H Majdoubi

; T Boulard
email: boulard@sophia.inra.fr

; H Fatnassi

L Bouirden

Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study

Keywords: Greenhouse, CFD, Modelling, Airflow, Climate distribution, Insect screens, Tomato

This study presents an analysis of air circulation and microclimate distribution during daytime in a 1-hectare Canary type tomato greenhouse in the coastal area of southern Morocco. The investigation of the climate inside the greenhouse is based on a numerical simulation using a finite volumes method to solve the mass, momentum and energy conservation equations. The main novelty of this simulation lies in the realism of the 3D modelling of this very large agricultural structure with (i) a coupling of convective and radiative exchanges at the surface of the plastic roof cover, (ii) simulation of the dynamic influence of the insect screens and tomato crop on airflow movement, using the concept of porous medium, (iii) simulation, in each grid cell of the crop canopy, of the sensible and latent heat exchanges between the greenhouse air and the tomato crop, and (iv) detailed simulation of climate parameters in a 1-hectare real-scale commercial greenhouse.

The model simulations were first validated with respect to temperature and relative humidity fields measured inside the experimental greenhouse for fairly steady-state outside conditions marked by a prevailing sea breeze around the solar noon. A good agreement was observed between the measured and simulated values for inside air temperatures and specific humidity. It was next used for exploring the details of the inside air temperature and humidity fields and plant microclimates and transpiration fluxes throughout the greenhouse space. Simulation for a wind direction perpendicular to the side and roof openings shows that the insect screen significantly reduced inside air velocity and increased inside temperature and humidity, especially in the vicinity of the crop canopy. It revealed the details of the flow field within the greenhouse. At the windward end of the greenhouse, the flow field was marked by a strong windwise air current above the tomato canopy which was fed by the wind ward side vent, and a slow air stream flowing within the tomato canopy space. Then, from the first third of the greenhouse to the leeward end, the flow field was marked by the combination of wind and buoyancy forces, with warmer and more humid inside air which was evacuated through the upper roof vents, while colder and dryer air was penetrated through the upper roof vent openings. Based on these simulations, design studies of the greenhouse crop system were performed to improve inside air temperature and humidity conditions by simple modifications of orientation of the crop rows.

Introduction

In recent years, a variety of new greenhouse designs have been introduced to meet requirements for agricultural production in Mediterranean regions. The use of Canary type greenhouses [START_REF] Majdoubi | Natural ventilation performance of a large scale canary greenhouse equipped with insect screens[END_REF] has been spreading rapidly in the Canary Island, along the Mediterranean coast of Spain and on the Atlantic coast of Morocco. With a total of about 100,000 hectares (Jouet, 2006), this type of greenhouse is now one of the most widely used in the world. However, because of its design and its use in subtropical regions, this type of structure has a very heterogeneous inside climate, which can be damaging to crop activity, particularly transpiration and photosynthesis. These greenhouses are often very large (1 hectare or more), and sidewall ventilation is more commonly used than roof ventilation; these factors intensify the climatic heterogeneity. Moreover, to reduce the need for pesticide application the vent openings have to be equipped with fine-mesh insect screens. They act as mechanical barriers to insects but also to air and significantly reduce the ventilation rate and thus tend in turn to rise inside air temperature and humidity.

Computational fluid dynamics (CFD) have also been increasingly used to study greenhouse ventilation, whether driven by wind (Mistriotis et al., 1997;Haxaire, 1999) or buoyancy forces, (Lamrani, 1997;Boulard et al., 1999), in both greenhouses and tunnels (Boulard and Wang, 2000).

Recently, Bartzanas et al. (2002) have characterised and numerically modelled the effects of insect screens on ventilation and inside climate in a tunnel greenhouse.

The effects of fine insect screens on the vent openings of Spanish 'Parral' greenhouses or Moroccan 'Canary type' greenhouses have been studied in details and simulated by means of CFD packages. Campen et al. (2003) and [START_REF] Molina-Aiz | Measurement and simulation of climate inside Almeria-type greenhouses using computational fluid dynamics[END_REF] have analysed the effect of wind speed on natural ventilation openings in a Spanish 'Parral' type greenhouse equipped with screened top and side ventilation, using a three-dimensional and a two-dimensional CFD simulation respectively. In both studies, the greenhouse-dependent ventilation characteristics and the influence on ventilation rate of wind direction and the geometry of the openings were numerically assessed.

For a commercial Canary type greenhouse of 0.5 ha, equipped with insect screens on side and roof openings, Fatnassi et al.(2003 and[START_REF] Ould Khaoua | Modélisation de l'aération naturelle et du microclimat des serres en verre de grande portée sous climat tempéré océanique[END_REF] have characterised the inside climate and the air exchange by means of tracer gas measurements, and then used these data to validate a three-dimensional simulation model using a commercial CFD software package (CFD2000).

As they increase in size, and with insect screening now being used systematically on their roof and side openings, Parral and Canary type greenhouses are looking increasingly similar to the large screenhouses used for horticultural production, which are also spreading rapidly in the subtropical and Mediterranean regions. Several studies have recently stressed the similarities between the two types of structure concerning their heat and mass balances [START_REF] Tanny | Airflow charateristics, energy balance and eddy covariance measurements in a banana screenhouse[END_REF] and the mechanisms involved in developing their inside climates [START_REF] Tanny | Screenhouse microclimate and ventilation: an experimental study[END_REF].

Although the Parral or Canary type greenhouse has been intensively studied in last decade, these studies still show some deficiencies which raise questions as to the realism of their climate simulations. The most crucial defect is that they fail to consider the interactions between canopy and air, and to couple convective and radiative exchanges, particularly at the level of the greenhouse roof cover. Similarly, few studies have been based on threedimensional studies for the greenhouses larger than 1 hectare which constitute the majority of the greenhouse area in Morocco.

In a previous study performed in the same greenhouse, [START_REF] Majdoubi | Natural ventilation performance of a large scale canary greenhouse equipped with insect screens[END_REF] showed that the overall ventilation performance of this type of shelter was hindered both by the insect screening of the vents and the orientation of the tomato rows perpendicularly to the prevailing wind. Therefore, the aim of the present study was to analyse in details the distribution of inside airflow and temperature and humidity fields in interaction with crop rows distribution.

3

We combined an experimental study and a modelling study of the micro-climate inside a real large scale Moroccan Canary type greenhouse of 1.12 ha equipped with insect screens (called 20/10 for 20 meshes cm -1 along the width and 10 meshes cm -1 along the length), across the roof and sidewall ventilation openings. The greenhouse numerical climate model is based on a commercial CFD (CFD2000®/ Storm) simulation of convective transfers, completed by a simulation, in each grid cell of the canopy, of the sensible and latent heat exchanges between the tomato crop and the greenhouse air (Boulard and Wang, 2000), together with the combination of radiative and convective transfers at roof level [START_REF] Montero | Computational Fluid Dynamic modelling of night-time energy fluxes in unheated greenhouses[END_REF][START_REF] Ould Khaoua | Modélisation de l'aération naturelle et du microclimat des serres en verre de grande portée sous climat tempéré océanique[END_REF].

The model was first validated by the measured data and then used to explore the details of air flow, temperature and humidity distribution. This CFD-assisted exploration of the inside climate and air circulation allows for a better assessment of the overall climate and plant activity and more rapid progress towards ways of improving them.

The numerical method 2.1. The numerical approach

The classical mass, momentum, energy and concentration equations can be represented for a steady-state, three-dimensional flow with the following conservation equation:

φ φ ∂ ∂φ Γ ∂ ∂ φ ∂ ∂ ∂ ∂φ S ) x ( x ) u ( t j j j j x + = (1)
where φ stands for the variables of interest, i.e. the three velocity components u i (m.s -1 ), the temperature T (K), and the specific humidity w (kg H2O .kg moistair -1 ). Γ φ and S φ represent the diffusion coefficient and source term of φ and a description of their forms can be found in Boulard & Wang (2000). The system of equations built with these variables is numerically solved with the finite volume method. Algorithms and methods for the resolution of this system of equations can be found in the CFD 2000 (2004) user's guide and will not be repeated here. To model the turbulent constraints using the standard k-ε turbulence model (Launder and Spalding, 1974), in equation (1) φ stands also for the turbulent kinetic energy k (m 2 s -2 ) and the dissipation of the turbulent kinetic energy ε (m 2 s -3 ), The Boussinesq model was also activated to take account of the gravity effect (Launder & Spalding, 1974), which means that the buoyancy force due to air density differences is added as a source term of the momentum equation [START_REF] Boulard | Convective and ventilation transfers in greenhouses, Part 1: The greenhouse considered as a perfectly stirred tank[END_REF].

Modeling of flow through insect screens and plants

For an air speed u, the drag forces induced by the insect screens and the crop that correspond to the term S φ for the equation of motion is included into our CFD study by means of the porous medium approach governed by the Darcy-Forchheimer equation:

( ) (
)

) u K C u K ( S p F p 2 ρ μ φ + - = (2)
where ρ (kg m -3 ) and μ (kg s -1 m -1 ) are respectively air density and dynamic viscosity. Values of both coefficients, K p (m 2 ) the permeability of the porous medium and C F (-) the nonlinear momentum loss, are deduced from the screen geometric properties (see Appendix 1).

For a crop canopy one considers only the second member (quadratic term) of relation (2). The sink of momentum is then proportional to leaf density and may be expressed by unit volume of the canopy using an other form, the following commonly used formula (Bruse, 1998):

2 u C I S D LAv ρ φ - = (3)
where LAv I (m 2 .m -3 ) is the leaf area index per volume and C D is the drag coefficient of the entire crop canopy. For a mature greenhouse tomato crop, Haxaire (1999) has found 0.32

= D C
, using wind tunnel facilities.

4

In summary, for the crop and the range of air speeds observed inside the canopy, the term in u of equation ( 2) can be omitted in front of the quadratic term and the non-linear momentum loss coefficient C F and the permeability K p of the medium can be deduced from the combination of equations ( 2) and (3):

D LAv p F C I K C = (4)
For our simulations, in a fist step, the tomato crop canopy was assimilated to a single 2.6 m high stand of porous medium with the same length and width as the greenhouse and with a leaf area index per volume, LAv I equal to 2.3 m 2 m -3 (corresponding to a LAs I (m 2 m -2 ) of 3). In a second step (see Appendix 4), we have parameterized the equivalent porous medium parameters to take into account the case where the leaf area index was arranged, as in the reality, according to rows and inter rows perpendicular to the air direction.

Computational meshes and boundary conditions

The 3D computational domain (Fig. 1) was 43.5 m high and included the modeled greenhouse (125m long x 90 m wide x 5.5 m high), the greenhouse situated leeward and considered as a blockage and their surroundings: the free space windward (30 m), leeward (90 m) and along the sides (30m to the north and 38m to the south) of the greenhouse. After several trials with different grid resolutions, the computational grid was set to the less time consuming: a 192 by 44 by 112 grid using Cartesian Body Fitted Coordinates (BFC) with finer resolutions imposed near the ground, walls and roof, where the thermal gradients were steeper (Fig 1).

The dynamics boundary conditions prescribed a nil vertical pressure gradient in the air at the upper limit of the computational domain, an inlet at the Western lateral limit (see Fig. 1.) and an outlet at the Eastern lateral limit whereas we have only considered conditions of flux conservation at the Southern and Northern sides. The inlet condition at the western side corresponds to the outside air conditions which direction was perpendicular to the West sidewall ventilation openings. A logarithmic profile corresponding to a wind speed profile similar to the one described by Haxaire (1999) for Southern France wind conditions was also specified at the Western boundary.

The thermal boundary conditions at the limits of the studied domain were basically of two types: (i) imposed temperature values equal to those measured during the experiment for the whole air profile at the Western lateral limit and at external soil surface and (ii) an imposed flux at internal soil surface.

Air speed and direction and air and soil surface temperatures and soil surface fluxes together with air humidity were continuously monitored and used as boundary conditions for the numerical simulation (see Table 1).

Simulation regime

The simulation was performed for steady state boundary conditions. For meeting this status, we have considered imposed climate and boundary conditions corresponding to measured data performed under clear sky conditions between 10 and 14 h (solar time) over 3 days (29/09/05 to 01/10/05). In these specific conditions, as sun position is almost fix in the sky and as sea breeze remains almost constant in speed and direction during the measurement period (Demrati et al., 2001), one can thus consider that the boundary conditions remain in quasi permanent regime. Their average and standard deviation values are summarized in Table 1.

Coupling thermal and water vapour exchanges between crop canopy and air

The coupled sensible and latent heat balances were considered at the scale of each mesh of the crop cover (approximately 0.3 3 m 3 ) by means of equations describing in permanent [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01.002: .01.002: Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 5 regime, the sensible and latent heat exchanges between the air and a virtual "big leaf". Following [START_REF] Boulard | Convective and ventilation transfers in greenhouses, Part 1: The greenhouse considered as a perfectly stirred tank[END_REF], the radiative net flux net R (Wm -2 ) reaching each mesh of the crop canopy was assimilated to a "volume heat source boundary condition" and partitioned into convective sensible sen Q (Wm -2 ) and latent heat fluxes Lat Q (Wm -2 ), depending on the heat and water exchanges between the air and this virtual "big leaf" characterised by its surface temperature (T v ):

0 = - - lat sen net Q Q R (5)
Where the sensible heat flux Sen Q was expressed with respect to the temperature difference between inside air and canopy [START_REF] Roy | CFD based determination of temperature and humidity at leaf surface[END_REF]:

( ) (
)

a i v LAv p Sen r T T I C Q - = ρ (6)
with T v and T i the canopy and inside air temperatures (K), p C (Jkg -1 K -1 ) the specific heat of air at constant pressure, LAv I (m 2 m -3 ) the leaf area index per volume and a r (sm -1 ) the leaf aerodynamic resistance.

Lat

Q

was deduced from a similar relation with respect to air humidity difference between w v * (kgkg -1 ), the saturated water content of the air at canopy temperature and i w (kgkg -1 ), the specific humidity of the air:

( )( ) (
)

s a i v LAv e v Lat r r w w I L L Q + - = * 3 1 ρ (7)
with the Lewis number Le=0.86, L v (J kg -1 ) the latent heat of water vaporization and s r (sm -1 ) the leaf stomatal resistance. The details of the equations and the method used to customise the commercial CFD model are described in Appendix 2.

Coupling convective and radiative exchanges at greenhouse roof level

Except for very recent studies [START_REF] Montero | Computational Fluid Dynamic modelling of night-time energy fluxes in unheated greenhouses[END_REF][START_REF] Ould Khaoua | Modélisation de l'aération naturelle et du microclimat des serres en verre de grande portée sous climat tempéré océanique[END_REF], most authors have considered imposed temperature conditions (measured values) at the greenhouse roof and walls, i.e. inside the studied domain. These imposed boundary conditions inside and not at the boundaries of the simulated domain are in contradiction with the very purpose of the simulation, which is to deduce the state variable inside the studied domain knowing only its boundary conditions. In fact this was due to the failure to couple radiative and convective exchanges, particularly at the roof and walls. In the present study, the contributions of solar and atmospheric radiation have been included in the model by simulating the roof cover energy balance and deducing the resulting roof temperature.

As the plastic cover is very thin (≈ 200 μm), one can consider that internal and external roof surfaces have approximately the same temperature and that the energy balance of the roof can be simplified to the following form:

0 H H Q Q ) P ( Q P Q Cce Cci R R v R v c c , sky c , si c , v = - - + - + + 1 (8)
Where Q c (Wm -2 ) represents the short wavelengths radiative flux absorbed by the roof cover (direct and reflected solar radiation);

c v R Q , (Wm -2
) is the long wavelength exchanged between the canopy and the roof cover, pondered by P v, the portion of crop canopy seen by the roof cover; (Wm -2 ) is the convective flux exchange between the outer surface of the plastic cover and outside air.

c si R Q , (
Expressing Eq.( 8) with respect to the roof temperature T c and the temperatures of the other elements in the greenhouse system, i.e. tomato canopy temperature T v , internal soil surface temperature T si ,, internal and external air temperatures T i and T e , and sky temperature T sky , together with their geometrical, optical and thermal properties, one can deduce the plastic cover temperature T c (see Appendix 3). This temperature computation is introduced in the numerical model using the "user defined" options available in the CFD 2000 software (see Appendix 3).

For each trial, about 10 days of uninterrupted calculation time were needed on average before converging to a solution for the greenhouse climate numerical simulation, using a 2.5 GHz frequency computer with a 512 MBytes random access memory (RAM).

Materials and methods

The greenhouse

The studied greenhouse (Fig. 2) is a commercial Canary type plastic greenhouse covered with a 200 µm thick single-layer polyethylene sheet. The dimensions of this 1.125-ha area greenhouse are: 90 m in length, 125 m in width, 5 m in height at the gutter and 5.5 m at the ridges. The spans and the tomato crop rows were oriented North-South, i.e., perpendicular to the direction of the prevailing sea breeze. There is another greenhouse nearby, leeward with respect to the prevailing wind and connected to the first one by an insect screened space (Fig. 2).

The greenhouse was provided with natural ventilation by means of seventeen roof vent openings (0.6×125 m² each, i.e. a total of 1275 m 2 ) covered with insect screens (20 meshes cm -1 in width, 10 meshes cm -1 in length, with a wire diameter of 0.28 mm). The sidewall ventilation openings were equipped with similar insect screens and the maximum opening areas were 875 m² on the West-East sides and 630 m² on the North-South sides. During the experiment, the total roof opening area and total sidewall opening area were maintained throughout at 1275 m² and 1505 m 2 respectively (Fig. 2).

Experimental conditions

The parameters described in Table 2 were systematically recorded, in order (i) to characterise the inside microclimate, (ii) to determine the simulation boundary conditions and (iii) to validate the simulation model. All these measurements were taken every 5 seconds and the data were then averaged and stored every fifteen minutes in two data loggers (Models 21 X and CR23, Campbell Scientific, Inc., Logan, USA).

The greenhouse was occupied by a tomato crop (Solanum Lycopersicum, cv. Gabriella) planted on July 18th, 2005, with a plant density of 1.8 plant m -2 in North-South oriented rows, i.e. perpendicular to the direction of the prevailing Westerly sea breeze (Fig. 2), and with a leaf area index LAs I (m 2 m -2 ) of 3 at the time of the measurements. An explicit description of the spatial variation (rows and between rows of plants) of plant density resolved in the model is presented in Appendix 4. The crop was watered by a drip irrigation system and soil surface was considered as a dry surface.

Results and discussion

Model validation 4.1.1. Distributed inside climate

Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. ( 2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural andForest Meteorology, 149 (6-7), 1050-1062 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009 1) shows (Fig. 3) that the difference between the simulated and measured air temperature profiles from West to East of the greenhouse ranges between 0.1 and 0.95°C, with an average difference of 0.61°C. Examination of the simulated and measured specific air humidity inside the experimental greenhouse at 1 and 4 m above the soil surface (Fig. 4), demonstrates that the measured and predicted values are very close, the difference ranging between 0 and 0.9 g kg -1 , with a root mean square error of 0.1 g kg -1 .

Greenhouse ventilation rate and crop transpiration flux

The overall air exchange rate had already been determined and modelled for this particular greenhouse using the overall heat and water vapour balance of the whole greenhouse space [START_REF] Majdoubi | Natural ventilation performance of a large scale canary greenhouse equipped with insect screens[END_REF]. This approach is similar to the tracer gas method, which minimum accuracy (Ducarme et al., 1994) is estimated to about 30%. Using this approach, and for the same vent opening area and wind speed conditions as those used as boundary conditions for our simulation (Table 1), one can estimate G (m 3 s -1 ) the ventilation flow rate in the greenhouse, G=131.25 m 3 s -1 .

Averaging T r the transpiration flux value which was deduced from the numerical model using equation ( 7) over the whole canopy space

v Lat r L Q T =
(9) One finds Tr =178.51 g h -1 m -2 . This computed value can be compared with an estimation, based on the determination of the overall ventilation rate G, of the overall water vapour flux extracted from the greenhouse by ventilation, e , Li Q /L v , with :

) w w )( A G ( Q i e si e , Li - = ρ (10)
where A si (m 2 ) is the greenhouse soil area. As the tomato plants are watered by drip irrigation, one can consider that soil evaporation is negligible and that the water vapour extracted per m 2 from the greenhouse by ventilation e , Li Q /L v must be equal to the canopy transpiration Tr per m 2 of greenhouse soil.

Application for our experimental conditions using the measured climatic data and the estimated overall ventilation rate G gives e , Li Q /L v = 169.61 gh -1 m -2 , which is close to the average computed transpiration flux Tr =178.51 gh -1 m -2 deduced from the numerical model.

Detailed indoor climate and air movement analysis

The main interest of this numerical simulation lies in the opportunity it provides details on air speed, temperature and humidity distributions throughout the greenhouse domain. We have thus systematically explored the longitudinal and vertical distributions for the main state variables and vectors derived from the model, inside the greenhouse.

Flow field

For the same wind direction as the prevailing sea breeze, which blows perpendicularly to the vent openings on the West side, we have plotted a vertical cross-section of the velocity vector field across the middle of the greenhouse (Fig. 5). This shows that air speed in the space between the top of the crop canopy and the greenhouse roof is approximately half that of the outside wind, with the same direction (from West to East). Air velocity is much lower inside the crop canopy than outside (1/10 to 1/15 of outside wind velocity U ext ) and it flows systematically in a reverse direction, i.e. from the leeward end (East) to the windward end (West) of the greenhouse. A similar observation, but confined to the downstream part of the greenhouse, has already been reported by Fatnassi et al. (2003) for a quite similar greenhouse (5000 m 2 ) equipped with coarser insect screens (6 by 6 meshes) and with roof ventilation [START_REF] Wang | Measurement and modelling of natural ventilation in a large Venlo type greenhouse[END_REF] has also evidenced this reverse flow in the whole volume of a 1600 m 2 Venlo type greenhouse equipped with discontinuous roof vent openings without insect screens. Based on computational fluid dynamics simulation and validation of the pressure distribution on the roof of a 100 m long commercial multi-span Venlo type glasshouse, Reichrath & Davies (2001) have also corroborated this phenomenon and shown that it was due to a static pressure difference between the windward and leeward parts of the inside greenhouse air, in relation with the static pressure field due to wind pressure at the outside surface of the greenhouse cover.

Fig. 5 looks more closely at air circulation along one period (it corresponds to one span with alternatively upper and lower vent openings) of the flow pattern inside the greenhouse; it reveals air circulation loops due to buoyancy forces with horizontal axes which develop perpendicularly to the wind direction. In fact, a detailed examination of the cross-section of the vertical flow fields at the level of the roof vents, alternately positioned at the roof ridges and in the dips (not shown here) has also clearly shown that these loops were fed by (cold and dry) outside air coming through the lower roof openings (acting as air inlets) and (warm and humid) inside air exiting through the upper roof openings situated at ridges (acting as air outlets).

The air velocity distribution in a vertical plan situated in the centre of the greenhouse along the West-East axis (Fig. 6) allows us to determine the inside air speed more precisely: 0.2 to 0.5 U ext between the top of the canopy and the roof, and 0 to -0.1 U ext inside the crop canopy. This considerable reduction in air speed, from 0.2 -0.5 U ext to 0 -0.1 U ext is essentially due to the drag effect of the tomato plant rows, which are exactly perpendicular to the airflow. This observed air speed reduction is consistent with the previous estimation of [START_REF] Majdoubi | Natural ventilation performance of a large scale canary greenhouse equipped with insect screens[END_REF] for the same greenhouse, of a strong reduction in the overall air exchange rate between inside and outside owing to the drag effect of the canopy.

The air velocity profiles across the middle of the greenhouse at heights of 1, 3 and 4 m (Fig. 7) show that inside air velocity at 3 and 4 m above soil level first strongly decreases in the first 10m, then increases progressively from the windward end to the leeward end, from approximately 0.25U ext to 0.73U ext . A similar trend is observed at 1m above soil level in the crop canopy, the inside air velocity varying approximately from -0.07U ext to -0.2U ext . One can also see that a periodicity in the air speed profiles at the 1 and 3m heights is due to a combination of wind and buoyancy forces imposed by the succession of roof vent openings alternately positioned at the roof ridges and in the dips, thus acting as outlets for the warm and light inside air and as inlets for the cold and heavy outside air. One must stress that similar observations of loops of warm air going out of the greenhouse through the upper vent openings while cold air was entering through the lower vent openings has systematically been reported by most of the authors who have experimentally and numerically studied greenhouse ventilation. Thus, Mistriotis et al.(1997a) and Boulard et al. (1999) have describe these loops for pure free convection in model scales greenhouses, whereas [START_REF] Sase | Wind tunnel testing on airflow and temperature distribution of a naturally ventilated greenhouse[END_REF] and [START_REF] Mistriotis | Analysis of the efficiency of greenhouse ventilation using Computational Fluid Dynamics[END_REF] also describe it for a combination of free and forced convection for both model scale and real size greenhouses.

Overall, one can summarise the complex inside air current as follows: (i) a strong air current fed by the outside wind passes through the greenhouse from the windward to the leeward side openings just above the crop canopy and below the greenhouse roof, and (ii) with a reverse direction and a much lower speed within the crop canopy. In addition, (iii) air loops fed by buoyancy forces due to the inside air temperature differences develop perpendicularly between the top (exit of warm and light air) and bottom (entry of cold and heavy air) roof vent openings and the canopy volume.

Temperature and humidity patterns 9

The vertical profile of air temperature in the centre of the greenhouse (Fig. 8), shows that high temperatures are confined to the immediate vicinity of all the solid surfaces intercepting the solar radiative flux, but which also slow down the air flow, as it is the case for the roof and soil surfaces and the crop surfaces within the canopy. Overall, the air temperature rise is very sharp, from 303K (30°C) outside the greenhouse and in the inner space between the roof and the top of the canopy to 311K (38°C) at the roof and soil surfaces. Within the crop canopy, air temperature rises from 305K (32°C) at its top to 311K (38°C) near the soil surface.

The longitudinal windwise profile of air temperature across the middle of the greenhouse at heights of 1, 3 and 4 m (Fig. 3), shows that overall air temperature values are significantly higher near the windward end than at the leeward end. This is in agreement with our analysis of the air circulation pattern showing that air speed and air exchange rate, which evacuate the heat, are higher near the leeward end of the greenhouse than at the windward one. The wide periodic variations in air temperature (approximately 3 to 4 K) recorded at a height of 1 m explain very well the formation of convective loops at this height, induced by buoyancy forces caused by important temperature differences over short vertical and horizontal distances (Fig. 3). Higher above ground level, the temperature field becomes more homogeneous, as is the case at heights of 3 and 4 m (difference at same height approximately 0.7 K) and its absolute value tends to the value of the outside temperature.

The vertical profile of specific air humidity in the centre of the greenhouse (Figure 9) does not exhibit any peaks at these levels as was the case for the temperature profile because plastic roof cover and soil surface do not exchange any water vapour. However, one can state an increase of about 4 g kg -1 in specific humidity from the inside greenhouse air just above the canopy to the lower part of the crop canopy.

The longitudinal windwise profiles of specific air humidity at different heights above the ground (Figure 4) don't exhibit, contrary to the air speed and temperature patterns, any strong gradient between the upstream and downstream ends of the greenhouse. The cyclic variations of air humidity, due to the convective loops, are also shown to be much weaker than for air temperature.

In summary, a strong, dry, cold outside air current fed by the wind is confined inside the greenhouse below the roof and just above the crop canopy, inside air speed being greater and air temperature and humidity lower near the upstream end of the greenhouse. As one penetrates down the crop canopy from its top to the soil surface, one can observe a weak reverse flow with respect to the wind direction, with very high air temperature and humidity conditions.

One can reconstitute the main characteristics of air circulation and heat and mass transfers within the greenhouse as follows (Fig 10 a): cold, dry air penetrates through the windward side vent opening. This air is first warmed up, its saturation deficit increasing automatically, allowing an increase in transpiration flux within the canopy and then an increase in specific humidity. This warmer and more humid air is then evacuated by buoyancy forces through the upper roof vent openings (Fig 10 b), it is replaced by colder and dryer outside air which penetrates through the lower roof vent openings. Always owing to buoyancy forces between inside warm and humid air and outside cold and dry air, one observes near the leeward end of the greenhouse (Fig 10 c) a cold and dry reverse flow which penetrates into the greenhouse through the screened space separating the two greenhouses.

Canopy transpiration flux

Under diurnal conditions, canopy transpiration is certainly the most important energy dissipation mechanism in the greenhouse agrosystem, maintaining inside climate conditions in accordance with plant needs. Following [START_REF] Stanhill | Solar radiation and water loss from glasshouse roses[END_REF] The latent heat flux according to canopy height below a lower roof opening where cold air penetrates is presented in Fig. 11. It shows that the latent heat of the transpiration flux decreases from about 180 Wm -2 at the top of the canopy to 135 Wm -2 near the soil surface. This 25% drop, already measured for a greenhouse tomato crop by Boulard et al. (1991), is attributed to two main causes: (i) the reduction in the absorption of overall radiation from the top to the base of the canopy, described in our model by a Beer Lambert law (see equations 2.4 and 2.5 in Appendix 2) and (ii) an increase in the saturation deficit at the base of the canopy, due to temperature increasing more than specific humidity (see Figures 6 and8); which in turn induces an increase in the stomatal resistance of the canopy leaves (see equation 2.3 in Appendix 2) and reduces transpiration flux.

The longitudinal latent heat flux distribution from West to East at 1m above ground (Fig. 12) revealed a considerable spatially cyclic heterogeneity with values ranging between 140 and 155Wm -2 . Comparing this cyclic evolution with similar ones evidenced for air speed, temperature and humidity (Figs. 3,4,7) allows us to conclude that generally, areas with low transpiration rates also correspond to areas of low air speed, high air temperature and high saturation deficit, both trends significantly increasing stomatal and aerodynamic leaf resistance and reducing crop transpiration rate proportionately.

Sensibility study: effect of plant row orientation on inside climate

In our study, the crop canopy has been treated as a single, large porous-medium block occupying the entire greenhouse soil area, whereas in reality the crops stands occupied only the half of the soil surface. For simulation purposes, the equivalent leaf area index per volume attributed to the plants in the porous-medium block has therefore been divided by two, to compensate for the artificial doubling of the canopy volume.

As the simulation results have underlined the strong influence of the crop canopy on flow and the associated climate fields, we have tried to make the crop row modelling more realistic and to test the effect of plant row orientation on inside climate. The originality of the approach, designed to keep the model simple, is that it preserves a single, large parallelepiped block of canopy occupying the whole greenhouse soil area, with overall porosity properties accounting for the row orientation with respect to the general air circulation.

Three different cases have been considered: (i) the case where the whole leaf area of the canopy is dispersed through the entire volume of the block occupying the whole greenhouse soil area, (ii) the actual case, where the crop canopy rows are perpendicular to the outside wind direction and occupy only the half of the soil surface (leaf surface density per volume of crop row is twice that of case (i)), and finally (iii) a case where the plant rows are parallel to the outside wind (leaf surface density per volume is also twice that of case (i)). Appendix 4 provides, for each case, the equivalent porous-medium properties to be considered for the canopy block.

The results of the simulations presenting the horizontal windwise greenhouse air velocity distribution from West to East at 1m height for the three cases of plant distribution considered show (Fig. 13) that with the most realistic case, i.e. case (ii), air speed at 1m height within the canopy is even lower than for case (i), and tends to 0. Conversely, orienting the rows in the direction of flow (case (iii)) substantially decreases the drag of the cover and increases air speed at this height. On the contrary, with the crop rows perpendicular to the wind direction (case (ii)), simulations at 1m height in the canopy show that the increase of the canopy drag effect induces with respect to the other two cases respectively a temperature rise of about 1°C (Fig. 14) and a humidity rise of about 1g kg -1 (Fig 15).

Conversely, from a practical point of view, it means that temperature and humidity rise at the level of the plant canopy can be reduced by about 25% by simply orienting the crop rows parallel to the direction of air flow.

Conclusion

We have seen in this study that an understanding of the air exchange mechanisms between the inside and outside of the greenhouse is crucial for determining the distributed climate of the greenhouse air and canopy. The CFD model of the greenhouse climate, including coupling with the long wave radiative transfers and the effects of plant canopy and insect screens, makes it possible to obtain a true picture of the whole greenhouse volume, including at canopy level. After being verified against the observed data for air, the CFD model can then be used as a powerful tool to explore the climate inside the whole greenhouse volume. One can thus present the continuous distribution of the main state and flux variables of microclimate and crop activity and highlight the factors that shape the inside climate.

In particular, the study shows that even with low outside wind speed as in our case ( ext U =1.3 m s -1 ), the outside wind governs in turn the inside air flow direction, inducing a strong windwise air current above the canopy and a very slow reverse flow inside the crop canopy. The weak air exchange within the canopy governs the climate at this level, with a major increase in air temperature and a more moderate increase in specific humidity. It also shows how buoyancy forces, induced by air temperature and humidity increases, give rise to air loops between the canopy and the roof vents, which in turn tend to accelerate the rate of heat and water vapour evacuation and to improve indoor climate conditions.

As our study of the influence of tomato crop row orientation shows, once the roles of the different mechanisms determining the inside microclimate have been identified in this way, it is possible to test virtual changes to the system so as to quickly identify solutions that will substantially improve its functioning. [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01. The values of the aerodynamic proprieties (K p and C F ) of the screen have been deduced from the literature using [START_REF] Miguel | Air through porous screens: from theory to practical considerations[END_REF] approach based on their correlations with the porosity of the net: (1.2) where α is the screen porosity (-) deduced from the dimensions of the thread [START_REF] Miguel | Air through porous screens: from theory to practical considerations[END_REF]:

(

)

) d W )( d L ( LxW + + = α (1.
3) with L= 0.788 mm and W= 0.255 mm are respectively meshes length and width, and d=0.28 mm, is the wire diameter.

The computed values used in equations (1.1), (1.2) and ( 1.3) for determining the aerodynamic proprieties (K p and C F ) of the (20x10) screen used in the studied greenhouse are α= 0.35, K=6.4 10 -10 , C F =0.402.

Appendix 2: Sensible and latent heat coupling between air and canopy

The sensible and latent heat transfer coupling between air and canopy was considered by "customizing" the CFD code by means of an appropriate "Source model" (Haxaire, 1999) available with the CFD software :

) iable var Dependent value ( Coef Source - × =
(2.1) where the terms in this equation were identified to the terms of the sensible and latent heat transfer equations between plant and air within each mesh of the canopy, i.e. for the temperature : The value of the aerodynamic resistance a r was deduced from the air speed within each mesh of the canopy using a relation pertaining for greenhouse crop conditions [START_REF] Boulard | Convective and ventilation transfers in greenhouses, Part 1: The greenhouse considered as a perfectly stirred tank[END_REF]:

( ) 5 . 0 0.288 U d C r v p a r ν λ ρ = (2.2)
where v d (m) is the mean characteristic length of the leaf; and U (m s -1 ) is the interior air speed within each mesh of the crop canopy domain; λ (W m -1 K -1 ) is air thermal conductivity and ν (m 2 s -1 ) is air viscosity.

Tomato leaf stomatal resistance was deduced from air temperature and saturation deficit values using Boulard et al (1991) formulation: Because of light interception within the canopy, we have expressed the solar radiation received at an height z (m) by means of a Beer's law according to the following equation:

⎪ ⎭ ⎪ ⎬ ⎫ ⎪ ⎩ ⎪ ⎨ ⎧ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ - - + = + )
( ) ) exp( ) ( H z H I k R z R LAs c gi - - = (2.4) ( ) ( ) ) ( 2 1 z dR z R z R R abs = - =
(2.5) Where R gi (W.m -2 ) is the global radiation inside the greenhouse and over the canopy; k c is the extinction coefficient of radiation, set to 0.75 for a tomato crop, H (m) is the total height of the canopy, LAs I is the crop stand leaf area index (m 2 m -2 ) and z 1 and z 2 are two arbitrary heights .

Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. ( 2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01. Finally, neglecting the long waves radiations in front of solar ones, tomato crop temperature (T v ) can be calculated according to the following equations:

⎥ ⎦ ⎤ ⎢ ⎣ ⎡ - - + = + = ) I r w wi L dz ) z ( dR ( I 2 1 C r T C C r T T LAv t a v LAv p a i p a i v ρ ρ ρ
(2.6) Appendix 3: Coupling the convective and radiative transfers at roof level.

Q c (W.m -2 ) used in Eq. ( 8), corresponds to the radiative balance of the plastic cover in the domain of the short wave lengths. It can be expressed according to:

ge v Sc Ss Sc ge v Sc Sv Sc ge Sc c R P R P R Q ) 1 ( - + + = τ ρ α τ ρ α α (3.1)
with Sc α and Sc τ the short wavelength absorption and transmission coefficients of the plastic cover respectively; ge R (W m -2 ) is the outside radiation; Sv ρ is the short waves reflection coefficient of the canopy and Ss ρ is the short waves reflection coefficient of the soil surface. Note that the optical insect proof nets properties are not considered in this equation because they represent less than 10% of the roof area and also because their properties are very similar to these of the plastic film.

The long wavelength radiative flux exchanged between the canopy and the roof cover

c , Rv Q (W.m -2
) used in Eq. ( 8) can be expressed as [START_REF] Majdoubi | Natural ventilation performance of a large scale canary greenhouse equipped with insect screens[END_REF]:

) 1 1 ( 1 ) ( 4 4 , - + -
= c c v v c v R A A T T Q c v ε ε σ (3.2) with v ε the crop emissivity; v A / c
A being the view factor from the crop cover to the roof with v A (m 2 ) and c A (m 2 ) the vegetation and the roof cover surface area respectively. Likewise, c , si R Q (W.m -2 ) used in Eq. ( 8) is the long wavelength radiative flux exchanged between the soil surface and the roof cover can be expressed as :

) 1 1 ( 1 ) ( 4 4 , - + - = c c si si c si R A A T T Q c si ε ε σ (3.3)
with si ε the soil surface emissivity and si A / c A the view factor from the soil surface to the roof, with si A (m 2 ) the soil surface area. c , sky R Q (W.m -2 ) used in Eq. ( 8) is the long wavelength radiative flux exchanged between the sky, considered as a black body, and the roof cover :

) T T ( Q c sky c R c , sky 4 4 - = σ ε (3.4) with c
ε the plastic cover emissivity; σ = 5.67 10 -8 (W m -2 K -4 ) the Stefan-Boltzmann constant. Sky temperature, sky T (K), can be approximated from the external air temperature according to [START_REF] Swinbank | Long wave radiation from clear skies[END_REF] (3.5)

Following [START_REF] Sacadura | Initiation aux transferts thermiques[END_REF], for "small" temperature differences between the elements of the radiative exchange system ( Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009 )

) ( A A T T h Q c c v v c v 1 r R c , v 1 1 1 - + - = ε ε (3.6) with: 3 1 4 v v r T h σε = ) ( A A ) T T ( h Q c c si si c si 2 r R c , si 1 1 1 - + - = ε ε (3.7) with : 3 2 4 si si r T h σε = ) T T ( h Q c sky c 3 r R c , sky - = ε (3.8) with, 3 3 4 sky r T h σ = Cci
H the sensible flux which is exchanged at the level of the internal face of the plastic roof cover can be given by:

) ( i c ci Cci T T K H - = (3.9)
Cce H represents the convective exchange at the level of the external face of the plastic roof cover and can be expressed as follow :

) (

e c ce Cce T T K H - =
(3.10) where ci K and ce K are respectively the convective exchange coefficients of the two faces of the plastic roof cover (Wm -2 .K -1 ). If we take in first approximation

5 1 5 4 38 . 5 d U K K ext ce ci = =
for a turbulent forced convection regime (Roy et al., 2002), the plastic cover temperature (T c ) can then finally be expressed as:

( ) 2 r 1 r 3 r e i ce si 2 r v 1
) ( A A P A c c v v v 1 1 1 - + = ε ε and (
)

) ( A A P 1 B c c si si v 1 1 1 - + - = ε ε
Appendix 4: Equivalent porous medium parameters value corresponding to different plant distributions with respect to the prevailing airflow.

For the calculation of the equivalent porous medium parameters value corresponding to the three different plants distributions with respect to the prevailing airflow, we have considered the combinations of resistances to air flow given in Fig. 16. R v is the resistance of the plant row to air flow, R 0.5v is the same resistance for the case where the plant canopy is dispersed on the whole greenhouse soil area, and R air is the resistance of the inter rows to air flow. Following Bernoulli's approach, one can express the pressure drop ΔP of a porous medium as a function of a discharge coefficient Al as follows:

2 2 U Al 5 . 0 P ρ Δ = (4.1)
If Al v is the discharge coefficient of air through the plant canopy arranged in rows, with respect to Eq. ( 4.1.) the corresponding pressure drop is ΔP v with 1/Al v 2 =R v . Likewise if Al a the discharge coefficient of air crossing the inter rows, the corresponding pressure drop is air P Δ and 1/Al a 2 =R air .

In the case (i) where the leaf area is dispersed into the whole canopy block, air must only overcome the pressure drop of the canopy considered with a half actual Lav I value and the total pressure drop is given by : In the case (ii) where the leaf area index is arranged according to rows perpendicular to the air direction, air must overcome the resistances of a stand of vegetation and of an inter row considered in serials, the total pressure drop can then be given by:

2 2 e 2 2 v 2 a tot U Al 5 . 0 U Al 1 Al 1 5 . 0 P ρ ρ Δ = ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ + = (4.3)
where Al e is the equivalent discharge coefficient of air for the row and inter row arrangement. In the case (iii) where the leaf area index is arranged according to rows parallel to the air direction, air must overcome the resistances of stands of vegetation and of an inter rows considered in parallel and the total pressure drop can be given by:

2 2 e 2 2 v 2 a 2 v 2 a tot U Al 5 . 0 U Al 1 Al 1 Al . Al 1 5 . 0 P ρ ρ Δ = ⎥ ⎥ ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎢ ⎢ ⎣ ⎡ + = (4.4)
Considering that Al a value is similar to the pressure drop coefficient of air through a vent opening (i.e. Al a = 0.7, Boulard and Baille (1995) [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009 [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01. [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009 [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01. [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01. Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01. [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01. [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01. Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01.

  . DOI : 10.1016/j.agrformet.2009.01.002

  of the aerodynamic proprieties (K p and C F ) of the screen.

  002: Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 15

  one can linearize the relations (3.2), (3.3) and (3.4) as follows: Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  Fig.1. View of the computational grid of the whole studies 3D domain for the CFD simulation. Gridcell dimensions inside the greenhouse vary between (0.26, 0.44, 0.032) near the soil and walls and (0.75, 1.25, 0.1) at2.5 m high.

  Fig.3. Simulated and measured air temperature profiles from West to East of the greenhouse, halfway along its North-South length: Measured values 1 m (▲) 4 m (♦) above soil level Simulated values 1 m ( ); 3 m ( ); 4 m ( )

  Fig.7. Modelled horizontal air velocity profiles across the middle of the greenhouse along W-E direction at 3 different heights:

  Fig.8. Modelled vertical profile of air temperature in the centre of the greenhouse

  . Reconstitution of air circulation within the greenhouse from W to E, halfway along N-S direction. a: at the windward end of the greenhouse b: in the middle of the greenhouse c: at the leeward end of the greenhouse Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  Fig.11. Modelled vertical distribution of the latent heat of the canopy transpiration flux in the centre of the greenhouse

  Fig.12. Modelled longitudinal transpiration flux distribution from West to East at 1m above ground in the middle of the greenhouse

  Fig. 13: Modelled horizontal windwise greenhouse air velocity distributions from West toEast at 1m height for the three cases of plant distribution considered i) Canopy considered as a single block ( ) ii) Canopy rows perpendicular to prevailing wind direction ( ) iii) Canopy rows parallel to the prevailing wind direction ( )

  Fig. 15: Modelled horizontal air humidity distributions from West to East at 1m height for the three cases of plant distribution considered i) Canopy considered as a single block ( ) ii) Canopy rows perpendicular to the prevailing wind direction ( ) iii) Canopy rows parallel to the prevailing wind direction ( )

  Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology,

  .01.002:Validation of the numerical model with respect to experimental data measured for the same boundary conditions (Table

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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and ForestMeteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01.002:

	openings which were parallel to the prevailing wind direction.
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Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  one can even say that through Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and ForestMeteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01.002: , the crop modify deeply its local climate, a modification which in turn influences strongly transpiration. Our additions to the CFD programme, for coupling aerial transfers and crop transpiration, allowed us to model this mechanism and thus simulate transpiration flux throughout the canopy.

	the transpiration mechanism
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Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and ForestMeteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01.002:

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Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology,

Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  .01.002:

	(
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  ) and substituting this value and those of Al v and Al 0.5v in relations (4.2 to 4.4) one finds Al e values corresponding to the porous medium properties for respectively the cases (i) to (iii). Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002 Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology,

		Case (i)	Case (ii)	Case (iii)
	Al e	0.96	0.52	1.05

Table 1 :

 1 .01.002: Experimental measurement (mean and standard deviation) performed outside the greenhouse between 10 and 14 h (solar time) every 15 minutes during 3 days(29, 30 September and 01 October 2005) and used as boundary conditions for the numerical simulation.

			TABLES
	Manuscrit d'auteur / Author manuscript	Parameters Outside temperature e T (°C) Outside relative humidity e RH (%) Sky temperature T sky (°C) Outside soil surface temperature si T (°C) Wind direction D v (degree) Wind speed ext U (m s -1 ) Net radiation R net (W m -2 ) Inside soil heat flux F s (W m -2 ) Table 2: Measured parameters, sensors used and locations Mesured parameters Sensors name	Mean 29.74 45.08 17.83 36,67 105.73 1.3 298.75 50	Locations	S.D 0.95 2.6 1.15 0.88 7.45 0.27 24.3 1.11
		Inside net radiation R net (W m -2 ) Net radiometer Q-7	between the top of the crop
				and the roof cover
	Manuscrit d'auteur / Author manuscript	Soil heat flux exchange at ground surface F S (W m -2 ) Inside air temperature i T (K) and relative humidity	Soil heat flux HFT3	1mm below the soil surface.
	Manuscrit d'auteur / Author manuscript
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  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and ForestMeteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01.002: Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology,

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Comment citer ce document : Majdoubi, H.,

[START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF]

. Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study.

Agricultural and Forest Meteorology, 149 (6-7), 1050-1062

. DOI : 10.1016/j.agrformet.2009.01.002 20 Fig.2. Schematic view of the studied greenhouse, its ventilation system and the surrounding (note that in this scheme, the West direction is on the right) Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology,

Comment citer ce document : Majdoubi, H., Boulard, T., Fatnassi, H., Bouirden, L. (2009). Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

Comment citer ce document : Majdoubi, H., [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF]. Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study. Agricultural and Forest Meteorology, 149 (6-7), 1050-1062. DOI : 10.1016/j.agrformet.2009.01.002

Version définitive du manuscrit publié dans / Final version of the manuscript published in : Agricultural and Forest Meteorology, 2009, vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009 [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009 [START_REF] Majdoubi | Airflow and microclimate patterns in a one-hectare Canary type greenhouse: an experimental and CFD assisted study[END_REF], vol.149, no.6-7, DOI : 10.1016/j.agrformet.2009.01..01.


Jane Roche

Marion Alignan

Andrée Bouniols

Muriel Cerny

Zephirin Mouloungui

Félicity Vear

Dr Othmane Merah
email: othmane.merah@ensiacet.fr

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions Suggestion for an abbreviated running title: Genotypes and environment effects on sterol contents in sunflower seeds

Keywords: sunflower seeds, phytosterols, genetic variability, genotype*environmental conditions interactions, fatty acid profiles

Phytosterols play essential roles in many plant cell mechanisms. They are of industrial interest since, as part of the diet, they can reduce Low Density Lipoprotein cholesterol. An increase in plant sterol contents by improved crop varieties or crop management could help to answer industrial demands and also to develop environmentally friendly extraction methods. The aim of this study was to evaluate genotypic variability for sterol content in cultivated sunflower and effects of sowing date, in particular sowing date. Results showed large variability among a collection of sixteen sunflower inbred lines and hybrids. Total sterols varied almost twofold between extreme genotypes. A delay of sowing, giving higher temperatures during seed formation, induced a general increase in total sterol concentration by up to 35 per cent, as well as sterol composition but this varied according to genotype. These results are discussed with the aim of improving sterol content by sunflower breeding programmes.

Introduction

Phytosterols are naturally present in plants, playing important roles in membrane fluidity and permeability [START_REF] Schaller | The role of sterols in plant growth and development[END_REF], embryogenesis [START_REF] Clouse | Molecular genetic studies confirm the role of brassinosteroids in plant growth and development[END_REF], and as precursors of brassinosteroid hormones involved in plant growth and development [START_REF] Lindsey | Importance of plant sterols in pattern formation and hormone signalling[END_REF]. During the last decade, there has been new interest in sterols due to their potential benefits for human health. Their role in reducing Low Density Lipoprotein cholesterol (LDL) [START_REF] Ostlund | Phytosterols, cholesterol absorption and healthy diets[END_REF] have been clearly established. Several studies have highlighted other interesting properties such as anti-cancer and anti-oxidation activities (Van Rensburget, Daniels, Van Zyl & Taljjard, 2000;[START_REF] Awad | Beta-sitosterol, a plant sterol induces apoptosis and activates key caspases in MDA-MB -231 human breast cancer cells[END_REF] Consequently, these bioactive molecules are now used for various industrial applications, particularly in nutrition as functional food, for example in enriched margarines [START_REF] Moreau | Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses[END_REF]. By chemical modification, phytosterols could also be used as raw materials in pharmaceutical production as a source of steroids [START_REF] Van Dansik | Phytosterols[END_REF] or in cosmetics [START_REF] Folmer | Sterol surfactants: from synthesis to applications[END_REF]. This diversification in use of sterols has led to an increase in industrial demand.

Phytosterols are present in different plant parts, mostly in seeds. Their level depends on species and sunflower seeds contain a relatively high concentration (Mouloungui, Roche & Bouniols, 2006). However, sterols are present at low concentration rendering their extraction rather difficult. Improvement of phytosterol concentration in seed could increase their accessibility and thus help for development of soft methods of sterol extraction.

Adapted crop management could also improve phytosterol content, comparable with fatty acid composition where both environmental and genetic factors have been shown to be sources of considerable variability of oil quality in sunflower seed. [START_REF] Roche | Management of environmental crop conditions to produce useful sunflower oil components[END_REF] demonstrated that the choice of genotype associated with adapted cultural practices could be used to orientate fatty acid biosynthesis. Genotypic

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., [START_REF] Roche | Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions[END_REF].

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 variability for sterol content has been reported in soybean (Yamaya, Endo, Fujimoto & Kitamur, 2007) and in cereals (Nurmi, Nyström, Edelman, Lampi & Piironen 2008;[START_REF] Alignan | Effects of genotype and sowing date on -phytostanols -phytosterols content and agronomic traits in wheat under organic agriculture[END_REF]. Effects of genotype on sunflower seed oil and protein contents have been widely reported, but, so far, there have been no studies of genotypic variability of sterol content. No information is available on interactions between genetic factors and environmental conditions affecting sterol concentration and composition in sunflower seed. Knowledge of the genetic variability available is essential to make possible heredity and heritability studies and mapping of genes controlling sterol content. Although [START_REF] Roche | Management of environmental crop conditions to produce useful sunflower oil components[END_REF] described the behaviour of genetic variability of total sterol contents, only 3 genotypes were analyzed in interaction with water stress. This work did not study the sterol composition. Considering the large genetic potentialities of sunflower, it therefore appears crucial to enlarge representative genetic backgrounds and to study genotype*environment interactions.

The aim of this study was to determine differences in sterol content and composition among sixteen sunflower lines and hybrids with different fatty acid profiles since this is the main quality trait currently important in breeding programmes.

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., [START_REF] Roche | Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions[END_REF].

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Materials and methods

Plant material

Sterol contents were studied in 13 inbred lines and 3 hybrids listed in table 1, chosen for their variation in genetic origin and fatty acid profile (high oleic, low oleic, low stearic or low palmitic acid contents). The line HA300 was provided as having high sterol content. The line HA821 was a reselection from HA300. 59259 was a dwarf line. 83HR4 and 83HR4OL were near isogenic lines differing only in their fatty acid profile.

Trial environmental conditions

The trial was conducted in south-western France at the Regional Centre for Organic Agriculture Trials, at Auch (32) in 2006, on a deep limestone clay silt soil with high waterholding capacity (Table 2). Two sowing dates were used to generate temperature variations during seed development. The trial was sown at a density of 7.1x10 4 plants/ha on April, 26 th for the conventional sowing date (CSD) and on June, 1 st for the late sowing dates (LSD). The development of the crop was monitored weekly using the main phenological stages E, R1, R5.1, R6, R8 and R9 as described by [START_REF] Schneiter | Description of sunflower growth stages[END_REF]. The trial was conducted as a complete randomised block design with 32 treatments (16 genotypes x 2 sowing dates) with 3 replications. Plot size was 16 m², 10 plants in each plot were bagged before flowering to obtain seed by self-pollination.

Extraction and determination of sterol contents

The small-scale sample extraction method optimized in our laboratory for reliable and economic analysis of sterols in sunflower seeds described in Roche, Alignan, Bouniols, Cerny, Mouloungui & Merah (in press, doi:10.1016/j.foodchem.2009.09.026) was used to extract and determine sterol content. Seed samples were bulks of all the bagged heads for Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., [START_REF] Roche | Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions[END_REF].

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 each treatment. Sterols analyzed were desmethylsterols (including β-sitosterol and campesterol), methylsterols and dimethylsterols.

Statistical data analysis

Phytosterol data are expressed as a weight percentage of seed dry matter (mg of sterol per 100g of seed dry matter). Statistical analyses were carried out on all the plots with 3 analytical replications (9 replications per treatment). Analyses of variance and Student-Newman & Keuls test were applied to experimental results to determine the significance between genotypes and sowing dates. The statistical analyses were performed using statistical package (Sigmastat version 2.0, USA). The different genotypes, sowing date treatment and their interaction on phytosterol contents were analyzed separately.

Results

Characterization of trial environment

Delaying sowing led to increased mean temperatures during the vegetative ([S-E] and [E-R5.1]) (+2.6 and 4 °C respectively) and the reproductive ([R5.1-R6] and [R6-R9]) phases (2.5 and 1.8 °C, respectively) compared with the conventional sowing date.

Late sowing received 70 mm less rainfall than the conventional sowing, with a pronounced drought period during ripening [R5.1-R6].

Effect of genotype Significant differences were observed between genotypes for all variables (tables 3 and 4).

The inbred line XRQ showed the highest mean total sterol content followed by HA300 and HA821.LP1. The lowest sterol contents were recorded for the dwarf line 59259, the oleic hybrid Trisun and then the oleic inbred lines OF and OPA3.

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., [START_REF] Roche | Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions[END_REF].

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

The genotype XRQ also had the highest mean content of the main sterol category desmethylsterols including β-sitosterol and campesterol, and of the precursor category dimethylsterols but not the intermediate methylsterol category. HA 300 and HA 821.LP1 had slightly lower desmethylsterol contents than XRQ but higher methylsterol contents. The two hybrids Olbaril and Santiago II had similar and intermediate mean values for all sterol categories.

Total sterol mean value of the high oleic genotypes was slightly lower than that of standard genotypes (217.0 and 236.7 mg/100 g of SDM respectively) but the two comparable nearisogenic lines 83HR4 and 83HR4OL had similar values for total sterols and desmethylsterols, with a slightly higher values for the high oleic line (table 4). There were significant differences only for β-sitosterol and campesterol, again with the high oleic line having greater sterol contents.

Effect of sowing date

Delayed sowing had a significant effect on total sterols as consequence of its impact on desmethylsterols, the main category (tables 3 and4). Except for dimethylsterol contents for which a significant decrease was observed (-8%), a delay of sowing was associated with an increased content of all sterol categories (+11%, +12%, +16% for total, desmethyl-and methylsterol contents).

To gain insight into the effects of temperature on sterol composition, the relation between two major pathway end-products was evaluated (figure 1). The ratio showed a positive and highly significant correlation (r²=0.84) between β-sitosterol and campesterol for all the genotypes whatever the sowing date. This positive correlation was conserved across the two sowing dates and closer for LSD (r²= 0.78, df = 30) than for CSD (r²=0.56,df = 14). The slope of the linear regression of late sowing date (5.2) was almost 50% greater than for conventional Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 sowing date (3.8), indicating a tendency to produce more β-sitosterol (+ 12%) rather than more campesterol (+7%).

Sowing date*genotype interaction

Although there were strong main factors effects (genotype and sowing date), sowing date affected sterol contents differently according to genotype (table 4). Nine of the 16 genotypes showed significant increases in total sterol content after late sowing. The most striking increases were for XRQ (+148%), HA383LS21 (+138%) and for the hybrid Trisun, which had the lowest sterol content after conventional sowing and a moderate value after late sowing (+189%), whereas HA300 and HA821.LP1 had the highest sterol contents from conventional sowing date and lower values after late sowing (85-90%). The lines 83HR4 and 83HR4OL also followed this pattern as did OPA3. The general increase or decrease according to genotype was the same for all sterol categories, except for dimethylsterol contents in HA383.LS2.

Discussion

Sterols may act as protector agents of sunflower embryo against environmental stresses Phytosterol contents varied according to sowing date. The highest levels of total sterols were obtained with delayed sowing giving the highest mean temperatures during flowering and seed ripening (+ 4 °C between seed emergence (E) and flowering (R5.1); and + 2.5 °C flowering and seed ripening onset (R6) compared to the conventional sowing date). This result confirms those obtained in our previous studies [START_REF] Roche | Management of environmental crop conditions to produce useful sunflower oil components[END_REF]. The same increase in total sterol content were observed in soybean seeds submitted to increased temperatures in greenhouse (20/10 °C, 27/17 °C, 35/25 °C day/night) (Vlahakis & Hazebroek, 2000) and in the field (Yamaya et al., 2007). In fact, phytosterols may be involved in mechanisms of plant Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 adaptation to temperature related to their ability to resist to oxidation within the cellular membranes [START_REF] Piironen | Plant sterols: biosynthesis, biological function and their importance to human nutrition[END_REF]. Due to their role in the regulation of membrane fluidity and permeability [START_REF] Schaller | The role of sterols in plant growth and development[END_REF], our results suggest that an increased accumulation of phytosterols may be a response of sunflower seed to high temperatures. This accumulation occurred mostly during the first stages of seed filling (until 40 % of SDW) (Roche et al., in press doi:10.1016(Roche et al., in press doi:10. /j.foodchem.2009.09.026.09.026). This is supported by higher temperatures recorded in LSD from R5.1 to R6 stages compared to CSD (table 2).

These results reinforce the hypothesis that modification of sterol content in mature sunflower seeds may be an adaptive strategy to seed desiccation reducing membrane fluidity to limit the effects of critically high temperatures. [START_REF] Peng | Sitosterol-β-glucoside as primer for cellulose synthesis in plants[END_REF] suggest that, in cotton membranes, β-sitosterol may be involved in cellulose biosynthesis as a primer for glucan chain elongation that also contributes to provide strength and flexibility to plant tissues.

High temperature during grain filling modifies sterol composition in sunflower seeds Higher temperatures during flowering modified the desmethylsterol biochemical composition of mature sunflower seeds, such as β-sitosterol/campesterol ratio. Although an increase of temperature induced an increase in both desmethylsterols components, the reduction of the ratio under high temperatures translates a more efficient biosynthesis of β-sitosterol to the expense of campesterol. These data are in disagreement with those reported by Yamaya et al.

(2007) on soybean but in accordance with our previous results on several sunflower genotypes submitted to more severe weather conditions [START_REF] Roche | Management of environmental crop conditions to produce useful sunflower oil components[END_REF]. The significant correlation may represent the biochemical balance between the sterol precursors of brassinosteroid biosynthesis through campesterol and their role as essential membrane components through βsitosterol. This balance is regulated by sterol methyl transferases (SMT1 and SMT2) enzymes

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 involved in sterol metabolism. Previous genetic and biochemical studies on Arabidopsis thaliana mutants overexpressing the enzyme SMT2 responsible of gramisterol methylation, led to an over accumulation of β-sitosterol, suggesting its implication in the modification of the β-sitosterol/campesterol ratio [START_REF] Lindsey | Importance of plant sterols in pattern formation and hormone signalling[END_REF]. The new sterol content could affect the biosynthetic interface between sterols and brassinosteroids, known to be involved in regulation of growth and development and response to environmental stresses [START_REF] Krishna | Brassinosteroid-mediated stress responses[END_REF]. The changing ratio of the β-sitosterol/campesterol observed in our experimental conditions may be explained by a modification of SMT2 activity under higher temperatures.

The amount of other sterols remained almost unchanged, particularly stigmasterol, which is thought to be the product of the desaturation at C22 of β-sitosterol (for review [START_REF] Benveniste | Sterol biosynthesis[END_REF], supporting our hypothesis. Although a sterol-22-desaturase in plants has not yet been characterized in terms of enzymatic activity, this metabolic step could be either considered as a rate-limiting enzymatic step in the pathway or a highly regulated point in the course of plant development for as yet unknown reasons. Whereas desmethyl-and methylsterol contents were higher when temperature increased (LSD), our results showed that dimethylsterol content was reduced by an increase of temperature. These results support the hypothesis that dimethylsterols as precursors of methyl-and desmethylsterols are regulated differently from the last two suggesting a different regulation of SMT1 which catalyses the conversion of cycloartenol into methylencycloartanol.

Genetic potentialities of sunflower seeds to produce sterols are affected by temperature

The 13 inbreds lines chosen as a first sample of the genetic variability for sterol contents present in modern inbred sunflower lines showed that there are highly significant differences.

It appears likely that a wider study could reveal even greater differences. The present study revealed a large variation and highly significant genotypic differences for each category of Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 sterols. The largest variation reached more than twice as much (58.5, 30.7, 38.8% for desmethyl, methyl-and dimethylsterols respectively). Although the variability of sterol content differed according to sterol categories and genotypes, some genotypes harbored distinguishable sterol contents. For example, XRQ contained the highest desmethyl-and dimethylsterol contents, whereas Trisun and 59259 contained the lowest contents of desmethyl-and methylsterols and OPA3 contained the lowest dimethylsterol content. Despite theses values represent a mean between conventional and late sowing dates, it appears that, the genotypes containing the lowest content of desmethylsterols generally also possessed the lowest content of dimethylsterols and the same behaviour was observed for the genotypes containing the highest content of desmethyl-and dimethylsterols. The inbred HA300, suggested as having high sterol contents confirmed this character, especially after the conventional sowing date. The significant genetic effects were confirmed by the similar behaviours of HA300 and HA821 which are related, and of 83HR4 and 83HR4OL which are nearly isogenic. This result showed no direct relation with saturated fatty acids (palmitic and stearic acids). The line XRQ had not been studied before for its sterol content, but should constitute a useful source for breeding for high sterol contents if the results of the present experiment are confirmed. Why some lines show decreases in sterol content after late sowing

as not yet explained. All the inbred lines had quite similar flowering dates, so the temperatures during their ripening periods should not have varied significantly. However, there were some variations in seed production between lines due to differences in self compatibility. Irregular seed size and development after self-pollination could be causes of different reactions to temperature.

Results from the literature on various species are contrasted. In soybean, only a weak genetic difference was found in sterol composition between Japanese and non Japanese seeds (Yamaya et al., 2007) whereas [START_REF] Nurmi | Phytosterols in Wheat Genotypes in the HEALTHGRAIN Diversity Screen[END_REF] and [START_REF] Alignan | Effects of genotype and sowing date on -phytostanols -phytosterols content and agronomic traits in wheat under organic agriculture[END_REF]

on wheat

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 reported a strong effect of genetic background. In the present study, the significant genetic variability of sterol contents showed that the different sterols were not accumulated at the same rate according to the genotypes and suggests different regulation related to genetic background. In addition, no difference was found between oleic and standard genotypes. The similarities between results for 83HR4 and 83HR4OL agree with the absence of any direct correlation between fatty acid and sterols biosynthesis although metabolic pathways of these two seed components are related through a common substrate: AcetylCoA.

Our results also revealed that the interaction between sowing date and genotypic potentialities could modify the stability of sterol content in sunflower seeds. The effect of high temperatures varies according to the genotype and the sterol category. Nevertheless, in more than 50% of the genotypes studied, there was an increase in total sterol, desmethylsterol and methylsterol contents after late sowing whereas the dimethylsterol content is lowered. This result supports the hypothesis that the character "high sterol content" may be under a genetic control [START_REF] Alignan | Variability and genetic analysis of sterols content in sunflower seeds[END_REF].

Conclusion

Choice of genotypes and effects of temperature could be used to orientate sterol biosynthesis to give sunflower seed production that would satisfy industrial requirements. The benefit of high crop temperature during seed filling on sterol concentrations could be used to increase sterol contents for industrial purposes through the delay of sowing in adapted zones. For example, the modification of the β-sitosterol/campesterol ratio under late sowing date could be a cultural practice useful to generate sunflower seeds enriched in campesterol as functional food ingredients. Moreover, our study highlights an effect of genetic variability on sterol content in the sunflower seeds. These results, in agreement with previous data on other Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 genotypes [START_REF] Roche | Management of environmental crop conditions to produce useful sunflower oil components[END_REF], suggest that sunflower genotypes should be screened to determine those that to fit industrial requirements. In food industries that require low concentrations of saturated fatty acids (C16:0 and C18:0) and a high concentration of oleic acid (C18:1) for healthy edible oils, a high concentration of sterols would provide increased functional properties. Our study suggests that sterol concentration in sunflower oil can be increased by integration of this new quality in breeding programmes.

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Van Rensburget, S. J., Daniels, W. M., Van Zyl, J. M., & Taljjard, J. J. (2000). A comparative study of the effects of cholesterol, beta-sitosterol, beta-sitosterol glucoside, dehydroepiandrosterone, sulphate and melatonin on in vitro lipid peroxidation. Metabolic Brain Disease, 15, 257-265. Vlahakis, C., & Hazebroek, J. (2000). Phytosterol accumulation in canola, sunflower, and soybean oils: effects of genetics, planting location, and temperature. Journal of American Oil Chemist 's Society, 77,49-53. Yamaya, A., Endo, Y., Fujimoto, K. & Kitamur, K. (2007). Effects of genetic variability and planting location on the phytosterol content and composition in soybean seeds. Food Chemistry, 102, 1071-1075.

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. ( 2010).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036 Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Figure caption Figure 1 .LSDFigure 1 .

 caption11 Figure caption Figure 1. Significant correlation between β-sitosterol and campesterol in whole seeds of 16 sunflower genotypes in conventional (CSD) and late (LSD) sowing dates in 2006. *** significant for p<000.1. CSD: conventional sowing date, LSD: late sowing date. SDM: Seed Dry Matter

  Hybrids are represented in italic. SDM: seed dry matter. CSD: Conventional sowing date. LSD: Late sowing date CSD and LSD values with same letter are not significantly different at the threshold of 5 %. Mean values of each sowing date and of each genotype with same letter are not significantly different at the threshold of 5 %.

Table 1 .

 1 Characteristics of sixteen genotypes studied (Inbred lines and hybrids) Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. (2010). Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

	Genotype	Characteristics	Type	Origin	C16:0 C18:0 C18:1 C18:2
	OF	High C18:1	LM	INRA, France	2.9	2.4	87.8	6.2
	83HR4OL	High C18:1	LR	INRA, France	5.3	3.3	76.5	13.8
	RHA345	High C18:1	LR	USDA, USA	3.7	4.3	84.9	6.0
	OEG	High C18:1	LM	INRA, France	4.9	5.3	85.4	3.0
	OPA3	High C18:1	LR	INRA, France	3.7	2.6	89.6	3.2
	Trisun 870	High C18:1	H	Mycogen,USA	3.2	4.8	89.1	1.5
	Olbaril	High C18:1	H	Pioneer Hi-Bred, USA	3.4	3.9	90.1	1.9
	83HR4	Standard C18:1	LR	INRA, France	8.6	3.6	16.1	70.7
	VHQ	Standard C18:1	LM	INRA, France	5.3	5.4	26.3	62.2
	XRQ	Standard C18:1	LM	INRA, France	5.9	3.3	25.4	64.9
	PSC8	Standard C18:1	LR	INRA, France	10.5	4.6	30.0	54.2
	HA300	High sterol content, standard C18:1	LM	USDA, USA	5.4	4.2	47.2	42.4
	HA821.LP1 Low C16:0, standard C18:1 LM	USDA, USA	5.4	6.4	22.0	65.6
	HA382.LS2 Low C18:0, standard C18:1 LM	USDA, USA	6.9	4.8	24.7	63.1
	59259	Dwarf, standard C18:1	LM	France	6.6	7.9	21.8	62.6
	Santiago II	Standard C18:1	H	Syngenta, France	5.7	5.8	27.7	59.6
	C16:0: palmitic acid content, C18:0: stearic acid content, C18:1: oleic acid content, C18:2: linoleic acid content
	expressed in % of total fatty acids
	LM: Inbred line cytoplasmic male sterility Maintainer, LR: Inbred line cytoplasmic male sterility Restorer, H:
	Hybrid

Table 2 .

 2 Rainfall and mean temperature during phenological periods of sunflower

	development in 2006
	Climate parameters	Sowing date	S-E	Phenological phases E-R5.1 R5.1-R6	R6-R9
	Mean temperature (°C)	CSD LSD	14.3 16.9	18.8 22.8	22.7 25.2	22.0 20.2
	Rainfall (mm)	CSD LSD	34.4 0.2	96 82.4	3.8 72.6	133 131.6
	CSD: conventional sowing date, LSD: late sowing date
	S: Sowing, E: emmergence, R5.1: beginning of flowering, R6: seed ripening onset, R8: end of seed ripening, and
	R9: physiological maturity, according to Schneiter and Miller (1981)

Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. (

2010

).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Table 3 .

 3 Mean square values of the effects of genotype, sowing date and their interactions on total sterols, desmethylsterols, methylsterols and dimethylsterols contents in 2006.

	Trait	Genotype	Sowing date	G*SD	Residues
	(mg/100gSDM)	(G)	(SD)
	Total sterol content	MS 46846.7 P ***	17731.4 ***	15050.0 ***	656.4
	Desmethylsterol	MS 42385.7 P ***	11730.8 ***	10138.6 ***	416.8
	Methylsterol	MS 548.4 P ***	438.7 ***	143.1 ***	19.0
	Dimethylsterol	MS 165.2 P ***	294.6 ***	195.6 ***	13.1
	β-sitosterol	MS 4748.2 P ***	18753.0 ***	4756.1 ***	176.8
	Campesterol	MS 137.6 P ***	119. 7 ***	183.5 ***	6.8

* : significant for p<00.5, ** : significant for p<0.01, *** : significant for p<0.001, NS : non significant MS : Mean Square, p : probability value, SDM : seed dry matter Comment citer ce document : Roche, J., Alignan, M., Bouniols, A., Cerny, M., Mouloungui, Z., Vear, F., MERAH, O. (

2010

).

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions. Food Chemistry, 121 (4), 990-995. , DOI : 10.1016/j.foodchem.2010.01.036

Table 4 .

 4 Effects of sowing date for each genotype on total sterol, desmethylsterols, methylsterols and dimethylsterols contents in 2006.

	Genotypes

Acknowledgment

We would like to thank the European Union for financially supporting this work (FEDER 7440 and 12688). Thanks are due to N. Hattry, F.-X. Boulanger and Q. Rivière for their technical support. The participation of Dr. O. Merah was supported by IUT Paul Sabatier, Toulouse (University of Toulouse III), France.