BioFactors 23 (2005) 179–187 IOS Press 179 Genomics of berry fruits antioxidant components E. D’Amico and G. Perrotta∗ ENEA Centro Ricerche Trisaia, Rotondella (MT), Italy Abstract. Reactive oxygen and nitrogen metabolites, which are side products of cell metabolism, can produce a lot of damage in biological macromolecules and tissues, producing a number of chronic illnesses. On the other hand, antioxidant metabolites usually accumulated in fruits and vegetables can provide an effective protection by neutralizing these reactive molecules. Among comestible vegetables, berry fruits are considered one of the richest sources of antioxidant metabolites; hence, they represent a good model for molecular and biochemical investigations about the biosynthesis and the functional role of antioxidants in plants. This review illustrates how recent developments in the fields of genomics and bioinformatics can provide powerful tools to better understand the molecular mechanisms that trigger biosynthesis and accumulation of antioxidant metabolites in berries. Keywords: Berry, antioxidants, flavonoids, genomics 1. Introduction Fruits and vegetables contain many antioxidant compounds such as vitamins (ascorbic acid, αtocopherol, folic acid); carotenoids (β -carotene); phenolic compounds (anthocyanins, flavones, proanthocyanidins and flavonols) and phenolic acids (cumaric, gallic, ellagic and cinnamic acid) [45]. In addition to their specific functions in cell metabolism, these compounds play a fundamental role protecting from free radicals responsible for the oxidation of lipids/proteins and for damages to the genomic DNA. Several studies have clearly established that the phytochemical antioxidant content in fruits has a positive impact on human health providing protection against some chronic and degenerative diseases [26–28,36]. Most of the investigations on plant antioxidants were initially focused on vitamins and carotenoids. However, after it has became clear that part of the total anti-oxidant activity in fruits is given by phenolic compounds, studies have been more orientated on the molecular mechanisms which modulate the biosynthesis and accumulation of phenolic compounds [18]. Among comestible vegetables, berries of the family of Rosaceae (Rubus and Fragaria) and those belonging the family of Ericaceae (Vaccinium) represent foods particularly rich in antioxidant compounds when compared to other fruit species [10,55,58]. Present knowledge over the molecular mechanisms that manage the biosynthesis of antioxidants in plants is still scarce, however recent advances in genomics and postgenomics of model plant species like Arabidopsis and rice will soon be extended in other species, including berries, providing powerful tools to investigate the biochemical networks, which are behind antioxidants biosynthesis, on a global scale. ∗ Address for correspondence: Gaetano Perrotta, ENEA Centro Ricerche Trisaia, S.S. 106 Ionica – Km 419,500, 75026 Rotondella (MT), Italy. Tel.: +39 0835 974 746/ +39 0835 974 453 (lab.); Fax: +39 0835 974 749; E-mail: gaetano.perrotta@ trisaia.enea.it. 0951-6433/05/$17.00  2005 – IOS Press and the authors. All rights reserved 180 E. D’Amico and G. Perrotta / Genomics of berry fruits antioxidant components 2. Antioxidants in berries Phenols represent a big class of compounds with variable size and structures [46] and with a highly fluctuant inter/intra-specific accumulation pattern [8,49]. The most abundant group of phenols in berries is usually represented by hydrolysable tannins (galloand ellagitannins) [37]. Oxydative bonds between galloyl groups convert gallotannins in ellagitannins that then can be hydrolysed in ellagic acid. Several reports indicate that hydrolysable tannins have strong antioxidant and antitumoral properties [12,13,22]. A second group of very abundant phenols in berries are anthocyanins, which pertain to the large family of flavonoids and are responsible of the fruit pigmentation. Anthocyanin pigments are mainly pelargonidin (red-magenta), cyanidin (red) and delphinidin (blue); in berry fruits they are mixed in variable quantities in order to confer specific colour gradations. Consequently strawberry (Fragaria x ananassa) accumulate more pelargonidin, while in blueberry and in bilberry delphinidin and cyanidin are more abundant [32]. However the actual biosynthesis anthocyanin derived compounds and the control of pigmentation in berries is by far a more complex process [21]. Flavonoids are involved in different biological processes: promote pigmentation, provide protection against UV radiations, are involved in defence mechanisms against pathogens and play a role in fertility and pollen germination [17,39]. Their chemical structure is typically composed by two aromatic rings connected each other through an eterocycle. Their precursors derivate from carbohydrate and phenylpropanoids biosynthesis pathway (Fig. 1). Among flavonoids, proanthocyanidins (PAs), or condensed tannins, represent a class of big branched molecules derived from the polymerization of flavan-3-ols. Flavan-3-ols are lateral intermediates of the main flavonoids pathway (Fig. 1) resulted by reduction of anthocyanidins and leucoanthocyanidins by the action of anthocyanidin reductase (anr) and leucoanthocyanidin reductase (lar) [60]. The branch of the pathway leading to PAs biosynthesis is less known and awaits further investigations. PAs are known to prevent human diseases like atherosclerosis, ulcer, large bowel cancer, cataracts, diabetes [9] and urinary tract infections [7,52]. Like other plant metabolites, PAs accumulation pattern can considerably change either in different plant species [16] and within the same species according to genetic and environmental cues. In a preliminary experiment we have measured the content of anthocyanins and flavan-3-ols/PAs in a number of cultivars (cv) and selections of Fragaria x ananassa sampled either in Northern and Southern Italian areas, using colorimetric essays. The analyses showed considerable differences in the accumulation pattern of either anthocyanins and flavan-3-ols/PAs not only among different genotypes but also within the same genotypes grown in different geographical areas (data not shown). The amount of anthocyanins and PAs in strawberry fruits seems therefore clearly dependent either from environmental and genetic factors. These factors await to be further characterized through the development of integrated high throughput analyses including genomics and proteomics tools. Finally, some vitamin molecules are crucial antioxidants. In plants the biosynthesis of the ascorbic acid (vitamin C) usually involves L-galactono-1,4-lactone [62]. However in some berries an alternative pathway is active in fruits starting from D-galacturonic acid [2] (Fig. 2A). The biological function of vitamin C is related to its elevated reducing power which neutralize many physiologically relevant reactive groups. However, it should be noted that present knowledge of antioxidant content in berry fruits is far to be complete. In a recent study in fact as many as twenty additional phenolic acids have been identified in different berries [63]. E. D’Amico and G. Perrotta / Genomics of berry fruits antioxidant components Phenylpropanoids PAL Phenylalanine 4CL C4H Cinnamic acid 4-Coumaric acid Dihydroflavonols: FLS Dihydroquercetin S C R B CHS F3 5 H Kaempferol Strawberry Cranberry Raspberry Bilberry Dihydromyricetin Dihydrokaempferol S C R B Quercetin Flavonols: 3 Malonyl-CoA 4-Coumaryl-CoA Chalcones CHI Flavanones F 3H F3 H 181 S R Myricetin C B DFR Leucoanthocyanidins: Leucocyanidin LeucoLeucopelargonidin delphinidin ANS Anthocyanidins: Cyanidin Pelargonidin Delphinidin FGT Anthocyanins: Cyanidin-3glucosyde ANR Flavan-3-ols: Epicatechin S C R B Pelargonidin-3glucosyde S R LAR ANR Catechin Delphinidin-3glucosyde B LAR Epiafzelechin AN R Afzelechin Polymerization Epigallocatechin LAR Gallocatechin ? Proanthocyanidins Fig. 1. Schematic overview of the major flavonoid pathway in berries. Enzymes are reported as follows: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone hydroxylase; F3’H, flavonoid-3’-hydroxylase; F3’5’H, flavonoid-3’,5’-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol-4-reductase; ANS, anthocyanidin synthase; FGT, flavonoids-3-glycosyltransferase; ANR, anthocyanidin reductase; LAR, leucoanthocyanidin reductase. 3. Genes and genomics Known genes involved in the productions of antioxidant compounds encode for enzymes (monoxygenases, dioxygenases, reductases, hydroxylases and transferases) and regulatory factors which promote the synthesis of secondary metabolites such as phenylpropanoids, flavonoids, vitamins and carotenoids. The isolation and characterization of these enzymes represents a very important step to understand the molecular interactions which regulate the biosynthesis of antioxidant compounds in berries. Dihydroflavonol-4-reductase (dfr), for instance, is known to be a key enzyme for biosynthesis of plant pigments, antioxidative, antifungal and antibacterial flavonoid compounds. Its enzymatic properties have been characterized using crude enzyme preparations [48]. Unfortunately, very little information is available about genes involved in antioxidant biosynthesis in berries. Only recently, after the discovery of the positive impact on human health, the scientific interest toward the study of the genetic and biochemical mechanisms involved in antioxidant production is boosted. Due to its relevant economic and commercial impact, strawberry is by far the most studied berry from the genomic point of view [4,5,15,47]. While only a few genes involved in antioxidant biosynthesis have been characterized in raspberry, cranberry and bilberry [32]. 182 E. D’Amico and G. Perrotta / Genomics of berry fruits antioxidant components A B Fig. 2. (A) Ascorbic acid biosynthesis pathways. The white background represents the alternative pathway occurring in berry fruits. (B) Reaction catalyzed by the ascorbate peroxidase (APX) enzyme. For the biosynthesis of antioxidant metabolites are usually requested either structural genes, which encode enzymes that directly participate to their biosynthesis, and regulatory genes which control the expression rate of structural genes [20]. It has been reported that transcriptional controls play an essential role in regulating the overall activity of flavonoid biosynthesis in response to different developmental and environmental signals [29,50,61]. 3.1. Genes related to antioxidant biosynthesis isolated and characterized in berries L-Phe ammonia-lyase (pal), ubiquitous in higher plants, is the first enzyme of the phenylpropanoids pathway (Fig. 1). It is upstream of several reactions which determine many fruit quality traits (antioxidants, colour, flavour, etc.) [30]. In berries, PAL gene has been characterized in strawberry, raspberry [24, 25,44] and partly in bilberry [32]. In raspberry, pal is encoded by two genes, RiPAL1 and RiPAL2, E. D’Amico and G. Perrotta / Genomics of berry fruits antioxidant components 183 which share over 70% of sequence identity. In ripening fruits, expression of RiPAL1 and RiPAL2 genes fluctuates independently suggesting the action of different regulatory signals [44]. In strawberry fruit the activity of pal enzyme shows two peaks, the first in the immature green stage and second a little before the full ripe stage, but it is not yet clear whether this can be the result of different gene expression of distinct PAL coding sequences. The first activity peak could, in principle, be connected to the biosynthesis of some flavonoids, like condensed tannins, and phenols which are abundant in the first stages of fruit development, while the second peak, near to the full ripe stage is certainly correlated to the anthocyanins accumulation [14]. Interestingly, the same pattern of pal activity has been reported for DFR and FGT genes of strawberry at the RNA level [51,53]. The biosynthesis of flavonoids starts with condensation of malonyl-CoA and p-coumaryl-CoA by the action of chalcone synthase (chs) (Fig. 1). Chs belongs to the polyketide synthase (pks) enzymes which are dimeric proteins that act directly on coenzyme A (CoA) thioesters of various carboxylic acids to extend the carbon backbone [57]. CHS gene was the first structural gene of the flavonoids biosynthesis pathway to be characterized. First reported in parsley [41], it has been then studied in many other plant species such in petunia where it is encoded by twelve different coding sequences, though only four of them seem to be functional [40]. Concerning berries, three CHS coding sequences showing distinct expression patterns in fruits have been isolated in Rubus. Indeed, both a fruit-ripening-dependent and independent model of gene expression coexist within the Rubus CHS gene family. Phylogenetic analysis placed the three Rubus pks in a single cluster, suggesting a recent duplication event. Thus, their distinct expression pattern suggests that their regulation and function(s), have evolved independently of the structural genes themselves [43]. A second quite investigated gene is DFR. Dihydroflavonol 4-reductase catalyzes the stereo specific reduction of dihydroflavonols to leucoanthocyanidins using NADPH as a cofactor [42]. Leucoanthocyanidins are the immediate precursors for the synthesis of anthocyanins and flavan-3-ols (Fig. 1). There are several reports showing that DFR gene expression is spatially and developmentally correlated with the anthocyanin accumulation pattern in different plant tissues [11,31,34,56,59]. In strawberry, DFR gene has been isolated by a subtractive cDNA library. Transcription patterns by northern blot analyses in different ripening stages have evidenced that DFR is much more expressed in immature green stage and in the nearly red ripe stage as well. Thus, as already discussed for PAL and FGT, the two DFR transcription peaks could also be connected with PAs/phenols and anthocyanins biosynthesis, respectively [51]. In higher plants DFR is probably encoded by a small gene family [11]. To further confirm the functional role of DFR, tobacco transgenic plants over-expressing the DFR gene of cranberry show a more intense pigmentation in flowers as a consequence of enhanced anthocyanins biosynthesis induced by the transgene [54]. In berries flavonoids biosynthesis seems to follow different patterns. While quercetin- and cyanidinderived anthocyanins are present in all berries, myricetin- and delphinidin-derived anthocyanins as well as kaempferol- and pelargonidin-derived anthocyanins are present only in some but not all berry species. The fact that fls and dfr enzymes can use alternatively dihydrokaempferol or dihydroquercetin as a substrate could explain why only small amount of kaempferol is present in berries where quercetin and myricetin are the predominant flavonoids. While dihydrokaempferol, precursor of either dihydroquercetin and dihydromyricetin, is present in all species [32] (Fig. 1). From literature data emerges a great variability in the composition and concentration of antioxidants in berries. This is mainly the effect of different genetic backgrounds which determine the biosynthesis of specific enzymes and regulation factors. Besides, developmental stages and environmental factors may 184 E. D’Amico and G. Perrotta / Genomics of berry fruits antioxidant components play a very important role for the differential accumulation of these metabolites. In bilberry it has been reported, as already known in other non berry species, that the expression of genes involved in flavonoids biosynthesis is boosted a consequence to an increased exposition to light [33]. Thus the establishing of these regulation mechanisms may be the result of the coordinated action of many regulation factors, mostly unknown, that finely modulate the abundance of specific metabolites on the basis of the plant physiological requirements. In strawberry has been cloned a gene (Fa-MYB1) probably involved in such modulation mechanisms. Fa-MYB1 codes for a 187 amino acids polypeptide belonging to the big family of myb transcription factors [35]. Fa-MYB1 transcription in fruits starts at the turning stage and reaches its max level at the full red ripe stage. Transgenic tobacco plants over-expressing Fa-myb1 show a drastic decrease of the ANS, FGT transcripts abundance and a remarkable reduction of anthocyanins and flavonols content, as well. These evidences suggest that Fa-myb1 in vivo may negatively influence the transcription of some genes involved in flavonoids biosynthesis in order to balance the pigment accumulation in late ripening stages and/or to modulate the accumulation of different flavonoid endproducts [6]. Very little is known about the molecular and genetic mechanisms involved in the biosynthesis of other antioxidants in berries and most of what is known has been investigated in strawberry, given the high economic importance and the elevated antioxidants content of this crop. Agius et al. [2] have isolated and characterised the GalUR gene in strawberry which codes for a NADPH-dependent D-galacturonate reductase. This enzyme is implicated in an alternative pathway for the biosynthesis of ascorbic acid (vitamin C) which involves the D-galacturonic acid, one of the main component of pectins in the cell wall (Fig. 2A). GalUR seems to be a key gene for the vitamin C biosynthesis in berry fruits since it has been confirmed that its transcription rate is strictly correlated in vivo with the actual content of vitamin C in different ripening stages. Furthermore, the promoter region of GalUR is of relevant biotechnological interest because of its strong activity, comparable to that of CaMV35S, and for the presence of light responsive elements [1,23]. Over-expression of strawberry GalUR in Arabidopsis thaliana induces a two-three fold increase of the vitamin C, confirming the fundamental role of GalUR for producing plants with higher antioxidant power [2]. A second key gene, cloned in strawberry, is APX which codes for the ascorbate peroxidase. This enzyme uses ascorbic acid as a substrate to reduce the l’H 2 O2 (Fig. 2B) providing protection of chloroplasts and other cell components from oxidative damages [38]. 3.2. Development of genomics tools The complexity of the metabolic pathways leading to the biosynthesis of antioxidant compounds makes investigations on the related gene functions very complicated and of problematic interpretation. Anyhow recent advances in plant genomics will hopefully provide the necessary knowledge to interpret the molecular basis of antioxidants biosynthesis. In strawberry, large scale comparative transcription profiling analyses have been performed in different tissues and ripening stages with the aim to isolate gene sequences involved in peculiar functions [3,5,6]. Collections of Expressed Sequence Tags (ESTs) and bioinformatics analyses represent useful tools to implement genomics investigations for discovery of new genes. At present a database containing over 3,700 EST sequences and related features from strawberry ripening fruits is available at http://fragariaest.trisaia.enea.it. A second EST collection from whole-plant tissues is available at ftp://ftp.genome.clemson.edu/pub/fragaria [19]. Comparative transcription profiling has been used to monitor the transcription changes during fruit ripening and in achenes and receptacles in strawberry. It has been found that 441 transcripts out of E. D’Amico and G. Perrotta / Genomics of berry fruits antioxidant components 185 1701 were altered between achenes and receptacles. Interestingly, most of the transcripts upregulated in achenes represent genes putatively involved in genetic/biochemical regulations (transcription factors, phosphatases, kinases) and in accumulation of storage products. While in the receptacle, altered transcripts were mostly related to fruit pigmentation and to cell wall metabolism. Consistently to its putative function, ANS gene shows a drastic upregulation during receptacle development, when the anthocyanin biosynthesis is boosted; at the same time, DFR transcripts are maintained quite stable during receptacle development. This is in agreement with the fact that dihydroflavonol reductase action is also required at early fruit ripening stages for the biosynthesis of PAs [5]. 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