Available online at www.sciencedirect.com Environmental and Experimental Botany 63 (2008) 216–223 Solar irradiance level alters the growth of basil (Ocimum basilicum L.) and its content of volatile oils Xianmin Chang a,∗ , Peter G. Alderson b , Charles J. Wright b a b Agronomy Institute, Orkney College, East Road, Kirkwall, Orkney KW15 1LX, UK Division of Agricultural and Environmental Sciences, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK Received 27 February 2007; received in revised form 3 October 2007; accepted 8 October 2007 Abstract The experiments were commenced in March 2003 and repeated in June 2003 at Sutton Bonington Campus, the University of Nottingham, UK, to investigate the effect of irradiance on plant growth and volatile oil content and composition in plants of basil. Four levels of irradiance were provided in the glasshouse, i.e. no shade (control), 25, 50 and 75% glasshouse irradiance. It suggested that basil grows well in full sun, however it can tolerate light shade. Heavy shading (75%) to provide a light integral of 5.3 moles m−2 d−1 resulted in shorter plants, lower weight, smaller leaf area, less shoots and higher specific leaf area, and also strongly reduced the rate of photosynthesis. There was no difference in CO2 assimilation rate between 24.9 moles m−2 d−1 light integrals (no shading) and 13.5 moles m−2 d−1 light integrals (25% shading). Shading effectively reduced leaf temperature when air temperature was less than 30 ◦ C, but heavy shading (75%) could not reduce leaf temperature when air temperature was above 36 ◦ C due to a limitation of free air convection. Consequently, leaf temperature increased. Heavy shading strongly reduced total volatile oil content in fresh leaves, especially in older plants (shading treatment applied at the 3 leaf-pair growth stage). There were three chemical compounds in basil leaves, namely linalool, eugenol and methyl eugenol, influenced by the shading treatments. Linalool and eugenol, which contribute to the characteristic taste of basil, were significantly increased by high daily light integrals, whereas methyleugenol was increased by lower daily light integrals. No differences in the relative content of 1,8-cineole, one of the key aromatic compounds of Ocimum species, were observed. © 2007 Elsevier B.V. All rights reserved. Keywords: Ocimum basilicum; Irradiance; Growth; Volatile oils 1. Introduction Light is the main factor controlling plant growth and development and, plants are affected in a complex manner by irradiance at all stages of their growth. The architecture of plants is dependent on the quantity, direction, duration and quality of light (Nilsen and Orcutt, 1996; Rich et al., 1987; Went, 1941). Photosynthesis is dependent on light for energy and for the induction of enzymatic processes. High irradiance affects plant processes directly through its effect on factors such as enzyme activity and photosynthesis, but it also affects plant physiological processes indirectly by its impact on thermal attributes of the tissues. Leaf temperature is regulated primarily by the influences of net radiation, latent heat exchange, conduction and convection, and air temperature. Some leaves may experience as much as 10–15 ◦ C ∗ Corresponding author. Tel.: +44 1856 569294; fax: +44 1856 569008. E-mail address: xianmin.chang@orknry.uhi.ac.uk (X. Chang). 0098-8472/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2007.10.017 difference between air and leaf temperature under certain conditions, however, most leaves experience a differential of 5 ◦ C or less (Nilsen and Orcutt, 1996). Plant responses to light include a variety of adaptations at physiological (photosynthesis, nutrient uptake) and biochemical (pigments, carbohydrates) levels. Such responses are translated into alterations of growth rate, plant architecture and morphological characteristics (Peralta et al., 2002). Exposure of plants to excessive light is a well-known cause of photoinhibition (Sorrentino et al., 1997). Light is a key factor in the ultimate production of many compounds because it supplies the energy needed to fix carbon. Light intensity plays an important role in the biosynthesis of medicinally important metabolites, for example camptothecin (CPT), an anti-cancer agent in the tree of joy (Camptotheca accuminata L.), is produced at higher levels in shade conditions (Kaufman et al., 1999). Plants of Rhynchelytrum repens, a tropical grass species popularly used for diabetes treatment, growing under natural irradiance showed increased total soluble carbohydrates X. Chang et al. / Environmental and Experimental Botany 63 (2008) 216–223 and higher fluctuations in starch content compared with plants cultivated under low irradiance (Souza et al., 2004). In contrast, plants of Aloe vera under high irradiance produced more axillary shoots resulting in higher dry matter but there were only minor effects on the concentration of soluble carbohydrates and aloin in the leaf exudates, suggesting that there were no substantial effects of irradiance on either primary or secondary carbon metabolites (Paez et al., 2000). For basil (Ocimum basilicum L.), Lee et al. (1994) used three different shading nets to study the effects of irradiance on plant size, weight and oil content in basil. Plants were either shaded with white cheesecloth (21% shading), black cheesecloth (50%) or Gariso (a black material also giving 50% shading) or were not shaded (control). Growth was generally better in plots shaded with white cheesecloth and in control plots than in the more heavily shaded plots. Plant height was greatest under black cheesecloth, but leaf area was greatest under white cheesecloth. No significant difference in total fresh weight (FW) was observed between plants under white cheesecloth and the controls. The essential oil content of the leaves and the total oil content/plant were higher in plots under white cheesecloth than in control plots. It was suggested that light shading was beneficial to growth and oil production. The results were different, however, when the same shade percentage was achieved with different shading materials probably due to the shading materials influencing the light quality, especially the R/FR ratio, which could lead to changes in plant morphology (Went, 1941; Morelli and Ruberti, 2002). Thus, it is difficult to conclude whether the effects observed were due to light quality or light intensity. The chemical composition of volatile oils in basil has been investigated since the 1930s, and by 1999 approximately 190 chemical compounds had been identified. Due to their high relative content in leaves and extensive uses in plant protection, food stocks and medicine during the past decades, eugenol, 1,8cineole and linalool have been studied extensively (Hiltunen and Holm, 1999). This paper reports the effect of irradiance on plant growth and volatile oil content and composition in plants of sweet basil using green ‘Rokolene’ shading nets with different percentage light transmissions, since Rokolene netting has been previously shown to have no effect on light quality (Wright and Sandrang, 1995). It was hypothesized that low irradiance would result in shorter plants with reduced dry matter, fewer leaves and shoots and a lower content and change in the composition of volatile oils. 2. Materials and methods 2.1. Plant materials Seeds of basil cv. ‘Basil Sweet Genovese’ obtained from Nickerson-Zwaan Ltd. (Lincolnshire, UK) were sown on the surface of Levington F2s compost (Fisons Horticulture Ltd.; Ipswich, UK) in plastic trays. Seedlings with one pair of unfolded leaves were transplanted to 12 cm diameter pots containing Levington M2A compost, and there was one plant per pot. The mean daily temperature in the glasshouse was set 217 at 21 ± 3 ◦ C for seed germination and plant raising. Between September and April, 16 h of supplementary lighting was provided by 400 W high-pressure sodium lamps (Poot Lichtenergie, Holland). A shading experiment in a glasshouse was commenced in March 2003 (first experiment) and repeated in June 2003 (second experiment). Shading treatment design and data collection: Three levels of reduced total irradiance were achieved by covering metal support frames (140 cm × 70 cm × 70 cm) with green ‘Rokolene’ shading net of different percentage light transmissions. In this way, four levels of irradiance were provided in the glasshouse, i.e. no shade (control), 25, 50 and 75% glasshouse irradiance, and a range of mean daily light integrals for each shade treatment was calculated (Table 1). Plants at the 1 and 3 leaf-pair growth stages were placed in the shade treatments for up to 2 weeks, and each treatment had 2 replicates with 36 plants. The irradiance and temperature for each treatment were measured 50 cm above the benches using a data logger (Campbell CR 10, Campbell Scientific, Inc. Logan, UT) at intervals of 60 min. Irradiance readings were subsequently converted to W m−2 . Daily light integral (DLI) (moles m−2 d−1 ), i.e. the total quantity of light delivered over the course of an entire day, was calculated by the formula (Faust, 2002): DLI (moles m−2 d−1 ) = [Sum of data logger reading each hour × 0.0036] × 2 where 0.0036 is derived from 60 s/min × 60 min/h ÷ 1,000,000 ␮moles mole−1 . Midday solar PPFD was also measured which was between 600 and 1600 ␮moles m−2 s−1 in the glasshouses without shade during the period of the experiment. 2.2. Leaf temperature An infrared thermometer (Kane-May KM823, Comark Ltd., England) was used to measure the temperature of the third and fourth pairs of leaves when shade treatment was applied at the one leaf-pair and three leaf-pair growth stages respectively. There were ten replicates (ten individual plants) for each treatment. 2.3. Photosynthesis An infrared gas analyser (CIRAS I, Scotrail and the University of Strathclyde, Scotland) was used to measure photosynthesis rate. Measurements were made on the 5th pair of leaves of plants that had received 2 weeks of shading treatments at the 3 leaf-pair growth stage. The rate was calculated from the difference between the CO2 concentration entering (Cin ) and leaving (Cout ) and the flow rate through the cuvette using the following formula: 11A = Cin × W − Cout × (W + E) = −[W × (Cout − Cin ) + Cout × E] 218 X. Chang et al. / Environmental and Experimental Botany 63 (2008) 216–223 Table 1 Mean daily light integrals (moles m−2 d−1 ) during shading treatments Experiment Date (2003) Control 25% shading 50% shading 75% shading First Second 09/April–06/May 02/July–23/July 19.8 24.9 12.2 13.5 8.8 11.3 4.3 5.3 From the known amounts of the pure chemicals and the peak areas, calibration formulae were established. For plants that received the shade treatment at the one leafpair and three leaf-pair growth stages, leaf samples for volatile oil analysis were collected from the third and fifth pairs of leaves respectively. Three samples were analysed for each replicate of the irradiance treatments. Chemical compounds were identified on the basis of relative retention times, using standard samples and comparing peaks with a Library (Wiley7n.L, supplied by Agilent Technologies, USA). where A is the rate of CO2 exchange in the cuvette (␮moles m−2 s−1 ), Cin the CO2 concentration of air into cuvette (␮moles mole−1 ), Cout the CO2 concentration of air leaving cuvette (␮moles mole−1 ), W the mass flow of dry air per unit leaf area (moles m−2 s−1 ), E the transpiration rate (moles m−2 s−1 ), and Cout − Cin is the CO2 difference (CO2 Diff.) that was calculated and displayed by the CIRAS. 2.4. Measurement of plant growth At the end of the treatments, 18 plants of each treatment were harvested for growth analysis and, plant height, plant weight (fresh and dry), leaf weight (fresh and dry), number of shoots, number of leaf-pairs on the main stem, leaf area and specific leaf area were recorded. 2.6. Statistical analysis Using Genstat, an analysis of variance (ANOVA) table was calculated for each growth parameter and for volatile oil content and composition. Differences between treatments were assessed using the F-test, and the Least Significant Difference (LSD) was calculated at the 0.05 probability level. 2.5. Extraction and identification of volatile oils Samples (5 g) of fresh leaf harvested from the third or fifth pair of leaves of plants were analysed for volatile oils using TD–GC/MS as described by Chang (2004) and Chang et al. (2005). External standards were used for calibration of the TD/GC. Standard solutions (0.025, 2.5, 5 and 7.5%) of 1,8-cineole, linalool plus eugenol were prepared by mixing, and ethyl acetate was selected as solvent. There were three replicates for each density and 1 ␮l was injected in each Universal tube (replicate). 3. Results 3.1. Plant growth Differences in plant morphology as well as plant and leaf weights (fresh and dry) were observed following 2 weeks of shading treatments. Since both experiments produced simi- Table 2 Growth parametersa of basil plants after 2 weeks of shading treatments applied at 1 and 3 leaf-pair growth stages Growth parameter Growth stage Mean daily light integral (moles m−2 d−1 ) 24.9 20.3 45.3 11.3 Plant height (cm) 1 3 Plant dry weight (g) 1 3 0.98 3.24 0.81 3.01 0.67 2.21 Leaf dry weight (g) 1 3 0.67 2.01 0.57 1.81 Number of axillary shoots 1 3 2.5 7.0 Number of leaf-pairs 1 3 Leaf area (cm2 ) Specific leaf area (cm2 g−1 ) a 21.0 43.0 13.5 19.1 40.7 SED P 5.3 10.6 24.5 1.377 1.577 <0.001 <0.001 0.21 0.88 0.067 0.149 <0.001 <0.001 0.45 1.39 0.15 0.67 0.040 0.079 <0.001 <0.001 1.5 6.5 1.5 6.0 0 2.3 0.408 0.465 <0.001 <0.001 4.0 6.0 4.0 6.0 4.0 6.0 3.4 5.2 0.102 0.118 <0.001 <0.001 1 3 237.1 700.0 233.7 714.0 213.7 629.0 110.4 356.0 18.690 37.900 <0.001 <0.001 1 3 351.8 363.5 409.3 393.8 477.0 453.5 731.0 533.7 49.200 36.300 <0.001 <0.001 Plant growth parameters are expressed on a per plant basis. SED: Standard error of the difference; P: probability. X. Chang et al. / Environmental and Experimental Botany 63 (2008) 216–223 lar results, the data presented is from the second experiment (Table 2). Plant height was increased by increasing the light integral from 5.3 to 11.3 moles m−2 d−1 , for plants treated at both the 1 and 3 leaf-pair growth stages. Light integrals greater than 11.3 moles m−2 d−1 applied at the 1 leaf-pair growth stage and 13.5 moles m−2 d−1 applied at the 3 leaf-pair growth stage did not increase plant height further, suggesting a light saturated response at these levels. The fresh and dry weights of plants and leaves were significantly increased with increases in light integral for 2 weeks at both plant ages, however, the differences between 24.9 and 13.5 moles m−2 d−1 were not significant, suggesting a saturation of the light level. Compared with treatments applied at the 1 leaf-pair growth stage, differences in the effects of the 11.3 and 13.5 moles m−2 d−1 light integrals applied at the 3 leaf-pair growth stage were significant. With increasing light integrals, numbers of axillary shoots increased by 2.5 and 4.7 when treatments were applied at the 1 and 3 leaf-pair growth stages respectively. Shoot numbers were significantly lower in plants under the 5.3 moles m−2 d−1 light integral compared with plants at the 24.9 moles m−2 d−1 light integral, and there was more than threefold difference when treatments were applied at the 3 leaf-pair growth stage. Differences between plants at the 24.9, 13.5 and 11.3 moles m−2 d−1 light integrals were not significant. The number of leaves on the plant main stem did not vary with light integrals from 11.3 to 24.9 moles m−2 d−1 , remaining constant at 4 pairs and 6 pairs of leaves with the treatments applied at the 1 and 3 leaf-pair growth stages respectively. However, differences in leaf number between 5.3 and 11.3 moles m−2 d−1 were significant and there was one leaf-pair less on plant main stems under 5.3 moles m−2 d−1 compared with the higher light integrals. Total leaf areas were enhanced significantly with increases of the light integrals; however, there was no difference between the 13.5 and 24.9 moles m−2 d−1 light integrals. Although leaf numbers per main stem were the same in the 11.3 and 24.9 moles m−2 d−1 treatments, the differences in shoot numbers, leaf numbers in shoots and individual leaf sizes resulted in differences in total leaf area between the 11.3 and 13.5 moles m−2 d−1 light integrals, especially when treatments were applied at the 3 leaf-pair growth stage. The areas of individual leaves were strongly affected by shading treatments, depending on when the treatments were applied. For treatments applied at the 1 leaf-pair growth stage, significant differences were detected from the 1st pair of leaves to the 4th pair of leaves, because the 1st pair of leaves were present but not fully expanded when the treatment started. When treatments were applied at the 3 leaf-pair growth stage, the 3rd pair of leaves was not fully expanded so, as expected, differences between the 3rd–5th pairs of leaves were significant. Specific leaf area was calculated by dividing total leaf area by total leaf dry weight. For treatments applied at the 1 leaf-pair growth stage, specific leaf area declined more than twofold when light integrals were increased from 5.3 to 24.9 moles m−2 d−1 . However, there was no difference between the 13.5 moles m−2 d−1 treatment and the 24.9 and 219 Fig. 1. Photosynthesis rate under different solar irradiances. 11.3 moles m−2 d−1 treatments. For treatments applied at the 3 leaf-pair growth stage, the trend was the same as with the treatments applied at the 1 leaf-pair growth stage, but the change was less. Although plants were at different growth stages, specific leaf areas for both sets of plants at the 24.9 moles m−2 d−1 light integral (control) were very similar; however, there was a big difference at the 5.3 moles m−2 d−1 light integral. This suggests that, in terms of specific leaf area, young plants were more sensitive to daily light integral than older plants. 3.2. Photosynthesis Photosynthesis was significantly reduced by shading (P < 0.001), with the CO2 assimilation rate ranging from 15.1 to 37.7 ␮moles m−2 s−1 between the 440 ␮moles m−2 s−1 (75% shading) and 1387 ␮moles m−2 s−1 (control) solar irradiance respectively (Fig. 1). The CO2 assimilation rate increased greatly with increase in irradiance from the lowest level, but this increase declined at higher levels of irradiance and there was no significant difference between the 1187 ␮moles m−2 s−1 (25% shading) and 1387 ␮moles m−2 s−1 (control) treatments. 3.3. Leaf temperature Data for leaf and air temperatures were collected at 10:00 a.m., 2:00 p.m. and 4:00 p.m. on April 16, April 22, July 9 and July 15. Since the highest air temperatures in the glasshouse on 16 and 22 April were less than 30 ◦ C, and the trend in changes in leaf temperature under different shade conditions was the same as for the data collected on 9 and 15 July before 2:00 p.m., the data from 15 July is presented. The mean air temperature from 9:00 to 10:00 a.m. was 19.4 ◦ C and there was no significant difference in leaf temperatures between shade treatments measured at 10:00 a.m. (Table 3). When leaf temperatures were measured at 2:00 p.m. while the mean air temperature was 30.3 ◦ C (from 1:00 to 2:00 p.m.), it was found that leaf temperature increased with increasing irradiance and there were differences between the shading treatments. 220 X. Chang et al. / Environmental and Experimental Botany 63 (2008) 216–223 Table 3 Leaf temperatures (◦ C) of basil plants under different shading conditions and the time of day Time of day (TAir ) 10.00 (19.4) 14.00 (30.3) 16.00 (36.0) Treatment (shading) Control 25% 50% 75% 24.8 27.6 30.7 25.1 27.5 29.1 24.0 26.7 28.7 24.7 26.0 30.8 SED P 0.484 0.613 0.465 0.271 0.050 <0.001 TAir : Air temperature (◦ C); SED: standard error of the difference; P: probability. At 4:00 p.m. when the mean air temperature was 36.0 ◦ C (from 3:00 to 4:00 p.m.), there was a highly significant difference in leaf temperatures under the different shading treatments. At the air temperature of 30.3 ◦ C, leaf temperature was decreased by high level of shading. In contrast, at 36 ◦ C air temperature, 75% shading resulted in leaf temperatures significantly higher than with 25 and 50% shading, but not higher than the control. At the lower air temperature of 19.4 ◦ C, leaf temperature was consistently higher than air temperature, however, at higher air temperatures, leaf temperature increased but was lower than the air temperature. At the high air temperature of 30 ◦ C, shading effectively reduced leaf temperature, however, at the higher air temperature of 36 ◦ C, heavy shading could not reduce leaf temperature. 3.4. Volatile oils Shading of basil plants led to a reduction in the oil content (P < 0.001) and changes in oil composition in the leaves. In treatments applied at the 3 leaf-pair growth stage, there was a fivefold reduction in volatile oil content with the 5.3 moles m−2 d−1 light integral (75% shading) compared with the 24.9 moles m−2 d−1 light integral (no shading) (Fig. 2). Treatments applied at the 1 leaf-pair growth stage showed no significant difference between 13.5 moles m−2 d−1 light integrals (25% shading) and 24.9 moles m−2 d−1 light integrals (no shading). The principal phenylpropanoids detected were eugenol and methyl eugenol, while the major terpenoids were 1,8-cineole, linalool, cis-ocimene and trans-␣-bergamotene. Small amounts of ␣-pinene, ␤-pinene, myrecene and camphor were also Fig. 2. Total volatile oil content in 5 g fresh leaf samples after 2 weeks of shading treatments applied at the 1 and 3 leaf-pair growth stages. Fig. 3. Relative content of selected volatile compounds after 2 weeks of shading treatments applied at the 1 leaf-pair growth stage. detected. When plants were treated at the 1 leaf-pair growth stage, shading led to a reduction in the relative contents of linalool and eugenol but methyl eugenol was markedly increased (Fig. 3). Linalool was reduced by a factor of 4 in plants grown under the 5.3 moles m−2 d−1 light integral compared with 24.9 moles m−2 d−1 light integral (no shading) (P = 0.007), and eugenol by a factor of 3 (P = 0.033). Differences with 13.5 moles m−2 d−1 light integrals (25% shading) and 11.5 moles m−2 d−1 light integrals (50% shading) were not significant. The biggest treatment effect was observed with methyl eugenol (P < 0.001), with sixfold enhancement by 5.3 moles m−2 d−1 light integral (75% shading) compared with 24.9 moles m−2 d−1 (no shading). For treatments applied at the 3 leaf-pair growth stage, differences in the relative contents of both linalool and eugenol with 13.5 (25% shading), 11.3 moles m−2 d−1 light integrals (50% shading) and 5.3 moles m−2 d−1 light integrals (75% shading) were not significant, however, with 24.9 moles m−2 d−1 light integral (no shading) their relative contents were higher (P < 0.05) (Fig. 4). Methyl eugenol was again enhanced by shading (P < 0.001), increasing more than fivefold from 0.34% with no shading to 1.89% with 5.3 moles m−2 d−1 light integral (75% shading), however, the relative content of methyl eugenol was significantly less than that in plants at the 1 leaf-pair growth stage. These results showed that shading of basil plants significantly affected the volatile oil composition and that young plants contained a higher relative content of methyl eugenol. In addi- Fig. 4. Relative content of selected volatile compounds after 2 weeks of shading treatments applied at the 3 leaf-pair growth stage. X. Chang et al. / Environmental and Experimental Botany 63 (2008) 216–223 221 as when treatments were applied at the 1 leaf-pair growth stage, however, the yields of the three oils were much higher (Fig. 6). 4. Discussion Fig. 5. Content of selected volatile oils after 2 weeks of shading treatments applied at the 1 leaf-pair growth stage. tion, plants at the younger growth stage were more sensitive to different shading conditions. Based on peak areas and calibration curves for selected individual chemicals, the yields of the three main chemicals (1,8-cineole, linalool and eugenol) were calculated (Fig. 5). Although differences in the relative content of 1,8-cineole in basil leaves when treatments were applied at the 1 leaf-pair growth stage were not significant, the quantity of 1,8-cineole in fresh leaves was significantly decreased by shading (P < 0.001), ranging from 1.29 ␮g g−1 with 5.3 moles m−2 d−1 light integral (75% shading) to 9.24 ␮g g−1 with 24.9 moles m−2 d−1 light integral (no shading), i.e. a sevenfold difference. For linalool, when treatments were applied at the 1 leaf-pair growth stage, due to the differences in relative content combined with the differences in total content, differences between the shading treatments were significant (P < 0.001), ranging from 0.25 ␮g g−1 with 5.3 moles m−2 d−1 light integral (75% shading) to 8.11 ␮g g−1 with 24.9 moles m−2 d−1 light integral (no shading), i.e. a factor of 33 times. However, differences between 13.5 moles m−2 d−1 light integrals (25% shading) and 11.3 moles m−2 d−1 light integrals (50% shading) were not significant. The yield of eugenol from fresh leaves increased by a factor of 9 from 0.19 ␮g g−1 with 5.3 moles m−2 d−1 light integral (75% shading) to 1.67 ␮g g−1 with 24.9 moles m−2 d−1 light integral (no shading). When shading treatments were applied at the 3 leaf-pair growth stage, a similar but less marked trend was observed Fig. 6. Content of selected volatile oils after 2 weeks of shading treatments applied at the 3 leaf-pair growth stage. The absence of any differences in plant height, weight, shoot number, leaf weight and leaf area between plants in the 24.9 moles m−2 d−1 (no shade) and 13.5 moles m−2 d−1 (25% shade) treatments may be explained by the latter possessing a greater conversion efficiency of light utilization for increasing plant size and dry matter. This is supported by previous reports for cineraria (Yeh, 1996) and strawberry (Wright and Sandrang, 1995). Commonly, dry matter accumulated by annual plants increases linearly with the amount of solar radiation intercepted, but shade-plants have a higher quantum efficiency due to a decrease in the ratio of the respiration rate to the rate of photosynthesis (Yeh, 1996). In general, plant responses to light include a variety of photosynthetic and biochemical adaptations that are translated into alterations of plant growth and architecture (Peralta et al., 2002). Also, reductions in irradiance lead to a reduction in the rate of photosynthesis and therefore a reduction in the rate of growth (Corree, 1983). This is supported by the current findings that the treatments at 11.3 moles m−2 d−1 light integrals (50% shade) and 5.3 moles m−2 d−1 light integrals (75% shade) significantly reduced plant size and weight, both in the young and old plants. As hypothesized, plant height, dry matter and leaf area increased with increasing irradiance between 5.4 moles m−2 d−1 light integrals (75% shading) and 13.5 moles m−2 d−1 light integrals (25% shading), which suggests that, although basil grows well under sunny conditions (Putievsky and Galambosi, 1999), it can tolerate light but not heavy shade. Specific leaf area decreased as daily light integral increased up to 13.5 moles m−2 d−1 , and thereafter remained fairly constant. This could be characterised as a sun-adaptation feature. The reduced thickness of the leaves in shaded conditions may be associated with smaller cell volume. The present results support the findings of Yeh (1996) for cineraria plants. As expected, photosynthesis in the basil plants increased with increasing irradiance. The initial part of the light response curve is linear because light is the dominant limiting factor. On reaching light saturation, the curve becomes horizontal because resources other than light become limiting (Nilsen and Orcutt, 1996). Leaf temperature is regulated primarily by the influence of net radiation, latent heat exchange, conduction and convection and air temperature (Nilsen and Orcutt, 1996). Any limitation of free convection reduces plant growth by limiting heat and gas exchanges between plant leaves and the ambient air (Kitaya et al., 2001). In this study, when the air temperature was 19.4 ◦ C at 10:00 a.m., the leaf temperature exceeded the air temperature by as much as 4.6–5.7 ◦ C because, during periods of intense irradiance and/or high tissue irradiance absorption, tissue temperature can increase dramatically compared with the air temperature (Nilsen and Orcutt, 1996). With the air temperature increasing to 30.3 ◦ C at 2:00 p.m., leaf temperature was effectively reduced by 222 X. Chang et al. / Environmental and Experimental Botany 63 (2008) 216–223 shading. When the air temperature was 36 ◦ C, light shading (25 and 50%) significantly reduced leaf temperature compared with no shading, but heavy shading (75%) resulted in increased leaf temperature. With the irradiance increasing during this period, leaf temperature was increased, and light shading reduced leaf temperature through its effect on light reduction and therefore reduced leaf temperature as stated above. Heavy shading (75%) restricted air movement due to the less porous shading net around the plants, with the consequent decrease in conductance of heat and gases in the leaf boundary layer resulting in higher leaf temperature; however, such critical air temperature conditions are not common during the growing season in England. The plants under 75% shading made less growth due to the reduced irradiance and also probably due to the restricted free air convection retarding heat and gas exchanges between leaves and the ambient air (Kitaya et al., 2001). This research suggested that further shade experiments should consider using clear netting rather than no netting as a control, or improving netting materials to prevent the restricted free air convection. The composition of volatile oils was strongly affected by light intensity. There were higher relative contents of linalool and eugenol under higher daily light integrals, and higher shading resulted in more methyl eugenol. Phenylalanine is the precursor for both eugenol and methyl eugenol and, under certain conditions, the methylation of eugenol to methyl eugenol, is catalysed by eugenol O-methyl transferase (EOMT) (Gang et al., 2001). In the present study, lower irradiance resulted in the accumulation of methyl eugenol, but how irradiance affects the activity of EOMT requires further investigation. Linalool is a common acyclic monoterpenoid compound that can be synthesized from ␣-pinene or ␤-pinene (Bauer et al., 2001). In plants, the enzyme linalool synthase catalyzes geranyl pyrophosphate (GPP) to linalool (Kaufman et al., 1999). In addition, GPP can be catalysed by 1,8-cineole synthase to 1,8-cineole (Gang et al., 2001; Kaufman et al., 1999) and ␣terpieol (Gang et al., 2001) catalysed by monoterpene cyclase to pinenes, 3-carene and limonene (Kaufman et al., 1999). In the present study, there were no differences in the relative contents of ␣-pinene, ␤-pinene, 3-carene and 1,8-cineole under different shading treatments. Significant differences were only found for linalool, suggesting that further research should be carried out to determine how the light intensity affects the activity of linalool synthase and thereby regulating the relative content of linalool. Some other chemicals maintained a relatively stable level under different conditions, for example the relative contents of pinenes, 3-carene and limonene did not change with different shading conditions, temperatures and supplementary UV-B or red light (Chang, 2004). The highest dry matter and volatile oil content were obtained under the highest daily light integrals, which suggests that, as secondary metabolites, the synthesis of volatile oils has a very close relationship with primary metabolism, i.e. the more photosynthates produced, the more secondary metabolites accumulated. Precursors are usually derived from basic metabolic pathways (Wink, 1999). Both phenylalanine and GPP are derived from sucrose catalysed by a series of enzymes, such as sucrose synthase, glucose-6-phosphate dehydrogenase, ger- anyl pyrophosphate synthase and shikimate kinase (Gang et al., 2001). 5. Conclusion This research suggested that heavy shading (75%) resulted in shorter plants, lower weight, smaller leaf area, less shoots and higher specific leaf area. Due to irradiance reduction, heavy shading (75%) strongly reduced photosynthesis rate and, effectively reduced leaf temperature when air temperature was less than 30 ◦ C. 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