Batavia lettuce plants ( Lactuca sativa var . capitata L.) cv Edurne (Syngenta) were germinated and grown in cubes (27 cm ³ ) of peat substrate (Profi-sustrat tray 50/50, Gramoflor GmbH, Vechta, Germany) in a tunnel greenhouse located on a farm in the Basque Country, northern Spain (lat: 43º 17’N, long: 2º 52’W, alt: 65 m a.s.l.). The climate in this region is Atlantic temperate (the mean annual values of temperature (T) for 2015 were T = 16.3ºC, T _max = 20.8ºC, T _min = 11.7ºC and monthly rainfall = 53 mm). Seedlings at the 4-5 leaves stage were transplanted on 1 September 2014, 22 October 2014, 20 February 2015, 26 June 2015 and 13 August 2015 in soil in each of two greenhouses co­vered with the plastic films being evaluated. Plantation density was 11.11 plants/m ² . Soil was covered with black plastic mulch. Plants were irrigated by aspersion through a delivery line located at 2.50 m above the ground. After each harvest, the plastic mulch was removed and fertilizers were incorporated into the soil with a rotavator at: N, 100 kg/ha; P, 20 kg/ha and K, 200 kg/ha. The growing cycles were: 1 September-17 October, 22 October-15 January, 20 February-24 April, 26 June-30 July and 13 August-16 September.

We investigated the effects on lettuce production and quality of two commercially available plastic horticultural films: a clear film (Lumisol Clear AF, bpi.visqueen, British Polythene Industries, London) yielding < 35% light diffusion, and a light-diffusing film (Lumisol Diffused AF, bpi.visqueen) yielding > 90% light diffusion. The values of light transmission were = 90 and = 88% for Lumisol Clear and Lumisol Diffused res­pectively, while UV transmission and thermicity were identical for both of the films (manufacturer’s data). Each plastic film was installed in a separate re­gular tunnel greenhouse (L = 44 m, W = 8.40 m and H = 3.10 m). Both greenhouses were aligned with the same longitudinal line in E-W direction, and each tunnel were spaced 7 m apart. Natural ventilation was made opening the doors manually according to the growers’ experience.

Global and diffuse photosynthetically active radia­tions (PAR, µmol photon/m ² ·s) were recorded using BF5 Sunshine Sensors connected to a GP1 data logger (Delta-T Devices, Cambridge, UK). An external sensor was located at a height of 2 m above the greenhouses, and those located in the middle of each tunnel (one per greenhouse) were located at 2.60 m above the ground and above the sprinklers. Data were recorded every 10 minutes as integral values (µmol photon/m ² ·s).

Air temperature was recorded every 10 minutes with HOBO U23 Pro v2 sensors (Onset Computer Corporation, MA, USA). An external sensor was located at a height of 2 m above the greenhouses and 3 sensors were placed along the centre of each greenhouse, spaced 4 m apart from each other at a height of 0.4 m above the ground.

Lettuces were harvested on five dates: 16 October 2014, 12 January 2015, 22 April 2015, 30 July 2015, and 16 September 2015, to meet local market demands. At predawn and within one hour, 24 lettuces from the middle of each greenhouse were collected for analysis. Individual fresh weights were recorded and two sub-samples of 12 lettuces were obtained: one for determining growth parameters and another for chemical analysis. Lettuces were kept in a cold-room at 10ºC until analysis, carried out within a maximum of 4 hours.

The number of leaves > 5 mm was recorded in 12 lettuces. Batches of 6 adult leaves per lettuce were photographed with a digital camera (Olympus, mod. SP-600UZ, Tokyo, Japan) and the leaf area was determined using image analysis software ASSESS v. 2.0. The leaves were then dried at 70ºC in a ventilated oven for 72 h (until constant weight). The specific leaf area (SLA) was calculated by dividing leaf area (cm ² ) by leaf dry weight (g). Light use efficiency (LUE, g/mol global PAR) was calculated as lettuce dry weight (g) divided by the sum of global PAR intercepted by a lettuce, assuming a constant lettuce surface area of 0.045 m ² during the growth period [1 m ² / (11.11 plants × 2)].

For analytical purposes, four batches of 3 lettuces per greenhouse were processed. Nitrates were quantified in 9 adult leaves from the inner of the lettuce per greenhouse (3 leaves per lettuce). Leaves were boiled in distilled water 1:6 (w:v) for 15 min and homogenized with a stick mixer (Princess, mod. The beast, Tilburg, The Netherlands) at 1000 W for one minute. The puree was boiled for another 15 min, with stirring, and filtered under vacuum through qualitative filter paper (Whatman nº 4, Healthcare, Buckinghamshire, UK). Distilled water was added to the filtrate to a final volu­me of 1 L. Nitrates were quantified with an ion-selective electrode (nº 9662, Crison, Barcelona, Spain) and a reference electrode (nº 5044, Crison) connected to a pH meter (Crison GLP22), following the manufacturer’s instructions. Filtrates were diluted to yield final nitrate concentrations below 60 mg/L.

Soluble solids content, pH and acidity were measu­red in lettuce juice. For this purpose, 3 half lettuces per batch were homogenized in a professional food blender (Palson, mod. Baly, Barcelona) at maximal speed (1200 W) for 1 min. The puree was centrifuged (Sorvall Le­gend XTR, Thermo Fischer Scientific, Madrid) at 7600 g for 15 min to produce a clear juice. Soluble solids content (ºBrix) was determined directly in the juice with a digital refractometer (Optic Ivymen System, Barcelona). Titratable acidity was determined according to AOAC method 942.15 ( AOAC, 1999 ) with a pH meter (model GLP22, Crison, Barcelona) and a Titrette® digital bottle-top burette (class A precision, Brand, Wertheim, Germany).

To determine the mineral contents, the remaining 3 half lettuces per batch were dried at 70ºC for 72 h (until constant weight). The dry material was ground and sieved through a 0.12 mm-mesh stainless steel. The ground material was homogenized, re-dried for at least 2 h at 80°C before weighing out 0.5 g samples for analysis. Samples of the dried homogenate were wet-digested in a mixture of 1% HNO ₃ + 2% HClO ₄ (85:15, v:v) under a temperature gradient ranging from ambient to 190ºC for 12 h. The mineral contents (K, Ca, Mg, Na, P, S, Fe, Mn, Cu and Zn) were determined and quantified by ICP-AES (Varian VISTA-MPX, Agilent Technologies, CA, USA). Calibration standards were prepared from Certipur® solutions (Merck, Germany) for all minerals.

The nitrogen and carbon contents of subsamples (100 mg) of the dried homogenate were measured in a TruSpec elemental analyzer (Leco, MI, USA). Calibration was carried out using standard reference material (orchard leaves, LECO®, Part. nº 50-055).

The organ construction costs were calculated as fo­llows ( Poorter, 1994 ):

CC = (-1.041 + 5.077 C) × (1-Ash) + (5.325 N) [1]

where CC is the total cost of producing one gram of plant biomass (g glucose per g dry weight), C is the carbon content of the organic biomass (g/g dw), and Ash and N are respectively the mineral and organic nitrogen contents of the total dry weight (g/g). Ash content provides a biased estimate of the mineral content because organic acids can be transformed into carbonates, and sodium, chloride or sulphur may also volatilize during burning of the organic matter at 450ºC ( Masle et al ., 1992 ). We therefore replaced the ash values with the sum of the macro and microelements measured in the samples.

Carotenoids and chlorophylls were quantified in 4 batches of 3 lettuces per greenhouse. In each lettuce, 3 leaf discs of 3.96 cm ² from individual leaf (1 disc per leaf, 3 discs per lettuce) were extracted (in DMSO) and quantified in duplicate. The test tubes were then incuba­ted at 70ºC for 3 h in the dark. After cooling the extract in the dark at ambient temperature, a 3 mL aliquot was analysed at 480, 649 and 665 nm (spectrophotometer DU730, Beckman Coulter, California, USA). The chlorophyll a , chlorophyll b and carotenoids concentrations were determined according to the equation proposed by Wellburn (1994) .

Data were subjected to one-way analysis of variance to determine any significant differences in the effects of the different plastic films. When ANOVA indicated a significant difference, a t -test for equality of means was used to identify differences between means. All statistical analyses were performed using SSPS 13 software.

The sums of global and diffuse PAR for each treatment and growth period are shown in Table 1 . The transmission coefficients for global PAR, as calculated by the ratio of inside to outside measured value of the parameter during each growth period, ranged from 76% to 85% for the clear plastic and from 68% to 77% for the light-diffusing plastic.

Lettuces grown under the light-diffusing plastic received between 5 and 15% less global PAR than those under clear plastic, depending on the growth period. The light-diffusing plastic yielded global PAR diffusion of between 88 and 92%, which represents an increase in the outside diffuse PAR of 1.7 to 2.0 times during the different growth periods. The clear plastic yielded global PAR diffusion of between 60 and 67%, which represents a 1.2 to 1.4-fold increase in the outside diffuse PAR.

On a daily basis, the amount of diffuse PAR transmi­tted inside the greenhouses by both plastics was dependent on global solar radiation ( Fig. 1 ). On sunny days, with 30% of diffuse PAR outside, the light-diffu­sing plastic transmitted 85% PAR diffusion, whereas the clear plastic transmitted 43% of diffuse PAR. On cloudy days, diffuse PAR was very similar outside and inside both greenhouses (between 96% and 99%), irres­pective of the type of plastic. This is attributed to the scattering of solar light by the clouds.

The mean daily air temperature and relative humi­dity (RH) inside the greenhouse during each growth period did not differ significantly in relation to the type of plastic ( Table 1 ). Values of inside temperature and RH were similar or slightly different from outside values. Differences in minimum and maximum temperature between inside and outside ranged from 0.8ºC (22 Oct-15 Jan 2015) to 2.6ºC (13 Aug-16 Sept 2015) and for relative humidity, from -4.4% (13 Aug-16 Sept 2015) to 3.1% (01 Sept-17 Oct 2014). On sunny days, the night-time temperature inside the greenhouse was slightly higher than the outside temperature (difference between 0.8 and 1.9ºC), irrespective of the type of plastic ( Fig. 2 ). During the hours of maximum solar radiation, the difference between temperatures inside and outside the greenhouses reached a maximum of 5ºC. On cold cloudy days, the temperature inside and outside the greenhouse was very similar.

The mean fresh weight (fw) of lettuces was strongly dependent on the harvest date ( Fig. 3A ) owing to the climatic variation throughout the different growth periods and the fact that the days of harvest were selec­ted to meet market demands. Irrespective of the type of plastic, the fw fluctuated between 712 ± 79 g and 254 ± 22 g. The light-diffusing plastic significantly reduced ( p < 0.001) the fw by between 12 to 36% on almost all harvest dates, except during the February–April growth period, when plants received a greater amount of global PAR (1022 and 1166 mol/m ² under light-diffusing and clear film respectively). Use of the light-diffusing plastic caused a significant increase in the dry weight (dw) of the lettuces between September 2014 and April 2015 ( Fig. 3B ), and in further growth periods, there were no differences in relation to the type of plastic film. The low fresh weights of lettuces grown under the light-diffusing plastic were due to the fewer leaves per lettuce and the smaller individual mean leaf areas ( Fig. 3C and 3D ). On almost all harvest dates, the number of leaves and the mean leaf areas were significantly lower (by up to 22% and 29%, respectively) in the lettuces grown under the light-diffusing plastic. However, the SLA ­va­lues were generally similar under both types of film, although the SLA value was significantly lower in plants grown under the light-diffusing plastic and harvested in January, when the global PAR was lowest ( Fig. 3E and Table 1 ).

The LUE in lettuces grown under the light-diffusing plastic was similar to or lower than in lettuces grown under the clear plastic (Fig 3F). The LUE decreased by up to 23 and 22% in the second and third growth periods respectively.

The type of plastic film significantly affected total chlorophylls (Chl a + b ), Chl a , and carotenoids (Car) contents in lettuces harvested between April and September, while the Chl b content, Chl a/b ratio and Chl a+b/ Car ratio were generally not affected ( Fig. 4 ). Irrespective of the type of plastic, the chlorophyll content of the lettuces was higher during the winter harvest dates than during spring-summer dates. The Chl a content decreased from 15.4 µg/cm ² on 16 Oct 2014 to 11.2 µg/cm ² on 16 Sept 2015, and Chl b decreased from 6.3 to 3.3 µg/cm ² between the same dates. The Chl a , Chl a+b and carotenoid contents of the lettuces grown under the light-diffusing plastic were similar to or lower than in lettuces produced under the clear plastic. The greatest reductions were 15% for Chl a , 8% for Chl a+b and 14% for carotenoids. Nevertheless, the reductions in pigment contents were not detectable by the human eye, and the light-diffusing film did not affect the visual appeal of the lettuces.

As values of the Chl a+b /Car ratio remained relatively constant, at around 4.5 µg/cm ² on all harvest dates, the plants were not stressed.

Irrespective of type of covering plastic and harvest dates, total macroelements (sum of Ca, Mg, Na, K, N, S and P) ranged from 133 ± 8 to 180 ± 7 g/kg dw ( Table 2 ). The major macroelements in lettuces were K and N, representing 52% and 28% of the sum of macro­elements. The type of plastic film strongly affected the sum of macroelements on four harvest dates. Total macroelement contents in the lettuces grown under the light-diffusing plastic were up to 10% higher in January 2015. The increase was mainly due to higher foliar K contents in the plants grown under the diffuse light (up to 19% higher for lettuces from the second harvest). By contrast, the P contents tended to decrease in lettuces grown under the light-diffusing plastic on the three harvest dates. Irrespective of type of plastic and harvest date, the Na and Mg contents fluctuated from 1.55 ± 0.23 to 3.46 ± 0.44 g/kg dw and from 2.56 ± 0.20 to 3.62 ± 0.34 g/kg dw respectively. The concentrations of the other macroelements in the lettuce were generally not affected by the type of plastic film.

Irrespective of treatments and harvest date, the sum of microelements (Fe, Cu, Zn and Mn) ranged from 148 ± 8 to 215 ± 19 mg/kg dw ( Table 3 ). The major microelements in lettuces were Fe and Zn, representing about 54% and 28% of the sum of microelements. The type of plastic generally did not affect the microelement composition of leaves, although total microelement concentrations in lettuces grown under light-diffusing plastic were significantly lower on the last two harvest dates. These differences were due, in one case, to a reduction of 34% in the Zn content and in the other, a reduction of 16% of the Fe content.

Nitrate contents tended to be higher (up to 23%) in lettuces grown under the light-diffusing plastic than in those grown under clear plastic ( Fig. 5 ). These differences were significant in lettuces grown in winter (harvested in December and April) with low light intensity. In all cases, the nitrate contents were below the maximum nitrate levels established for lettuce in EC Regulation No. 1258/2011 ( EC, 2011 ): 5000 mg/kg fw for lettuces harvested between 1 October and 31 March, and 4000 mg/kg fw for harvests between 1 April and 30 September. Irrespective of the type of plastic cover and harvest date, leaf NO ₃ content was strongly linearly correlated to the amount of global and diffuse PAR incident on the lettuces (Pearson coefficient, r = 0.65 and 0.67 respectively), while any consistent correlation was found between leaf NO ₃ content and direct PAR ( Fig. 6 ).

The type of greenhouse covering film did not affect the nitrogen content, carbon content, C/N ratio or the cons­truction cost (CC) of lettuces. Irrespective of treatment and harvest date, the mean N content values ranged from 3.97% dw ± 0.16 to 4.66% ± 0.09, the mean C content from 36% dw ± 0.3 to 40% ± 0.9, the mean C/N ratio from 7.9 ± 0.2 to 10.0 ± 0.8 and the mean CC from 0.91 ± 0.01 to 1.08 ± 0.04 (g/g dw). The CC represents the amount of glucose used to provide the carbon skeletons, reducing power (NADH or equivalent) and chemical energy (ATP or equivalent) required for synthesis of one unit of dry biomass.

Irrespective of the type of plastic film and harvest date, the soluble solids content (SSC) of the lettuce juice ranged from 2.93 ± 0.13 to 4.28 ± 0.10 ºBrix ( Fig. 7 ). The type of plastic significantly affected the SSC on the first three harvest dates. The SSC was higher (by up to 16%) in lettuces grown under the light-diffusing plastic than in plants produced under clear plastic.

The acidity tended to be higher and the pH lower in the lettuces grown under the light-diffusing plastic than in those grown under the clear plastic ( Fig. 7 ).