Harpin enhances antioxidant nutrient accumulation and decreases enzymatic browning in stored soybean sprouts

: Enzymatic browning causes quality losses in the soybean sprout industry. Herein, the e ﬀ ects of harpin, a proteinaceous bacterial elicitor isolated from Erwinia amylovora , in regulating enzymatic browning and anti - oxidant nutrient accumulation in stored soybean sprout were investigated. Enhanced soybean sprout growth ( eval - uated by sprout length and fresh weight ) occurred after spraying three times ( 0, 2, and 4 days after sowing ) with 30 mg L − 1 harpin during the growth stage. The decline in vitamin C and total phenolic contents and total antioxidant capacity ( evaluated by Fe 3 + reducing power ) were attenuated by harpin during storage. Harpin increased phenylalanine ammonia - lyase, NADPH oxidase ( NOX ) , superoxide dismu - tase, and catalase activities and inhibited polyphenol oxidase activity and enzymatic browning in soybean sprouts during storage. However, this harpin - promoted sprout growth, enhanced antioxidant accumulation and enzyme activity and improved sprout quality could be partly, but drastically, abolished using diphenyleneiodonium chloride, a speci ﬁ c inhibitor of NOX. Moreover, the mechanisms by which harpin in ﬂ uences antioxidant accumulation and enzymatic browning in soybean sprouts during storage were discussed from the perspective of NOX - mediated H 2 O 2 signalling.


Introduction
Germination improves the nutritional quality of legumes, which are an important source of food for many countries [1].Reports have shown that endoenzymes can hydrolyse macromolecules (e.g., starches, protein, and lipids) to produce free amino acids, available carbohydrates, dietary fibre, and bioactive compounds during seed germination [1,2].In general, vitamins and polyphenols are considered beneficial as antioxidants, and their levels and type can vary dramatically during germination [3].These antioxidants are required to scavenge reactive oxygen species (ROS), excessive amounts of which can lead to oxidative damage (e.g., lipid peroxidation) in plants [4].
Moreover, ROS are implicated in seed germination and postharvest vegetable senescence [5,6].High ROS production could lead to oxidative damage and promote plant senescence [4,7].However, ROS, such as H 2 O 2 , also act as signalling molecules at nanomolar levels in plants [8].NADPH oxidase (NOX), localized on the plasma membrane, is an important enzyme for ROS production and thus play key roles in many biological processes [9].Interestingly, this unique ROS-producing enzyme can integrate seed germination [10] and postharvest fruit senescence [11].However, the antioxidant system, which consists of enzymatic (e.g., superoxide dismutase (SOD) and catalase (CAT)) and non-enzymatic antioxidants (e.g., vitamin C), can efficiently scavenge the over-produced ROS [4].
Before consumption, sprouts are usually stored at low temperature to reduce microbiological contamination and retard sprout growth [12].However, enzymatic browning can cause great losses in the quality of vegetables and fruits after minimal processing during storage [13][14][15].Postharvest browning of vegetables and fruits is generally thought to involve rapid degradation of polyphenols by polyphenol oxidase (PPO, EC1.14.18.1), resulting in brown by-products [16][17][18][19].This is particularly relevant for soybean (Glycine max L. Merr.), which contains abundant polyphenols that are highly susceptible to enzymatic browning, causing a decline in polyphenol accumulation and thereby reducing nutritional quality [20].Thus, many methods have been developed to control the postharvest enzymatic browning in sprout [21].
Soybean is not only a source of oil (∼20%) and protein (∼40%) but also antinutritional compounds; however, these can be lost during postharvest processing [22].Soybeans are utilized in a variety of foods such as soymilk, soybean sprouts, and other fermented food products [23].
Harpin, a proteinaceous bacterial elicitor isolated from Erwinia amylovora, is commonly used in various biological agriculture practices [24,25].Treatment with harpin, which binds to plant receptors, induces a hypersensitive response and activates several biochemical pathways related to growth and resistance enhancement [26].Harpin can also induce phenylalanine ammonia-lyase (PAL, EC4.3.1.5)and PPO activities and enhance the accumulation of phenolics in plants [25,27].In addition, harpin could activate NOX and promote ROS production in plants [28,29].
Herein, harpin was applied to soybean seeds and sprouts, and its effects were investigated.First, whether harpin enhanced seed germination, sprout growth, and bioactive metabolites (e.g., vitamin C and phenolics) accumulation in soybean sprouts was assessed.Second, how harpin affects sprout browning and related enzyme activities during storage was determined.Finally, the possible associated mechanisms were investigated.

Experimental design
Soybean seeds were purchased from Luoyang Kechuang Seed Co. (Luoyang, China), sterilized, and then rinsed with distilled water at 25°C for 15 min.Healthy seeds of the same size were then sown evenly on trays filled with vermiculite and kept in a growth chamber under dark conditions at room temperature with 70% humidity.According to our preliminary experimental results (data not shown), 30 mg L −1 harpin (Messenger, Eden Bioscience Co., Bothell, WA, USA) or in combination with DPI (50 µM) was sprayed to treat soybean seeds and sprouts three times at 48 h intervals (0, 2, and 4 days after sowing).The control groups were sprayed with distilled water.Sprouts were collected at days 1, 3, 5, and 7 after sowing to analyse sprout growth.Meanwhile, harvested soybean sprouts (day 7 after sowing) were stored without light exposure at room temperature with 70% humidity and collected on postharvest days 0, 3, 6, and 9.All samples were frozen in liquid nitrogen immediately and kept in polyethylene bags at −70°C for subsequent analysis of H 2 O 2 content, lipid peroxidation, vitamin C levels, phenolic contents, total antioxidant capacity, and enzyme (NOX, SOD, CAT, PAL, and PPO) activities.For all assays, three replicates of 50 seeds were included for each treatment.

Sprout growth and fresh weight assay
Sprout length (excluding roots) was measured using vernier callipers and fresh weight (excluding roots) was measured using an electronic balance.

Browning index assay
The browning index of soybean sprouts was evaluated using a previously reported method [30].The absorbance was recorded at 420 nm using an ultraviolet (UV)-1280 spectrophotometer (Shimadzu, Kyoto, Japan).

Antioxidant content and antioxidant capacity assay
The titrimetric method with 2,6-DCPIP was used to evaluate the vitamin C content of soybean sprouts [27].
Total phenolics content was assayed using the Folin-Ciocalteu reagent method [31].The absorbance was recorded at 760 nm using a UV-visible (Vis) spectrophotometer (Shimadzu) and the results are expressed as gallic acid equivalents (mg g −1 of dry weight).
For total antioxidant capacity assay, the ferric reducing ability of plasma assay [32] was used.The absorbance of the reaction mixture was recorded at 593 nm using UV-Vis spectroscopy.

Phenolic-related enzyme activities assay
PAL activity was determined as the rate of conversion of L-phenyl alanine to trans-cinnamic acid at 290 nm as described previously [33].The absorbance was recorded at 290 nm and the enzyme activity is expressed as mmol trans-cinnamic acid min −1 mg −1 protein.
PPO activity was measured using a previously described method [14].The absorbance was measured at 420 nm and one unit (U) of activity was defined as the amount of enzyme that causes an increase of 0.001 absorbance per minute.
The soluble protein concentration was determined using the Bradford method [34], with bovine serum albumin as the standard.

Assays of ROS-related enzyme activities
The SOD activity was measured using the NBT method [35].The increase in absorbance due to formazan formation was read at 560 nm.One unit of SOD activity is defined as the amount of enzyme that inhibits NBT photoreduction by 50%.
CAT activity was measured using a previously described method [36].The activity was determined by following the consumption of H 2 O 2 at 240 nm for 3 min.
The NOX enzyme extraction and assay were performed using a Plant NOX assay Kit (GenMed Scientifics Inc., Arlington, MA, USA; GMS50096.3v.A.) in accordance with the manufacturer's instructions.The activity was calculated by monitoring the decrease in absorbance at 340 nm [37].
Hydrogen peroxide (H 2 O 2 ) accumulation in soybean sprouts was measured using the oxidation xylenol orange assay [39].The absorbance by the Fe(III)-xylenol orange complex was recorded at 560 nm.

Data analysis
Experiments were conducted in a completely randomized design.Three replicates were included for each treatment, except for the seed germination and sprout growth assays (five replicates).All data were analysed using Duncan's multiple range test (p < 0.05) using SPSS 13.0 software (IBM Cop., Armonk, NY, USA).
3 Results and analysis

Effects of harpin on sprout growth
Harpin significantly enhanced sprout length by approximately 24% and fresh weight by 17% at 7 days after sowing compared with those in the controls (Figure 1a and b; p < 0.05).However, this harpin-promoted sprout growth could be partly and significantly abolished using DPI (Figure 1; p < 0.05).Compared with the control, DPI addition reduced the sprout length and fresh weight by approximately 12 and 8%, respectively, in harpin-treated sprouts at 7 days after sowing (Figure 1; p < 0.05).

Effects of harpin on antioxidant accumulation and lipid peroxidation
Compared with those at 0 days, the vitamin C levels, the total phenolics content, and the total antioxidant capacity decreased by approximately 64, 36, and 43%, respectively, in soybean sprouts at 9 days after storage (Figure 2; p < 0.05).These effects were partly reversed by harpin in soybean sprouts during storage (Figure 2a-c; p < 0.05).Specifically, harpin increased vitamin C accumulation, the total phenolics content, and total antioxidant capacity in soybean sprouts by approximately 126, 49, and 52%, respectively, compared with those in the controls at 9 days after sowing (Figure 2a-c; p < 0.05).However, this harpin-promoted antioxidant accumulation could be partly and significantly reversed using DPI (Figure 2a-c; p < 0.05).
In detail, application of DPI decreased vitamin C accumulation, the total phenolics content, and the total antioxidant capacity by approximately 25, 14, and 16%, respectively, in harpin-treated sprouts after storage for 9 days (Figure 1a-c; p < 0.05).In addition, harpin profoundly changed the lipid peroxidation (as evaluated by TBARS) of soybean sprouts during storage (Figure 2d; p < 0.05).Compared with that in the control, harpin application reduced TBARS accumulation by approximately 18, 27, and 35% in sprouts at 3, 6, and 9 days after harvest, respectively (Figure 2d; p < 0.05).However, these effects of harpin on the TBARS content could be profoundly modified by DPI.In detail, DPI enhanced TBARS accumulation in soybean sprouts by approximately 8, 13, and 16% at 3, 6, and 9 days after harvest, respectively (Figure 2d; p < 0.05).

Effects of harpin on PAL and PPO activities and sprout browning
Compared with that after 0 days of storage, the PAL and PPO activities decreased by approximately 70 and 34%, respectively, after storage for 9 days (Figure 3a and b; p < 0.05).However, harpin inhibited this decrease in PAL activity and increase in PPO activity in soybean sprouts during storage.Treatment with harpin increased PAL activity by approximately 42% and decreased PPO activity by 8% relative to those in the controls after storage for 6 days (Figure 3a and b; p < 0.05).However, this harpininduced increase in PAL activity was significantly reversed by DPI (Figure 3a and b; p < 0.05).In detail, DPI + harpin reduced the PAL activity by approximately 17 and 24% compared with that in the harpin-treated sprouts after at 6 and 9 days after harvest, respectively (Figure 3a and b; p < 0.05).Similarly, the harpin-induced decrease in PPO activity was significantly reversed using DPI (Figure 3a and b; p < 0.05).In detail, DPI + harpin increased the PPO activity by approximately 14 and 17% compared with that in the harpin-treated sprouts at 3 and 6 days after harvest, respectively (Figure 3a and b; p < 0.05).
Harpin inhibited enzymatic browning in soybean sprouts during storage (Figure 3c; p < 0.05).Specifically, harpin decreased the browning index by approximately 63 and 66% in soybean sprouts relative to those in the controls after storage for 6 and 9 days, respectively (Figure 3c; p < 0.05).This harpin-induced inhibition of enzymatic browning was significantly reversed using DPI (Figure 3c and d; p < 0.05).In detail, DPI + harpin increased the browning index by approximately 97 and 80% compared with that in the harpin-treated sprouts at 6 and 9 days, respectively, after harvest (Figure 3c and d; p < 0.05).

Effects of harpin on ROS-related enzyme activities and H 2 O 2 content
Compared with those in the control, harpin significantly increased the activities of NOX, SOD, and CAT in soybean sprouts during storage stage (Figure 4a, c, and d; p < 0.05).In detail, treatment with harpin increased the NOX, SOD, and CAT activities by approximately 113, 105, and 77%, respectively, in soybean sprouts at 9 days after harvest (Figure 4a, c, and d; p < 0.05).However, these harpin-enhanced enzyme activities could be partly but significantly attenuated using DPI.In detail, DPI decreased the NOX, SOD, and CAT activities by approximately 24, 10, and 12%, respectively, in harpintreated sprouts at 3 days after harvest (Figure 4a, c, and d; p < 0.05).In addition, treatment with harpin or DPI changed the H 2 O 2 content significantly in soybean sprouts during storage (Figure 4b; p < 0.05).In detail, harpin application reduced H 2 O 2 accumulation by approximately 9 and 18% in soybean sprouts at 6 and 9 days after harvest, respectively, compared with that in the control (Figure 4b; p < 0.05).However, treatment with DPI + harpin enhanced the H 2 O 2 content by approximately 6 and 12% in soybean sprouts at 6 and 9 days after harvest, respectively, compared with that in the harpin group (Figure 4b; p < 0.05).

Discussion
In the present study, the effects of harpin on soybean sprout growth, antioxidant nutrient accumulation, and enzymatic browning were investigated.Application of harpin (30 mg L −1 ) markedly accelerated sprout growth (evaluated by sprout length and fresh weight) compared with that of the controls (Figure 1).These results partly agreed with those of a previous report showing that harpin-promoted Arabidopsis growth [40].According to the resource availability hypothesis [41], enhancement of plant growth always reduces defence compound accumulation [42].However, a previous study showed that expression of the harpin coding gene hrf1 enhanced drought tolerance in rice by increasing antioxidant enzyme activities [43].Thus, whether harpin decreases or elevates antioxidant accumulation (i.e., vitamin C and phenolics) in soybean sprouts remains contentious.Previous studies showed that harpin significantly increased phenolic compound accumulation and antioxidant capacity in lettuce varieties [44].Here, the decline in vitamin C and the total phenolic content, coupled with total antioxidant capacity, was drastically attenuated by harpin during storage (Figure 2).This is partly in accordance with previous reports showing that harpin markedly enhanced vitamin C levels in postharvest jujube fruit [27] and increased the accumulation of phenolics in lettuce [45] during storage.In another study, harpin reduced the extent of lipid peroxidation, which was used to evaluate postharvest senescence [38].This suggested that the harpin-induced delay in postharvest senescence of soybean sprouts could be partly attributed to enhanced antioxidant accumulation.Published data show that vitamin C could inhibit PPO [16], a key enzyme for the degradation of phenolics [15].Therefore, the effects of harpin on phenolic metabolism-related enzyme activities were also investigated.
The activities of PAL (a key enzyme for phenolics biosynthesis) [33] and PPO in harpin-treated soybean sprouts during storage were also determined (Figure 3a and b).The decline in PAL activity was significantly inhibited by harpin in soybean sprouts during storage, whereas harpin significantly inhibited the increase in PPO activity in soybean sprouts relative that in the controls following harvest.Thus, the harpin-induced increase in the total phenolics content in soybean sprouts could be partly attributed to the high PAL and low PPO activities during storage.How does increasing the phenolics content affects enzymatic browning in soybean sprouts during storage?
PPO is a key enzyme for enzymatic browning in postharvest fruit [16][17][18].In the present study, the content of phenolics in harpin-treated soybean sprouts was increased significantly compared with that in the controls.Subsequently, the effects of harpin in accelerating enzymatic browning in soybean sprouts were investigated by measuring the browning index in harpin-treated and control soybean sprouts.The results showed that harpin could significantly reduce the browning index (Figure 3c).Previous studies showed that an increase in vitamin C correlated with a decrease in PPO activity because vitamin C is a natural inhibitor of PPO [16,45].Thus, harpin might inhibit enzymatic browning partly by inhibiting PPO activity via vitamin C in soybean sprouts during storage.Herein, the enhancement of vitamin C and phenolics accumulation might be an efficient strategy by which harpin reduces enzymatic browning in postharvest soybean sprouts.Previous report showed that curcumin application could induce the biosynthesis and accumulation of antioxidants (e.g., vitamin C and CAT), which could decrease PPO activities and prevent this enzyme from reacting with phenolics, thereby reducing the occurrence of enzymatic browning [46].However, the mechanism that controls this harpin-induced antioxidant nutrient accumulation in soybean sprouts during storage is unknown.
Previous reports showed that harpin could activate NOX and induce H 2 O 2 production in plants [28,29].Moreover, studies showed that H 2 O 2 could stimulate plant growth [47], activate PAL activities and enhance phenolic compound accumulation in plants [48,49].In this study, harpin-promoted sprout growth, delayed senescence, enhanced antioxidant accumulation, and reduced enzymatic browning, which could be partly, but significantly, reversed using DPI (Figure 4a and b), a specific inhibitor of NOX [50].This suggested that the harpin-regulated postharvest soybean sprout quality is closely associated with NOX-originated H 2 O 2 .In addition, harpin enhanced NOX activities during storage (Figure 4a).However, harpin significantly reduced H 2 O 2 and TBARS accumulation in soybean sprouts during storage (Figures 2d and 4b).Moreover, our results showed significantly higher activities of SOD and CAT, two major ROS scavenging enzymes, in harpin-treated sprouts during storage (Figure 4c and d).Thus, the harpin-induced inhibition of peroxide accumulation in stored soybean sprouts could be attributed to the high activities of antioxidant enzymes (i.e., SOD and CAT) and the high accumulation of non-enzymatic antioxidants (vitamin C and phenolics).Moreover, this harpin-induced increase in NOX activities and H 2 O 2 production, as well as the elevated SOD and CAT activities, could be partly but significantly abolished using DPI (Figure 4).This partly agrees with the results of previous reports, which showed that NOX-mediated H 2 O 2 could further increase antioxidant enzyme (e.g., SOD and CAT) activities and non-enzymatic antioxidant accumulation (e.g., ascorbic acid) in plants under abiotic stress [51,52].Our results also showed that harpin could delay soybean sprout senescence (evaluated by the TBARS content) by elevating the enzymatic (i.e., SOD and CAT) and non-enzymatic (i.e., vitamin C and phenolics) antioxidant accumulation during storage.These data suggested that NOX-originated H 2 O 2 is required for harpin-induced promotion of soybean sprout growth, nonenzymatic antioxidant accumulation, and quality improvement during storage.

Conclusion
Harpin can promote soybean sprout growth, reduce lipid peroxidation, and increase the NOX-originated H 2 O 2 production.Harpin increased PAL, SOD, and CAT activities in soybean sprouts during storage.Harpin-induced antioxidant accumulation but inhibited the increase of PPO activity and reduced enzymatic browning in soybean sprouts during storage.

Figure 1 :
Figure 1: Sprout length and fresh weight.Effects of 30 mg L −1 harpin on sprout growth (a), sprout length (except root) (b) and fresh weight (c) of soybean sprouts after treatment for 7 days.Bars represent standard deviation of the mean (n = 5); means associated with the same letter are not significantly different (p < 0.05).DPI, diphenyleneiodonium chloride.

Figure 2 :
Figure 2: Antioxidant accumulation and lipid peroxidation in soybean sprouts.Effects of 30 mg L −1 harpin on vitamin C level (a), total phenolics content (b), total antioxidant capacity (c) and lipid peroxidation (d) in soybean sprouts during storage.Bars represent standard deviation of the mean (n = 3); means associated with the same letter are not significantly different during preharvest or postharvest stages (p < 0.05).DPI, diphenyleneiodonium chloride.

Figure 3 :
Figure 3: Enzyme activities and browning index.Effects of 30 mg L −1 harpin on PAL activity (a), PPO activity (b) and browning index (c) in soybean sprouts during storage.Bars represent standard deviation of the mean (n = 3); means associated with the same letter are not significantly different during preharvest or postharvest stages (p < 0.05).DPI, diphenyleneiodonium chloride; PAL, phenylalanine ammonia-lyase; PPO, polyphenol oxidase.

Figure 4 :
Figure 4: H 2 O 2 content and ROS-related enzyme activities.Effects of 30 mg L −1 harpin on NADPH oxidase activity (a), hydrogen peroxide content (b), superoxide dismutase activity (c) and catalase activity (d) in soybean sprouts during storage.Bars represent standard deviation of the mean (n = 3); means associated with the same letter are not significantly different during preharvest or postharvest stages (p < 0.05).DPI, diphenyleneiodonium chloride.