Temperature or Ethylene Regulate Browning in Lotus Root by Modulating Polyphenols and Starch Metabolism

Abstract

Browning is the major physiological cause of quality loss in lotus root. This study explored the effects of temperature (4 °C, 25 °C, 35 °C) or ethylene (ET) on quality, especially browning, as well as polyphenol and starch metabolism in lotus root. Low temperature (4 °C) reduced browning and color changes (L*, a*), while retaining water and vitamin C (Vc) content. ET maintained Vc and soluble protein, while high temperature (35 °C) promoted total soluble solids (TSS) and soluble sugar accumulation. ET or 35 °C upregulated polyphenol metabolism-related genes including NnPAL1/4, NnCHS1, NnF3H and NnANR, increased total phenolic and flavonoid content, and enhanced antioxidant capacity. Moreover, 35 °C increased PAL activity, and ET also upregulated NnUGT88B1. Furthermore, 4 °C downregulated NnGBE1-1/2, promoted starch accumulation, while ET upregulated NnSSI, downregulated NnGBE1-1/2, and delayed starch decline. Meanwhile, ET elevated NnETR and NnEBF1-2 and mediated ethylene signaling transduction. In conclusion, 4 °C storage was optimal for delaying browning and starch metabolism of lotus root. Meanwhile, ET treatment or 35 °C were more beneficial to obtain more phenolics and flavonoids.

1. Introduction

Lotus root (Nelumbo nucifera Gaertn.) is an important export vegetable in China, rich in dietary fiber, starch, sugars, protein, amino acids, minerals, vitamins, and bioactive substances such as polyphenols and polysaccharides [1]. Among them, polyphenols are important bioactive components that contribute to the nutritional quality and functional properties, while also acting as key substrates for enzymatic browning [2]. In plants, the biosynthesis of polyphenols initiates from the shikimate pathway, proceeding through specialized routes such as the phenylpropanoid and tyrosine pathways to form phenolic acids, flavonoids, tannins, and other secondary metabolites [3]. Key enzymes like phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate:CoA ligase (4CL) drive the phenylpropanoid pathway, providing precursors for downstream compounds including flavonoids [3]. The flavonoid pathway originates with chalcone synthase (CHS), which catalyzes chalcone skeleton formation, followed by sequential modifications via enzymes including chalcone isomerase, flavanone 3-hydroxylase (F3H), dihydroflavonol reductase, and glycosyltransferases such as UDP-glycosyltransferase (UGT), anthocyanidin reductase (ANR) and catechol-O-methyltransferase (COMT) to generate structural diversity. Meanwhile, starch is served as a predominant carbohydrate and vital energy reserve in lotus root, significantly contributing to its textural properties, culinary applications, and consumer acceptance [4]. Starch metabolism involves dynamic synthesis and decomposition mediated by enzymes such as starch synthase (SS), glycogen branching enzyme (GBE), and isoamylase starch debranching enzyme (ISA) [4]. Both polyphenol metabolism and starch metabolism determine the postharvest quality attributes of lotus root. However, postharvest browning—primarily driven by polyphenol oxidase (PPO) and peroxidase (POD)—severely limits the shelf-life and market value of lotus root [5]. Furthermore, vitamin C, a natural anti-browning compound, is closely associated with browning susceptibility, whereas TSS, a key ripeness indicator, also contributes to tissue browning [6,7].

To mitigate browning, various physical and chemical strategies have been investigated, predominantly in fresh-cut lotus root slices. For instance, high temperature was shown to upregulate browning-related genes (NnPAL1, NnPPO1 and NnPOD1/2/3/4/5/6) alongside increased enzyme activities and browning intensity [8]. Chemical treatments such as melatonin (40 mmol/L) and glutathione (1%) could modulate phenolic metabolism, enhancing PAL activity while suppressing PPO and/or POD activities, thereby reducing browning [5,9]. In fresh-cut lotus root slices, ET was reported to increase the activities of PAL, PPO, and POD along with total phenol accumulation, thus promoting browning development [10]. In another non-climacteric fruit, fresh-cut pitaya, ET was found to enhance phenylpropanoid pathway activity via ethylene signaling transduction, upregulating genes such as HuETR1/2, HuEIN3, and HuERF1, while downregulating the negative regulator HuCTR1, leading to the accumulation of phenolics and flavonoids [11]. Similarly, ET profoundly influences carbohydrate metabolism; it can accelerate starch degradation by upregulating α-amylase expression and activity, thereby reducing starch content and increasing soluble sugars [12], while also suppressing starch synthase genes to limit starch accumulation [13].

Despite these insights, existing studies have primarily focused on fresh-cut lotus root slices under single-factor treatments. The integrated effects of temperature and ethylene on whole lotus root, particularly the dynamic balance between browning progression and beneficial phytochemical accumulation, remain poorly understood. This gap hinders a comprehensive understanding of how storage conditions coordinately affect quality, metabolism, and shelf-life in intact lotus roots.

Therefore, the aim of this study was to explore the effects of temperature and ethylene on storage quality, browning, phenolic metabolism, starch metabolism, and related gene expression of intact lotus root during postharvest storage. Specific objectives were: (1) to evaluate their combined effects on browning and nutritional quality; (2) to analyze dynamic changes in polyphenol profiles and antioxidant capacity; (3) to investigate starch metabolism dynamics; and (4) to correlate these physiological and biochemical changes with the expression of key genes involved in polyphenol biosynthesis, starch metabolism, and ethylene signal transduction.

2. Materials and Methods

2.1. Materials and Treatment

Lotus roots (Nelumbo nucifera Gaertn. cv. Wuzhi No. 2) were harvested from Hankoubei, Hubei Province, China. The lotus roots with intact surfaces, no mechanical damage, uniform size, consistent growth period, similar weight and length were chosen. They were stored at 4 °C for 24 h, and then washed with tap water to remove sediment, and subsequently cut horizontally with a sterilized blade. The samples were randomly divided into four treatment groups: the first group was stored at 4 °C (4 °C), the second group was stored at 25 °C (25 °C), the third group was immersed in 4 g/L ethephon for 5 min, and then stored at 25 °C (25 °C + 4 g/L ET), and the fourth group was stored at 35 °C (35 °C). Based on lotus root postharvest research and preliminary screening experiments, 4 g/L ethephon was used for treatment, with 4 g/L showing a significant effect on browning delay without causing tissue damage. ET treatment was only performed at 25 °C, which represents the normal ambient storage condition for lotus root. This design was used to focus on the regulatory role of ethylene under room-temperature storage. Based on previous research, 35 °C was selected as the high-temperature treatment [8]. Each treatment group contained 36 intact lotus roots, with three biological replicates per treatment, a total of 108 fruits. Samples were collected after 0, 3, 6, 9, and 12 d of storage.

2.2. Color, Browning Degree, Water Content and Total Soluble Solids (TSS)

The color parameters (L*, a*, b*) were measured using the JZ-600 colorimeter (Jingzhu Instrument Company, China) at randomly selected homogeneous epidermal sites.

Browning degree was assessed as described by Min et al. [14] with modifications. Briefly, a tissue homogenate (3.0 g in 30 mL water) was centrifuged, and the absorbance of the supernatant was measured at 410 nm after incubation at 25 °C. The browning degree was calculated as A410 × 10.

The water content was determined according to Dong et al. [15] with modifications. Pre-weighed trays and lotus root slices were successively dried at 105 °C until constant weight (±2 mg) was achieved. The water content was calculated from the weight loss and expressed as a percentage based on triplicate measurements.

The content of TSS was quantitatively analyzed using a portable refractometer. Tissue (10 g) was homogenized under an ice bath and centrifuged (7826× g, room temperature, 5 min). The supernatant was directly used for measurement in triplicate.

2.3. Soluble Sugar, Starch, Soluble Protein, and Vitamin C (Vc)

The soluble sugar content was determined as described by Dong et al. [15] with modifications. Briefly, 3 g of sample was extracted twice with boiling water, and the combined supernatant was diluted to 50 mL. For analysis, 0.05 mL of the extract was reacted with 1.95 mL of water, 1 mL of 9% phenol, and 5 mL of concentrated sulfuric acid. After 30 min of incubation at room temperature, the absorbance was measured at 485 nm. All analyses were performed in triplicate. A standard curve was prepared using anhydrous glucose solutions. Soluble sugar content (%) was calculated as Equation (1):

Soluble sugar content (%) = (B × V_t × 100)/(m × V_s × 10⁶)

(1)

where B denotes sugar concentration from the standard curve in μg/mL, V_t signifies the total extract volume of 50 mL, V_s represents the assay aliquot volume of 0.05 mL, and m indicates the sample mass in grams.

The starch content was determined using the Micro Starch Assay Kit (Beijing Solebo Biotechnology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The result was expressed as mg/g FW. The soluble protein content was measured using the Total Protein Quantification Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and in accordance with the manufacturer’s protocol. The result was expressed as mg/g FW. The Vc content was determined using the Vitamin C Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The result was expressed as µg/g FW.

2.4. Total Phenolics Content (TPC), Total Flavonoid Content (TFC), and Monomeric Phenol Content

TPC was determined using the Folin–Ciocalteu method [16]. Tissues (3.0 g) were homogenized in 30 mL of 60% ice-cold ethanol and centrifuged. The supernatant (0.125 mL) was reacted with Folin–Ciocalteu reagent and 7% sodium carbonate solution. After 90 min of incubation in the dark, absorbance was measured at 760 nm. TPC was calculated from a gallic acid standard curve and expressed as mg GAE/100 g FW.

TFC was determined following Alhaithloul et al. [17] with modifications. Fresh tissue (5.0 g) was homogenized in 50 mL of 60% ice-cold ethanol and centrifuged. The supernatant (4 mL) was sequentially reacted with 5% NaNO₂, 10% Al(NO₃)₃, and 1 M NaOH, with incubations between each step. The final mixture was diluted to 10 mL with 80% ethanol, incubated in the dark for 15 min, and the absorbance was measured at 510 nm. TFC was calculated using a rutin standard curve and expressed as mg RE/100 g FW.

Phenolic compounds were extracted from lotus root (32 g) following Li et al. [11], which involved homogenization in 160 mL of ice-cold 40% (v/v) ethanol (pH 3.0, adjusted with HCl), followed by ultrasonication (40 kHz, 300 W, 72 min), and finally centrifugation at 4500× g for 10 min. The supernatant was filtered using Whatman No. 4 paper, and the residue was subjected to another ultrasonic treatment (10 min) and centrifugation in 200 mL of pH 3.0 ethanol solution. All the supernatants were mixed, subjected to rotary evaporation at 40 °C, and reconstituted to 10 mL of methanol. HPLC analysis was performed using a C₁₈ column (250 × 4.6 mm, 5 μm) with a diode array detector, detection at 280 nm, a flow rate of 1.0 mL/min, a column temperature of 30 °C, and an injection of 20 μL. Each extract was analyzed twice using two different HPLC elution programs to optimally separate two distinct sets of phenolic compounds: Method 1, designed for pyrogallic acid, gastrodin, p-coumaric acid, gallocatechin, catechol, catechin, chlorogenic acid, caffeic acid, epicatechin, quercetin, used methanol (A)/0.4% glacial acetic acid (B) with gradient: 5–25% A (0–40 min), 25–50% A (40–50 min), 50–70% A (50–65 min), 70–100% A (65–66 min), isocratic 100% A (66–72 min), 100–5% A (72–73 min), 5% A (73–80 min); Method 2, designed for gallic acid, rutin, hyperoside, resveratrol, used acetonitrile (A)/0.4% glacial acetic acid (B) with gradient: 5–25% A (0–10 min), 25–35% A (10–20 min), 35–100% A (20–21 min), isocratic 100% A (21–25 min), 100–5% A (25–26 min), 5% A (26–30 min).

2.5. DPPH Free Radical Scavenging Rate and ABTS Radical Scavenging Ability

DPPH free radical scavenging rate was determined as described by Dong et al. [15] with modifications. Briefly, tissue (2.0 g) was extracted with 25 mL of absolute ethanol using ultrasonication at 50 °C and centrifugation. The supernatant was diluted 10-fold, and 2 mL of the dilution was mixed with 2 mL of 0.2 mmol/L DPPH solution. After incubation in the dark for 30 min, the absorbance was measured at 517 nm. The result was expressed as the percentage of radical scavenging rate.

ABTS radical scavenging ability was determined using the ABTS assay kit (Beyotime Biotechnology, Shanghai, China). Fresh tissue (5.0 g) was homogenized in 25 mL of ice-cold phosphate buffer (0.1 M, pH 7.0) and centrifuged. The supernatant was analyzed according to the kit instructions, and results were expressed as mM Trolox/g.

2.6. Activity of PAL

Phenylalanine Ammonia-Lyase Activity Assay Kit (Solarbio, Beijing, China) was used to determine the PAL activity by monitoring the production of trans-cinnamic acid at 290 nm. One unit of enzyme activity was defined as the amount that produces 1 μmol of trans-cinnamic acid per hour at 37 °C, and results were expressed as U/g fresh weight.

2.7. Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA was extracted from frozen tissues using a modified CTAB method, followed by LiCl precipitation and purification [9]. RNA purity and concentration were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was further verified by 1.0% agarose gel electrophoresis. After assessing RNA quality and quantity, genomic DNA was removed with a TURBO DNA-free^TM Kit (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was synthesized from 1 μg of DNA-free RNA using the iScript cDNA Synthesis Kit. qRT-PCR was performed using SsoFast™ EvaGreen^® Supermix (Bio-Rad, Hercules, CA, USA) on a CFX96 Touch system with gene-specific primers. The PCR program was initiated for 3 min at 95 °C, then followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s, and completed with a melting curve from 95 °C for 10 s, 60 °C for 5 s, 95 °C for 5 s. The Actin-101 was used as the internal reference, and relative expression levels were calculated using the 2^−ΔΔCT method. The gene expression level at day 0 was set as 1 for normalization. For each treatment, the expression level at each storage time point was compared with its own day 0 value. All samples were analyzed with three biological replicates and three technical replicates. Primer sequences are listed in Table S1.

2.8. Statistical Analysis

All experiments were performed using a completely randomized design, repeated three times, and the results were expressed as the mean ± standard error. The data were analyzed using SPSS software (version 20.0, IBM Corporation, Armonk, NY, USA). The significance analysis was determined by the one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. p < 0.05 was considered statistically significant. Figures were generated using Origin software (version 2021, OriginLab Corporation, Northampton, MA, USA). For correlation analysis, Pearson correlation coefficients were calculated using the combined data from all treatments and time points to assess the association between groups of variables. Two-tailed tests and the same significance level (p < 0.05) were used to evaluate the significance of correlations.

3. Results

3.1. Effects of Temperature or ET on the Browning of Lotus Root During Storage

Storage at 4 °C significantly delayed browning, with minimal changes in color parameters (higher L* and lower a* values), while 35 °C accelerated browning (Figure 1a–e). ET treatment (25 °C + 4 g/L ET) reduced a* values compared to 25 °C alone, but no significant differences were observed in L* and b* values. Browning degree increased least at 4 °C (7.37% by day 12) and most at 35 °C (39.17%). These results demonstrated that low temperature (4 °C) delayed browning.

3.2. Effects of Temperature or ET on the Nutrient Composition of Lotus Root During Storage

Water content declined least during storage at 4 °C, while the most obvious decrease was observed at 35 °C (Figure 2a). For TSS and total soluble sugar contents, both components accumulated to higher levels under high-temperature treatment (35 °C), with the maximum value (1.76%) observed on day 12 at 35 °C. In contrast, low temperature (4 °C) significantly inhibited the accumulation of TSS and soluble sugar (Figure 2b,c). Vitamin C content decreased sharply at 35 °C, with a loss rate of 44.79% by day 12, while ET treatment at 25 °C effectively maintained higher Vc content, reaching a peak of 1177.20 μg/g on day 3 (Figure 2d). Soluble protein content exhibited biphasic changes during storage, and the highest peak (2.52 mg/g) was detected in the 4 °C treatment (Figure 2e). Overall, these results indicated that 4 °C and ET treatments were more effective in maintaining the main quality indexes of lotus seeds during storage.

3.3. Effects of Temperature or ET on the Polyphenols and Antioxidant Capacity of Lotus Root During Storage

Figure 3a showed that TPC significantly increased across all treatments (p < 0.05). High temperatures markedly enhanced TPC, with gains of 16.70% (25 °C), 21.85% (25 °C + 4 g/L ET), and 31.86% (35 °C) (p < 0.05). Crucially, ET treatment at 25 °C significantly elevated TPC by 5.15% compared with the 25 °C group (p < 0.05).

Figure 3b revealed total flavonoid accumulation in lotus roots across storage treatments, demonstrating progressive increases throughout the storage period. The 35 °C groups showed the highest total flavonoid accumulation, reaching 89.40 mg/100 g by day 12, equivalent to a 75% elevation from the initial 51.05 mg/100 g. Comparatively, flavonoid levels represented 21%, 34%, and 48% increases in the 4 °C, 25 °C, or 25 °C + 4 g/L ET groups after 12 days of storage, respectively. These data established that high temperatures and ET application substantially promoted flavonoid biosynthesis in lotus root.

Figure 3c,d illustrated that high temperature and ethephon treatment enhanced antioxidant capacity. On day 3, DPPH radical scavenging rate peaked at 45.21% and 48.87% for 25 °C or 25 °C + 4 g/L ET groups, significantly higher than that of 4 °C or 35 °C groups (p < 0.05). All treatments exhibited maximal ABTS radical scavenging ability during mid-to-late storage, with the 35 °C group attaining peak activity of 0.93 mM Trolox/g on day 9, significantly higher than that of other groups (p < 0.05).

Table 1. Changes in the individual phenolic content of lotus root during storage.

Phenolic Substance		Group	Storage Time
			0 d	3 d	6 d	9 d	12 d
Phenolic acids	gallic acid	4 °C	0.59 ± 0.01 C 1	0.56 ± 0.01 Dd 3	0.55 ± 0.01 Ed	0.81 ± 0.01 Bc	0.86 ± 0.01 Ab
		25 °C	0.59 ± 0.01 E	0.64 ± 0.01 Dc	0.80 ± 0.01 Ca	0.85 ± 0.01 Bb	0.92 ± 0.02 Aa
		25 °C + 4 g/L ET	0.59 ± 0.01 E	0.73 ± 0.01 Ca	0.77 ± 0.01 Bb	0.70 ± 0.01 Dd	0.95 ± 0.01 Aa
		35 °C	0.59 ± 0.01 D	0.70 ± 0.01 Cb	0.70 ± 0.01 Cc	0.97 ± 0.02 Aa	0.88 ± 0.01 Bb
	coumaric acid	4 °C	2.04 ± 0.01 D	2.19 ± 0.01 Cc	3.11 ± 0.01 Ba	1.49 ± 0.09 Ed	3.90 ± 0.01 Ac
		25 °C	2.04 ± 0.01 C	1.64 ± 0.02 Dd	2.43 ± 0.04 Bb	2.53 ± 0.03 Bc	4.47 ± 0.22 Ab
		25 °C + 4 g/L ET	2.04 ± 0.01 E	2.62 ± 0.04 Db	3.02 ± 0.06 Ca	3.74 ± 0.14 Bb	4.06 ± 0.10 Ac
		35 °C	2.04 ± 0.01 E	2.85 ± 0.01 Ca	2.48 ± 0.02 Db	4.87 ± 0.09 Ba	5.15 ± 0.06 Aa
	coffeic acid	4 °C	0.23 ± 0.01 D	0.26 ± 0.01 Dc	0.42 ± 0.03 Cd	0.81 ± 0.01 Bd	1.34 ± 0.06 Ab
		25 °C	0.23 ± 0.01 C	0.57 ± 0.01 Bb	0.55 ± 0.01 Bc	0.93 ± 0.03 Ac	0.99 ± 0.03 Ad
		25 °C + 4 g/L ET	0.23 ± 0.01 E	0.95 ± 0.01 Ca	0.68 ± 0.05 Db	1.12 ± 0.01 Bb	1.85 ± 0.01 Aa
		35 °C	0.23 ± 0.01 E	0.61 ± 0.01 Db	0.79 ± 0.01 Ca	2.05 ± 0.08 Aa	1.24 ± 0.08 Bc
	chlorogenic acid	4 °C	2.42 ± 0.14 C	1.95 ± 0.01 Dc	2.79 ± 0.09 Bb	4.20 ± 0.01 Ab	2.91 ± 0.10 Bc
		25 °C	2.42 ± 0.14 C	2.07 ± 0.01 Db	2.33 ± 0.02 Cc	3.83 ± 0.05 Bc	4.28 ± 0.08 Aa
		25 °C + 4 g/L ET	2.42 ± 0.14 D	1.74 ± 0.01 Ed	3.41 ± 0.16 Ca	4.91 ± 0.08 Aa	4.37 ± 0.03 Ba
		35 °C	2.42 ± 0.14 C	2.63 ± 0.10 Ca	2.58 ± 0.02 Cab	3.75 ± 0.03 Bc	4.03 ± 0.03 Ab
Flavonoids	quercetin	4 °C	0.27 ± 0.01 D	nd 2	0.70 ± 0.01 Bb	0.89 ± 0.01 Ab	0.56 ± 0.01 Cb
		25 °C	0.27 ± 0.01 E	0.38 ± 0.01 Dc	0.55 ± 0.01 Bc	0.58 ± 0.01 Ac	0.49 ± 0.01 Cd
		25 °C + 4 g/L ET	0.27 ± 0.01 E	1.03 ± 0.01 Ba	1.69 ± 0.01 Aa	0.57 ± 0.01 Dc	0.88 ± 0.02 Ca
		35 °C	0.27 ± 0.01 D	0.53 ± 0.01 Bb	0.46 ± 0.02 Cd	1.17 ± 0.01 Aa	0.52 ± 0.01 Bc
	rutin	4 °C	2.21 ± 0.03 C	2.91 ± 0.02 ABd	1.71 ± 0.01 Dc	2.76 ± 0.01 Bd	2.98 ± 0.12 Ad
		25 °C	2.21 ± 0.03 E	3.29 ± 0.06 Cc	2.43 ± 0.07 Db	4.77 ± 0.01 Aa	3.83 ± 0.01 Bc
		25 °C + 4 g/L ET	2.21 ± 0.03 E	5.62 ± 0.01 Aa	2.53 ± 0.02 Db	3.65 ± 0.01 Cc	5.15 ± 0.12 Bb
		35 °C	2.21 ± 0.03 D	3.65 ± 0.01 Cb	3.95 ± 0.04 Ca	4.59 ± 0.01 Bb	6.24 ± 0.23 Aa
	hyperoside	4 °C	0.84 ± 0.02 C	1.21 ± 0.01 Ac	1.20 ± 0.01 Ac	0.88 ± 0.01 Bc	0.89 ± 0.01 Bc
		25 °C	0.84 ± 0.02 E	1.08 ± 0.02 Dd	1.75 ± 0.01 Ab	1.43 ± 0.01 Ba	1.20 ± 0.04 Cb
		25 °C + 4 g/L ET	0.84 ± 0.02 E	1.47 ± 0.07 Ba	2.42 ± 0.01 Aa	1.30 ± 0.01 Db	1.43 ± 0.01 Cb
		35 °C	0.84 ± 0.02 E	1.34 ± 0.09 Cab	1.07 ± 0.01 Dd	1.44 ± 0.01 Ba	1.65 ± 0.06 Aa
	catechin	4 °C	49.57 ± 0.23 E	71.73 ± 0.15 Dc	147.04 ± 3.32 Aa	124.35 ± 0.18 Bb	95.74 ± 0.22 Cd
		25 °C	49.57 ± 0.23 B	45.41 ± 3.18 Bd	115.74 ± 0.52 Ab	112.66 ± 0.31 Ad	112.29 ± 0.l05 Ac
		25 °C + 4 g/L ET	49.57 ± 0.23 C	93.23 ± 0.52 Bb	121.37 ± 0.51 Ab	120.89 ± 0.28 Ac	120.31 ± 0.10 Ab
		35 °C	49.57 ± 0.23 E	112.14 ± 0.40 Ca	106.21 ± 2.19 Dc	141.40 ± 0.45 Ba	146.71 ± 0.64 Aa
Non-flavonoids	gastrodin	4 °C	119.69 ± 0.82 C	72.85 ± 5.58 Da	141.23 ± 4.56 BCb	156.54 ± 1.37 Bc	198.83 ± 18.04 Ab
		25 °C	119.69 ± 0.82 B	55.43 ± 0.55 Cb	184.08 ± 2.05 Aa	203.71 ± 7.41 Ab	188.19 ± 15.33 Ab
		25 °C + 4 g/L ET	119.69 ± 0.82 C	60.60 ± 0.53 Db	176.29 ± 9.87 Ba	161.58 ± 8.97 Bc	205.84 ± 2.01 Ab
		35 °C	119.69 ± 0.82 D	59.37 ± 1.59 Eb	168.41 ± 1.94 Ca	321.94 ± 4.63 Aa	259.02 ± 4.78 Ba
	catechol	4 °C	1.21 ± 0.01 C	0.99 ± 0.02 Db	2.72 ± 0.02 Bb	3.70 ± 0.04 Ab	3.75 ± 0.10 Ab
		25 °C	1.21 ± 0.01 D	0.90 ± 0.02 Db	1.95 ± 0.07 Cc	2.88 ± 0.11 Bc	3.77 ± 0.24 Ab
		25 °C + 4 g/L ET	1.21 ± 0.01 C	2.60 ± 0.07 Ba	3.43 ± 0.14 Aa	3.38 ± 0.08 Ab	3.45 ± 0.15 Ab
		35 °C	1.21 ± 0.01 D	2.67 ± 0.03 Ca	2.90 ± 0.04 Bb	4.39 ± 0.13 Aa	4.48 ± 0.08 Aa
	resveratrol	4 °C	nd	0.08 ± 0.01 Ac	0.07 ± 0.01 Bc	0.20 ± 0.01 C	0.10 ± 0.01 C
		25 °C	nd	nd	0.13 ± 0.01 b	nd	nd
		25 °C + 4 g/L ET	nd	0.10 ± 0.01 b	0.21 ± 0.01 a	nd	nd
		35 °C	nd	0.14 ± 0.01 a	nd	nd	nd
	pyrogallol	4 °C	nd	nd	9.54 ± 0.71 Bb	20.55 ± 0.75 Ab	20.87 ± 0.39 Aa
		25 °C	nd	nd	8.07 ± 0.30 Bb	23.21 ± 0.61 Ab	22.06 ± 0.59 Aa
		25 °C + 4 g/L ET	nd	nd	13.37 ± 1.21 Ca	14.45 ± 1.06 Bc	16.40 ± 0.64 Ab
		35 °C	nd	14.22 ± 0.94 C	16.46 ± 1.76 Ba	29.66 ± 1.16 Aa	17.19 ± 0.85 Bb

¹ Concentrations are expressed in μg/g. ² nd, not detected. ³ Uppercase letters indicate significant differences (p < 0.05) between storage days within the same treatment group. Lowercase letters indicate significant differences (p < 0.05) between treatment groups on the same storage day.

showed treatment-specific dynamics of monomeric phenolics in lotus roots during storage, revealing universal increases in phenolic acids with distinct accumulation patterns. Chlorogenic acid demonstrated the most pronounced response to storage duration, followed by coumaric acid. ET substantially enhanced chlorogenic acid accumulation, culminating in a peak concentration of 4.91 μg/g on day 9 that significantly higher than that of other treatment groups (p < 0.05). Among flavonoids, content of rutin and catechin in 35 °C group was significantly higher than that of the other groups (p < 0.05), indicating that high temperature (35 °C) increased their content. For non-flavonoids, gastrodin exhibited an initial decline followed by recovery, with high temperature promoting its accumulation; the 35 °C group reached peak gastrodin content of 321.94 μg/g on day 9, exceeding the lowest observed level by 5.42-fold. Resveratrol remained minimal or undetectable across treatments, while pyrogallol and catechol peaked during later storage stages. These monomeric phenolic accumulation patterns mirrored total phenolics and flavonoid trends, demonstrating that high temperature (35 °C) stimulates the biosynthesis of phenolics such as coumaric acid, rutin, hyperoside, catechin, gastrodin and catechol. ET promoted the formation of phenolics, such as coffeic acid, quercetin, rutin, and catechin. These results indicate that appropriate high temperature and ET treatment can enhance the phenolic metabolism and antioxidant capacity of lotus seeds.

3.4. Effects of Temperature or ET on the Phenylpropanoid Pathway During Lotus Root Storage

As shown in Figure 4, PAL activity was maintained at a relatively high level under 35 °C treatment, with an increase of 46.96% compared with the control. Notably, PAL activity peaked at 38.84 U/g under ET treatment on day 6, which was recognized as the most representative day and ET as the most representative treatment for PAL activity. In line with the changes in enzyme activity, the transcript levels of NnPAL1, NnPAL4, NnPOD, and NnCOMT-like were significantly upregulated by high temperature or ET treatment. In addition, NnPAL4 exhibited a specific response to cold stress. These coordinated changes between enzyme activity and gene expression suggest that these genes play important roles in regulating phenylpropanoid metabolism under different treatments.

3.5. Effects of Temperature or ET on the Flavonoid Metabolism During Storage

In 4 °C or 25 °C treated lotus roots, the expression of flavonoid biosynthetic genes, such as NnCHS1, NnF3H, NnANR, and NnUGT88B1, consistently displayed an initial increase followed by progressive decline during storage (Figure 5). In contrast, high temperature (35 °C) upregulated NnCHS1, NnF3H, and NnANR (3.08-, 5.40-, and 2.05-fold by day 12), while downregulated NnUGT88B1. Compared to the groups of 25 °C, ET elevated flavonoid biosynthesis genes expression at specific time points. Peak induction occurred for NnCHS1 on day 3 at 5.34 times, NnF3H on day 9 at 4.43 times, NnANR on day 6 at 7.48 times, and NnUGT88B1 on day 3 at 2.10 times (Figure 5). Collectively, these results demonstrated ET-mediated transcriptional activation of flavonoid pathway genes, driving enhanced flavonoid accumulation.

3.6. Effects of Temperature or ET on the Starch Metabolism of Lotus Root During Storage

As shown in Figure 6, starch content declined least at 4 °C, reaching a peak value of 146.93 mg/g on day 12, while the most obvious degradation was observed at 25 °C. ET treatment initially promoted starch degradation but delayed its decline by day 12. The most representative day for starch content changes was day 12, and the most representative treatment was 4 °C. In addition, starch biosynthetic genes including NnSSI, NnGBE1-1, and NnGBE1-2 were significantly upregulated at 35 °C but markedly suppressed under ET treatment. These results indicate that temperature and ET treatment effectively regulated starch metabolism by modulating the expression of starch synthesis-related genes.

3.7. Effects of Temperature or ET on the Ethylene Signal Transduction of Lotus Root During Storage

Expression of ETRs in the 4 °C group was significantly upregulated compared with the 25 °C or 35 °C groups, and 25 °C + 4 g/L ET group exhibited downregulated expression of these genes during storage compared with the 25 °C group (p < 0.05, Figure 7a–c). This indicated that high temperatures and ET promoted the binding of ET to the ETR component, thus facilitating ethylene signal perception and the initiation of downstream signal transduction.

CTR1 encoded a critical negative regulator of ethylene signaling and exhibited substantial temperature dependence throughout storage (Figure 7d). Both 4 °C or 35 °C groups significantly downregulated NnCTR1 compared with the 25 °C group, with maximal suppression observed on day 12 (p < 0.05). This bidirectional repression suggested thermal extremes universally attenuate CTR1-mediated ethylene signaling inhibition regardless of hypothermic or hyperthermic stress. The 25 °C + 4 g/L ET groups further accentuated this regulatory effect, suppressing NnCTR1 expression compared with 25 °C groups except day 9.

NnEIN3-1 in 25 °C + 4 g/L ET group was upregulated on days 6 and 12 and NnEIN3-2 on days 6 and 9 compared with the 25 °C group, indicating that ET upregulated EIN3 (Figure 7e,f). Concurrently, EIN3-1 and EIN3-2 in the 4 °C group were significantly downregulated compared with the 25 °C group (p < 0.05).

On day 3, the expression levels of NnEBF1-1/2/3 in the 25 °C groups were respectively 6.54 times, 2.76 times, and 2.18 times higher than those in the 25 °C + 4 g/L ET group (Figure 7g–i). Thus, ET significantly downregulated EBF1-related genes during the early storage (p < 0.05), suppressing EBF1 expression and promoting EIN3 expression. Notably, at the end of storage, the expression levels of NnEBF1-2 in the 25 °C + 4 g/L ET group were significantly higher compared with the 25 °C group (p < 0.05).

3.8. Correlation Analysis of TPC, TFC, Antioxidant Capacity, Polyphenols Metabolism and Starch Metabolism in Lotus Root During Storage

Polyphenols metabolism genes, such as NnPAL1/3/4, NnCHS1, NnF3H, and NnANR exhibited highly correlations with TPC, TFC, PAL activity, and antioxidant capacity. Specifically, both high temperature and ET significantly promoted the expression of these genes (p < 0.05), thereby enhancing the accumulation of phenolics and flavonoids during storage, indicating that temperature and ET may regulate polyphenols metabolism in lotus root by modulating the expression of these genes. However, NnUGT88B1, NnPOD, and NnCOMT-like appeared uninvolved in temperature-regulated polyphenols metabolism.

A distinct regulatory pattern was observed at low temperature (4 °C), and the expression of NnPAL4 and NnCOMT-like (polyphenols synthesis genes) was negatively correlated with NnPOD, which reflected a synthesis-degradation balance and may account for the suppressed polyphenols accumulation (Figure 3a) and browning (Figure 1e) under low temperature.

Under 35 °C, NnF3H expression showed a significant positive correlation with TFC (p < 0.05), the expression level of NnANR was positively correlated with TPC, and the expression level of NnCHS1 was positively correlated with the ABTS radical scavenging ability. These results suggested that high temperature may promote polyphenols accumulation and enhance antioxidant capacity in lotus root during storage by inducing the expression of NnF3H, NnANR and NnCHS1.

Compared with the 25 °C group, the 25 °C + 4 g/L ET group demonstrated more significant correlations between TPC, TFC, antioxidant capacity, PAL activity, and the expression of NnPAL1/3/4, NnCHS1, NnF3H and NnANR, indicating that ET may upregulate these genes to promote polyphenols accumulation during storage (Figure 8).

4. Discussion

4.1. Low Temperature (4 °C) Optimally Preserves Lotus Root Quality by Delaying Browning and Regulating Starch Metabolism

This study demonstrates that storage at 4 °C represents the most effective condition for preserving the overall quality of lotus root, as it significantly delayed browning, maintained color stability (evidenced by higher L* and lower a* values). Concurrently, the better retention of water and Vc content under this condition not only contributes to nutritional value but, in the case of Vc, also provides an enhanced antioxidant defense against browning [18]. The underlying mechanism also involves the coordinated regulation of starch metabolism and polyphenol-related pathways. Specifically, 4 °C storage downregulated the expression of starch branching enzyme genes (NnGBE1-1/2), promoting starch accumulation and suppressing the conversion of polysaccharides to soluble sugars. This observation aligns with Gong et al. [4], who reported that 4 °C storage delayed amylopectin reduction and water dissipation in lotus roots. Moreover, low temperature maintained lower phenylalanine ammonia-lyase (PAL) activity and modulated the balance between polyphenol synthesis (e.g., NnPAL4, NnCOMT-like) and degradation (e.g., NnPOD), thereby reducing substrate availability for PPO/POD-mediated oxidation and ultimately inhibiting browning. This finding is consistent with Min et al. [19], who demonstrated that 4 °C effectively suppressed phenolic oxidation and associated enzyme activities in fresh-cut lotus root. In contrast to high-temperature treatments, storage at 4 °C avoids excessive activation of the phenylpropanoid pathway, thereby preventing the unnecessary depletion of nutritional components such as Vc and helping to maintain tissue integrity. The agreement between our results and previous studies [4,19] confirms that low-temperature storage is a universally effective strategy for quality preservation in lotus root, with its regulatory influence on starch and polyphenol metabolism being conserved across different postharvest handling conditions (intact vs. fresh-cut).

4.2. High Temperature (35 °C) Enhances Phenolic and Flavonoid Accumulation by Activating the Phenylpropanoid/Flavonoid Pathway

A central finding of this study is that storage at 35 °C markedly enhanced the accumulation of total phenolics (TPC), total flavonoids (TFC), and specific monomeric phenolics-such as coumaric acid, rutin, and catechin-while concurrently strengthening antioxidant capacity, as reflected in elevated DPPH and ABTS radical scavenging activities. This phenomenon can be mechanistically explained by the pronounced upregulation of key genes involved in the phenylpropanoid and flavonoid biosynthesis pathways. Specifically, 35 °C storage upregulated NnPAL1/4 (critical genes regulating phenolic synthesis), NnCHS1 (encoding chalcone synthase, the gateway enzyme to flavonoid production), NnF3H (flavanone 3-hydroxylase), and NnANR (anthocyanidin reductase), collectively accelerating the flux toward phenolic and flavonoid end-products. Supporting this transcriptional activation, PAL activity was sustainably elevated under 35 °C, showing a 46.96% increase over initial levels, which directly contributed to the observed TPC increase. This aligns with Min et al. [14], who reported that high temperature upregulates browning-related genes (including NnPAL1, NnPPO1, and several NnPODs) alongside increased enzyme activities in fresh-cut lotus root. Notably, although 35 °C storage significantly increased total phenolic content, total flavonoid content, and antioxidant capacity in lotus root, it also led to the most severe browning. Browning is mainly attributed to the oxidation and condensation of phenolic compounds. In the present study, only total phenolics and total flavonoids were determined, which cannot fully reflect the dynamic changes in specific browning-related phenolic substrates. Therefore, the elevated total phenolic content at high temperature does not contradict the accelerated browning. Even though total phenolics accumulated, the rapid oxidation and polymerization of individual phenolic compounds could still promote the browning process.

The thermal induction of phenolic metabolism observed here is consistent with reports in other species. For instance, high temperature promoted phenolic and flavonoid accumulation in peppers [20] and tomato seedlings [17], suggesting that heat-induced activation of the phenylpropanoid pathway represents a conserved stress response across plants. Furthermore, the substantial increase in soluble sugar content (reaching 1.76% by day 12) under 35 °C storage corroborates findings by Holland et al. [21], who associated elevated temperature with enhanced activity of sucrose-metabolizing enzymes and altered sugar profiles. However, unlike the moderate temperature effect described in tomato seedlings, our study demonstrates that a relatively high postharvest temperature (35 °C) exerts a more robust inductive effect on secondary metabolism in lotus root. This discrepancy may be attributed to differences in physiological status (seedlings versus postharvest storage organs) and exposure duration, where detached lotus roots—as sink tissues—may redirect carbon resources toward secondary metabolites as part of an acute stress adaptation mechanism.

4.3. Ethylene Treatment Modulates Bioactive Compound Accumulation, Nutrient Retention, and Starch Metabolism in Lotus Root

This study demonstrated that exogenous ethylene (25 °C + 4 g/L ET) exerted multifaceted regulatory effects on postharvest lotus root. Specifically, ET treatment effectively maintained Vc and soluble protein contents, promoted the accumulation of specific phenolics (e.g., chlorogenic acid, caffeic acid) and flavonoids (e.g., quercetin, rutin), and ultimately delayed starch degradation. The mechanism underlying Vc retention aligns with findings in strawberry, where ethylene application similarly enhanced Vc accumulation [22]; this is consistent with our observation of a peak Vc content (1177.20 μg/g) on day 3 in the ET-treated group. Notably, this transient Vc accumulation holds significant implications for browning control. As a natural anti-browning agent, Vc not only acts as a reducing agent that converts o-quinones back to their phenolic precursors but also directly inhibits PPO activity [6,7]. Therefore, the early-stage surge in Vc content in ET-treated fruit likely establishes a robust antioxidant barrier, mitigating browning initiation by maintaining phenolic substrates in their reduced form.

The enhanced phenolic and flavonoid accumulation can be attributed to the ethylene-induced upregulation of key biosynthetic genes (NnCHS1, NnF3H, NnANR, NnUGT88B1), which likely increased metabolic flux through the phenylpropanoid pathway. This finding corroborates the work of Wang et al. [10] on fresh-cut lotus root. Furthermore, the observed peak in chlorogenic acid (4.91 μg/g on day 9) suggests a potential synergistic interaction between ethylene and other hormones, such as abscisic acid, in regulating phenolic biosynthesis [23]. It is noteworthy that ethylene’s effect on total phenolic content (TPC) is species-specific. While it promoted TPC in lotus root and mung bean sprouts [24], it suppressed TPC in water chestnuts [25], highlighting divergent regulatory networks in the phenylpropanoid pathway across species.

A particularly interesting finding was the dual role of ethylene in starch metabolism: it initially promoted degradation but ultimately maintained a higher starch content at the end of storage compared to the 25 °C control. The initial degradation phase is consistent with reports in sand pear and rice, where ethylene upregulates amylase activity [12,26]. However, the subsequent inhibition of starch decline, associated with the upregulation of NnSSI and downregulation of NnGBE1-1/2, suggests a more complex, species-specific regulatory pattern. Similar suppression of starch synthase genes by ethylene has been observed in apples and wheat [12,13]. This discrepancy may reflect the distinct physiological roles of starch: in storage organs like lotus root, starch retention is crucial for tissue integrity, whereas in climacteric fruits, its degradation is integral to the ripening process. Starch metabolism plays a crucial role in maintaining cellular energy status and delaying senescence in postharvest lotus root, which is closely associated with the occurrence and development of browning. Adequate intracellular energy supply contributes to preserving membrane integrity and reducing the oxidation of phenolic substrates, thereby alleviating enzymatic browning. Starch degradation can provide soluble sugars and available energy for various physiological metabolic processes, further affecting the activities of browning-related enzymes and phenolic accumulation [27]. Therefore, monitoring starch metabolism contributes to a more comprehensive understanding of the mechanisms underlying postharvest quality deterioration and browning in intact lotus root during storage.

4.4. Ethylene Regulates Lotus Root Metabolism Through a Conserved Signal Transduction Cascade

This study further elucidated that the ethylene-mediated regulation of polyphenol and starch metabolism operates through a canonical signal transduction pathway. Upon perception by ethylene receptors (NnETR), exogenous ethylene initiated a signaling cascade characterized by the downregulation of negative regulators (NnCTR1, NnEBF1-1/3) and the concomitant upregulation of positive regulators (NnEIN3-1/2). This aligns with the established signaling paradigm wherein CTR1 inactivation promotes EIN2 stabilization and nuclear translocation, ultimately activating downstream ethylene response factors (ERFs) [28]. The critical role of NnEIN3-1/2 upregulation is underscored by its function in directly binding to the promoters of ERFs to regulate metabolic target genes [29], thereby explaining the transcriptional activation of key genes such as NnCHS1, NnF3H, and NnSSI.

The observed downregulation of NnCTR1 under both 4 °C and 35 °C storage, compared to 25 °C, suggests that thermal extremes universally attenuate CTR1-mediated inhibition of ethylene signaling. This finding is consistent with Hao et al. [30], who demonstrated that CTR1 destabilization modulates ethylene responses to ambient temperature fluctuations. Conversely, the specific downregulation of NnEIN3-1/2 in the 4 °C group corroborates reports in tomato, where low temperature suppresses SlEIN3 expression to delay senescence [31].

The consistency of this regulatory framework is supported by studies in other species: ethylene upregulates HuETR1/2 and HuEIN3 to enhance phenolic accumulation in fresh-cut pitaya [11], while in wheat, ethylene signaling modulates ABA/GA balance to govern seedling growth [32]. Furthermore, the ET-induced upregulation of NnETR resonates with findings in rice, where ETR2 overexpression alters starch accumulation by regulating downstream genes [33], suggesting a potential link between ethylene perception and starch metabolism in lotus root. This is further supported by reports that ethylene suppresses starch synthase genes (e.g., SSIIa) in wheat [13], highlighting conserved regulatory nodes across species.

A feedback mechanism is indicated by the upregulation of NnEBF1-2 at the end of storage in the ET group, which may function to prevent excessive ethylene responses, consistent with the role of EBF1 in modulating EIN3/EIL1 stability [30]. While the precise cross-talk mechanism—particularly how NnEIN3 directly regulates starch metabolism genes like NnSSI and NnGBE—requires further validation, our results provide a foundational framework linking ethylene signal transduction to primary and secondary metabolic remodeling in postharvest lotus root.

5. Conclusions

In conclusion, postharvest temperature and exogenous ethylene treatment effectively regulated the quality maintenance, phenolic metabolism, and starch metabolism of lotus roots. Low temperature (4 °C) was conducive to maintaining appearance quality, water content, and Vc, thereby delaying browning. Low temperature (4 °C) maintained, and low PAL activity, preserved a high starch content and downregulated the expression of NnGBE1-1/2. In contrast, high temperature (35 °C) and ethylene treatment could enhance the accumulation of TPC, TFC and antioxidant capacity by activating the phenylpropanoid pathway. Ethylene signaling was involved in the transcriptional regulation of key structural genes related to polyphenol synthesis, which further promoted the accumulation of total phenolics, flavonoids, and monomeric phenols. Meanwhile, ethylene also modulated the expression of genes related to starch synthesis and degradation, thereby maintaining relatively high starch content. Based on the present results, low temperature (4 °C) is recommended to maintain the overall quality of lotus roots, while ethylene treatment at room temperature can be used as a practical strategy to improve the functional quality of lotus roots. This study only evaluated ET treatment at 25 °C; future research should explore the effect of ET at 4 °C and 35 °C to clarify whether temperature modulates ET’s regulatory role. Additionally, the molecular mechanism of cross-talk between ethylene signaling and starch metabolism requires further validation using gene silencing or overexpression techniques.