Brassinosteroid analogues effect on yield and quality parameters of field-grown lettuce (Lactuca sativa L.)

Scientia Horticulturae 143 (2012) 29–37

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Brassinosteroid analogues effect on yield and quality parameters of field-grown lettuce (Lactuca sativa L.) Mario Serna a , Francisca Hernández b , Francisco Coll c , Asunción Amorós a,∗ a

Department of Applied Biology, Escuela Politécnica Superior de Orihuela (Miguel Hernández University of Elche), Ctra. Beniel Km 3.2, 03312 Orihuela, Spain Department of Vegetal Production, Escuela Politécnica Superior de Orihuela (Miguel Hernández University of Elche), Ctra. Beniel Km 3.2, 03312 Orihuela, Spain c Centro de Estudios de Productos Naturales, Facultad de Química, Universidad de la Habana, Zapata y G, Vedado cp 10 400, Cuba b

a r t i c l e

i n f o

Article history: Received 16 March 2012 Received in revised form 4 May 2012 Accepted 22 May 2012 Keywords: Spirostanic brassinosteroid analogue Lettuce Yield Quality parameters

a b s t r a c t Lettuce is a common crop in Spain. It is the most widely used fresh-cut vegetable product for salads in Europe, so increasing its yield is of great interest. The effects from two brassinosteroid (BR) analogues, DI-31 (BB16) and DI-100, were evaluated at concentrations of 4, 8 and 12 ppm together with a seaweed extract and amino acids called Tomex Amin (2.5 l/ha) to enhance their activity. These were sprayed fourfold to foliage of Lactuca sativa L. var. Beliva grown under field conditions. All treatments with BR analogues increased production. Treatments with DI-31 and DI-100 at a dose of 12 ppm (30 mg/ha) resulted in the highest production increases, which were 25.93% and 31.08%, respectively, relative to the control with Tomex Amin (T02). We could not correlate the slight increase in net photosynthesis with increasing yield produced by both BR analogues, except for treatment at the highest concentration of DI-100, which produced the maximum yield increase and also the biggest net photosynthesis with significant differences with respect to the control. This increased yield was caused by an increase in weight lettuce (which causes diameter and length increases) caused by increase of water absorption. These treatments produced no significant changes in the organoleptic quality of lettuce. The chemical variables related to lettuce quality, such as humidity, carbon and nitrogen content, sugar and organic acid content, total antioxidant activity (TAA) and phenolic content, were similar in the control and treated lettuces. The results showed that sprayed brassinosteroid analogues may play an important role in increasing the yield of field grown lettuces due to an increase in lettuce size without any undesirable effects on their nutritive and organoleptic properties. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Brassinosteroids are steroid plant hormones that play important regulatory roles in various physiological processes, including growth, differentiation, root and stem elongation, pollen tube growth, leaf bending and epinasty, xylem differentiation, disease resistance, stress tolerance and senescence (Clouse and Sasse, 1998; Nemhauser and Chory, 2004). This group of plant steroids includes more than 70 compounds that seem to be ubiquitously distributed throughout the plant kingdom. BRs have been detected and isolated from seeds, fruits, leaves, galls and pollen (Bajguz and Tretyn, 2003). The use of BRs has also been investigated in agricultural production. Several studies have demonstrated that BRs influence plant growth, seed germination, nitrogen fixation, senescence, leaf abscission and stress tolerance (Bajguz, 2000; Khripach et al., 2000; Anuradha and Rao, 2001; Arteca and Arteca, 2001; Yu et al., 2002;

∗ Corresponding author. Tel.: +34 96 6749686; fax: +34 96 6749678. E-mail address: [email protected] (A. Amorós). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.05.019

Nakashita et al., 2003; Howell et al., 2007; Kartal et al., 2009; Bajguz and Hayat, 2009). As a consequence, extensive research has been undertaken to develop BRs as plant growth regulators for agricultural production (Ikekawa and Zhao, 1991; Vardhini et al., 2006; Divi and Krishna, 2009). There is a promising prospect to increase crop yield and food production through the application of BR-derived, growth-promoting substances in modern agrosystems (Wu et al., 2008). Furthermore, BRs are a type of non-toxic (Muthuraman and Srikumar, 2010; Murkunde and Murthy, 2010; Steigerová et al., 2010; Sysa et al., 2010; Esposito et al., 2011) and environmentally friendly hormone (Kang and Guo, 2011). In an attempt to reduce the cost of BR synthesis and increase BR stability when applied, many types of brassinosteroid analogues have been produced (Zullo and Adam, 2002), including the spirostane ana˜ et al., 2003; Mazorra et al., logues of BR, like BB6 and MH5 (Núnez 2004). These brassinosteroid analogues are characterized by the presence of a spiroketalic ring instead of the typical BR side chain, and these chemicals possess BR-like activity (Mazorra et al., 2004). However, few treatments have been performed in the field under real growing conditions; most have been conducted with

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M. Serna et al. / Scientia Horticulturae 143 (2012) 29–37

Table 1 Selected meteorological data for the 2008–2009 experimental seasons. Month

February March April May

Average air temperature (◦ C)

Total precipitation (l/m2 )

2008

2009

2008

2009

2008

2009

10.20 13.38 15.19 16.58

9.47 11.70 13.82 18.58

10.53 16.90 21.22 18.59

11.04 15.88 19.70 25.69

72.33 47.29 47.83 63.14

69.59 65.43 63.80 62.45

plants grown under controlled environmental conditions in the laboratory. Many BRs and BR analogues that showed high biological activity in bioassays or controlled-environment experiments failed to stimulate plants grown under field conditions (Holá et al., 2010). This can be explained by various reasons. The timing of BR application has been shown to have an important effect on plant response ˜ et al., 2003). The length of plant expoto the BR treatment (Núnez sure to BRs, the application frequency, the application mode and the type and dose of BRs can also substantially affect the growth/yield promoting activity of these compounds (Holá et al., 2010). The lettuce plant is the principal green vegetable cultivated in Spain, and therefore increasing its production can be very beneficial economically. With increased interest in fresh-cut vegetable production, lettuce is the vegetable used most in fresh-cut salads in Europe, so increasing this vegetable’s production is very interesting for both fresh consumption as well as industrial products. The aim of this study was to evaluate the effects from exogenous application of two spirostane BR analogues, DI-31 (BB-16) and DI-100, on the growth, yield and quality of field grown lettuce plants and to examine the dependence of this effect on the concentration of BRs. This is the first report of DI-100 application.

2. Materials and methods

Humidity (%)

We mixed BRs with the commercial fertilizer Tomex Amin, which is formulated with 50% algae extract of Ascophyllum nodosum and 50% hydrolysed protein, whose composition of l-amino acids of plant origin is shown in Table 2. The DI-31 and DI-100 solutions were each applied as treatments at concentrations of 4, 8 and 12 ppm (10, 20 and 30 mg/ha) with Tomex Amin 2.5 l/ha, with water alone applied as control one (T01) and Tomex Amin 2.5 l/ha alone as control two (T02). Each solution was sprayed on the lettuces once before transplantation and three times afterwards (one application every three weeks). In all cases, the solution was applied with a manual pump. The experiment was arranged in a completely random design with 4 replicates per treatment and 50 lettuces for treatment and repetition. The other culture conditions were what the commercial farm did. 2.2. Measured parameters 2.2.1. Photosynthetic parameters The measurements of net photosynthesis (␮mol/m2 s) and internal C concentration in the leaves (ppm) were made with a TPS-1 photosynthesis instrument on the day following each treatment in the field. Analyses were applied to two leaves from each of 10 randomly chosen plants from each treatment, and two measurements were taken from each leaf. In all, 40 bits of data were obtained per treatment and date.

2.1. Plant material and synthetic BR treatments The experiment was carried out at a commercial plantation using Lactuca sativa L. var. Beliva (Nunhems) located in Agost, 18 Km west of Alicante (38◦ 26 21 N 0◦ 38 22 W). Its climate is temperate. The meteorological conditions for each experimental season are shown in Table 1. Cultivation took place between February and May of 2008 and 2009. Lettuce seedlings were transplanted to the field when they had a true leaf. Planting was carried out in 50 cm banks separated by 1 m axes on which 2 rows of lettuce were planted to a plantation of 0.3 m × 0.25 m, equivalent to 66,000 lettuce plants/ha. The BR analogues used in the present work are spirostanic analogues of brassinosteroids that were prepared by the Laboratory of Productos Naturales at La Habana University, Cuba. DI-31, a diosgenin derivative, with global formula C27 O5 H42 (commercially known as Biobras-16) is the (25R), 3␤, 5␣, dihydroxy-spirostan-6one (Jomarrón et al., 2000). The effects from BR treatment in the field are of short duration (Sasse, 1997; Symons and Reid, 2004; Symons et al., 2006) because they degrade quickly, so in this experiment we used DI-100. This compound is an acrylamide polymer obtained from DI-31 3␤-hemi maleate and acrylamide polymerization. This polymer releases the active compound, DI 31, more slowly and may have a physiological effect in the longer term. This research team was the first to use DI-100. Studies on the application of mineral fertilizers combined with BRs (Pirogovskaya et al., 1996) have demonstrated that this increases production and enhances the quality of crops. Terry et al. (2001) increased the yield of tomatoes using DI-31 (BB16) with N fertilization in the form of urea, phosphorus and potassium, as well as bio-based mycorrhizal fungi (Glomus clarum and Azospirillum brasiliense).

2.2.2. Weight, size and yield All lettuce was harvested when it was commercially ready, and weighed to calculate total yield. Then in the laboratory, 100 lettuces per treatment were selected (25 lettuce per treatment and repetition) as the sample size for the physical and chemical analysis to study the lettuce quality. These physical parameters were analyzed on 100 lettuces: average weight (using a KERN 6K1 CB digital balance with 0.01 g precision), length (measured with a 500 mm ruler), and equatorial diameter using a digital diameter device with a precision Table 2 Aminogram of Tomex Amin (g amino acid/100 g protein). Amino acid

%

Glycine Valine Proline Alanine Aspartic acid Methionine Leucine Lysine Arginine Glutamic acid Histidine Isoleucine Phenylalanine Threonine Tyrosine Tryptophan Cystine Serine

4.18 4.96 5.06 4.26 11.5 1.29 8.14 6.25 7.64 18.95 2.57 4.86 5.16 3.67 3.77 1.38 1.3 5.16

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of 0.01 mm. Compactness was calculated as the weight/diameter ratio. 2.2.3. Moisture content and total carbon and nitrogen The moisture content was determined by drying at 55 ◦ C in a BINDER oven until reaching constant weight and expressed as a percentage. The total carbon and nitrogen content were determined by burning the material at 1000 ◦ C with a Leco TruSpec CN elemental analyzer expressed as g/100 g of dry weight. 2.2.4. Soluble solids and acidity Soluble solids were measured using an ATAGO N20 refractometer with a scale from 0 to 32 ◦ Brix with ±0.2 accuracy. Concentrations are expressed in ◦ Brix at 20 ◦ C. Acidity was determined by potentiometric titration with 0.1 N NaOH to pH 8.1 with a METROHM 702 pH meter. The results are expressed as grams of malic acid per 100 g of fresh weight. 2.2.5. Sugars and organic acids Three grams of leaves from each subsample were homogenized with 10 ml of deionised water using a polytron homogenizer (IKA Labotechnik, Staufen, Germany) and centrifuged at 15,000 × g for 20 min at 4 ◦ C. A 10 ␮l aliquot of the supernatant was used to quantify organic acids and sugars using an HPLC system (HewlettPackard, series 1100, Waldbrom, Germany) equipped with a SUPELCOGEL C-610H (30 cm × 7.8 mm) column (at 30 ◦ C), a refractive index detector (for sugar analysis), and an absorbance detector (210 nm UV for acid analysis). The elution system consisted of 0.1% H3 PO4 , running isocratically at a flow rate of 0.5 ml min−1 (Amorós et al., 2003). A calibration curve was used to determine the concentration of individual organic acids and sugars in the samples. The following standard curves were used: sucrose, fructose and glucose, and malic, succinic, citric and tartaric acids from Sigma (Poole, Dorset, UK). Results were expressed as grams per 100 g of fresh weight (%). 2.2.6. Total antioxidant activity and phenolic compounds The same supernatant used for quantification of sugars and organic acids was used for total antioxidant activity and total phenolic compound quantification. The TAA was determined according to Cano et al. (1998) using the enzymatic system composed of chromophore 2,2 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), the horseradish peroxidase enzyme (HRP), and its oxidant substrate (hydrogen peroxide), in which ABTS• + radicals are generated. The assay temperature was 25 ◦ C, and the reaction was monitored at 414 nm until a stable absorbance was obtained using a UNICAM Helios ␣ spectrophotometer (Cambridge, UK). After this, a suitable amount of lettuce extract was added, and the observed decrease in absorbance was determined. A calibration curve was performed with Trolox ((R)(þ)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid), as standard antioxidant for TAA, and the results are expressed as milligrams of Trolox equivalents per 100 g fresh weight. The generation of ABTS• + radicals before the addition of the lettuce extract prevented interference from compounds. Total phenolic compounds were quantified according to the method described by Wood et al. (2002) using the Folin-Ciocalteu reagent. A calibration curve was performed with gallic acid and the results were expressed as mg of gallic acid equivalents per 100 g fresh weight. 2.3. Statistical analysis The data were analyzed by analysis of variance (ANOVA) and means were compared by Tukey’s multiple range test at p < 0.05. The data were analyzed with statistical package Statgraphics Plus 3.0.

Fig. 1. Effects of brassinosteroid analogue foliar sprays on lettuce plant yields. T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

3. Results 3.1. Lettuce yield The average production of control T02 (36,797.8 ± 1176.5 kg lettuce/ha) was superior to that of the lettuce without Tomex Amin, T01 (34,324 ± 1036.7 kg lettuce/ha), confirming that the increased nitrogen fertilizer led to an increase in production, although this difference was not significant (Fig. 1). All the BR analogue treatments assayed produced high lettuce yields. DI-100 yielded more than DI-31 at the same concentration but without statistical differences. The yields for each BR analogue were dose-dependent; the lettuce production increased as did the doses. Thus, the DI-31 and DI-100 treatments, at 12 ppm, produced a 35% (46,339.7 ± 1318.0 kg/ha) and 40.52% (48,234.6 ± 1466.8 kg/ha) increase in yield, respectively, with respect to control T01, and they produced a 25.93% and 31.08% increase in yield when compared to control T02 with the Tomex Amin treatment. The other BR analogue treatments also achieved significant increases over the controls. Therefore, we can say that treatment with BR analogues produced the expected effect of increased lettuce production. 3.2. Photosynthetic parameters The smallest net photosynthesis corresponded to control lettuce T01 with an average value of 4.05 ± 0.75 ␮mol/m2 s (Fig. 2a), which is the same treatment producing the smallest yield (Fig. 1). Treatment T3 with DI-100 produced the maximum photosynthetic level with an average value of 9.30 ± 1.75 ␮mol/m2 s and was the treatment producing the greatest yield (Fig. 1) as well. Regarding internal CO2 lettuce leaf concentrations, all the treatments maintained similar values except for treatment T3 with BR DI-100; this had the highest internal CO2 value of 820 ± 40 ppm average internal leaf CO2 (Fig. 2b). The smallest value came from control T02 with an average of 640 ± 25 ppm average internal leaf CO2 . All the cultivated lettuces had similar levels of internal leaf CO2 for conducting photosynthesis. 3.3. Physical parameters All treatments with BR analogues produced lettuce with greater weights and sizes than the control lettuce. The DI-100 treatments

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Fig. 2. Effects of brassinosteroid analogue foliar sprays on the net photosynthetic rate (␮mol CO2 /m2 s) (a) and intercellular CO2 concentration content of lettuce leaves (b). T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

produced lettuce larger and heavier than the DI-31 treatment at the same concentration but without significant differences. In both cases, the increases produced by the BRs were positive dosedependent (Table 3). The greatest weights and largest sizes were obtained with 12 ppm treatments for both DI-100 (751.50 ± 22.27 g/lettuce) and DI-31 (732.19 ± 22.82 g/lettuce) without statistically significant differences between them. The application of DI-100 and DI-31 produced an increase in weight with respect to control treatment T01 (538.04 ± 15.79 g/lettuce) of 39.67% for DI-100 and 36.08% for DI31. The increase was 29.93% for DI-100 and 26.60% for DI-30 with respect to control treatment T02 (578.35 ± 18.78 g/lettuce). The largest diameter was obtained with the DI-100 treatment at 12 ppm doses, 15.19 ± 0.32 cm, and the longest was obtained with the DI100 treatment at a 12 ppm dose, reaching a value of 14.18 ± 0.31 cm. The maximum compactness was obtained with the highest dosis of DI-100, with an average value of 52.31 ± 2.70, which was significantly different with respect to controls T01 as well as T02. The remaining treatments with BRs presented compactness values superior to the controls, although they were not significant. The lowest compactness value was obtained in control T02 with an average compactness value of 44.09 ± 1.60. These lettuce compactness increases corresponded to treatment with BRs because the T02 containing Tomex Amin without BRs resulted in a compactness value inferior to the treatments with BRs. 3.4. Moisture content, total carbon and total nitrogen Moisture from the control T02 lettuces was 96.68 ± 0.18%, and this parameter was similar in the DI-31 treated lettuces

(97.06–97.12%) without significant differences (P < 0.05) from the controls (Table 4). The moisture content from the DI-100 treatment lettuces (T2 and T3) was significantly lower (P < 0.05) than the control lettuces. Total carbon content was similar in lettuce from the control and treated plants (39.92–41.67 g/100 g DW), showing that the BR applications did not modify this parameter. All BR treatments slightly decreased the total N content in lettuce. The decreases were greater with increasing doses applied and decreases were greater with the DI-100 (3.70–3.47 g/100 g DW) than with the DI-31 (3.81–3.68 g/100 g DW) applications. 3.5. Chemical parameters The total soluble solid content in lettuce was not modified by the brassinosteroid application (Fig. 3a). The highest value was obtained with the DI-100 treatment at a dose of 4 ppm (3.46 ± 0.10 ◦ Brix) without significant differences with respect to T01 (3.23 ± 0.07 ◦ Brix). Fructose (between 1.199 and 1.505 g per 100 g FW) and glucose (0.925–1.220 g per 100 g FW) were the prominent sugars in lettuce followed by sucrose at lower concentrations (0.104–0.168 g per 100 g FW). No treatments with BR analogues modified the content of reducing sugars (glucose and fructose), although the sucrose content did increase with respect to the controls (Table 5), but by being the smallest sugar it did not modify the content of total soluble solids. The total acid content (Fig. 3b) was very low in the lettuce so that treatments with BRs practically did not change the total acidity. Malic acid was the prominent organic acid in lettuce (0.088–0.119 g per 100 g FW) and consequently, the main one for total acidity

Table 3 Effects of brassinosteroid analogue foliar sprays on the weight, diameter, length and compactness of lettuce plants. T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. Treatments

Fresh weight (g)a

T01 T02 T1-31 T2-31 T3-31 T1-100 T2-100 T3-100

538.04 578.35 677.77 698.27 732.19 710.30 736.55 751.50

± ± ± ± ± ± ± ±

15.79 a 18.78 a 18.05 b 21.66 bc 22.82 c 18.99 bc 18.87 bc 22.27 c

Diameter (mm)a 13.16 13.55 14.52 14.72 14.81 14.61 15.01 15.19

± ± ± ± ± ± ± ±

0.36 a 0.35 a 0.22 b 0.26 b 0.31 b 0.30 b 0.27 b 0.32 b

Length (mm)a 11.47 11.92 13.11 13.47 13.61 13.76 13.96 14.18

± ± ± ± ± ± ± ±

0.42 a 0.40 a 0.38 b 0.31 bc 0.34 bc 0.36 bc 0.31 bc 0.31 c

Mean of 100 lettuces for each treatment. a The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

Compactness (FW/D)a 44.34 44.09 47.48 47.56 48.37 49.01 47.72 52.31

± ± ± ± ± ± ± ±

1.83 a 1.60 a 2.06 ab 2.27 ab 2.37 ab 2.21 ab 2.44 ab 2.70 b

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Table 4 Effects of brassinosteroid analogue foliar sprays (DI-31 and DI-100) on the moisture, total carbon and total nitrogen content of lettuce plants. T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. Treatments

Moisture (%)a

T01 T02 T1-31 T2-31 T3-31 T1-100 T2-100 T3-100

96.74 96.68 97.09 97.12 97.06 97.27 97.38 98.03

± ± ± ± ± ± ± ±

Total carbon (g/100 g DW)a

0.20 a 0.18 a 0.18 ab 0.18 ab 0.20 ab 0.11 ab 0.30 bc 0.30 c

40.60 40.76 41.49 41.10 41.67 40.24 39.92 41.39

± ± ± ± ± ± ± ±

1.45 a 1.66 a 1.49 a 1.65 a 1.62 a 1.64 a 1.72 a 1.41 a

Total nitrogen (g/100 g DW)a 3.74 3.99 3.81 3.80 3.68 3.70 3.51 3.47

± ± ± ± ± ± ± ±

0.10 abc 0.10 c 0.07 bc 0.15 bc 0.07 ab 0.09 ab 0.08 a 0.12 a

Mean of 100 lettuces for each treatment. a The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

Fig. 3. Effects of brassinosteroid analogue foliar sprays on the soluble solids content of lettuce plants (a) and total titratable acidity content of lettuce plants (b). T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

(Table 6). Succinic and citric acids were present at concentrations of 0.039–0.077 g per 100 g FW, with tartaric acid at a lower concentration, about 0.01 g per 100 g FW. No significant (P < 0.05) differences were found in sugar or organic acid contents among the control and treated lettuces.

but only showed significant differences from the T02 treatment at the highest DI-100 concentration. All BR treatments decreased the total phenol contents, with greater decreases occurring with increasing BR concentrations applied (Fig. 4b). The decreases were greatest in the lettuce treated with DI-31 with significant differences.

3.6. Total antioxidant activity and phenol content The T02 treatment with Tomex Amin (10.85 mg Trolox eq/100 g FW) produced a slight decrease in TAA with respect to the T01 control lettuce (11.48 mg Trolox eq/100 g FW) but this was not significant (Fig. 4a). BR treatments produced slight decreases in TAA Table 5 Effects of brassinosteroid analogue foliar sprays (DI-31 and DI-100) on the fructose, glucose and sucrose sugar content of lettuce plants. T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. Treatments

Fructose (%)a

T01 T02 T1-31 T2-31 T3-31 T1-100 T2-100 T3-100

1.338 1.272 1.199 1.505 1.414 1.439 1.442 1.399

± ± ± ± ± ± ± ±

0.064 a 0.058 a 0.050 a 0.090 a 0.059 a 0.043 a 0.065 a 0.046 a

Glucose (%)a 1.084 0.972 0.925 1.220 1.108 1.148 1.126 1.094

± ± ± ± ± ± ± ±

0.056 a 0.051 a 0.042 a 0.085 b 0.051 a 0.037 a 0.053 a 0.040

Sucrose (%)a 0.118 0.104 0.130 0.154 0.144 0.156 0.168 0.130

± ± ± ± ± ± ± ±

0.004 ab 0.004 a 0.006 abc 0.009 bcd 0.007 bcd 0.007 cd 0.008 d 0.005 abc

Mean of 100 lettuces for each treatment. a The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

4. Discussion Tomex Amin (T02) (Fig. 1) produced a light, non-significant increase (P < 0.05) in lettuce production with respect to the control plants (T01). This indicates that the control plants were well nourished and an increase in the nitrogen fertilizer did not significantly increase production (Marscher, 2012). Both brassinosteroid analogues significantly increased the lettuce production in the field, and DI-100 at 12 ppm achieved the highest yield increase. This may be because DI-100 is formed by DI-31 with an acrylamide polymer cover that releases the active compound more slowly and may have a physiological effect in the longer term since the effects from BR treatment in the field are short duration (Sasse, 1997; Symons and Reid, 2004; Symons et al., 2006) because they degrade quickly, and so in this experiment we used DI-100 for the first time in a commercial crop. The increased lettuce production may be because BRs stimulate elongation, cell division and differentiation that promote growth (Howell et al., 2007; Kartal et al., 2009; Kang and Guo, 2011). Arteca and Arteca (2001) have also found that treatment with BRs increases Arabidopsis plant growth by cell divisions in the apical meristem in independent action by other hormones such as auxin, gibberellins and ethylene. This effect can be supported that BRs

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Fig. 4. Effects of brassinosteroid analogue foliar sprays on the total antioxidant activity content of lettuce plants (a) and total phenol contents of lettuce plants (b). T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

stimulate the flow of assimilates from source to sink (Wu et al., 2008), and in lettuce, the apical meristem is a stronger sink (Veit, 2009). Brassinosteroid applications in different crops have resulted ˜ in yield increases for potatoes (Torres and Núnez, 1997; Ramraj ˜ et al., 1998), wheat (Holá et al., 2010), et al., 1997), onions (Núnez soybean and common beans (Zullo and Adam, 2002), rice, mustard, and cotton (Ramraj et al., 1997), cladodes cactus pear (Cortes et al., 2003), groundnut (Vardhini and Rao, 1998), yellow passion fruit (Gomes et al., 2006), and Coleus plants (Swamy and Rao, 2011), among others. Increases in lettuce yields from BR treatments were directly correlated with increases in lettuce weight, diameter, and length (Table 3). The results from the present study clearly demonstrate the effectiveness of DI-100 and DI-31 in the improvement of the overall vegetative growth of lettuce plants. Similar growth promotion due to BR application was reported earlier in increased growth of the coffee plant (Isaac, 2002), geranium plant (Swamy and Rao, ˜ 2008), Coleus plant (Swamy and Rao, 2011), Vicia faba plants (Pinol and Simon, 2009), and field-grown maize (Holá et al., 2010), among others. Increases in the size of the lettuce leaves could be due directly to the effect from BRs, since Nakaya et al. (2002) found that mutants of Arabidopsis thaliana (det2) with known defects in the perception of brassinosteroids develop small leaves. Treatment of the det2 mutants with brassinosteroids reversed the mutation and restored the potential for growth to that of the wild type. BRs also play a dual role in regulating cell expansion and cell proliferation in the leaf (Kim et al., 2008; Oh et al., 2011) due to activation of the CycD3 gene (Hu et al., 2000). Both the weight and diameter of lettuce increased with BRs treatments, but the weight increase

was greater, resulting in an increase of the lettuce compactness (Table 3). This increased compactness could be due to a greater number of leaves. This research did not evaluate the number of leaves per lettuce plant due the difficulty doing this would have been with the Iceberg variety. However, other studies have shown that treatments with BRs cause an increase in the number of leaves ˜ and Simon, 2009), cucumber (Jian et al., 2012), geraof bean (Pinol nium (Swamy and Rao, 2008), Coleus (Swamy and Rao, 2011) and coffee plants (Isaac, 2002), among other crops. The light compactness increase might have been due to the leaves being heavier and therefore more compact, due to the cellular proliferation increase the BRs caused in them, just like Nakaya et al. (2002) demonstrated in A. thaliana. This could also be due to an increase in the foliar area, just like Swamy and Rao (2011) found in Coleus, and Jian et al. (2012) did so in cucumber leaves, both of these treated with 24epibrassinolide. Increases in lettuce production due to treatment with BRs were directly correlated with increases in lettuce weight, diameter, and length (Table 3). Thus, DI-100 and DI-31 in the field did not alter the lettuce morphology, only their growth rate. These findings corroborate those of Khripach et al. (1999) and Cortes et al. (2003), who reported that exogenous BR applications to plants do not alter their morphology. There was no correlation between the increase in lettuce yields treated with BRs and an increase in net photosynthesis (r2 = 0.56), although both parameters increased significantly (P < 0.05) in the T3 treatment with DI-100, reaching maximum yield. Other authors did find a strong relationship between increased production and increased photosynthetic induced BRs, such as Yu et al. (2004) in cucumber, Fariduddin et al. (2008) in mung bean, Swamy

Table 6 Effects of brassinosteroid analogue foliar sprays (DI-31 and DI-100) on the malic, succinic, citric and tartaric acids content of lettuce plants. T01 is the control, T02 is the control with Tomex Amin, T1-31 is 4 ppm DI-31 treatment, T2-31 is 8 ppm DI-31 treatment, T3-31 is 12 ppm DI-31 treatment, T1-100 is 4 ppm DI-100 treatment, T2-100 is 8 ppm DI-100 treatment and T3-100 is 12 ppm DI-100 treatment. Treatments

Malic (%)a

T01 T02 T1-31 T2-31 T3-31 T1-100 T2-100 T3-100

0.110 0.101 0.119 0.100 0.113 0.092 0.088 0.109

± ± ± ± ± ± ± ±

0.012 a 0.010 a 0.016 a 0.010 a 0.011 a 0.008 a 0.010 a 0.013 a

Succinic (%)a 0.058 0.039 0.077 0.065 0.054 0.059 0.053 0.062

± ± ± ± ± ± ± ±

0.007 ab 0.010 a 0.019 b 0.010 ab 0.006 ab 0.009 ab 0.008 ab 0.015 ab

Citric (%)a 0.040 0.046 0.055 0.045 0.050 0.051 0.044 0.045

± ± ± ± ± ± ± ±

0.003 a 0.004 a 0.009 a 0.004 a 0.004 a 0.010 a 0.004 a 0.003 a

Mean of 100 lettuces for each treatment. a The values followed by the same letter show no statistically significant differences (P < 0.05). Mean values ± standard error.

Tartaric (%)a 0.017 0.015 0.015 0.015 0.016 0.016 0.016 0.020

± ± ± ± ± ± ± ±

0.001 ab 0.001 a 0.001 a 0.001 a 0.001 a 0.001 a 0.001 ab 0.002 b

M. Serna et al. / Scientia Horticulturae 143 (2012) 29–37

and Rao in geranium (2008) and Coleus (2011), Xia et al. (2009) ˜ and Simon (2009) and Jian et al. (2012) in cucumber, and Pinol in V. faba, among other crops. Our lower correlation might be due to us measuring net photosynthesis 24 h after treatment with BR analogues, while Yu et al. (2004) observed net photosynthesis increases 3 h after treating cucumber plants with 24-epibrassinolide. As the effects from BR are of short duration, their effect might be drastically diminished at 24 h, and therefore an attenuated effect is observed, and a photosynthesis increase is still observed in the treatments at higher BR concentrations. However, all these studies were made in very young plants cultivated under controlled conditions so caution should be taken when extrapolating these results to plants grown under natural conditions in the field. Like us, Koˆcová et al. (2010) studied photosynthetic parameters in leaves of field grown-maize, finding that treatment with 24-epibrassinolide significantly affected neither the efficiency of photosynthetic electron transport nor the content of chlorophylls or carotenoids. On the other hand, treatment with BR analogues held internal CO2 concentration in the leaves (Fig. 2b) and only significantly increased in the T3 treatment with DI-100, which was the treatment that did increase net photosynthesis (Fig. 2a). The yield increase in lettuce treated with BR analogues can also be due, in part, to increased water absorption since the treated lettuce with higher moisture content was significant in the DI-100 treatment, which achieved the highest lettuce production (Table 4). ˜ (2003), The same results were also obtained by Mazorra and Núnez who tried tomato plants with a similar BR, Biobrás-6, and obtained significant increases in relative humidity in tomato plants. However, this effect depends upon the concentration of BRs applied (Isaac, 2002). The light increase in the water content of lettuces treated with BRs might be due to the implication of the BRs in the H+ pump (Khripach et al., 2003), which increases water absorption (Sairam, 1994), and which has also been related by Hayat et al. (2007) treating brown mustard plants with 28-homobrassinolide, also increasing their humidity percentage. Furthermore, Haubrick et al. (2006) also observed that brassinolide treatments induced stomatal closure by inhibiting stomatal opening, which in turn produced an increase in the cellular water content. It could be that lettuces have higher humidity contents due to greater water absorption by an increase in their radicular system, just like Kartal et al. (2009) found in barley treated with homobrassinolide. In turn, Kandelinskaya et al. (2007) also observed that BR treatments produced changes in the phytohormone balance in lupins, with increases in the auxin concentration and decreases in the ABA concentration which, bound to also increasing the amino acid concentration and protein systems of low molecular weight, caused an increase in the osmotic pressure of lupins, manifesting itself with greater water absorption, and therefore, the weight of the lupin seeds. However, Lisso et al. (2006) found that tomatoes from dwarf mutant plants, which cannot synthesize BRs, produced tomatoes with inferior dry matter percentages, an effect reversed by treatment with BRs. The increased weight in lettuce treated with BR analogues did not influence the total carbon content (Table 4). However, the total nitrogen percentage in the lettuces decreased slightly with the BR treatment, especially in those treatments producing greater fresh plant weights and, furthermore, greater relative humidity contents (treatments with DI-100). It is well documented that treatment with BRs causes increased NO3 − absorption (Mai et al., 1989) and increases in nitrate reductase activity (Ali et al., 2006; Fariduddin et al., 2008; Hayat et al., 2007; Hayat and Ahmad, 2003; Mazorra and ˜ Nunez, 2003) because they promote the transcription and/or translation of the nitrate reductase gene (Kalinich et al., 1985; Bajguz, 2000). This led to increase the N content necessary for the plant’s growth, but as an increase in such growth was produced and the

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BRs also caused an increase in the relative humidity of the lettuces, the result was a decrease in the total nitrogen content in the lettuces treated versus the controls. This decrease in N was also found by Khripach et al. (1996) in potato plants treated with the natural BR, brassinolide (BL), 28-homobrassinolide (28 HB) and 24epibrassinolide (24 epi). In any case, and despite the increase in the fresh weight of the lettuces treated with BRs, the total C and N contents were found in normal quantities for lettuces (Broadley et al., 2003), and thus they were of the same quality as untreated lettuces. Physico-chemical properties related to lettuce quality, such as soluble solid content, total acidity, and sugar and organic acid contents (Fig. 3 and Tables 5 and 6) were similar in the controls and treated lettuces. Jian et al. (2012) found that treatment with BRs in cucumber maintained the soluble sugar content. However, Vicentini et al. (2009) have characterized a gene whose transcript is a component of the BR receptor located within the plasmatic membrane of cells in the perivascular sheath of sugar cane (plant C4), confirming that it is directly involved in sucrose accumulation in such cells. These results were also corroborated by Laxmi et al. (2004) with mutant Arabidopsis bls1 plants, whose transcript is involved in the regulation of endogenous BR levels, which induces imbalances in sugars. Furthermore, and in in vitro cultures, Bajguz and Asami (2005) obtained sugar increases in Wolffia arriza plants. In the case of the lettuces, it is possible that this sugar redistribution occurred and the plant used them to increase its growth, one reason that neither the soluble solid contents nor the reducing sugars were modified, although an increase was observed in the sucrose percentage in treated lettuces. On the other hand, the BR treatments did not significantly modify the total acid content in the lettuces (Fig. 3) or that of the malic, succinic, citric or tartaric organic acids (Table 6) treated with BRs, although Khripach et al. (1996) did find ascorbic acid increases caused by treatments with natural BRs in potatoes. Generally, the TAA and total phenol content decreased lightly but not significantly with the exceptions of treatment T3 with DI100 (Fig. 4) for TAA and treatments T2 and T3 with DI-31 for the total phenol content. The TAA data found for lettuce were less than those found by other authors for other lettuce varieties, like Lollo Rosso (García-Macías et al., 2007) or Romaine (Blasco et al., 2008), surely due to Llorach et al. (2004) and Kang and Salveit (2002) demonstrating that the Baby and Romaine lettuce varieties possess more TAA than does Iceberg. The total phenol quantities that we obtained in lettuces, treated or not, were low if we compare them with other lettuce varieties, like Lollo Rosso, which is due to these lettuces being red in colour in their commercial state (García-Macías et al., 2007; Rajapakse et al., 2009), while our data had approximately double the total phenols as does Romaine lettuce (Blasco et al., 2008). These differences, therefore, could be due to varietal differences, something corroborated by other authors who found smaller total phenol quantities in Iceberg lettuce than in others, like Green Batavia, Romaine, Green and Red Oak Leaf Lettuce, Lollo Biondo and Lollo Rosso (Crozier et al., 1997; Dupont et al., 2000; Llorach et al., 2004), while Kang and Salveit (2002) report greater total phenol quantities in Iceberg lettuce than in Romaine. It is well documented that treatment with BRs in stressed plants provokes an increase in certain enzymatic activities that are antioxidants, such as catalase, superoxide dismutase and peroxidase, which provoke a resistance to stress in the plants treated with brassinosteroids with respect to unstressed plants. The same also occurs with the total phenol content, as its content grew when plants exposed to different types of stress were treated with BRs (Korableva et al., 1999). However, the behavior of BRs upon the antioxidant or total phenolic activity in unstressed plants has not been studied. In any case, the lettuces showed very little antioxidant activity and total phenol content if they are compared to fruits in general (Wolfe et al., 2008), and so their contribution to the diet is minor.

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5. Conclusion In conclusion, the DI-31 and DI-100 lettuce plant spray treatments were effective in increasing total yield due to increasing their growth without any undesirable effects on the physical or organoleptic qualities of the treated lettuce. The optimal concentration was found to be 12 ppm for both compounds, and the DI-100 at 12 ppm treatment increased lettuce production the most. This increase has been achieved in field treatments with two repetitions in consecutive years. Khripach et al. (2000) envisaged a great role for brassinosteroids in 21st century agriculture. The results of the present study present a case for the use of brassinosteroids to improve lettuce plants. Acknowledgement The authors are grateful to the Consellería de Educación of the Generalitat Valenciana for financial support (GV07/016). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.scienta.2012.05.019. References Ali, B., Hayat, S., Aiman Hasan, S., Ahmad, A., 2006. Effect of root applied 28homobrassinolide on the performance of Lycopersicon esculentum. Sci. Hortic. 110, 267–273. Amorós, A., Zapata, P., Pretel, M.T., Botella, M.A., Serrano, M., 2003. Physico-chemical and quality and physiological changes during fruit development and ripening of five loquat (Eriobotrya japonica Lindl.) Cultivars. Food Sci. Technol. Int. 9 (1), 43–51. Anuradha, S., Rao, S.S.R., 2001. Effects of brassinosteroid son salinity stress induced inhibition of seed germination and seedling growth of rice (Oryza sativa L.). Plant Growth Regul. 33, 151–153. Arteca, J.M., Arteca, R.N., 2001. Brassinosteroid-induce exaggerated growth in hydroponically grown Arabidopsis plants. Physiol. Plantarum 112, 104–112. Bajguz, A., 2000. Effect of brassinosteroids on nucleic acids and protein content in cultured cells of Chorella vulgaris. Plant Physiol. Biochem. 38, 209–215. Bajguz, A., Asami, T., 2005. Suppression of Wolffia arrhiza growth by brassinazole, an inhibitor of brassinosteroid biosynthesis and its restoration by endogenous 24-epibrassinolide. Phytochemistry 66, 1787–1796. Bajguz, A., Hayat, S., 2009. Effects of brassinosteroids on the plant responses to environmental stress. Plant Physiol. Biochem. 47, 1–8. Bajguz, A., Tretyn, A., 2003. The chemical characteristic and distribution of brassinosteroids in plants. Phytochemistry 63, 1027–1046. Blasco, J.J., Rios, L.M., Cervilla, E., Sanchez-Rodríguez, J.M., Ruiz, J.M., Romero, L., 2008. Iodine biofortification and antioxidant capacity of lettuce: potential benefits for cultivation and human health. Ann. Appl. Biol. 152, 289–299. Broadley, M.R., Seginer, I., Burns, A., Escobar-Gutiérrez, A.J., Burns, I.G., White, P.J., 2003. The nitrogen and nitrate economy of butterhead lettuce (Lactuca sativa Var. capitata L.). J. Exp. Bot. 54, 2081–2090. Cano, A., Hernández-Ruíz, J., García-Canovas, F., Acosta, M., Arnao, M.B., 1998. And end-point method for estimation of the total antioxidant activity in plant material. Phytochem. Anal. 9, 196–202. Clouse, S.D., Sasse, J.M., 1998. Brassinosteroids: essential regulators of plant growth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 427–451. Cortes, P.A., Terrazas, T., León, T., Larqué-Saavedra, A., 2003. Brassinosteroid effects on the precocity and yield of cladodes of cactus pear (Opuntia ficus-indica (L) Mill.). Sci. Hortic. 97, 65–73. Crozier, A., Lean, M.E.J., McDonald, M.S., Black, C., 1997. Quantitative analysis of the flavonoid content of commercial tomatoes, onions, lettuce and celery. J. Agric. Food Chem. 45, 590–595. Divi, U.K., Krishna, P., 2009. Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance. New Biotechnol. 26, 131–136. Dupont, M.S., Mondin, Z., Williamson, G., Price, K.H., 2000. Effect of variety, processing, and storage on the flavonoid glycoside content and composition of lettuce and endive. J. Agric. Food Chem. 48, 3957–3964. Esposito, D., Komarnytsky, S., Shapses, S., Raskin, I., 2011. Anabolic effect of plant brassinosteroid. FASEB J. 25, 3708–3719. Fariduddin, Q., Hasan, S.A., Ali, B., Hayat, S., Ahmad, A., 2008. Effect of modes of application of 28-homobrassinolide on mung bean. Turk. J. Biol. 32, 17–21. García-Macías, P., Ordidge, M., Vysini, E., Waroonphan, S., Battey, N.H., Gordon, M.H., Hadley, P., Philip, J., Lovegrove, J.A., Wagstaffe, A., 2007. Changes in the flavonoid and phenolic acid contents and antioxidant activity of red leaf lettuce (Lollo

Rosso) due to cultivation under plastic films varying in ultraviolet transparency. J. Agric. Food Chem. 55, 10168–19172. Gomes, M.M.A., Compostrini, E., Rocha, N., Pio, A., Massi, T., Siqueira, L., Carriello, ˜ R.C., Torres, A., Nunez, M., Zullo, M.A., 2006. Brassinosteroid analogue effects on the yield of yellow passion fruit plants (Passiflora edulis f. flavicarpa). Sci. Hortic. 110, 235–240. Haubrick, L.L., Torsethaugen, G., Assmann, S.M., 2006. Effect of brassinolide, alone and in concert with abscisic acid, on control of stomatal aperture and potassium currents of Vicia faba guard cell protoplasts. Physiol. Plantarum 128, 134–143. Hayat, S., Ahmad, A., 2003. Soaking seeds of Lens culinaris with 28-homobrassinolide increased nitrate reductase activity and grain yield in the field in India. Assoc. Appl. Biol. 143, 121–124. Hayat, S., Ali, B., Hasan, A., Ahmad, A., 2007. Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea. Environ. Exp. Bot. 60, 33–41. Holá, D., Rothová, O., Koˆcová, M., Kohout, L., Kvasnica, M., 2010. The effect of brassinosteroids on the morphology, development and yield of field-grown maize. Plant Growth Regul. 61, 29–43. Howell, W.M., Keller III, G.E., Kirkpatrick, J.D., Jenkins, R.L., 2007. Effects of the plant steroidal hormone, 24-epibrassinolide, on the mitotic index and growth of onion (Allium cepa) root tips. Genet. Mol. Res. 6, 50–58. Hu, Y., Bao, F., Li, J., 2000. Promotive effect of brassinosteroids on cell division involves a distinct CycD3-induction pathway in Arabidopsis. Plant J. 24, 693–701. Ikekawa, N., Zhao, Y-J., 1991. Application of 24-epibrassinolide in agriculture. In: Cutler, H.G., Yokota, T., Adam, G. (Eds.), Brassinosteroids: Chemistry, Bioactivity and Applications, 24. American Chemical Society, Washington, pp. 280–291 (ACS Symposium Series 474). Isaac, E., 2002. Resultados de las aplicaciones de brasinoesteroides en plántulas de cafeto en la fase de aclimatación. Centro Nacional de Electromagnetismo Aplicado (CNEA) Universidad de Oriente. Anexo II, 1–14. Jian, Y.P., Cheng, F., Zhou, Y.H., Xia, X.J., Shi, K., Yu, J.Q., 2012. Interactive effects of CO2 enrichment and brassinosteroid on CO2 assimilation and photosynthetic electron transport in Cucumis sativus. Environ. Exp. Bot. 75, 98–106. Jomarrón, I., Coll, F., Robaina, R., Alonso, E., Cabrera, M.T., 2000. Polyhydroxispirostannones as plant growth regulators. European Patent Office Number 1020477, Bulletin number 2000/29. Kalinich, J.F., Mandava, N.B., Todhunter, J.A., 1985. Relationship of nucleic acid metabolism on brassinolide-induced responses in beans. Plant Physiol. 120, 207–214. Kandelinskaya, O.L., Topunov, A.F., Grishchenko, E.R., 2007. Biochemical aspects of growth-stimulating effects of steroid phytohormones on Lupine plants. Appl. Biochem. Microbiol. 43, 324–331. Kang, Y.Y., Guo, S.R., 2011. Role of brassinosteroids on horticultural crops. In: Hayat, S., Ahmad, A. (Eds.), Brassinosteroids: A Class of Plant Hormone. Springer, pp. 269–288. Kang, H.M., Salveit, M.E., 2002. Antioxidant capacity lettuce leaf tissue increases after wounding. J. Agric. Food Chem. 50, 7536–7541. Kartal, G., Temel, A., Arican, E., Gozukirmizi, N., 2009. Effects of brassinosteroids on barley root growth, antioxidant system and cell division. Plant Growth Regul. 58, 261–267. Khripach, V.A., Zhabinskii, V., De Groot, A.E., 1999. A New Class of Plant Hormones. Academic Press, San Diego. Khripach, V.A., Zhabinskii, V., De Groot, A.E., 2000. Twenty years of brassinosteroids: steroidal plant hormones warrant better crops for the XXI Century. Ann. Bot. 86, 441–447. Khripach, V.A., Zhabinskii, V., Khripach, N.B., 2003. New practical aspects of brassinosteroids and results of their 10 year agricultural use in Russia and Balarus. In: Hayat, S., Ahmad, A. (Eds.), Brassinosteroids: Bioactivity and Crop Productivity. Kluwer Academic Publisher, Dordrecht, pp. 189–230. Khripach, V.A., Zhabinskii, V., Litvinovskaya, R.P., Zavadskaya, M.I., Saveleva, E.A., Karas, I.I., Vakulenko, V.V., 1996. A method of increasing of potato food value. Pat. Appl. BY 960, 345. Kim, S.-L., Lee, Y., Lee, S.-H., Kim, S.-H., Han, T.-J., Kim, S.-K., 2008. Brassinolide influences the regeneration of adventitious shoots from cultured leaf discs of tobacco. J. Plant Biol. 51, 221–226. Korableva, N.P., Platonova, T.A., Dogonadze, M.Z., Bibick, N.D., 1999. A stability change of potato to premature germination and diseases under the brassinosteroid action. In: Shevelucha, V.S., Karlov, G.I., Karsunkina, N.P., Salnikova, E.I., Skorobogatova, I.V., Siusheva, A.G. (Eds.), Regulators of Plant Growth and Development, vol. 5. Agricultural Academy, Moscow, pp. 102–103. Koˆcová, M., Rothová, O., Holá, D., Kvasnica, M., Kohout, L., 2010. The effects of brassinosteroids on photosynthetic parameters in leaves of two field-grown maize inbred lines and their F1 hybrid. Biol. Plantarum 54, 785–788. Laxmi, A., Paul, L.K., Peters, J.L., Khurana, J.P., 2004. Arabidopsis constitutive photomorphogenic mutant, bls1, displays altered brassinosteroid response and sugar sensitivity. Plant Mol. Biol. 56, 185–201. Lisso, J., Altmann, T., Müssing, C., 2006. Metabolic changes in fruits of the tomato dx mutant. Phytochemistry 67, 2232–2238. Llorach, R., Tomás-Barberán, F.A., Ferreres, F., 2004. Lettuce and chicory byproducts as a source of antioxidant phenolic extracts. J. Agric. Food Chem. 52, 5109–5116. Mai, Y.Y., Lin, J.M., Zeng, X.L., Pan, R.J., 1989. Effect of homobrassinolide of the activity of nitrate reductase in rice seedlings. Plant Physiol. Commun. 2, 50–52. Marscher, P., 2012. Marschner’s Mineral nutrition of higher plants. Academic Press. ˜ M., 2003. Influencia de análogos de brasinoesteroides en la Mazorra, L.M., Núnez, respuesta de plantas de tomate a diferentes estrés ambientales. Cultivos Trop. 24, 35–40.

M. Serna et al. / Scientia Horticulturae 143 (2012) 29–37 ˜ Mazorra, L.B., Núnez, M., Nápoles, M.C., Yoshida, S., Robaina, C., Coll, F., Asami, T., 2004. Effects of structural analogs of Brassinosteroids on the recovery of growth inhibition by a specific brassinosteroid biosynthesis inhibitor. Plant Growth Regul. 44, 183–185. Muthuraman, P., Srikumar, K., 2010. Induction of hexokinase I expression in normal and diabetic rats by a brassinosteroid isoform. Eur. J. Pharm. Sci. 41, 1–9. Murkunde, Y.V., Murthy, P.B., 2010. Developmental toxicity of homobrassinolide in wistar rats. Int. J. Toxicol. 29, 517–522. Nakashita, H., Yasuda, M., Nitta, T., Asami, T., Fujioka, S., Arai, Y., Sekimata, K., Takatsuto, S., Yamaguchi, I., Yoshida, S., 2003. Brassinosteroid functions in a broad range of diseases resistance in tobacco and rice. Plant J. 33, 887–898. Nakaya, M., Tsukawa, H., Murakami, N., Kato, M., 2002. Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana. Plant Cell Physiol. 43, 239–244. Nemhauser, J.L., Chory, J., 2004. Bring it on: new insights into the mechanism of brassinosteroid action. J. Exp. Bot. 55, 265–270. ˜ M., Robaina, C., Coll, F., 2003. Synthesis and practical applications of brassiNúnez, nosteroid analogs. In: Hayat, S., Ahmad, A. (Eds.), Brassinosteroids: Bioactivity and Crop Productivity. Kluwer Academic, The Netherlands. ˜ M., Sosa, J.L., Alfonso, J.L., Coll, F., 1998. Influencia de dos nuevos biorreguNúnez, ladores cubanos en el rendimiento de plantas de cebolla (Allium cepa) cv. red creole. Cultivos Trop. 19, 21–24. Oh, M.H., Sun, J., Oh, D.H., Zielinski, R.E., Clouse, S.D., Huber, S.C., 2011. Enhancing Arabidopsis leaf growth by engineering the BRASSINOSTEROID INSENSITIVE1 receptor kinase. Plant Physiol. 157, 120–131. ˜ Pinol, R., Simon, E., 2009. Effect of 24-epibrassinolide on chlorophyll fluorescence and photosynthetic CO2 assimilation in Vicia faba plants treated with the photosynthesis-inhibiting herbicide terbutryn. J. Plant Growth Regul. 28, 97–105. Pirogovskaya, G.V., Bogdevitch, I.M., Naumova, G.V., Khripach, V.A., Azizbekyan, S.G., Krul, L.P., 1996. New forms of mineral fertilizers with additives of plant growth regulators. Proc. Plant Growth Regul. Soc. Am. 23, 146–151. Rajapakse, N.C., He, C., Cisneros-Zevallos, L., Davis Jr., F.T., 2009. Hypobaria and hypoxia affects growth and phytochemical contents of lettuce. Sci. Hortic. 122, 171–178. Ramraj, V.M., Vyas, B.N., Godrej, N.B., Mistry, K.B., Swami, B.N., Singh, N., 1997. Effects of 28-homobrassinolide on yields of wheat, rice, groundnut, mustard, potato and cotton. J. Agric. Sci. 128, 405–413. Sairam, R.K., 1994. Effects of homobrassinolide application on plant metabolism and grain yield under irrigated and moisture stress conditions of two wheat varieties. Plant Growth Regul. 14, 173–181. Sasse, J.M., 1997. Recent progress in brassinosteroid research. Physiol. Plantarum 100, 696–701. Steigerová, J., Okleˇst’ková, J., Levková, M., Rárová, L., Koláˇr, Z., Strnad, M., 2010. Brassinosteroids cause cell cycle arrest and apoptosis of human breast cancer cell. Chem.-Biol. Interact. 188, 487–496. Swamy, K.N., Rao, S.S.R., 2008. Influence of 28-homobrassinolide on growth, photosynthesis metabolite and essential oil content of geranium [Pelargonium graveolens (L.) Herit]. Am. J. Plant Physiol. 3, 173–174.

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Swamy, K.N., Rao, S.S.R., 2011. Effect of brassinosteroids on the performance of Coleus (Coleus forskohlii), Journal of Herbs. Spices Med. Plants 17, 12–20. Symons, G.M., Davies, C., Shavrukov, Y., Dry, I.B., Reid, J.B., Thomas, M.R., 2006. Grapes on steroids. Brassinosteroids are involved in grape berry ripening. Plant Physiol. 140, 150–158. Symons, G.M., Reid, J.B., 2004. Brassinosteroids do not undergo long distance transport in pea: implications for the regulation of endogenous brassinosteroid levels. Plant Physiol. 135, 2196–2206. Sysa, A.G., Kiselev, P.A., Zhabinskii, V.N., Khripach, V.A., 2010. Effect of the structure of the brassinosteroid side chain on monooxygenase activity of liver microsomes. Appl. Biochem. Microbiol. 46, 23–27. Terry, E., Núnez, M., Pino, M., Medina, N., 2001. Efectividad de la combinación Biofertilizantes-Análogo de brasinoesteroides en la nutrición del tomate (Lycopersicon escullentum Mill). Cultivos Trop. 22, 59–65. ˜ M., 1997. The application of biobras-6 and its effect on potato Torres, W., Núnez, (Solanum tuberosum L.) yields. Cultivos Trop. 18, 8–10. Vardhini, B.V., Anuradha, S., Rao, S.S.R., 2006. Brassinosteroids-new class of plant hormones with potential to improve crop productivity. Indian J. Plant Physiol. 11, 1–12. Vardhini, B.V., Rao, S.S.R., 1998. Effect of brassinosteroids on growth, metabolite content and yield of Arachis hypogaea. Phytochemistry 48, 927–930. Veit, B., 2009. Hormone mediated regulation of the shoot apical meristem. Plant Mol. Biol. 69, 397–408. Vicentini, R., Felix, J.D.M., Carnier, M., Menossi, M., 2009. Characterization of a sugarcane (Saccharum spp.) gene homolog to the brassinosteroid insenstive1associated receptor kinase 1 that is associated to sugar content. Plant Cell Rep. 28, 481–491. Wolfe, K.L., Kang, X., He, X., Dong, M., Zhang, Q., Liu, R.H., 2008. Cellular antioxidant activity of common fruits. J. Agric. Food Chem. 56, 8418–8426. Wood, J.E., Senthilmohan, S.T., Peskin, A.V., 2002. Antioxidant activity of procyanidin-containing plant extracts at different pHs. Food Chem. 77, 155–161. Wu, C.Y., Trieu, A., Radhakrishnan, P., Know, S.F., Harris, S., Zhyang, K., Wang, J.L., Wan, J.M., Zhai, H.Q., Takatsuto, S., Matsumoto, S., Fujioka, S., Feldmann, K.A., Pennell, R.I., 2008. Brassinosteroids regulate grain filling in rice. Plant Cell 20, 2130–2145. Xia, X.J., Huang, L.F., Zhou, Y.H., Mao, W.H., Shi, K., Wu, J.X., Asami, T., Chen, Z., Yu, J.Q., 2009. Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus. Planta 230, 1185–1196. Yu, J.Q., Huang, L.F., Hu, W.H., Zhou, Y.H., Mao, W.H., Ye, S.F., Nogues, S., 2004. A role for brassinosteroids in the regulation of photosynthesis in Cucumis sativus. J. Exp. Bot. 55, 1135–1143. Yu, J.Q., Zhou, Y.H., Ye, S.F., Huang, L.F., 2002. 24-epibrassinolide and abscisic acid protect cucumber seedlings from chilling injury. J. Hortic. Sci. Biotechnol. 77, 470–473. Zullo, M.A.T., Adam, G., 2002. Brassinosteroid phytohormones—structure, bioactivity and applications. Braz. J. Plant Physiol. 14, 83–121.