A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants.

BACKGROUND: Hydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits. RESULTS: The putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine. CONCLUSION: SrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism.

Calcium/calmodulin alleviates substrate inhibition in a strawberry UDP-glucosyltransferase involved in fruit anthocyanin biosynthesis.

BACKGROUND: UDP-glucosyltransferase (UGT) is a key enzyme for anthocyanin biosynthesis, which by catalyzing glycosylation of anthocyanidins increases their solubility and accumulation in plants. Previously we showed that pre-harvest spray of CaCl2 enhanced anthocyanin accumulation in strawberry fruit by stimulating the expression of anthocyanin structural genes including a fruit specific FvUGT1. RESULTS: To further understand the regulation of anthocyanin biosynthesis, we conducted kinetic analysis of recombinant FvUGT1 on glycosylation of pelargonidin, the major anthocyanidin in strawberry fruit. At the fixed pelargonidin concentration, FvUGT1 catalyzed the sugar transfer from UDP-glucose basically following Michaelis-Menten kinetics. By contrast, at the fixed UDP-glucose concentration, pelargonidin over 150 µM exhibited marked partial substrate inhibition in an uncompetitive mode. These results suggest that the sugar acceptor at high concentration inhibits FvUGT1 activity by binding to another site in addition to the catalytic site. Furthermore, calcium/calmodulin specifically bound FvUGT1 at a site partially overlapping with the interdomain linker, and significantly relieved the substrate inhibition. In the presence of 0.1 and 0.5 µM calmodulin, V max was increased by 71.4 and 327 %, respectively. CONCLUSIONS: FvUGT1 activity is inhibited by anthocyanidin, the sugar acceptor substrate, and calcium/calmodulin binding to FvUGT1 enhances anthocyanin accumulation via alleviation of this substrate inhibition.

Differential expression of calcium/calmodulin-regulated SlSRs in response to abiotic and biotic stresses in tomato fruit.

Calcium has been shown to enhance stress tolerance, maintain firmness and reduce decay in fruits. Previously we reported that seven tomato SlSRs encode calcium/calmodulin-regulated proteins, and that their expressions are developmentally regulated during fruit development and ripening, and are also responsive to ethylene. To study their expressions in response to stresses encountered during postharvest handling, tomato fruit at the mature-green stage was subjected to chilling and wounding injuries, infected with Botrytis cinerea and treated with salicylic acid or methyl jasmonate. Gene expression studies revealed that the seven SlSRs differentially respond to different stress signals. SlSR2 was the only gene upregulated by all the treatments. SlSR4 acted as a late pathogen-induced gene; it was upregulated by salicylic acid and methyl jasmonate, but downregulated by cold treatment. SlSR3L was cold- and wound-responsive and was also induced by salicylic acid. SlSR1 and SlSR1L were repressed by cold, wounding and pathogen infection, but were upregulated by salicylic acid and methyl jasmonate. Overall, results of these expression studies indicate that individual SlSRs have distinct roles in responses to the specific stress signals, and SlSRs may act as a coordinator(s) connecting calcium-mediated signaling with other stress signal transduction pathways during fruit ripening and storage.

Characterization of a calcium/calmodulin-regulated SR/CAMTA gene family during tomato fruit development and ripening.

BACKGROUND: Fruit ripening is a complicated development process affected by a variety of external and internal cues. It is well established that calcium treatment delays fruit ripening and senescence. However, the underlying molecular mechanisms remain unclear. RESULTS: Previous studies have shown that calcium/calmodulin-regulated SR/CAMTAs are important for modulation of disease resistance, cold sensitivity and wounding response in vegetative tissues. To study the possible roles of this gene family in fruit development and ripening, we cloned seven SR/CAMTAs, designated as SlSRs, from tomato, a model fruit-bearing crop. All seven genes encode polypeptides with a conserved DNA-binding domain and a calmodulin-binding site. Calmodulin specifically binds to the putative targeting site in a calcium-dependent manner. All SlSRs were highly yet differentially expressed during fruit development and ripening. Most notably, the expression of SlSR2 was scarcely detected at the mature green and breaker stages, two critical stages of fruit development and ripening; and SlSR3L and SlSR4 were expressed exclusively in fruit tissues. During the developmental span from 10 to 50 days post anthesis, the expression profiles of all seven SlSRs were dramatically altered in ripening mutant rin compared with wildtype fruit. By contrast, only minor alterations were noted for ripening mutant nor and Nr fruit. In addition, ethylene treatment of mature green wildtype fruit transiently stimulated expression of all SlSRs within one to two hours. CONCLUSIONS: This study indicates that SlSR expression is influenced by both the Rin-mediated developmental network and ethylene signaling. The results suggest that calcium signaling is involved in the regulation of fruit development and ripening through calcium/calmodulin/SlSR interactions.

Antioxidant glucosylated caffeoylquinic acid derivatives in the invasive tropical soda apple, Solanum viarum.

Eggplant and related Solanum species contain abundant caffeoylquinic acid (CQA) derivatives. Fruit of the invasive species Solanum viarum Dunal contain numerous complex CQA derivatives, but only a few have been identified. The structures of two new compounds isolated from methanolic extracts of S. viarum fruit by C(18)-HPLC-DAD were determined using 2D NMR and MS data. Both include two 5-CQA molecules joined by glucose via ester and glycosidic linkages. The structures of compounds 1 and 2 (viarumacids A and B) are, respectively, 5-caffeoyl- and 3-malonyl-5-caffeoyl-[4-(1ß-[6-(5-caffeoyl)quinate]glucopyranosyl)]quinic acid. The antioxidant activities determined by ABTS(•+) and DPPH(•) assays were in the order 1 > 2 > 5-CQA.

Penicillium solitum produces a polygalacturonase isozyme in decayed Anjou pear fruit capable of macerating host tissue in vitro.

A polygalacturonase (PG) isozyme was isolated from Penicillium solitum-decayed Anjou pear fruit and purified to homogeneity with a multistep process. Both gel filtration and cation exchange chromatography revealed a single PG activity peak, and analysis of the purified protein showed a single band with a molecular mass of 43 kDa, which is of fungal origin. The purified enzyme was active from pH 3.5-6, with an optimum at pH 4.5. PG activity was detectable 0-70 C with 50 C maximum. The purified isozyme was inhibited by the divalent cations Ca(2+), Mg(2+), Mn(2+) and Fe(2+) and analysis of enzymatic hydrolysis products revealed polygalacturonic acid monomers and oligomers. The purified enzyme has an isoelectric point of 5.3 and is not associated with a glycosylated protein. The PG isozyme macerated fruit tissue plugs in vitro and produced ~1.2-fold more soluble polyuronides from pear than from apple tissue, which further substantiates the role of PG in postharvest decay. Data from this study show for the first time that the purified PG produced in decayed Anjou pear by P. solitum, a weakly virulent fungus, is different from that PG produced by the same fungus in decayed apple.

Purification and biochemical characterization of polygalacturonase produced by penicillium expansum during postharvest decay of 'Anjou' pear.

A polygalacturonase (PG) was extracted and purified from decayed tissue of 'Anjou' pear fruit inoculated with Penicillium expansum. Ammonium sulfate precipitation, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. PG enzyme activity from healthy and wounded pear tissue was undetectable, which supports the claim that the purified PG is of fungal origin. The purified enzyme had a molecular mass of 41 kDa and a pI of 7.8. Activity of the PG was not associated with a glycosylated protein. The enzyme was active over a broad pH range from 3 to 6, with optimal activity at 4.5 in sodium citrate and sodium acetate buffers. The optimal temperature for activity was 37 degrees C but the enzyme was also active at 0, 5, 10, 20, and 50 degrees C. Thin-layer chromatographic analysis of PG hydrolysis products showed that the enzyme exhibits endo- and exo-activity. The purified enzyme macerated tissue in vitro causing approximately 30% reduction in mass of pear plugs compared with approximately 17% reduction for apple. Additionally, it produced 1.5-fold more soluble polyuronides on pear than apple tissue. This work shows for the first time the production of a PG by P. expansum during postharvest decay of pear fruit is different from the previously described PG produced in decayed apple fruit by the same pathogen.

Isolation, purification, and characterization of a polygalacturonase produced in Penicillium solitum-decayed 'Golden Delicious' apple fruit.

Polygalacturonase (PG) was extracted and purified from decayed 'Golden Delicious' apple fruit inoculated with Penicillium solitum. Ammonium sulfate, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. The purified PG most likely originates from the fungus because PG activity from healthy and wounded apple tissue was undetectable. Analysis of cation exchange-purified material using sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 50-kDa band. The enzyme was active over a broad pH range (3 to 7), with optimal activity between pH 4 and 5. PG was highly active at 20 and 37 degrees C but was also detectable at 2, 50, and 75 degrees C. Divalent cations affected PG enzyme activity; Mg and Fe increased, whereas Ca and Mn reduced activity in vitro. Thin-layer chromatographic separation of hydrolysis products and data from a PG plate activity assay based on staining with ruthenium red showed that the enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit.

Ripening-specific stigmasterol increase in tomato fruit is associated with increased sterol C-22 desaturase (CYP710A11) gene expression.

Phytosterol content and composition and sterol C-22 desaturase (LeSD1; CYP710A11) transcript levels in pericarp tissue of 'Rutgers' tomato fruit were compared in the wild-type (wt) and isogenic lines of the nonripening mutants nor and rin at four stages of ripening/aging. Wild-type fruit were harvested at the mature-green (MG), breaker (BK), breaker plus 3 days (B + 3), and breaker plus 6 days (B + 6) stages, whereas nor and rin fruits were harvested at comparable chronological ages (days after pollination). At the MG stage, wt and mutant fruits had closely similar sterol contents, compositions, and conjugations, with >91% of the total sterols in the acylated steryl glycoside plus steryl glycoside (ASG + SG) fraction. During ripening/aging, there were substantial increases in total sterols and the percentage of sterols in the free plus esterified (FS + SE) fraction. Both changes were greater in wt than in nor or rin. In fruit of wt, rin, and nor, respectively, the increases in total sterols between MG and B + 6 were 2.1-, 1.9-, and 1.5-fold, and at B + 6 the percentages of total sterols in FS + SE were 42, 21, and 24. Among all sterol lipids (ASG, SG, FS, and SE), the ratio of stigmasterol (stigmasta-5,22-dien-3beta-ol) to beta-sitosterol (stigmast-5-en-3beta-ol), the two major sterols in tomato, increased 2.3-fold during ripening of wt fruit but declined slightly during comparable aging of nor and rin fruits. In accord with these changes, the abundance of LeSD1 transcript increased 4-fold in pericarp of ripening wt fruit, peaking at B + 3, whereas transcript levels in nor and rin fruits fluctuated but never exceeded the abundance in wt fruit at the MG stage. These findings indicate that the ripening-specific increase in stigmasterol in wt fruit results from a marked increase in LeSD1 transcription and translation, which accelerates C-22 desaturation of the precursor sterol, beta-sitosterol.

Phytosterols and their derivatives: Structural diversity, distribution, metabolism, analysis, and health-promoting uses.

Phytosterols (plant sterols) occur in the cells of all plants. They are important structural components that stabilize the biological membranes of plants. Sterols can occur in the "free" unbound form or they can be covalently bound via an ester or glycosidic bond. Since our previous 2002 review on phytosterols and phytosterol conjugates, phytosterol glucosides have been found to be important structural components in the lipid rafts of the plasma membrane of plant cells, where they are thought to be essential to the function of plasma membrane enzymes and perhaps other proteins. Phytosterols also serve as precursors in the synthesis of important bioactive compounds such as steroidal saponins, steroidal glycoalkaloids, phytoecdysteroids, and brassinosteroids. Methods for the analysis of phytosterols range from traditional gas chromatography of free phytosterols to modern sophisticated forms of mass spectrometry which have been used for the new field of sterol lipidomics, sometimes called "sterolomics." Phytosterol-enriched functional foods first appeared about twenty years ago and many clinical studies have confirmed the low density lipoprotein (LDL) cholesterol-lowering properties of various types of phytosterols. In recent years additional clinical studies and more than ten important meta-analyses have provided insights to better understand the cholesterol-lowering and other biological effects of plant sterols.

Oxidation products of alpha-farnesene associated with superficial scald development in d'Anjou pear fruits are conjugated trienols.

Conjugated triene (CT) oxidation products of the acyclic sesquiterpene (E,E)-alpha-farnesene are thought to induce development of the physiological storage disorder superficial scald in apple and pear fruits of susceptible cultivars. CTs that accumulate in peel tissues of Granny Smith and Delicious apples after several weeks of cold storage are known to be conjugated trienols (CTols) rather than the corresponding hydroperoxides produced by in vitro autoxidation of alpha-farnesene. Here, it is shown that CTols are also the predominant in vivo oxidation products of alpha-farnesene in cold-stored pear fruit of the highly scald-susceptible d'Anjou cultivar. Analysis by octadecylsilane reversed phase high-performance liquid chromatography with diode array detection, gas chromatography with flame ionization detection, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance spectroscopy identified 3,7,11-trimethyldodeca-1,3(E),5(E),10-tetraen-7-ol as the major CT in hexane extracts of peel tissue from d'Anjou pears stored for 3-5 months in air at -1 degrees C. The possible origins of CTols in apples and pears and the hypothesized role of these oxidation products of alpha-farnesene in the induction of scald are discussed. .

Ethylene and alpha-farnesene metabolism in green and red skin of three apple cultivars in response to 1-methylcyclopropene (1-MCP) treatment.

Relationships among alpha-farnesene synthesis and oxidation, ethylene production and perception, antioxidative enzyme activities, and superficial scald development in fruit of three commercial apple cultivars were investigated at the biochemical and gene transcriptional levels. Scald-susceptible Cortland and Law Rome and scald-resistant Idared apples were untreated or treated with the ethylene action inhibitor 1-methylcyclopropene (1-MCP) and stored for up to 25 weeks at 0.5 degrees C. Separate blushed (red) and unblushed (green) peel tissue samples were taken at harvest and after 2, 4, 6, 10, 15, 20, and 25 weeks of storage. Large increases in peel tissue concentrations of alpha-farnesene and its conjugated trienol (CTol) oxidation products occurred in untreated Cortland and Law Rome and were about 4-9-fold greater than those in Idared. In both Cortland and Law Rome, accumulation of CTols in green peel was nearly twice that in red peel. 1-MCP treatment delayed and attenuated alpha-farnesene and CTol accumulation in each cultivar. Activities of peroxidase (POX) and catalase (CAT) were lower in red peel than in green peel, with the exception of CAT in Law Rome, whereas no effects of 1-MCP on enzyme activities were detected except for Cortland. In control fruit, internal ethylene concentrations (IECs) increased during the first 4-6 weeks to reach highest levels in Cortland, intermediate levels in Law Rome, and low levels in Idared. In 1-MCP-treated fruit, IECs increased gradually to modest levels by 25 weeks in Cortland and Law Rome but were almost nil in Idared. Expression patterns of the alpha-farnesene synthase gene MdAFS1, the ethylene receptor gene MdERS1, and the ethylene biosynthetic genes MdACS1 and MdACO1 were generally in accord with the patterns of alpha-farnesene and ethylene production. In particular, MdAFS1 and MdACS1 showed similar patterns of expression in each cultivar. Among the controls, transcript levels increased more rapidly in Cortland and Law Rome than in Idared during the first few weeks of storage. In 1-MCP-treated fruit, transcript abundance in Cortland and Law Rome rose to untreated control levels after 10-15 weeks but remained low in Idared. Scald symptoms were restricted to unblushed skin, and the incidence in controls after 25 weeks was nearly 100% in Cortland and Law Rome compared with 1% in Idared. 1-MCP treatment reduced scald incidence to 14, 3, and 0% in Cortland, Law Rome, and Idared, respectively. Overall, the results support the proposed role of CTols in scald induction and indicate that alpha-farnesene synthesis is tightly regulated by ethylene. However, gene transcription alone does not account for the big differences in ethylene and alpha-farnesene production in Cortland, Law Rome, and Idared apples.

Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses.

Phytosterols (plant sterols) are triterpenes that are important structural components of plant membranes, and free phytosterols serve to stabilize phospholipid bilayers in plant cell membranes just as cholesterol does in animal cell membranes. Most phytosterols contain 28 or 29 carbons and one or two carbon-carbon double bonds, typically one in the sterol nucleus and sometimes a second in the alkyl side chain. Phytostanols are a fully-saturated subgroup of phytosterols (contain no double bonds). Phytostanols occur in trace levels in many plant species and they occur in high levels in tissues of only in a few cereal species. Phytosterols can be converted to phytostanols by chemical hydrogenation. More than 200 different types of phytosterols have been reported in plant species. In addition to the free form, phytosterols occur as four types of "conjugates," in which the 3beta-OH group is esterified to a fatty acid or a hydroxycinnamic acid, or glycosylated with a hexose (usually glucose) or a 6-fatty-acyl hexose. The most popular methods for phytosterol analysis involve hydrolysis of the esters (and sometimes the glycosides) and capillary GLC of the total phytosterols, either in the free form or as TMS or acetylated derivatives. Several alternative methods have been reported for analysis of free phytosterols and intact phytosteryl conjugates. Phytosterols and phytostanols have received much attention in the last five years because of their cholesterol-lowering properties. Early phytosterol-enriched products contained free phytosterols and relatively large dosages were required to significantly lower serum cholesterol. In the last several years two spreads, one containing phytostanyl fatty-acid esters and the other phytosteryl fatty-acid esters, have been commercialized and were shown to significantly lower serum cholesterol at dosages of 1-3 g per day. The popularity of these products has caused the medical and biochemical community to focus much attention on phytosterols and consequently research activity on phytosterols has increased dramatically.

Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal.

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that have an important role in plant development and adaptation to environmental changes. In this study, we identified and characterized a spermidine hydroxycinnamoyl transferase (SHT) from eggplant (Solanum melongena) and its wild relative S. richardii, designated as SmSHT and SrSHT, respectively. SmSHT was abundant in flowers and fruits, whereas the level of SrSHT was remarkably low in all tissues. Heat-shock/drought treatment stimulated the expression of SmSHT in both leaves and fruits, indicating its involvement in plant stress response. Both SHT polypeptides had extremely high identity with just five amino-acid substitutions. Recombinant SmSHT catalyzed the synthesis of mono-, bi- and tri- acylated polyamines. Using caffeoyl-CoA as the acyl donor, SmSHT preferred spermidine as the acyl acceptor. When spermidine was the acyl acceptor, the donor preference order for SmSHT was caffeoyl-CoA>feruloyl-CoA>ρ-coumaroyl-CoA. SrSHT exhibited the same substrate specificity as SmSHT, yet exhibited significantly higher catalytic activity than SmSHT. For example, under caffeoyl-CoA and spermidine, Kcat of SrSHT was 37.3% higher than SmSHT. Molecular modeling suggests that five amino-acid substitutions in SrSHT result in four alterations in their predicted 3D structures. In particular, the conserved Lys402 adjacent to the DFGWG motif, and Cys200 in the crossover loop in SmSHT were replaced by Glu and Ser in SrSHT. These substitutions may contribute to the enhanced activity in SrSHT. Our study provides a platform to generate HCAA rich fruits for eggplant and other solanaceous crops.

Parallel reductions in phenolic constituents resulting from the domestication of eggplant.

Crop domestication is often accompanied by changes in metabolite compositions that alter traits such as flavor, color, or other beneficial properties. Fruits of eggplants (Solanum melongena L.) and related species are abundant and diverse in pharmacologically interesting phenolic compounds, particularly hydroxycinnamic acid (HCA) conjugates such as the antioxidant caffeoylquinic acids (CQA) and HCA-polyamine amides (HCAA). To understand metabolite variability through the lens of natural and artificial selection, HPLC-DAD was used to generate phenolic profiles for 32 compounds in fruits from 93 accessions representing 9 Solanum species. Profiles were used for identification of species-level and infraspecific chemical patterns across both genetic distance and landscape. Sampling of plant lines included the undomesticated progenitor of eggplant and Asian landraces with a genetic background associated with three Asian regions near proposed separate centers of domestication to test whether chemical changes were convergent despite different origins. Results showed ten compounds were unique to species, and ten other compounds varied significantly in abundance among species. Five CQAs and three HCA-polyamine conjugates were more abundant in wild (undomesticated) versus domesticated eggplant, indicating that artificial selection may have led to reduced phenolic levels. No chemical abundance patterns were associated with site-origin. However, one genetically distinct lineage of geographically-restricted SE Asian eggplants (S. melongena subsp. ovigerum) had a higher HCAA content and diversity than other lineages, which is suggested to be related to artificial selection for small, firm fruit. Overall, patterns show that fruit size, palatability and texture were preferentially selected over health-beneficial phytochemical content during domestication of several nightshade crops.

Cloning, characterization and functional expression of a phospholipase Dalpha cDNA from tomato fruit.

Phospholipase D (PLD; EC 3.1.4.4) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Phosphatidic acid (PA), the product of PLD hydrolysis of PL, increases in pericarp tissue during ripening of tomato (Lycopersicon esculentum Mill.) fruit, suggesting that increased PLD activity may be involved in loss of membrane function associated with ripening. However, a recent report showed a decline in soluble PLD activity in both normal and nonripening mutant fruit over the span that encompasses full ripening. To directly assess the role of PLD in tomato ripening, we have initiated a molecular genetic approach. Using a PLDalpha cDNA from castor bean as a probe, a PLDalpha cDNA (LEPLD2) was isolated from a tomato fruit library. It has an open reading frame of 2 421 nucleotides, predicted to encode a polypeptide of 807 amino acids, with a molecular mass of 91.9 kDa. These values are close to those of PLDalphas from 11 plant species and LEPLD2 has >/=73% nucleotide sequence identity with PLDalpha cDNAs from castor bean and tobacco, as well as another tomato cDNA. LEPLD2 transcript was detected in all tissues of the tomato plant by RNA gel-blot analysis. Levels were very low in roots, low in stems, moderate in leaves, high in flowers and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded phosphatidylcholine-hydrolyzing enzyme, and cells transformed with a pFLAG-MAC vector construct produced a FLAG-PLDalpha fusion protein that migrated close to the calculated 94 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Distribution of hydroxycinnamic acid conjugates in fruit of commercial eggplant (Solanum melongena L.) cultivars.

There is gathering evidence that antioxidant phytonutrients in fruits and vegetables have health-promoting effects. Eggplant fruit have a high content of antioxidant phenolic compounds. We evaluated the main class of eggplant phenolics, hydroxycinnamic acid conjugates, in the fruit of seven commercial cultivars. Fourteen conjugates were quantified and identified by high-performance liquid chromatography, ES(-)-MS, and (1)H NMR data. Significant differences in their content and composition were evident among cultivars and in tissue from stem, middle, and blossom end segments. Chlorogenic acid (5-O-caffeoylquinic acid) was the predominant compound, and its 3-O-, 4-O-, and 5-O-cis isomers were also present. The 10 other phenolics fell into four groups, including 3,5- and 4,5-dicaffeoylquinic acid isomers, four amide conjugates, two unknown caffeic acid conjugates, and 3-O-acetyl esters of 5-O- and 4-O-caffeoylquinic acid. Dicaffeoylquinic and 3-O-acetyl chlorogenic acids were most variable among the cultivars. Dicaffeoyquinic acids were most abundant in the blossom end, whereas 3-O-acetyl esters were highest in the midsection.

A new liquid chromatography-mass spectrometry-based strategy to integrate chemistry, morphology, and evolution of eggplant (Solanum) species.

This study presents a strategy based on repeatable reversed-phase LC-TOF-MS methods and statistical tools, including untargeted PCA and targeted PLS/OPLS-DA models, to analyze 31 accessions representing 24 species in the eggplant genus Solanum (Solanaceae), including eight species whose metabolic profiles were studied for the first time. Sixty-two Solanum metabolites were identified after detailed analysis of UV absorbance spectra, mass spectral fragmentation patterns, NMR spectra, and/or co-injection experiments with authentic standards. Among these were two new 5-O-caffeoylquinic acid derivatives that were identified by analyzing their MS/MS fragmentation. Based on these results, a Solanum metabolic database (SMD) and a detailed biosynthetic pathway of Solanum metabolites were created. Results of analyses identified seven marker metabolites that distinguish four Solanum sections, and revealed species-specific chemical patterns. Combining LC-MS data with multivariate statistical analysis was proven effective in studying the metabolic network within the large genus Solanum, allowing for integration of complicated chemistry, morphology, and evolutionary relationships.

Two new antioxidant malonated caffeoylquinic acid isomers in fruits of wild eggplant relatives.

Fruits of the cultivated eggplant species Solanum melongena and its wild relative Solanum incanum have a high content of hydroxycinnamic acid conjugates, which are implicated in the human health benefits of various fruits and vegetables. Monocaffeoylquinic acid esters, in particular 5-O-(E)-caffeoylquinic acid, are usually predominant in solanaceous fruits and tubers. Two closely related caffeoylquinic acid derivatives with longer C(18) HPLC retention times than those of monocaffeoylquinic acids are minor constituents in cultivated eggplant fruit. In a prior study, the two compounds were tentatively identified as 3-O-acetyl- and 4-O-acetyl-5-O-(E)-caffeoylquinic acids and composed ≤2% of the total hydroxycinnamic acid conjugates in fruit of most S. melongena accessions. It was recently found that the pair of these caffeoylquinic acid derivatives can compose 15-25% of the total hydroxycinnamic acid conjugates in fruits of S. incanum and wild S. melongena. This facilitated C(18) HPLC isolation and structural elucidation using (1)H and (13)C NMR techniques and HR-ToF-MS. The isomeric compounds were identified as 3-O-malonyl-5-O-(E)-caffeoylquinic acid (isomer 1) and 4-O-(E)-caffeoyl-5-O-malonylquinic acid (isomer 2). Both exhibited free radical scavenging activity, albeit about 4-fold lower than that of the flavonol quercetin dihydrate. By contrast, the iron chelation activities of isomers 1 and 2, respectively, were about 3- and 6-fold greater than that of quercetin dihydrate. Reports of malonylhydroxycinnamoylquinic acids are rare, and only a few of these compounds have been structurally elucidated using both NMR and MS techniques. To the authors' knowledge, these two malonylcaffeoylquinic acid isomers have not previously been reported.

Ripening, storage temperature, ethylene action, and oxidative stress alter apple peel phytosterol metabolism.

The chilling conditions of apple cold storage can provoke an economically significant necrotic peel disorder called superficial scald (scald) in susceptible cultivars. Disorder development can be reduced by inhibiting ethylene action or oxidative stress as well as intermittent warming. It was previously demonstrated that scald is preceded by a metabolomic shift that results in altered levels of various classes of triterpenoids, including metabolites with mass spectral features similar to ß-sitosterol. In this study, a key class of phytosterol metabolites was identified. Changes in peel tissue levels of conjugates of ß-sitosterol and campesterol, including acylated steryl glycosides (ASG), steryl glycosides (SG) and steryl esters (SE), as well as free sterols (FS), were determined during the period of scald development. Responses to pre-storage treatment with the ethylene action inhibitor, 1-methylcyclopropene, or an antioxidant (diphenylamine), rapid temperature elevation, and cold acclimation using intermittent warming treatments were evaluated. Diphenylamine, 1-MCP, and intermittent warming all reduced or prevented scald development. ASG levels increased and SE levels decreased in untreated control fruit during storage. Removing fruit from cold storage to ambient temperature induced rapid shifts in ASG and SE fatty acyl moieties from unsaturated to saturated. FS and SG levels remained relatively stable during storage but SG levels increased following a temperature increase after storage. ASG, SE, and SG levels did not increase during 6 months cold storage in fruit subjected to intermittent warming treatment. Overall, the results show that apple peel phytosteryl conjugate metabolism is influenced by storage duration, oxidative stress, ethylene action/ripening, and storage temperature.