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Effects of humic acid derived from sediments on the postharvest vase life extension in cut chrysanthemum flowers
Postharvest Biology and Technology 101 (2015) 82–87
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effects of humic acid derived from sediments on the postharvest vase life extension in cut chrysanthemum flowers Hong-mei Fan, Tian Li, Xia Sun, Xian-zhi Sun, Cheng-shu Zheng ∗ State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agriculture University, No. 61, Daizong Street, Tai’an 271018, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 7 July 2014 Received in revised form 14 September 2014 Accepted 14 September 2014 Keywords: Humic acid Chrysanthemum Postharvest vase life Net photosynthetic rate Antioxidant enzymes
a b s t r a c t Previous research has shown that humic acid can extend the vase life of cut flowers. However, the mechanisms responsible for this effect are unclear. In this study, the physiological mechanisms of foliar humic acid fertilizer on cut chrysanthemum flower postharvest vase life were investigated. Seedlings of chrysanthemum were sprayed with the same volume of distilled H2 O, inorganic NPK fertilizer and organic foliar humic acid fertilizer every 15 days (15, 30, 45, 60 days after transplanting). The results showed that foliar application of humic acid improved the chlorophyll content, the net photosynthetic rate (Pn ), contents of soluble sugars and soluble protein in the leaves of chrysanthemum, and increased the flower size, fresh weight, vase life, activities of antioxidant enzymes, and decreased the malondialdehyde (MDA) content in cut chrysanthemum flowers. It was concluded that the responses of the foliar humic acid fertilizer on postharvest vase life extension of cut chrysanthemum flowers could be related with the higher chlorophyll content, Pn , contents of soluble sugars and soluble protein in the leaves, the greater flower size, fresh weight, activities of antioxidant enzymes, and the lower MDA content in cut chrysanthemum flowers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chrysanthemum (Chrysanthemum morifolium R.) cut flowers and play an important role in the florist trade (Zhang et al., 2013). Since beauty is the main reason why cut flowers are sold, much effort has been made to lengthen flower lifespan (Arrom and Munné-Bosch, 2012; Asrar, 2012; Gul and Tahir, 2013). Senescence is a programmed process that does not occur in all floral organs at the same time. According to their specific biological function, petals are the first tissues showing signs of senescence (Arrom and Munné-Bosch, 2012). Currently, postharvest senescence is a major limitation to the marketing of cut flowers, as shown by petal in-rolling and discoloration due to reactive oxygen species (ROS) after harvest (Trippi and Paulin, 1984). Considerable effort is needed therefore to develop postharvest handling to suppress ROS and extend vase life of cut flowers. Many studies have been conducted to extend flower longevity with scientific and technological
∗ Corresponding author. Tel.: +86 13695386893. E-mail addresses: [email protected] (H.-m. Fan), [email protected], [email protected], [email protected] (C.-s. Zheng). http://dx.doi.org/10.1016/j.postharvbio.2014.09.019 0925-5214/© 2014 Elsevier B.V. All rights reserved.
advances (Halevy and Mayak, 1979, 1981; Van Doorn, 1997; Scariot et al., 2014). Humic acid (HA) is the fraction of naturally occurring organic materials commonly found in soils, sediments and natural waters, which derive from the decomposition of plant and animal residues (Bandiera et al., 2009). Some authors have proposed that humic acid promotes photosynthesis, respiration (Heil, 2005) and chlorophyll content (Xu et al., 2012), thus improving plant carbohydrate contents, which will directly influence the quality and life of flowers. Cordeiro et al. (2011) have also reported that humic acid has effects on antioxidative defense mechanisms, reporting the stimulation of catalases (CAT) and generation of ROS. Garcíaa et al. (2012) reported that humic acid could play a major role in resisting oxidative stress by enhancing antioxidative activity and improving membrane stability. These facts suggest that humic acid could be beneficial for postharvest quality, if applied preharvest to the chrysanthemum plant. Therefore, the present study was conducted to investigate the mechanisms by which preharvest applied humic acid could influence postharvest quality and vase life from the viewpoint of chlorophyll content, photosynthesis, contents of soluble sugars and soluble protein in the leaves, and the flower size, fresh weight,
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antioxidative capacity and MDA content in cut chrysanthemum flowers.
set as follows: PAR = 1000 mol m−2 s−1 , leaf temperature = 25 ◦ C, CO2 = 450 ppm.
2. Materials and methods
2.5. Measurements of the contents of soluble sugars and soluble protein in the leaves of chrysanthemum
2.1. Humic acid extraction Compost derived humic acid was obtained in a valley filled with sediments of plant and animal residues in China (Hohhot, Inner Mongolia). The compost was extracted using an alkali/acid fractionation procedure (Valdrighi et al., 1996). The compost was digested in 0.1 N KOH (1:10 w/v) for 24 h at 25 ◦ C. The undigested bulk residue was then separated from the solute fraction by centrifugation at 8000 rpm for 20 min followed by filtration through a glass wool layer. The filtered supernatant was then acidified at pH 2.0 with 6.0 N H2 SO4 and maintained in the dark at 25 ◦ C for 24 h in order to obtain flocculation of humic acid. Finally, the humic acid was collected by centrifuging at 8000 rpm for 20 min, and resuspended in 0.1 N KOH. 2.2. Growth conditions of plants, humic acid application The experiment was conducted in a greenhouse at Horticultural Station of Shandong Agricultural University, located in Tai’an, Shandong (35◦ 38 N, 116◦ 02 E). Cutting seedlings of chrysanthemum (C. morifolium cv. Jinba) of similar height and diameter were cultivated on July 17th, 2013. Rooted seedlings were transplanted into plots with 150 plants per plot (10 m2 ) on August 1st, 2013 with a relative humidity (RH) 65–75%, temperatures 18–25 ◦ C, and the mean daily photosynthetically active radiation (PAR) 1000 Mol m−2 day−1 . On August 16th (15 days after transplanting), the seedlings were sprayed with the same volume of distilled H2 O (Control), inorganic NPK fertilizer (N:P2 O5 :K2 O = 16:6:20) at 0.3% (w/v) concentration and foliar humic acid (FHA) fertilizer at 1:600 (v/v) diluted concentration respectively every 15 days (15, 30, 45, 60 days after transplanting). Each treatment was replicated three times in a randomized complete block design. The concentration of the FHA used was determined from a preliminary experiment. The concentration of the NPK fertilizer was determined on the total content of NPK in the 1:600 (v/v) diluted FHA fertilizer at the optimum concentration range used in the production to ensure that any differences in flower life and quality responses were humic acidmediated. 2.3. Measurements of chlorophyll content in the leaves of chrysanthemum The 4th–5th fully expanded leaves from the top of the chrysanthemum were selected at 0, 15, 30, 45, 60 DAS (days after treatment). The leaves were soaked in 80% acetone for 12–24 h, and then centrifuged at 5000 rpm for 10 min. The supernatant was collected. Then the absorbance of the supernatant was read at 645 and 663 nm, respectively. The content of chlorophyll was calculated according to the equation: 20.2A645 + 8.02A663 (Lichtenthaler and Lester Packer, 1987). 2.4. Measurements of the Pn in the leaves of chrysanthemum The net photosynthetic rate (Pn ) was measured from 9:00 a.m. to 11:00 a.m. at 0, 15, 30, 45, 60 DAS on the 4th–5th fully expanded leaves from the top of the chrysanthemum, using a CIRAS-2 infrared gas analyzer (PP-System, Hitchin, UK) with a Parkinson’s Automatic Universal Leaf Cuvette equipped with 2.5-cm2 area cuvette inserts. Environmental conditions inside the cuvette were
The leaves of chrysanthemum were selected at 0, 15, 30, 45, 60 DAS for measurement. Soluble sugar and soluble protein measurements were followed the procedures that were described by Frohlich and Kutscherah (1995). Soluble sugar was extracted with anthrone. Samples (0.30 g) of fresh leaves were put into test tubes with 10 mL distilled water and sealed. The tubes were incubated in a water bath at 90 ◦ C for 30 min, then the tubes were removed and the volume set at 25 mL. 0.5 mL supernatant was collected and mixed with 1.5 mL distilled water, 0.5 mL anthrone and 5 mL concentrated sulfuric acid. The mixed solution was read at 620 nm for soluble sugars measurement. Soluble protein was extracted with coomassie brilliant blue. Samples (0.50 g) of fresh leaves were ground in a mortar with 5 mL phosphate buffer solution and then transferred into centrifuge tubes. The solutions were centrifuged at 5000 rpm for 15 min and the supernatant extracted. 1 mL supernatant was mixed with 5 mL coomassie brilliant blue and then read at 595 nm for soluble protein measurement. 2.6. Vase life test Flowers were collected at the pre-opening stage (October 28th) with a similar maturity, then re-cut in water to about 75 cm stem length, and finally inserted into vases with same volume of distilled water and renewed every day throughout the holding time. There were three flowers per flask and 20 flasks per treatment. Throughout the vase period, the flowers were held in a room at 22 ± 1 ◦ C and 65 ± 3% of relative humidity (RH) and an 8 h light period per day under irradiance of 13 W m−2 above the flowers using fluorescent tubes. 2.7. Measurements of the flower size, fresh weight and vase life Flower size was defined as the maximum width of each flower and measured with a ruler every two days. Fresh weight of the capitulum was measured every two days throughout the vase life period. The average vase life was assessed to be terminated when 80% flowers had senesced, which was characterized by loss of turgor followed by petal wilting. 2.8. Measurements of the antioxidant enzymes activities in the cut chrysanthemum flowers Petals were selected from each treatment at 0, 2, 4, 6, 8 days of inserting for measurement. 0.5 g of flower tissue was suspended in 5 mL of ice-cold HEPES buffer (25 mM, pH 7.8) containing 0.5 mM EDTA and 2% PVP. The homogenate was centrifuged at 4 ◦ C and 5000 × g for 15 min and the resulting supernatants were used for the determination of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) (Ramiro et al., 2006). The determination of SOD activity was performed at 560 nm following Hwang et al. (1999). One unit of SOD activity was defined as the amount of enzyme that causes a 50% inhibition of the rate of nitroblue tetrazolium reduction. POD activity was determined at 470 nm by measuring peroxidation of hydrogen peroxide with guaiacol as an electron donor (Chance and Maehly, 1955). CAT activity was assayed at 240 nm by measuring the conversion rate of hydrogen peroxide to water and oxygen molecules (Beers and Sizer, 1952).
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Fig. 1. Effects of different fertilizers on the chlorophyll content in the leaves of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
Fig. 2. Effects of different fertilizers on the net photosynthetic rate (Pn ) in the leaves of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
2.9. Measurements of malonaldehyde (MDA) content in the cut chrysanthemum flowers
3.3. Effects of FHA on the contents of soluble sugar and soluble protein in the leaves of chrysanthemum
Petals were selected from each treatment at 0, 2, 4, 6, 8 days for MDA content measurement. MDA was extracted with 10% trichloroacetic acid and assayed at 450, 532 and 600 nm following the procedures that were described by Dhindsa et al. (1981) and modified by Xu et al. (2008).
The contents of soluble sugar (Fig. 3A) and soluble protein (Fig. 3B) increased with the increasing time of treatment in the leaves. The contents of soluble sugar and soluble protein were higher in the FHA treatment than those in other treatments at all days. At 60 DAS, the contents of soluble sugars were 65.7% and 26.1%, and soluble protein 62.2% and 29.5%, higher respectively upon application of FHA fertilizer than those of the control and the NPK fertilizer. The results indicated that FHA fertilizer improved the contents of soluble sugars and soluble protein in the leaves of chrysanthemum.
2.10. Statistical analysis Data were expressed as means ± standard errors. Differences were tested with one-way ANOVA and the least significant difference (LSD) using SPSS 17.0. P-values of <0.05 were considered to be significant.
3. Results 3.1. Effects of FHA on chlorophyll content in the leaves of chrysanthemum The content of chlorophyll is illustrated in Fig. 1. Among the three treatments, the content of chlorophyll increased within the time of treatment and there was no significant difference before 30 DAS. However, the content of chlorophyll was 43.6% and 23.8% higher upon application of FHA fertilizer than that of the control and the NPK fertilizer at 60 DAS and significant differences (P < 0.05) were observed (Fig. 1). The results indicated that FHA fertilizer significantly improved the chlorophyll content in the leaves of chrysanthemum.
3.4. Effects of FHA on the flower size, fresh weight and vase life The flower size (Fig. 4A) and fresh weight (Fig. 4B) of the cut chrysanthemum flowers in the FHA treatment were the greatest among the three treatments throughout the holding time. The flower size increased within the first 4 days and reached the maximum diameter of 11.8 cm in the FHA treatment, 10 cm in the control treatment and 10.5 cm in the NPK treatment respectively at 4 days in the vase, then decreased slowly. However, FHA delayed the decrease of flower size during the late vase stage. The fresh weight increased within the first 4 days and then decreased gradually. FHA delayed the decrease of fresh weight during the late vase stage. The vase life in the cut flowers is shown in Table 1. The vase life was 61.2% and 33.0% greater than that of the control and NPK treatments, and significant difference was observed (P < 0.05). The result showed that the vase life of cut chrysanthemum flowers was extended by being sprayed with FHA fertilizer.
3.2. Effects of FHA on the Pn in the leaves of chrysanthemum The net photosynthesis rate (Pn ) improved significantly in the leaves of chrysanthemum sprayed with FHA fertilizer (Fig. 2). The Pn was not significantly different among the three treatments before 30 DAS, yet it was 49.8% and 27.6% higher respectively in the leaves of chrysanthemum sprayed with FHA fertilizer than that of the control and the NPK fertilizer at 60 DAS, and significant differences were observed (P < 0.05). It was found that the Pn was increased obviously upon application of FHA fertilizer.
Table 1 Effects of different fertilizers on vase life in the cut chrysanthemum flowers. Treatments
Vase life (d)
Control NPK FHA
8.5 ± 0.3c 10.3 ± 0.1b 13.7 ± 0.2a
Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. The results are the mean ± SE (n = 20). Different letters indicate significant differences between treatments at 0.05 level.
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Fig. 3. Effects of different fertilizers on the contents of soluble sugar (A) and soluble protein and (B) in the leaves of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
3.5. Effects of FHA on antioxidant enzyme activities in the cut chrysanthemum flowers The antioxidant enzymes activities in chrysanthemum flowers in the vase are shown in Fig. 5. The SOD (Fig. 5A), POD (Fig. 5B), and CAT (Fig. 5C) activities increased within the first 4 days and no significant difference was observed among the three treatments, with decreases afterwards. The SOD activity in the FHA treatment was 116.7% and 23.8%, POD 25.2% and 22.9%, and CAT 40.7% and 23.6% respectively higher at 8 days than that of the control and NPK treatments, and significant differences were observed (P < 0.05). The results indicated that FHA fertilizer increased the SOD, POD, and CAT activities in the chrysanthemum flowers. 3.6. Effects of FHA on MDA content in the cut chrysanthemum flowers The MDA content in the vase flowers are shown in Fig. 6. The MDA content in the three treatments increased after 2 days of inserting. However, it was much lower in FHA treatment in comparison with that of the control and NPK treatments. The results indicate that FHA fertilizer decreased the MDA content in the chrysanthemum flowers. 4. Discussion Plant carbohydrate contents such as the contents of soluble sugars and soluble protein directly influence the quality and lifespan of a flower. Flower maturation and senescence has been shown to be
accompanied by a decline in total carbohydrate content (Lay-yee et al., 1992; Gulzar et al., 2005). Postharvest life of cut flowers has been shown to be dependent on the carbohydrate status (Emongor, 2004; Kazemi et al., 2010). The high carbohydrate contents are based on high photosynthesis rates resulting from high contents of chlorophyll. In present study, the chlorophyll content (Fig. 1), the Pn (Fig. 2) and contents of soluble sugars and soluble protein (Fig. 3) in the leaves of chrysanthemum increased significantly after being sprayed with FHA fertilizer compared with those of the control and NPK fertilizer. The result indicated that FHA fertilizer can improve the chlorophyll content and photosynthesis, thus improve the stimulation of carbohydrate content, which will directly influence the flower quality and vase life of cut chrysanthemum flowers. Enhancement of vase life of cut flowers bears a positive relation with the stimulation of carbohydrate content in the leaves and the delay in loss of flower size and fresh weight (Zuliana et al., 2008; Chutichudet et al., 2011; Gul et al., 2012). In the present study, we observed that flower size and fresh weight (Fig. 4A and B) were higher in FHA treatment than those of the control and NPK treatment at all vase life days. The decrease of flower size and fresh weight at the late holding time might be due to nutrient absorption decreasing gradually when the cut flowers were isolated from the whole plants for a long period, and the nutrients could not meet plant consumption. However, FHA treatment delayed the decreases in flower size and fresh weight, which indicated that FHA fertilizer can increase flower size and fresh weight and delay the loss in flower size and fresh weight in cut chrysanthemum flowers due to higher contents of soluble sugar and soluble protein in the leaves,
Fig. 4. Effects of different fertilizers on flower size (A), fresh weight (B) in the cut chrysanthemum flowers during vase life. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
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Fig. 6. Effects of different fertilizers on MDA content in the cut flowers of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
Fig. 5. Effects of different fertilizers on SOD activity (A), POD activity (B), CAT activity (C) in the cut flowers of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
and thereby substantially extended the vase life of cut chrysanthemum flowers. Aging of petals is accompanied by morphological, biochemical and biophysical deterioration (Bartoli et al., 1996). Upon senescence, membrane permeability increases rapidly, which is combined with an increase in lipid peroxidation (Trippi and Paulin, 1984). Bartoli et al. (1995) reported that both lipid peroxidation and membrane permeability increased in chrysanthemum petals during aging. This information is consistent with the hypothesis that disruption of cellular membrane integrity is an inherent feature of senescence in petals (Bartoli et al., 1997). Free radicals have been implicated in programmed cell death (Bailly et al., 2001). Lipid
peroxidation involves a series of free radicals in a chain mechanism due to producing ROS such as O2 •− and H2 O2 (Garcíaa et al., 2012). The production of ROS might increase in plants, eventually inducing the oxidation of certain cellular components when the ROS level is high. Activities of SOD, POD and CAT represent antioxidant defense systems that can reduce lipid peroxidation and protect plants from ROS damage (Panavas and Rubinstein, 1998; Singh et al., 2012). In the present study, activities of SOD, POD and CAT increased within the first 4 days. The possible reasons for the increases are that the growth environment changed suddenly when the flowers were cut from the whole plants, inducing the production of ROS. Thus the activities of SOD, POD and CAT were stimulated to protect the flowers from ROS damage. The decrease of the activities of SOD, POD and CAT at late vase time (Fig. 5A–C) might be due to due to accumulation of ROS which in turn inhibited the ability of the antioxidant defense system, thus the activities of SOD, POD, CAT decreased. However, FHA treatment could improve the activities of SOD, POD and CAT to delay the accumulation of O2 •− and H2 O2 during the later vase stage of cut chrysanthemum flowers. Different results were reported by Bartoli et al. (1995) where SOD, POD and CAT activity increased at the beginning of petal senescence in chrysanthemum. The possible reasons for the different results may be the different chrysanthemum cultivars with different mechanisms of senescence or different test environments. MDA is an important index that reflects the extent of damage in plants faced with stress or senescence. The MDA content increased within senescence accompanied with a rapid increase in membrane permeability (Gao et al., 2010). In the present study, the MDA content in the three treatments increased at the late vase stage. FHA treatment reduced the increase in MDA content (Fig. 6), which indicated lower tissue damage and slower senescence in the FHA-treated chrysanthemum flowers. 5. Conclusion Foliar application of humic acid increased the chlorophyll content, photosynthesis and contents of soluble sugar and soluble protein in the leaves of chrysanthemum, and thus improved the flower size, fresh weight and delayed the loss in flower size, fresh weight, and maintained a longer period of vase time in cut chrysanthemum flowers. Foliar humic acid also enhanced activities of antioxidant enzymes, reduced MDA content, thus delayed flower senescence and extended the vase life of cut chrysanthemum flowers.
H.-m. Fan et al. / Postharvest Biology and Technology 101 (2015) 82–87
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Effects of humic acid derived from sediments on the postharvest vase life extension in cut chrysanthemum flowers Hong-mei Fan, Tian Li, Xia Sun, Xian-zhi Sun, Cheng-shu Zheng ∗ State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agriculture University, No. 61, Daizong Street, Tai’an 271018, Shandong, PR China
a r t i c l e
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Article history: Received 7 July 2014 Received in revised form 14 September 2014 Accepted 14 September 2014 Keywords: Humic acid Chrysanthemum Postharvest vase life Net photosynthetic rate Antioxidant enzymes
a b s t r a c t Previous research has shown that humic acid can extend the vase life of cut flowers. However, the mechanisms responsible for this effect are unclear. In this study, the physiological mechanisms of foliar humic acid fertilizer on cut chrysanthemum flower postharvest vase life were investigated. Seedlings of chrysanthemum were sprayed with the same volume of distilled H2 O, inorganic NPK fertilizer and organic foliar humic acid fertilizer every 15 days (15, 30, 45, 60 days after transplanting). The results showed that foliar application of humic acid improved the chlorophyll content, the net photosynthetic rate (Pn ), contents of soluble sugars and soluble protein in the leaves of chrysanthemum, and increased the flower size, fresh weight, vase life, activities of antioxidant enzymes, and decreased the malondialdehyde (MDA) content in cut chrysanthemum flowers. It was concluded that the responses of the foliar humic acid fertilizer on postharvest vase life extension of cut chrysanthemum flowers could be related with the higher chlorophyll content, Pn , contents of soluble sugars and soluble protein in the leaves, the greater flower size, fresh weight, activities of antioxidant enzymes, and the lower MDA content in cut chrysanthemum flowers. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chrysanthemum (Chrysanthemum morifolium R.) cut flowers and play an important role in the florist trade (Zhang et al., 2013). Since beauty is the main reason why cut flowers are sold, much effort has been made to lengthen flower lifespan (Arrom and Munné-Bosch, 2012; Asrar, 2012; Gul and Tahir, 2013). Senescence is a programmed process that does not occur in all floral organs at the same time. According to their specific biological function, petals are the first tissues showing signs of senescence (Arrom and Munné-Bosch, 2012). Currently, postharvest senescence is a major limitation to the marketing of cut flowers, as shown by petal in-rolling and discoloration due to reactive oxygen species (ROS) after harvest (Trippi and Paulin, 1984). Considerable effort is needed therefore to develop postharvest handling to suppress ROS and extend vase life of cut flowers. Many studies have been conducted to extend flower longevity with scientific and technological
∗ Corresponding author. Tel.: +86 13695386893. E-mail addresses: [email protected] (H.-m. Fan), [email protected], [email protected], [email protected] (C.-s. Zheng). http://dx.doi.org/10.1016/j.postharvbio.2014.09.019 0925-5214/© 2014 Elsevier B.V. All rights reserved.
advances (Halevy and Mayak, 1979, 1981; Van Doorn, 1997; Scariot et al., 2014). Humic acid (HA) is the fraction of naturally occurring organic materials commonly found in soils, sediments and natural waters, which derive from the decomposition of plant and animal residues (Bandiera et al., 2009). Some authors have proposed that humic acid promotes photosynthesis, respiration (Heil, 2005) and chlorophyll content (Xu et al., 2012), thus improving plant carbohydrate contents, which will directly influence the quality and life of flowers. Cordeiro et al. (2011) have also reported that humic acid has effects on antioxidative defense mechanisms, reporting the stimulation of catalases (CAT) and generation of ROS. Garcíaa et al. (2012) reported that humic acid could play a major role in resisting oxidative stress by enhancing antioxidative activity and improving membrane stability. These facts suggest that humic acid could be beneficial for postharvest quality, if applied preharvest to the chrysanthemum plant. Therefore, the present study was conducted to investigate the mechanisms by which preharvest applied humic acid could influence postharvest quality and vase life from the viewpoint of chlorophyll content, photosynthesis, contents of soluble sugars and soluble protein in the leaves, and the flower size, fresh weight,
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antioxidative capacity and MDA content in cut chrysanthemum flowers.
set as follows: PAR = 1000 mol m−2 s−1 , leaf temperature = 25 ◦ C, CO2 = 450 ppm.
2. Materials and methods
2.5. Measurements of the contents of soluble sugars and soluble protein in the leaves of chrysanthemum
2.1. Humic acid extraction Compost derived humic acid was obtained in a valley filled with sediments of plant and animal residues in China (Hohhot, Inner Mongolia). The compost was extracted using an alkali/acid fractionation procedure (Valdrighi et al., 1996). The compost was digested in 0.1 N KOH (1:10 w/v) for 24 h at 25 ◦ C. The undigested bulk residue was then separated from the solute fraction by centrifugation at 8000 rpm for 20 min followed by filtration through a glass wool layer. The filtered supernatant was then acidified at pH 2.0 with 6.0 N H2 SO4 and maintained in the dark at 25 ◦ C for 24 h in order to obtain flocculation of humic acid. Finally, the humic acid was collected by centrifuging at 8000 rpm for 20 min, and resuspended in 0.1 N KOH. 2.2. Growth conditions of plants, humic acid application The experiment was conducted in a greenhouse at Horticultural Station of Shandong Agricultural University, located in Tai’an, Shandong (35◦ 38 N, 116◦ 02 E). Cutting seedlings of chrysanthemum (C. morifolium cv. Jinba) of similar height and diameter were cultivated on July 17th, 2013. Rooted seedlings were transplanted into plots with 150 plants per plot (10 m2 ) on August 1st, 2013 with a relative humidity (RH) 65–75%, temperatures 18–25 ◦ C, and the mean daily photosynthetically active radiation (PAR) 1000 Mol m−2 day−1 . On August 16th (15 days after transplanting), the seedlings were sprayed with the same volume of distilled H2 O (Control), inorganic NPK fertilizer (N:P2 O5 :K2 O = 16:6:20) at 0.3% (w/v) concentration and foliar humic acid (FHA) fertilizer at 1:600 (v/v) diluted concentration respectively every 15 days (15, 30, 45, 60 days after transplanting). Each treatment was replicated three times in a randomized complete block design. The concentration of the FHA used was determined from a preliminary experiment. The concentration of the NPK fertilizer was determined on the total content of NPK in the 1:600 (v/v) diluted FHA fertilizer at the optimum concentration range used in the production to ensure that any differences in flower life and quality responses were humic acidmediated. 2.3. Measurements of chlorophyll content in the leaves of chrysanthemum The 4th–5th fully expanded leaves from the top of the chrysanthemum were selected at 0, 15, 30, 45, 60 DAS (days after treatment). The leaves were soaked in 80% acetone for 12–24 h, and then centrifuged at 5000 rpm for 10 min. The supernatant was collected. Then the absorbance of the supernatant was read at 645 and 663 nm, respectively. The content of chlorophyll was calculated according to the equation: 20.2A645 + 8.02A663 (Lichtenthaler and Lester Packer, 1987). 2.4. Measurements of the Pn in the leaves of chrysanthemum The net photosynthetic rate (Pn ) was measured from 9:00 a.m. to 11:00 a.m. at 0, 15, 30, 45, 60 DAS on the 4th–5th fully expanded leaves from the top of the chrysanthemum, using a CIRAS-2 infrared gas analyzer (PP-System, Hitchin, UK) with a Parkinson’s Automatic Universal Leaf Cuvette equipped with 2.5-cm2 area cuvette inserts. Environmental conditions inside the cuvette were
The leaves of chrysanthemum were selected at 0, 15, 30, 45, 60 DAS for measurement. Soluble sugar and soluble protein measurements were followed the procedures that were described by Frohlich and Kutscherah (1995). Soluble sugar was extracted with anthrone. Samples (0.30 g) of fresh leaves were put into test tubes with 10 mL distilled water and sealed. The tubes were incubated in a water bath at 90 ◦ C for 30 min, then the tubes were removed and the volume set at 25 mL. 0.5 mL supernatant was collected and mixed with 1.5 mL distilled water, 0.5 mL anthrone and 5 mL concentrated sulfuric acid. The mixed solution was read at 620 nm for soluble sugars measurement. Soluble protein was extracted with coomassie brilliant blue. Samples (0.50 g) of fresh leaves were ground in a mortar with 5 mL phosphate buffer solution and then transferred into centrifuge tubes. The solutions were centrifuged at 5000 rpm for 15 min and the supernatant extracted. 1 mL supernatant was mixed with 5 mL coomassie brilliant blue and then read at 595 nm for soluble protein measurement. 2.6. Vase life test Flowers were collected at the pre-opening stage (October 28th) with a similar maturity, then re-cut in water to about 75 cm stem length, and finally inserted into vases with same volume of distilled water and renewed every day throughout the holding time. There were three flowers per flask and 20 flasks per treatment. Throughout the vase period, the flowers were held in a room at 22 ± 1 ◦ C and 65 ± 3% of relative humidity (RH) and an 8 h light period per day under irradiance of 13 W m−2 above the flowers using fluorescent tubes. 2.7. Measurements of the flower size, fresh weight and vase life Flower size was defined as the maximum width of each flower and measured with a ruler every two days. Fresh weight of the capitulum was measured every two days throughout the vase life period. The average vase life was assessed to be terminated when 80% flowers had senesced, which was characterized by loss of turgor followed by petal wilting. 2.8. Measurements of the antioxidant enzymes activities in the cut chrysanthemum flowers Petals were selected from each treatment at 0, 2, 4, 6, 8 days of inserting for measurement. 0.5 g of flower tissue was suspended in 5 mL of ice-cold HEPES buffer (25 mM, pH 7.8) containing 0.5 mM EDTA and 2% PVP. The homogenate was centrifuged at 4 ◦ C and 5000 × g for 15 min and the resulting supernatants were used for the determination of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) (Ramiro et al., 2006). The determination of SOD activity was performed at 560 nm following Hwang et al. (1999). One unit of SOD activity was defined as the amount of enzyme that causes a 50% inhibition of the rate of nitroblue tetrazolium reduction. POD activity was determined at 470 nm by measuring peroxidation of hydrogen peroxide with guaiacol as an electron donor (Chance and Maehly, 1955). CAT activity was assayed at 240 nm by measuring the conversion rate of hydrogen peroxide to water and oxygen molecules (Beers and Sizer, 1952).
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Fig. 1. Effects of different fertilizers on the chlorophyll content in the leaves of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
Fig. 2. Effects of different fertilizers on the net photosynthetic rate (Pn ) in the leaves of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
2.9. Measurements of malonaldehyde (MDA) content in the cut chrysanthemum flowers
3.3. Effects of FHA on the contents of soluble sugar and soluble protein in the leaves of chrysanthemum
Petals were selected from each treatment at 0, 2, 4, 6, 8 days for MDA content measurement. MDA was extracted with 10% trichloroacetic acid and assayed at 450, 532 and 600 nm following the procedures that were described by Dhindsa et al. (1981) and modified by Xu et al. (2008).
The contents of soluble sugar (Fig. 3A) and soluble protein (Fig. 3B) increased with the increasing time of treatment in the leaves. The contents of soluble sugar and soluble protein were higher in the FHA treatment than those in other treatments at all days. At 60 DAS, the contents of soluble sugars were 65.7% and 26.1%, and soluble protein 62.2% and 29.5%, higher respectively upon application of FHA fertilizer than those of the control and the NPK fertilizer. The results indicated that FHA fertilizer improved the contents of soluble sugars and soluble protein in the leaves of chrysanthemum.
2.10. Statistical analysis Data were expressed as means ± standard errors. Differences were tested with one-way ANOVA and the least significant difference (LSD) using SPSS 17.0. P-values of <0.05 were considered to be significant.
3. Results 3.1. Effects of FHA on chlorophyll content in the leaves of chrysanthemum The content of chlorophyll is illustrated in Fig. 1. Among the three treatments, the content of chlorophyll increased within the time of treatment and there was no significant difference before 30 DAS. However, the content of chlorophyll was 43.6% and 23.8% higher upon application of FHA fertilizer than that of the control and the NPK fertilizer at 60 DAS and significant differences (P < 0.05) were observed (Fig. 1). The results indicated that FHA fertilizer significantly improved the chlorophyll content in the leaves of chrysanthemum.
3.4. Effects of FHA on the flower size, fresh weight and vase life The flower size (Fig. 4A) and fresh weight (Fig. 4B) of the cut chrysanthemum flowers in the FHA treatment were the greatest among the three treatments throughout the holding time. The flower size increased within the first 4 days and reached the maximum diameter of 11.8 cm in the FHA treatment, 10 cm in the control treatment and 10.5 cm in the NPK treatment respectively at 4 days in the vase, then decreased slowly. However, FHA delayed the decrease of flower size during the late vase stage. The fresh weight increased within the first 4 days and then decreased gradually. FHA delayed the decrease of fresh weight during the late vase stage. The vase life in the cut flowers is shown in Table 1. The vase life was 61.2% and 33.0% greater than that of the control and NPK treatments, and significant difference was observed (P < 0.05). The result showed that the vase life of cut chrysanthemum flowers was extended by being sprayed with FHA fertilizer.
3.2. Effects of FHA on the Pn in the leaves of chrysanthemum The net photosynthesis rate (Pn ) improved significantly in the leaves of chrysanthemum sprayed with FHA fertilizer (Fig. 2). The Pn was not significantly different among the three treatments before 30 DAS, yet it was 49.8% and 27.6% higher respectively in the leaves of chrysanthemum sprayed with FHA fertilizer than that of the control and the NPK fertilizer at 60 DAS, and significant differences were observed (P < 0.05). It was found that the Pn was increased obviously upon application of FHA fertilizer.
Table 1 Effects of different fertilizers on vase life in the cut chrysanthemum flowers. Treatments
Vase life (d)
Control NPK FHA
8.5 ± 0.3c 10.3 ± 0.1b 13.7 ± 0.2a
Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. The results are the mean ± SE (n = 20). Different letters indicate significant differences between treatments at 0.05 level.
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Fig. 3. Effects of different fertilizers on the contents of soluble sugar (A) and soluble protein and (B) in the leaves of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
3.5. Effects of FHA on antioxidant enzyme activities in the cut chrysanthemum flowers The antioxidant enzymes activities in chrysanthemum flowers in the vase are shown in Fig. 5. The SOD (Fig. 5A), POD (Fig. 5B), and CAT (Fig. 5C) activities increased within the first 4 days and no significant difference was observed among the three treatments, with decreases afterwards. The SOD activity in the FHA treatment was 116.7% and 23.8%, POD 25.2% and 22.9%, and CAT 40.7% and 23.6% respectively higher at 8 days than that of the control and NPK treatments, and significant differences were observed (P < 0.05). The results indicated that FHA fertilizer increased the SOD, POD, and CAT activities in the chrysanthemum flowers. 3.6. Effects of FHA on MDA content in the cut chrysanthemum flowers The MDA content in the vase flowers are shown in Fig. 6. The MDA content in the three treatments increased after 2 days of inserting. However, it was much lower in FHA treatment in comparison with that of the control and NPK treatments. The results indicate that FHA fertilizer decreased the MDA content in the chrysanthemum flowers. 4. Discussion Plant carbohydrate contents such as the contents of soluble sugars and soluble protein directly influence the quality and lifespan of a flower. Flower maturation and senescence has been shown to be
accompanied by a decline in total carbohydrate content (Lay-yee et al., 1992; Gulzar et al., 2005). Postharvest life of cut flowers has been shown to be dependent on the carbohydrate status (Emongor, 2004; Kazemi et al., 2010). The high carbohydrate contents are based on high photosynthesis rates resulting from high contents of chlorophyll. In present study, the chlorophyll content (Fig. 1), the Pn (Fig. 2) and contents of soluble sugars and soluble protein (Fig. 3) in the leaves of chrysanthemum increased significantly after being sprayed with FHA fertilizer compared with those of the control and NPK fertilizer. The result indicated that FHA fertilizer can improve the chlorophyll content and photosynthesis, thus improve the stimulation of carbohydrate content, which will directly influence the flower quality and vase life of cut chrysanthemum flowers. Enhancement of vase life of cut flowers bears a positive relation with the stimulation of carbohydrate content in the leaves and the delay in loss of flower size and fresh weight (Zuliana et al., 2008; Chutichudet et al., 2011; Gul et al., 2012). In the present study, we observed that flower size and fresh weight (Fig. 4A and B) were higher in FHA treatment than those of the control and NPK treatment at all vase life days. The decrease of flower size and fresh weight at the late holding time might be due to nutrient absorption decreasing gradually when the cut flowers were isolated from the whole plants for a long period, and the nutrients could not meet plant consumption. However, FHA treatment delayed the decreases in flower size and fresh weight, which indicated that FHA fertilizer can increase flower size and fresh weight and delay the loss in flower size and fresh weight in cut chrysanthemum flowers due to higher contents of soluble sugar and soluble protein in the leaves,
Fig. 4. Effects of different fertilizers on flower size (A), fresh weight (B) in the cut chrysanthemum flowers during vase life. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
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Fig. 6. Effects of different fertilizers on MDA content in the cut flowers of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
Fig. 5. Effects of different fertilizers on SOD activity (A), POD activity (B), CAT activity (C) in the cut flowers of chrysanthemum. Control, spraying with distilled water; NPK, spraying with inorganic NPK fertilizer; FHA, spraying with foliar humic acid (FHA) fertilizer. Bars represent standard errors.
and thereby substantially extended the vase life of cut chrysanthemum flowers. Aging of petals is accompanied by morphological, biochemical and biophysical deterioration (Bartoli et al., 1996). Upon senescence, membrane permeability increases rapidly, which is combined with an increase in lipid peroxidation (Trippi and Paulin, 1984). Bartoli et al. (1995) reported that both lipid peroxidation and membrane permeability increased in chrysanthemum petals during aging. This information is consistent with the hypothesis that disruption of cellular membrane integrity is an inherent feature of senescence in petals (Bartoli et al., 1997). Free radicals have been implicated in programmed cell death (Bailly et al., 2001). Lipid
peroxidation involves a series of free radicals in a chain mechanism due to producing ROS such as O2 •− and H2 O2 (Garcíaa et al., 2012). The production of ROS might increase in plants, eventually inducing the oxidation of certain cellular components when the ROS level is high. Activities of SOD, POD and CAT represent antioxidant defense systems that can reduce lipid peroxidation and protect plants from ROS damage (Panavas and Rubinstein, 1998; Singh et al., 2012). In the present study, activities of SOD, POD and CAT increased within the first 4 days. The possible reasons for the increases are that the growth environment changed suddenly when the flowers were cut from the whole plants, inducing the production of ROS. Thus the activities of SOD, POD and CAT were stimulated to protect the flowers from ROS damage. The decrease of the activities of SOD, POD and CAT at late vase time (Fig. 5A–C) might be due to due to accumulation of ROS which in turn inhibited the ability of the antioxidant defense system, thus the activities of SOD, POD, CAT decreased. However, FHA treatment could improve the activities of SOD, POD and CAT to delay the accumulation of O2 •− and H2 O2 during the later vase stage of cut chrysanthemum flowers. Different results were reported by Bartoli et al. (1995) where SOD, POD and CAT activity increased at the beginning of petal senescence in chrysanthemum. The possible reasons for the different results may be the different chrysanthemum cultivars with different mechanisms of senescence or different test environments. MDA is an important index that reflects the extent of damage in plants faced with stress or senescence. The MDA content increased within senescence accompanied with a rapid increase in membrane permeability (Gao et al., 2010). In the present study, the MDA content in the three treatments increased at the late vase stage. FHA treatment reduced the increase in MDA content (Fig. 6), which indicated lower tissue damage and slower senescence in the FHA-treated chrysanthemum flowers. 5. Conclusion Foliar application of humic acid increased the chlorophyll content, photosynthesis and contents of soluble sugar and soluble protein in the leaves of chrysanthemum, and thus improved the flower size, fresh weight and delayed the loss in flower size, fresh weight, and maintained a longer period of vase time in cut chrysanthemum flowers. Foliar humic acid also enhanced activities of antioxidant enzymes, reduced MDA content, thus delayed flower senescence and extended the vase life of cut chrysanthemum flowers.
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