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MAP  >  Technology in Horticulture
2024 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Plant materials, growth conditions, and stress treatment
Vector construction
Chlorophyll and anthocyanin content determination
Physiological analysis
RNA isolation and qRT-PCR analysis
Results
Screening and characterization of lncRNAs
Generation of lncRNA overexpression lines
Effect of lncRNA overexpression on heat tolerance in Arabidopsis
Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis
Effect of lncRNA overexpression on drought tolerance in Arabidopsis
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
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References
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ARTICLE   Open Access    

Doses of aminoethoxyvinylglycine (AVG) in tomato postharvest storage

  • 1.

    Faculty of Agronomic Sciences, São Paulo State University, Botucatu, São Paulo 18610, Brazil

  • 2.

    Montes Claros State University, Janaúba, Minas Gerais 39440, Brazil

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  • Tomatoes are one of the main vegetables found daily in world cuisine and are highly perishable. The aim of this study was to evaluate the quality of 'Débora' tomato fruits over the post-harvest period in refrigerated storage, after immersion in solutions with different concentrations of aminoethoxyvinylglycine (AVG). The experimental design was entirely randomized in a 4 × 5 double factorial scheme, with four treatments, AVG doses 0 (control), 500, 1,000, and 1,500 mg·L−1, and five days of evaluation during the 28 d of refrigerated storage (days 0, 7, 14, 21 and 28) with three replications. Physicochemical analyses were carried out on weight loss, respiration rate, firmness, hydrogen potential (pH), soluble solids, titratable acidity and external and internal fruit color parameters, luminosity, chroma, and °hue. Two-way ANOVA, Tukey's mean test (p < 0.05), and multivariate statistical analysis by principal components (PCA) were carried out. The PCA allowed us to infer that, as weight loss increased, firmness decreased, an effect that was minimized with AVG at a dose of 1,500 mg·L−1, which may be related to the inhibition of the fruit's ethylene production rate. AVG delayed the post-harvest ripening of tomato fruit, reduced the respiration rate of the fruit, and the changes in external and internal chroma. The doses of AVG did not affect the luminosity and pH of the fruit pulp.

  • Introduction

    Plants are continuously subjected to unpredictable environmental conditions and encounter a multitude of stressors throughout their growth and development, posing a significant challenge to global crop production and food security[1]. Heat and drought are undoubtedly the two most important stresses that have a huge impact on crops. Both elicit a wide array of biochemical, molecular, and physiological alterations and responses, impacting diverse cellular processes and ultimately influencing crop yield and quality[2].

    A primary physiological consequence of both stresses is the diminished photosynthetic capacity, partially resulting from the degradation of chlorophyll due to leaf senescence under stress conditions. Chlorophyll accumulation was diminished in numerous plants subjected to drought or heat stress conditions[3,4]. Various environmental stresses prompt excessive generation of reactive oxygen species (ROS), initiating oxidative damage that compromises lipids, and proteins, and poses a serious threat to cellular functions[2]. To mitigate oxidative stress and minimize damage, plants have developed various protective mechanisms to neutralize ROS. Several antioxidant enzymes, such as SOD, POD, and CAT, are integral to cellular antioxidative defense mechanisms. Additionally, antioxidants such as anthocyanins and proline serve as crucial ROS scavengers[5,6]. The elevation in temperature typically induces the transient synthesis of heat shock proteins (Hsps), which function as molecular chaperones in protecting proteins from denaturation and aggregation, with their activity primarily regulated at the transcriptional level by heat shock factors (Hsfs)[7]. The significance of Hsps and Hsfs in all organisms, including plants, has been assessed in various stress conditions that could disrupt cellular homeostasis and result in protein dysfunction[7]. Drought stress can also trigger the transcription of a suite of marker genes, including RD29A, RD29B, NCED3, AREB1, Rab18, etc., which assist plants in mitigating cellular damage during dehydration and bolstering their resilience to stress[8−10].

    Previous research efforts focusing on the regulatory control of stress-related genes have largely centered around protein-coding genes. In recent years, non-protein-coding transcripts have emerged as important regulatory factors in gene expression. Among them, long non-coding RNAs (lncRNAs) lncRNAs have been identified as implicated in various abiotic stresses[11,12]. LncRNAs are a class of non-coding RNAs (ncRNAs) exceeding 200 nucleotides in length. They possess minimal or no protein-coding potential[13]. In plants, lncRNAs are specifically transcribed by RNA polymerases Pol IV, Pol V, Pol II, and Pol III[14,15]. LncRNAs exhibit low abundance and display strong tissue and cellular expression specificity relative to mRNAs. Moreover, sequence conservation of lncRNAs is was very poor across different plant species[13,16,17]. The widespread adoption of high-throughput RNA sequencing technology has revealed lncRNAs as potential regulators of plant development and environmental responses. In cucumber, RNA-seq analysis has predicted 2,085 lncRNAs to be heat-responsive, with some potentially acting as competitive endogenous RNAs (ceRNAs) to execute their functions[18]. In radish, a strand-specific RNA-seq (ssRNA-seq) technique identified 169 lncRNAs that were differentially expressed following heat treatment[19]. In Arabidopsis, asHSFB2a, the natural antisense transcript of HSFB2a was massively induced upon heat stress and exhibited a counteracted expression trend relative to HSFB2a. Overexpression of asHSFB2a entirely suppressed the expression of HSFB2a and impacted the plant's response to heat stress[20]. For drought stress resistance, 244 lncRNAs were predicted in tomatoes to be drought responsive probably by interacting with miRNAs and mRNAs[21]. Under drought stress and rehydration, 477 and 706 lncRNAs were differentially expressed in drought-tolerant Brassica napus Q2 compared to drought-sensitive B. napus, respectively[22]. In foxtail millet and maize, 19 and 644 lncRNAs, respectively, were identified as drought-responsive[23,24]. Despite the identification of numerous lncRNAs by high-throughput sequencing, which suggests their potential involvement in various abiotic stress processes, only a minority have been experimentally validated for function.

    In our previous study, we characterized 1,229 differentially expressed (DE) lncRNAs in Chinese cabbage as heat-responsive, and subsequent bioinformatics analysis reduced this number to 81, which are more likely associated with heat resistance[25]. lnc000283 and lnc012465 were selected from among them for further functional investigation. The findings indicated that both lnc000283 and lnc012465 could be promptly induced by heat shock (HS). Overexpression of either lnc000283 or lnc012465 in Arabidopsis plants enhanced their capacity to tolerate heat stress. Additionally, both lnc000283 and lnc012465 conferred drought tolerance to transgenic Arabidopsis.

    Materials and methods

    Plant materials, growth conditions, and stress treatment

    The lncRNA sequences examined in this study were from Chiifu-401-42 Chinese cabbage and all Arabidopsis plants were of the Col-0 background. Transgenic plants expressing lnc000283 and lnc012465 were generated using the Agrobacterium tumefaciens-mediated floral dip method[26]. Single-copy and homozygous T3 plants were identified through genetic segregation on an agar medium supplemented with kanamycin. The T3 generation plants, or their homozygous progeny, were utilized in the experiments.

    For phenotypic assessment, Arabidopsis seeds were initially sown on filter paper moistened with ddH2O and placed in a 4 °C freezer for 2 d. Subsequently, they were evenly planted in nutrient-rich soil and transferred to a growth chamber operating a 16-h day/8-h night cycle, with day/night temperatures of 22 °C/18 °C and a light intensity of 250 μmol·m−2·s−1. After 10 d of growth, Arabidopsis plants with uniform growth were transferred to 50-hole plates. Arabidopsis plants grown in Petri dishes were firstly seed-sterilized and then sown on 1/2 MS medium supplemented with 10 g·L−1 sucrose. The seeds were then placed in a 4 °C refrigerator for 2 d in the dark before transferring them to a light incubator. The day/night duration was set to 16 h/8 h, the day/night temperature to 21 °C/18 °C, and the light intensity to 100 μmol·m−2·s−1.

    For heat treatment, 3-week-old seedlings were subjected to 38 °C for 4 d within a light incubator, subsequently transferred to their original growth conditions under the same light/dark cycles. For drought treatment, 3-week-old Arabidopsis seedlings were deprived of water for 10 d, followed by rehydration to facilitate a 2-d recovery period. Plants were photographed and surveyed both before and after treatment.

    Vector construction

    The lncRNA sequences (lnc000283 and lnc012465) were chemically synthesized based on RNA-seq data, with restriction sites for BamH1 and Kpn1 engineered upstream and downstream. The resultant lncRNA constructs were subcloned into the pCambia2301 binary vector, incorporating a cauliflower mosaic virus (CaMV) 35S promoter. The recombinant vectors were transformed into Escherichia coli TOP10 competent cells (Clontech), incubated at 37 °C overnight, after which single clones were selected for PCR verification, and the confirmed positive colonies were submitted for sequencing. Following verification, the correct plasmids were introduced into A. tumefaciens strain GV3101 using the freeze-thaw method and subsequently transformed into Arabidopsis wild-type (Col) plants.

    Chlorophyll and anthocyanin content determination

    To quantify the chlorophyll content, the aerial portions of wild-type and transgenic Arabidopsis plants, grown in Petri dishes were weighed, minced, and then subjected to boiling in 95% ethanol until fully decolorized. Aliquots of 200 μL from the extract were transferred to a 96-well plate and the absorbance at 663 nm and 645 nm was measured via spectrophotometry by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Three biological replicates were analyzed for WT and each transgenic line. Chlorophyll content was determined according to the formula of the Arnon method[27]: Chlorophyll a = (12.72A663 − 2.59A645) v/w, Chlorophyll b = (22.88A645 − 4.67A663) v/w, Total chlorophyll = (20.29A645 + 8.05A663) v/w.

    The quantification of anthocyanin was performed as follows: aerial parts of wild-type and transgenic Arabidopsis plants, cultivated in Petri dishes, were weighed and ground to powder in liquid nitrogen. Subsequently, the samples were incubated in 600 μL of acidified methanol (containing 1% HCl) at 70 °C for 1 h. Following this, 1 mL of chloroform was added, and the mixture was vigorously shaken to remove chlorophyll. The mixture was then centrifuged at 12,000 rpm for 5 min, after which the absorbance of the aqueous phase was determined at 535 nm using a spectrophotometer (Shimadzu, Kyoto, Japan). Three biological replicates were analyzed for WT and each transgenic line. The relative anthocyanin content was calculated according to anthocyanin concentration and extraction solution volume. One anthocyanin unit is defined as an absorption unit at a wavelength of 535 nm in 1 mL of extract solution. In the end, the quantity was normalized to the fresh weight of each sample.

    Physiological analysis

    Three-week-old transgenic and WT A. thaliana plants, subjected to normal conditions or varying durations of heat or drought stress, were utilized for subsequent physiological assessments. All assays were performed in accordance with the method described by Chen & Zhang[28]. In brief, 0.1 g of fresh leaf tissue was homogenized in 500 μL of 100 mM PBS (pH 7.8) while chilled on ice. The homogenate was then centrifuged at 4 °C, and the resultant supernatant was employed for further analysis. For the determination of MDA content, 100 μL of the supernatant was combined with 500 μL of a 0.25% thiobarbituric acid (TBA) solution (which was prepared by dissolving 0.125 g of TBA in 5 mL of 1 mol·L−1 NaOH before being added to 45 mL of 10% TCA) and boiled for 15 min. Following a 5 min cooling period on ice, the absorbance was measured at 532 nm and 600 nm. The activity of POD was determined as follows: initially, 28 μL of 0.2% guaiacol and 19 μL of 30% H2O2 were sequentially added to 50 mL of 10mM PBS (pH 7.0), after thorough heating and mixing, 1 mL was transferred into a cuvette, then 50 μL of the supernatant was added to the cuvette and the absorbance at 470 nm was monitored every 15 s for 1 min. To determine the proline content, a reaction solution was prepared by mixing 3% sulfosalicylic acid, acetic acid, and 2.5% acidic ninhydrin in a ratio of 1:1:2, then 50 μL of the supernatant was added to 1 mL of the reaction solution, which was then subjected to a boiling water bath for 15 min (the solution turned red after the boiling water bath). Following cooling on ice, the absorbance at 520 nm was recorded. For the quantification of proline, an L-proline standard curve was prepared by dissolving 0, 5, 10, 15, 20, 25, and 30 μg of L-proline in 0.5 mL of ddH2O, followed by the addition of 1 mL of the reaction solution and measuring the absorbance at 520 nm. The proline content in the samples was then determined based on the L-proline standard curve.

    RNA isolation and qRT-PCR analysis

    Total RNA was isolated from the aerial parts of Arabidopsis using the TaKaRa MiniBEST Plant RNA Extraction Kit, followed by purification and reverse transcription using the PrimeScript RT reagent Kit with gDNA Eraser (Takara). The cDNA product was diluted 10 times and real-time PCR was conducted in triplicate for each biological replicate using SYBR PCR Master Mix (Applied Biosystems) on the ABI 7500 system under the following conditions: 98 °C for 3 min, followed by 40 cycles of 98 °C for 2 s and 60 °C for 30 s. The relative expression levels of each gene were normalized against the transcript abundance of the endogenous control UBC30 (At5g56150) and calculated using the 2−ΔCᴛ method. The specific primers employed for qRT-PCR are detailed in Supplemental Table S1.

    Results

    Screening and characterization of lncRNAs

    In our prior investigation, dozens of lncRNAs associated with the heat stress response in Chinese cabbage were identified through informatics analysis. Two lncRNAs (lnc000283 and lnc012465) were chosen for genetic transformation in Arabidopsis to elucidate their functions comprehensively. Transcriptome data analysis indicated that the expression of lnc000283 and lnc012465 in Chinese cabbage were both induced by HS. To verify the accuracy, the expression patterns of lnc000283 and lnc012465 were confirmed through quantitative real-time PCR (qRT-PCR), and the results from qRT-PCR were consistent with those obtained from RNA-seq (Fig. 1a). The corresponding homologous genes in Arabidopsis were identified as CNT2088434 and CNT2088742, exhibiting sequence similarities of 88% and 87%, respectively (Supplemental Fig. S1). Subcellular localization predictions using the lnclocator database (www.csbio.sjtu.edu.cn/bioinf/lncLocator) suggested that both lncRNAs are localized within the nucleus (Supplemental Table S2). Bioinformatics analysis was conducted using the CPC tool (http://cpc.cbi.pku.edu.cn/) indicated that lnc000283 and lnc012465 are noncoding sequences, with coding probabilities of 0.0466805 and 0.0432148, respectively comparable to the well-characterized lncRNAs COLDAIR and Xist, but significantly lower than those of the protein-coding genes UBC10 and ACT2 (Fig. 1b).

    Figure 1.  Characteristics of lnc000283 and lnc012465. (a) Expression level of lnc000283 and lnc012465 in Chinese cabbage leaves treated at 38 °C at different time points, as determined by qRT-PCR and RNA-seq. CK is a representative plant before heating, and T1, T4, T8, and T12 denote plants that were subjected to 38 °C for 1, 4, 8, and 12 h, respectively. The expression levels were normalized to the expression level of Actin. (b) Analysis of coding potential for lnc000283 and lnc012465. The coding potential scores were calculated using the CPC program. UBC10 (At5g53300) and ACT2 (At3g18780) are positive controls that encode proteins. COLDAIR (HG975388) and Xist (L04961) serve as negative controls, exhibiting minimal protein-coding potential.
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    Generation of lncRNA overexpression lines

    To elucidate the role of lnc000283 and lnc012465 in response to abiotic stress, overexpression vectors were constructed for these lncRNAs, driven by the CaMV 35S promoter, and they were introduced into Arabidopsis thaliana (Col-0 ecotype). Through PCR identification and generational antibiotic screening, two homozygous positive lines for lnc012465 and lnc000283 were obtained. The relative expression levels of these lncRNAs were assessed using qRT-PCR (Fig. 2a). When plants were grown in 1/2 MS medium, with the consumption of nutrients, and reduction of water, the leaves of WT began to turn yellow, but the lnc000283 and lnc012465 overexpression lines developed a deep purple color of leaf veins (Fig. 2b). Examination of chlorophyll and anthocyanin contents in the plants revealed that both overexpression lines had higher levels of chlorophyll and anthocyanin compared to the WT, suggesting that the transgenic plants might possess enhanced resistance to nutritional or water stress (Fig. 2c, d).

    Figure 2.  Arabidopsis plants overexpressing lnc000283 and lnc012465 had higher anthocyanins and chlorophyll content. (a) The relative expression level of lnc000283 and lnc012465 in WT and different transgenic lines. UBC10 (At5g53300) was used as an internal control. Each value is mean ± sd (n = 3). (b) The phenotype of WT and Arabidopsis overexpressing lnc000283 or lnc012465 grown on 1/2 MS medium 50 d after sowing. The (c) anthocyanin and (d) chlorophyll content of WT and transgenic Arabidopsis overexpressing lnc000283 or lnc012465. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01).
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    Effect of lncRNA overexpression on heat tolerance in Arabidopsis

    Given that lnc000283 and lnc012465 were highly induced by heat, the thermotolerance of the overexpressing (OE) plants were compared to that of the wild type. Arabidopsis plants were initially exposed to a an HS treatment at 38 °C for 4 d, followed by recovery at room temperature. The death caused by HS was processive. Post-severe HS challenge for 4 d, OE plants initially appeared similar to WT, but upon recovery, their leaves started to fold or curl, followed by a transition to yellow, white, and eventually drying out (Fig. 3a). OE lnc000283 and OE lnc012465 plants exhibited enhanced thermotolerance compared to WT, with lnc012465 showing particularly strong tolerance (Fig. 3a; Supplemental Fig. S2a). After 5 d of recovery, leaf coloration indicated that transgenic plants maintained a significantly higher percentage of green leaves and a lower percentage of bleached leaves compared to WT (Fig. 3b; Supplemental Fig. S2b). Under non-heat-stress conditions, WT and OE plants possessed comparable water content. However, following heat stress, the fresh-to-dry weight ratio of OE lnc000283 and lnc012465 lines was significantly greater than that of WT (Fig. 3c; Supplemental Fig. S2c). Abiotic stresses frequently trigger the production of excessive reactive oxygen species (ROS), which are believed to cause lipid peroxidation of membrane lipids, leading to damage to macromolecules. Leaf MDA content is commonly used as an indicator of lipid peroxidation under stress conditions; therefore, the MDA content in both transgenic and WT plants was assessed. Figure 3d shows that the MDA content in WT plants progressively increased after heat treatment, whereas in the two lines overexpressing lnc012465, the MDA content increased only slightly and remained significantly lower than that in WT at all time points. In plants overexpressing lnc000283, the MDA content did not significantly differ from that of WT before heat stress. However, after 4 d of heat treatment, the MDA content was significantly lower compared to WT (Supplemental Fig. S2d). The results suggested that the expression of both lnc012465 and lnc000283 can mitigate injury caused by membrane lipid peroxidation under heat-stress conditions. Peroxidase (POD) is a crucial antioxidant enzyme involved in ROS scavenging. Figure 3e and Supplemental Fig. S2e demonstrate that POD activity increased in both transgenic and WT plants after heat treatment. However, the increase in WT plants was modest, whereas OE lnc000283 and OE lnc012465 plants exhibited consistently higher POD activity. As anticipated, proline levels were induced in response to stress in all studied plants (Fig. 3f; Supplemental Fig. S2f). However, under normal conditions and 2 d post-heat stress treatment, the proline content in OE lnc000283 and OE lnc012465 plants did not exhibit significant changes compared to WT (Fig. 3f; Supplemental Fig. S2f). Moreover, after 4 d of heat stress, the proline content in OE lnc012465 lines was significantly lower than in WT, and the OE lnc000283 transgenic line 12-6 also showed a marked decrease in proline content compared to WT (Fig. 3f; Supplemental Fig. S2f). The results indicated that the thermotolerance of plants overexpressing either lnc000283 or lnc012465 was independent of proline accumulation.

    Figure 3.  Overexpressing lnc012465 lines are more tolerant to heat stress. (a) Phenotypes of WT and OE lnc012465 plants were assessed before and after exposure to heat stress. The heat treatment was applied to 25-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh-to-dry weight ratio of Arabidopsis leaves was measured before and after 38 °C heat treatment. (d)−(f) depict the MDA content, POD activity, and proline content in Arabidopsis leaves at varying durations of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on the expression quantity of genes related to high-temperature response in Arabidopsis

    To elucidate the molecular mechanisms by which lncRNAs enhance thermotolerance in Arabidopsis, the expression of the Hsf gene HsfA7a and three Hsps (Hsp25.3, Hsa32, and Hsp18.1-CI) in OE lnc000283, OE lnc012465, and WT Arabidopsis plants were investigated at various time points following heat treatment. As shown in Fig. 4 and Supplemental Fig. S3, both Hsf and Hsps exhibited a rapid response to heat stress with strong induction. Notably, the transcripts of HsfA7a and Hsp25.3 were significantly upregulated at 1 h after heat exposure, then experienced a sharp decrease. Hsa32 and Hsp18.1-CI were highly induced at 1 h and, unlike the other proteins, sustained high expression levels at 3 h (Fig. 4; Supplemental Fig. S3). At 1 h post-heat treatment, the transcript levels of Hsa32 and HsfA7a in OE lnc000283 did not significantly differ from those in WT. However, by 3 h, Hsa32 expression was roughly 50% of the WT level, while HsfA7a expression was approximately double that of WT (Supplemental Fig. S3). The overexpression of lnc000283 did not significantly affect the transcript level of Hsp25.3 at any of the tested time points. Notably, Hsp18.1-CI expression in both lines overexpressing lnc000283 was significantly induced at all three detection points post-heat treatment, reaching approximately 4-9-fold higher levels than in the WT (Supplemental Fig. S3). In Arabidopsis plants with elevated expression of lnc012465, the expression patterns of all Hsp and Hsf genes were similar to those in plants overexpressing lnc000283, with the notable exception of Hsa32. Unlike the WT, Hsa32 did not show a trend of down-regulation at 3 h post-heat treatment (Fig. 4). The findings suggest that the substantial induction of Hsp18.1-CI may play a role in enhancing the thermotolerance of Arabidopsis plants overexpressing lnc000283 and lnc012465.

    Figure 4.  The expression of HSF and HSP genes in lnc012465 overexpressing lines before and after different heat treatment times. Gene expression levels were quantified using RT-qPCR and normalized to UBC10 (At5g53300). Each value represents the mean ± standard deviation (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Effect of lncRNA overexpression on drought tolerance in Arabidopsis

    Prior research has implicated a significant proportion of genes in conferring resistance to various abiotic stresses. To elucidate the functions of lnc000283 and lnc012465 more thoroughly, WT and transgenic plants were subjected to drought stress by depriving them of water for 9 d. It was noted that the majority of leaves in WT plants withered and dried, whereas the OE lnc000283 and OE lnc012465 plants exhibited reduced withering, with only a minority displaying dryness (Fig. 5a; Supplemental Fig. S4a). Eight days post-rewatering, a negligible fraction of WT seedlings exhibited recovery, whereas the overwhelming majority of transgenic plants regained vigorous growth (Fig. 5a; Supplemental Fig. S4a). The transgenic plants demonstrated a significantly higher survival rate compared to the WT plants. Following 9 d of water deficit treatment, less than 40% of the WT plants survived, whereas the OE 012465 lines 8-7 and 9-1 exhibited survival rates of 100% and 95%, respectively, and the OE 000283 lines 11-10 and 12-6 had survival rates of 87% each. (Fig. 5b; Supplemental Fig. S4b). Water loss serves as a critical metric for assessing plant drought tolerance, hence the fresh-to-dry weight ratio of excised leaves was assessed via desiccation analysis. Following 4 d of drought treatment, the fresh-to-dry weight ratio for WT plants was reduced to 43%, whereas for OE lnc000283 lines 11-10 and 12-6, it was reduced to 73% and 75%, respectively. For OE 012465 lines 8-7 and 9-1, the ratios were reduced to 67% and 62%, respectively (Fig. 5c; Supplemental Fig. S4c). The findings indicated that lnc000283 and lnc012465 endow the transgenic plants with drought tolerance.

    Figure 5.  Overexpressing lnc012465 lines are more tolerant to drought stress. (a) Phenotype of WT and OE lnc012465 plants before and after subjecting to drought stress. Drought treatment was carried out on 20-day-old Arabidopsis plants. (b) The percentage of leaves with different colors in Arabidopsis after heat treatment and recovery for 5 d. (c) The fresh weight to dry weight ratio of Arabidopsis leaves before and after undergoing 38 °C heat treatment. (d)−(f) MDA content, POD activity, and proline content in Arabidopsis leaves under different times of heat stress. The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001)
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    MDA content in leaves is a standard biomarker for assessing the extent of drought stress-induced damage. Prior to drought stress exposure, MDA levels in WT and transgenic plants were comparable. However, following 7 and 9 d of water deficit, the MDA content in the transgenic plants was markedly reduced compared to the WT, suggesting a less severe degree of membrane lipid peroxidation in the transgenic plants (Fig. 5d; Supplemental Fig. S4d). Oxidative stress frequently coincides with drought stress, hence the activity of POD was assessed to evaluate the ROS scavenging ability. The findings indicated that as the duration of drought treatment increased, POD activity progressively rose. Before drought exposure, the POD activity in lines 11-10 and 12-6 of OE 000283 was 2.4-fold and 2.2-fold higher than that of the WT, respectively (refer to Supplemental Fig. S4e). Following drought treatment, the POD activity in the transgenic lines remained significantly elevated compared to the wild type, although the enhancement was less pronounced than before the treatment (Supplemental Fig. S4e). In the OE 012465 plants, the POD activity in lines 8-7 and 9-1 significantly surpassed that of the wild type, with the discrepancy being more pronounced during drought stress (Fig. 5e). The proline content in WT and OE 000283 plants exhibited no significant differences before and after 7 d of treatment. However, after 9 d of drought, the proline content in OE 000283 plants was significantly lower compared to that in the WT (Supplemental Fig. S4f). OE 000465 plants showed no significant difference from the wild type before and after drought treatment (Fig. 5f). The findings were consistent with those under heat stress, indicating that the enhanced stress resistance due to the overexpression of lnc000283 and lnc012465 in Arabidopsis is not reliant on proline accumulation.

    Following drought stress treatment, the expression levels of drought-related genes such as RD29A, RD29B, NCED3, AREB1, and Rab18 were significantly elevated in plants overexpressing lnc000283 and lnc012465 compared to WT plants. These findings suggest that lnc000283 and lnc012465 modulate Arabidopsis drought tolerance by regulating the expression of genes associated with the drought stress response (Fig. 6; Supplemental Fig. S5).

    Figure 6.  The expression of drought-responsive genes in lnc012465 overexpressing lines before and after different drought treatment time. Gene expression levels were determined by qRT-PCR normalized against UBC10 (At5g53300). Each value is mean ± sd (n = 3). The asterisks above the bars indicate statistical significance using Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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    Discussion

    The integrity of global food security is under threat due to the confluence of rapid population expansion and profound climatic shifts[29]. Amidst the shifting climatic landscape, heat and drought stress have emerged as primary limitations to crop yield and global food security. Understanding how plants detect stress cues and acclimate to challenging conditions is a pivotal biological inquiry. Moreover, enhancing plant resilience to stress is essential for maintaining agricultural productivity and fostering environmental sustainability[2]. Concurrently, the advancement of next-generation sequencing (NGS) technology has led to the identification of a substantial number of lncRNAs that participate in diverse stress responses, with functional analyses having been conducted on several of these molecules.[30] For instance, in the case of potatoes, the lncRNA StFLORE has been identified to modulate water loss through its interaction with the homologous gene StCDF1[31]. LncRNA TCONS_00021861 can activate the IAA biosynthetic pathway, thereby endowing rice with resistance to drought stress[32]. In wheat, the expression of TalnRNA27 and TalnRNA5 was upregulated in response to heat stress[33]. Our prior investigation identified a total of 81 lncRNAs in Chinese cabbage that engage in intricate interactions with their respective mRNA targets across various phases of heat treatment[25]. Two lncRNAs, lnc000283 and lnc012465, were chosen for subsequent functional analysis. Findings confirmed that these lncRNAs endow transgenic Arabidopsis plants with enhanced tolerance to both heat and drought, thereby offering novel resources for enhancing stress resistance through genetic engineering.

    Abiotic stresses frequently trigger the synthesis of anthocyanins, serving as natural antioxidants that mitigate oxidative damage by neutralizing surplus reactive oxygen species (ROS), thereby protecting plants from growth inhibition and cell death, allowing plants to adapt to abiotic stresses[34,35]. For instance, during chilling stress, the accumulation of anthocyanins within leaves can mitigate oxidative damage, thereby enhancing the photosynthetic rate[36]. Consequently, the level of abiotic stress tolerance can be inferred from the concentration of anthocyanins. The reduction of photosynthetic ability is one of the key physiological phenomena of stresses, which is partly due to the degradation of chlorophyll caused by leaf senescence during stress. The reduced accumulation of chlorophyll in the plants was seen in many plants when exposed to drought or heat stress conditions. The current investigation revealed that lncRNA-overexpressing plants cultivated in Petri dishes exhibited increased accumulation of both chlorophyll and anthocyanins in advanced growth phases, indicating that these transgenic plants, overexpressing lnc000283 and lnc012465, demonstrated enhanced stress tolerance and superior growth performance relative to WT (Fig. 2c, d).

    Upon exposure to heat stress, there is a marked induction of transcription for numerous genes that encode molecular chaperones in plants, with the vast majority of these genes contributing to the prevention of protein denaturation-related damage and the augmentation of thermotolerance[37−39]. The present investigation identified multiple heat-inducible genes in plants overexpressing lnc000283 and lnc012465, as well as in WT (Fig. 4; Supplemental Fig. S3). The findings indicated that of the four HSP or HSF genes examined, Hsp18.1-CI exhibited a significantly greater abundance in both OE lnc000283 and OE lnc012465 plants compared to the WT following heat treatment for several days. Hsp18.1-CI, formerly referred to as Hsp18.2 has been the subject of investigation since 1989.[40] Following the fusion of the 5' region of Hsp18.2 in frame with the uidA gene of Escherichia coli, the activity of GUS, serving as the driver gene was observed to increase upon exposure to HS[40]. The Arabidopsis hsfA2 mutant exhibited diminished thermotolerance after heat acclimation, with the transcript levels of Hsp18.1-CI being substantially reduced compared to those in wild-type plants following a 4-h recovery period[41]. The findings revealed that the upregulation of Hsp18.1-CI protein is a critical mechanism by which plants achieve enhanced protection against heat stress in adverse environmental conditions, thereby bolstering their thermotolerance.

    Plants cultivated in natural settings are often subjected to concurrent multiple abiotic stresses, which can exacerbate threats to their routine physiological functions, growth, and developmental processes[42,43]. Elucidating the molecular mechanisms underlying plant responses to abiotic stress is crucial for the development of new crop varieties with enhanced tolerance to multiple abiotic stresses. Previous research has indicated that the overexpression of certain protein-coding genes can endow plants with resistance to a variety of abiotic stresses. For instance, tomatoes with robust expression of ShCML44 demonstrated significantly enhanced tolerance to drought, cold, and salinity stresses[44]. Overexpression of PeCBF4a in poplar plants confers enhanced tolerance to a range of abiotic stresses[45]. With respect to lncRNAs, transgenic Arabidopsis plants that overexpress lncRNA-DRIR displayed marked increased tolerance to salt and drought stresses compared to the wild-type[46]. In the present study, both overexpression lines of lnc000283 and lnc012465 exhibited resistance to heat and drought stresses, thereby contributing to the enhancement of plant resilience against multiple stresses (Figs 3, 5; Supplemental Figs S2, S4).

    The number of genes implicated in plant drought resistance is regulated by both ABA-dependent and ABA-independent pathways[47,48]. It is well established that the expression of RD29A exhibits a high level of responsiveness to drought stress, operating through both ABA-dependent and ABA-independent mechanisms[49]. RD29B, AREB1, and RAB18 are governed by an ABA-dependent regulatory pathway[10,49,50]. NCED3 is involved in ABA biosynthesis[51]. In the present study, the transcript levels of RD29A, RD29B, NCED3, AREB1, and RAB18 were significantly elevated in OE lnc000283 and OE lnc012465 plants compared to those in the WT plants (Fig. 6; Supplemental Fig. S5). The findings indicated that the drought tolerance imparted by OE lnc000283 and OE lnc012465 plants is contingent upon an ABA-dependent mechanism.

    Prior research has indicated that certain long non-coding RNAs (lncRNAs) can assume analogous roles across diverse biological contexts. For example, the lncRNA bra-miR156HG has been shown to modulate leaf morphology and flowering time in both B. campestris and Arabidopsis[52]. Heterogeneous expression of MSL-lncRNAs in Arabidopsis has been associated with the promotion of maleness, and similarly, it is implicated in the sexual lability observed in female poplars[53]. In the present study, lnc000283 and lnc012465 were induced by heat in Chinese cabbage, and their heterologous expression was found to confer heat tolerance in Arabidopsis. Additionally, sequences homologous to lnc000283 and lnc012465 were identified in Arabidopsis (Supplemental Fig. S1). The data suggest that these sequences may share a comparable function to that of heat-inducible sequences, potentially accounting for the conservation of lnc000283 and lnc012465'os functionality across various species.

    Conclusions

    In conclusion, the functions of two heat-inducible lncRNAs, lnc000283 and lnc012465 have been elucidated. Transgenic Arabidopsis lines overexpressing these lncRNAs accumulated higher levels of anthocyanins and chlorophyll at a later stage of growth compared to the WT when grown on Petri dishes. Furthermore, under heat and drought stress conditions, these OE plants exhibited enhanced stress tolerance, with several genes related to the stress resistance pathway being significantly upregulated. Collectively, these findings offer novel insights for the development of new varieties with tolerance to multiple stresses.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and supervision: Li N, Song X; experiment performing: Wang Y, Sun S; manuscript preparation and revision: Wang Y, Feng X, Li N. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    All data generated or analyzed during this study are included in this published article and its supplementary information files.

    Acknowledgments

    This work was supported by the National Natural Science Foundation of China (32172583), the Natural Science Foundation of Hebei (C2021209019), the Natural Science Foundation for Distinguished Young Scholars of Hebei (C2022209010), and the Basic Research Program of Tangshan (22130231H).

  • The authors declare that they have no conflict of interest.

  • Supplemental Fig. S1 Correlations by color map cluster of twelve parameters evaluated in tomato fruit treated with doses of AVG and stored (15 ± 1 °C and RH 90 ± 5 %) for 28 days.
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    Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H, Gaona Acevedo AF, Lopes Vieites R. 2024. Doses of aminoethoxyvinylglycine (AVG) in tomato postharvest storage. Technology in Horticulture 4: e011 doi: 10.48130/tihort-0024-0008
    Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H, Gaona Acevedo AF, Lopes Vieites R. 2024. Doses of aminoethoxyvinylglycine (AVG) in tomato postharvest storage. Technology in Horticulture 4: e011 doi: 10.48130/tihort-0024-0008
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Doses of aminoethoxyvinylglycine (AVG) in tomato postharvest storage

  • 1.

    Faculty of Agronomic Sciences, São Paulo State University, Botucatu, São Paulo 18610, Brazil

  • 2.

    Montes Claros State University, Janaúba, Minas Gerais 39440, Brazil

  • Received: 20 January 2024
  • Revised: 20 March 2024
  • Accepted: 27 March 2024
  • Published online: 15 April 2024
Technology in Horticulture  4,  Article number: e011  (2024)  |  Cite this article

Abstract: Tomatoes are one of the main vegetables found daily in world cuisine and are highly perishable. The aim of this study was to evaluate the quality of 'Débora' tomato fruits over the post-harvest period in refrigerated storage, after immersion in solutions with different concentrations of aminoethoxyvinylglycine (AVG). The experimental design was entirely randomized in a 4 × 5 double factorial scheme, with four treatments, AVG doses 0 (control), 500, 1,000, and 1,500 mg·L−1, and five days of evaluation during the 28 d of refrigerated storage (days 0, 7, 14, 21 and 28) with three replications. Physicochemical analyses were carried out on weight loss, respiration rate, firmness, hydrogen potential (pH), soluble solids, titratable acidity and external and internal fruit color parameters, luminosity, chroma, and °hue. Two-way ANOVA, Tukey's mean test (p < 0.05), and multivariate statistical analysis by principal components (PCA) were carried out. The PCA allowed us to infer that, as weight loss increased, firmness decreased, an effect that was minimized with AVG at a dose of 1,500 mg·L−1, which may be related to the inhibition of the fruit's ethylene production rate. AVG delayed the post-harvest ripening of tomato fruit, reduced the respiration rate of the fruit, and the changes in external and internal chroma. The doses of AVG did not affect the luminosity and pH of the fruit pulp.

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    Introduction

    • Tomato (Solanum lycopersicum L.), is one of the main vegetables found daily in the world's cuisine. It can be eaten both fresh and processed. The heightened demand for this fruit contributes significantly to job creation within the tomato production sector, and it plays a significant role in agribusiness[1]. In addition to its aroma, flavor, and texture, tomatoes are widely accepted for their many benefits for human health. It is a food rich in lycopene, vitamins A and C, and minerals such as potassium, phosphorus, and magnesium, which are important for human nutrition[2].

      Climacteric fruits, like tomatoes, are highly perishable. They show a rapid and significant increase in respiration during ripening, with a series of biochemical and visual changes occurring[3]. Post-harvest losses of fruit and vegetables begin at harvest and continue throughout the marketing stage until consumption, i.e. during packaging, transportation, storage and at the consumer level.

      Ethylene is the main hormone responsible for ripening and its control is one of the main factors in reducing post-harvest losses. There are several inhibitors used in the conservation of climacteric fruits, which control the action and/or synthesis of ethylene, extending the shelf life of the product[4]. One form of control, for example, is the compound aminoethoxyvinylglycine (AVG).

      AVG suppresses ethylene biosynthesis by inhibiting the enzymatic activity responsible for converting S-adenosyl methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC)[5,6]. Several studies have used AVG to delay ripening and maintain post-harvest quality in climacteric fruits such as bananas[7] and apples[8]. In pears, AVG treatment suppressed the rate of fruit respiration, reduced the loss of firmness, reduced internal browning, senescence disorders, and consequently effectively delayed fruit ripening[9].

      Considering the quality and shelf life of the product, there are several damages caused to tomatoes, including handling, transportation, mechanical damage and exposure to high temperatures[3], resulting in rapid ripening with intensification of the red color. As such, tomatoes require a great deal of care and technology for their preservation.

      As the effect of AVG can vary depending on the dose and the type of fruit[10], and as there are few studies with AVG in tomatoes, more detailed studies are needed to better understand the issues of the feasibility of using AVG at an appropriate dose. In this context, the aim of this work was to evaluate the quality of tomato fruit cultivar 'Débora' over various postharvest times in refrigerated storage, after immersion in solutions with different concentrations of aminoethoxyvinylglycine (AVG).

    Materials and methods

      Plant material

    • Tomato fruits (Solanum lycopersicum L. Cultivar 'Débora') obtained from commercial cultivation were used. The fruits were harvested at the salad tomato stage of ripeness, green and ripe, and transferred to the Fruit and Vegetable PostHarvest Laboratory at the Universidade Estadual Paulista 'Júlio de Mesquita Filho', Faculdade de Ciências Agronômicas, Campus de Botucatu, São Paulo, Brazil, where they were selected to standardize the batch, eliminating those with physical damage (dented and/or cracked) and biological damage (diseases and/or pests). The fruits were randomly divided, immersed for 15 min in a solution of hypochlorite with 2% active chlorine, diluted to 7%, washed in running water, and dried in the air, remaining at rest for 24 h to remove the field heat.

      Treatment with aminoethoxyvinylglycine (AVG)

    • Each treatment consisted of immersing the fruit for 5 min in a solution of AVG at four concentrations: 0 (control), 500, 1,000, and 1,500 mg·L−1. Each treatment consisted of three replicates. The commercial product used was the plant regulator ReTain®.

      After applying the treatments, the fruit was packed in expanded polystyrene trays (two fruits) covered with polyvinyl chloride film (thickness 0.020 mm), stored in a cold room at 15 ± 1 °C and 90% ± 1% RH (average of 31 °C outside) and assessed for quality every 7 d for 28 d.

      Weight loss

    • To determine weight loss, a semi-analytical scale (Owa labor model) was used with a maximum load of 2,000 g and a division of 10 mg. The results were calculated as the percentage (%) of weight loss at the start of the experiment and at different intervals during storage using the equation: PM (%) = (Pi ‒ Pj/Pi) × 100, where PP = Weight loss (%); Pi = Initial weight of the fruit (g); Pj = Weight of the fruit in the period following Pi (g)[11].

      Respiration rate

    • The respiration rate was determined by quantifying CO2 production in a respirometer, according to a methodology adapted from Bleinroth et al.[12], using the equation: TCO2 = 2.2 × (V0 ‒ V1) × 10/P × T, where, TCO2 = Respiration rate (mL CO2 kg−1·h−1); V0 = Volume spent of HCl for potassium hydroxide titration − standard before CO2 absorption (mL); V1 = Volume spent of HCl for potassium hydroxide titration after CO2 absorption from respiration (mL); P = Fruit weight (kg−1); T = Respiration time (h−1); 2.2 = Inherent CO2 (mL) equivalent (44/2), multiplied by the concentration of hydrochloric acid at 0.1 N; 10 = Adjustment for total potassium hydroxide used in CO2 absorption (mL).

      Firmness

    • Firmness was measured at two different points on each fruit and determined using a Texturometer (STEVENS - LFRA Texture Analyzer), with a penetration distance of 20 mm and a speed of 2.0 mm·s−1, using a TA 9/1000 tip. The results were expressed in Newton (N).

      Hydrogen potential (pH), Total Soluble Solids (TSS) and Titratable Acidity (TA)

    • The hydrogen potential (pH) of the tomato pulp obtained with the aid of a mixer was determined using a tabletop digital pH meter (model DMPH - 2), according to the methodology of AOAC[11].

      The total soluble solids (TSS) content of the extracted tomato pulp was measured using a tabletop digital refractometer (Digital Refractometer DR 202) by direct refractometric reading, according to the methodology of AOAC[11] and the results were expressed in °Brix.

      Titratable acidity (TA) was determined by titrimetry. Three milliliters of extracted tomato juice were diluted to 100 mL with distilled water and titrated with 0.1 N sodium hydroxide solution (NaOH) at pH 8.2. The results were expressed as a percentage of citric acid in the fresh pulp, according to the methodology recommended by AOAC[11]. The calculation was made using the formula: AT (%) = [(V × N × meq)/Y] × 100, where V = Volume of sodium hydroxide used in ml, N = Normality of sodium hydroxide and meq = 0.064, Y = Volume of bulk fruit juice mL[11].

      External and internal instrumental color

    • The color of the tomato peel and pulp was determined using a Konica Minolta colorimeter (Chroma meter, CR 400) where L*, expressed as a percentage, indicates luminosity values (0% = black and 100% = white), C* is represented by Chroma which defines color intensity. The Hue angle is the value in degrees corresponding to the three-dimensional color diagram and ranges from: 0° to 18° for red-violet, 19° to 54° for red, 55° to 90° for orange, 91° to 126° for yellow, 127° to 162° for yellow-green, 163° to 198° for green, 199° to 234° for blue-green, 235° to 270° for blue, 271° to 306° for blue-violet and 307° to 342° for violet, 343° to 360° red-violet, making 360°[13]. For the approximate reproduction of the color profile, determined by the Konica Minolta colorimeter, the Luminosity, Chroma and Hue angle values of the fruit peel were used to feed into the colorizer.org platform, topic HSL(A), according to the CIELAB diagram.

      Experimental design

    • The experimental design was entirely randomized in a 4 × 5 double factorial scheme, with four treatments being the doses of AVG 0 (control), 500, 1,000, and 1,500 mg·L−1 and 5 d of evaluation during the 28 d of refrigerated storage (0, 7, 14, 21, and 28 d) with three repetitions (n = 3).

      Statistical analysis

    • The data obtained was subjected to the Shapiro-Wilk normality test using GraphPad Prism software version 8.0.1 (San Diego, CA, USA). With all the data showing normality, it was subjected to analysis of variance (Two-Way ANOVA), and the means were compared using the Tukey test, p < 0.05; and at p < 0.01; using the Sisvar software version 5.6 (Lavras, MG, Brazil). The results were presented as means with standard deviation (mean ± SD). Principal component analysis and correlations was carried out using JMP 10 statistical software (SAS Institute Inc., USA).

    Results and discussion

    • The results of the analysis of variance with the doses of AVG (0, 500, 1,000, and 1,500 mg·L−1), storage time (days 0, 7, 14, 21, and 28) and the interaction between these factors for the physicochemical parameters assessed at the post-harvest stage of the tomato fruit are presented in Table 1. Except for pH, all the parameters showed significant differences depending on the doses and storage time. There was an interaction between the factors, except for pH and total soluble solids.

      Table 1.  Results of the analysis of variance (Two-Way ANOVA) of the effect of AVG doses, storage time and the interaction of these factors on the physicochemical parameters evaluated in tomato fruits.

      Cause of variationD.F.Weight lossRespiratory rateFirmnessTotal soluble solidspHTritrable acidity
      Dose30.000**0.000**0.027*0.042*0.778ns0.046*
      Time (d)40.000**0.000**0.000**0.049*0.579ns0.000**
      Dose × time120.004**0.000**0.000**0.219ns0.988ns0.002**
      Residue400.1503.2640.0330.1220.0690.000
      ns represents not significant; * represents p ≤ 0.05 and ** represents p ≤ 0.01.

      There was an increase in weight loss in tomato fruit at all doses during storage (Fig. 1a). The control fruit (0 mg·L−1) showed less weight loss compared to the fruit treated with AVG, and on day 28 the 1,000 and 1,500 mg·L−1 doses showed greater weight loss compared to the other doses. In a similar previous study, the weight loss of 'Grando F1' tomatoes increased with longer storage time and reached 1.38% in the AVG-treated fruit and 1.21% in the control fruit after 20 d at 12 °C[14], so there seems to be no consistent effect of AVG treatments on weight loss. Different results were obtained in apple cultivar 'Eva', where the doses of 500 and 1,500 mg·L−1 were responsible for the lowest weight loss from the 21st and 28th d[15]. Although positive effects have been observed in some fruit varieties, opposite results have been obtained in other fruits[10].

      Figure 1. 

      (a) Weight loss, (b) respiration rate and (c) firmness of tomato fruit treated with doses of aminoethoxyvinylglycine (AVG) and stored (15 ± 1 °C and RH 90% ± 5 %) for 28 d. Lowercase letters differ the doses in a single storage time and capital letters differ the storage times in a single dose, according to the Tukey test (p < 0.05).

      Weight loss is a process that occurs due to the loss of water, stimulated by the process of respiration and transpiration due to the vapor pressure deficit on the surface of the fruit[16]. The loss of water leads to softening of the tissues, affecting the texture, color, and flavor of the fruit, reducing its time on the market. As reported by Taiz et al.[17], AVG also acts to inhibit enzymes that use pyridoxal phosphate as an enzyme cofactor and when applied in high doses, this may have occurred in this study by increasing weight loss, inhibiting various aspects of the plant's metabolism, especially interfering with the rind tissue.

      There was an increase in the respiration rate of the fruit on the 7th day of evaluation, with a gradual increase over time until the maximum climacteric peak of 55.31 mL CO2 kg−1·h−1 on the 14th day for the control fruit, followed by a decline on the other days (Fig. 1b). From the 7th to the 28th day, the control had a significantly higher respiration rate, 55% to 64% higher on the 28th day compared to the AVG-treated fruit, showing a positive effect of this compound. The respiration rate can be reduced using various technologies that reduce the metabolic activity of the fruit, such as refrigerated storage and inhibitors of ethylene action[6], like AVG. The continuation of the metabolic process gradually alters the composition of the product, leading to senescence, which was delayed with AVG in this study.

      Over time, there was a decrease in the firmness of the fruit from day 14 onwards, which was gradual until day 28 (Fig. 1c). AVG provided greater fruit firmness compared to the control at a dose of 1,500 mg·L−1 on day 7 and 1,000 mg·L−1 on day 21, and on the other days there was no difference between the doses. The difference in firmness between treated and untreated fruit disappears over time[18], which probably occurred on day 28. When applied as a postharvest dip treatment, AVG reduced the rate of fruit softening in 'Grando F1' tomatoes[14], 'Huangguan' pears[9] and sweet orange Citrus sinensis (L.)[19] during storage or shelf life, as a consequence of ethylene inhibition.

      The total soluble solids (TSS) content of the control fruit (0 mg·L−1) decreased over time, and for the other doses there was stability, with no significant difference (Fig. 2a). On day 28, the 1,000 mg·L−1 dose provided a 1.0 °Brix increase in SS content compared to the control. The SS content is used as an indirect estimate of sugars, as well as other compounds that are present in the vascular sap, such as vitamins, phenolics and pectin's. Like this study[20], working with 'Tommy Atkins' mangoes at room temperature, observed stability in the SS content when treated with AVG during the 12 d of storage. However, this was probably due to the ripening process of the fruit with the conversion of starch to sugar.

      Figure 2. 

      (a) Total soluble solids, (b) pH and (c) titratable acidity of tomato fruit treated with doses of aminoethoxyvinylglycine and stored (15 ± 1 °C and RH 90% ± 5 %) for 28 d. Lowercase letters differ the doses in a single storage time and capital letters differ the storage times in a single dose, according to the Tukey test (p < 0.05).

      The pH showed no statistical difference between the treatments (Fig. 2b). A previous study,[15] also found no significant difference for the doses of AVG, but found a significant effect for the storage time of the 'Eva' apple cultivar. The author observed higher pH values on the 0th and 7th day of storage, with a decrease in value from the 14th day onwards. This reported decrease was linked to the release of organic acids resulting from the degradation of the cell wall, a fact that did not occur in this study under the conditions in which the experiment was carried out.

      Titratable acidity (TA) remained stable in the fruit treated with the 1,500 mg·L−1 dose during storage; on the other hand, there was a reduction in the fruit treated with the other doses (Fig. 2c). In the fruit treated with the 1,500 mg·L−1 dose on day 28, the acidity was 0.08% and 0.05% higher, respectively, compared to the two lower doses, 0 and 500 mg·L−1. Corroborating this work, dos Santos et al.[20], working with 'Kent' mango, reported that the 300 mg·g−1 concentration of AVG resulted in fruit with a higher acidity content. Titratable acidity analysis is used to quantify acidity through a predominant acid according to the plant material, determining the percentage of organic acids.

      Corroborating the present study, it was previously reported that TA decreased over time and no significant differences were found in the pH of the pulp of 'Grando F1' tomatoes between fruit treated and untreated with 1,000 mg·L−1 AVG at −30 kPa during storage at 12 °C for 20 d and a further 2 d at 20 °C[14]. However, unlike the present study, these authors found no significant differences in SS and TA between fruit treated and not treated with AVG, reporting that changes in organic acids and sugars in tomatoes were not controlled by ethylene[21], a positive effect that certainly occurred in the present study due to the inhibition of ethylene by AVG at a dose of 1,500 mg·L−1, as it kept TA and SS stable.

      The results of the Two-Way ANOVA with the doses of AVG (0, 500, 1,000, and 1,500 mg·L−1), storage time (days 0, 7, 14, 21, and 28) and the interaction between these factors for the external and internal color parameters, assessed at post-harvest of the tomato fruit, are shown in Table 2. Except for the effect of the doses and the interaction between the factors for internal luminosity (pulp), all the other parameters showed significant differences. Storage time was significant for all parameters.

      Table 2.  Results of the analysis of variance (Two-Way ANOVA) of the effect of AVG doses, storage time and the interaction of these factors on the external and internal color parameters evaluated in tomato fruits.

      Cause of variationD.F.L peelC peelh° peelL pulpC pulph° pulp
      Dose30.001**0.000**0.000**0.866ns0.030*0.045 *
      Time (d)40.000**0.000**0.000**0.000**0.000**0.000**
      Dose × time120.038*0.010*0.000**0.695ns0.014*0.000**
      Residue401.7804.4023.2947.0972.50712.640
      ns represents not significant; * represents p ≤ 0.05 and ** represents p ≤ 0.01.

      As for peel color, external luminosity decreased over time (Fig. 3a), and on day 28, the 500 mg·L−1 dose provided significantly lower luminosity compared to the 0 mg·L−1 dose, indicating lower brightness of the fruit periderm (Fig. 3b), but both did not differ from the other doses. There was an increase in external chroma values over time, and on day 28 the AVG doses showed lower chroma compared to the control. Corroborating the present study, in a previous study with 'Grando F1' tomatoes treated with 1,000 mg·L−1 of AVG at −30 kPa during storage at 12 °C for 20 d and a further 2 d at 20 °C after refrigerated storage[14], it was reported that AVG reduced the rate of ethylene production and delayed ripening changes in peel color (L*, C* and h° values, chlorophyll and lycopene content). Therefore, C* values increased while h° and L* values decreased at a lower rate in AVG-treated fruit than in control fruit during storage and shelf life.

      Figure 3. 

      External instrumental color (peel), (a) luminosity, (b) chroma and (c) hue angle of tomato fruits treated with doses of AVG and stored (15 ± 1 °C and RH 90% ± 5 %) for 28 d. Lowercase letters differ the doses in a single storage time and capital letters differ the storage times in a single dose, according to the Tukey test (p < 0.05).

      The external °hue decreased until day 14 and remained stable for the rest of the time (Fig. 3c). This behavior leads to a change from green to red, which can possibly be attributed to the degradation of chlorophyll and the biosynthesis of lycopene, responsible for the tomato's red color. On day 14, the doses of AVG showed lower °hue compared to the control, and on day 21 there was no difference between the doses, and on day 28 the highest doses (1,000 and 1,500 mg·L−1) provided color maintenance compared to the control (Fig. 4), indicating that AVG reduced fruit ripening.

      Figure 4. 

      Approximate reproduction of the color profile of the peel of tomato fruit treated with doses of AVG and stored (15 ± 1 °C and RH 90% ± 5%) for 28 d, determined using a Konica Minolta colorimeter, with Luminosity, Chroma and °Hue values fed into the colorizer.org platform.

      For internal color, there was no statistical difference between AVG doses only for brightness, showing that there was no negative effect of AVG on fruit brightness. In relation to time, there was a decrease over the days, indicating a loss in the internal brightness of the fruit (Fig. 5a). On the 28th day, the internal chroma was higher in the 0 mg·L−1 control and the doses did not differ (Fig. 5b). There was an increase in color intensity on days 14 and 21. The internal ºhue gradually decreased until day 14 and remained stable on the other days (Fig. 5c), and there was no significant difference between the doses on days 14, 21 and 28. Aglar[22] reported that spraying 225 mg·L−1 of AVG on 'Li' jujube trees pre-harvest and keeping the fruit in cold storage for 45 d at 0 ± 0.5 °C and 90% ± 5% relative humidity (RH) reduced the development of fruit color.

      Figure 5. 

      Internal instrumental color (pulp), (a) luminosity, (b) chroma and (c) hue angle of tomato fruit treated with doses of AVG and stored (15 ± 1 °C and 90% ± 5% RH) for 28 d. Lowercase letters differ the doses in a single storage time and capital letters differ the storage times in a single dose, according to the Tukey test (p < 0.05).

      Principal component analysis (PCA) of 12 parameters evaluated in tomato fruit treated with doses of AVG (0, 500, 1,000, and 1,500 mg·L−1) and stored for 28 d (day 0, 7, 14, 21, and 28), allowed the general observation of the data in a smaller dimension, separated by treatment in different quadrants (Fig. 6). The variability was explained by two principal components (PC) with eigenvalues > 1.0. These two significant PCs (PC1 and PC2) accumulated 76.9% of the total variation.

      Figure 6. 

      (a) Score plot and (b) load plot of the principal component analysis of 12 parameters evaluated in tomato fruit treated with doses of AVG and stored (15 ± 1 °C and RH 90% ± 5%) for 28 d. Score graph (a): 0 mg·L−1 dose on days 0 (A), 7 (E), 14 (B), 21 (C) and 28 (D); 500 mg·L−1 dose on days 0 (P), 7 (T), 14 (Q), 21 (R) and 28 (S); 1,000 mg·L−1 dose on days 0 (F), 7 (J), 14 (G), 21 (H) and 28 (I); and 1,500 mg·L−1 dose on days 0 (K), 7 (O), 14 (L), 21 (M) and 28 (N). Load graph (b): weight loss (WL), respiratory rate (Resp), firmness (Firm), total soluble solids (SS), hydrogen potential (pH), titrable acidity (TA), lightness external (L peel), chroma external (C peel), hue angle external (°h peel), lightness internal (L pulp), chroma internal (C pulp) and hue angle internal (°h pulp).

      PC1 was responsible for 61.6% of the total variation and was effective in separating the treatments on days 0 and 7 into positive scores and the treatments on the remaining days (14, 21, and 28) into negative scores (Fig. 6a). Analysis of the PC1 loadings (Fig. 6b) suggests that this separation is mainly due to the analyses of firmness, external and internal luminosity and external and internal °h which have strong positive loadings (> 0.80) and the analyses of external and internal chroma with strong negative loadings in the PCA (< 0.75). The PC1 scores and loadings showed a greater effect for time than for AVG doses.

      PC2 in the score graph (Fig. 6a) was important mainly for separating dose 0 on day 28 (D), since this treatment had a positive score, from doses 1,000 and 1,500 on day 28 (I, N) which had the strongest negative scores, corroborating the respiration rate mean test (Fig. 1b). PC2 represented 15.3% of the total variation and was mainly related to positively charged respiration rate and negatively charged weight loss (Fig. 6b). Therefore, principal component analysis was effective in confirming the results presented here.

      By grouping the dependent variables via correlation, PCA indicated that there were positive correlations between weight loss, external and internal chroma, which correlated negatively with firmness, external and internal luminosity, external and internal hue angle, total soluble solids, and titratable acidity, and all the latter correlated positively with each other (Supplemental Fig. S1). This infers that, as weight loss increased, firmness decreased, an effect that was minimized with AVG, which may be related to the inhibition of the fruit's ethylene production rate and the expression of genes associated with chlorogenic acid metabolism, perception and signal transduction and membrane breakdown in the central tissue, as well as a decrease in malondialdehyde activity and polyphenol oxidase enzyme activity[9].

    Conclusions

    • Under the conditions in which the experiment was carried out, it can be concluded that immersing 'Débora' tomato fruit in AVG solution at a dose of 1,500 mg·L−1 delayed ripening, reduced the fruit's respiration rate, and the changes in external and internal chroma, and it did not reduce weight loss. The doses of AVG did not negatively affect the luminosity and pH of the fruit pulp. The untreated fruit became soft and ripe red 7 d earlier than the treated fruit during the 28 d of storage at 15 ± 1 °C and 90% ± 1% RH. The feasibility of using AVG to market tomato fruit to distant markets or to local or retail markets for longer was demonstrated. More studies are needed on the use of AVG in different conditions and with other tomato cultivars.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Aparecida dos Santos J, Rocha Lacerda V; methodology: Rocha Lacerda V, Aparecida dos Santos J, Gaona Acevedo AF, Lopes Vieites R; software: Rocha Lacerda V; validation: Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H, Gaona Acevedo AF, Rocha Lacerda V; formal analysis: Rocha Lacerda V, Aparecida dos Santos J, Gaona Acevedo AF; investigation: Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H; resources: Rocha Lacerda V; data curation: Rocha Lacerda V, Aparecida dos Santos J; draft manuscript preparation: Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H; final manuscript preparation: Rocha Lacerda V, Sílvia Angélica de Oliveira H, Lopes Vieites R; visualization: Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H, Gaona Acevedo AF; supervision: Lopes Vieites R; project administration: Aparecida dos Santos J; funding acquisition: Lopes Vieites R. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article, and are available from the corresponding author on reasonable request.

      Acknowledgments

      • This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Financing Code 001. To the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, for the financial support for this work.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Supplementary information
      • Supplemental Fig. S1 Correlations by color map cluster of twelve parameters evaluated in tomato fruit treated with doses of AVG and stored (15 ± 1 °C and RH 90 ± 5 %) for 28 days.
      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (6)  Table (2) References (22)
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    Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H, Gaona Acevedo AF, Lopes Vieites R. 2024. Doses of aminoethoxyvinylglycine (AVG) in tomato postharvest storage. Technology in Horticulture 4: e011 doi: 10.48130/tihort-0024-0008
    Rocha Lacerda V, Aparecida dos Santos J, Sílvia Angélica de Oliveira H, Gaona Acevedo AF, Lopes Vieites R. 2024. Doses of aminoethoxyvinylglycine (AVG) in tomato postharvest storage. Technology in Horticulture 4: e011 doi: 10.48130/tihort-0024-0008
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  • Introduction
  • Materials and methods

  • Plant material

  • Treatment with aminoethoxyvinylglycine (AVG)

  • Weight loss

  • Respiration rate

  • Firmness

  • Hydrogen potential (pH), Total Soluble Solids (TSS) and Titratable Acidity (TA)

  • External and internal instrumental color

  • Experimental design

  • Statistical analysis

  • Results and discussion
  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Supplementary information
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