Rubus idaeus RiACS1 Gene Is Involved in Ethylene Synthesis and Accelerates Fruit Ripening in Solanum lycopersicum

Abstract

Raspberry is a berry whose fruit is not tolerant to storage; breeding varieties with extended storage time and high comprehensive quality are significant for raspberries in cold regions. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is a limiting enzyme in the ethylene synthesis process, which plays essential roles in fruit ripening and softening in plants. In this study, the RiACS1 gene in raspberry (Rubus idaeus L.) variety ‘Polka’ was cloned. The RiACS1 gene overexpression vector was constructed and transformed into tomato plants using the Agrobacterium tumefaciens infection method to verify its function in their reproductive development. The RiACS1 gene, with a total length of 1476 bp, encoded a protein with 491 amino acids. The subcellular localization analysis of the RiACS1 protein in the tobacco transient expression system revealed that the RiACS1-GFP fusion protein was mainly located in the nucleus. Compared with the control, the flowering time and fruit color turning time of transgenic strains were advanced, and the fruit hardness was reduced. Overexpression of RiACS1 increased the activity of ACC synthase, ethylene release rate, and respiration rate during the transchromic phase. It changed the substance content, increased the content of vitamin C and anthocyanin in the fruit ripening process, and decreased the content of chlorophyll and titrable acid at the maturity stage. In addition, RiACS1 increased the relative expression levels of ethylene synthesis-related genes such as SlACS4, SlACO3, and SlACO1 in the fruit ripening process, while it decreased the expression levels of SlACS2 at the maturity stage. These results suggested that the RiACS1 gene could promote early flowering and fruit ripening in tomato plants. This study provided a basis for further modifying raspberry varieties using molecular biology techniques.

1. Introduction

Ethylene is an essential plant hormone that regulates plant growth and development. In addition to playing an important role in plant flower bud differentiation, fruit ripening and softening, and other growth and development processes [1,2,3], ethylene also regulates signal transduction and metabolic reactions in abiotic stresses such as waterlogging, drought, and low temperature [4,5]. Under average growth and development of plants, the ethylene content in various tissues is relatively low. However, after the plants are subjected to stress, ethylene is rapidly synthesized, and a large amount of ethylene quickly diffuses and transfers to various tissues of the plant, causing a series of reactions and weakening the impact of stress on the plant [6,7]. Ethylene is, therefore, an important ‘bridge’ between changes in the external environment and the plant’s intrinsic developmental response [8]. Raspberry fruit has very high nutritional value and health functions, making it particularly suitable for fresh consumption and processing [9], but it is prone to softening after maturity. Hormones have always been considered the main factor regulating fruit growth and development [10], among which ethylene plays a significant role in fruit development. Raspberries are generally considered non-climacteric fruits [11], but the raspberry variety ‘Harriet′s’ should belong to the climacteric fruit of respiration [12]. Most previous studies suggest that abscisic acid plays a significant regulatory role in the maturation of non-climacteric fruits, while ethylene mainly regulates climacteric fruits [13]. Recent studies have shown that ethylene also plays a crucial role in the development of non-climacteric fruits.

The methionine pathway for ethylene biosynthesis was first demonstrated in 1984 [14,15,16,17,18,19]. In the ethylene biosynthesis process, ACS catalyzes ACC generation from SAM, which is the rate-limiting step [20]. The ACS gene was initially obtained from the zucchini (Cucurbita) fruit and subsequently cloned and validated in various plants [21]. At present, ACS has been cloned from plants such as the tomato (Solanum lycopersicum), grape (Vitis vinifera L.), and Khasi mandarin (Citrus reticulata Blanco) [22,23,24]. Fourteen ACS genes have been identified in Solanum lycopersicum [25]. The expression of 4 out of 13 ACS genes in pear (Pyrus ussuriensis) was found to be induced by ethylene and inhibited by 1-MCP [26]. This multi-gene coding characteristic further ensures that the ACS gene can accurately regulate the ethylene concentration in the plant body to make the plant grow and develop normally. The members of the ACS gene family have diverse functions. They can play important roles in various growth and development processes such as flower bud differentiation, fruit abscission, and fruit ripening and softening in plants. MdACS6, MdACS3a and MdACS1, are mainly responsible for ethylene production during apple fruit maturation [27]. AtACS2 affects stomatal density in Arabidopsis leaf epidermis [28]. All-male cucumber strains (Cucumis sativus L.) were controlled by CsACS11, which directly inhibited the expression of the ACC oxidase gene (CsACO2) and inhibited the development of cucumber seedpods [29]. CiACS4 is involved in plant height regulation in lemon (Citrus limon L. Burm) by inhibiting gibberellin biosynthesis [30].

During the ripening process of raspberry fruit, ethylene production increases. Increased ethylene production and respiration rates can be detected during the ginkgo stage and continue until the fruit is fully ripe [31]. Studies have shown that the ethylene concentration of raspberry fruit may increase by 75 times during ripening [32]. Therefore, ethylene production in raspberry fruits is very different from that in other non-climacteric fruits. In addition, exogenous ethylene can enhance respiration, reduce the hardness, and promote fruit ripening [33]. Therefore, ethylene can regulate the growth and development of raspberry fruit. RiACS1 expression increased gradually during the ripening process of raspberry fruit. It was higher in the red fruit stage and overripe stage, and in the overripe stage, the expression level of RiACS1 in the receptacle was more than three times higher than that in the drupelets [31].

Our previous studies showed that after ethylene treatment of raspberry fruits, the contents of soluble sugar, anthocyanin, and soluble pectin were increased, the contents of titrable acid, chlorophyll and protopectin were decreased, the fruit hardness was decreased, and the expression of RiACS1 was increased, while the effect of 1-MCP was opposite, indicating that the RiACS1 gene of raspberry is involved in fruit ripening. Therefore, the cloning and analysis of the raspberry RiACS1 gene was carried out in this study to further promote the research on the ripening mechanism of raspberry fruit. This study used the raspberry variety ‘Polka’ as the experimental material, designed specific primers to clone the RiACS1 gene from the raspberry fruit, and overexpressed the gene in tomato plants. The changes in relevant physiological indicators and gene expression levels were measured to verify the function of the RiACS1 gene. In this study, the role of the RiACS1 gene in ethyl synthesis and ripening of raspberry fruits was clarified, and the possible mechanism of RiACS1 regulation on ripening was discussed, which is helpful to enrich the molecular and physiological studies of raspberry fruits after harvest and provide a scientific theoretical basis for genetic breeding and variety improvement. It has a very positive significance to further promote and improve the industrial development of raspberry.

2. Materials and Methods

2.1. Plant Material and Growing Conditions

The raspberry variety ‘Polka’ was used as the experimental material. The roots, stems, leaves, and fruits of 4 different maturity stages (green fruit stage, ginkgo stage, coloring stage, and maturity stage) were collected from Xiangyang Farm of Northeast Agricultural University in August 2020. The row spacing of raspberries was 2 m, and the spacing of plants was 1 m [34]. After picking the experimental materials, they were quickly placed in a liquid nitrogen tank and brought back to the laboratory for cryopreservation. The tobacco used for subcellular localization was Nicotiana benthamiana, and the tomato materials used for genetic transformation was ‘Micro-Tom’ (Solanum lycopersicum).

2.2. Cloning of RiACS1 Gene

RNA was extracted from mature raspberry fruits using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China). The cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Based on the CDS interval of the raspberry RiACS1 gene as a reference sequence, the specific primers were designed using Premier 5.0 (Supplementary Table S1), synthesized by the Beijing Genomics Institute (Beijing, China). PCR amplification of RiACS1 using cDNA obtained from reverse transcription was used as a template. The target fragment was separated and cut by agarose gel electrophoresis [35], and the DNA Clean-up Kit (Kangweishiji, Beijing, China) was used to purify DNA fragments. The pEASY®-T5 vector (TransGen Biotech, Beijing, China) was used to link the target fragment [36].

2.3. RiACS1 Bioinformatics Analysis

Species with high amino acid sequence homology with RiACS1 were searched for by NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 2 June 2024), and DNAMIN6.0.3.99 performed sequence comparison. Mega11.0 was used to construct the phylogenetic tree of the RiACS1 gene. ExPASy online tools were used (https://web.expasy.org/protparam/, accessed on 5 June 2024) to predict the RiACS1 protein of the physical and chemical properties. The RiACS1 protein domain was predicted using the SMART (http://smart.embl-heidelberg.de/, accessed on 2 June 2024) domain analysis website.

2.4. Subcellular Localization of RiACS1 Protein

The full-length sequence of RiACS1 without stop codon was inserted between the two restriction sites of BamHI and SalI of pCAMBIA 1300s to obtain p35S: RiACS1-GFP. The forward primer containing the BamHI enzyme site was 5’-AGCTCGGTACCCGGGGATCCAT GGGCTCGAACTCAGCACT-3’, and the reverse primer containing the SalI enzyme site was 5’-GCCCTTGCTCACCATGTCGACATTTGCTCGAAGTAGAGGCGAG-3’. The p35S: RiACS1-GFP and p35S: GFP vectors were transformed into Agrobacterium strain GV3101. The bacterial solution with $O{D}_{600}=0.4$ was injected into tobacco leaves growing for 5–6 weeks with a syringe and cultured under low light for 48–60 h. The fluorescence position was then observed under a confocal laser microscope [37].

2.5. Quantitative Real-Time PCR (qPCR) Analysis of RiACS1

The conserved sequence of RiACS1 was found by sequence alignment, and q-PCR primers were designed (Supplemental Table S1). Using the cDNAs of raspberry roots, stems, leaves, and four fruits of different ripening stages (green fruit stage, ginkgo stage, coloring stage, and ripening stage) as templates, specific methods are described in Section 2.2, q-PCR was performed using BlazeTaq™SYBR®Green qPCR Mix 2.0 (GeneBio, Guangzhou, China). The PCR reaction procedure was as follows: predenaturation at 94 ℃ for 30 s; then denatured at 95 ℃ for 5 s and annealed at 54 ℃ for 40 s; extended for 30 s at 72 ℃, one cycle, repeated 34 times; finally, kept at 72 ℃ for 10 min. The 18 s gene was used as a reference gene in a quantitative study. Quantitative data were analyzed using the $2^{-\Delta \Delta CT}$ method [38].

2.6. Genetic Transformation of Tomatoes

The leaf disk method was used for tomato genetic transformation [39]. The cotyledons of sterile tomato seedlings were cut into 0.5cm squares and placed on the medium (4.4 g L⁻¹ MS + 30 g L⁻¹ sucrose + 1 mg L⁻¹ Zeatin + 0.1 mg L⁻¹ IAA + 7.5 g L⁻¹ Agar), which was cultured in the dark for 48 h. Then, it was infected with the bacterial solution of Agrobacterium strain GV3101, $O{D}_{600}=0.5$, which contained the pCAMBIA1300-RiACS1 vector, for 15 min. It was then cultured in the dark for 48 h. Then, the cotyledons were transferred to a screening medium (4.4 g L⁻¹ MS + 30 g L⁻¹ Sucrose + 2 mg L⁻¹ Zeatin + 0.1 mg L⁻¹ IAA + 50 mg L⁻¹ Kan + 400 mg L⁻¹ Tim + 7.5 g L⁻¹ Agar) to screen resistant buds. The culture conditions for explants are 16 h of light and 8 h of darkness [40]. The resistant buds were transferred to shoot elongation medium (4.4 g L⁻¹ MS + 30 g L⁻¹ Sucrose + 0.2 mg L⁻¹ Zeatin + 0.2 mg L⁻¹ IAA + 50 mg L⁻¹ Kan + 300 mg L⁻¹ Tim + 7.5 g L⁻¹ Agar) and rooting medium (4.4 g L⁻¹ MS + 30 g L⁻¹ Sucrose + 4 mg L⁻¹ IBA + 200 mg L⁻¹ Tim + 7.5 g L⁻¹ Agar). When the root grew to 5–8 cm, the medium wrapped with the root was washed and planted in the nutrient soil for further cultivation. The T1 transgenic tomato was obtained. Transgenic tomatoes of the T1 generation continued to be cultured and the seeds were collected. The seeds were screened with a seed screening medium (4.4 g L⁻¹ MS + 30 g L⁻¹ Sucrose + 50 mg L⁻¹ Kan + 7.5 g L⁻¹ Agar) until transgenic tomatoes of the T3 generation were obtained.

2.7. Determination of Physiological Indicators Related to Transgenic Tomatoes

The fruit hardness was determined by the Texture Analyzer TA (Baosheng, Shanghai, China) [41] using a probe. A Fruit and Vegetable Respiration Measuring Instrument (Yuntang, Shandong, China) was used to determine the respiration rate [42]. Then, 5 g fruit was put into a 250 mL container, and the carbon dioxide concentration was measured every 15 min three times. ACC synthase activity was determined by high-performance liquid chromatography [31]. Culture 0.06 mM S-adenosylmethionine (SAM) was placed in a 100 mM potassium phosphate buffer containing 0.1 mM PLP. The purified enzyme was added for 20 min and monitored at 260 nm. The ethylene production rate was determined by gas chromatography [43]. The 5g fruit was incubated in a 100 mL container at room temperature for 2 h, and 1 mL of gas was extracted with a 1 mL syringe for determination by a gas chromatograph with a flame ionization detector. The chlorophyll content was determined by the acetone method [44]. The fruit was ground with liquid nitrogen and extracted with 80% acetone for 5 min. The results were determined by an Ultraviolet UV-3600i Plus (Shimadzu Corporation of Japan, Tokyo, Japan) spectrometer at 663 nm and 645 nm. The content of anthocyanins was determined by the HCl–methanol method [45]. The powder after grinding with liquid nitrogen was extracted for 30 min with 1% hydrochloric acid–methanol solution, which was measured at 530 nm using an Ultraviolet UV-3600i Plus (Shimadzu, Kyoto, Japan) spectrometer. Titrable acidity was determined by the method of acid-base titration [46], and the sample solution was titrated with 0.1 mol/L NaOH to the bromothymol blue endpoint (blue-green). Vitamin C content was determined by the method of 2,6-DichlorophenolIndophenol [47]. The sample was diluted ten times and titrated with a labeled 2, 6-dichlorophenol indophenol solution until the solution was reddish.

2.8. Expression Analysis of Ethylene Synthesis-Related Genes in Tomato Fruits with Transgenic Tomatoes

The genes SLACS2, SLACS4, SLACO1, and SLACO3 (Supplementary Table S1) related to ethylene synthesis in Micro-Tom tomatoes were amplified by q-PCR with Actin as the internal reference gene. The methods are described in Section 2.5.

2.9. Statistical Analysis

The test results were all repeated three times. SPSS27.0.1 software was used for data analysis, and Student’s t-test was used for comparison of the difference significance, with * p ⩽ 0.05, ** p ⩽ 0.01.

3. Results

3.1. Property and Sequence Analysis of RiACS1 Protein

The gene sequence of RiACS1 is 1476 bp long and encodes 491 amino acids (Figure S1). The predicted theoretical molecular mass (MW) of RiACS1 protein is 6.168076 kDa, the theoretical isoelectric point (pI) is 9.15, and the mean hydrophilic coefficient is −0.272 (Figure S2).

Eleven ACS amino acid sequences from other species were found on the NCBI website, and multiple sequence alignment was performed between them and the RiACS1 protein sequence. The amino acid sequences of RiACS1 protein were basically consistent with those of other ACS protein conserved domains, and the conserved sequences were highly similar (Figure 1A). The phylogenetic tree showed that the RiACS1 protein was closely related to Fragaria × ananassa (Figure 1B).

3.2. Subcellular Localization of RiACS1 Protein

The RiACS1-GFP fusion protein and control GFP protein were expressed instantaneously in tobacco mesophyll cells and observed under confocal laser microscopy. The green fluorescent protein in the control group was distributed throughout the cell, while the fluorescence of the RiACS1-GFP fusion protein was only in the nucleus (Figure 2), indicating that the RiACS1 protein was located in the nucleus.

3.3. Expression Analysis of RiACS1 Gene in Different Tissues and Fruit Ripening Stages of Raspberry

The expression of the RiACS1 gene in different tissues and fruit development stages of raspberry was analyzed by Real-Time PCR. The result showed that the expression level of the RiACS1 gene in the roots and fruits was significantly higher than in the stems and leaves. In addition, with the ripening of raspberry fruits, the relative expression level of the RiACS1 gene increased significantly, with the highest expression level in the ripening stage (Figure 3).

3.4. Phenotypic Observation and Identification of Transgenic Tomato Plants

In order to analyze the function of the RiACS1 gene, it was overexpressed in tomato plants. Through verification, three transgenic strains (R1, R2, R3) were obtained (Figure 4C). The phenomenon of premature flowering of transgenic strains was observed with WT (wild type) and ULs (unloading lines) as controls (Figure 4A). After 30 days of planting, the growth of transgenic plants and control plants was basically the same, but most transgenic plants had flower bud differentiation at this time. After 40 days of planting, it was observed that the transgenic plants had entered the flowering period, while some control plants had observed a flowering phenomenon. From planting to flowering, the control took an average of 39 days, while the transgenic strain took 32 days (Figure 4B). This phenomenon suggests that the RiACS1 gene plays a role in promoting flowering in tomato plants. The expression of the RiACS1 gene in transgenic strains was detected by qRT-PCR (Figure 4D), and it was significantly higher than that in control.

3.5. Overexpression of RiACS1 Promotes Tomato Fruit Ripening

The phenotypes of tomato fruits were observed 28, 37, and 45 days after flowering, respectively. During the transition period, the transgenic tomato fruit was darker than the control fruit. At 37 days after flowering, the color rate of the transgenic fruits reached more than 90%, while that of the control fruits was less than 90%. The results showed that the tomato plants with RiACS1 overexpression entered the color-turning stage and red-ripening stage in advance (Figure 5A), indicating that RiACS1 overexpression may promote tomato fruit ripening.

The relative expression level of the RiACS1 gene increased during the ripening of the transgenic tomato lines. It rose slowly from 28 D to 37 D after anthesis and rapidly from 37 D to 45 D after anthesis (Figure 5B) while the controls showed only a slight increase. The hardness of the whole fruit showed a downward trend. The hardness of the transgenic lines was significantly lower than that of the WT and UL (Figure 5C), indicating that the RiACS1 gene could promote fruit softening. The ACC synthase activity in the transgenic tomatoes and controls increased first and then decreased. The ACC synthase activity of the transgenic lines was significantly higher than that of the WT at 37 days after anthesis; the results indicated that the RiACS1 promoted ACC synthase activity, and the difference in ACC synthase activity was mainly reflected in 37 days after anthesis, that is, the color transformation period (Figure 5D).

During the ripening process of tomato fruits, the ethylene release rate and respiration rate showed a trend of first increasing and then decreasing. Among them, transgenic strains’ ethylene release rate and respiration rate showed a significant increase from 28 to 37 days after flowering. It was significantly different from the controls. In comparison, the change was not significant at 45 days after flowering (Figure 5E,F).

3.6. Material Changes in Tomato Fruits at Different Ripening Stages

To further analyze the effect of the RiACS1 gene on tomato fruit ripening, the contents of titrable acid (TA), vitamin C (Vc), anthocyanin, and chlorophyll in different growth stages of the fruits were measured (Figure 6). The contents of TA, Vc, and anthocyanin increased gradually with the continuous ripening of tomato fruits. The contents of Vc and anthocyanin in the transgenic strains were significantly higher compared with the control, while TA was slightly higher than that of the wild type. The chlorophyll content of both the transgenic tomatoes and control plants was continuously decreased. The chlorophyll content of the transgenic tomato fruits was significantly higher than that of the wild-type plants at 28 d after flowering. Then, it decreased rapidly and was lower than wild-type plants at 37 d after flowering. These results indicated that the overexpression of the RiACS1 gene could promote the accumulation of Vc, and anthocyanin and the decomposition of chlorophyll in tomato fruits.

3.7. Analysis of Gene Expression Related to Ethylene Synthesis

In order to further explore the relationship between RiACS1 and ethylene biosynthesis, the expression levels of SlACO1, SlACO3, SlACS2, and SlACS4 in WT, VL, and RiACS1 overexpressed tomato fruits were measured (Figure 7). It can be found that SlACO1 and SlACO3 continue to increase during tomato ripening, while SlACS2 and SlACS4 increase first and then decrease. The difference was that the expression levels of SlACO1, SlACO3, SlACS2, and SlACS4 in transgenic strains were significantly different compared with the control at different development stages, and the expression levels of each gene were significantly higher than those of the wild type at the color transfer stage. The expression levels of SlACO1, SlACO3, and SlACS4 at maturity were higher than those of the wild type while the SlACS2 was lower than the controls. These results suggest that overexpression of the RiACS1 gene affects the expression of genes related to ethylene biosynthesis in tomato fruits.

4. Discussion

4.1. RiACS1 Gene Sequence and Subcellular Localization Analysis

Ethylene is a simple, small-molecule organic compound, but its complex and diverse physiological effects make it indispensable for plant growth and development [48]. As the key enzyme in ethylene synthesis, ACS has been cloned from crops such as tomato, grape, cucumber, and lemon [17,18,24,25]. The physicochemical properties of the RiACS1 protein were consistent with the bioinformatics analysis results of wheat (Triticum aestivum L.), Cucurbita maxima, and other plants [49,50]. The RiACS1 protein is highly similar to other ACS family proteins in conserved sequences, with seven conserved domains, which are similar to the conserved domains of PpyACS1 in Sand Pear (Pyrus pyrifolia), AtACS11 in Arabidopsis and PgACS1 in guava (Psidium guajava L.) [51,52,53].

The role of protein in the cell varies with its location, so subcellular localization can provide evidence for exploring the specific role of protein. For example, CsACS9 in sweet orange (Citrus sinensis) is located in chloroplasts, and CsACS1 is in cytoplasm [54]. It is suggested that the CsACS gene might be essential in ethylene synthesis during fruit development. In this study, RiACS1 was located in the nucleus (Figure 2), and it may have some functions similar to those of nuclear proteins, which play a role in the nucleus. This finding further proves that this gene has a wide range of functions.

4.2. Relationship Between RiACS1 Gene and Ripening and Softening of Raspberry Fruit

The RiACS1 gene was expressed in the roots, stems, leaves, and fruits of raspberries, and its expression increased significantly from the ginkgo stage to the maturity stage and reached the highest level at the maturity stage (Figure 3), indicating that this gene was closely related to the ripening and softening of raspberries. The effect of ACS on fruit ripening has been demonstrated in many plants. Silencing SlACS1A, SlACS2, and SlACS6 genes in Solanum lycopersicum delayed the softening and decay of tomato fruits [55]. AdACS1 expression in kiwi (Actinidia deliciosa) fruit has been shown to promote fruit ripening [56]. ACS2 is a key enzyme for ethylene synthesis in tomato, and the mutant ACS2-1 showed faster fruit ripening [57]. In this study, the RiACS1 gene was overexpressed in tomatoes, and similar results were obtained.

4.3. Overexpression of RiACS1 Gene Promoted Early Flowering and Fruit Softening in Tomato

Flower bud differentiation is the proof of plant transition from vegetative to reproductive growth [58]. Inhibiting the expression of the ACACS2 gene in pineapple (Ananas comosus (L.) Merr.) significantly delayed the flowering period of the transgenic lines [59]. However, overexpression of the lemon (Citrus limon L. Burm) 1-aminocyclopropane-1-carboxylate synthase gene (CiACS4) in tobacco resulted in late flowering [60]. The results indicated that although the ACS gene plays a regulatory role in plant flowering, it may play a different role because of different species or gene family members. In this experiment, the flowering time of the transgenic strains was earlier than that of the control (Figure 4A,B), indicating that the RiACS1 gene in raspberries can promote early flowering in tomatoes.

Fruit ripening is a complex process regulated by many genes, resulting in changes in color, starch/sugar metabolism, fruit softening, texture changes, and aroma volatiles synthesis [61,62,63]. Therefore, determining the content of chlorophyll, titratable acid, anthocyanins, and vitamin C in plant fruits is necessary to determine their effect. In this study, from days 28 to 37 after flowering, the fruit coloring degree, ACC synthase activity, ethylene release rate, respiratory rate, titrable acid (TA), vitamin C (Vc), and anthocyanin contents of transgenic Solanum lycopersicum were increased significantly, while chlorophyll and fruit hardness were decreased significantly. The same conclusion has been confirmed in citrus, ethylene regulates respiration and chlorophyll breakdown in citrus [64]. Exogenous ethylene increased the content of anthocyanin in grapefruit by increasing the synthesis of anthocyanin-related genes [65]. During the ripening of the tomato fruit, the massive production of ethylene is related to the high expression of ACS2 and ACS4 induced by ethylene through a positive feedback regulation [66,67]. The expression of ACO3 is induced but transitory at the breaker stage, while ACO1 expression is sustained during ripening [68]. In this study, the relative expressions of genes related to ethylene synthesis, SlACS4, SlACS2, SlACO3, and SlACO1, were significantly increased in transgenic tomato fruits. These changes were consistent with the fruit ripening mechanism. However, from days 37 to 45 after flowering, the ACC synthase activity, ethylene release rate, and respiration rate showed a decreasing trend, and the decline was faster in transgenic Solanum lycopersicum. The transfer of the justice gene would inhibit the expression of endogenous homologous genes [69]. This study also showed that the RiACS1 gene promoted the expression of SlACS4, SlACO3, and SlACO1 but inhibited the expression of SlACS2. SlACS2 is a crucial gene regulating ethylene synthesis in tomatoes, and it is mainly responsible for ethylene synthesis in fruit ripening [66]. Therefore, the large expression of the RiACS1 gene may inhibit the expression of the SlACS2 gene and the activity of ACC synthase in tomatoes, thus reducing the ethylene release.

5. Conclusions

Overexpression of RiACS1 in tomatoes promoted fruit ripening by significantly increasing ACC synthase activity and up-regulating expression levels of genes related to ethylene synthesis before the color transition. This study laid a foundation for further research on whether inhibiting the synthesis of ethylene can delay the ripening and senescence of raspberry fruit. In the next step, the genetic transformation system of raspberry can be established to transform the RiACS1 gene, and the regulatory network of RiACS1 in the development of raspberry fruit can be further explored by combining gene silencing techniques. It not only has great significance for fruit quality control and variety popularization and application, but also helps to enrich the theoretical system of fruit quality and provide a scientific theoretical basis for genetic breeding and variety improvement.