Introduction

Owing to higher phytochemicals, kiwifruit has received worldwide attention, and its consumption benefits human health. As a climacteric fruit, kiwifruit has a short shelf life, causing nutritional quality and commodity value to deteriorate during storage at 25 °C. Cold storage has been widely used for delaying senescence and preserving quality of kiwifruit. During cold storage, kiwifruit suffers from chilling injury, lessening its economic and nutritional value. Therefore, developing approaches for alleviating chilling injury and preserving quality in kiwifruit during cold storage in indispensable1,2,3,4.

During cold storage, insufficient intracellular ATP suppling arising from lower H+-ATPase, Ca2+-ATPase, succinic dehydrogenase (SDH), and cytochrome c oxidase (CCO) enzymes activity, intracellular reactive oxygen species (ROS) overaccumulation arising from ineffective ROS avoidance and scavenging systems activity and membrane integrity deterioration representing by lower membrane unsaturated/saturated fatty acids (unSFA/SFA) accumulation owing to higher oleic (C18:1), stearic (C18:0) and palmitic (C16:0) acids accumulation accompanied by lower linoleic (C18:2) and linolenic (C18:3) acids accumulation could be responsible for accelerating chilling injury symptoms development in kiwifruit5,6,7,8. By exogenous melatonin1,3, 1-methylcyclopropene (1-MCP)9,10,11, γ-aminobutyric acid (GABA)4, hot water treatment (HWT)12, sodium nitroprusside (SNP; exogenous NO donor)2, salicylic acid13, putrescine7, low-temperature conditioning (LTC, 12 °C for 3 days)14, hydrogen-rich water (HRW)15, abscisic acid (ABA)16 and phytosulfokine α (PSKα)17 application, alleviating chilling injury in kiwifruit during cold storage could be attributed to enhancing endogenous and salicylic acid biosynthesis and signaling, preventing O2− generation and H2O2 accumulation, improving ascorbate acid (AA) and glutathione (GSH) accumulation, and enhancing phenols and flavonoids accumulation, all are valuable in membrane integrity preservation evidenced by lower electrolytes leakage and malondialdehyde (MDA) accumulation.

By tyrosine supplying from the shikimate pathway, tyrosine decarboxylase (TyrDC) enzyme activity is responsible for tyramine biosynthesis from tyrosine, while monophenol hydroxylase (MH) enzyme activity is responsible for dopamine biosynthesis from tyramine. In addition, tyrosine hydroxylase (TyrH) enzyme activity is responsible for dihydroxyphenylalanine (DOPA) biosynthesis from tyrosine, while DOPA decarboxylase (DDC) enzyme activity is responsible for dopamine biosynthesis from DOPA18. Triggering TyrDC gene expression is crucial for enhancing endogenous dopamine accumulation in plants during environmental stimulus, which can serve as a defense strategy by exhibiting potential ROS scavenging capacity19. Wang et al.20 indicated that exogenous dopamine application promoted endogenous dopamine accumulation by triggering TyrDC gene expression, which could be responsible for enhancing flavonoids, anthocyanins, and sucrose accumulation, thereby improving apple fruit quality. Ali et al.21 reported that exogenous dopamine application alleviated chilling injury in banana fruits by promoting endogenous dopamine accumulation arising from triggering TyrDC gene expression, enhancing endogenous glycine betaine accumulation arising from triggering choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) genes expression accompanied by improving superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzymes activity giving rise to lower H2O2 accumulation, which could be crucial for membrane integrity maintenance as shown by lower electrolyte leakage and MDA accumulation. Recently, Nazari et al.22 reported that exogenous dopamine application alleviated chilling injury in banana fruits by triggering phenylalanine ammonia-lyase (PAL) gene expression and enzyme activity concurrent with suppressing polyphenol oxidase (PPO) gene expression and enzyme activity responsible for improving DPPH, FRAP, and ABTS radicals scavenging activity by promoting phenols and flavonoids accumulation, promoting endogenous proline accumulation by triggering pyrroline-5-carboxylate synthetase (P5CS) and ornithine δ-aminotransferase (OAT) genes expression and enzymes activity concurrent with suppressing proline dehydrogenase (ProDH) gene expression and enzyme activity and enhancing endogenous γ-aminobutyric acid (GABA) accumulation by triggering glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) genes expression and enzymes activity.

To the best of our knowledge, this is the first report regarding the effects of exogenous dopamine application in alleviating chilling injury in banana fruits during storage at 1 °C for 120 days by analyzing post-translational proteins SUMOylation responsive SUMO E3 ligase (SIZ1), post-translational proteins PARylation responsive poly(ADP-Ribose) polymerase 1 (PARP1) and post-translational proteins phosphorylation responsive target of rapamycin (TOR) and non-fermenting 1-related kinase 1 (SnRK1) genes expression, endogenous salicylic acid and proline accumulation, intracellular ATP and NADPH accumulation, endogenous H2O2 accumulation, endogenous phenols and flavonoids accumulation by PAL enzyme activity and ascorbic acid accumulation by CAT and SOD enzymes activity contributing in DPPH scavenging capacity and PLD and LOX genes expression along with electrolyte leakage and MDA accumulation representing membrane integrity.

Materials and methods

Kiwifruits and dopamine treatments

Kiwifruits (Actinidia deliciosa cv. Hayward) at commercial maturity (TSS 6.3%) were harvested from an orchard in Astara, Gilan province, Iran. Selecting healthy, uniformly sized fruits without disease or injuries, we randomly divided 1200 fruits into 5 lots, with three replicates of 80 fruits each. The fruits underwent immersion in solutions containing 0 (ddH2O as control), 50, 100, 150, and 200 µM dopamine for 20 min at 20 °C. Subsequently, fruits were air-dried at room temperature for 2 h and then transferred to cold storage at 1 ± 0.5 °C with 80% relative humidity for 120 days. Chilling injury and electrolyte leakage were evaluated every 30 days using 10 fruits, and flesh tissue from another 10 fruits was stored at − 80 °C for subsequent biochemical and gene expression analyses.

Chilling injury

The chilling injury was evaluated based on a 1–5 hedonic scale according to Jiao et al.3, 0 = without visible disorder, 1 = 0 〈 visible disorder covering ≤ 1/4, 2 = 1/4 < visible disorder covering ≤ 1/3, 3 = 1/3 < visible disorder covering ≤ 1/2, and 4 = visible disorder covering 〉 1/2. The scale considered the intensity of pulp lignification, grainy, water-soaking, or pitting. The chilling injury incidence (%) was calculated as Σ [(chilling injury level) × (number of fruit at the chilling injury level)] / (4 × total number of fruit) × 100.

Electrolyte leakage and MDA accumulation

Electrolyte leakage (%) was determined according to the procedures described by Aghdam et al.23. For assaying of MDA accumulation using thiobarbituric acid (TBA) method according to the procedures described by Aghdam et al.23, one gram of frozen powder was homogenized with 25 mL of 5% (w/v) trichloroacetic acid (TCA). Then, centrifuged for 10 min at 10,000×g. TBA reactivity was determined by adding 2.5 mL of 0.5% TBA in 1 5% TCA to 1.5 mL of the supernatant. The reaction solution was held for 30 min in boiling water, then cooled quickly and centrifuged at 12,000×g for 10 min. Absorbance was measured at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm, calculated with an extinction coefficient of 1.55 nmol L− 1 m− 1. MDA accumulation was expressed as nmol g− 1 fresh weight (FW).

CAT, and SOD activity, ascorbic acid and H2O2 accumulation

CAT and SOD enzymes activity (U mg− 1 protein), as well as ascorbic acid (mg kg− 1 FW) and H2O2 accumulation (µmol g− 1 FW) were assayed as stated by Jiao et al.3. For assaying of CAT and SOD enzymes activity, one gram of frozen powder was homogenized with 15 mL of 50 mM phosphate buffer (pH 7.8) containing 0.2 mM EDTA and 2% PVP. The homogenate was centrifuged at 4 °C at 12,000×g for 20 min. The supernatant was filtered and used for enzymes activity assay. For CAT, 100 µL of the enzyme extract was added to 2.9 mL of reaction mixture containing 15 mM H2O2 and 50 mM phosphate buffer (pH 7). The degradation of H2O2 was measured by the decrease in absorbance at 240 nm for 1 min. One unit of CAT activity was defined as a decrease in absorbance at 240 nm of 0.01 per minute. For SOD, the reaction mixture contained 50 mM phosphate buffer (pH 7), 5 mM methionine, 100 µM EDTA and 65 µM nitroblue tetrazolium (NBT). To 2.9 mL of the reaction mixture, 60 µL of enzyme extract and 40 µL of 0.15 mM riboflavin. The tubes were then placed in a fluorescent light incubator (40 W, 10 min) and the formation of blue formazan was monitored by recording the absorbance at 560 nm. One unit of SOD activity is defined as the enzyme that causes a 50% inhibition of NBT reduction under assay conditions. Protein content in the enzyme extracts was determined using the Bradford (1976) method with bovine serum albumin as standard. For assaying of ascorbic acid accumulation using ascorbic acid oxidation with 2,6-dichlorophenolindophenol (DCIP) dye, one gram of frozen powder was homogenized with 20 mL of 6% metaphosphoric acid in 2 M acetic acid. The mixture was centrifuged at 17,000×g for 15 min at 4 °C. One milliliter of supernatant was mixed with 0.05 mL of 0.2% DCIP and the solution was incubated at room temperature for 1 h. After that, 1 mL of 2% thiourea in 5% metaphosphoric acid and 0.5 mL of 2% dinitrophenyl hydrazine (DNPH) in 4.5 M sulfuric acid were added to the solution and then incubated at 60 °C for 3 h. The reaction was stopped by placing the tubes in an ice bath and slowly adding 2.5 mL of cold 90% sulfuric acid. Total ascorbic acid was measured by absorbance at 540 nm using a standard curve. For assaying of H2O2 accumulation, one gram of frozen powder was homogenized with 5 ml of acetone at 0 °C. After centrifugation for 15 min at 6000×g at 4 °C, the supernatant was collected. The supernatant (1 ml) was mixed with 0.1 ml of 5% titanium sulfate and 0.2 ml ammonia and then centrifuged for 10 min at 6000×g at 4 °C. The pellets were dissolved in 3 ml of 10% (v/v) H2SO4 and centrifuged for 10 min at 5000×g at 4 °C. Absorbance of the titanium-peroxide complex was measured at 410 nm.

PAL activity, phenols and flavonoids accumulation, and DPPH scavenging capacity

PAL enzyme activity (U mg− 1 protein) was determined according to Nguyen et al.24. Phenols accumulation (mg GAE kg− 1 FW) was assessed using the Folin-Ciocalteu method23, while flavonoids accumulation (g kg− 1 FW) was measured using the AlCl3 colorimetric procedure as stated by Sharafi et al.25. DPPH scavenging capacity (%) was determined following the protocol of Aghdam et al.23. For assaying of PAL enzyme activity, one gram of frozen powder was homogenized with 20 mL of 50 mM borate buffer (pH 8.5) containing 5 mM β-mercaptoethanol and 0.5 g polyvinylpolypyrrolidone. Then, centrifuged at 18,000×g for 20 min at 4 °C. PAL activity was determined in the supernatant, 0.3 mL of which was added to a reaction mixture containing 0.7 mL of 100 mM l-phenylalanine and 3 mL of 50 mM borate buffer (pH 8.5). After incubation of the mixture at 40 °C for 1 h, the reaction was stopped by adding 0.1 mL of 5 mM HCl. PAL enzyme activity was calculated from the absorbance of the assay mixture at 290 nm. For assaying of phenols and flavonoids accumulation, one gram of frozen powder was homogenized with 8 mL of methanol and extracted for 24 h in the dark. For assaying of phenols accumulation, 0.1 mL of extract was mixed with 2 ml of 2% Na2CO3 and allowed to stand for 2 min at room temperature. For each sample 0.1 mL of 50% (v/v) Folin–Ciocalteu reagent was added with mixing and allowed to stand for 30 min, and the absorbance was measured at 720 nm. For assaying of flavonoids accumulation, 75 µL of aqueous NaNO2 (5%) was added to 0.25 ml of extracts. After 5 min, 0.15 mL of 10% aqueous AlCl3 was added and was vortexed. The mixture was allowed to stand for 6 min at room temperature. Then 0.5 mL of 1 mol L− 1 NaOH was added to this mixture. The final volume was adjusted to 2.5 ml with deionized water. The absorbance was measured against the blank at 507 nm. For assaying of DPPH scavenging capacity, one gram of frozen powder was homogenized with 8 mL of methanol and extracted for 24 h in the dark. Then the homogenate was centrifuged at 12,000×g for 20 min at 4 °C. Fifty µL of the supernatant was added to 1.0 mL of 6 × 10− 5 M DPPH radical in methanol. The mixture was shaken and left at room temperature for 30 min; the absorbance was measured at 515 nm.

Endogenous ATP, NADPH, salicylic acid and proline accumulation

Endogenous ATP (nmol g− 1 FW), NADPH (µg kg− 1 FW), and salicylic acid (µg kg− 1 FW) accumulation were analyzed by Plant ATP ELISA Kit, Plant NADPH ELISA Kit and Plant SA ELISA Kit (Shanghai YuDuoBio, Shanghai, China) at 450 nm, following the manufacturer’s protocol. For assaying of endogenous proline accumulation by the acid ninhydrin method as described by Aghdam et al.23, one gram of frozen powder was homogenized with 10 mL of 3% (v/v) sulfosalicylic acid. Endogenous proline accumulation was expressed in mmol kg− 1 on a fresh weight basis.

Genes expression assay by RT-qPCR

Total RNA extraction and cDNA synthesis were carried out as stated by Jiao et al.3. Stress-responsive genes expression was analyzed by the ABI StepOne system (Applied Biosystems) with the SYBR Green PCR Master Mix (Jena Bioscience, Germany) with three technical replicates. Normalization to the Actin Ct value was performed (Table 1), and the relative gene expression was calculated using the 2−ΔΔCt formula as Livak and Schmittgen26.

Table 1 Primers used in the qRT-PCR analysis of gene expression.
Full size table

Statistical analysis

The experiment employed a factorial design within a completely randomized design (CRD), featuring two primary factors: treatments (0 and 150 µM dopamine) and storage time (30, 60, 90, and 120 days). Each combination of treatment and storage time was replicated three times, with each replication consisting of 80 fruits. Twenty fruits were sampled at each of the specified storage times within a replication. Data are presented as means ± standard errors (S.E.) derived from three biological replications. A two-way analysis of variance (ANOVA) was conducted to assess the impact of dopamine concentration and storage time. Duncan’s test were performed to compare differences between mean values at a 5 and 1% level of significance (P < 0.05 and P < 0.01). The analysis was carried out using SPSS software, version 20.

Results

Chilling injury

As shown in Fig. 1, kiwifruits treated with dopamine at concentrations of 0, 50, 100, 150, and 200 µM exhibited chilling injury percentages of 54, 45, 50, 35, and 48%, respectively, after 120 days of storage at 1 °C. Therefore, kiwifruits subjected to 150 µM dopamine were selected for further biochemical and gene expression analysis.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
Full size image

Chilling injury in kiwifruits treated with dopamine at concentrations of 0, 50, 100, 150, and 200 µM during storage at 1 °C for 120 days. Mean values (n = 3) are presented, and error bars denote standard errors of the means. Different letters indicate significant differences determined by Duncan’s test at P < 0.05.

Electrolyte leakage and MDA accumulation

As shown in Fig. 2, kiwifruits treated with 150 µM dopamine demonstrated significantly lower electrolyte leakage (Fig. 2A; P < 0.05) and MDA accumulation (Fig. 2B; P < 0.01) compared to the control during storage at 1 °C for 120 days.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.
Full size image

Electrolyte leakage (A) and malondialdehyde accumulation (B) in kiwifruits treated with dopamine at concentrations of 0 and 150 µM during storage at 1 °C for 120 days. Data displayed as mean values (n = 3), with error bars representing standard errors. Different letters indicate significant differences determined by Duncan’s test at P < 0.05.

SIZ1, PARP1, TOR and SnRK1 genes expression

During storage at 1 °C for 120 days, kiwifruits displayed higher PARP1, SIZ1, and SnRK1 genes expression while lower TOR gene expression. As shown in Fig. 3, kiwifruits treated with 150 µM dopamine displayed higher SIZ1 (Fig. 3A; P < 0.01) and TOR (Fig. 3B; P < 0.01) genes expression along with lower PARP1 (Fig. 3C; P < 0.01) and SnRK1 (Fig. 3D; P < 0.01) genes expression compared to the control during cold storage.

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
Full size image

Gene expression levels of SIZ1 (A), TOR (B), PARP1 (C) and SnRK1 (D) in kiwifruits treated with dopamine at concentrations of 0 and 150 µM during storage at 1 °C for 120 days. Mean values (n = 3) are presented, and error bars denote standard errors of the means. Different letters indicate significant differences determined by Duncan’s test at P < 0.05.

SOD and CAT enzymes activity along with ascorbic acid and H2O2 accumulation

In Fig. 4, it is evident that kiwifruits treated with 150 µM dopamine displayed elevated CAT enzyme activity (Fig. 4A; P < 0.01) compared to the control during storage at 1 °C for 120 days. There was no significant difference between control and dopamine treatment in SOD activity (Fig. 4B; P < 0.01). Furthermore, this increased CAT enzyme activity corresponded to reduced H2O2 accumulation (Fig. 4C; P < 0.05) and heightened ascorbic acid levels (Fig. 4D; P < 0.01) compared to the control during the storage period.

Fig. 4
Fig. 4The alternative text for this image may have been generated using AI.
Full size image

Activity of SOD (A) and CAT (B) enzymes along with H2O2 (C) and ascorbic acid (D) accumulation in kiwifruits treated with dopamine at concentrations of 0 and 150 µM during storage at 1 °C for 120 days. Mean values of n = 3 are displayed, with error bars indicating standard errors of the means. Different letters denote significant differences based on Duncan’s test at P < 0.05.

PAL activity, phenols and flavonoids accumulation, and DPPH scavenging capacity

As shown in Fig. 5, kiwifruits treated with 150 µM dopamine exhibited higher PAL enzyme activity (Fig. 5A; P < 0.01) compared to the control during cold storage. Higher PAL enzyme activity in kiwifruits treated with 150 µM dopamine was accompanied by higher phenols (Fig. 5B; P < 0.05) and flavonoids (Fig. 5C; P < 0.05) accumulation along with higher DPPH scavenging capacity (Fig. 5D; P < 0.01) compared to the control.

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
Full size image

PAL enzyme activity (A) along with accumulation of phenols (B) and flavonoids (C), and DPPH scavenging capacity (D) in kiwifruits treated with dopamine at concentrations of 0 and 150 µM during storage at 1 °C for 120 days. Data displayed as mean values (n = 3), with error bars representing standard errors. Different letters indicate significant differences determined by Duncan’s test at P < 0.05.

PLD and LOX genes expression

As shown in Fig. 6, kiwifruits treated with 150 µM dopamine displayed lower PLD (Fig. 6A; P < 0.05) and LOX (Fig. 6B; P < 0.01) genes expression compared to the control during storage at 1 °C for 120 days.

Fig. 6
Fig. 6The alternative text for this image may have been generated using AI.
Full size image

Expression of PLD (A), and LOX (B) genes in kiwifruits treated with dopamine at 0 and 150 µM during storage at 1 °C for 120 days. Mean values (n = 3) are presented, with error bars indicating standard errors. Different letters denote significant differences based on Duncan’s test at P < 0.05.

Endogenous ATP, NADPH, salicylic acid and proline accumulation

As shown in Fig. 7, kiwifruits treated with 150 µM dopamine demonstrated heightened levels of ATP (Fig. 7A; P < 0.01), NADPH (Fig. 7B; P < 0.05), and salicylic acid accumulation (Fig. 7C; P < 0.05) compared to the control during storage at 1 °C for 120 days. As shown in Fig. 7D, kiwifruits treated with 150 µM dopamine displayed significantly increased proline accumulation (P < 0.01) compared to the control during cold storage.

Fig. 7
Fig. 7The alternative text for this image may have been generated using AI.
Full size image

Accumulation of endogenous ATP (A), NADPH (B), salicylic acid (C) and proline (D) in kiwifruits treated with dopamine at concentrations of 0 and 150 µM during storage at 1 °C for 120 days. Mean values (n = 3) are presented, with error bars representing standard errors. Different letters indicate significant differences determined by Duncan’s test at P < 0.05.

Discussion

Kiwifruits treated with dopamine at 150 µM exhibited the lowest chilling injury during storage at 1 °C. Concomitant with chilling injury symptoms appearance during storage at 1 °C for 120 days, kiwifruits displayed higher post-translational proteins PARylation responsive PARP1, post-translational proteins phosphorylation responsive SnRK1, and post-translational proteins SUMOylation responsive SIZ1 genes expression along with lower post-translational proteins phosphorylation responsive TOR gene expression, which could be responsible for insufficient intracellular energy (ATP) and reducing power (NADPH) supplying leading to ineffective CAT and SOD enzymes activity leading to higher endogenous H2O2 accumulation along with lower ascorbic acid accumulation, lower phenols and flavonoids accumulation leading to lower DPPH scavenging capacity, and higher PLD and LOX genes expression responsible for accelerating membrane integrity deterioration as evidenced by higher electrolyte leakage and MDA accumulation.

By 150 µM dopamine application, kiwifruits exhibited lower chilling injury during storage at 1 °C for 120 days, which was associated with higher SUMO E3 ligase SIZ1 gene expression, accompanied by higher membrane integrity as shown by lower electrolyte leakage and MDA accumulation. During low-temperature storage, peach fruits displayed higher SIZ1 gene expression. By exogenous melatonin application, triggering SIZ1 gene expression was accompanied by higher chilling tolerance in peach fruits27. Jiao et al.28 reported that tomato fruits displayed higher WRKY31 transcription factor expression and higher SIZ1 gene and protein expression. Jiao et al.28 reported the transactivation of the SIZ1 promoter by WRKY31 protein through the binding of WRKY31 to the W-box binding motif in the SIZ1 promoter. Jiao et al.28 reported that SIZ1 is responsible for alleviating chilling injury in tomato fruits by enhancing CAT, SOD, APX and GR genes expression and enzymes activity leading to suppressing ROS accumulation. Accordingly, lower endogenous H2O2 accumulation, higher ascorbic acid accumulation along with higher CAT enzyme activity in kiwifruits treated with dopamine could be attributed to triggering SIZ1 genes expression.

As well as SIZ1 gene expression, alleviating chilling injury in kiwifruits treated with 150 µM dopamine during storage at 1 °C for 120 day was associated with higher TOR gene expression and lower SnRK1 gene expression. Concomitant with chilling injury symptoms appearance, insufficient intracellular ATP supplying, intracellular ROS overaccumulation, and insufficient intracellular sucrose and glucose supplying could be responsible for SnRK1 signaling pathway activation while TOR signaling pathway suppression29. By exogenous trehalose application, alleviating chilling injury in guava fruits could be attributed to higher endogenous trehalose accumulation accompanied by lower endogenous sucrose accumulation, which is accountable for inhibiting SnRK1 gene expression and activity in guava fruits30. By employing nuclear magnetic resonance (NMR)-based metabolomics analysis, Wang et al.31 reported that conferring chilling injury tolerance, as evidenced by lower internal browning in peach fruits by exogenous trehalose application, was accompanied by higher proline accumulation. By exogenous trehalose application, endogenous trehalose accumulation may be responsible for suppressing SnRK1 gene expression. By suppressing SnRK1 gene expression, enhancing endogenous proline accumulation in peach fruits by exogenous trehalose application could be attributed to suppressing ProDH gene expression and enzyme activity31. By enhancing the SnRK1-bZIP63 signaling pathway during energy starvation response, proline could be employed by proline dehydrogenase (ProDH) enzyme activity to support electrons for mitochondria electron transport system activity and participate in energy maintenance32. In addition, the TOR-S6K signaling pathway is responsible for supporting proline biosynthesis by P5CS and P5CR phosphorylation33. In plants, the glucose-TOR signaling pathway is responsible for the transcriptional reprogramming glycolysis (EMP) pathway, tricarboxylic acid (TCA) cycle, and electron transport system for sufficient ATP, NADH, and carbon skeletons supplying oxidative pentose phosphate (OxPP) pathway for sufficient NADPH and carbon skeletons supplying; shikimate pathway for sufficient phenylalanine, tyrosine and tryptophan supplying; phenylpropanoid-flavonoid pathway for phenols, flavonoids and anthocyanins biosynthesis33,34. In addition, the glucose-TOR signaling pathway directs transcriptional networks for suppressing branched-chain valine, leucine, and isoleucine degradation, proline degradation, phospholipids degradation, and mitochondrial alternative electron transferring system activity33,34. Therefore, higher intracellular energy (ATP) and reducing power (NADPH) supply in kiwifruits treated with dopamine could be attributed to lower SnRK1 gene expression and higher TOR gene expression. By triggering TOR/SnRK1 gene expression, enhancing endogenous proline accumulation could be advantageous for attenuating kiwifruit chilling injury by dopamine treatment.

In peach fruits, attenuation of chilling injury through salicylic acid combined with ultrasound treatment is associated with higher endogenous salicylic acid accumulation and higher CAT, APX, MDHAR, DHAR, and GR enzyme activity35. In cucumber fruits (a non-climacteric fruit), salicyloyl chitosan treatment alleviated chilling injury by promoting endogenous salicylic acid accumulation, enhancing CAT, SOD, APX, and GR enzymes activity, improving ascorbic acid accumulation, accompanied by preserving membrane integrity36. Gao et al.37 reported that exogenous melatonin application alleviated chilling injury in peach fruits by promoting OxPP pathway activity representing by higher glucose-6-phosphatase dehydrogenase (G6PDH) enzyme activity responsible for sufficient reducing power NADPH and carbon skeleton erythrose 4-phosphate Er4P supplying for promoting shikimic acid pathway activity representing by higher shikimate dehydrogenase (SKDH) enzyme activity responsible for supplying sufficient phenylalanine for promoting phenylpropanoid pathway activity representing by higher PAL enzyme activity responsible for endogenous salicylic acid accumulation. In addition to endogenous salicylic acid accumulation, higher phenols accumulation in peach fruits by exogenous melatonin application could be attributed to higher PAL/PPO enzymes activity. Gao et al.37 reported that attenuating chilling injury in peach fruits by exogenous melatonin application could be attributed to higher membrane unsaturated/saturated fatty acids (unSFA/SFA) accumulation may arise from sufficient NADPH supplying. By exogenous melatonin application, Guo et al.1 attenuating chilling injury in kiwifruits could be attributed to higher endogenous salicylic acid accumulation arising from higher PAL and benzoic acid 2-hydroxylase (BA2H) enzymes activity. By enhancing endogenous salicylic acid accumulation, enhancing CAT, SOD, and APX enzymes activity leading to lower O2− generation and H2O2 accumulation advantageous for membrane integrity preservation as evidenced by lower electrolyte leakage and MDA accumulation1. Hence, higher endogenous salicylic acid accumulation in kiwifruits treated with dopamine could be attributed to triggering TOR/SnRK1 gene expression, which enhances shikimate and phenylpropanoid pathways activity34. In addition to endogenous salicylic acid accumulation, enhancing shikimate and phenylpropanoid pathways activity by triggering TOR/SnRK1 gene expression is crucial for enhancing phenols and flavonoids accumulation, leading to higher DPPH scavenging capacity in kiwifruits treated with dopamine.

Hu et al.38 reported that exogenous 24-epibrassinolide application attenuated chilling injury in peach fruit by triggering CBF5 transcription factor expression while suppressing PLD and LOX genes expression, which could be responsible for higher unSFA/SFA accumulation, thereby preserving membrane integrity, as evidenced by lower MDA accumulation. Kong et al.39 reported that exogenous melatonin application alleviated chilling injury in green bell pepper fruits by suppressing NAC1 transcription factor expression while suppressing PLD and LOX genes expression and enzymes activity,, which could be responsible for higher unSFA/SFA accumulation, thereby preserving membrane integrity, as evidenced by lower electrolyte leakage and MDA accumulation. Wang et al.6 reported that higher chilling tolerance in mature kiwifruit during cold storage could be ascribed to lower O2− generation and H2O2 accumulation arising from higher CAT, SOD, APX, and GR enzymes activity accompanied by sufficient ATP supplying arising from higher H+-ATPase, Ca2+-ATPase, CCO, and SDH enzymes activity, both are beneficial for preserving membrane integrity, as evidenced by lower electrolyte leakage and MDA accumulation. He et al.40 reported that sufficient ATP supplying is crucial for alleviating chilling injury in banana fruits by suppressing PLD and LOX enzymes activity. By exogenous melatonin application, delaying lotus seeds senescence delaying could be attributed to sufficient ATP supplying arising from higher H+-ATPase, Ca2+-ATPase, CCO, and SDH enzymes activity accompanied by suppressing PLD and LOX enzymes activity, which have benefits in membrane integrity preservation41. Li et al.42 reported that banana fruits during chilling stress, displayed higher PLD and LOX genes expression and enzymes activity along with lower fatty acid desaturase (FAD) gene expression and enzyme activity, which is crucial for lower unSFA/SFA accumulation, thereby membrane integrity deteriorating as evidenced by higher electrolyte leakage and MDA accumulation. Therefore, suppressing PLD and LOX genes expression in kiwifruits treated with dopamine could be responsible for membrane integrity preservation, as evidenced by lower electrolyte leakage and MDA accumulation.

ROS overaccumulation during stresses enhances PARP1 gene expression and enzyme activity, which is accountable for poly(ADP)-ribosylation of nuclear target proteins. By utilization of NAD+, insufficient NAD+ and ATP supply during stresses could be attributed to higher PARP1 enzyme activity. In addition to intracellular NADP+ supply, providing intracellular NAD+ is essential for NADH production and is crucial for a sufficient intracellular ATP supply. Therefore, higher PARP1 enzyme activity during stresses is responsible for insufficient NADH, NADPH and ATP provision accompanied by ROS overaccumulation43,44. Vanderauwera et al.44 reported that suppressing PARP1 gene expression and enzyme activity could be advantageous for stress tolerance by promoting NADH and ATP provision. By ozone application, strawberry fruits displayed lower PARP1 gene expression accompanied by sufficient intracellular NAD+ and ATP supply. By ozone application, sufficient intracellular NAD+ supply is accompanied by higher NADK enzyme activity, which provides sufficient intracellular NADPH by enhancing G6PDH enzyme activity. By providing intracellular ATP and NADPH, enhancing GSH biosynthesis is crucial for suppressing ROS accumulation and improving DPPH scavenging capacity, thereby delaying senescence45. Piechowiak et al.46 reported that competitive PARP inhibitor nicotinamide (NAM) treatment attenuates oxidative stress in strawberry fruit by suppressing PARP1 gene expression and improving NAD+ metabolism. Oxidative stress resulting from UV-C treatment in strawberry fruits was accompanied by higher PARP-1 gene and protein expression while attenuating oxidative stress in strawberry fruits as evidenced by higher ABTS scavenging capacity by NAM treatment could be attributed to suppressing PARP1 gene and protein expression. By suppressing PARP1 gene and protein expression, strawberry fruits treated with NAM displayed higher NAD+ accumulation accompanied by higher SDH protein expression and enzyme activity, contributing to sufficient intracellular ATP supply. By enhancing NAD+ kinase enzyme activity in strawberry fruits treated with nicotinamide (NAM), sufficient NADP+ supplying could be employed by G6PDH enzyme activity for sufficient intracellular NADPH supplying46. Therefore, sufficient intracellular ATP and NADPH supplying in kiwifruits treated with dopamine could be attributed to suppressing PARP1 gene expression, which could be beneficial for suppressing membrane integrity deterioration, as evidenced by lower electrolyte leakage and MDA accumulation.

Conclusion

In summary, dopamine treatment emerged as a promising strategy for mitigating chilling injury in kiwifruits by preserving membrane integrity, as shown by lower electrolyte leakage and MDA accumulation during cold storage. Alleviating chilling injury in kiwifruits by dopamine treatment may be ascribed to triggering TOR and SIZ1 genes expression along with suppressing SnRK1 and PARP1 genes expression leading to enhancing endogenous salicylic acid and proline accumulation, enhancing phenols and flavonoids accumulation leading to higher DPPH scavenging capacity, suppressing endogenous H2O2 accumulation along with enhancing ascorbic acid accumulation arising from higher CAT/SOD enzyme activity. Hence, triggering TOR and SIZ1 gene expression and suppressing SnRK1 and PARP1 gene expression may be the prevailing molecular mechanism hired by dopamine treatment for alleviating chilling injury in kiwifruits. However, further studies at biochemical and molecular levels are needed for an illustration of dopamine effects on endogenous salicylic acid biosynthesis (PAL, BA2H and ICS genes expression and enzymes activity) and signaling (NPR1, TGA and PRs genes expression), endogenous proline accumulation (P5CS, OAT and ProDH genes expression and enzymes activity), and phenylpropanoid pathway (PAL, 4CL, C4H and CHS genes expression and enzymes activity) of kiwifruits during cold storage.