and bundling protein like the CRP1 protein in animal [START_REF] Thomas | Tobacco WLIM1 is a novel F-Actin binding protein involved in actin cytoskeleton remodeling[END_REF][START_REF] Tran | Cysteine-rich protein 1 (CRP1) regulates actin filament bundling[END_REF]. More recently, it has been shown that lily LlLIM1 and all Arabidopsis LIM domain proteins also regulate the actin cytoskeleton organization and dynamics [START_REF] Papuga | Arabidopsis LIM proteins: a family of actin bundlers with distinct expression patterns and modes of regulation[END_REF][START_REF] Wang | An actin-binding protein, lllim1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes[END_REF].

[C-X 2 -C-X 17 - H-X 2 -C]-X 2 -[C-X 2 -C-X 17 -C-X 2 -H]
Since the discovery of SF3/PLIM1, the first gene coding for a plant LIM domain protein [START_REF] Baltz | Characterization of a pollenspecific cDNA from sunflower encoding a zinc finger protein[END_REF][START_REF] Baltz | A LIM motif is present in a pollen-specific protein[END_REF], an increased number of LIM genes has been identified in a wide range of plant species including Arabidopsis, rice and poplar [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF][START_REF] Eliasson | Molecular and expression analysis of a LIM protein gene family from flowering plants[END_REF]. Whereas both Arabidopsis thaliana and Oryza sativa genomes contain six gene models, the Populus trichocarpa genome contains at least twelve PtLIM gene models resulting from the duplication of six ancestral genes [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. These duplicated genes probably appeared during the "salicoid" whole-genome duplication event [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF][START_REF] Tuskan | The genome of black cottonwood, Populus trichocarpa (Torr. & Gray)[END_REF]. The plant LIM domain protein family has been divided into four groups: αLIM1, βLIM1, γLIM2, δLIM2 containing monophyletic subgroups differing according to their classification in plant taxonomic class or subclass and/or in their expression specificities [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. In sunflower, tobacco and Arabidopsis, genes belonging to the WLIM1 and WLIM2 subgroups are widely expressed in plant, whereas genes belonging to the PLIM1 and PLIM2 subgroups are preferentially expressed in pollen [START_REF] Eliasson | Molecular and expression analysis of a LIM protein gene family from flowering plants[END_REF][START_REF] Papuga | Arabidopsis LIM proteins: a family of actin bundlers with distinct expression patterns and modes of regulation[END_REF][START_REF] Baltz | Differential localization of the LIM domain protein PLIM-1 in microspores and mature pollen grains from sunflower[END_REF]. However, the expression pattern of genes classified into the βLIM1 group and the XLIM1 and PLIM2-like subgroups remains unknown and there is no Arabidopsis and rice orthologs within these newly identified monophyletic groups [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF].

Although EST distribution analysis in cDNA libraries from different poplar tissues may be indicative of their expression profile, the expression pattern of PtLIM genes has not been investigated. Using EST and microarray data, some poplar LIM genes (e.g. PtWLIM1a and b and PtXLIM1a and b) appear highly expressed in wood or in tension wood [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF][START_REF] Andersson-Gunneras | Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis[END_REF][START_REF] Déjardin | Expressed sequence tags from poplar wood tissues -a comparative analysis from multiple libraries[END_REF]. In trees, the formation of wood (or secondary xylem) is an important developmental process involving different steps: firstly cell division from the cambial meristem followed by xylem differentiation that include cell expansion, secondary wall synthesis, lignification and finally programmed cell death [START_REF] Mellerowicz | Unravelling cell wall formation in the woody dicot stem[END_REF]. Produced in response to mechanical stresses, tension wood is a peculiar cellulose-rich and poorly lignified wood characterized by specific mechanical and structural properties that allow the tree to re-orientate its axis [START_REF] Timell | The chemical composition of tension wood[END_REF].

In this article, the expression of the twelve poplar PtLIM genes was investigated in different reproductive and vegetative tissues. Some PtLIM genes were ubiquitously expressed whereas others showed a preferential expression in pollen, cottony hairs or secondary xylem. Interestingly a subset of PtLIM genes was up-regulated in tension wood area and all duplicated genes exhibited distinct expression pattern suggesting subfunctionalisations. From this expression analysis and considering the previous phylogenetic classification we suggest a simplified nomenclature for the poplar LIM domain family.

Findings

Expression of poplar LIM genes in vegetative and reproductive organs

Previous EST distribution analyses indicated that PtXLIM1a and b and PtWLIM1a and b transcripts were mostly found in wood forming tissues whereas PtWLIM2a and b genes seemed to be ubiquitously expressed [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. However, only a few PtLIM1a and b ESTs were found in undifferentiated cells and vegetative buds cDNA libraries. Moreover, PtPLIM2a, b, c and d transcripts were not or only poorly represented in poplar cDNA libraries preventing any inference on their expression profile. Therefore, the expression level of the twelve PtLIM genes was measured in different vegetative and reproductive tissues by semi quantitative RT-PCR.

The PtXLIM1a and b and PtWLIM1a and b genes were strongly expressed in poplar stem particularly in wood, whatever the age of the tree (Figure 1 and Additional file 1). Moreover, they also showed a strong expression in the root differentiating xylem. These genes, except PtWLIM1a, were not or only weakly expressed in leaves, calli, and reproductive organs indicating a preferential expression in vascular tissues. PtXLIM1a expression appeared higher in developing and mature xylem (DX and MX) than in cambial zone (CZ) and phloem, suggesting specificity toward secondary xylem differentiation. Its duplicated gene PtXLIM1b as well as PtWLIM1b were more expressed in DX than in MX both in stem and root. Although the expression of PtWLIM1a gene was stronger in the stem, it seemed to be widely expressed in the whole tree except in pollen. From these results, genes from the XLIM1 and WLIM1 subgroups may play an important function during wood formation in poplar.

The duplicated PtLIM1a and b genes displayed distinct expression patterns. Whereas PtLIM1a gene was expressed in a wide range of tissues, the PtLIM1b gene appeared specifically expressed in reproductive organs including fruit, male and female flowers and to a lesser extent pollen (Figure 1). Interestingly, like PtWLIM1a, PtLIM1a expression is the highest in calli as well as in cottony hair (F). The expression pattern of the duplicated PtWLIM2a and b genes were strikingly different although their expression appeared rather weak in most poplar tissues. Indeed, PtWLIM2a was ubiquitously expressed in the plant when PtWLIM2b was mostly expressed in leaf and stem tissues. The PtWLIM1a, PtLIM1a and PtWLIM2a and b genes were also expressed in young roots collected from in vitro poplar plantlets, indicative of a preferential expression in tissues at primary growth stage (data not shown). Thus, PtLIM1a, PtWLIM1a and PtWLIM2a appeared widely expressed in poplar whereas the expression of their respective duplicated genes seemed more restricted to particular tissues.

PtPLIM2a and b exhibited a very strong expression in stamens, mature pollen and especially in germinated pollen. Interestingly, the duplicated PtPLIM2c and d genes, closely related to PtPLIM2a and b, were also highly expressed in the same tissues. The rather weak expression of these four genes in stamens at an early developmental stage compared to older stamens, pollen and germinating pollen suggests an expression in microspores rather than in filament, anther wall, rachis or peduncle supporting stamens. Surprisingly, these four genes were also highly expressed in isolated cottony hairs and in the capsules bearing these fibers, indicative of a dual expression in both pollen and cotton fibers. The PtPLIM2a gene was also expressed, but to a lower extent, in DX and MX, and PtPLIM2d transcript was detected in all parts of leaves and stems. These results indicate that PtPLIM2a and b genes and their paralogs PtPLIM2c and d may have redundant functions during pollen tube growth as well as in cotton fiber development.

Comparison of poplar LIM gene expression between opposite and tension wood

Previously, PtXLIM1a ESTs were shown to be more abundant in tension wood DX than in opposite wood DX [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF][START_REF] Déjardin | Expressed sequence tags from poplar wood tissues -a comparative analysis from multiple libraries[END_REF] and microarray analyses indicated a higher expression of PtXLIM1b in tension wood than in normal wood [START_REF] Andersson-Gunneras | Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis[END_REF]. This prompted us to investigate more precisely the expression pattern of the PtLIM genes during tension wood differentiation on artificially tilted poplar trees. Tension wood, formed at the upper side of the tilted stem, is easily recognizable as a bright whitish crescent, and its presence was confirmed by the amplification of PopFLA1 gene (data not shown) a marker of tension wood formation [START_REF] Lafarguette | Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood[END_REF].

Unexpectedly, the semi quantitative RT-PCR results showed that a number of poplar PtLIM genes: PtXLIM1a and b, but also PtWLIM1b, PtWLIM2a, PtPLIM2a and PtPLIM2d exhibited a higher expression in tension wood compared to opposite wood (Figure 2). These results confirmed the increased accumulation of PtXLIM1a and b transcripts in DX from tension wood but also revealed a high expression level in tension wood MX. PtPLIM2a expression in tension wood appeared to be restricted to DX and MX. By contrast, the accumulation of PtWLIM1b transcript in tension wood was mainly localized in DX, CZ and phloem/bark. The accumulation of PtWLIM2a and PtPLIM2d in DX was only slightly higher in tension wood compared to opposite wood and the other PtLIM genes did not exhibit any tension wood preferential up-regulation. The induction of PtXLIM1a gene in tension wood was supported at the protein level by western blot analysis using purified anti-6His-PtXLIM1a antibodies. A 29 kDa polypeptide corresponding to the expected size of the PtXLIM1a protein was detected from the CZ to the MX but not in leaves and reproductive organs (Additional file 2, data not shown). The PtXLIM1a protein was particularly more expressed in DX from tension wood compared to opposite wood. In summary, PtXLIM1a and b as well as PtWLIM1b and PtPLIM2a are unambiguously tension wood-induced genes and their expression appears to fluctuate depending on xylem differentiation.

Discussion

Expression of LIM duplicated genes in poplar, toward a simplified nomenclature

The previous classification and nomenclature of plant LIM domain protein was complex because of the lack of expression data concerning genes belonging to newly identified XLIM1, PLIM2-like subgroups and βLIM1 group [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. Furthermore, the expression of poplar genes from WLIM1, WLIM2 and PLIM2 subgroups was predicted from EST analysis and phylogenetic comparison. Thus, the expression pattern of PtLIM genes was analyzed on an exhaustive set of poplar tissues covering most of the tree developmental stages. From our results, the 12 PtLIM genes were all expressed in poplar indicating that pseudogenization events did not occur. Poplar PtLIM genes are organized in six pairs of duplicated genes, and the high sequence similarity between duplicated PtLIM genes, ranging from 85 to 95% of amino acid identity, suggests that they share similar functions [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. However, a few pair of genes exhibit distinct expression patterns suggesting a subfunctionalisation in specific tissues or possibly a neofunctionalisation [START_REF] Lynch | The evolutionary fate and consequences of duplicate genes[END_REF]. For example, the PtWLIM1a and PtLIM1a genes almost constitutively expressed may be involved in a general cellular function, whereas the corresponding duplicated genes probably evolved towards specific functions such as xylem development for PtWLIM1b and eventually reproductive processes for PtLIM1b. The finding that PtXLIM1a and b genes were preferentially expressed in xylem strengthens their classification into the XLIM1 monophyletic subgroup distinct from the WLIM1 subgroup (Additional file 3) [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. XLIM1 genes probably result from an older duplication of the ancestral WLIM1 gene with a further specialization towards new vascular specific functions potentially important for tree physiology. From our results, we have now strong evidence supporting a pollen preferential expression for the PtPLIM2c and d genes classified into the PLIM2-like subgroup phylogenetically close to the pollen specific PLIM2 subgroup (Additional file 3) [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. It is thus likely that orthologous genes from sunflower and tobacco classified within the PLIM2-like subgroup also share the same pollen preferential expression. According to their expression profile and phylogeny, we propose a new nomenclature for the poplar LIM domain protein family (Table 1).

Expression pattern of LIM genes in differentiating secondary xylem and tension wood

According to the expression study, the PtXLIM1a and b and PtWLIM1b genes were preferentially expressed in differentiating secondary xylem both in root and shoot, and were up-regulated in tension wood (Figures 1 and Figure 2, Additional file 1). Wood formation involves cell expansion and deposition of a highly lignified secondary cell wall [START_REF] Déjardin | Expressed sequence tags from poplar wood tissues -a comparative analysis from multiple libraries[END_REF][START_REF] Mellerowicz | Unravelling cell wall formation in the woody dicot stem[END_REF]. The tobacco NtWLIM1 ortholog probably acts as a transcription factor activating the expression of PAL, 4CL and CAD genes coding for enzymes of the lignin biosynthesis pathway [START_REF] Kawaoka | Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis[END_REF]. Interestingly, this protein also acts on the stabilization of actin bundles [START_REF] Thomas | Tobacco WLIM1 is a novel F-Actin binding protein involved in actin cytoskeleton remodeling[END_REF]. Actin cables, longitudinally oriented in vessels and fibers, certainly play an essential role in the cytoplasmic streaming and the distribution of cellulose and lignin precursors throughout these very elongated cells [START_REF] Chaffey | Understanding the role of the cytoskeleton in wood formation in angiosperm trees: hybrid aspen (Populus tremula × P. tremuloides) as the model species[END_REF]. Taken together, both expression patterns and potential functions suggest the involvement of PtXLIM1a and b and PtWLIM1b in the differentiation of xylem cells, during cell elongation and secondary cell wall formation. Although some variations do exist, the common up-regulation of several PtLIM genes in tension wood may be indicative of a functional redundancy during tension wood formation. We know that tension wood is enriched in cellulose and contains less lignin than opposite or normal wood [START_REF] Timell | The chemical composition of tension wood[END_REF], and in accordance, the expression of PAL, 4CL and CAD genes involved in lignin biosynthesis is lower in poplar tension wood [START_REF] Andersson-Gunneras | Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis[END_REF]. However, this merely applies during the deposition of the cellulose rich gelatinous layer (G-layer), specific to tension wood fibers, that is possibly devoid of any lignin, whereas the sub layers (S1 and S2) deposited before the G-layer contains regular amount of lignin [START_REF] Pilate | Lignification and tension wood[END_REF]. Therefore, we cannot rule out that poplar LIM domain proteins act as transcriptional activators of lignin biosynthetic genes as proposed for the NtWLIM1 protein [START_REF] Kawaoka | Functional analysis of tobacco LIM protein Ntlim1 involved in lignin biosynthesis[END_REF]. Interestingly, it was demonstrated that muscle specific CRP2 and CRP3 proteins are involved in the stretch response and the regulation of cell contractile force by interacting with actin stress fibers [START_REF] Jr | Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo[END_REF][START_REF] Knoll | The cardiac mechanical stretch sensor machinery involves a z disc complex that is defective in a subset of human dilated cardiomyopathy[END_REF]. The actin binding and bundling functions between plant LIM domain proteins and the related animal CRP proteins is highly conserved [START_REF] Thomas | Tobacco WLIM1 is a novel F-Actin binding protein involved in actin cytoskeleton remodeling[END_REF][START_REF] Tran | Cysteine-rich protein 1 (CRP1) regulates actin filament bundling[END_REF][START_REF] Papuga | Arabidopsis LIM proteins: a family of actin bundlers with distinct expression patterns and modes of regulation[END_REF][START_REF] Wang | An actin-binding protein, lllim1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes[END_REF]. According to the high expression of several PtLIM genes in tension wood, it is tempting to speculate that poplar LIM proteins may also be implicated in the perception/transduction of mechanical stresses through interactions with actin cables just as proposed for CRP in muscle cells [START_REF] Kadrmas | The LIM domain: from the cytoskeleton to the nucleus[END_REF][START_REF] Jr | Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo[END_REF]. However, such an involvement of actin bundles in tensile or contractile strain still needs to be demonstrated in tree wood fibers.

Plant LIM domain proteins and anisotropic cell elongation

Poplar PLIM2 genes are preferentially expressed in mature and germinating pollen. These genes are potentially involved in pollen tube growth as already suggested for their orthologs in tobacco, sunflower and Arabidopsis. Indeed, NtPLIM1, NtPLIM2, HaPLIM2, AtPLIM2a, b and c genes are highly expressed in pollen and HaPLIM1-SF3 protein strongly accumulates in germinating pollen cones [START_REF] Eliasson | Molecular and expression analysis of a LIM protein gene family from flowering plants[END_REF][START_REF] Papuga | Arabidopsis LIM proteins: a family of actin bundlers with distinct expression patterns and modes of regulation[END_REF][START_REF] Baltz | Differential localization of the LIM domain protein PLIM-1 in microspores and mature pollen grains from sunflower[END_REF]. Poplar PLIM2 genes together with the PtWLIM1a and PtLIM1a genes were also strongly expressed in cottony fibers and capsules. Poplar cottony fibers resemble to cotton fibers from Gossypium sp. even though they originate from placenta and are borne by the capsule base [START_REF] Boes | Floral phenology and morphology of black cottonwood, Populus trichocarpa (Salicaceae)[END_REF]. Cotton fibers are unicellular trichomes composed of 95% The old nomenclature of LIM gene and phylogenetic subgroups [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF] is indicated under bracket cellulose that undergo considerable cell elongation with thick longitudinally oriented actin filaments [START_REF] Li | The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation[END_REF][START_REF] Ruan | Rapid cell expansion and cellulose synthesis regulated by plasmodesmata and sugar: insights from the single-celled cotton fibre[END_REF].

Remarkably a high number of EST homologous to LIM domain protein has been detected in cotton fibers libraries [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. Both our data and previous studies indicate that most of the plant LIM genes are expressed in cells with anisotropic elongation, such as xylem fibers, pollen tubes, and cottony fibers. This expression pattern is in agreement with the capacity of plant LIM proteins to bind and bundle actin filaments in order to favor the formation of thick actin cables [START_REF] Thomas | Tobacco WLIM1 is a novel F-Actin binding protein involved in actin cytoskeleton remodeling[END_REF][START_REF] Papuga | Arabidopsis LIM proteins: a family of actin bundlers with distinct expression patterns and modes of regulation[END_REF][START_REF] Wang | An actin-binding protein, lllim1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes[END_REF]. Indeed longitudinally-oriented thick actin cables may be very important for effective tip growth and anisotropic cell expansion. Further genetics, biochemical and cell biological studies are required to characterize the function of plant LIM domain protein during cell elongation.

Methods

Sampling of poplar tissues

Vegetative tissues have been harvested on Populus tremula x P. alba, INRA clone 717-1-B4, poplar trees. Coppiced poplar trees kept at 4°C during the winter were transferred mid-March in the greenhouse, potted in compost (3 L) and individually supplied with water and fertilizers by a drip system. Straight stems and leaves were collected on three individual 3 month-old young poplar trees. Differentiating xylem (DX) and mature xylem (MX) samples were sequentially scraped from the debarked stem with a scalpel (Additional file 4). Cambial zone (CZ) was first lightly scraped from the peeled bark, while the remaining tissues consisted of the phloem and bark (PB) samples. Young stem (YS) samples enriched in primary growth tissues were the green stem portions collected below the apex. Following the sampling procedure described above, tension and opposite wood samples were collected, respectively at the upper and lower sides of the stems of three trees tilted for one month. Petiole (Pe) and main vessels (V) were separated from the limb (L) of leaves with a scalpel.

A number of samples were also collected during the spring, when cambium was very active, on a four year-old mature poplar tree cultivated in the tree nursery. CZ samples were lightly scraped from the peeled bark of the trunk. Then, phloem (Ph) samples were peeled from bark (B) samples. DX samples were first gently scraped from the debarked stem, while MX was sampled as wood chips from the remaining stem. The different samples from the main root were harvested following the same procedure, except that the peeled cortex contains both secondary phloem and root epidermis (PE), and after DX removal, the root MX consisted of the entire central cylinder including the pith. Undifferentiated cells were collected from calli (INRA clone 717-1-B4) cultivated for 2 weeks at 24°C in the dark on MB5 agar plates [START_REF] Gamborg | Nutrient requirements of suspension cultures of soybean root cells[END_REF].

Because Populus tremula x P. alba trees from INRA 717-1-B4 clone were female hybrid clones, female and male flowers have been collected on Populus nigra and P. nigra var. Italica poplar trees. Excised branches bearing immature male or female flower catkins from trees growing on the Loire riverside were harvested in early April 2006. Female and male branches were respectively transferred in compost and water. One month later, after anthesis, the rachis, peduncles and bracts were removed from female flowers with a forceps and a scalpel. The stigmas (Sg) were separated from the ovaries and styles (OS) (See Additional file 4). After removal of protecting calyx, stamens with peduncles and rachis were collected from male flowers at early (St1) or late (St2) developmental stages. St1 corresponded to young and small partiallyopen male flowers, bearing sessile stamens on the rachis, whereas at St2, just before anthesis, male flowers were fully expanded and bore elongated rachis and peduncles with spreader stamens. Mature pollen (P) was spread on modified MS/2 media [START_REF] Leplé | Transgenic poplars; expression of chimeric genes using four different constructs[END_REF][START_REF] Murashige | A revised medium for rapid growth and bio assays with tobacco tissue cultures[END_REF] and germinated pollen (GP) with elongated pollen tubes was harvested 24 h after germination. In order to produce and collect seeds, female flowers were manually fecundated with a paintbrush; one month later, the cottony hairs or fibers (F) were separated from seeds (S) and capsules (Ca).

Samples were immediately frozen upon collection, ground in liquid nitrogen and stored at -80°C before use. Samples collected from leaves and stem of 3 different greenhouse-grown poplar trees were pooled after grinding and before RNA extraction.

RNA extraction and semi quantitative RT-PCR analyses

Total RNAs were prepared using the method described by Chang and colleague [START_REF] Chang | A simple and efficient method for isolating RNA from pine trees[END_REF]. After overnight precipitation with LiCl, RNAs were further purified using QIAGEN RNEasy kit ® according to manufacturer's instructions. RNA concentration and quality were determined by OD measurement at 260 nm and 280 nm. Two micrograms of total RNA were reverse transcribed using 500 ng of oligo (dT)12-18 and 200 units of SuperScript™ II RT in a total volume of 20 μl following the manufacturer's instructions (Invitrogen™, Life technologies). After first-strand cDNA synthesis, RNA complementary to the cDNA was removed by incubating the reaction with 2 units of E. coli RNase H at 37°C for 20 min. Gene-specific PCR primers were designed to amplify PtLIM genes in Populus trichocarpa Nisqually genotype as well as in P. tremula × P. alba (INRA clone 717-1-B4) when cDNA sequences (PtXLIM1a and b, PtWLIM1a and b, PtWLIM2a and PtPLIM2a) were available [START_REF] Arnaud | Genomewide analysis of LIM gene family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa[END_REF]. Primers were located at both ends of gene coding sequences in order to amplify the four introns and therefore detect any genomic contamination. PCR amplification was done with 1/60 of the first-strand cDNA as template, 0,5 unit of rTaq DNA polymerase (Invitrogen™, Life technologies), 1,25 mM MgCl 2 and 0,2 μM of gene specific primers (Additional file 5) in a total volume of 20 μl. For each primer pair, gene-specific amplification was optimized and verified on available PtLIM cDNA clones used as template. For PtLIM genes, the cycling conditions were 94°C for 5 min for one initial step followed by 94°C for 45 s, 65 or 60°C for 45 s and 72°C for 1 min, for 25 to 40 cycles (See Additional file 5). For 18S rRNA, the cycling conditions were 94°C for 5 min for one initial step followed by 94°C for 45 s, 55°C for 1 min and 72°C for 1 min 30, for 20 cycles [START_REF] Lafarguette | Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood[END_REF]. The PCR was terminated with one extra step at 72°C for 10 min and hold at 4°C. The amplification was done using a Gen-eAmp ® PCR System 9700 (Applied Biosystems). Ten microliters of each PCR product were electrophoresed on a 1% agarose gel that was scanned under UV light after ethidium bromide staining. Results presented are the representative illustration of at least three independent semi quantitative RT-PCR experiments. The identity of each PCR product was checked by sequencing.

Production of 6His-PtXLIM1a protein, protein extraction and Western blot

See Additional file 6 for detailed description of the procedure.

Figure 1

 1 Figure 1 Expression of PtLIM genes using semi quantitative RT-PCR analysis in poplar trees. A. Vegetative organs: Three straight young poplars (Populus tremula × P. alba) producing normal wood were cultivated in the greenhouse for three months. Phloem and bark (PB), cambial zone (CZ), developing xylem (DX) and mature xylem (XM) were scraped from the stem. To study primary growth, young green portions of stems (YS) were collected just below the apex. Leaf vascular tissues like petioles (Pe) and main vessels (V) were separated from limbs (L). Two-weeksold dark-grown calli (C) were also harvested. B. Reproductive organs: female flowers were dissected from mature poplar trees (Populus nigra) growing in the nursery and the stigma (Sg) were separated from the ovary and style (OS). Two different developmental stages were analyzed on male flowers: partially-open flowers (St1) at an early developmental stage, and fully-open flowers at pre-anthesis stage (St2). Mature pollen (P) and germinated pollen (GP) were also analyzed, as well as seeds (S) separated from both the cottony fibers (F) and the capsule protecting seeds (Ca). 18S rRNA signals are indicative of the total RNA quantity in each sample

Figure 2

 2 Figure 2 Expression of PtLIM genes using semi quantitative RT-PCR analysis in opposite and tension wood. Three young poplars (Populus tremula × P. alba) were cultivated in the greenhouse for two months, and tilted for one month to induce tension wood formation. Phloem and bark (PB), cambial zone (CZ), developing xylem (DX) and mature xylem (XM) samples were scraped from the upper side (tension wood) and the lower side (opposite wood) of tilted stems. 18S rRNA signals are indicative of the total RNA quantity in each sample

Table 1

 1 Phylogenetic classification and nomenclature of poplar PtLIM genes

	Group	Subgroup	Poplar	Arabidopsis	Rice	Sunflower	Expression
							profile
	αLIM1	XLIM1	PtXLIM1a (PtGLIM1a)	not found	not found	not found	secondary
		(FLIM1)	PtXLIM1b (PtGLIM1b)				xylem
		WLIM1	PtWLIM1a	AtWLIM1	OsWLIM1	HaWLIM1	ubiquitous
			PtWLIM1b
		PLIM1	not found	not found	not found	HaPLIM1a-SF3	pollen
						HaPLIM1b
	βLIM1	βLIM1	PtLIM1a (PtβLIM1a)	not found	not found	HaLIM1 (HaβLIM1)	ubiquitous
			PtLIM1b (PtβLIM1b)
	γLIM2	WLIM2	PtWLIM2a	AtWLIM2a	OsWLIM2	HaWLIM2	ubiquitous
			PtWLIM2b	AtWLIM2b
	δLIM2	PLIM2	PtPLIM2a	AtPLIM2a	OsPLIM2a	HaPLIM2a	pollen
			PtPLIM2b	AtPLIM2b	OsPLIM2b
				AtPLIM2c	OsPLIM2c
		PLIM2-like	PtPLIM2c (PtδLIM2a)	not found	not found	HaPLIM2b (HaδLIM2a)	pollen
		(δLIM2)	PtPLIM2d (PtδLIM2b)			HaPLIM2c (HaδLIM2b)

Acknowledgements

DA was supported by a PhD grant from the 'Région Centre'. The authors acknowledge Alain Roussel and Franck Coste (CNRS-CBM Orléans) for their help to produce the recombinant PtXLIM1a protein.

Availability of supporting data

The data sets supporting the results of this article are included within the article and its additional files.

Additional material

Additional file 1: PtLIM genes expression in wood tissues of an adult poplar tree. Expression of PtLIM genes using semi quantitative RT-PCR analysis in the trunk and main root of a four-year old poplar tree.

Additional file 2: Expression of PtXLIM1a protein in opposite and tension wood. Immunodetection of PtXLIM1a protein in total protein extracts collected from tilted trunk of four-year old poplar trees.

Additional file 3: Phylogenetic tree of plant LIM domain proteins. Phylogenetic tree of poplar, Arabidopsis, rice, tobacco and sunflower LIM domain proteins.

Additional file 4: Sampling of wood tissues and reproductive organs. Illustration of samples collected from wood forming tissues on stems and description of male and female flowers used in the study.

Additional file 5: Primers used in semi-quantitative RT-PCR. Forward and reverse primer sequences (5' to 3') for all PtLIM genes analyzed and PCR conditions used for semi-quantitative RT-PCR.

Additional file 6: Supplemental methods for protein extraction and western blot. Method used for protein extraction, western-blot analysis, production and purification of 6His-PtXLIM1a recombinant protein.

Author details 1 INRA, UR0588 Amélioration, Génétique et Physiologie Forestières, CS 40001 Ardon, F-45075 Orléans Cedex 2, France. 2 CNRS, UPR4301, Centre de Biophysique Moléculaire, Equipe «Signalisation cellulaire et neurofibromatose», F-45000 Orléans, France.

Authors' contributions DA participated in the conception and design of the study, carried out semi-quantitative RT-PCR experiments as well as the production and purification of recombinant protein, and wrote the manuscript. AD and J-CL participated in the design of the study, manuscript revising and provided helpful discussions. M-CL-D performed sequencing of PCR products. MV participated in the collection of reproductive samples. NB carried out protein extraction, antibodies purification and western blot. HB coordinated and contributed to production and purification of recombinant protein. GP coordinated the study, participated in its conception and design as well as revisions to the manuscript. All authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.