番茄(Solanum lycopersicum)类胡萝卜素降解关键基因筛选

类胡萝卜素衍生挥发物对提升番茄风味至关重要。为筛选调控类胡萝卜素衍生挥发物合成的关键基因,以90个番茄自交系中香气寡淡的TI4001和香气浓郁的CI1005为材料,分析了番茄类胡萝卜素裂解双加氧酶(SlCCDs)基因在不同组织及不同发育期果实中的表达量,果实不同成熟期类胡萝卜素及其衍生挥发物的含量。发现在7个SlCCDs基因中,SlCCD1A和SlCCD1B基因在番茄果实中表达量最高,且随着果实发育成熟表达量显著升高。果实中类胡萝卜素及其衍生挥发物含量也显著升高。SlCCD1A和SlCCD1B基因表达量与类胡萝卜素及其衍生挥发物含量之间极显著正相关。推测SlCCD1A和SlCCD1B基因是裂解类胡萝卜素合成挥发物的关键基因。     

The carotenoid cleavage dioxygenase 1 enzyme has broad substrate specificity, cleaving multiple carotenoids at two different bond positions

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').     

NON-SMOKY GLYCOSYLTRANSFERASE1 Prevents the Release of Smoky Aroma from Tomato Fruit

Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed “smoky.” Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. Using a combinatorial omics approach, we identified the NON-SMOKY GLYCOSYLTRANSFERASE1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.     

Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon

Carotenoids are nutritionally important tetraterpenoid pigments that upon oxidative cleavage give rise to apocarotenoid (norisoprene) aroma volatiles. beta-Carotene is the predominant pigment in orange-fleshed melon (Cucumis melo L.) varieties, reaching levels of up to 50 microg/gFW. Pale green and white cultivars have much lower levels (0-10 microg/gFW). In parallel, beta-ionone, the 9,10 cleavage product of beta-carotene, is present (12-33ng/gFW) in orange-fleshed melon varieties that accumulate beta-carotene, and in much lower levels (0-5 ng/gFW) in pale green and white fleshed varieties. A search for a gene putatively responsible for the cleavage of beta-carotene into beta-ionone was carried out in annotated melon fruit EST databases yielding a sequence (CmCCD1) highly similar (84%) to other plant carotenoid cleavage dioxygenase genes. To test its function, the clone was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. We show here that the CmCCD1 gene product cleaves carotenoids at positions 9,10 and 9',10', generating geranylacetone from phytoene; pseudoionone from lycopene; beta-ionone from beta-carotene, as well as alpha-ionone and pseudoionone from delta-carotene. CmCCD1 gene expression is upregulated upon fruit development both in orange, pale-green and white melon varieties, despite the lack of apocarotenoid volatiles in the later. Thus, the accumulation of beta-ionone in melon fruit is probably limited by the availability of carotenoid substrate.     

Effect of suppression of ethylene biosynthesis on flavor products in tomato fruits

To elucidate the role of ethylene in the production of flavor compounds by tomato fruits, wild-type tomato (Lycopersicon esculentum L., cv. Lichun) and its transgenic antisense LeACS2 line with suppressed ethylene biosynthesis were used. The metabolism of individual sugars was ethylene-independent. However, citric acid and malic acid were under ethylene regulation. The content of these acids was higher in transgenic tomato fruits and returned to normal level after transgenic fruits were treated with ethylene. Because most of amino acids, which are important precursors of volatiles, were shown to be correlated with ethylene, we surmise that amino acid-related aroma volatiles were also affected by ethylene. Headspace analysis of volatiles showed a significant accumulation of aldehydes in wild-type tomato fruits during fruit ripening and showed a dramatic decrease in most aroma volatiles in transgenic tomato fruits as compared with wild-type fruits. The production of hexanal, hexanol, trans-2-heptenal, cis-3-hexanol, and carotenoid-related volatiles, except β-damascenone and β-ionone, was inhibited by suppression of ethylene biosynthesis. No remarkable differences were observed in the concentrations of cis-3-hexenal and trans-2-hexenal between transgenic and wild-type tomato fruits, indicating these two volatiles to be independent of ethylene. Thus, there are various regulation patterns of flavor profiles in tomato fruits by ethylene. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 92–101. The text was submitted by the authors in English. Both authors equally contributed to this work.     

Identification of a specific isoform of tomato lipoxygenase (TomloxC) involved in the generation of fatty acid-derived flavor compounds

There are at least five lipoxygenases (TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE) present in tomato (Lycopersicon esculentum Mill.) fruit, but their role in generation of fruit flavor volatiles has been unclear. To assess the physiological role of TomloxC in the generation of volatile C6 aldehyde and alcohol flavor compounds, we produced transgenic tomato plants with greatly reduced TomloxC using sense and antisense constructs under control of the cauliflower mosaic virus 35S promoter. The expression level of the TomloxC mRNA in some transgenic plants was selectively reduced by gene silencing or antisense inhibition to between 1% and 5% of the wild-type controls, but the expression levels of mRNAs for the four other isoforms were unaffected. The specific depletion of TomloxC in transgenic tomatoes led to a marked reduction in the levels of known flavor volatiles, including hexanal, hexenal, and hexenol, to as little as 1.5% of those of wild-type controls following maceration of ripening fruit. Addition of linoleic or linolenic acid to fruit homogenates significantly increased the levels of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than with the wild-type control. Confocal imaging of tobacco (Nicotiana tabacum) leaf cells expressing a TomloxC-GFP fusion confirmed a chloroplast localization of the protein. Together, these results suggest that TomloxC is a chloroplast-targeted lipoxygenase isoform that can use both linoleic and linolenic acids as substrates to generate volatile C6 flavor compounds. The roles of the other lipoxygenase isoforms are discussed.     

Effect of the Cnr mutation on carotenoid formation during tomato fruit ripening

The characteristic pigmentation of ripe tomato fruit is due to the deposition of carotenoid pigments. In tomato, numerous colour mutants exist. The Cnr tomato mutant has a colourless, non-ripening phenotype. In this work, carotenoid formation in the Cnr mutant has been studied at the biochemical level. The carotenoid composition of Ailsa Craig (AC) and Cnr leaves was qualitatively and quantitatively similar. However, Cnr fruits had low levels of total carotenoids and lacked detectable levels of phytoene and lycopene. The presence of normal tocopherols and ubiquinone-9 levels in the ripe Cnr fruits suggested that other biosynthetically related isoprenoids were unaffected by the alterations to carotenoid biosynthesis. In vitro assays confirmed the virtual absence of phytoene synthesis in the ripe Cnr fruit. Extracts from ripe fruit of the Cnr mutant also revealed a reduced ability to synthesise the carotenoid precursor geranylgeranyl diphosphate (GGPP). These results suggest that besides affecting the first committed step in carotenoid biosynthesis (phytoene synthase) the Cnr mutation also affects the formation of the isoprenoid precursor (GGPP).     

Characterization of a novel carotenoid cleavage dioxygenase from plants

The plant hormone abscisic acid is derived from the oxidative cleavage of a carotenoid precursor. Enzymes that catalyze this carotenoid cleavage reaction, nine-cis epoxy-carotenoid dioxygenases, have been identified in several plant species. Similar proteins, whose functions are not yet known, are present in diverse organisms. A putative cleavage enzyme from Arabidopsis thaliana contains several highly conserved motifs found in other carotenoid cleavage enzymes. However, the overall homology with known abscisic acid biosynthetic enzymes is low. To determine the biochemical function of this protein, it was expressed in Escherichia coli and used for in vitro assays. The recombinant protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions. In most instances, the enzyme cleaves the substrate symmetrically to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrate. Based upon sequence similarity, orthologs of this gene are present throughout the plant kingdom. A similar protein in beans catalyzes the same reaction in vitro. The characterization of these activities offers the potential to synthesize a variety of interesting, natural products and is the first step in determining the function of this gene family in plants.     

Role of Ethylene in the Biosynthetic Pathways of Aroma Volatiles in Ripening Fruit

During the past decade, fruit aroma biosynthetic pathways were established in some climacteric fruits, such as tomato, apple, and melon. Inhibition of ethylene biosynthesis or its action in these fruits can reduce the production of fruit volatiles. Furthermore, ethylene partially regulates expression of a few important enzyme genes in fruit volatile biosynthetic pathways. The aim of this review is to bring together recent advances for understanding the regulatory role of ethylene in the biosynthesis of aroma volatiles in some fruits.     

Identification of loci affecting flavour volatile emissions in tomato fruits

Fresh tomato fruit flavour is the sum of the interaction between sugars, acids, and a set of approximately 30 volatile compounds synthesized from a diverse set of precursors, including amino acids, lipids, and carotenoids. Some of these volatiles impart desirable qualities while others are negatively perceived. As a first step to identify the genes responsible for the synthesis of flavour-related chemicals, an attempt was made to identify loci that influence the chemical composition of ripe fruits. A genetically diverse but well-defined Solanum pennellii IL population was used. Because S. pennellii is a small green-fruited species, this population exhibits great biochemical diversity and is a rich source of genes affecting both fruit development and chemical composition. This population was used to identify multiple loci affecting the composition of chemicals related to flavour. Twenty-five loci were identified that are significantly altered in one or more of 23 different volatiles and four were altered in citric acid content. It was further shown that emissions of carotenoid-derived volatiles were directly correlated with the fruit carotenoid content. Linked molecular markers should be useful for breeding programmes aimed at improving fruit flavour. In the longer term, the genes responsible for controlling the levels of these chemicals will be important tools for understanding the complex interactions that ultimately integrate to provide the unique flavour of a tomato.     

A Role for Differential Glycoconjugation in the Emission of Phenylpropanoid Volatiles from Tomato Fruit Discovered Using a Metabolic Data Fusion Approach

A role for differential glycoconjugation in the emission of phenylpropanoid volatiles from ripening tomato fruit (Solanum lycopersicum) upon fruit tissue disruption has been discovered in this study. Application of a multiinstrumental analytical platform for metabolic profiling of fruits from a diverse collection of tomato cultivars revealed that emission of three discriminatory phenylpropanoid volatiles, namely methyl salicylate, guaiacol, and eugenol, took place upon disruption of fruit tissue through cleavage of the corresponding glycoconjugates, identified putatively as hexose-pentosides. However, in certain genotypes, phenylpropanoid volatile emission was arrested due to the corresponding hexose-pentoside precursors having been converted into glycoconjugate species of a higher complexity: dihexose-pentosides and malonyl-dihexose-pentosides. This glycoside conversion was established to occur in tomato fruit during the later phases of fruit ripening and has consequently led to the inability of red fruits of these genotypes to emit key phenylpropanoid volatiles upon fruit tissue disruption. This principle of volatile emission regulation can pave the way to new strategies for controlling tomato fruit flavor and taste.More than 7,000 metabolites, including volatiles, have already been identified in plant-based foods and beverages (Goff and Klee, 2006). Volatile organic compounds constitute a significant part of the plant metabolome, and the number of individual volatiles already described for various plants is approaching 2,000 (Dudareva et al., 2006). Significant progress has been made on the functional characterization of these plant volatiles over the past decades. For example, volatiles have been shown to play an important role in the interaction between plants and their environment. They are involved in the defense of plants against pathogens, where they serve as airborne signaling molecules to induce a defense response in other plant parts or neighboring plants (Shulaev et al., 1997). They also act as direct repellents of herbivorous pests or as attractants of the predators of these pests as part of the “cry for help” response (Dudareva et al., 2004; Kappers et al., 2005; Baldwin et al., 2006). In addition, flower volatiles are important for the attraction of pollinators (Dudareva et al., 2004), while fruit volatiles may have a role in attracting seed dispersers (Goff and Klee, 2006; Schwab et al., 2008). Besides their physiological and ecological functions, plant volatiles are also important determinants of consumer quality traits in flowers, fruits, and vegetables as well as the processed products derived from them.Tomato (Solanum lycopersicum) is one of the most important vegetable crops worldwide, and its fresh fruits and processed products are consumed and appreciated in every society. Volatiles are considered as major determinants of tomato fruit flavor (Buttery et al., 1987; Buttery and Ling, 1993; Baldwin et al., 1998, 2000; Tandon et al., 2000; Krumbein et al., 2004; Ruiz et al., 2005; Tieman et al., 2006; Kovács et al., 2009; Zanor et al., 2009). Several hundred tomato fruit volatile compounds have been described in the literature (Petro-Turza, 1987), but only a small part of this diversity is believed to have an impact on tomato fruit organoleptic properties (Buttery and Ling, 1993; Baldwin et al., 2000). We have previously screened red-ripe fruits for variation in their volatile metabolome using a collection of 94 tomato cultivars representing the current diversity within commercial germplasm (Tikunov et al., 2005). In that study, three phenylpropanoid (PhP) volatiles, methyl salicylate (MeSA), guaiacol, and eugenol, were found to be discriminatory within this germplasm collection and roughly divided the cultivars into two groups. Fruits from one group had the capacity to emit significant amounts of these three PhP volatiles upon fruit tissue disruption (blending), while fruits from the other group emitted none or hardly any.The considered relevance of these findings relates to their potential importance in consumer perception of fruit taste differences. It has been proposed previously that PhP volatiles likely have an impact on tomato fruit aroma. MeSA, the methyl ester of salicylic acid, is a potent odor component of wintergreen (Gaultheria procumbens). MeSA content has been shown to be negatively correlated with typical tomato flavor (Krumbein and Auerswald, 1998). Guaiacol is also a well-known flavoring compound and has been associated with a so-called “pharmaceutical” aroma in tomato fruits (Causse et al., 2002). Likewise, eugenol is a well-known odorant that gives the distinctive, pungent flavor to cloves (Syzygium aromaticum) and significantly contributes to the aroma of cinnamon (Cinnamomum verum). Although a potential physiological role for these PhP volatiles in tomato fruits remains unclear, they have been implicated to have a signaling and/or defense function (Shulaev et al., 1997; Koeduka et al., 2006; Sasso et al., 2007). Therefore, there is clear potential for the release of these compounds to influence, either negatively or positively, tomato flavor.In plants, PhP volatiles are primarily derived from Phe (Dudareva and Pichersky, 2000). Cinnamic acid, directly derived from Phe by a deamination catalyzed by Phe ammonia lyase, can either be β-oxidatively or nonoxidatively converted into benzoic acid. This can be further hydroxylated into salicylic acid by benzoic acid 2-hydroxylase. Recently, genetic studies in Arabidopsis (Arabidopsis thaliana) revealed the existence of an alternative pathway for the production of salicylic acid from isochorismate, thus bypassing Phe and its derivatives (Wildermuth et al., 2001). Salicylic acid is the immediate precursor of the volatile MeSA, through the action of salicylic acid methyl transferase (Boatright et al., 2004). Cinnamic acid can also be converted to other phenolic acids: p-coumaric acid, caffeic acid, and ferulic acid. Ferulic acid can be converted into coniferyl alcohol and further to eugenol (Gang, 2005). The biochemical origin of guaiacol in plants is not completely known. However, its chemical structure clearly points to the same PhP origin, as has already been demonstrated for bacteria (Chang and Kang, 2004).Glycosylation is a common means to conjugate plant secondary metabolites, in order to facilitate their transport and storage and to reduce their reactivity by blocking reactive hydroxyl groups. In tomato fruit, many volatile compounds, including PhP volatiles, are bound as glycosides, thus representing an aroma reserve (Buttery et al., 1990; Marlatt et al., 1992; Ortiz-Serrano and Gil, 2007). Such glycosidically bound volatiles can be liberated when cell compartmentation is destroyed, as happens on consumption of fresh fruits or industrial processing or as may happen during late ripening stages. As a consequence of this disruption, the contents of different cell compartments can mix and stored volatile glycosides become exposed to endogenous or exogenous cleavage enzymes, such as glycosyl hydrolases (glycosidases), which leads to glycoside cleavage and volatile emission. Thus, understanding the biochemical processes leading to the formation and/or cleavage of volatile glycoconjugates may provide tools to exploit more efficiently the aroma reserve present in tomato fruit in order to improve tomato fruit flavor.In order to gain greater insight into the volatile compound variation in tomato fruit, we previously analyzed the volatile metabolites using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) in a broad screening of 94 contrasting tomato genotypes representing the variation present in the germplasm of commercial tomato varieties (Tikunov et al., 2005; Van Berloo et al., 2008). This nontargeted metabolomics approach enabled the detection and putative identification of 322 volatiles. Subsequent multivariate analysis revealed (1) differences between tomato types (cherry versus round tomatoes) driven by the accumulation of phenolic-derived volatiles such as phenylethanol and phenylacetaldehyde, and (2) that the PhP-derived volatiles MeSA, guaiacol, and eugenol roughly split the set of genotypes into two distinct groups, independent of tomato fruit (pheno)type, where fruits of one of these groups emitted considerable amounts of these three PhP volatiles but fruits of the other emitted little or none.In this paper, we describe the investigation into the biochemical basis underlying this difference in capacity to emit PhP volatiles. A multiinstrumental metabolomics platform was used to profile fruits of the same broad tomato germplasm collection for both volatile and nonvolatile metabolites. Metabolic data fusion of both liquid chromatography (LC)-MS and GC-MS data sets, followed by multivariate analyses, suggested a principle for the regulation of PhP volatile emission in tomato fruit through differential volatile-sugar conjugation patterns. Subsequent series of quantitative biochemical experiments proved an important role of this process in regulating the emission of PhP volatiles from tomato fruit.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.