Rosmarinic Acid Delays Tomato Fruit Ripening

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Zhangjian Hu	+ 2773 word(s)	2773	2021-11-18 04:35:43	\|
2	format correct	Vivi Li	+ 9 word(s)	2782	2021-12-22 04:07:57	\|

This entry is adapted from the peer-reviewed paper 10.3390/antiox10111821

Fruits are excellent sources of essential vitamins and health-boosting minerals. Recently, regulation of fruit ripening by both internal and external cues for the improvement of fruit quality and shelf life has received considerable attention. Rosmarinic acid (RA) is a kind of natural plant-derived polyphenol, widely used in the drug therapy and food industry due to its distinct physiological functions.

rosmarinic acid fruit ripening tomato flavor quality ethylene antioxidant system

1. Introduction

Tomato is regarded as an important horticultural crop worldwide. As a rich source of carbohydrates, vitamins, dietary fiber, and antioxidants, fleshy tomato fruits provide humans with numerous health benefits ^[1]. Fruit ripening is a well-orchestrated and important development process in the life cycle of tomatoes. Traditionally, fruit ripening has been classified into two types, such as the climacteric type and the nonclimacteric type. Tomatoes belong to typical climacteric fruits, showing a characteristic burst in respiration rates and ethylene production at or just before the onset of ripening coinciding with massive transcriptional changes ^[2]. Understanding the underlying mechanisms of fruit ripening is extremely essential for enhancing our scientific knowledge to improve fruit quality, nutritional values, and shelf life towards reducing fruit waste and economic losses in the tomato industry.

Generally, tomato fruit ripening is characterized by softening, color changes, and accumulations of flavor and aromatic compounds ^[3]. Fruit softening is regarded as a consequence of cell wall disassembly and the changes in cellular turgor ^[4]. The fruit color change from green to red is associated with the degradation of chlorophylls and the simultaneous accumulation of carotenoids such as lycopene, β-carotene, and lutein. Among these carotenoids, lycopene is considered a major component that contributes to the formation of red color in tomato fruits ^[5]. Meanwhile, fruit ripening triggers a substantial accumulation of carbohydrates, that provide energy for fruit developmental changes and also trigger ripening-associated sugar signal pathways. The major form of carbohydrates imported into fruits from the photosynthetic organs is sucrose, which is also synthesized in the cytosol, vacuole, and apoplast of fruit cells ^[6]. During ripening, sucrose can be irreversibly hydrolyzed by invertase into glucose and fructose, or cleaved by sucrose synthase into fructose and uridine diphosphate-(UDP-) glucose in the presence of UDP ^[7]. These distinct sugar metabolic processes change the content and component of sugars, and remodel fruit flavor and total soluble solids. Besides, carbohydrate accumulation, dramatic changes in free amino acids, organic acids, and volatiles occur during tomato fruit ripening ^[8].

Ethylene production is regarded as a critical indicator of fruit ripening, and also is the major cue controlling most aspects of ripening in climacteric fruits. To avoid the postharvest losses caused by the early onset of ripening, it is a good strategy to either block or inhibit ethylene synthesis. The key enzymes involved in ethylene synthesis include 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase ACO. ACS contributes to ACC production from S-adenosylmethionine (SAM), and ACO is responsible for the conversion of ACC into ethylene. Genetic mutations in ethylene biosynthesis genes affect tomato fruit ripening ^[3]. Meanwhile, exogenous application of the potent ethylene perception inhibitor 1-methylcyclopropene (1-MCP) can remarkably delay fruit ripening ^[9]. However, the inhibition of fruit ripening by the above-mentioned approaches also results in an accompanying loss in flavor quality of tomato fruits.

Except for the ethylene burst, increased respiration rates during tomato fruit ripening are associated with oxidative processes, producing reactive oxygen species (ROS), such as ephemeral singlet oxygen, superoxide radical (O₂^•−), and hydrogen peroxide (H₂O₂). At low and moderate concentrations, ROS can function as a second messenger to transduce signaling cascades that mediate many plant physiological responses during fruit ripening ^[10]. However, high ROS accumulation promoted by water loss, ethylene production, and respiration induces lipid peroxidation in cells during postharvest storage, exacerbating oxidative stress and protein damages. Therefore, plants develop a complex antioxidative defense system, which includes enzymatic and non-enzymatic components. The enzymatic components, such as superoxide dismutase (SOD), catalase (CAT), and the enzymes involved in the ascorbate-glutathione cycle, including ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR), constitute the first layer of defense against oxidative stress ^[11]. Meanwhile, non-enzymatic antioxidants such as ascorbate, glutathione, carotenoids, tocopherols and polyphenols work in concert with antioxidant enzymes to maintain a steady state of redox homeostasis ^[12]. Among the non-enzymatic antioxidants, polyphenols are the most abundant antioxidant in plant fruits, and can directly capture oxidative free radicals ^[13].

Rosmarinic acid (RA), named after rosemary (Rosmarinus officinaliss L.) plants, is a classical type of polyphenol, widely found in various plant species, ranging from the hornworts (Anthocerotaceae, one of the earliest land plant families) to eudicotyledonous plants (many species from the families Lamiaceae and Boraginaceae) ^[14]. Biosynthesis of RA initiates from the aromatic amino acids L-phenylalanine and L-tyrosine, which is transformed to the intermediary precursors 4-coumaroyl-CoA by the general phenylpropanoid pathway and 4-hydroxyphenyllactic acid, respectively. Then, the two intermediary precursors are coupled to form 4-coumaroyl-4′-hydroxyphenyllactic acid (4C-pHPL) by rosmarinic acid synthase, and the hydroxyl groups of RA are eventually added by cytochrome P450-relied monooxygenase reactions ^[14]. However, whether RA can be biosynthesized in tomato has not been characterized yet. RA has a number of potentially beneficial pharmacological functions, and it exhibits antiallergic, anti-inflammatory, anticarcinogenic, antimicrobial, and neuroprotective properties ^[15]. In addition, RA can scavenge free radicals, chelate pro-oxidant ions, and prevent lipid peroxidation. Therefore, RA is regarded as a natural antioxidant with a variety of valuable applications in the food industry ^[16]. However, the role of RA in fruit ripening remains poorly understood, which limits its application in fruit postharvest storage.

2. Effect of RA Treatment on the Sensory Quality of Tomato Fruits

Tomato fruits were collected at 0 d, 4 d, 8 d, and 12 d after soaking treatments with different RA concentrations or H₂O as control. As illustrated in Figure 1A, 0.25 mM, 0.5 mM, and 0.75 mM RA soaking treatments inhibited tomato fruit color change during storage at 4 and 8 d, compared with the control treatment. In addition, RA treatment also maintained higher firmness than control during storage from 4 d to 12 d (Figure 1B). These results indicated that RA treatment retarded the color change and ripening of tomato fruits.

Figure 1. Rosmarinic acid (RA) postponed tomato fruit ripening. (A) The representative phenotype of tomato fruits at different days after RA treatment. Tomato fruits at the mature green stage were treated with 0.25, 0.5, and 0.75 mM RA, or H₂O control. Bar = 2 cm. (B) Effect of RA on the fruit firmness at 0 d, 4 d, 8 d, 12 d after treatment. The data in (B) are presented as mean values ± SD; n = 9. Asterisks in (B) indicate statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01) compared with control under the same time point, as determined by Student’s t-test.

3. Effect of RA Treatment on Ripening-Induced Ethylene Production

Ethylene has long been implicated in ripening initiation, especially in climacteric fruits ^[5]. In order to address whether RA treatment affected ripening-induced ethylene biosynthesis, we quantified the ethylene production at different postharvest time points. As shown in Figure 2A, tomato fruits with control exhibited a typical ethylene burst after the onset of ripening, whereas RA treatments substantially inhibited ethylene production at 4 d, 8 d, and 12 d, especially with 0.5 mM RA. In line with the ethylene production, RA largely suppressed ripening-induced transcript abundance of ethylene biosynthesis genes, such as ACS2, ACS4, ACO1, and ACO4 at 4 d after treatment (Figure 2B). Collectively, these results strongly suggest that RA inhibited ethylene production.

Figure 2. RA inhibited ripening-induced ethylene biosynthesis signaling. Effects of RA treatment on (A) ethylene production, and (B) transcript abundance of ethylene biosynthesis genes ACS2, ACS4, ACO1, and ACO4. The transcript abundance of each gene under control at 0 d was defined as 1. Tomato fruits at the mature green stage were treated with 0.25 mM, 0.5 mM, 0.75 mM RA, or H₂O control. The fruit samples were collected at the indicated time points for ethylene content quantification and qRT-PCR analysis. The data are presented as mean values ± SD; n = 4 in (A), 3 in (B). Asterisks in (B) indicate statistically significant differences (** p ≤ 0.01) compared with control under the same time point, as determined by Student’s t-test.

4. Effects of RA Treatment on Carotenoid Compositions

Carotenoid metabolism is a major fruit ripening trait, which primarily determines tomato fruit color. To explore whether RA treatment affected carotenoid biosynthesis during fruit ripening, we measured the accumulation of three typical carotenoids, including lycopene, lutein, and β-carotene. Time-course analysis revealed that the lycopene contents in tomato fruits gradually increased during fruit ripening, whereas ripening-induced lycopene accumulation was significantly inhibited at 4, 8, and 12 d after RA treatment compared to control (Figure 3A). In agreement with the deceased lycopene accumulation by RA treatment, the ripening-induced transcripts abundance of Phytoene Desaturase (PDS), Zeta-Carotene Desaturase (ZDS), and Carotene Isomerase (CRTISO), the genes involved in lycopene biosynthesis, was substantially suppressed at 4 d after RA treatment (Figure S1 available online at https://www.mdpi.com/article/10.3390/antiox10111821/s1). In contrast, RA significantly prevented ripening-mediated lutein content reduction at both 4 and 8 d after RA (Figure 3B). Meanwhile, RA treatment did not affect β-carotene accumulation during fruit ripening (Figure 3C). Overall, RA differentially regulated carotenoid compositions and negatively affected lycopene accumulation, which was in accordance with the delayed color change in RA-treated fruits.

Figure 3. RA affected carotenoid biosynthesis during fruit ripening. Effects of RA treatment on (A) lycopene content, (B) lutein content, and (C) β-carotene content during fruit ripening. Tomato fruits at the mature green stage were treated with 0.5 mM RA, or H₂O control. The fruit samples were collected at the indicated time points for carotenoid content quantification. The data are presented as mean values ± SD; n = 3. Asterisks indicate statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01) compared with control under the same time point, as determined by Student’s t-test.

5. Effects of RA Treatment on Sugar and Organic Acid Accumulation

Tomato fruit ripening generally entails a dynamic metabolism of sugar and organic acid, which remarkably contribute to flavor ^[6]. To investigate the effects of RA treatment on fruit sugar accumulation, we determined the concentrations of sucrose, glucose, and fructose in tomato fruits with RA or control during fruit ripening. As illustrated in Figure 4A, RA largely prevented the decline in sucrose content during fruit ripening. Conversely, the ripening-induced monosaccharide accumulations of glucose and fructose were significantly inhibited (Figure 4B,C). Following the quantification assays, we also found that RA inhibited the decrease in malic acid, one of the primary acids in tomato fruits, during ripening (Figure 4D). However, RA did not affect critic acid content during ripening (Figure 4E). Taken together, these results indicated that RA treatment modestly influenced sugar and organic acid metabolism in fruits during ripening.

Figure 4. RA influenced sugar and organic acid accumulation during fruit ripening. Effects of RA treatment on (A) sucrose content, (B) glucose content, (C) fructose content, (D) malic acid content, and (E) citric acid content. Tomato fruits at the mature green stage were treated with 0.5 mM RA, or H₂O control. The fruit samples were collected at the indicated time points for sugar and organic acid content quantification. The data are presented as mean values ± SD; n = 3. Asterisks indicate statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01) compared with control under the same time point, as determined by Student’s t-test.

6. Effects of RA Treatment on Antioxidant System

At early ripening stages, the increasing respiratory rates in tomato fruits are typically accompanied by a ROS burst ^[17]. The activation of antioxidant enzymes such as APX (ascorbate peroxidase), CAT (catalase), GR (glutathione reductase), and SOD (superoxide dismutase) could protect fruits against oxidative stress during fruit ripening and senescence ^[2]. To better understand the effects of RA treatment on the fruit antioxidant system, we analyzed the activity of antioxidant enzymes. As illustrated in Figure 5A, ripening-induced increase in the activity of APX, CAT, GR, and SOD was further promoted by RA treatment, and RA-mediated higher antioxidant enzyme activity could be still distinguished at 12 d after treatment. Consistent with the enzyme activity, the transcript abundance of genes encoding APX, CAT, POD, and SOD was also largely induced by RA treatment at 4 d of the early ripening stage (Figure 5B).

Figure 5. RA promoted transcript abundance and enzyme activity of antioxidant enzymes during fruit ripening. Effects of RA treatment on (A) antioxidant enzyme activity and (B) transcript abundance of antioxidant enzyme encoding genes. The transcript abundance of each gene under control at 0 d was defined as 1. APX, ascorbate peroxidase; CAT, catalase; GR, glutathione reductase; SOD, superoxide dismutase. Tomato fruits at the mature green stage were treated with 0.5 mM RA, or H₂O control. The fruit samples were collected at the indicated time points for qRT-PCR and enzyme activity analysis. The data are presented as mean values ± SD; n = 4 in (A), 3 in (B). Asterisks indicate statistically significant differences (* p ≤ 0.05, ** p ≤ 0.01) compared with control under the same time point, as determined by Student’s t-test.

Beside diverse antioxidant enzymes, antioxidant metabolites also play an indispensable role in the antioxidant system, among which ascorbate and glutathione act as the heart of the cytosol redox hub regulating redox homeostasis ^[18]. Generally, the changes in the ratios of reduced ascorbate to dehydroascorbate (AsA/DHA) and glutathione to glutathione disulfide (GSH/GSSG) reflect the alterations in redox status. The AsA content reduced and the DHA increased in H₂O-treated fruits at 4 d of the early ripening stage, which led to a decline in the AsA/DHA ratio (Figure 6A–C). Notably, RA treatment was able to prevent the decline in AsA content, and inhibit the DHA accumulation, eventually keeping a higher level of the AsA/DHA ratio in fruits compared to that with control treatments (Figure 6A–C). Similarly, RA treatment also significantly induced the GSH content and substantially suppressed the GSSG content, protecting the GSH/GSSG ratio from further reduction during ripening (Figure 6D–F). These findings indicate that RA plays a positive role in strengthening the antioxidant system by activating antioxidant enzyme activity and regulating the redox status of antioxidant metabolite during tomato fruit ripening.

Figure 6. RA affected the redox status of antioxidant contents during fruit ripening. Effects of RA treatment on (A) reduced ascorbate (AsA) content, (B) dehydroascorbate (DHA) content, (C) redox status of ascorbate, (D) glutathione (GSH) content, (E) glutathione disulfide (GSSG) content, and (F) redox status of glutathione. Tomato fruits at the mature green stage were treated with 0.5 mM RA, or H₂O control. The fruit samples were collected at the indicated time points for antioxidant quantification analysis. The data are presented as mean values ± SD; n = 4. Asterisks indicate statistically significant differences (** p ≤ 0.01) compared with control under the same time point, as determined by Student’s t-test.

7. Effects of RA Treatment on Amino Acid Biosynthesis

During fruit ripening, a variety of metabolites are synthesized and degraded. To further clarify the global effects of RA on fruit ripening, we carried out an untargeted metabolomic analysis to recognize metabolite accumulation patterns of tomato fruits in response to RA treatment at 4 and 8 d. A total of 1328 metabolites were identified at two ripening stages (Table S2). The principal component analysis (PCA) revealed that the samples were divided into four groups, each associated with a treatment at a specific time point (Figure 7A). We identified 200 metabolites (80 upregulated and 120 downregulated in the RA-treated fruits compared with the H₂O-treated fruits at 4 d), and 168 metabolites (55 upregulated and 113 downregulated in the RA-treated fruits compared with the H₂O-treated fruits at 8 d) were differentially accumulated in positive or negative mode (VIP > 1, p ≤ 0.05), respectively (Figure 7B, Table S3). Strikingly, enrichment analysis of KEGG categories revealed that the differentially accumulated metabolites (DAMs) were mainly clustered into the pathways of ABC transports, the biosynthesis of amino acids, and the aminoacyl-tRNA biosynthesis both at 4 and 8 d (Figure 7C, Table S4). A close look into these enriched KEGG pathways showed that 10 out of 19 items in the ABC transports pathway, 13 out of 17 items in the biosynthesis of amino acids pathway, and 13 out of 13 items in the aminoacyl-tRNA biosynthesis pathway were associated with amino acids, peptides, and analogues (Table S5). According to the heat map of the accumulations of DAMs in these three pathways, the amino acids of D-glutamine, DL-serine, DL-valine, histidine, isoleucine, L-aspartic acid, L-proline, DL-tyrosine, and DL-tryptophan were enhanced by RA-treatment both at 4 and 8 d, whereas DL-glutamic acid and L-methionine were declined by exogenously applied RA (Figure 7D). Overall, the data revealed the specific roles of RA in tomato ripening and flavor formation potentially by regulating amino acid metabolism.

Figure 7. Effects of RA treatment on fruit metabolome. (A) PCA of metabolites as influenced by RA treatment at 4 and 8 d. (B) Number of metabolites increased or decreased by RA treatment at 4 and 8 d, respectively. (C) Top 5 enriched KEGG pathways based on RA-regulated differentially accumulated metabolites (DAMs) at 4 and 8 d, respectively. (D) Heatmap indicates the relative metabolite abundance of RA-regulated DAMs within the top 3 enriched KEGG pathways of ABC transporters, the biosynthesis of amino acids, and the aminoacyl-tRNA biosynthesis. The original metabolite abundance was subjected to data adjustment by normalization using an R package. Red and blue lines indicate the up- and down-regulated metabolites by RA treatment at 4 and 8 d, respectively. The color scale on the right indicates relative metabolites abundance.

References

Chen, T.; Qin, G.; Tian, S. Regulatory network of fruit ripening: Current understanding and future challenges. New Phytol. 2020, 228, 1219–1226.
Zuccarelli, R.; Rodriguez-Ruiz, M.; Lopes-Oliveira, P.J.; Pascoal, G.B.; Andrade, S.C.S.; Furlan, C.M.; Purgatto, E.; Palma, J.M.; Corpas, F.J.; Rossi, M.; et al. Multifaceted roles of nitric oxide in tomato fruit ripening: NO-induced metabolic rewiring and consequences for fruit quality traits. J. Exp. Bot. 2021, 72, 941–958.
Liu, M.C.; Pirrello, J.; Chervin, C.; Roustan, J.P.; Bouzayen, M. Ethylene control of fruit ripening: Revisiting the complex network of transcriptional regulation. Plant Physiol. 2015, 169, 2380–2390.
Seymour, G.B.; Chapman, N.H.; Chew, B.L.; Rose, J.K.C. Regulation of ripening and opportunities for control in tomato and other fruits. Plant Biotechnol. J. 2013, 11, 269–278.
Li, S.; Chen, K.S.; Grierson, D. Molecular and hormonal mechanisms regulating fleshy fruit ripening. Cells 2021, 10, 1136.
Qin, G.Z.; Zhu, Z.; Wang, W.H.; Cai, J.H.; Chen, Y.; Li, L.; Tian, S.P. A tomato vacuolar invertase inhibitor mediates sucrose metabolism and influences fruit ripening. Plant Physiol. 2016, 172, 1596–1611.
Wan, H.J.; Wu, L.M.; Yang, Y.J.; Zhou, G.Z.; Ruan, Y.L. Evolution of sucrose metabolism: The dichotomy of invertases and beyond. Trends Plant Sci. 2018, 23, 163–177.
Klee, H.J.; Giovannoni, J.J. Genetics and control of tomato fruit ripening and quality attributes. Annu. Rev. Genet. 2011, 45, 41–59.
Watkins, C.B. The use of 1-methylcyclopropene (1-MCP) on fruits and vegetables. Biotechnol. Adv. 2006, 24, 389–409.
Waszczak, C.; Carmody, M.; Kangasjarvi, J. Reactive oxygen species in plant signaling. Annul. Rev. Plant Biol. 2018, 69, 209–236.
Foyer, C.H.; Shigeoka, S. Understanding oxidative stress and antioxidant functions to enhance photosynthesis. Plant Physiol. 2011, 155, 93–100.
Sharma, P.; Jha, A.B.; Dubey, R.S.; Pessarakli, M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 2012, 217037.
Cui, X.J.; Lin, Q.L.; Liang, Y. Plant-derived antioxidants protect the nervous system from aging by inhibiting oxidative stress. Front. Aging Neurosci. 2020, 12, 209.
Petersen, M.; Abdullah, Y.; Benner, J.; Eberle, D.; Gehlen, K.; Hucherig, S.; Janiak, V.; Kim, K.H.; Sander, M.; Weitzel, C.; et al. Evolution of rosmarinic acid biosynthesis. Phytochemistry 2009, 70, 1663–1679.
Sik, B.; Kapcsandi, V.; Szekelyhidi, R.; Hanczne, E.L.; Ajtony, Z. Recent advances in the analysis of rosmarinic acid from herbs in the Lamiaceae family. Nat. Prod. Commun. 2019, 14, 1934578X19864216.
Marchev, A.S.; Vasileva, L.V.; Amirova, K.M.; Savova, M.S.; Koycheva, I.K.; Balcheva-Sivenova, Z.P.; Vasileva, S.M.; Georgiev, M.I. Rosmarinic acid—From bench to valuable applications in food industry. Trends Food Sci. Tech. 2021.
Kumar, V.; Irfan, M.; Ghosh, S.; Chakraborty, N.; Chakraborty, S.; Datta, A. Fruit ripening mutants reveal cell metabolism and redox state during ripening. Protoplasma 2016, 253, 581–594.
Foyer, C.H.; Noctor, G. Ascorbate and glutathione: The heart of the redox hub. Plant Physiol. 2011, 155, 2–18.

© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.

Upload a video for this entry

Information

Subjects: Food Science & Technology

Contributor MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Zhangjian Hu

View Times: 596

Update Date: 22 Dec 2021

Table of Contents

Video Upload Options

Confirm