Keywords

1 Introduction

Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops in agriculture, both domestically and worldwide. Because this vegetable can be adapted for cultivation in various environments ranging from tropical to nearly alpine regions, its production area is now expanding worldwide. On the other hand, in the traditional cultivation area, which is concentrated around the Mediterranean Sea and in the southern and western parts of the United States (USA) because of the warm and dry climate that is favorable for tomato cultivation, yield loss from salt injury has arisen as a serious problem in 19.5 % of the irrigated land area and in the irrigation water (Flowers and Yeo 1995; Cuartero and Fernandez-Munoz 1999; Foolad 2004). Additionally, 2 million hectares (ha), equivalent to 1 % of the world’s tomato cultivation areas, are lost every year because of environmental factors such as salinity, drought, and soil erosion. In some cases, poor cultivation techniques can also promote the damage caused by environmental stresses (Ashraf and Foolad 2007).

It has been suggested that moderate salinity and drought stress improve the nutrient quality in tomato fruits by increasing sugars, amino acids, carotenoids, and other substances (Adams 1991; Gao et al. 1998; Ho et al. 1987; Krauss et al. 2006; Tal et al. 1979; De Pascale et al. 2001; Saito et al. 2008a). Recently, these cultivation conditions have been introduced in hydroponic cultivation techniques such as the nutrient film technique (NFT), and high-Brix tomatoes are being produced in Japan (Sakamoto et al. 1999; Saito et al. 2006) and in northern European countries (Adams 1991). Such high-Brix fruits are referred to as “fruit tomatoes” and have become popular in the Japanese consumer market because the fruit flavor is much better than that of a tomato cultivated under ordinary conditions. Until recently, the development of “fruit tomatoes” was thought to result from a “concentration effect” caused by the suppression of fruit size caused by salt stress (Ehret and Ho 1986; Ho et al. 1987; Sakamoto et al. 1999). However, evidence uncovered during the past decade indicates that other physiological and molecular alterations might be involved in this phenomenon.

This chapter describes the effects of salt stress on various aspects of tomato growth and on the content of the nutritive components of the fruit, with a specific focus on sugars, and discusses the possible mechanisms that are responsible for the formation of high-Brix fruit.

2 Effect of Salinity on Plant Growth and Yield

In Mediterranean countries, tomatoes have also been studied as a model crop in salinized or dry land agriculture, and a large amount of data about the effects of salinity on the growth of tomato plants has been produced. Most commercial cultivars are moderately sensitive to salinity at all stages of plant development, including seed germination, vegetative growth, and fruit production (Ashraf and Foolad 2007). Even at NaCl concentrations as low as 80 mM, salt stress suppresses seed germination (Cuartero and Fernandez-Munoz 1999), vegetative growth, leaf area, and root weight (Papadopoulos and Rendig 1983; Van Ieperen 1996). Predictably, fruit yield is also negatively affected by salt stress because of a decrease in average fruit weight or the number of fruits produced by a single plant (Cuartero and Fernandez-Munoz 1999). At relatively low electrical conductivity (EC), fruit weight rather than fruit number tends affect the fruit yield. In contrast, at an elevated EC, fruit yield is mainly affected by reduction in fruit number (Van Ieperen 1996). In salt-stressed fruit, fruit enlargement is suppressed during the cell expansion phase because water uptake into the fruit, which is the motive force for cellular expansion, is suppressed by the increased salinity (Ehret and Ho 1986). Regarding marketable yield, increased blossom-end rot (BER) hampers tomato production. It has been suggested that BER is a physiological disorder caused by calcium deficiency in certain parts of the fruit (Adams and Ho 1992; Chretien et al. 2000; Franco et al. 1994; Willumsen et al. 1996) because Ca2+ uptake by the roots and its transport through the xylem to the fruits are both decreased under severe salt stress conditions (Belda et al. 1996; Ho et al. 1993). However, the detailed mechanism underlying BER is still unclear, and an effective method for combating BER has remained elusive.

3 Effect of Salt Stress on Fruit Qualities

As described in Sect. 1.2, salt stress has unfavorable effects on both plant growth and fruit enlargement in tomatoes. On the other hand, it has been reported that moderate salinity improves fruit quality by affecting the levels of soluble solids, such as sugars and acids, as well as the pH value; these are key factors in quality evaluations of fruit sold in markets, and salt stress generally improves fruit quality by increasing the content of those substances. This phenomenon has been attributed to a “concentration effect” that results from the suppression of fruit enlargement in plants exposed to salt stress. However, during the past decade, increasing evidence has indicated that alterations in assimilatory metabolism and the translocation of assimilates into the fruit are likely to be involved in the increase in soluble solids and other components.

3.1 Organic Acids

Acids largely affect fruit flavor by interacting with sugars and by creating acidity (Stevens et al. 1977), and organic acids account for approximately 13 % of the fruit dry matter (Davies and Hobson 1981). Malic acid and citric acid are the most abundant organic acids in fruit (4 % malic acid and 9 % citric acid on a dry matter basis); citric acid is more important for the sour flavor of the fruit than malic acid. Moderate salt stress (EC 8.0 dS m−1) enhances the accumulation of citric and malic acids by 1.7- and 2.5 fold, respectively, compared to control conditions (EC 2.5 dS m−1) at the red-ripe stage (Saito et al. 2008a). Transcriptional analyses showed that the expression of genes involved in organic acid metabolism, such as phosphoenolpyruvate carboxykinase (PEPCK), malate dehydrogenase (MDH), malic enzyme (ME), and pyruvate kinase (PK), is upregulated by both moderate (EC 8.0 dS m−1) and severe (EC 15.0 dS m−1) salt stress in ripening fruit (Saito et al. 2008a; Yin et al. 2010a). These results suggest that a shunt of the tricarboxylic acid (TCA) cycle (malate–oxaloacetate–PEP–pyruvate–citrate) is involved in organic acid metabolism and is stimulated by salt stress. Davies (1964) suggested that the accumulation of organic acids in the fruit counterbalances excessive cations to maintain the fruit pH. The difference between the cation and anion level tends to be greater in salt-stressed fruits, which leads to a higher concentration of organic acids in these fruits (Cuartero and Fernandez-Munoz 1999).

3.2 Carotenoids

Tomato fruits contain various carotenoids that are a major source of the antioxidant lycopene in the human diet, although the quantity, distribution, and antioxidant effects vary among cultivars (Minoggio et al. 2003). Fresh tomato fruit and its processed products provide approximately 85 % of the lycopene in the human daily diet (Canene-Adams et al. 2005). Tomatoes are also a notable source of other carotenoids, such as β-carotene, lutein, phytoene, phytofluene, and ζ-carotene. Because the carotenoid content is an important trait of tomato, many studies have reported the effects of salinity on the carotenoid content in tomato fruits. Generally, salt stress enhances lycopene and β-carotene accumulation; however, some reports indicated that the increase was observed only on a fresh weight and not a dry weight basis. Thus, these authors concluded that the increase should be attributed to a concentration effect caused by the suppression of fruit expansion (Krauss et al. 2006; Dumas et al. 2003; Shi and Le Maguer 2000). On the other hand, De Pascale et al. (2001) reported that the total carotenoid and lycopene content increased on both a fresh weight and dry weight basis under moderate salt stress (EC 4.0 dS m−1) and suggested that metabolic alteration is involved in the increase along with the concentration effect. Our previous study also supports this observation. A key enzyme involved in carotenoid biosynthesis that is produced by the phytoene synthase gene was upregulated by moderate salinity stress (Saito et al. 2008a). Krauss et al. (2006) suggested that reduced leaf area caused by the growth suppression under salt stress and the increased exposure of fruit to sunlight resulted in increased carotenoid accumulation because carotenoid biosynthesis is regulated by light and increased exposure of the fruit to sunlight. Further investigation is required to elucidate the mechanisms underlying the effect of salinity on carotenoid accumulation.

3.3 Amino Acids

Tomato fruit flavor largely depends on the types and quantity of free amino acids present in the fruit. Several amino acids, such as glutamate, γ-aminobutyric acid (GABA), glutamine, and aspartic acid, account for approximately 80 % of the total free amino acids in the fruit (Kader et al. 1978). Among these amino acids, glutamate is the principal free amino acid in red-ripe fruit in commercial cultivars and it confers the characteristic “umami” flavor to tomato (Sorrequieta et al. 2010). It has been reported that salt stress increases the total amino acid content in tomato fruit, and the effect of the stress is greater in the pericarp than in the columella tissue (Zushi and Matsuzoe 2006; Yin et al. 2010a). Importantly, the proline content is increased markedly in both pericarp and columella tissue. Proline is a well-known indicator that responds to abiotic stresses such as salinity and drought in higher plants and most likely functions as an osmoprotectant in fruit exposed to salt stress (Ashton and Verma 1993; Claussen 2005).

Glutamate and GABA are the most abundant amino acids in tomato fruits (Inaba et al. 1980; Rolin et al. 2000). Tomato accumulates higher amounts of GABA in its edible parts than most other vegetable crops (Matsumoto et al. 1997). GABA is a four-carbon, nonprotein amino acid commonly found in diverse organisms, and it serves as a major inhibitory neurotransmitter in vertebrates (Zhang and Jackson 1993). It has been known to play a role in reducing blood pressure in the human body (Inoue et al. 2003). GABA is synthesized from glutamate by glutamate decarboxylase (GAD), which is activated by Ca2+/CaM (Aurisano et al. 1995; Snedden et al. 1995; Turano and Fang 1998) and acidic pH (Johnson et al. 1997; Snedden et al. 1996). Salt stress also enhances GABA accumulation in the fruit at the red stage (Saito et al. 2008a, b). However, although salinity enhances fruit acidity and stimulates cellular Ca2+ in plant cells (Sanders et al. 1999), we cannot determine whether these factors directly promote GABA accumulation in tomato fruit because our previous data showed that glutamic acid decarboxylase (GAD) activity was unchanged in salt-stressed fruit; in fact, our data indicated that only the protein content increased (Yin et al. 2010a). Consequently, substrate availability and an increased concentration of GAD would promote GABA accumulation in salt-stressed tomato fruit. In fact, the GABA metabolic pathway is TCA cycle shunt (Bouchè and Fromm 2004). GABA accumulation reaches a maximum in mature green fruit and is rapidly degraded during fruit ripening (Rolin et al. 2000; Akihiro et al. 2008). Tracer analyses utilizing [14C]-labeled GABA demonstrated that GABA in mature green fruit is converted to malate, citrate, cis-aconitate, isocitrate, and 2-oxoglutarate in red-ripe fruit; furthermore, [14C] was also detected in the CO2 gas fraction during ripening (Yin et al. 2010a). Those results indicate that dissimilated GABA flows back into the TCA cycle, is metabolized to organic acids, and is utilized as a substrate for respiration during climacteric ripening of fruit.

It was also reported that GABA accumulation in the fruit was enhanced 1.6- to 1.9 fold during the postharvest storage of red-ripe fruit under anaerobic (low O2) conditions compared with normal air conditions, regardless of the CO2 concentration (Mae et al. 2012). This increase was caused by increased GAD activity and decreased GABA transaminase (GABA-T) activity.

3.4 Ascorbic Acid

Ascorbic acid (ASA), which is present in tomato fruit, has an important role in the maintenance of human health by serving as an antioxidant that scavenges reactive oxygen species (ROS). In addition, ASA-related antioxidant systems, including the ascorbate–glutathione cycle (in which ascorbate peroxidase is involved), prevent the accumulation of toxic ROS levels under environmental stress conditions (Zhang 2013). Fruit ASA content is affected by cropping season, cultivar, and environmental stress (Dumas et al. 2003). However, the effect of salinity on fruit ASA content is actually not as clear as its effects on other metabolites such as sugars and amino acids. Several studies have shown positive results (Krauss et al. 2006), negative results (Zushi and Matsuzoe 2009), or no change (Fanasca et al. 2007) in the ASA content, and an interaction with other factors such as cultivars and growth conditions (e.g., temperature and light intensity) was suggested. Zushi et al. (2014) examined the combined effect of salt stress and light intensity on ASA content and the activity of ASA-related antioxidant enzymes in tomato fruits using tissue culture experiments. The results revealed that changes in the ASA content and the activity of antioxidant enzymes under salt stress conditions could not be explained only by salinity, because light intensity could also be involved in the regulation of antioxidant enzyme activity.

4 Effect of Salinity on Carbohydrate Allocation to Fruits

Salinity stress inhibits water uptake by the roots and water influx into the fruits as a result of high osmotic pressure around the root system, resulting in a decrease in fruit size and an increase in quality constituents. As described in Sect. 1.3, enhanced metabolite accumulation in salinity-stressed fruit has been attributed to a “concentration effect.” However, our previous studies revealed that the ratio of sucrose to the total sugar content in salt-stressed fruits was increased compared with that of the control (Saito et al. 2006), and the effect of salt stress on the sugar concentration was apparently more severe than its suppressive effect on fruit weight (Yin et al. 2010b). Gao et al. (1998) reported that salinity enhanced the transport of assimilates from leaves to adjacent fruits. These results indicate that the increase in sugar content from salinity is not simply a result associated with the suppression of fruit growth; rather, it is also caused by alterations in sugar metabolism or sugar translocation into the fruit.

To examine alterations in carbohydrate partitioning in plants exposed to salt stress, Saito et al. (2009) investigated the distribution of carbohydrates under moderate salt stress conditions (EC 8.0 dS m−1) in tomato plants by feeding [13C]-labeled CO2 to source leaves at 16 days after anthesis, which is the time when a tomato plant shows the greatest transport activity of photoassimilates from the source leaves to the fruits (Ho 1986). Although both photosynthetic and transpiration ratios were reduced by approximately 60 % under salt stress, the content of [13C]-labeled assimilate to fruits was 28 % higher compared with that observed in the control (Fig. 1.1). The distribution ratio of [13C]-labeled assimilate in fruits was 32 % higher under saline conditions than under control conditions (Fig. 1.1). A similar result was also observed in the [13C] tracer analyses performed under different salt stress conditions (EC 15.0 dS m−1), in which the carbohydrate influx into the fruit was strongly enhanced by salinity in immature and mature green fruits, even though the stress severely suppressed plant growth (Yin et al. 2010b). These results indicate that salt stress alters the allocation of photoassimilate in the entire plant and enhances the sink strength of fruits.

Fig. 1.1
figure 1

Effect of salinity treatment on 13C content and distribution in tomato plants 48 h after feeding 13CO2. Bars indicate SE (n = 3)

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5 Salinity Stress Enhances Starch and Sugar Accumulation in Fruit

5.1 Sugar Metabolism

In addition to the enhancement of photoassimilate translocation and the sink strength of fruit, salt stress increases the sugar content in fruit. This sugar mainly consists of hexoses produced by the cleavage of sucrose. Interestingly, sucrolytic activity is also thought to be an index of sink strength (Yamaki 2010). There are two notable enzymes involved in the cleavage of sucrose to monosaccharides: sucrose synthase and invertase. Sucrose synthase (Susy) converts sucrose into fructose and UDP-glucose. By contrast, invertase irreversibly catalyzes the hydrolytic cleavage of sucrose into glucose and fructose. In tomato, Susy was considered to be a major factor determining the fruit sink strength because a strong correlation among Susy activity, ADP-glucose pyrophosphorylase activity, and starch accumulation in early developing fruit was found (Robinson et al. 1988; Yelle et al. 1988). Moreover, antisense transgenic plants in which Susy activity was suppressed displayed reduced fruit setting and sucrose import capacity in young fruit (D’Aoust et al. 1999). However, the suppression of the Susy-encoding gene in tomato did not lead to remarkable alterations in starch and sugar accumulation in the fruit (Chengappa et al. 1999). There is minimal evidence that sucrose synthase is directly involved in the control of fruit sugar content and composition in tomato.

On the other hand, during the past two decades, increasing evidence has indicated that invertase is an essential factor involved in the regulation of sugar content in tomato fruit. In plants, invertases are classified into three isozyme types according to their solubility, subcellular localization, isoelectric point (pI), and optimal pH (Sturm 1999): cell wall invertase (CWIN), vacuolar invertase (VIN), and cytoplasmic invertase (CIN). Among these isozyme types, CWIN and VIN are characterized as acid invertases because of their acidic optimal pH, whereas CIN is characterized as a neutral invertase because of its neutral optimal pH. Several studies have revealed the diverse roles of invertases in the plant life cycle, including their participation in various responses to abiotic and biotic stresses such as drought, hypoxia, high temperature, wounding, and pathogen infection (Roitsch and González 2004).

The relationship between fruit sugar content and abiotic stress suggests that the most important invertase is CWIN. During the past decade, CWIN has attracted attention as an essential enzyme for determination of the total soluble solids level in tomato (Fridman et al. 2002, 2004; Zanor et al. 2009). Among the genes encoding CWIN (LINs), LIN6 responds to various biotic and abiotic stimuli, including wounding, pathogen infection, and sugars (Godt and Roitsch 1997; Ohyama et al. 1998; Sinha et al. 2002). Additionally, LIN7 was suggested to be involved in heat stress tolerance because its expression was specifically promoted by heat stress in heat-tolerant varieties (Li et al. 2012). Li et al. (2012) also suggested that the increased ability of young fruits to import sucrose contributes to the heat tolerance of the variety and is likely to be governed by LIN7 expression. However, reports that fruit VIN and CWIN activities were not affected by salt stress (Carvajal et al. 2000; Saito et al. 2009) suggest that these two isozymes are most likely not involved in the phenomenon. In contrast, there was a positive correlation between CIN activity and hexose levels in the fruit of plants exposed to salinity stress (Balibrea et al. 1996, 2006). However, minimal information is available on CIN in tomato, and its physiological function has yet to be elucidated. These results suggest that CIN functions under specific environmental conditions such as salinity stress.

5.2 Starch Biosynthesis

Several studies have reported that salt stress enhances starch accumulation in early-developing tomato fruit (Balibrea et al. 1996; Gao et al. 1998; Yin et al. 2010b). In fact, this phenomenon involves an increase in ADP-glucose pyrophosphorylase (AGPase) activity. AGPase plays a role in the regulation of starch accumulation in early-developing fruit (Schaffer and Petreikov 1997; Schaffer et al. 2000). This enzyme catalyzes the synthesis of ADP-glucose from glucose-1-phosphate and ATP, which is the first regulatory step in starch biosynthesis in plants (Tsai and Neleson 1966; Lin et al. 1988; Stark et al. 1992). Plant AGPase is a heterotetrameric enzyme composed of two small and two large subunits (Morell et al. 1987). In tomato, four AGPase-encoding genes were isolated: one encodes the small subunit (AgpS1) and the other three genes encode the large subunit (AgpL1, L2, and L3) (Chen et al. 1998; Park and Chung 1998). Among these genes, AgpL1 and AgpS1 are predominantly expressed in fruit, and both genes show the highest expression during early developmental stages (Petreikov et al. 2006; Yin et al. 2010b). It has been reported that plant AGPase-encoding genes are regulated at the transcriptional level by phosphates, nitrates, and sugars (Müller-Röber et al. 1990; Scheible et al. 1997; Nielsen et al. 1998; Sokolov et al. 1998; Li et al. 2002). Additionally, our previous work showed that AgpS1 and AgpL1 expression was specifically enhanced by salt stress in early developing fruits in an ABA- and osmotic stress-independent manner (Yin et al. 2010b). Detailed expression analyses utilizing detached fruits revealed that the response of AgpL1 expression to salt stress is a sugar-mediated response. This observation was indirectly supported by the [13C] tracer analyses, in which the carbohydrate influx into the fruit was notably enhanced under salinity stress conditions in immature green fruits (Yin et al. 2010b). The observation that starch biosynthesis in fruit is dependent on the sugar supply is consistent with the results of N’tchobo et al. (1999). It has been reported that total soluble sugar contents and sucrose phosphate synthase (SPS) activity were increased (Carvajal et al. 2000) and the expression of the sucrose transporter gene LeSUT1 was enhanced (Yin et al. 2010b) by salinity stress in leaves. Considering the results of the tracer analyses, which showed that the allocation of photosynthetic [14C]/[13C] to fruits and roots was increased under saline conditions (Gao et al. 1998; Saito et al. 2009), those responses are most likely a result of a systemic response to salt stress that promotes assimilate accumulation in sink organs. Salt stress expands carbohydrate availability in the developing fruits and promotes AGPase gene expression and consequent starch biosynthesis, followed by starch breakdown during ripening, which results in a high sugar content in red-ripe fruit. Similar phenomena were observed in different germplasms, such an Solanum pennellii-derived introgression line possessing the Brix9-2-5 allele, in which enhanced starch accumulation in young fruit results in a higher content of total soluble solids in red-ripe fruit compared with normal tomato cultivars (Robinson et al. 1988; Baxter et al. 2005). These observations support the view that salt stress enhances sugar accumulation in red-ripe fruit through activation of starch biosynthesis in immature green fruit.

6 Conclusion

In this chapter, we introduced the concept of active alteration of fruit metabolism in response to salinity stress and discussed the proposed molecular mechanisms underlying the development of the “fruit tomato.” As described in Sect. 1.5 and elsewhere, increased sugar levels cannot be explained only by a “concentration effect” caused by the suppression of fruit enlargement. This insight indicates that some metabolic pathways, at least those related to sugar and starch biosynthesis, have functions that are independent from that of the suppression of fruit enlargement. Generally, moderate salt stress simultaneously causes a reduction in fruit yield with a concomitant improvement in fruit quality. However, utilizing current biotechnological techniques, we are interested in producing a high-Brix fruit without yield reduction; this could be accomplished, for example, by manipulating the expression of starch biosynthesis genes. Another possibility entails the use of a reverse genetic approach to obtain a mutant in which a metabolic pathway is modified by knockout of a protein that degrades a particular metabolite (for example, GABA-T) utilizing a targeted mutant screen, such as the TILLING (targeting-induced local lesions in genomes) technique. Additionally, starch accumulation under salt stress would be a useful marker for identifying candidates that can be used in breeding varieties with high-Brix fruit. We hope that the information described in this chapter will be useful for researchers studying salt stress responses and those who are interested in the development of novel, high-value-added fruit crop varieties.