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Endogenous hydrogen gas delays petal senescence and extends the vase life of lisianthus cut flowers
Postharvest Biology and Technology 147 (2019) 148–155
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Endogenous hydrogen gas delays petal senescence and extends the vase life of lisianthus cut flowers
T
Jiuchang Sua, Yang Niea, Gan Zhaoa, Dan Chenga, Ren Wangb, Jun Chenc, Shihai Zhangd, ⁎ Wenbiao Shena, a
College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China c Shennongjia Shi Zhen Water Structure Co., Ltd, Shennongjia 442400, China d Jiangsu Huiwanjia Agricultural Science and Technology Industrial Co., Ltd, Nanjing 210000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Endogenous H2 Petal Senescence Vase life Lisianthus cut flowers Redox homeostasis
Exogenous hydrogen-rich water (HRW) improves vase life and quality of cut flowers, but the effects of endogenous hydrogen gas (H2) are not known. During the vase of cut lisianthus flowers, endogenous H2 concentrations decreased and redox homeostasis was impaired. The supplementation with HRW containing 0.078 mmol L−1 H2 blocked, but 2,6-dichlorophenolindophenol (DCPIP; a putative inhibitor of H2 synthesis) increased, endogenous H2 production. Senescence of cut flowers was delayed by H2, but accelerated by DCPIP. Also, decreased vase life by DCPIP was blocked by H2 administration. These beneficial roles of H2 were associated with less lipid peroxidation, and the increased activities of superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase. Compared with DCPIP alone, the soluble protein, total chlorophyll, and proline contents were elevated when H2 and DCPIP were added together. Overall, endogenous H2 prolongs vase life of lisianthus cut flowers in part by maintaining redox homeostasis.
1. Introduction Hydrogen gas (H2) is an important molecular messenger in animal (Wang, 2014), and plant (Li et al., 2018) cells. H2 administration, in the form of hydrogen-rich water (HRW), not only induces root organogenesis (Lin et al., 2014; Zhu et al., 2016; Cao et al., 2017), but also enhances plant tolerance against various adverse environmental stresses, such as paraquat toxicity (Jin et al., 2013), salinity (Xu et al., 2013), heavy metal exposure (Cui et al., 2013), and water-deficit (Xie et al., 2014; Su et al., 2018). In addition, the application of H2 delays ripening and senescence of kiwifruit and mushrooms (Hu et al., 2014, 2018; Chen et al., 2017), and prolongs vase life of cut lily and rose flowers (Ren et al., 2017), via improving postharvest quality and reducing oxidative damage. Bacteria and algae can produce H2 by hydrogenase activity (Ghirardi et al., 2007; Bothe et al., 2010). The potential to metabolize H2 may exist in higher plants, as endogenous H2 production or emission has been detected in rice, Arabidopsis, and other plant species (Cao et al., 2017; Jin et al., 2013; Xu et al., 2013; Xie et al., 2014; Hu et al., 2018; Renwick et al., 1964; Zeng et al., 2013). Whether a hydrogenaselike protein is present in higher plants however, remains to be
⁎
determined. Additionally, the activities of a hydrogenase from Scenedesmus obliquus was dramatically reduced by 2,6-dichlorophenolindophenol (DCPIP), an inhibitor of the photosynthetic electron flow (Florin et al., 2001). The results above suggested that there could be a synthetic source of endogenous H2 metabolism, which was associated with the alteration of photosynthetic electron flow. Therefore, it is considered that DCPIP could be a putative inhibitor of H2 synthesis. In higher plants, senescence mainly occurs in an age-dependent manner, and triggered by certain phytohormones and environmental stresses (Ma et al., 2018). In fact, senescence is a complex and highly coordinated process (Wu et al., 2017), during which cells undergo active degenerative processes, including the loss of water, degradation of chloroplasts, reduction of proteins, and lipid peroxidation (Prochazkova et al., 2001; Lim et al., 2003; López-Fernández et al., 2015). The terminal phase of cut flower life in a vase is also a senescent process that is characterized by a time-dependent petal wilting, or flower withering. Current results confirmed that senescence is generally accompanied by the excessive production of reactive oxygen species (ROS), including the overproduction of superoxide anion radical and hydrogen peroxide (H2O2) (Rogers and Munné-Bosch, 2016; Jędrzejuk
Corresponding author at: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. E-mail address: [email protected] (W. Shen).
https://doi.org/10.1016/j.postharvbio.2018.09.018 Received 25 May 2018; Received in revised form 25 September 2018; Accepted 26 September 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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et al., 2018). Oxidative stress is one of the important factors resulting in plant aging, since it is able to accelerate the aging process of cut flowers to reduce ornamental value (Ren et al., 2017). During senescence, chloroplasts undergo plentiful destruction and finally collapse, followed by reducing production of chlorophyll (López-Fernández et al., 2015). In addition, proline metabolism may influence ROS signal pathways that alter the progress of senescence (Zhang and Becker, 2015). It has also been observed in previous investigations that an increase of proline content delayed the senescence of cut roses (Kumar et al., 2009, 2010). Lisianthus (Eustoma grandiflorum), an important ornamental flower that is becoming one of the most highly valued cut flowers in worldwide flower market (Bahrami et al., 2013; Kawabata et al., 2009). Since postharvest longevity is an important indicator for ornamental cut flowers value (Olsen et al., 2015), there is significant scope for studies to delay the senescence of postharvest flowers. Generally, the postharvest life and lasting quality could be prolonged in cut flowers by selecting the optimal temperature for transport and storage (Rafdi et al., 2014), and using humic acid (Fan et al., 2015) and nitric oxide (Naing et al., 2017). Exogenous H2 application can have many effects on plants. We have hypothesized that endogenous H2 plays a role in delaying senescence of cut flowers. To assess this hypothesis, the effects of HRW and DCPIP on senescence of lisianthus cut flowers were investigated and compared.
For further experiments, cut flowers were incubated in treatment solutions (100 mL) containing 0.078 mmol L−1 H2 and 500 μmol L−1 2,6-dichlorophenolindophenol (DCPIP; an inhibitor of the photosynthetic electron flow (Florin et al., 2001), a putative inhibitor of H2 synthesis) alone and in combination for the vase life treatments. All treatment solutions were renewed daily, and the experimental conditions were kept at 25 ± 1 °C under a 14:10 h light/dark cycle for the indicated time points. The sample tissue used for analysis was the flower petals.
2. Materials and methods
2.5. Determination of hydrogen peroxide (H2O2) content and corresponding histochemical staining
2.4. Changes of vase life, fresh weight, and maximum flower diameter The end of vase life was determined as the time in which 50% of the open flower petals had wilted. The vase life of cut flowers was determined by the number of days from the day that cut flowers were put in the treated solutions (Ren et al., 2017) until they had no ornamental value (underwent color change, wilt, or loose turgidity). The mean value of vase life of all flowers in each vase was calculated as average vase life for each treatment. For the measurement of the fresh weight, all petals from new flowers were weighed every day at a fixed time. The maximum flower diameter was defined as the maximum width of each flower, and measured by Vernier caliper (Ren et al., 2017).
2.1. Preparation of hydrogen-rich water (HRW) According to previous method (Gu et al., 2017), H2O2 content was measured by the spectrophotography with a minor modification. After the assay reagent was incubated for 45 min, the absorbance of the Fe3+xylenol-orange complex was determined at 560 nm A standard curve was obtained by adding variable amount of H2O2. In situ detection of H2O2 was performed as described previously (Gu et al., 2017). The petals of cut flowers were stained with 0.1% 3,3-diaminobenzidine (DAB; Sigma-Aldrich) at room temperature for 12 h in the dark, after which the decolorized petals were observed under a light microscope.
Hydrogen-rich water (HRW) was prepared by a modified previous method (Su et al., 2018; Xie et al., 2014). Purified hydrogen gas (H2, 99.99% [v/v]) generated from H2 generator (SHC-300; Saikesaisi Hydrogen Energy Co., Ltd., Shandong, China) was bubbled into 500 mL distilled water at a rate of 150 mL min−1 for 30 min. The concentration (0.78 mmol L−1) of H2 in the saturated HRW was determined by gas chromatography (GC-see below). Afterwards, saturated HRW was immediately diluted to 10% and 50% saturated levels (0.078 and 0.39 mmol L−1 H2, respectively) for further experiments. We discovered that the H2 concentration in solutions was stable at 25 °C for at least 12 h (Jin et al., 2013; Su et al., 2018).
2.6. Assay of thiobarbituric acid reactive substances (TBARS) content Lipid peroxidation was determined by measuring the contents of thiobarbituric acid reactive substances (TBARS) following the method previously described (Hodges et al., 1999). TBARS content was expressed as mmol kg−1 fresh weight (FW).
2.2. Determination of H2 concentration Endogenous H2 concentration was measured by the method of Jin et al., (2013) with some modifications. For this method, gas chromatography (Tianmei GC7900 equipped with a thermal conductivity detector, Tianmei Scientific Instrument Co., Ltd., Shanghai, China), was used. Before the headspace was analyzed, the gas chromatographic bottle was heated at 70 °C for 1 h to liberate H2 from HRW and plant tissues, and allowed to cool at room temperature. N2 was used as carrier gas, and air pressure was 0.2 MPa to convey the H2 to the thermal conductivity detector.
2.7. Determination of proline content Proline content was determined by ninhydrin assay as previously described (Bates et al., 1973). The absorbance of the liquid phase was read at 520 nm using a spectrophotometer. A standard curve was obtained by adding variable amount of L-proline. 2.8. Assay of antioxidant enzyme activity
2.3. Plant material and treatments Frozen petal samples (0.3 g) from the cut flowers were ground finely in liquid nitrogen and then homogenized in 3 mL of 50 mmol L−1 phosphate buffer (pH 7.0) containing 1 mmol L−1 EDTA and 1% polyvinylpyrrolidone (PVP) for superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT) assay, or together with the addition of 1 mmol L−1 ascorbic acid (ASC) for the ascorbate peroxidase (APX) assay. SOD activity was estimated by monitoring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Jin et al., 2013). The amount of enzyme required to cause 50% inhibition of the reduction rate of NBT was defined as one unit (U). POD activity was determined by detecting the oxidation of guaiacol (extinction coefficient 26.6 mm−1 cm−1) at 470 nm (Cui et al., 2013). CAT activity was assayed by monitoring the reduction of H2O2 (extinction coefficient
Flowers of lisianthus (Eustoma grandiflorum) without defects and physical damage from a commercial grower, were harvested early in the morning, and quickly transferred to the laboratory. Lisianthus stems were re-cut to a uniform length of 25 cm under distilled water to avoid air embolism, and the stems were then used for further experiments. First, the stems of cut flowers were incubated with treatment solutions (100 mL) containing 0.078, 0.39, and 0.78 mmol L−1 H2. A pilot experiment showed that the effects of 0.078 mmol L−1 H2 on delaying cut flower senescence were the most obvious, so that concentration was applied in the subsequent tests. For the entire experiment, all stems were continuously kept in the treatment solution until the end of the experiment was reached. 149
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39.4 mm−1 cm−1) for at least 2 min at 240 nm (Su et al., 2018). APX activity was determined by monitoring the decrease at 290 nm (extinction coefficient 2.8 mm−1 cm−1) (Cui et al., 2013). The protein concentration of petal extracts was estimated as outlined previously (Bradford, 1976).
either delayed or accelerated senescence were observed in cut flowers when supplemented with H2 and DCPIP, respectively. In parallel, the concentration of endogenous H2 in cut flowers was analyzed. The timecourse experiment showed that the endogenous H2 concentration increased slightly until 3 d, followed by the severe reduction up to 7 d (Fig. 2C). The results indicated that H2 levels were changed during senescence. By contrast, the addition of exogenous H2 could delay the reduction in endogenous H2 levels, correlating these data with those from phenotypic parameters, including flower diameter and fresh weight (Fig. 2D and E). Importantly, the beneficial response to exogenous H2 were abolished by the presence of DCPIP at 500 μmol L−1 (Fig. 2C), a concentration expected to be effective in the inhibition of endogenous H2 production. Thus, 500 μmol L−1 DCPIP was regarded as a putative inhibitor of H2 synthesis, at least in our experimental conditions. These results further indicated that a close relationship might exist between maintaining H2 levels and delayed senescence in lisianthus cut flowers.
2.9. Determination of total chlorophyll content Total chlorophyll content was measured followed the methods as previously reported (Liu et al., 2017). 2.10. Experimental design Referring to previous reports (In et al., 2017; Naing et al., 2017) with minor modification, all experiments were arranged in a randomized complete block design. Bunches of 15 flower stems per vase were packed in a glass jar with treatment solutions and placed in the growth chamber. Here, the experiment was done 3 times with triplicates per experiment, and three replicates (each replicate consisting of 15 flowers per vase) included 45 flowers (15 × 3) for each time. For the determination of vase life, fresh weight, and maximum flower diameter, 15 flowers per replicate were selected for the evaluation at each sample day, and the total flowers in triplicate was 45 (15 × 3) for each time. DAB-dependent histochemical staining was from six petals per treatment, and representative phenotypes were photographed. For the measurement of other parameters, including H2 concentration, H2O2 levels, TBARS content, enzyme activities, soluble protein content, total chlorophyll content, and proline accumulation, two flowers per replicate were used, and the total flowers in triplicate was 6 (2 × 3) for each time.
3.3. H2-delayed senescence and extended vase life were dependent on the reestablishment of redox homeostasis As expected, a gradual increase of DAB-dependent staining was observed in control samples during senescence up to 7 d (Fig. 3A). Similar results were observed in the changes of endogenous H2O2 levels determined with spectrophotography (Fig. 3B), confirming that redox homeostasis was impaired during senescence. Subsequent time-course analysis revealed that with respect to the control petals, reduced staining appeared in the petals with H2, especially at 6 d (Fig. 3A). Alone, DCPIP brought about more dark brown color precipitates, compared to the control at 6 d. Changes in endogenous H2O2 levels showed a similar pattern (Fig. 3B). Combined with corresponding changes in endogenous H2 levels (Fig. 2C), we further postulate that the reestablishment of redox homeostasis was closely associated with endogenous H2 production. Subsequently, lipid peroxidation in cut flower petals was measured by determining TBARS content. As expected, TBARS content was gradually increased during process of senescence (Fig. 3C). Compared to the controls, delayed TBARS level increases were found in cut flowers in the presence of 0.078 mmol L−1 H2. Alone, DCPIP aggravation of lipid peroxidation was observed, which was abolished by co-incubation with H2. Considering the changes in phenotypic parameters (Fig. 2), above results confirmed that H2-delayed senescence and -extended vase life were dependent on the reestablishment of redox homeostasis. To further discern the contribution of endogenous H2, changes in enzymatic activities of several antioxidant enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (POD), and catalase (CAT), were determined. The results shown in Fig. 4 revealed that during senescence, the activities of SOD, APX, POD, and CAT in petals were initially increased, and thereafter declined to minimal levels. These changes in enzymatic activities were weakened by DCPIP. Meanwhile, contrasting phenomena were observed as a result of H2 treatment. However, when H2 was added together with DCPIP, the changes in enzymatic activities were impaired or delayed. Consistently, we noticed that the enzymatic changes were matched with a corresponding alteration in lipid peroxidation (Fig. 3). Combined, the results further confirmed a central role of redox homeostasis with respect to the beneficial effects of H2.
2.11. Statistical analysis All data are expressed as the mean ± SE from three independent experiments with three replicates for each. Statistical analysis was performed by using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) and differences among treatments were analyzed by one-way analysis of variance (ANOVA), taking P < 0.05 as significant, according to Duncan's multiple range test. 3. Results 3.1. H2 delays petal senescence to extend the vase life of lisianthus cut flowers Cut flowers were incubated with treatment solutions containing 0.078, 0.39, and 0.78 mmol L−1 H2. Afterwards, corresponding phenotypes of cut flowers during senescence were showed in Fig. 1A. The vase life of cut flowers incubated with 0.078 mmol L−1 H2 was increased to about 11 d, compared to about 7 d for the chemical-free control plants (Fig. 1B). Correspondingly, treatments of 0.39 and 0.78 mmol L−1 H2 prolonged the vase life for just 2 and 1 d, respectively. Similarly, the reduction in the flower opening index (especially diameter) and biomass (especially fresh weight) during vase was significantly blocked by the 0.078 mmol L−1 H2 administration (Fig. 1C and D). Since the 0.078 mmol L−1 H2 treatment provided the most improved vase life, a treatment solution containing 0.078 mmol L−1 H2 was subsequently used for the remainder of the study. 3.2. Decreased H2 levels and delayed senescence were sensitive to DCPIP
3.4. High levels of total chlorophyll, soluble protein, and proline are maintained by H2, but weaken or blocked by DCPIP
To verify whether endogenous H2 was involved in delaying senescence and extending flower vase life, both H2 and DCPIP, alone and in combination, were applied, so that the treatment effects could be compared. DCPIP has previously been shown to be an inhibitor of the photosynthetic electron flow (Florin et al., 2001). As shown in Fig. 2A,
A time-course analysis revealed progressive decreased soluble protein (Fig. 5A) and total chlorophyll (Fig. 5B) contents, in comparison with the increased proline content up to 7 d (Fig. 5C), in cut flowers during vase storage. Interestingly, the H2 treatment was able to preserve the higher levels of total chlorophyll, soluble protein, and proline 150
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Fig. 1. Representative photographs (A), vase life (B), fresh weight (C), and maximum flower diameter (D) in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing the indicated concentrations of H2 during cut flower vase storage. Error bars represent the standard error (SE). There were 45 flowers (15 × 3) in three replicates per time, and the experiments were conducted for 3 times (B–D). Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests. Scale bar = 1 cm.
slightly increased and then dramatically declined (Fig. 2C). This observation was linked to the process of senescence which resulted in the decrease of H2 metabolism in cut flowers over time in a vase. Although we have not investigated the detailed synthetic pathway(s) of endogenous H2 production/loss from the cut flower petal tissues of lisianthus, a test with H2 synthesis inhibitor (DCPIP) suggested that the alterations of H2 production in vivo in petals were closely associated with the process of senescence. This deduction was initially confirmed by improvements in cut flower vase life, flower diameter, and fresh weight (Fig. 2B, D, and E), when exogenous H2 and DCPIP were used independently or in combination. Consistent with the previous studies (Ren et al., 2017), the supplementation of vase water with H2 could increase petal endogenous H2 levels, resulting in the extension of the vase life of cut flowers (Fig. 2). The results also suggest that the persistence of H2 levels was critical for delaying flower senescence. To further investigate the role of endogenous H2 in extending flower vase life, DCPIP, a putative H2 synthesis inhibitor (Florin et al., 2001), was used, and the inhibitory role was confirmed in our experimental
than the control or other treatments. In contrast, the DCPIP treatment inhibited or decreased all of above parameters, particularly at 5 d. In view of the changes in endogenous H2 levels (Fig. 2C), these results clearly confirmed that endogenous H2 plays a crucial role in preserving higher levels of total chlorophyll, soluble protein, and proline. 4. Discussion Recently, a beneficial role for exogenous H2 has been observed in extending cut flowers vase life (Ren et al., 2017). In this report, the use of histochemical staining and biochemical approaches, showed that endogenous H2 was required to delay petal senescence and extending the vase life of lisianthus cut flowers, at least partially, by modulating cellular redox homeostasis. Generally, the endogenous H2 concentration in plants is relatively stable, but can be altered during senescence (Hu et al., 2018; Li et al., 2018). Here, we observed for the first time that flowers with an increased vase life had an endogenous H2 concentration that initially 151
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Fig. 2. Representative photographs (A), vase life (B), H2 concentration (C), maximum flower diameter (D), and fresh weight (E) in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during vase flower storage. Error bars represent the standard error (SE). There were 45 (15 × 3 for B, D, and E) or 6 (2 × 3 for C) flowers in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests. Scale bar = 1 cm.
that H2 synthesis catalyzed by hydrogenase-like proteins should be a high priority for future investigation in higher plants. During the senescence process in cut flowers, ROS (especially H2O2) levels have been observed to gradually increase (Bahrami et al., 2013; Hossain et al., 2006; Kumar et al., 2007). As a in vivo counter to ROS, it has been suggested that the intracellular antioxidant defense could be enhanced by H2 both in animals and plants (Li et al., 2018; Jin et al., 2013; Xu et al., 2013; Cui et al., 2013; Xie et al., 2014; Su et al., 2018). This study has observed that during the normal senescence of cut flowers, the contents of H2O2 and TBARS progressively increased (Fig. 3B and C), indicating that the redox homeostasis was being disrupted. Changes in histochemical staining showed a similar pattern (Fig. 3A). Several observations have further indicated that the redox homeostasis was reestablished by endogenous H2. Firstly, senescence-induced H2O2 accumulation was impaired by H2 administration (Fig. 3A
conditions (Fig. 2C). As expected, Fig. 2 showed that DCPIP not only inhibited endogenous H2 production, but also accelerated cut flowers senescence. By contrast, the effects elicited by DCPIP were negated by the addition of H2. A similar delay in kiwifruit ripening was observed after a fumigation test with H2 gas directly (Hu et al., 2018) or HRW (Hu et al., 2014). Considering the danger of H2 being flammable and explosive, the application of H2 was carried out by using HRW in these experiments. However, it should be considered that the dissolved oxygen might be reduced in HRW so as cause hypoxic effects (Xie et al., 2014; Su et al., 2018). This effect of hypoxia on cut flowers can not be easily excluded. Combined, the results suggested that endogenous H2 plays an important role in delaying senescence of cut flowers. The production of endogenous H2 by the green alga S. obliquus has linked the H2 producing hydrogenase to the photosynthetic electron transport chain (Florin et al., 2001). However, the biosynthetic source and pathway for H2 in higher plants have yet to be defined. We identify 152
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Fig. 3. Representative stained photographs (A), H2O2 concentration (B) and TBARS levels (C) in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during cut flower vase storage. Afterward, 6 petals were stained with DAB, and represent picture was immediately photographed (A; Scale bar = 2 mm). Error bars represent the standard error (SE). There were 6 flowers (2 × 3 for B and C) in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests.
down the increase of TBARS content, which was partially reversed by DCPIP (Fig. 3C). Similar findings were also observed in a previous study (Hu et al., 2014), which showed that H2 protected the kiwifruit against oxidative damage during storage. Finally, the activities of antioxidant enzymes including SOD, APX, POD, and CAT, in H2-treated cut flowers were significantly higher than those in controls (Fig. 4), similar to the
and B). The lower H2O2 levels triggered by H2 were shown to be beneficial in delaying senescence, at least partially. These differences reflected that the physiological roles of H2 might be linked to the varied concentrations of H2O2. Second, endogenous H2-reestablished redox homeostasis in cut flowers was confirmed by the assessment of TBARS levels. For example, the results showed that H2 administration slowed
Fig. 4. SOD (A), APX (B), POD (C), and CAT (D) activities in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during cut flower vase storage. The activities of antioxidant enzyme were expressed on a protein mass basis. Error bars represent the standard error (SE). There were 6 (2 × 3) flowers in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests.
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Fig. 6. Schematic model describing the endogenous H2-delayed senescence in lisianthus cut flowers.
vase life. However, since a reduction of proline levels was beneficial for delaying the petal senescence (Kazemi et al., 2011), it has yet to be understood whether the increased proline content delays petal senescence or whether it was a symptom of senescence. A schematic model describing the endogenous H2-delayed senescence in lisianthus cut flowers is shown in Fig. 6, and the contribution of reestablishment of redox balance is schematically outlined. Our results suggest that H2 inclusion in vase water may be a useful strategy for prolonging flower postharvest life, as well as maintaining cut flowers in attractive open states. It is worth noting that since the solubility of H2 in water is very low, the use of HRW is safe in practical application. Indeed, both HRW and H2 gas have been previously used to improve the storage of cut flowers, fruits and mushrooms (Chen et al., 2017; Hu et al., 2014, 2018; Ren et al., 2017). Hu et al., (2018) extended this understanding by showing that H2 extends the shelf life of kiwifruit by decreasing ethylene biosynthesis. Finally, the effects of hormonal levels and other gaseous signals on senescence (Olsen et al., 2015; Naing et al., 2017; Cubría-Radío et al., 2017), the interplay among H2, phytohormones (such as ethylene, abscisic acid, etc.), and other gaseous signals, requires urgent investigation.
Fig. 5. Soluble protein (A), total chlorophyll (B), and proline (C) concentrations in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during cut flower vase storage. Error bars represent the standard error (SE). There were 6 (2 × 3) flowers in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests.
previous reports (Jin et al., 2013; Su et al., 2018). Consistently, the changes triggered by H2 were sensitive to DCPIP. Taken together, the lower contents of H2O2 and TBARS in H2-treated cut flowers might be ascribed to the increased activities of SOD, APX, POD, and CAT. To date, other studies have also confirmed that H2 plays an antioxidant role in the delay of senescence (Chen et al., 2017; Hu et al., 2014; Ren et al., 2017). Regrettably, no research has yet defined the antioxidant mechanism of H2 (Li et al., 2018). The next study in H2 effects is envisaged to solve the questions raised in this study, since the involvement of nitric oxide in H2-triggered osmotic stress tolerance has been observed previously (Su et al., 2018). Other evidence has been observed, supporting the hypothesis that endogenous H2 was beneficial in delaying the senescence of cut flowers. Generically, the cessation of photosynthesis and degeneration of cellular structures combined with strong losses of proteins (Lim et al., 2003) and chlorophyll (López-Fernández et al., 2015) are a prominent characteristic of senescence in plants. This study observed that higher levels of H2 delayed the loss of chlorophyll and soluble proteins during senescence (Fig. 5A and B). In a previous report, it was confirmed that proline content of cut roses gradually increased during the process of senescence (Kumar et al., 2009). Based on the visible phenotypes (Figs. 1 and 2) and proline content (Fig. 5C), we suggest that the elevated proline levels induced by H2 may be beneficial in extending the
5. Conclusions This investigation revealed that endogenous H2 prolonged lisianthus cut flower life by delaying the senescence. Trials were conducted to investigate the effect of altering endogenous H2 levels on the vase life of cut flowers, indicating that endogenous H2 may be critical in delaying senescence. Our results further confirmed that redox homeostasis was reestablished by vase water supplementation with H2, which might contribute to an increase in the antioxidant potential of the cut flowers. Decreased soluble protein, increased total chlorophyll and proline contents were also modulated by H2 and its antagonist, DCPIP. These findings are practically significant for both postharvest physiology and applied biology.
Conflict of interest statement The authors declare that they have no conflict of interest. 154
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Acknowledgements
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Endogenous hydrogen gas delays petal senescence and extends the vase life of lisianthus cut flowers
T
Jiuchang Sua, Yang Niea, Gan Zhaoa, Dan Chenga, Ren Wangb, Jun Chenc, Shihai Zhangd, ⁎ Wenbiao Shena, a
College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China c Shennongjia Shi Zhen Water Structure Co., Ltd, Shennongjia 442400, China d Jiangsu Huiwanjia Agricultural Science and Technology Industrial Co., Ltd, Nanjing 210000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Endogenous H2 Petal Senescence Vase life Lisianthus cut flowers Redox homeostasis
Exogenous hydrogen-rich water (HRW) improves vase life and quality of cut flowers, but the effects of endogenous hydrogen gas (H2) are not known. During the vase of cut lisianthus flowers, endogenous H2 concentrations decreased and redox homeostasis was impaired. The supplementation with HRW containing 0.078 mmol L−1 H2 blocked, but 2,6-dichlorophenolindophenol (DCPIP; a putative inhibitor of H2 synthesis) increased, endogenous H2 production. Senescence of cut flowers was delayed by H2, but accelerated by DCPIP. Also, decreased vase life by DCPIP was blocked by H2 administration. These beneficial roles of H2 were associated with less lipid peroxidation, and the increased activities of superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, and catalase. Compared with DCPIP alone, the soluble protein, total chlorophyll, and proline contents were elevated when H2 and DCPIP were added together. Overall, endogenous H2 prolongs vase life of lisianthus cut flowers in part by maintaining redox homeostasis.
1. Introduction Hydrogen gas (H2) is an important molecular messenger in animal (Wang, 2014), and plant (Li et al., 2018) cells. H2 administration, in the form of hydrogen-rich water (HRW), not only induces root organogenesis (Lin et al., 2014; Zhu et al., 2016; Cao et al., 2017), but also enhances plant tolerance against various adverse environmental stresses, such as paraquat toxicity (Jin et al., 2013), salinity (Xu et al., 2013), heavy metal exposure (Cui et al., 2013), and water-deficit (Xie et al., 2014; Su et al., 2018). In addition, the application of H2 delays ripening and senescence of kiwifruit and mushrooms (Hu et al., 2014, 2018; Chen et al., 2017), and prolongs vase life of cut lily and rose flowers (Ren et al., 2017), via improving postharvest quality and reducing oxidative damage. Bacteria and algae can produce H2 by hydrogenase activity (Ghirardi et al., 2007; Bothe et al., 2010). The potential to metabolize H2 may exist in higher plants, as endogenous H2 production or emission has been detected in rice, Arabidopsis, and other plant species (Cao et al., 2017; Jin et al., 2013; Xu et al., 2013; Xie et al., 2014; Hu et al., 2018; Renwick et al., 1964; Zeng et al., 2013). Whether a hydrogenaselike protein is present in higher plants however, remains to be
⁎
determined. Additionally, the activities of a hydrogenase from Scenedesmus obliquus was dramatically reduced by 2,6-dichlorophenolindophenol (DCPIP), an inhibitor of the photosynthetic electron flow (Florin et al., 2001). The results above suggested that there could be a synthetic source of endogenous H2 metabolism, which was associated with the alteration of photosynthetic electron flow. Therefore, it is considered that DCPIP could be a putative inhibitor of H2 synthesis. In higher plants, senescence mainly occurs in an age-dependent manner, and triggered by certain phytohormones and environmental stresses (Ma et al., 2018). In fact, senescence is a complex and highly coordinated process (Wu et al., 2017), during which cells undergo active degenerative processes, including the loss of water, degradation of chloroplasts, reduction of proteins, and lipid peroxidation (Prochazkova et al., 2001; Lim et al., 2003; López-Fernández et al., 2015). The terminal phase of cut flower life in a vase is also a senescent process that is characterized by a time-dependent petal wilting, or flower withering. Current results confirmed that senescence is generally accompanied by the excessive production of reactive oxygen species (ROS), including the overproduction of superoxide anion radical and hydrogen peroxide (H2O2) (Rogers and Munné-Bosch, 2016; Jędrzejuk
Corresponding author at: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. E-mail address: [email protected] (W. Shen).
https://doi.org/10.1016/j.postharvbio.2018.09.018 Received 25 May 2018; Received in revised form 25 September 2018; Accepted 26 September 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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et al., 2018). Oxidative stress is one of the important factors resulting in plant aging, since it is able to accelerate the aging process of cut flowers to reduce ornamental value (Ren et al., 2017). During senescence, chloroplasts undergo plentiful destruction and finally collapse, followed by reducing production of chlorophyll (López-Fernández et al., 2015). In addition, proline metabolism may influence ROS signal pathways that alter the progress of senescence (Zhang and Becker, 2015). It has also been observed in previous investigations that an increase of proline content delayed the senescence of cut roses (Kumar et al., 2009, 2010). Lisianthus (Eustoma grandiflorum), an important ornamental flower that is becoming one of the most highly valued cut flowers in worldwide flower market (Bahrami et al., 2013; Kawabata et al., 2009). Since postharvest longevity is an important indicator for ornamental cut flowers value (Olsen et al., 2015), there is significant scope for studies to delay the senescence of postharvest flowers. Generally, the postharvest life and lasting quality could be prolonged in cut flowers by selecting the optimal temperature for transport and storage (Rafdi et al., 2014), and using humic acid (Fan et al., 2015) and nitric oxide (Naing et al., 2017). Exogenous H2 application can have many effects on plants. We have hypothesized that endogenous H2 plays a role in delaying senescence of cut flowers. To assess this hypothesis, the effects of HRW and DCPIP on senescence of lisianthus cut flowers were investigated and compared.
For further experiments, cut flowers were incubated in treatment solutions (100 mL) containing 0.078 mmol L−1 H2 and 500 μmol L−1 2,6-dichlorophenolindophenol (DCPIP; an inhibitor of the photosynthetic electron flow (Florin et al., 2001), a putative inhibitor of H2 synthesis) alone and in combination for the vase life treatments. All treatment solutions were renewed daily, and the experimental conditions were kept at 25 ± 1 °C under a 14:10 h light/dark cycle for the indicated time points. The sample tissue used for analysis was the flower petals.
2. Materials and methods
2.5. Determination of hydrogen peroxide (H2O2) content and corresponding histochemical staining
2.4. Changes of vase life, fresh weight, and maximum flower diameter The end of vase life was determined as the time in which 50% of the open flower petals had wilted. The vase life of cut flowers was determined by the number of days from the day that cut flowers were put in the treated solutions (Ren et al., 2017) until they had no ornamental value (underwent color change, wilt, or loose turgidity). The mean value of vase life of all flowers in each vase was calculated as average vase life for each treatment. For the measurement of the fresh weight, all petals from new flowers were weighed every day at a fixed time. The maximum flower diameter was defined as the maximum width of each flower, and measured by Vernier caliper (Ren et al., 2017).
2.1. Preparation of hydrogen-rich water (HRW) According to previous method (Gu et al., 2017), H2O2 content was measured by the spectrophotography with a minor modification. After the assay reagent was incubated for 45 min, the absorbance of the Fe3+xylenol-orange complex was determined at 560 nm A standard curve was obtained by adding variable amount of H2O2. In situ detection of H2O2 was performed as described previously (Gu et al., 2017). The petals of cut flowers were stained with 0.1% 3,3-diaminobenzidine (DAB; Sigma-Aldrich) at room temperature for 12 h in the dark, after which the decolorized petals were observed under a light microscope.
Hydrogen-rich water (HRW) was prepared by a modified previous method (Su et al., 2018; Xie et al., 2014). Purified hydrogen gas (H2, 99.99% [v/v]) generated from H2 generator (SHC-300; Saikesaisi Hydrogen Energy Co., Ltd., Shandong, China) was bubbled into 500 mL distilled water at a rate of 150 mL min−1 for 30 min. The concentration (0.78 mmol L−1) of H2 in the saturated HRW was determined by gas chromatography (GC-see below). Afterwards, saturated HRW was immediately diluted to 10% and 50% saturated levels (0.078 and 0.39 mmol L−1 H2, respectively) for further experiments. We discovered that the H2 concentration in solutions was stable at 25 °C for at least 12 h (Jin et al., 2013; Su et al., 2018).
2.6. Assay of thiobarbituric acid reactive substances (TBARS) content Lipid peroxidation was determined by measuring the contents of thiobarbituric acid reactive substances (TBARS) following the method previously described (Hodges et al., 1999). TBARS content was expressed as mmol kg−1 fresh weight (FW).
2.2. Determination of H2 concentration Endogenous H2 concentration was measured by the method of Jin et al., (2013) with some modifications. For this method, gas chromatography (Tianmei GC7900 equipped with a thermal conductivity detector, Tianmei Scientific Instrument Co., Ltd., Shanghai, China), was used. Before the headspace was analyzed, the gas chromatographic bottle was heated at 70 °C for 1 h to liberate H2 from HRW and plant tissues, and allowed to cool at room temperature. N2 was used as carrier gas, and air pressure was 0.2 MPa to convey the H2 to the thermal conductivity detector.
2.7. Determination of proline content Proline content was determined by ninhydrin assay as previously described (Bates et al., 1973). The absorbance of the liquid phase was read at 520 nm using a spectrophotometer. A standard curve was obtained by adding variable amount of L-proline. 2.8. Assay of antioxidant enzyme activity
2.3. Plant material and treatments Frozen petal samples (0.3 g) from the cut flowers were ground finely in liquid nitrogen and then homogenized in 3 mL of 50 mmol L−1 phosphate buffer (pH 7.0) containing 1 mmol L−1 EDTA and 1% polyvinylpyrrolidone (PVP) for superoxide dismutase (SOD), guaiacol peroxidase (POD), and catalase (CAT) assay, or together with the addition of 1 mmol L−1 ascorbic acid (ASC) for the ascorbate peroxidase (APX) assay. SOD activity was estimated by monitoring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Jin et al., 2013). The amount of enzyme required to cause 50% inhibition of the reduction rate of NBT was defined as one unit (U). POD activity was determined by detecting the oxidation of guaiacol (extinction coefficient 26.6 mm−1 cm−1) at 470 nm (Cui et al., 2013). CAT activity was assayed by monitoring the reduction of H2O2 (extinction coefficient
Flowers of lisianthus (Eustoma grandiflorum) without defects and physical damage from a commercial grower, were harvested early in the morning, and quickly transferred to the laboratory. Lisianthus stems were re-cut to a uniform length of 25 cm under distilled water to avoid air embolism, and the stems were then used for further experiments. First, the stems of cut flowers were incubated with treatment solutions (100 mL) containing 0.078, 0.39, and 0.78 mmol L−1 H2. A pilot experiment showed that the effects of 0.078 mmol L−1 H2 on delaying cut flower senescence were the most obvious, so that concentration was applied in the subsequent tests. For the entire experiment, all stems were continuously kept in the treatment solution until the end of the experiment was reached. 149
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39.4 mm−1 cm−1) for at least 2 min at 240 nm (Su et al., 2018). APX activity was determined by monitoring the decrease at 290 nm (extinction coefficient 2.8 mm−1 cm−1) (Cui et al., 2013). The protein concentration of petal extracts was estimated as outlined previously (Bradford, 1976).
either delayed or accelerated senescence were observed in cut flowers when supplemented with H2 and DCPIP, respectively. In parallel, the concentration of endogenous H2 in cut flowers was analyzed. The timecourse experiment showed that the endogenous H2 concentration increased slightly until 3 d, followed by the severe reduction up to 7 d (Fig. 2C). The results indicated that H2 levels were changed during senescence. By contrast, the addition of exogenous H2 could delay the reduction in endogenous H2 levels, correlating these data with those from phenotypic parameters, including flower diameter and fresh weight (Fig. 2D and E). Importantly, the beneficial response to exogenous H2 were abolished by the presence of DCPIP at 500 μmol L−1 (Fig. 2C), a concentration expected to be effective in the inhibition of endogenous H2 production. Thus, 500 μmol L−1 DCPIP was regarded as a putative inhibitor of H2 synthesis, at least in our experimental conditions. These results further indicated that a close relationship might exist between maintaining H2 levels and delayed senescence in lisianthus cut flowers.
2.9. Determination of total chlorophyll content Total chlorophyll content was measured followed the methods as previously reported (Liu et al., 2017). 2.10. Experimental design Referring to previous reports (In et al., 2017; Naing et al., 2017) with minor modification, all experiments were arranged in a randomized complete block design. Bunches of 15 flower stems per vase were packed in a glass jar with treatment solutions and placed in the growth chamber. Here, the experiment was done 3 times with triplicates per experiment, and three replicates (each replicate consisting of 15 flowers per vase) included 45 flowers (15 × 3) for each time. For the determination of vase life, fresh weight, and maximum flower diameter, 15 flowers per replicate were selected for the evaluation at each sample day, and the total flowers in triplicate was 45 (15 × 3) for each time. DAB-dependent histochemical staining was from six petals per treatment, and representative phenotypes were photographed. For the measurement of other parameters, including H2 concentration, H2O2 levels, TBARS content, enzyme activities, soluble protein content, total chlorophyll content, and proline accumulation, two flowers per replicate were used, and the total flowers in triplicate was 6 (2 × 3) for each time.
3.3. H2-delayed senescence and extended vase life were dependent on the reestablishment of redox homeostasis As expected, a gradual increase of DAB-dependent staining was observed in control samples during senescence up to 7 d (Fig. 3A). Similar results were observed in the changes of endogenous H2O2 levels determined with spectrophotography (Fig. 3B), confirming that redox homeostasis was impaired during senescence. Subsequent time-course analysis revealed that with respect to the control petals, reduced staining appeared in the petals with H2, especially at 6 d (Fig. 3A). Alone, DCPIP brought about more dark brown color precipitates, compared to the control at 6 d. Changes in endogenous H2O2 levels showed a similar pattern (Fig. 3B). Combined with corresponding changes in endogenous H2 levels (Fig. 2C), we further postulate that the reestablishment of redox homeostasis was closely associated with endogenous H2 production. Subsequently, lipid peroxidation in cut flower petals was measured by determining TBARS content. As expected, TBARS content was gradually increased during process of senescence (Fig. 3C). Compared to the controls, delayed TBARS level increases were found in cut flowers in the presence of 0.078 mmol L−1 H2. Alone, DCPIP aggravation of lipid peroxidation was observed, which was abolished by co-incubation with H2. Considering the changes in phenotypic parameters (Fig. 2), above results confirmed that H2-delayed senescence and -extended vase life were dependent on the reestablishment of redox homeostasis. To further discern the contribution of endogenous H2, changes in enzymatic activities of several antioxidant enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (POD), and catalase (CAT), were determined. The results shown in Fig. 4 revealed that during senescence, the activities of SOD, APX, POD, and CAT in petals were initially increased, and thereafter declined to minimal levels. These changes in enzymatic activities were weakened by DCPIP. Meanwhile, contrasting phenomena were observed as a result of H2 treatment. However, when H2 was added together with DCPIP, the changes in enzymatic activities were impaired or delayed. Consistently, we noticed that the enzymatic changes were matched with a corresponding alteration in lipid peroxidation (Fig. 3). Combined, the results further confirmed a central role of redox homeostasis with respect to the beneficial effects of H2.
2.11. Statistical analysis All data are expressed as the mean ± SE from three independent experiments with three replicates for each. Statistical analysis was performed by using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) and differences among treatments were analyzed by one-way analysis of variance (ANOVA), taking P < 0.05 as significant, according to Duncan's multiple range test. 3. Results 3.1. H2 delays petal senescence to extend the vase life of lisianthus cut flowers Cut flowers were incubated with treatment solutions containing 0.078, 0.39, and 0.78 mmol L−1 H2. Afterwards, corresponding phenotypes of cut flowers during senescence were showed in Fig. 1A. The vase life of cut flowers incubated with 0.078 mmol L−1 H2 was increased to about 11 d, compared to about 7 d for the chemical-free control plants (Fig. 1B). Correspondingly, treatments of 0.39 and 0.78 mmol L−1 H2 prolonged the vase life for just 2 and 1 d, respectively. Similarly, the reduction in the flower opening index (especially diameter) and biomass (especially fresh weight) during vase was significantly blocked by the 0.078 mmol L−1 H2 administration (Fig. 1C and D). Since the 0.078 mmol L−1 H2 treatment provided the most improved vase life, a treatment solution containing 0.078 mmol L−1 H2 was subsequently used for the remainder of the study. 3.2. Decreased H2 levels and delayed senescence were sensitive to DCPIP
3.4. High levels of total chlorophyll, soluble protein, and proline are maintained by H2, but weaken or blocked by DCPIP
To verify whether endogenous H2 was involved in delaying senescence and extending flower vase life, both H2 and DCPIP, alone and in combination, were applied, so that the treatment effects could be compared. DCPIP has previously been shown to be an inhibitor of the photosynthetic electron flow (Florin et al., 2001). As shown in Fig. 2A,
A time-course analysis revealed progressive decreased soluble protein (Fig. 5A) and total chlorophyll (Fig. 5B) contents, in comparison with the increased proline content up to 7 d (Fig. 5C), in cut flowers during vase storage. Interestingly, the H2 treatment was able to preserve the higher levels of total chlorophyll, soluble protein, and proline 150
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Fig. 1. Representative photographs (A), vase life (B), fresh weight (C), and maximum flower diameter (D) in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing the indicated concentrations of H2 during cut flower vase storage. Error bars represent the standard error (SE). There were 45 flowers (15 × 3) in three replicates per time, and the experiments were conducted for 3 times (B–D). Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests. Scale bar = 1 cm.
slightly increased and then dramatically declined (Fig. 2C). This observation was linked to the process of senescence which resulted in the decrease of H2 metabolism in cut flowers over time in a vase. Although we have not investigated the detailed synthetic pathway(s) of endogenous H2 production/loss from the cut flower petal tissues of lisianthus, a test with H2 synthesis inhibitor (DCPIP) suggested that the alterations of H2 production in vivo in petals were closely associated with the process of senescence. This deduction was initially confirmed by improvements in cut flower vase life, flower diameter, and fresh weight (Fig. 2B, D, and E), when exogenous H2 and DCPIP were used independently or in combination. Consistent with the previous studies (Ren et al., 2017), the supplementation of vase water with H2 could increase petal endogenous H2 levels, resulting in the extension of the vase life of cut flowers (Fig. 2). The results also suggest that the persistence of H2 levels was critical for delaying flower senescence. To further investigate the role of endogenous H2 in extending flower vase life, DCPIP, a putative H2 synthesis inhibitor (Florin et al., 2001), was used, and the inhibitory role was confirmed in our experimental
than the control or other treatments. In contrast, the DCPIP treatment inhibited or decreased all of above parameters, particularly at 5 d. In view of the changes in endogenous H2 levels (Fig. 2C), these results clearly confirmed that endogenous H2 plays a crucial role in preserving higher levels of total chlorophyll, soluble protein, and proline. 4. Discussion Recently, a beneficial role for exogenous H2 has been observed in extending cut flowers vase life (Ren et al., 2017). In this report, the use of histochemical staining and biochemical approaches, showed that endogenous H2 was required to delay petal senescence and extending the vase life of lisianthus cut flowers, at least partially, by modulating cellular redox homeostasis. Generally, the endogenous H2 concentration in plants is relatively stable, but can be altered during senescence (Hu et al., 2018; Li et al., 2018). Here, we observed for the first time that flowers with an increased vase life had an endogenous H2 concentration that initially 151
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Fig. 2. Representative photographs (A), vase life (B), H2 concentration (C), maximum flower diameter (D), and fresh weight (E) in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during vase flower storage. Error bars represent the standard error (SE). There were 45 (15 × 3 for B, D, and E) or 6 (2 × 3 for C) flowers in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests. Scale bar = 1 cm.
that H2 synthesis catalyzed by hydrogenase-like proteins should be a high priority for future investigation in higher plants. During the senescence process in cut flowers, ROS (especially H2O2) levels have been observed to gradually increase (Bahrami et al., 2013; Hossain et al., 2006; Kumar et al., 2007). As a in vivo counter to ROS, it has been suggested that the intracellular antioxidant defense could be enhanced by H2 both in animals and plants (Li et al., 2018; Jin et al., 2013; Xu et al., 2013; Cui et al., 2013; Xie et al., 2014; Su et al., 2018). This study has observed that during the normal senescence of cut flowers, the contents of H2O2 and TBARS progressively increased (Fig. 3B and C), indicating that the redox homeostasis was being disrupted. Changes in histochemical staining showed a similar pattern (Fig. 3A). Several observations have further indicated that the redox homeostasis was reestablished by endogenous H2. Firstly, senescence-induced H2O2 accumulation was impaired by H2 administration (Fig. 3A
conditions (Fig. 2C). As expected, Fig. 2 showed that DCPIP not only inhibited endogenous H2 production, but also accelerated cut flowers senescence. By contrast, the effects elicited by DCPIP were negated by the addition of H2. A similar delay in kiwifruit ripening was observed after a fumigation test with H2 gas directly (Hu et al., 2018) or HRW (Hu et al., 2014). Considering the danger of H2 being flammable and explosive, the application of H2 was carried out by using HRW in these experiments. However, it should be considered that the dissolved oxygen might be reduced in HRW so as cause hypoxic effects (Xie et al., 2014; Su et al., 2018). This effect of hypoxia on cut flowers can not be easily excluded. Combined, the results suggested that endogenous H2 plays an important role in delaying senescence of cut flowers. The production of endogenous H2 by the green alga S. obliquus has linked the H2 producing hydrogenase to the photosynthetic electron transport chain (Florin et al., 2001). However, the biosynthetic source and pathway for H2 in higher plants have yet to be defined. We identify 152
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Fig. 3. Representative stained photographs (A), H2O2 concentration (B) and TBARS levels (C) in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during cut flower vase storage. Afterward, 6 petals were stained with DAB, and represent picture was immediately photographed (A; Scale bar = 2 mm). Error bars represent the standard error (SE). There were 6 flowers (2 × 3 for B and C) in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests.
down the increase of TBARS content, which was partially reversed by DCPIP (Fig. 3C). Similar findings were also observed in a previous study (Hu et al., 2014), which showed that H2 protected the kiwifruit against oxidative damage during storage. Finally, the activities of antioxidant enzymes including SOD, APX, POD, and CAT, in H2-treated cut flowers were significantly higher than those in controls (Fig. 4), similar to the
and B). The lower H2O2 levels triggered by H2 were shown to be beneficial in delaying senescence, at least partially. These differences reflected that the physiological roles of H2 might be linked to the varied concentrations of H2O2. Second, endogenous H2-reestablished redox homeostasis in cut flowers was confirmed by the assessment of TBARS levels. For example, the results showed that H2 administration slowed
Fig. 4. SOD (A), APX (B), POD (C), and CAT (D) activities in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during cut flower vase storage. The activities of antioxidant enzyme were expressed on a protein mass basis. Error bars represent the standard error (SE). There were 6 (2 × 3) flowers in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests.
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Fig. 6. Schematic model describing the endogenous H2-delayed senescence in lisianthus cut flowers.
vase life. However, since a reduction of proline levels was beneficial for delaying the petal senescence (Kazemi et al., 2011), it has yet to be understood whether the increased proline content delays petal senescence or whether it was a symptom of senescence. A schematic model describing the endogenous H2-delayed senescence in lisianthus cut flowers is shown in Fig. 6, and the contribution of reestablishment of redox balance is schematically outlined. Our results suggest that H2 inclusion in vase water may be a useful strategy for prolonging flower postharvest life, as well as maintaining cut flowers in attractive open states. It is worth noting that since the solubility of H2 in water is very low, the use of HRW is safe in practical application. Indeed, both HRW and H2 gas have been previously used to improve the storage of cut flowers, fruits and mushrooms (Chen et al., 2017; Hu et al., 2014, 2018; Ren et al., 2017). Hu et al., (2018) extended this understanding by showing that H2 extends the shelf life of kiwifruit by decreasing ethylene biosynthesis. Finally, the effects of hormonal levels and other gaseous signals on senescence (Olsen et al., 2015; Naing et al., 2017; Cubría-Radío et al., 2017), the interplay among H2, phytohormones (such as ethylene, abscisic acid, etc.), and other gaseous signals, requires urgent investigation.
Fig. 5. Soluble protein (A), total chlorophyll (B), and proline (C) concentrations in untreated (control) and H2-treated vase cut flowers. Lisianthus cut flowers were incubated in treatment solutions containing 0.078 mmol L−1 H2 and/or 500 μmol L−1 DCPIP during cut flower vase storage. Error bars represent the standard error (SE). There were 6 (2 × 3) flowers in three replicates per time, and the experiments were conducted for 3 times. Bars with different letters are significantly different (P < 0.05) according to Duncan’s multiple tests.
previous reports (Jin et al., 2013; Su et al., 2018). Consistently, the changes triggered by H2 were sensitive to DCPIP. Taken together, the lower contents of H2O2 and TBARS in H2-treated cut flowers might be ascribed to the increased activities of SOD, APX, POD, and CAT. To date, other studies have also confirmed that H2 plays an antioxidant role in the delay of senescence (Chen et al., 2017; Hu et al., 2014; Ren et al., 2017). Regrettably, no research has yet defined the antioxidant mechanism of H2 (Li et al., 2018). The next study in H2 effects is envisaged to solve the questions raised in this study, since the involvement of nitric oxide in H2-triggered osmotic stress tolerance has been observed previously (Su et al., 2018). Other evidence has been observed, supporting the hypothesis that endogenous H2 was beneficial in delaying the senescence of cut flowers. Generically, the cessation of photosynthesis and degeneration of cellular structures combined with strong losses of proteins (Lim et al., 2003) and chlorophyll (López-Fernández et al., 2015) are a prominent characteristic of senescence in plants. This study observed that higher levels of H2 delayed the loss of chlorophyll and soluble proteins during senescence (Fig. 5A and B). In a previous report, it was confirmed that proline content of cut roses gradually increased during the process of senescence (Kumar et al., 2009). Based on the visible phenotypes (Figs. 1 and 2) and proline content (Fig. 5C), we suggest that the elevated proline levels induced by H2 may be beneficial in extending the
5. Conclusions This investigation revealed that endogenous H2 prolonged lisianthus cut flower life by delaying the senescence. Trials were conducted to investigate the effect of altering endogenous H2 levels on the vase life of cut flowers, indicating that endogenous H2 may be critical in delaying senescence. Our results further confirmed that redox homeostasis was reestablished by vase water supplementation with H2, which might contribute to an increase in the antioxidant potential of the cut flowers. Decreased soluble protein, increased total chlorophyll and proline contents were also modulated by H2 and its antagonist, DCPIP. These findings are practically significant for both postharvest physiology and applied biology.
Conflict of interest statement The authors declare that they have no conflict of interest. 154
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Acknowledgements
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