Tomato (Solanum lycopersicum) is a popular horticulture crop consumed as fresh fruit or raw material for the food industry. Tomato production has increased worldwide every year. Tomato contains high micro and macronutrients such as vitamins, minerals, fiber, and other beneficial compounds for human health. Furthermore, it is a model for studying fruit biology, fruit development, softening, ripening, and fruit metabolism (Brummell and Harpster, 2001; Giovannoni, 2004; Carrari and Fernie, 2006), because it has a small genome size (950 Mb), a relatively short life cycle, and stable genetic transformation (Osorio et al., 2011; Kumar et al., 2012). Tomato belongs to climacteric fruit. Thus postharvest handling is essential during shipment and marketing. In climacteric fruits, ethylene accelerates fruit ripening and softening. Moreover, ethylene affects leaf abscission, stem or root elongation, root hair development, epinasty, and flower fading (Abeles et al., 1992).

In developing countries, the loss of horticultural products during postharvest handling reached 50% due to storage, transportation, and packaging conditions (Kitinoja and Kader, 2015). Moreover, the presence of ethylene directly affects the lost fruit quality. Several methods have been developed to prevent the ethylene effect in reducing postharvest tomato fruit quality, such as inhibiting ethylene biosynthesis and perception by chemical compounds, atmosphere modification, and genetic modification. In climacteric fruits such as tomatoes, the inhibition of ethylene perception is more effective than ethylene biosynthesis due to the limitation in the perception of ethylene to its receptor. 1-Methylcyclopropene (1-MCP) is a non-toxic chemical compound that effectively prevents the binding process of ethylene to the receptor. Therefore, the ethylene effect can be minimized. However, this method can be more laborious and impracticable to apply to the farmers. Recently, the genetic modification approach has been widely used to develop prolonged fruit shelf life by down-regulated the ethylene biosynthesis and perception gene. However, this method needs to be supported and acceptable in some countries. The mutation method would be a practical approach for generating new ethylene-insensitive cultivars. Mutation in the ethylene receptor gene has successfully generated several insensitive tomato mutants, such as Sletr1-1, Sletr1-2, and Sletr4-1 (Okabe et al., 2011; Mubarok et al., 2015; Mubarok et al., 2019). This review discusses the commercial use of the ethylene-insensitive mutants, Nr, Sletr1-1, Sletr1-2, and Sletr4-1, as potential breeding material to generate new prolonged shelf life for cultivated tomatoes. It also highlights the prospect and problems associated with using the mutants.

Fruit ripening is regulated by ethylene. Ethylene biosynthesis and signaling are modulated during the development of plant tissue and are responsible for inducing many biochemical processes (Abeles et al., 1992). Ethylene biosynthesis is subject to both positive and negative feedback regulation (Kende, 1993). Ethylene biosynthesis in higher plants has been well-characterized. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) are enzymes of ethylene biosynthesis that have been recognized as the rate-limiting step (Yang and Hoffman, 1984; Kende, 1993). ACS activity is the critical step in controlling ethylene production, whereas ACO activity is constitutive (Yang and Hoffman, 1984; Theologis et al., 1993). The genes encoding ACS and ACO have been studied in more detail than other enzymes in the ethylene pathway. In higher plants, ACS and ACO are encoded by multigene families. Eight ACS genes (LeACS1A, LeACS1B, and LeACS2-7) (Zarembinski and Theologis, 1994; Oetiker et al., 1997; Shiu et al., 1998) and five ACO genes have been identified in tomatoes (Van-der-Hoeven et al., 2002).

The receptor is the crucial factor for ethylene action. A copper cofactor mediates the binding process of ethylene to the receptor (Rodríguez et al., 2010). The absence of copper cofactor caused less capability to bind ethylene. The binding site for copper could be replaced by any other metal, such as silver, due to a strong affinity issue. Silver is commonly used to inhibit ethylene perception by replacing the site of copper. This situation impedes conformational change that is typically found in the presence of copper cofactor in the receptor site. There were three domains classification of ethylene receptor protein based on its structure, i.e., sensor domain, kinase domain, and response regulator domain (Ciardi and Klee, 2001). Both amino-terminal ethylene-binding and the most highly conserved GAF are reported subdomains of the sensor domain (Aravind and Ponting, 1997).

In tomatoes, at least six ethylene receptor genes (LeETR1–6) were identified, and LeETR3 is denoted as NR (Payton et al., 1996). The expression of each tomato receptor is different in temporal and spatial patterns depending on the development stage and external stimuli (Alexander and Grierson, 2002). LeETR1 and LeETR2 are expressed constitutively in all tissues throughout development, NR is up-regulated at anthesis, and both NR and LeETR4 are up-regulated during ripening, senescence, abscission (Payton et al., 1996; Tieman et al., 2000), and pathogen infection (Ciardi et al., 2000). LeETR5 is expressed in fruit, flowers, and during pathogen infection (Tieman and Klee, 1999).

The binding of ethylene to receptors causes conformational changes in a receptor or inactivates a receptor, resulting in the inactivation of a negative regulator of downstream ethylene signaling such as CTR1 (Kieber et al., 1993). Suppression of CTR1 activates ETHYLENE INSENSITIVE (EIN2) to act as an essential positive regulator of the ethylene signaling pathway (Wang et al., 2002). Genetic epinasty analysis of ethylene response mutants has shown that EIN2 acts downstream of CTR1 and positively signals upstream of EIN3 (Alonso et al., 1999; Wang et al., 2002). EIN3 is both necessary and sufficient for the activation of ethylene-responsive target genes and, in particular, for ERF1 (Solano et al., 1998). ERF1 belongs to a large family of plant-specific transcription factors referred to as ethylene response element-binding proteins (EREBPs) (Carrari and Fernie, 2006). Transcription factor ERF1 and other EREBPs can interact with the GCC box, which causes ethylene responses in plants (Yamamoto et al., 1999).

Ethylene has become a central problem in postharvest horticultural products. Several strategies are needed to manipulate the adverse ethylene effects leading to the maintenance of the postharvest quality of the horticultural product, including tomatoes. Developing new cultivars by mutation is one strategy for obtaining tomato mutants with long fruit shelf-life, such as ripening-inhibitor (rin), colorless non-ripening (Cnr), non-ripening (nor), green-ripe (Gr) and Nr.

Targeting induced local lesions in genomes (TILLING) is a general method to identify induced point mutations in the genomes of any organism. This method accelerates identifying the modified function of desired genes and selecting mutants rather than conventional mutation breeding. TILLING method has identified some mutants, for instance, SleIF4E1 of tomato mutant, which showed potyvirus resistance (Piron et al., 2010), CmACO1 of melon mutant, which produced long shelf-life fruit (Dahmani-Mardas et al., 2010), Sletr1-1, Sletr1-2, and Sletr4-1, which show in the reduction of ethylene sensitivity (Okabe et al., 2011; Mubarok et al., 2019).

The expression analysis of related genes of ethylene biosynthesis and perception has been widely investigated in tomato mutants. This analysis showed that each mutant has a different location where the mutation occurred. In the Nr mutant, a mutation occurred in the ethylene-binding domain of the NR ethylene receptor; therefore, ethylene cannot be perceived, and its response cannot be expressed (Lanahan et al., 1994; Wilkinson et al., 1995). In the ripening inhibitor (rin) mutant, the mutation occurred in the RIN transcription factor; therefore, autocatalytic ethylene production does not show, and the ethylene signal downstream cannot be transmitted (Vrebalov et al., 2002).

In the novel ethylene receptor mutants, in the Sletr1-1 and Sletr1-2 tomato mutant of ‘Micro-Tom’, the mutations occurred in the first and second transmembrane domain in the ethylene receptor, respectively. The location of mutation of Sletr1-1 (P51) and Nr (P36) are similar in the first transmembrane domain; however, they have different ethylene sensitivity (Wilkinson et al., 1995; Okabe et al., 2011). In the Sletr4-1, there has an amino acid substitution, G154S, that occurs between the transmembrane and GAF domains, whereas the Sletr5-1 tomato mutant has the amino acid substitution, R278Q, within the GAF domain (Mubarok et al., 2019) ( Figure 1 ).

The ethylene receptor gene plays an important role in ethylene action. Mutation in SlETR1, SlETR4, and SlETR5 results in altered ethylene sensitivity, showing the different changes in ethylene triple response and fruit shelf life. From the seedlings’ ethylene triple response assay, the four new ethylene receptor mutants, Sletr1-1, Sletr1-2, Sletr4-1, and Sletr5-1, exhibited a different ethylene sensitivity. Sletr1-1, Sletr1-2, Sletr4-1, and Sletr5-1 display completely ethylene insensitive, moderate ethylene insensitive, low ethylene sensitivity, and high ethylene sensitivity, respectively (Okabe et al., 2011; Mubarok et al., 2015; Mubarok et al., 2019).

Tomato fruit development could be divided into three phases, namely (i) the main phase with rapid and active cell division; (ii) the phase with a stable increase in size due to cell expansion, and the fruit ripening phase (Pirrello et al., 2012). During the ripening process, the tomato fruit experienced specific changes in appearance, color, texture, taste, and aroma (Giovannoni, 2004). The ripening process in tomato fruits was further divided into three phases, i.e., mature green, breaker, and red. Tomato fruit discoloration during the ripening process occurred due to the increased lycopene and beta carotene content; and chlorophyll degradation during the transition from chloroplasts to chromoplasts. The mature green stage is the final fruit formation stage, as indicated by fully expanded fruit size. In this stage, seed formation began. In a later phase, the breaker, the fruit starts to rip as characterized by specific metabolites degradation and the initiation of ethylene production spike and respiration as a sign of climacteric characteristics. While in the red phase, the fruit is considered ripe, with optimum metabolite content, and also experiences the beginning of the senescence phase (Fraser et al., 1994; Osei et al., 2017).

The fruit shelf life is one of the essential characteristics of the postharvest quality of horticulture crops. In climacteric fruit such as tomatoes, fruit shelf life is commonly affected by ethylene, which accelerates fruit ripening. Therefore, to improve fruit shelf life, the response of ethylene must be minimized. Mutation in ethylene receptor genes significantly underestimated the ethylene response by extending fruit shelf life. Improving the fruit shelf life of tomatoes by developing a new cultivar is an excellent way to get a significant aim in tomato breeding because it can provide various benefits for both tomato producers and consumers. Besides long fruit shelf life, other traits must be improved in tomatoes, such as fruit performance and fruit nutrient, because it is crucial factors for fruit quality and the human diet.

Several mutant alleles, such as Sletr1-1, Sletr1-2, and Nr, reduced ethylene sensitivity, impacting fruit development and ripening. In both homozygous and heterozygous Sletr1-1 mutants, there is a disturbance in the process of petals withering, while the effect is weaker for Nr in both homozygous and heterozygous conditions. However, this condition is influenced by parental background (Okabe et al., 2011). An earlier study by Okabe et al. (2011) reported that petal flowers of Sletr1-1 mutants still stick to the fruit even up to 60 days after pollination (DAP), while in WT-MT and Sletr1-2, the petal withered at 3 and 5 DAP, respectively.

The delay in petal abortion can be used as one of the indicators related to ethylene sensitivity that further affects fruit development, ripening, and postharvest fruit shelf life. The ripening phenotypes in Sletr1-1 were different between homozygous and heterozygous plants. The homozygous Sletr1-1 displayed yellow and orange color (Okabe et al., 2011) whereas heterozygous Sletr1-1 fruits showed reddish-orange color. Homozygous Sletr1-1 and Nr showed similar fruit ripening phenotypes due to imperfect ripening processes (Lanahan et al., 1994; Okabe et al., 2011). Phenotypic differences in fruit ripening were not detected between homozygous and heterozygous Sletr1-2, where the fruit showed perfect ripening. Crossing commercial tomato cultivars with several mutants, such as Nr, Sletr1-1, and Sletr1-2, might facilitate the development of a commercial F1 hybrid line. However, not all ethylene-insensitive mutants can be used as genetic material in the breeding program of long fruit shelf-life tomatoes, for example, Nr and Sletr1-1. Although both Nr and Sletr1-1 had low sensitivity to ethylene, these two mutants displayed an incomplete maturation phenotype, even though they had an insufficient red color in heterozygous form.

The fruit shelf life of the mutant differs from one another either in homozygote or heterozygous form. In homozygous conditions, the fruits of Sletr1-1 and Sletr1-2 are still intact. They do not show any damage characterized by the absence of a black spot on the fruit surface during the 60 days of storage in a sealed chamber at 25°C, while the WT-MT fruit shows some damage at the age of 20-25 days after harvesting (Okabe et al., 2011). However, the increase in fruit storage resistance is not very strongly shown in the homozygous Sletr4-1 (Mubarok et al., 2019). It was likely that the fruit shelf life could be dramatically extended, and post-harvest fruit damage could be inhibited. The use of Sletr1-2 as breeding material to form a hybrid generation has been carried out. The Sletr1-2 has a strong inheritance pattern in increasing the shelf life of fruits in all commercial parental backgrounds. However, the length of fruit shelf-life resistance is different in each parental background of ‘Aichi First’, ‘Ailsa Craig’, ‘Moneymaker’, and ‘M82’, with an average increase in shelf-life resistance ranging from 4−5 days longer in open room conditions at a storage temperature of 20 ± 2°C and relative humidity of 80% (Mubarok et al., 2015).

Mutations in the SlETR1, SLETR4, and SlETR5 genes do not affect plant external appearance, especially in vegetative organs. However, there is an alteration in plant sensitivity to abiotic stress. The Sletr1-1 is a promising genotype since it has low sensitivity to ethylene and shows dominant inheritance during a breeding program in increasing the shelf life of tomato fruits (Okabe et al., 2011). However, some undesirable characteristics are found in the mutant Sletr1-1, i.e., stress sensitivity response. The F1 generations of Sletr1-1 experience withering and disease attacks during the transplanting process to the NFT system. The wilting plant is caused by root damage when transplanted from the nursery (Mubarok et al., 2015). The inability of the F1 Sletr1-1 to recover the damaged root system and the inhibition of new root formation cause secondary threats, such as the pathogen attack to the root and stem base. In contrast, the F1 Sletr1-2 are not susceptible to biotic and abiotic stress, whereas all hybrid of F1 Sletr1-2 shows similar characteristics to F1 WT-MT, i.e., healthy and white roots without roots rot after transplanting (Mubarok et al., 2015).

Heterozygous Sletr1-1 shows increased susceptibility to infections of diseases caused by Fusarium oxysporum. A similar finding was reported in the Atetr1-1 mutant of Arabidopsis thaliana that showed an increase in disease infection by certain pathogens such as Botrytis cinerea, Fusarium solani, Fusarium oxysporum f. sp. matthiolae, Xanthomonas campestris pv. Campestris, and Pythium spp (O'Donnell et al., 2003; Agarwal et al., 2012). In addition, Nr mutants cannot produce adventitious roots in waterlogging conditions (Visser and Voesenek, 2004; Vidoz et al., 2010) and are susceptible to some pathogens (Francia et al., 2007; Kavroulakis et al., 2007; Cantu et al., 2009).

Aside from the increased susceptibility to biotic and abiotic stress, another undesirable characteristic in the ethylene mutant is a change in fruit color as the implication of the pigment reduction, especially lycopene and beta carotene. In tomatoes, the fruit color can be used to estimate maturity level ( Table 1 ). The delay in fruit ripening occurs in the mutant Sletr1-1. This mutant Sletr1-1 undergoes yellow or orange discoloration 7 to 10 days later than WT-MT. Moreover, this mutant mostly does not produce full red fruit color nor does the F1 generation (Okabe et al., 2011; Mubarok et al., 2015). The inability to produce full red color is also observed in the mutant Nr (Lanahan et al., 1994). On the opposite, this phenomenon is not found in the mutant Sletr1-2 or its F1 generation. Sletr1-2 fruits can produce the normal red fruit color as its wild type even in their F1 generation (Okabe et al., 2011; Mubarok et al., 2015). Although the mutants Sletr1-1 and Nr produce fruits with a long shelf life, they have yet to be widely used in breeding programs due to their susceptibility to biotic and abiotic stress, and the imperfection of the fruit ripening process leads to less red color (Francia et al., 2007; Kavroulakis et al., 2007; Cantu et al., 2009)

The change or mutation in related genes in ethylene biosynthesis and action may regulate the gene transcription and ultimately affect the metabolite contents of the tomato fruit. In the rin mutant, the mutation in the RIN gene inhibits carotenoid biosynthesis, aroma, production of flavor compounds, and softening (Herner and Sink, 1973; Tigchelaar et al., 1978; Knapp et al., 1989; Vrebalov et al., 2002; Kumar et al., 2012). The novel insights into the molecular biology of ethylene-mediated ripening regulatory networks in tomato during fruit development has been revealed by analyzing nor, rin, and Nr mutant at transcriptomic, proteomic, and metabolomic levels (Osorio et al., 2011). Recently, a new investigation on the effect of the mutation in rin mutant showed that the RIN mutation results in a profound change in fruit transcriptome during ripening that is similar to other spontaneous mutations, such as Nr, hp-2dg, and cnr (Eriksson et al., 2004; Alba et al., 2005; Osorio et al., 2011; Rohrmann et al., 2011; Kumar et al., 2012; Mubarok et al., 2021).

The presence of ethylene hormone can affect the growth, yield, and quality of horticultural commodity yields. In post-harvest handling, the presence of ethylene can have both positive and negative effects depending upon the purpose of its use. For storage and transportation purposes, especially in climacteric fruits such as tomatoes, ethylene accelerates the fruit ripening, leading to shorter fruit shelf life. Mutant with ethylene gene receptor modification can be used as an alternative solution because the negative influence of the ethylene hormone in this genotype can be minimized.

These mutants can be used as elders in plant breeding programs to produce new superior tomato cultivars with longer fruit shelf life (Mubarok et al., 2015; Wiguna et al., 2021). With the production of this shelf-resistant commercial tomato cultivar, the post-harvest problem in tomato fruits can be solved. The use of these mutant tomatoes for breeding programs will be more effective when compared to other ethylene-inhibition methods, such as controlled atmospheric storage with high-cost disadvantages. In the future, these mutants will have a considerable function, especially in plant breeding programs to assemble tomatoes for fresh consumption. With the knowledge of these mutants, it is hoped that the breeding program can run well and that new superior tomato cultivars can be produced, especially for the raw consumed tomatoes such as beef and cherry tomatoes.

Ethylene is one of the critical problems in the post-harvest handling of climacteric fruits such as tomatoes. Developing tomato cultivars that are insensitive to ethylene is one of the effective ways to control the negative influence of ethylene in accelerating fruit damage. The TILLING method has successfully obtained mutant tomatoes less sensitive to ethylene, including Nr, Sletr1-1, Sletr1-2, and Sletr4-1. The Sletr1-2 mutant is the most promising genotype for further development among the four mentioned mutant genotypes. The Sletr1-2 mutant is less sensitive to environmental stress and can produce red fruits, unlike the Sletr1-1 mutant, which only has yellow fruits. In its F1 generation, the mutation in the Sletr1-2 allele shows a less significant effect on the nutritional content of the fruit, which is very important for human health. Therefore, the Sletr1-2 mutant is a potential mutant used in breeding programs for assembling new superior tomato cultivars with long fruit shelf life.

All authors contributed to the article and approved the submitted version.