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Comparison of antioxidant activities and endogenous hormone levels between bush and vine-type tropical pumpkin (Cucurbita moschata Duchesne)
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Scientia Horticulturae 116 (2008) 27–33 www.elsevier.com/locate/scihorti
Comparison of antioxidant activities and endogenous hormone levels between bush and vine-type tropical pumpkin (Cucurbita moschata Duchesne) Tao Wu, Jiashu Cao *, Yafeng Zhang Institute of Vegetable Science, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, China Received 6 June 2007; received in revised form 11 November 2007; accepted 14 November 2007
Abstract A bush-type plant was selected from a cultivar ‘Cga’ of tropical pumpkin (Cucurbita moschata Duchesne) in order to study and compare the bush and vine habit of C. moschata in terms of various physiological and biochemical characteristics. During the internode development of the bush and vine-type plants in C. moschata, the metabolism of reactive oxygen species (hydrogen peroxide, H2O2; and superoxide anion radical, O2), the activities of some antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; and ascorbate peroxidase, APX), and the levels of some endogenous hormones (gibberellin A3, GA3; gibberellin A4, GA4; and indole-3-acetic acid, IAA) were compared. Bush plants were found to have a significantly higher O2 level than vine plants at 28 days after germination (DAG) and onwards. Moreover, there was no significant difference of H2O2 level between the two genotypes until 32 DAG, in which the difference was significant at 32 DAG. In both genotypes, no significant differences of SOD activity were observed, and this was opposite to APX activity in which significant differences were observed during almost the whole sampling stages. What’s more, APX activity of bush plants was significantly higher than that of vine plants at 24, 28, 32, and 36 DAG. CAT activity showed the same trend for the two genotypes. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) indicated that the internodes of bush plants contain lower levels of GA3, IAA, and GA4 than those of vine plants in C. moschata. In addition, GA3 deficiency was also found in the leaves and roots of bush plants. In conclusion, this work provided some new information about different levels of endogenous chemicals in pumpkins with differing growth habits. # 2007 Elsevier B.V. All rights reserved. Keywords: Ascorbate peroxidase; Cell elongation; ELISA; Gibberellin; Internode
1. Introduction Cucurbita moschata Duchesne is one of the most important species of pumpkin in traditional agricultural systems in the world. Most cultivars of Cucurbita species are large and trailing plants. Some plants of the developed cultivars have short vines, which give them a bushy appearance (Shifriss, 1947; Denna and Munger, 1963; Cao et al., 2005). The vine elongation of plants is a very complex process consisting of complicated physiological and biochemical modifications. However, there are only a few studies focusing on the physiological and biochemical differences between bush and vine plants of pumpkin. A suggested role for reactive oxygen species (ROS), (superoxide anion radical, O2; hydrogen peroxide, H2O2;
* Corresponding author. Tel.: +86 571 86971188; fax: +86 571 86971188. E-mail address: [email protected] (J. Cao). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.11.003
and hydroxyl radicals, OH) during plant growth and morphogenesis is involvement as stress signal molecules. However, they have also been proposed to possess a decisive role on cell wall loosening and other processes required for cell elongation (Schopfer, 2001). It has been shown that H2O2 may function as a developmental signaling molecule in the differentiation of secondary walls in cotton fibers (Potikha et al., 1999). High ROS production was observed in the expansion zone of maize leaves, and a certain concentration of H2O2 was found to be necessary for leaf elongation (Rodrı´guez et al., 2002). ROS play an important role in root hair growth and may provide insight into the mechanism of plant cell growth in general (Carol and Dolan, 2006). In addition to the ROS function as messengers in plant cells, principle antioxidant enzymes such as superoxide dismutases (SOD; EC 1.15.1.1), catalases (CAT; EC 1.11.1.6), and ascorbate peroxidase (APX; EC 1.11.1.11), which help decomposing ROS, are monitored as indicators of redox homeostasis.
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The important role of endogenous hormones in plant growth and development has been studied for many years. Plant hormones can modulate gene expression and protein synthesis, and subsequently modulate the physiological and biochemical process of plants (Chen et al., 1993). The bush-type plant used in this study was selected from a cultivar ‘Cga’ of tropical pumpkin (C. moschata Duchesne). Bush plants were characterized by shorter internodes, earlier flowering, a higher ration of female to male flowers and smaller fruit than vine ‘Cga’ plants. Genetic analysis indicated monogenic inheritance with the bush genotype dominant (Wu et al., 2007). The results of Cao et al. (2005) revealed that the bush plant in C. moschata was a gibberellin-responsive mutant, and the bushy appearance was the result of inhibition of cell elongation. However, the endogenous levels of hormones in pumpkin bush plants were not investigated in that paper. Thus, the investigation of differences in endogenous hormones between bush and vine plants can reflect the internal development of pumpkin plants. To this aim, the present study was undertaken to characterize and compare ROS metabolism, activities of antioxidant enzymes (SOD, CAT, and APX), and selected endogenous hormones (GA3, GA4, and IAA) levels during different growth periods of bush and vine plants in C. moschata, and to determine the relationship between the antioxidant activities, the hormone levels in plants and the phenotype difference in C. moschata. It was hoped that this study would provide some new information about different levels of endogenous compounds in pumpkins with differing growth habits. 2. Materials and methods 2.1. Plant material The bush-type plant was selected from a cultivar ‘Cga’ of tropical pumpkin (C. moschata Duchesne). Seedlings of bush and vine plants could be distinguished at third leaf stage. Bush plants had short internodes length (4.1 0.4 cm vs. 10.2 0.6 cm), short vine length (14.5 4.9 cm vs. 175.6 28.2 cm) and small number of internode (3.5 0.7 vs. 17.2 1.6) compared with vine plants at 60 days after planting. Vine plants produced more male flowers (21.1 4.3) than bush plants (13.7 3.3). In contrast, bush plants flowered eight days earlier (40.6 2.9 days vs. 48.6 2.7 days) than vine plants. Although bush plants had a higher rate of fruit abortion than vine plants, they had a larger total number of fruit than vine plants in a plant basis (Wu et al., 2007). Seeds of bush (CgaBu) and vine (CgaV) parental plants were sown on 16th March 2006, with the seedlings grown in a mixture of 1 vermiculite:1 perlite (v/v) on the research farm of the Institute of Vegetable Science at Zhejiang University, Hangzhou, China. At the twoor three-leaf stage, seedling roots were washed gently in water, and then four plants were transplanted into a plastic pot containing 8 L of the nutrient solution of Japan Yuanshi composition (Yu and Matsui, 1997). The Japan Yuanshi composition nutrient solution comprised of the following elements: macronutrients (in mM): N, 14 [4.0 KNO3, 4.0 Ca(NO3)2, and 2.0 NH4H2PO4]; P, 2.0 (NH4H2PO4); K, 4.0
(KNO3); Ca, 4.0 [Ca(NO3)2]; and Mg, 2.0 (MgSO4); and micronutrients (in mg L1): Fe, 3.0 (Fe-EDTA); Mn, 0.5 (MnSO4); B, 0.5 (H3BO3); Zn, 0.05 (ZnSO4); Mo, 0.05 [(NH4)6Mo7O24]; and Cu, 0.02 (CuSO4). The pH of the nutrient solution was adjusted to 5.8 with 1 M NaOH and the solutions were changed weekly. Polyurethane foam and a plastic mesh supported the seedlings, which grew under controlled environmental conditions. Plants were grown under supplementary lights with an irradiance of 90 W m2 and a 12-h photoperiod. The air temperature was 22–24 8C and relative humidity was 75–90%. Samples (internodes, leaves, and roots) were randomly collected from 10 seedlings of each type at various times after germination (20, 24, 28, 32, and 36 DAG). The first internode of bush and vine plants at different development stages was collected. Experiments were carried out with the leaves of the middle (the third) story of the bush and vine-type plants at different stages of their development. Primary roots of the bush and vine plants were also collected at different stages of their development. Samples were weighed, immediately frozen in liquid nitrogen, and then were stored at 80 8C until use. 2.2. Enzyme extraction Enzyme extracts were prepared by homogenizing samples (500 mg) in a prechilled mortar and pestle nestled in ice along with 5% polyvinylpyrrolidone and 1 mL of chilled extraction buffer consisting of 50 mM potassium phosphated (pH 7.5) buffer and 1 mM EDTA. For APX activity 5 mM ascorbic acid was included in the grinding medium. The homogenate was centrifuged (ULTRA80, Sorval, Thermo Fisher Scientific, Waltham, MA, USA) at 12,000 g for 15 min at 4 8C. Supernatant was collected for subsequent protein and enzyme assays immediately following extraction. Protein concentration was determined spectrophotometrically at 595 nm using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, California; catalogue no. 500-0006) using a method described by Bradford (1976). Enzyme activities based on total protein were determined spectrophotometrically (LAMBAD-40, PerkinElmer, Boston, MA, USA) at 25 8C. 2.2.1. Measurement of SOD activity SOD activity was measured according to Beauchamp and Fridovich (1971) with some modifications. The assay mixture (1 mL) contained 100 mM sodium phosphate buffer (pH 7.8), 57 mM nitroblue tetrazolium (NBT, Wako Pure Chemical Industries, Japan), 0.025% of Triton-X-100, 0.11 mM of EDTA, 0.01 M methionine, 1.3 mM riboflavin and 50 mg of total protein. One enzyme unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT measured at 560 nm on a spectrophotometer. The specific activity of SOD was expressed as unit per mg protein. 2.2.2. Measurement of CAT activity CAT activity was assayed in a 1 mL of reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 1 mM
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EDTA, and 60 mM H2O2 following a protocol of Aebi (1984). The reaction was initiated by the addition of 50 mg of total protein and CAT activity was determined by following the decomposition of H2O2 at 240 nm. 2.2.3. Measurement of APX activity APX activity was determined using the method described by Nakano and Asada (1981). The assay mixture (1 mL) contained 90 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.65 mM ascorbate and 1 mM H2O2. The reaction was initiated by adding 50 mg of total protein. APX activity was determined by following the H2O2 dependent decomposition of ascorbate at 290 nm. 2.2.4. O2 detection The detection of O2 was measured according to hydroxylamine oxidation (Wang and Luo, 1990). Enzyme extracts were prepared by homogenizing samples (1.5 g) in a prechilled mortar and pestle nestled in ice along with 5% polyvinylpyrrolidone and 3 mL of chilled extraction buffer consisting of 50 mM potassium phosphated (pH 7.5) buffer and 1 mM EDTA. The homogenate was centrifuged (ULTRA80, Sorval, Thermo Fisher Scientific, Waltham, MA, USA) at 12,000 g for 15 min at 4 8C. Supernatants were collected for O2 detection. A mixture of 0.9 mL 50 mM potassium phosphate buffer (pH 7.8), 1 mL enzyme extract, and 0.1 mL 10 mM hydroxylation was incubated at 25 8C for 1 h, and then this was mixed with 1 mL 17 mM p-aminobenzene sulfonic acid and 1 mL 7 mM a-naphthylamine solution, at 25 8C for 20 min. The reaction was measured spectrophotometrically at 530 nm. The O2 level was obtained using a linear calibration curve of NaNO2. 2.2.5. H2O2 detection Internodes were placed in titanium sulfate and the total H2O2 was detected according to Patterson et al. (1984). Enzyme extracts were prepared by homogenizing samples (0.3 g) in a prechilled mortar and pestle nestled in ice along with 5 mL of chilled acetone. The homogenate was centrifuged (ULTRA80, Sorval, Thermo Fisher Scientific, Waltham, MA, USA) at 3000 g for 10 min at 4 8C. Supernatants were collected for H2O2 detection. One milliliter of acetone extraction was added through a solution consisting of 0.1 mL hydrochloric acid (containing 20% TiCl4) and 0.2 mL 17 M ammonia solution. The homogenate was centrifuged at 3000 g for 10 min at 4 8C. The precipitate was washed three times with acetone, and was centrifuged at 3000 g for 10 min at 4 8C at each time. This precipitate was dissolved in 3 mL 1 M sulfuric acid, and then was measured spectrophotometrically at 410 nm. H2O2 concentration was determined by a standard graph from 5 to 1 mM H2O2 constructed by direct addition of H2O2 to the titanium solution. 2.3. Phytohormone quantification Endogenous phytohormones were extracted from the internode, leaf, and root of pumpkin bush and vine plants at
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various DAG. They were quantified by ELISA technique as described in Chen et al. (1996) using reagents from China Agricultural University (China). The main steps of extraction and purification of plant hormones prior to immunoassay were: extraction of homogenized samples in cold 80% (v/v) aqueous methanol at a rate of 5 mL g1 fresh weight overnight at 4 8C with 1 mM butylated hydroxytoluene added to prevent oxidation. The supernatant was collected after centrifugation at 10,000 g (4 8C) for 15 min. Then, the crude extracts were passed through a C18 Sep-Pak cartridge (Waters, Milford. MA, USA). The efflux was collected and 400 mL of it was taken out and dried in N2. The residue was dissolved in 400 mL phosphate-buffered saline (PBS, 0.01 M, pH 7.5, containing 0.1% (v/v) Tween-20 and 0.1% (w/v) glutin) for determining the contents of GA3, GA4, and IAA. Another 600 mL aliquot of the filtrate was taken and dried in N2, and the residue dissolved in 200 mL PBS (0.01 M, pH 9.2) and adjusted to pH 8.5, before partitioned three times with an equal volume of ethyl acetate. The remaining aqueous phase was adjusted to pH 2.5 and extracted three times with an equal volume of ethyl acetate again. The extracts (ethyl acetate phase) were pooled and dried in N2; the residue was either redissolved in 200 mL PBS (0.01 M, pH 7.4) to analyze for GA3 and GA4, or redissolved in 200 mL 100% methanol for methylation with freshly synthesized ethereal diazomethane, and taken up with 400 mL PBS for analyses of IAA. The main steps of indirect competitive ELISA measurement using polyclonal antibodies against IAA are as follows. Microtitration plates (Nunc. Denmark) were coated with synthetic hormone–ovalbumin conjugates in NaHCO3 buffer (50 mM, pH 9.6) and left overnight at 37 8C. The solution was decanted and ovalbumin solution (10 mg mL1) was added to each well for blocking the nonspecific binding. After incubation for 30 min at 37 8C, authentic hormones or samples and antibodies were added and incubated 45 min at 37 8C. Then horseradish peroxidase-labeled goat anti-rabbit immunoglobulin was added to each well and incubated 1 h at 37 8C. Finally, the buffered enzyme substrate (H2O2 and ortho-phenylenediamine) was added, and the enzyme reaction was carried out in the dark at 37 8C for 15 min, then terminated using 3 M H2SO4, and the absorbance was recorded at 490 nm. The main steps of direct competitive ELISA measurement based on a monoclonal antibody of high specificity for GA3 and GA4 are as follows. Microtitration plates were precoated overnight at 4 8C with rabbit anti-mouse immunoglobulin. Then the wells were coated with suitable amounts of monoclonal antibodies in PBS (0.01 M, pH 7.4) at 37 8C for 70 min. Authentic hormones or samples were added 1 h before the addition of horseradish peroxidase-labeled hormones. After 2 h incubation at 25 8C, the wells were washed five times with PBS containing 0.1% Tween-20. Then the substrate for the enzyme (H2O2 and ortho-phenylene-diamine) was added, and the enzyme reaction was carried out in the dark at 37 8C for 15 min, and then terminated using 3 M H2SO4, and the absorbance was recorded at 490 nm. The need to validate immunoassays for plant hormones has been well documented (Pengelly, 1985). In this study, the
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percentage recovery of each hormone was calculated by adding known amounts of authentic hormone to split extracts. Percentage recoveries were all above 90%, and all sample extract dilution curves paralleled the standard curves, indicating the absence of nonspecific inhibitors in the extracts. Each hormone was determined four times on the same extract, and all samples were assayed four times. The standard error (S.E.) was calculated. 2.4. Statistical analyses The experiments were setup in a completely randomized design. Each replicate contained mixed sample randomly collected from 10 seedlings of each type. Four replications per plant type were used with measurement date as a block. Data means and standard errors (S.E.) were then averaged from four repeated experiments. The data were analyzed by an analysis of variance (ANOVA) using DPS (version 7.05) (Ruifeng Info Technology Ltd., Hangzhou, China), and significant differences among treatment means were calculated by Duncan’s multiple range test (P < 0.05).
Fig. 2. Activities of catalase (CAT) (A), superoxide dismutase (SOD) (B), and ascorbate peroxidase (APX) (C) during the development of bush and vine plants in Cucurbita moschata Duchesne. The internodes of bush and vine plants were selected from seedlings 20, 24, 28, 32, and 36 days after germination, respectively. The values are means of four replicates standard error (S.E.). *, Significant at P < 0.05; **, significant at P < 0.01.
3. Results 3.1. Comparisons of O2 and H2O2 levels in pumpkin bush and vine-type plants Levels of O2 and H2O2 in bush and vine-type plants were determined and compared during different development periods. The results showed that, although it was delayed at 28 DAG, a gradual increase of O2 level had been observed in vine plants. In bush plants, however, an increased O2 level was found at 28 DAG, which then decreased at the latter stage. In addition, bush plants had a significantly higher O2 level than vine plants at 28 and 36 DAG (P < 0.01 and P < 0.05) (Fig. 1A). Moreover, there was no significant difference of H2O2 levels between the two genotypes until 32
DAG, in which the difference became significant (P < 0.05) (Fig. 1B). 3.2. Comparisons of antioxidant enzymes activities in pumpkin bush and vine-type plants The results of CAT activity determination indicated that CAT activity dropped between 20 and 28 DAG and then rose to a peak at 36 DAG with both treatments. However, CAT activity was appreciably higher (2–3-fold) in bush than in vine plants during the early stages of seedling development (Fig. 2A). Both genotypes displayed an increased trend of SOD activity along with time. However, there was no significant difference observed between the two genotypes (Fig. 2B), which is the opposite of APX activity, whose decreasing trend was observed in both genotypes. In addition, significant differences of APX activity were observed between bush and vine plants (P < 0.05 and P < 0.01) (Fig. 2C). Particularly, APX activity in bush plants was significantly higher than that of vine plants at 24, 28, 32, and 36DAG. 3.3. Different levels of endogenous GA3, GA4 and IAA in pumpkin bush and vine-type plants
Fig. 1. Levels of superoxide radical (O2) (A) and hydrogen peroxide (H2O2) (B) in different growth periods of bush and vine plants in Cucurbita moschata Duchesne. The internodes of bush and vine plants were selected from seedlings 20, 24, 28, 32, and 36 days after germination, respectively. The values are means of four replicates standard error (S.E.). *, Significant at P < 0.05; **, significant at P < 0.01.
The endogenous GA3 and GA4 levels of bush and vine plants were compared in this current report. A time-course study revealed that the level of GA3 in the internodes of bush plants was lower than that in vine plants. The difference of GA3 level between vine and bush plants was relatively small (1.1-fold) in emerging seedlings (20 DAG), in which the bush phenotype was not yet apparent but became more marked with increasing plant age as the characteristic bush phenotype developed. The resulting difference in GA3 level between vine and bush plants could be up to 7.3-fold at 28 DAG (Fig. 3A). The quantification
T. Wu et al. / Scientia Horticulturae 116 (2008) 27–33
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and 36 DAG (2.7, 1.7, and 1.8-fold, respectively) (P < 0.01) (Fig. 3C). 4. Discussion
Fig. 3. Changes in endogenous GA3 (A), GA4 (B), and IAA (C) levels in the internodes of vine and bush plants in Cucurbita moschata Duchesne. The internodes of bush and vine plants were selected from seedlings 20, 24, 28, 32, and 36 days after germination, respectively. The values are means of four replicates standard error (S.E.). *, Significant at P < 0.05; **, significant at P < 0.01.
of endogenous GA3 levels in various plant organs revealed that GA3 deficiency in bush plants was widespread (except for levels in root at 20 and 24 DAG), and was most severe in internodes (Table 1; Fig. 3A). In vine plants, the GA4 level decreased from 135.8 12.2 ng g1 FW at 20 DAG to 72.8 15.2 ng g1 FW at 28 DAG, and then increased to 130.2 14.2 ng g1 FW at 36 DAG. Moreover, the GA4 level in bush plants increased from 50.9 10.1 ng g1 FW at 20 DAG to 88.0 13.6 ng g1 FW at 24 DAG, and then decreased to 55.7 8.8 ng g1 FW at 36 DAG. The GA4 level in bush-type plants was 37.5% of that in vine-type plants at 20 DAG, and this was the most apparent difference of GA4 level between bush and vine plants (Fig. 3B). The level of IAA in this report showed a different trend for the two phenotypes. A gradual decrease was observed in bush plants, before increasing at the latter sampling stages. Although delayed at 28 DAG, an increased IAA level was found along with the development of vine plants. In addition, vine plants had a significantly higher IAA level than bush plants at 24, 28,
A previous study of microscopic observation showed that the bush stature in C. moschata might be the result of cell elongation inhibition (Cao et al., 2005). It has been hypothesized that the biochemical mechanism of cell growth involving cell wall loosening can be induced by OH (Liszkay et al., 2004). As one of the precursors of OH, a high O2 level will help generate a high OH level, which can in turn promote the growth of plant cells. However, the result of this report was in conflict with the hypothesis mentioned above. The high O2 level of bush plants (Fig. 1A) indicated that the high O2 concentration might have a negative effect on the elongation of pumpkin internodes. APX is an enzyme possibly involved in the regulation of the meristematic activity in plant tissues (De Gara et al., 1996). APX as well as ascorbate oxidase (AOX), generates ascorbate free radical (AFR) that is known to stimulate cell cycle progression and to break the cell quiescence (Hidalgo et al., 1989; De Cabo et al., 1996). The high level of APX activity accompanied by increased content of ascorbate (ASC) is characteristic for the cells that are actively dividing, while during cell differentiation APX activity decreases (De Gara et al., 1996; Pinto et al., 2000). It was suggested that the role of APX in cell division and elongation might be due to its effect on the availability of H2O2 for other peroxidases and of ascorbate for prolyl-hydroxylase (De Gara et al., 1991; De Tullio et al., 1999). In addition, APX is a major consumer of ascorbic acid (AsA), generating monodehydroxyascorbate in the reduction of H2O2 to water. It is hypothesized that AsA can stimulate cell elongation via electron transport across the plasma membrane (Smirnoff, 1996). In this report, APX activity in bush plants was found to be significantly higher than that of vine plants at 24, 28, 32, and 36 DAG (Fig. 2C), suggesting that APX may have a negative effect on the elongation of pumpkin internodes. Gibberellins (GAs) are isoprenoid phytohormones required for shoot elongation in higher plants. The essential role of GAs in shoot elongation has been demonstrated clearly by the isolation of mutants deficient in GA biosynthesis in a number of
Table 1 Changes in endogenous GA3 levels of leaves and roots in vine and bush plants in Cucurbita moschata Duchesne Measurements
Tissue type (ng g1 FW) Leaf
Root
Vine 20 24 28 32 36
DAG DAG DAG DAG DAG
Bush **
3282.6 97.5 1634.6 96.8** 733.2 110.0 399.5 125.4* 174.1 120.9
1762.8 94.9 776.7 104.1 602.4 250.9 212.1 99.0 101.2 75.4
Vine
Bush **
235.0 47.5 202.8 46.8 445.4 30.0** 560.0 25.4** 395.0 50.9**
445.8 34.9 230.8 24.1 164.7 25.9 119.1 29.0 139.4 35.4
Note: The differences are presented in the same row, in the same organ between the two growth types. The values are means of four replicates standard error (S.E.). DAG, days after germination. * Significant at P < 0.05. ** Significant at P < 0.01.
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plant species, including barley, tomato, Arabidopsis, and rice (Hentrich et al., 1985; Koornneef et al., 1985; Jones, 1987; Matsukura et al., 1998). The mutants in all of these species typically have shortened internodes, resulting in a dwarf phenotype. The quantification of endogenous GA3 levels in various plant organs revealed that GA3 deficiency in bush plants was widespread (Table 1; Fig. 3A). This is parallel to the results by Cao et al. (2005), which showed that the pumpkin dwarf plant was a GA3-related mutant. In that paper, the internode length of bush plants could be elongated by the exogenous application of 0.6 mM GA3. The GA4 contents of vine plants were higher than those of bush plants during all of the sampling stages (Fig. 3B). The severe deficiency of GA4 content in the internodes of bush plants suggested that GA3 might not be the only physiologically active GA responsible for the internode elongation of bush pumpkin plants. Thus, a further application of exogenous GA4 with different levels to bush seedlings is needed to determine whether or not there is any relationship between GA4 and the internode elongation of pumpkin bush plant. In higher plants, IAA has been shown to influence a wide variety of growth processes (Lincoln et al., 1990). Evidence from physiological studies indicates that IAA affects such diverse processes as cell expansion during shoot elongation (Jacobs and Ray, 1976), and apical dominance (Phillips, 1975). Many mutants that appear to affect IAA levels cause morphological anomalies (Symons et al., 2002). In this report, vine plants had a significantly higher IAA level than bush plants at 24, 28, and 36 DAG (2.7, 1.7, and 1.8-fold respectively) (P < 0.01) (Fig. 3C). Though the high levels of IAA may promote the internode elongation of pumpkin, the exogenous application of IAA had no effect on the internode elongation of pumpkin bush plants (Cao et al., 2005). The increasing number of mutations or transgenes that affect IAA content, transport, or sensitivity, especially in Arabidopsis, provides a different approach to investigate the role of IAA in apical dominance, and in particular which stages of lateral shoot development it regulates in vivo (Stirnberg et al., 1999). Plants expressing the IAA biosynthetic genes from Agrobacterium tumefaciens have high endogenous IAA levels and increased apical dominance (Klee et al., 1987; Romano et al., 1993, 1995). The iaaL gene from Pseudomonas savastanoi, encoding an enzyme that conjugates IAA to Lys, has been transformed into tobacco, resulting in reduced levels of free IAA and reduced apical dominance (Romano et al., 1991). Although there are some studies regarding on the involvement of IAA in the control of apical dominance based on the bushy phenotype of mutant plants (Estelle and Somerville, 1987; Lincoln et al., 1990), the plant height should not be the direct result of apical dominance (Romano et al., 1991). The phenotype difference between bush and vine plant in C. moschata was compared in Wu et al. (2007), and the significant difference was the vine length. What’s more, bush plant had fewer lateral shoots than that of vine plant (data not shown). Based on this point, it is hypothesized that apical dominance should not be the reason of the bush growth habit in C. moschata. A further study with a different application method and a different level of exogenous IAA is thereby needed to determine whether or not there is any
relationship between IAA and vine elongation of tropical pumpkin plant. 5. Conclusions In conclusion, the present study has shown that two phenotypes of tropical pumpkin differing in their vine length exhibit differences in their O2 levels, APX activities and endogenous levels of GA3, GA4, and IAA. However, direct relationship of H2O2 content, CAT activity, and SOD activity with vine development in C. moschata Duchesne was not found. In addition, we may infer that high O2 concentration might have a negative effect on the elongation of pumpkin internodes. Also, the results of this report suggest that APX might play a negative effect on the elongation of pumpkin internodes. GA3 might be an important factor in the vine elongation of tropical pumpkin plants and further research should be designed to elucidate the mechanisms of GA4 and IAA associated with vine elongation of tropical pumpkin. Acknowledgement This work was supported by the Natural Science Foundation of China (no. 30270909). References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assay and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Cao, J.S., Yu, H.F., Ye, W.Z., Yu, X.L., Liu, L.C., Wang, Y.Q., Xiang, X., 2005. Identification and characterization of a gibberellin-related dwarf mutant in pumpkin (Cucurbita moschata). J. Hortic. Sci. Biotechnol. 80, 29–31. Carol, R.J., Dolan, L., 2006. The role of reactive oxygen species in cell growth: lessons from root hairs. J. Exp. Bot. 57, 1829–1834. Chen, C.M., Jin, G., Andersen, B.R., Ertl, J.R., 1993. Modulation of plant gene expression by cytokinins. Aust. J. Plant Physiol. 20, 609–619. Chen, J.G., Du, X.M., Zhao, H.Y., Zhou, X., 1996. Fluctuation in levels of endogenous plant hormones in ovules of normal and mutant cotton during flowering and their relation to fiber development. J. Plant Growth Regul. 15, 173–177. De Cabo, R.C., Gonza0 les-Reyes, J.A., Co0 rdoba, F., Navas, P., 1996. Rooting hastened by ascorbate and ascorbate free radical. J. Plant Growth Regul. 15, 53–56. De Gara, L., de Pinto, M.C., Paciolla, C., Cappetti, V., Arrigoni, O., 1996. Is ascorbate peroxidase only a scavenger of hydrogen peroxide? In: Obinger, C., Burner, U., Ebermann, R., Penel, C., Greppin H. (Eds.), Plant Peroxidases Biochemistry and Physiology IV. University of Geneva, International Symposium, pp. 157–162. De Gara, L., Tommasi, F., Liso, R., Arrigoni, O., 1991. Ascorbic acid utilisation by prolyl hydroxylase in vivo. Phytochemistry 30, 1397–1399. Denna, D.W., Munger, H.M., 1963. Morphology of the bush and vine habits and the allelism of the bush genes in Cucurbita maxima and C. pepo squash. Proc. Am. Soc. Hortic. Sci. 82, 370–377. De Tullio, M.C., Paciolla, C., Vecchia, F.D., Rascio, N., D’Emerico, S., De Gara, L., Liso, R., Arrigoni, O., 1999. Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation. Planta 209, 424–434. Estelle, M.A., Somerville, C., 1987. Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Genet. 206, 200–206.
T. Wu et al. / Scientia Horticulturae 116 (2008) 27–33 Hentrich, W., Bergner, C., Sembdner, G., 1985. Characterization of a gibberellin sensitive dwarf mutant of barley (Hordeum vulgare L., Mut. Dornburg 576). Plant Growth Regul. 3, 103–110. Hidalgo, A., Gonzales-Reyes, J.A., Navas, P., 1989. Ascorbate free radical enhances vacuolization in onion root meristems. Plant Cell Environ. 12, 455–460. Jacobs, M., Ray, P., 1976. Rapid auxin-induced decrease in free space pH and its relationship to auxin-induced growth in maize and pea. Plant Physiol. 58, 203–209. Jones, M.G., 1987. Gibberellins and the procera mutant of tomato. Planta 172, 280–284. Klee, H.J., Horsch, R.B., Hinchee, M.A., Hoffmann, N.L., 1987. The effects of overproduction of two Agrobacterium tumefaciens T-DNA auxin biosynthesis gene products in transgenic petunia plants. Genes Dev. 1, 86–96. Koornneef, M., Elgersma, A., Hanhart, C.J., van Loenen-Martinet, E.P., van Rijn, L., Zeevaart, J.A.D., 1985. A gibberellin insensitive mutant of Arabidopsis thaliana. Physiol. Plant. 65, 33–39. Lincoln, C., Britton, J.H., Estelle, M., 1990. Growth and development of the axr1 mutants of Arabidopsis. Plant Cell 2, 1071–1080. Liszkay, A., van der Zalm, E., Schopfer, P., 2004. Production of reactive oxygen intermediates (O2, H2O2, and OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiol. 136, 3114–3123. Matsukura, C., Itoh, S., Nemoto, K., Tanimoto, E., Yamaguchi, J., 1998. Promotion of leaf sheath growth by gibberellin acid in a dwarf mutant of rice. Planta 205, 145–152. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Patterson, B.D., Mackae, E.A., Ferguson, I.B., 1984. Estimation of hydrogen peroxide in plant extracts using titanium (IV). Anal. Biochem. 139, 487–492. Pengelly, W.L., 1985. Validation of immunoassays. In: Bopp, M. (Ed.), Plant Growth Substances. Springer, Berlin, pp. 35–43. Phillips, I.D., 1975. Apical dominance. Annu. Rev. Plant Physiol. 26, 341–367. Pinto, M.C., Tommasi, F., De Gara, L., 2000. Enzymes of the ascorbate biosynthesis and ascorbate–glutathione cycle in cultured cells of tobacco Bright Yellow 2. Plant Physiol. Biochem. 38, 541–550.
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Potikha, T.S., Collins, C.C., Johnson, D.I., Delmer, D.P., Levine, A., 1999. The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers. Plant Physiol. 119, 849–858. Rodrı´guez, A.A., Grunberg, K.A., Taleisnik, E.L., 2002. Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension. Plant Physiol. 129, 1627–1632. Romano, C.P., Cooper, M.L., Klee, H.J., 1993. Uncoupling auxin and ethylene effects in transgenic tobacco and Arabidopsis plants. Plant Cell 5, 181–189. Romano, C.P., Hein, M.B., Klee, H.J., 1991. Inactivation of auxin in tobacco transformed with the indoleacetic acid–lysine synthetase gene of Pseudomonas savastanoi. Genes Dev. 5, 438–446. Romano, C.P., Robson, P.R.H., Smith, H., Estelle, M., Klee, H., 1995. Transgene-mediated auxin overproduction in Arabidopsis: hypocotyls elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants. Plant Mol. Biol. 27, 1071–1083. Schopfer, P., 2001. Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth. Plant J. 28, 679–688. Shifriss, O., 1947. Developmental reversal of dominance in Cucurbita pepo. Proc. Am. Soc. Hortic. Sci. 50, 330–346. Smirnoff, N., 1996. The function and metabolism of ascorbic acid in plants. Ann. Botany 78, 661–669. Stirnberg, P., Chatfield, S.P., Ottoline Leyser, H.M., 1999. Axr1 acts after lateral bud formation to inhibit lateral bud growth in Arabidopsis. Plant Physiol. 121, 839–847. Symons, G.M., Ross, J.J., Murfet, I.C., 2002. The bushy pea mutant is IAAdeficient. Physiol. Plant. 116, 389–397. Wang, A.G., Luo, G.H., 1990. Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiol. Commun. 16, 55–57 (In Chinese). Wu, T., Zhou, J.H., Zhang, Y.F., Cao, J.S., 2007. Characterization and inheritance of a bush-type in tropical pumpkin (Cucurbita moschata Duchesne). Sci. Hortic. 114, 1–4. Yu, J.Q., Matsui, Y., 1997. Effects of root exudates of cucumber (Cucumis sativus L.) and allelochemicals on ion uptake by cucumber seedling. J. Chem. Ecol. 23, 817–827.
Scientia Horticulturae 116 (2008) 27–33 www.elsevier.com/locate/scihorti
Comparison of antioxidant activities and endogenous hormone levels between bush and vine-type tropical pumpkin (Cucurbita moschata Duchesne) Tao Wu, Jiashu Cao *, Yafeng Zhang Institute of Vegetable Science, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, China Received 6 June 2007; received in revised form 11 November 2007; accepted 14 November 2007
Abstract A bush-type plant was selected from a cultivar ‘Cga’ of tropical pumpkin (Cucurbita moschata Duchesne) in order to study and compare the bush and vine habit of C. moschata in terms of various physiological and biochemical characteristics. During the internode development of the bush and vine-type plants in C. moschata, the metabolism of reactive oxygen species (hydrogen peroxide, H2O2; and superoxide anion radical, O2), the activities of some antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; and ascorbate peroxidase, APX), and the levels of some endogenous hormones (gibberellin A3, GA3; gibberellin A4, GA4; and indole-3-acetic acid, IAA) were compared. Bush plants were found to have a significantly higher O2 level than vine plants at 28 days after germination (DAG) and onwards. Moreover, there was no significant difference of H2O2 level between the two genotypes until 32 DAG, in which the difference was significant at 32 DAG. In both genotypes, no significant differences of SOD activity were observed, and this was opposite to APX activity in which significant differences were observed during almost the whole sampling stages. What’s more, APX activity of bush plants was significantly higher than that of vine plants at 24, 28, 32, and 36 DAG. CAT activity showed the same trend for the two genotypes. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) indicated that the internodes of bush plants contain lower levels of GA3, IAA, and GA4 than those of vine plants in C. moschata. In addition, GA3 deficiency was also found in the leaves and roots of bush plants. In conclusion, this work provided some new information about different levels of endogenous chemicals in pumpkins with differing growth habits. # 2007 Elsevier B.V. All rights reserved. Keywords: Ascorbate peroxidase; Cell elongation; ELISA; Gibberellin; Internode
1. Introduction Cucurbita moschata Duchesne is one of the most important species of pumpkin in traditional agricultural systems in the world. Most cultivars of Cucurbita species are large and trailing plants. Some plants of the developed cultivars have short vines, which give them a bushy appearance (Shifriss, 1947; Denna and Munger, 1963; Cao et al., 2005). The vine elongation of plants is a very complex process consisting of complicated physiological and biochemical modifications. However, there are only a few studies focusing on the physiological and biochemical differences between bush and vine plants of pumpkin. A suggested role for reactive oxygen species (ROS), (superoxide anion radical, O2; hydrogen peroxide, H2O2;
* Corresponding author. Tel.: +86 571 86971188; fax: +86 571 86971188. E-mail address: [email protected] (J. Cao). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.11.003
and hydroxyl radicals, OH) during plant growth and morphogenesis is involvement as stress signal molecules. However, they have also been proposed to possess a decisive role on cell wall loosening and other processes required for cell elongation (Schopfer, 2001). It has been shown that H2O2 may function as a developmental signaling molecule in the differentiation of secondary walls in cotton fibers (Potikha et al., 1999). High ROS production was observed in the expansion zone of maize leaves, and a certain concentration of H2O2 was found to be necessary for leaf elongation (Rodrı´guez et al., 2002). ROS play an important role in root hair growth and may provide insight into the mechanism of plant cell growth in general (Carol and Dolan, 2006). In addition to the ROS function as messengers in plant cells, principle antioxidant enzymes such as superoxide dismutases (SOD; EC 1.15.1.1), catalases (CAT; EC 1.11.1.6), and ascorbate peroxidase (APX; EC 1.11.1.11), which help decomposing ROS, are monitored as indicators of redox homeostasis.
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The important role of endogenous hormones in plant growth and development has been studied for many years. Plant hormones can modulate gene expression and protein synthesis, and subsequently modulate the physiological and biochemical process of plants (Chen et al., 1993). The bush-type plant used in this study was selected from a cultivar ‘Cga’ of tropical pumpkin (C. moschata Duchesne). Bush plants were characterized by shorter internodes, earlier flowering, a higher ration of female to male flowers and smaller fruit than vine ‘Cga’ plants. Genetic analysis indicated monogenic inheritance with the bush genotype dominant (Wu et al., 2007). The results of Cao et al. (2005) revealed that the bush plant in C. moschata was a gibberellin-responsive mutant, and the bushy appearance was the result of inhibition of cell elongation. However, the endogenous levels of hormones in pumpkin bush plants were not investigated in that paper. Thus, the investigation of differences in endogenous hormones between bush and vine plants can reflect the internal development of pumpkin plants. To this aim, the present study was undertaken to characterize and compare ROS metabolism, activities of antioxidant enzymes (SOD, CAT, and APX), and selected endogenous hormones (GA3, GA4, and IAA) levels during different growth periods of bush and vine plants in C. moschata, and to determine the relationship between the antioxidant activities, the hormone levels in plants and the phenotype difference in C. moschata. It was hoped that this study would provide some new information about different levels of endogenous compounds in pumpkins with differing growth habits. 2. Materials and methods 2.1. Plant material The bush-type plant was selected from a cultivar ‘Cga’ of tropical pumpkin (C. moschata Duchesne). Seedlings of bush and vine plants could be distinguished at third leaf stage. Bush plants had short internodes length (4.1 0.4 cm vs. 10.2 0.6 cm), short vine length (14.5 4.9 cm vs. 175.6 28.2 cm) and small number of internode (3.5 0.7 vs. 17.2 1.6) compared with vine plants at 60 days after planting. Vine plants produced more male flowers (21.1 4.3) than bush plants (13.7 3.3). In contrast, bush plants flowered eight days earlier (40.6 2.9 days vs. 48.6 2.7 days) than vine plants. Although bush plants had a higher rate of fruit abortion than vine plants, they had a larger total number of fruit than vine plants in a plant basis (Wu et al., 2007). Seeds of bush (CgaBu) and vine (CgaV) parental plants were sown on 16th March 2006, with the seedlings grown in a mixture of 1 vermiculite:1 perlite (v/v) on the research farm of the Institute of Vegetable Science at Zhejiang University, Hangzhou, China. At the twoor three-leaf stage, seedling roots were washed gently in water, and then four plants were transplanted into a plastic pot containing 8 L of the nutrient solution of Japan Yuanshi composition (Yu and Matsui, 1997). The Japan Yuanshi composition nutrient solution comprised of the following elements: macronutrients (in mM): N, 14 [4.0 KNO3, 4.0 Ca(NO3)2, and 2.0 NH4H2PO4]; P, 2.0 (NH4H2PO4); K, 4.0
(KNO3); Ca, 4.0 [Ca(NO3)2]; and Mg, 2.0 (MgSO4); and micronutrients (in mg L1): Fe, 3.0 (Fe-EDTA); Mn, 0.5 (MnSO4); B, 0.5 (H3BO3); Zn, 0.05 (ZnSO4); Mo, 0.05 [(NH4)6Mo7O24]; and Cu, 0.02 (CuSO4). The pH of the nutrient solution was adjusted to 5.8 with 1 M NaOH and the solutions were changed weekly. Polyurethane foam and a plastic mesh supported the seedlings, which grew under controlled environmental conditions. Plants were grown under supplementary lights with an irradiance of 90 W m2 and a 12-h photoperiod. The air temperature was 22–24 8C and relative humidity was 75–90%. Samples (internodes, leaves, and roots) were randomly collected from 10 seedlings of each type at various times after germination (20, 24, 28, 32, and 36 DAG). The first internode of bush and vine plants at different development stages was collected. Experiments were carried out with the leaves of the middle (the third) story of the bush and vine-type plants at different stages of their development. Primary roots of the bush and vine plants were also collected at different stages of their development. Samples were weighed, immediately frozen in liquid nitrogen, and then were stored at 80 8C until use. 2.2. Enzyme extraction Enzyme extracts were prepared by homogenizing samples (500 mg) in a prechilled mortar and pestle nestled in ice along with 5% polyvinylpyrrolidone and 1 mL of chilled extraction buffer consisting of 50 mM potassium phosphated (pH 7.5) buffer and 1 mM EDTA. For APX activity 5 mM ascorbic acid was included in the grinding medium. The homogenate was centrifuged (ULTRA80, Sorval, Thermo Fisher Scientific, Waltham, MA, USA) at 12,000 g for 15 min at 4 8C. Supernatant was collected for subsequent protein and enzyme assays immediately following extraction. Protein concentration was determined spectrophotometrically at 595 nm using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, California; catalogue no. 500-0006) using a method described by Bradford (1976). Enzyme activities based on total protein were determined spectrophotometrically (LAMBAD-40, PerkinElmer, Boston, MA, USA) at 25 8C. 2.2.1. Measurement of SOD activity SOD activity was measured according to Beauchamp and Fridovich (1971) with some modifications. The assay mixture (1 mL) contained 100 mM sodium phosphate buffer (pH 7.8), 57 mM nitroblue tetrazolium (NBT, Wako Pure Chemical Industries, Japan), 0.025% of Triton-X-100, 0.11 mM of EDTA, 0.01 M methionine, 1.3 mM riboflavin and 50 mg of total protein. One enzyme unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT measured at 560 nm on a spectrophotometer. The specific activity of SOD was expressed as unit per mg protein. 2.2.2. Measurement of CAT activity CAT activity was assayed in a 1 mL of reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 1 mM
T. Wu et al. / Scientia Horticulturae 116 (2008) 27–33
EDTA, and 60 mM H2O2 following a protocol of Aebi (1984). The reaction was initiated by the addition of 50 mg of total protein and CAT activity was determined by following the decomposition of H2O2 at 240 nm. 2.2.3. Measurement of APX activity APX activity was determined using the method described by Nakano and Asada (1981). The assay mixture (1 mL) contained 90 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.65 mM ascorbate and 1 mM H2O2. The reaction was initiated by adding 50 mg of total protein. APX activity was determined by following the H2O2 dependent decomposition of ascorbate at 290 nm. 2.2.4. O2 detection The detection of O2 was measured according to hydroxylamine oxidation (Wang and Luo, 1990). Enzyme extracts were prepared by homogenizing samples (1.5 g) in a prechilled mortar and pestle nestled in ice along with 5% polyvinylpyrrolidone and 3 mL of chilled extraction buffer consisting of 50 mM potassium phosphated (pH 7.5) buffer and 1 mM EDTA. The homogenate was centrifuged (ULTRA80, Sorval, Thermo Fisher Scientific, Waltham, MA, USA) at 12,000 g for 15 min at 4 8C. Supernatants were collected for O2 detection. A mixture of 0.9 mL 50 mM potassium phosphate buffer (pH 7.8), 1 mL enzyme extract, and 0.1 mL 10 mM hydroxylation was incubated at 25 8C for 1 h, and then this was mixed with 1 mL 17 mM p-aminobenzene sulfonic acid and 1 mL 7 mM a-naphthylamine solution, at 25 8C for 20 min. The reaction was measured spectrophotometrically at 530 nm. The O2 level was obtained using a linear calibration curve of NaNO2. 2.2.5. H2O2 detection Internodes were placed in titanium sulfate and the total H2O2 was detected according to Patterson et al. (1984). Enzyme extracts were prepared by homogenizing samples (0.3 g) in a prechilled mortar and pestle nestled in ice along with 5 mL of chilled acetone. The homogenate was centrifuged (ULTRA80, Sorval, Thermo Fisher Scientific, Waltham, MA, USA) at 3000 g for 10 min at 4 8C. Supernatants were collected for H2O2 detection. One milliliter of acetone extraction was added through a solution consisting of 0.1 mL hydrochloric acid (containing 20% TiCl4) and 0.2 mL 17 M ammonia solution. The homogenate was centrifuged at 3000 g for 10 min at 4 8C. The precipitate was washed three times with acetone, and was centrifuged at 3000 g for 10 min at 4 8C at each time. This precipitate was dissolved in 3 mL 1 M sulfuric acid, and then was measured spectrophotometrically at 410 nm. H2O2 concentration was determined by a standard graph from 5 to 1 mM H2O2 constructed by direct addition of H2O2 to the titanium solution. 2.3. Phytohormone quantification Endogenous phytohormones were extracted from the internode, leaf, and root of pumpkin bush and vine plants at
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various DAG. They were quantified by ELISA technique as described in Chen et al. (1996) using reagents from China Agricultural University (China). The main steps of extraction and purification of plant hormones prior to immunoassay were: extraction of homogenized samples in cold 80% (v/v) aqueous methanol at a rate of 5 mL g1 fresh weight overnight at 4 8C with 1 mM butylated hydroxytoluene added to prevent oxidation. The supernatant was collected after centrifugation at 10,000 g (4 8C) for 15 min. Then, the crude extracts were passed through a C18 Sep-Pak cartridge (Waters, Milford. MA, USA). The efflux was collected and 400 mL of it was taken out and dried in N2. The residue was dissolved in 400 mL phosphate-buffered saline (PBS, 0.01 M, pH 7.5, containing 0.1% (v/v) Tween-20 and 0.1% (w/v) glutin) for determining the contents of GA3, GA4, and IAA. Another 600 mL aliquot of the filtrate was taken and dried in N2, and the residue dissolved in 200 mL PBS (0.01 M, pH 9.2) and adjusted to pH 8.5, before partitioned three times with an equal volume of ethyl acetate. The remaining aqueous phase was adjusted to pH 2.5 and extracted three times with an equal volume of ethyl acetate again. The extracts (ethyl acetate phase) were pooled and dried in N2; the residue was either redissolved in 200 mL PBS (0.01 M, pH 7.4) to analyze for GA3 and GA4, or redissolved in 200 mL 100% methanol for methylation with freshly synthesized ethereal diazomethane, and taken up with 400 mL PBS for analyses of IAA. The main steps of indirect competitive ELISA measurement using polyclonal antibodies against IAA are as follows. Microtitration plates (Nunc. Denmark) were coated with synthetic hormone–ovalbumin conjugates in NaHCO3 buffer (50 mM, pH 9.6) and left overnight at 37 8C. The solution was decanted and ovalbumin solution (10 mg mL1) was added to each well for blocking the nonspecific binding. After incubation for 30 min at 37 8C, authentic hormones or samples and antibodies were added and incubated 45 min at 37 8C. Then horseradish peroxidase-labeled goat anti-rabbit immunoglobulin was added to each well and incubated 1 h at 37 8C. Finally, the buffered enzyme substrate (H2O2 and ortho-phenylenediamine) was added, and the enzyme reaction was carried out in the dark at 37 8C for 15 min, then terminated using 3 M H2SO4, and the absorbance was recorded at 490 nm. The main steps of direct competitive ELISA measurement based on a monoclonal antibody of high specificity for GA3 and GA4 are as follows. Microtitration plates were precoated overnight at 4 8C with rabbit anti-mouse immunoglobulin. Then the wells were coated with suitable amounts of monoclonal antibodies in PBS (0.01 M, pH 7.4) at 37 8C for 70 min. Authentic hormones or samples were added 1 h before the addition of horseradish peroxidase-labeled hormones. After 2 h incubation at 25 8C, the wells were washed five times with PBS containing 0.1% Tween-20. Then the substrate for the enzyme (H2O2 and ortho-phenylene-diamine) was added, and the enzyme reaction was carried out in the dark at 37 8C for 15 min, and then terminated using 3 M H2SO4, and the absorbance was recorded at 490 nm. The need to validate immunoassays for plant hormones has been well documented (Pengelly, 1985). In this study, the
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percentage recovery of each hormone was calculated by adding known amounts of authentic hormone to split extracts. Percentage recoveries were all above 90%, and all sample extract dilution curves paralleled the standard curves, indicating the absence of nonspecific inhibitors in the extracts. Each hormone was determined four times on the same extract, and all samples were assayed four times. The standard error (S.E.) was calculated. 2.4. Statistical analyses The experiments were setup in a completely randomized design. Each replicate contained mixed sample randomly collected from 10 seedlings of each type. Four replications per plant type were used with measurement date as a block. Data means and standard errors (S.E.) were then averaged from four repeated experiments. The data were analyzed by an analysis of variance (ANOVA) using DPS (version 7.05) (Ruifeng Info Technology Ltd., Hangzhou, China), and significant differences among treatment means were calculated by Duncan’s multiple range test (P < 0.05).
Fig. 2. Activities of catalase (CAT) (A), superoxide dismutase (SOD) (B), and ascorbate peroxidase (APX) (C) during the development of bush and vine plants in Cucurbita moschata Duchesne. The internodes of bush and vine plants were selected from seedlings 20, 24, 28, 32, and 36 days after germination, respectively. The values are means of four replicates standard error (S.E.). *, Significant at P < 0.05; **, significant at P < 0.01.
3. Results 3.1. Comparisons of O2 and H2O2 levels in pumpkin bush and vine-type plants Levels of O2 and H2O2 in bush and vine-type plants were determined and compared during different development periods. The results showed that, although it was delayed at 28 DAG, a gradual increase of O2 level had been observed in vine plants. In bush plants, however, an increased O2 level was found at 28 DAG, which then decreased at the latter stage. In addition, bush plants had a significantly higher O2 level than vine plants at 28 and 36 DAG (P < 0.01 and P < 0.05) (Fig. 1A). Moreover, there was no significant difference of H2O2 levels between the two genotypes until 32
DAG, in which the difference became significant (P < 0.05) (Fig. 1B). 3.2. Comparisons of antioxidant enzymes activities in pumpkin bush and vine-type plants The results of CAT activity determination indicated that CAT activity dropped between 20 and 28 DAG and then rose to a peak at 36 DAG with both treatments. However, CAT activity was appreciably higher (2–3-fold) in bush than in vine plants during the early stages of seedling development (Fig. 2A). Both genotypes displayed an increased trend of SOD activity along with time. However, there was no significant difference observed between the two genotypes (Fig. 2B), which is the opposite of APX activity, whose decreasing trend was observed in both genotypes. In addition, significant differences of APX activity were observed between bush and vine plants (P < 0.05 and P < 0.01) (Fig. 2C). Particularly, APX activity in bush plants was significantly higher than that of vine plants at 24, 28, 32, and 36DAG. 3.3. Different levels of endogenous GA3, GA4 and IAA in pumpkin bush and vine-type plants
Fig. 1. Levels of superoxide radical (O2) (A) and hydrogen peroxide (H2O2) (B) in different growth periods of bush and vine plants in Cucurbita moschata Duchesne. The internodes of bush and vine plants were selected from seedlings 20, 24, 28, 32, and 36 days after germination, respectively. The values are means of four replicates standard error (S.E.). *, Significant at P < 0.05; **, significant at P < 0.01.
The endogenous GA3 and GA4 levels of bush and vine plants were compared in this current report. A time-course study revealed that the level of GA3 in the internodes of bush plants was lower than that in vine plants. The difference of GA3 level between vine and bush plants was relatively small (1.1-fold) in emerging seedlings (20 DAG), in which the bush phenotype was not yet apparent but became more marked with increasing plant age as the characteristic bush phenotype developed. The resulting difference in GA3 level between vine and bush plants could be up to 7.3-fold at 28 DAG (Fig. 3A). The quantification
T. Wu et al. / Scientia Horticulturae 116 (2008) 27–33
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and 36 DAG (2.7, 1.7, and 1.8-fold, respectively) (P < 0.01) (Fig. 3C). 4. Discussion
Fig. 3. Changes in endogenous GA3 (A), GA4 (B), and IAA (C) levels in the internodes of vine and bush plants in Cucurbita moschata Duchesne. The internodes of bush and vine plants were selected from seedlings 20, 24, 28, 32, and 36 days after germination, respectively. The values are means of four replicates standard error (S.E.). *, Significant at P < 0.05; **, significant at P < 0.01.
of endogenous GA3 levels in various plant organs revealed that GA3 deficiency in bush plants was widespread (except for levels in root at 20 and 24 DAG), and was most severe in internodes (Table 1; Fig. 3A). In vine plants, the GA4 level decreased from 135.8 12.2 ng g1 FW at 20 DAG to 72.8 15.2 ng g1 FW at 28 DAG, and then increased to 130.2 14.2 ng g1 FW at 36 DAG. Moreover, the GA4 level in bush plants increased from 50.9 10.1 ng g1 FW at 20 DAG to 88.0 13.6 ng g1 FW at 24 DAG, and then decreased to 55.7 8.8 ng g1 FW at 36 DAG. The GA4 level in bush-type plants was 37.5% of that in vine-type plants at 20 DAG, and this was the most apparent difference of GA4 level between bush and vine plants (Fig. 3B). The level of IAA in this report showed a different trend for the two phenotypes. A gradual decrease was observed in bush plants, before increasing at the latter sampling stages. Although delayed at 28 DAG, an increased IAA level was found along with the development of vine plants. In addition, vine plants had a significantly higher IAA level than bush plants at 24, 28,
A previous study of microscopic observation showed that the bush stature in C. moschata might be the result of cell elongation inhibition (Cao et al., 2005). It has been hypothesized that the biochemical mechanism of cell growth involving cell wall loosening can be induced by OH (Liszkay et al., 2004). As one of the precursors of OH, a high O2 level will help generate a high OH level, which can in turn promote the growth of plant cells. However, the result of this report was in conflict with the hypothesis mentioned above. The high O2 level of bush plants (Fig. 1A) indicated that the high O2 concentration might have a negative effect on the elongation of pumpkin internodes. APX is an enzyme possibly involved in the regulation of the meristematic activity in plant tissues (De Gara et al., 1996). APX as well as ascorbate oxidase (AOX), generates ascorbate free radical (AFR) that is known to stimulate cell cycle progression and to break the cell quiescence (Hidalgo et al., 1989; De Cabo et al., 1996). The high level of APX activity accompanied by increased content of ascorbate (ASC) is characteristic for the cells that are actively dividing, while during cell differentiation APX activity decreases (De Gara et al., 1996; Pinto et al., 2000). It was suggested that the role of APX in cell division and elongation might be due to its effect on the availability of H2O2 for other peroxidases and of ascorbate for prolyl-hydroxylase (De Gara et al., 1991; De Tullio et al., 1999). In addition, APX is a major consumer of ascorbic acid (AsA), generating monodehydroxyascorbate in the reduction of H2O2 to water. It is hypothesized that AsA can stimulate cell elongation via electron transport across the plasma membrane (Smirnoff, 1996). In this report, APX activity in bush plants was found to be significantly higher than that of vine plants at 24, 28, 32, and 36 DAG (Fig. 2C), suggesting that APX may have a negative effect on the elongation of pumpkin internodes. Gibberellins (GAs) are isoprenoid phytohormones required for shoot elongation in higher plants. The essential role of GAs in shoot elongation has been demonstrated clearly by the isolation of mutants deficient in GA biosynthesis in a number of
Table 1 Changes in endogenous GA3 levels of leaves and roots in vine and bush plants in Cucurbita moschata Duchesne Measurements
Tissue type (ng g1 FW) Leaf
Root
Vine 20 24 28 32 36
DAG DAG DAG DAG DAG
Bush **
3282.6 97.5 1634.6 96.8** 733.2 110.0 399.5 125.4* 174.1 120.9
1762.8 94.9 776.7 104.1 602.4 250.9 212.1 99.0 101.2 75.4
Vine
Bush **
235.0 47.5 202.8 46.8 445.4 30.0** 560.0 25.4** 395.0 50.9**
445.8 34.9 230.8 24.1 164.7 25.9 119.1 29.0 139.4 35.4
Note: The differences are presented in the same row, in the same organ between the two growth types. The values are means of four replicates standard error (S.E.). DAG, days after germination. * Significant at P < 0.05. ** Significant at P < 0.01.
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plant species, including barley, tomato, Arabidopsis, and rice (Hentrich et al., 1985; Koornneef et al., 1985; Jones, 1987; Matsukura et al., 1998). The mutants in all of these species typically have shortened internodes, resulting in a dwarf phenotype. The quantification of endogenous GA3 levels in various plant organs revealed that GA3 deficiency in bush plants was widespread (Table 1; Fig. 3A). This is parallel to the results by Cao et al. (2005), which showed that the pumpkin dwarf plant was a GA3-related mutant. In that paper, the internode length of bush plants could be elongated by the exogenous application of 0.6 mM GA3. The GA4 contents of vine plants were higher than those of bush plants during all of the sampling stages (Fig. 3B). The severe deficiency of GA4 content in the internodes of bush plants suggested that GA3 might not be the only physiologically active GA responsible for the internode elongation of bush pumpkin plants. Thus, a further application of exogenous GA4 with different levels to bush seedlings is needed to determine whether or not there is any relationship between GA4 and the internode elongation of pumpkin bush plant. In higher plants, IAA has been shown to influence a wide variety of growth processes (Lincoln et al., 1990). Evidence from physiological studies indicates that IAA affects such diverse processes as cell expansion during shoot elongation (Jacobs and Ray, 1976), and apical dominance (Phillips, 1975). Many mutants that appear to affect IAA levels cause morphological anomalies (Symons et al., 2002). In this report, vine plants had a significantly higher IAA level than bush plants at 24, 28, and 36 DAG (2.7, 1.7, and 1.8-fold respectively) (P < 0.01) (Fig. 3C). Though the high levels of IAA may promote the internode elongation of pumpkin, the exogenous application of IAA had no effect on the internode elongation of pumpkin bush plants (Cao et al., 2005). The increasing number of mutations or transgenes that affect IAA content, transport, or sensitivity, especially in Arabidopsis, provides a different approach to investigate the role of IAA in apical dominance, and in particular which stages of lateral shoot development it regulates in vivo (Stirnberg et al., 1999). Plants expressing the IAA biosynthetic genes from Agrobacterium tumefaciens have high endogenous IAA levels and increased apical dominance (Klee et al., 1987; Romano et al., 1993, 1995). The iaaL gene from Pseudomonas savastanoi, encoding an enzyme that conjugates IAA to Lys, has been transformed into tobacco, resulting in reduced levels of free IAA and reduced apical dominance (Romano et al., 1991). Although there are some studies regarding on the involvement of IAA in the control of apical dominance based on the bushy phenotype of mutant plants (Estelle and Somerville, 1987; Lincoln et al., 1990), the plant height should not be the direct result of apical dominance (Romano et al., 1991). The phenotype difference between bush and vine plant in C. moschata was compared in Wu et al. (2007), and the significant difference was the vine length. What’s more, bush plant had fewer lateral shoots than that of vine plant (data not shown). Based on this point, it is hypothesized that apical dominance should not be the reason of the bush growth habit in C. moschata. A further study with a different application method and a different level of exogenous IAA is thereby needed to determine whether or not there is any
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