Abstract
NAC transcription factors have been characterized in numerous plants, and the NAC gene has been shown to be involved not only in plant growth and development, but also in plant responses to abiotic and biological stresses, such as drought, high salinity, low temperature, and anaerobic/hypoxic stress. Creating an environment of anaerobic/hypoxic stress has been shown to be one of the effective storage methods for delaying the browning of fresh-cut lotus (Nelumbo nucifera) root. However, whether NAC is associated with lotus root browning under anaerobic stress has not been studied. In this study, vacuum packaging (VP; anaerobic/hypoxic stress) effectively delayed the browning of fresh-cut lotus root. The changes in the expressions of NnPAL1, NnPPOA, and NnPOD2/3 were consistent with phenylalanine aminolase, polyphenol oxidase (PPO), and peroxidase (POD) enzyme activity changes and lotus root browning. Using RNA sequencing, five NnNAC genes were isolated and studied. Transcriptional analysis indicates that the NnNAC genes showed different responses to VP. The expressions of NnNAC1/4 were inhibited by VP, which was consistent with the observed change in the degree of fresh-cut lotus root browning. However, NnNAC2 messenger RNA (mRNA) levels were upregulated, and the expressions of NnNAC3/5 showed no clear differences under different packaging scenarios. Thus, NnNAC1/4 were identified as promising candidates for further transcriptional regulation analysis in lotus root to understand more fully the molecular mechanism of browning under anaerobic/anoxic stress.
With the increasing interest in healthy and nutritious diets, and continuing changes in consumer lifestyles, the consumption of fresh-cut fruits and vegetables has become increasingly popular (Chen et al., 2016; Sipahi et al., 2013). However, the short shelf-lives and decline in the postprocessing of fresh-cut fruits and vegetables limit their production for retail. Lotus (Nelumbo nucifera) root is an important aquatic vegetable in China. Fresh-cut lotus root is a popular vegetable, but browning is one of the major factors limiting its quality for sale. Thus, browning control is a critical issue in the lotus root industry.
Artificial treatments have been developed successfully to delay the browning of fresh-cut lotus roots, including modified-atmosphere packaging (MAP) (Cheng et al., 2015), VP in low-temperature storage (Min et al., 2017, 2019; Xie et al., 2018), heat treatment (Tsouvaltzis et al., 2011), and so on. Among the various treatments, VP in low-temperature storage has been most successful in maintaining quality and prolonging the shelf life of fresh-cut lotus root (Aquino-Bolanos et al., 2000; Sharma and Rao, 2017; Toivonen and Brummell, 2008). Total phenolic content, oxygen, PPO, and POD are known to be the main factors that cause browning (Du et al., 2009; Walker and Ferrar, 1998). Our previous research reported that NnPAL1, NnPPOA, and NnPOD2/3 are the most promising candidates for genes to target to control fresh-cut lotus root browning with MAP and VP in low-temperature storage (Min et al., 2017, 2018, 2019).
Although the anaerobic/hypoxic environment produced by VP creates a kind of unfavorable stress to plants, it has a remarkable effect on delaying fresh-cut lotus root browning. In our previous study, we explored the role of ethylene response factor (ERF) in delaying lotus root browning in VP under low temperatures and found that NnERF4/5 could be important candidates for regulators of fresh-cut lotus root browning (Min et al., 2018). However, the relationship between other transcription factors and fresh-cut lotus root browning has rarely been reported. In addition to ERFs, NAC genes are among the important transcription factors reported to be involved in anaerobic/hypoxic responses, which indicate their potential to be involved in lotus root browning under VP. NAC (NAM, ATAF1/2, CUC2) transcription factors are a class of transcriptional regulators unique to higher plants. More than 100 NAC genes have been identified and characterized in the model plant Arabidopsis thaliana (Nuruzzaman et al., 2010). Among them, ANAC019, ANAC055, and ANAC072 enhance tolerance to drought stress in A. thaliana (Tran et al., 2004), and ANAC2 is involved in the response to plant hormones (He et al., 2005). In addition, ANAC102 is induced by hypoxia stress (0.1% oxygen), and the expression of some genes related to hypoxia stress is enhanced in transgenic plants overexpressing ANAC102. After knocking out the ANAC102 gene, the seed germination rate under hypoxia stress was shown to be significantly decreased (Christianson et al., 2009).
In addition, some studies have also shown that there is a correlation between NAC and phenylalanine ammonia lyase (PAL), PPO, and POD enzymes, and coding genes. NAC and the PAL gene showed overlapping expression patterns in the response of Citrus sinensis to cold exposure (Crifò et al., 2012). The expressions of NAC and PAL were upregulated simultaneously during gibberellic acid (GA3) treatment in GA3-induced xylem development in Betula (Guo et al., 2015). Overexpression of ThNAC13 in Tamarix hispida induced POD activities, and ThNAC13 induced the expression of PODs in transgenic A. thaliana (Wang et al., 2017b). DgNAC1-overexpressed transgenic Chrysanthemum eticuspe showed greater activities of superoxide dismutase, POD, and catalase under salt stress (Wang et al., 2017a). This suggested that NAC transcription factors have the potential to be involved in lotus root browning, but experimental evidence is lacking in lotus root.
In our study, five NnNAC genes were isolated from lotus root based on the RNA sequencing (RNA-Seq) database and a National Center for Biotechnology Information (NCBI) database. The effect of VP on browning, total phenol, PPO, PAL, and POD enzyme activity; and PAL, PPO, POD, and NAC gene expression changes were analyzed. Some PAL, PPO, POD, and NnNAC genes were found to correlate positively with lotus root browning, and the possible roles of these and other NnNAC genes are discussed here.
Materials and Methods
Sample preparation and treatments.
Lotus roots (cv. Wuzhi 2) were purchased from the Caidian District, Wuhan, China, in 2016 and were shipped immediately to the laboratory. Lotus roots that were not wounded and were of uniform size were selected for treatment. After the lotus roots were stored at 4 °C for 24 h, they were rinsed gently with tap water by hand to remove any silt. Next, the lotus roots were peeled and cut into 5-mm-thick slices. The slices of lotus root were divided into two groups for different treatments: a) VP and b) atmospheric pressure packaging (AP). Finally, the samples were stored at 4 ± 1 °C for 35 d, and sampling was performed every 7 d. Fifty kilograms lotus root was purchased for each experiment and ≈20 kg sliced root was packaged. The size of the vacuum packages was 200 × 280 mm and the material was polyethylene. The size of atmospheric pressure packages was 180 × 120 × 25 mm and the materials were polypropylene pallet and polyvinyl chloride plastic wrap. One hundred fifty replications samples were available for each treatment, and 20 replications samples were use during each 7-d sampling time. The experiment repeated three times over time.
Browning degree (BD) and total phenolic content.
The BD and total phenolic content were determined according to the methods described in our previous study (Min et al., 2017). Briefly, the BD was determined as follows: Lotus root tissue (3.0-g slices) was homogenized with 30 mL distilled water at 4 °C and then centrifuged for 5 min at 10,000 gn. Next, the supernatant was collected and incubated for 5 min in a water bath at 25 °C. The absorbance was measured at 410 nm using a spectrophotometer, and the BD was expressed as A410 × 10.
The total phenolic content was determined as follows: Lotus root tissue (3.0-g slices) was homogenized with 30 mL ethanol (60%) and then centrifuged for 5 min at 10,000 gn. The supernatant (10 mL) was collected and mixed with 40 mL 60% ethanol to obtain an extract. The extract (0.125 mL) was mixed with 0.625 mL distilled water, and then 0.125 mL Folin phenol reagent was added. After fully mixing the solution, the mixture was left at room temperature for 3 min. Then, 1.25 mL Na2CO3 (7%) and distilled water (1.0 mL) were added. The mixture was incubated at 25 °C in the dark for 90 min, after which the absorbance was measured at 760 nm using a spectrophotometer. The standard curve of gallic acid was used to quantify the total phenol content. The result was the expression of milligrams gallic acid equivalents per kilogram fresh weight, and all treatments were repeated three times.
PAL, PPO, and POD activity.
PAL, PPO, and POD were extracted and analyzed according to the method described in our previous study (Min et al., 2017). The PAL enzyme was extracted as follows: In an ice bath, 0.1 g lotus root was added to 1 mL reagent 1 and centrifuged for 10 min at 10,000 gn at 4 °C. The supernatant was then collected as a crude extract. The PAL enzyme activity unit (U) was defined by spectrophotometry at 290 nm, and 1 U was defined spectrophotometrically as a change of 0.1 in absorbance per min.
The PPO enzyme was extracted as follows: Fresh-cut lotus root (3.0 g) was homogenized with 50 mL phosphate buffered saline (0.05 mol·L–1, pH 7.0) in and ice mortar and then centrifuged at 3820 gn for 15 min at 4 °C. The PPO enzyme activity unit was defined by spectrophotometry at 420 nm; 1 U was described as the amount of enzyme leading to a change in absorbance of 0.001 per min.
The POD enzyme was extracted as follows: Fresh-cut lotus root was homogenized in 5.0 mL 0.2 mol·L–1 extraction buffer [pH 7.0, 1 mol·L–1 polyethylene glycol, 4% (w/v) polyvinyl polypyrrolidone, 1% (w/v) nonionic surfactant] (Triton X-100; Amresco, Solon, OH) and the solution was centrifuged at 15,290 gn for 30 min at 4 °C. The POD enzyme activity unit was defined by spectrophotometry at 470 nm; 1 U was defined spectrophotometrically as an increase of 0.01 in absorbance per min.
RNA extraction and complementary DNA (cDNA) synthesis.
Total RNA was prepared according to the method described in our previous study (Min et al., 2017). TURBO Dnase (Ambion, Austin, TX) was used to remove the DNA traces in the total RNA that contaminated the genome. The iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) was used for cDNA synthesis of 1.0 mg RNA. At each sampling point, three biological replicates were used for RNA extraction experiments.
Gene isolation and sequence analysis.
The NAC genes were isolated based on the RNA-Seq and NCBI databases. RNA sequencing was conducted by the Beijing Genome Institute (Shenzhen, China) according to the method described previously (Min et al., 2014). The sequences of primers for gene cloning are listed in Table 1. Phylogenetic trees of NAC genes were generated using ClustalX (version 1.81; Min et al., 2018) and calculated using FigTree (version 1.3.1; University of Edinburgh, Edinburgh, UK). The deduced amino acid sequences of homologous genes of A. thaliana were obtained from The Arabidopsis Information Resource.
Sequences of the primers used for NAC genes partial coding sequences clone.
Oligonucleotide primers and real-time polymerase chain reaction (PCR) analysis.
Oligonucleotide primers were designed using primer 3 for real-time PCR analysis (Min et al., 2017). The specificity of primers was determined by melting curves and PCR product resequencing, as described in our previous study (Min et al., 2012). The sequences of oligonucleotide primers are listed in Table 2.
Sequences of the primers for real-time polymerase chain reaction (PCR) of NAC genes.
Ssofast EvaGreen Supermix kit (Bio-Rad Laboratories) and CFX96 fluorescence PCR were used for real-time PCR analysis for gene expression study according to the method described in our previous work (Min et al., 2017).
Statistical analysis.
Figures were drawn using Origin software (version 8.0; OriginLab, Northampton, MA). Statistical analysis of differences was conducted using the least significant difference via DPS7.05 software (Zhejiang University, Hangzhou, China).
Results and Discussion
BD and total phenol content.
The fresh-cut lotus root used in our research had a mean BD value of 0.2150 at day 0 (Fig. 1). VP promoted an increase in the BD value to 0.29 after 35 d, whereas BD showed a greater growth in AP (0.61 at day 35) (Fig. 1). As shown in Fig. 2, the effects of the two different packaging methods on the total phenol content were clearly different during storage. Slower changes were found in the samples subjected to VP, changing from 96.89 mg·kg–1 at day 0 to 108.98 mg·kg–1 at day 35. However, the samples with AP showed faster growth, from 96.89 mg·kg–1 at day 0 to 159.25 mg·kg–1 at day 35.
Effects of two different packaging methods on browning degree of fresh-cut ‘Wuzhi 2’ lotus root. Browning degree was chosen as the main index of fruit and vegetable browning. Fresh-cut lotus root was packaged by vacuum packaging [VP (circles)] and atmospheric pressure packaging [AP (squares)] separately. Error bars represent se from three biological replicates. lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
Effects of two different packaging methods on total phenol content of fresh-cut ‘Wuzhi 2’ lotus root. The total phenol was the main substrate for enzymatic browning in fruits and vegetables. Fresh-cut lotus root was packaged by vacuum packaging [VP (circles)] and atmospheric pressure packaging [AP (squares)] separately. Error bars represent se from three biological replicates. FW = fresh weight; lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
BD value was chosen as the main index for evaluating browning. By determining the BD in storage after using two different packaging methods, it was clear that the BD of the samples subjected to VP (anaerobic stress) was significantly less than that of the control group. These results confirmed and extended our previous finding (Min et al., 2019).
PAL, PPO, and POD activities.
PAL, PPO, and POD enzyme activities under VP and AP were significantly different during storage. As shown in Fig. 3, the PAL activity of the samples undergoing VP and AP showed different magnitudes of change, from the initial value of 12.802 to 12.225 U/g (VP) and 26.354 U/g (AP) at day 35.
Effects of two different packaging methods on phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activity of fresh-cut ‘Wuzhi 2’ lotus root. PAL, PPO, and POD have been proposed as being involved in enzymatic browning. Fresh-cut lotus root was packaged by vacuum packaging [VP (circles)] and atmospheric pressure packaging [AP (squares)] separately. Error bars represent se from three biological replicates. lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
The PPO activity of VP increased slowly, from the initial value of 0.396 to 0.604 U/g at day 35. However, PPO activity increased rapidly under AP, from the initial value of 0.396 to 1.18 U/g at day 35. The POD activity of the samples of VP increased slowly, from the initial value of 0.044 to 0.085 U/g at day 35, but POD activity increased rapidly when subjected to AP, from the initial value of 0.044 to 0.146 U/g at day 35.
It has been suggested in previous work that PAL, PPO, and POD mainly involve enzymatic browning of lotus root (Soliva-Fortuny and Martıìn-Belloso, 2003; Zheng et al., 2016). Changes in PAL, PPO, and POD enzymatic activity in storage under two different packaging methods were analyzed. These results showed that PAL, PPO, and POD activity were inhibited during VP, which occurred in parallel with less significant browning during storage, which was consistent with previous reports (Min et al., 2019),
NnPAL, NnPPO, and NnPOD gene expression.
There was an obvious differential response to different packaging methods for the two PAL, two PPO, and seven POD genes. As shown in Fig. 4, compared with AP, NnPAL1 expression in ‘Wuzhi 2’ was inhibited by VP, and the expression of NnPAL2 was induced by VP. The expressions of NnPPOA and NnPPOC were inhibited by VP during the storage process, of which NnPPOA was more obviously inhibited by VP (Fig. 5). Moreover, the relative expression was less than that of AP, which was consistent with changes in the PPO enzyme activity. The expressions of NnPOD2 and NnPOD3 in VP decreased by more than 50 times. The expressions of NnPOD1 and NnPOD5 were also inhibited by VP during the early stage, but the inhibition multiples were not as obvious as those of NnPOD2 and NnPOD3 (Fig. 6). NnPOD4 and NnPOD6 were induced by VP, especially NnPOD6 (Fig. 6). NnPOD7 was downregulated during storage, but there was no significant difference between the two groups (Fig. 6). In our previous study, NnPAL1, NnPPOA, and NnPOD2/3 of fresh-cut lotus root (‘Elian 5’) with high-concentration CO2 MAP were also more obviously downregulated than other genes (Xie et al., 2018).
Messenger RNA amounts from phenylalanine ammonia lyase (PAL) genes in response to different packaging methods. Fresh-cut ‘Wuzhi 2’ lotus root was packaged by vacuum packaging [VP (black)] and atmospheric pressure packaging [AP (white)] separately. Zero-day sample values were set at 1. Error bars represent se from three biological replicates. lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
Messenger RNA amounts from polyphenol oxidase (PPO) genes in response to different packaging methods. Fresh-cut ‘Wuzhi 2’ lotus root was packaged by vacuum packaging [VP (black)] and atmospheric pressure packaging [AP (white)] separately. Error bars represent se from three biological replicates. lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
Messenger RNA amounts from peroxidase (POD) genes in response to different packaging methods. Fresh-cut ‘Wuzhi 2’ lotus root was packaged by vacuum packaging [VP (black)] and atmospheric pressure packaging [AP (white)] separately. Error bars represent se from three biological replicates. lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
The experimental results based on two cultivars showed that NnPAL1, NnPPOA, and NnPOD2/3 were significantly downregulated by VP (low oxygen). In addition, changes in the expressions of NnPAL1, NnPPOA, and NnPOD2/3 were consistent with PAL, PPO, and POD enzyme activity changes. Previous studies and the current results further prove that NnPAL1, NnPPOA, and NnPOD2/3 may be important candidates for genes to target to control fresh-cut lotus root browning (Min et al., 2017, 2018, 2019).
NAC gene isolation and phylogenetic analysis.
Five NAC cDNAs (NnNAC1-XP_010243336.1, NnNAC2-XP_010249131.1, NnNAC3-XP_010275272.1, NnNAC4-XP_010257746.1, and NnNAC5-XP_010269977.1) were isolated from ‘Wuzhi 2’ lotus root using the RNA-Seq and NCBI databases. To study the phylogenetic relationship between the NAC proteins in lotus root and A. thaliana, a phylogenetic tree was constructed based on their translated amino acid sequences using ClustalX 2.1 and FigTree 3.1 software. As shown in Fig. 7, NnNAC1/3/4 are closer to ANAC063, and this type of NAC gene plays an important role in drought resistance and salt tolerance (Yokotani et al., 2009). ONAC063 expression was not induced by high-salinity in Oryza sativa roots, and the seeds of ONAC063-expressing transgenic A. thaliana showed enhanced tolerance to high salinity and osmotic pressure.
Phylogenetic tree of NAC genes. Lotus root NnNAC genes are highlighted in red in the online article. The amino acid sequences of the Arabidopsis thaliana NAC family were obtained from The Arabidopsis Information Resource (TAIR) and National Center for Biotechnology Information (NCBI) databases.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
NnNAC2 belongs to SENU5 in Group I. It has been reported that this type of NAC gene plays an important role in high salt conditions, drought, and high-temperature stress (Dong, 2014). NnNAC5 belongs to the subfamily of ANAC011 in Group I. It has been reported that this type of NAC gene plays an important role in the reaction of ethylene and jasmonic acid (Kang et al., 2014). Collectively, these data suggest that NnNAC1–5 may be involved in diverse functions.
NAC gene expression.
The five NAC genes exhibited different expression patterns (Fig. 8). The expressions of NnNAC1/4 were continuously inhibited by VP, with their mRNA decreasing in abundance ≈12- and 60-fold, respectively. In contrast, transcripts of NnNAC2 increased under VP storage at day 21. Unlike the other NnNAC genes, the expressions of NnNAC3/5 showed no clear difference between the different packaging methods.
Messenger RNA amounts from NAC genes in response to vacuum packaging (VP) and atmospheric pressure packaging (AP) treatments. Fresh-cut ‘Wuzhi 2’ lotus root was stored by VP (circles) and AP (squares) at 4 °C separately. Fresh-cut lotus root was packaged by VP (black) and AP (white) separately. Error bars represent se from three biological replicates. lsd = least significant difference.
Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 1; 10.21273/JASHS04806-19
One of most interesting results is that NAC1/4 were downregulated by anaerobic or hypoxic environments (VP). Phylogenetic analysis showed that ANAC063, which is closer to NAC1/4, is not only responsive to drought resistance, but also to salt tolerance (Yokotani et al., 2009), which tells us that NAC exhibit diversity in nonbiological stress responses. And the NAC1/4 expression change pattern occurred concomitantly with increase in NnPAL1, NnPPOA, and NnPOD2/3 gene expressions and BD. This indicates that, in lotus root, the expression of NAC1/4 correlated highly with browning, and the relationships of NAC1/4 and NnPAL1, NnPPOA, and NnPOD2/3 should be the subjects of further research.
In addition to the two aforementioned NnNAC genes, NnNAC2 expression also showed interesting features. Unlike the positively regulated NAC genes, NnNAC2 mRNA levels were inversely related to browning, which was upregulated by the anaerobic or hypoxic environments (VP) during the storage period.
It is well known that the processing of fresh-cut fruits and vegetables promotes faster physiological and biochemical changes and microbial growth, which may reduce the quality of their flavor and texture (Toivonen and Brummell, 2008). In addition, fresh-cut peppers in VP were found to have more noticeable ethanol and acetaldehyde contents (González-Aguilar et al., 2004). Therefore, NnNAC2 may be related to quality loss in fresh-cut fruits and vegetables, especially with respect to flavor. In similar studies, DkNAC1/3/5/6 were found to be associated positively with the persimmon deastringency process, which involves acetaldehyde and ethanol synthesis (Min et al., 2015).
Conclusions
The BD; PAL, PPO, and POD enzyme activities; and gene expression assay results in our study show that VP was an effective method for delaying fresh-cut lotus root browning. Five NnNAC genes were isolated, and fresh-cut lotus root and NnNAC1/4 correlated highly with fresh-cut lotus root browning. Finally, NnNAC1/4 were identified as major candidates for further transcriptional regulation analysis in lotus root to understand more fully the molecular mechanism of browning under anaerobic/hypoxic stress.
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