Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls.

BACKGROUND AND AIMS: The softening of ripening fruit involves partial depolymerization of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated 'fingerprint' product (tetrasaccharide) of RGL action. KEY RESULTS: In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide ('ΔUA-Rha-GalA-Rha'), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose, etc.) by electrophoresis at pH 2, then separated from ΔUA-GalA (the fingerprint of pectate lyase action) by thin-layer chromatography. The 'ΔUA-Rha-GalA-Rha' was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The 'fingerprint' : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1 : 72 (mol/mol), indicating that ~1.4 % of the backbone Rha→GalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action. CONCLUSIONS: The results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Prevention of Enzymatic Browning by Natural Extracts and Genome-Editing: A Review on Recent Progress.

Fresh fruits and vegetable products are easily perishable during postharvest handling due to enzymatic browning reactions. This phenomenon has contributed to a significant loss of food quality and appearance. Thus, a safe and effective alternative method from natural sources is needed to tackle enzymatic browning prevention. The capabilities of natural anti-browning agents derived from plant- and animal-based resources in inhibiting enzymatic activity have been demonstrated in the literature. Some also possess strong antioxidants properties. This review aims to summarize a recent investigation regarding the use of natural anti-browning extracts from different sources for controlling the browning. The potential applications of genome-editing in preventing browning activity and improving postharvest quality is also discussed. Moreover, the patents on the anti-browning extract from natural sources is also presented in this review. The information reviewed here could provide new insights, contributing to the development of natural anti-browning extracts and genome-editing techniques for the prevention of food browning.

Milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits on individual caseins.

In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from ß-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.

Chitosan Augments Tetramycin against Soft Rot in Kiwifruit and Enhances Its Improvement for Kiwifruit Growth, Quality and Aroma.

In this study, the co-application of chitosan and tetramycin against kiwifruit soft rot and its effects on the disease resistance, growth, quality and aroma of kiwifruit were investigated. The results show that chitosan could effectively enhance tetramycin against soft rot of kiwifruit with the field control efficacy of 85.33% for spraying chitosan 100 time + 0.3% tetramycin AS 5000-time dilution liquid, which was higher than 80.99% for 0.3% tetramycin AS 5000-time dilution liquid and significantly (p < 0.01) higher than 40.66% for chitosan 100-time dilution liquid. Chitosan could significantly (p < 0.05) improve the promoting effects of tetramycin on total phenolics, total flavonoids, SOD activity of kiwifruit compared to tetramycin during storage for 0-28 days and enhance the disease resistance of kiwifruit. Moreover, the co-application of chitosan and tetramycin was more effective than tetramycin or chitosan alone in enhancing fruit growth, improving fruit quality and increasing fruit aroma. This study highlights that chitosan can be used as an adjuvant to enhance tetramycin against soft rot of kiwifruit and promote tetramycin's improvement for the single fruit volume and weight, vitamin C, soluble sugar, soluble solid, dry matter, soluble protein, titratable acidity and aroma of kiwifruit.

Effect of gum Arabic, xanthan and carrageenan coatings containing antimicrobial agent on postharvest quality of strawberry: Assessing the physicochemical, enzyme activity and bioactive properties.

Effect of edible coatings of gum Arabic, carrageenan and xanthan gum containing lemon grass essential oil 1% w/v on postharvest quality of strawberry was studied under refrigeration for a period of 12 days. Results showed all the three coatings maintained fruit quality parameters during storage compared to control. Among all the coatings, carrageenan coated fruits showed delayed weight loss (10.1 to 8%), decay percentage (78.42 to 14.29%), retained ascorbic acid (0.15 to 0.27 g kg-1), antioxidant activity (18.17 to 25.85%), firmness (9.07 to 12.43 N), L* (32.38 to 40.42), a* (16.08 to 17.22) and b* (27.36 to 33.54). Carrageenan gum also showed lowest cellulase activity (0.03 units h-1 mg protein-1), pectin methylesterase activity (1.13 A620 min-1 mg protein-1) and ß-galactosidase activity (0.51 µmol min-1 mg protein-1), while showed maximum reduction in polygalacturonase activity (0.07 units h-1 mg protein-1) at the end of storage. Carrageenan gum was found effective in retention of anthocyanins and phenolic compounds during storage. Coatings loaded with antimicrobial agent inhibited psychrophilic bacteria, yeast and mold growth. It is concluded that carrageenan gum could better retain strawberry quality up to 12 days under refrigeration.

Purification and characterization of bromelain from pineapple (Ananas comosus L.) peel waste.

Bromelain is widely used in food industry and pharmaceutical products due to its strong antioxidant properties. Therefore, the extraction of bromelain from pineapple peel may improve the profitability and sustainability of pineapple industry. The aim of this work is to show the purification, stability, and kinetics of bromelain from pineapple peel. By studying the stability of purified bromelain (PB), we found that the activity of PB was inhibited by Fe3+ , Al3+ , methanol, ethanol, and n-butyl alcohol, while it was increased in the presence of Ca2+ , ethylenediamine tetra acetic acid, glucose, D-xylose, maltose, potassium sodium tartrate, sodium citrate, citric acid, and sodium nitrite. These stability tests will expand the application and space acquisition of bromelain. The kinetics study indicated that the thermal inactivation of PB was conforming to the first-order reaction and the half-life (t1/2 ) of PB under different temperature conditions (45, 55, 65, and 75 °C) was 81.54, 31.12, 10.28, and 5.23 min, respectively. Therefore, the inactivation time of PB can be predicted at different temperatures for food heating processing. PRACTICAL APPLICATION: The potential of utilizing pineapple peel for bromelain extraction might improve the profitability and sustainability of the pineapple industry.

Defining the role of a caffeic acid 3-O-methyltransferase from Azadirachta indica fruits in the biosynthesis of ferulic acid through heterologous over-expression in Ocimum species and Withania somnifera.

MAIN CONCLUSION: The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem (NCOMT) fruits has been isolated and heterologously expressed in E. coli. The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta. Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.

Physiological and metabolic analysis of winter jujube after postharvest treatment with calcium chloride and a composite film.

BACKGROUND: Ziziphus jujuba Miller cv. Dongzao is extremely susceptible to reddening, browning, nutritional loss, and perishability after harvest. In this study, we evaluated the mechanisms of calcium chloride and chitosan/nano-silica composite film treatments on the quality, especially in reddening, by physiological and metabolomic assays. RESULTS: The treatment delayed the decline of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and chalcone isomerase (CHI) activities. Meanwhile, the treated groups retarded the increases in anthocyanin and quercetin contents by inhibiting the gene expressions of flavonol synthase (ZjFLS), dihydroflavonol 4-reductase (ZjDFR), and anthocyanidin synthase (ZjANS), while promoting leucoanthocyanidin reductase (ZjLAR) expression, which leads to retardation of fruit reddening. Anthocyanins were found to be responsible for post-harvest winter jujube reddening through principal component analysis. Results from the technique for order preference by similarity to an ideal solution indicated that the treated group delayed the decline of the quality of 'Dongzao' and extended its shelf life. CONCLUSION: The treatment induced the heightening of flavonoids metabolism. They enhanced the nutritional value and the ability to resist stress by delaying the decline of PAL, CHS, and CHI activities. Meanwhile, the treated groups retarded the increase in anthocyanin and quercetin contents by inhibiting the gene expressions of ZjFLS, ZjDFR, and ZjANS and promoting ZjLAR expression, which leads to retardation of fruit reddening. Anthocyanins are responsible for post-harvest winter jujube reddening. Coating treatment effectively delayed the decline of winter jujube quality. © 2020 Society of Chemical Industry.

Influences of postharvest ATP treatment on storage quality and enzyme activity in sucrose metabolism of Malus domestica.

The respiratory metabolism of apples remains vigorous after harvest, which can accelerate the consumption of sugar, organic acid, and other substances, thus leading to a decline in quality. The influence of postharvest ATP treatment on the changes of quality parameters and sucrose metabolism-related enzyme activity in apples was investigated in this study. The results showed that applying ATP effectively repressed the respiratory rate and weight loss and maintained higher levels of soluble solids content and flesh firmness in apples. In addition, ATP treatment enhanced succinate dehydrogenase, cytochrome oxidase, sucrose phosphate synthase, and sucrose synthase synthesis activities and reduced neutral invertase, acid invertase, and sucrose synthase cleavage activities in apples. These findings suggest that applying ATP after harvest could improve the internal quality of apples by suppressing the respiratory rate and modulating sucrose metabolism.

The applicability of using a protease extracted from cashew fruits (Anacardium occidentale), as possible meat tenderizer: An experimental design approach.

Meat tenderness is one of the most important organoleptic properties in determining consumer acceptance in meat product marketability. Therefore, an effective meat tenderization method is sought after by exploring plant-derived proteolytic enzymes as meat tenderizer. In this study, a novel protease from Cashew was identified as a new alternative halal meat tenderizer. The extraction of cashew protease was optimized using response surface methodology (R2 = 0.9803) by varying pH, CaCl2 concentration, mixing time, and mass. pH 6.34, 7.92 mM CaCl2 concentration, 5.51 min mixing time, and 19.24 g sample mass were the optimal extraction conditions. There was no significant difference (n = 3; p < 0.05) between the calculated (6.302 units/ml) and experimental (6.493 ± 0.229 units/ml) protease activity. The ascending order of the effects was pH < mixing time < CaCl2 < sample mass. In meat tenderizing application, the meat samples treated with 9% (v/w) crude protease extract obtained the lowest shear force (1.38 ± 0.25 N) to cause deformation on the meat. An electrophoretic analysis showed that protein bands above ~49.8 kDa were completely degraded into protein bands below ~22.4 kDa. Scanning electron microscopy shows the disruption of the muscle fibers after being treated by the Cashew protease. The results of this study show the Cashew (Anacardium occidentale) crude extract can be used as an alternative of the animal and microbial protease as meat tenderizer and subsequently overcome the shortcoming of the halal industrial protease.

[Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida].

In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.

Determination of Quercetin and Flavonol Synthase in Boesenbergia rotunda Rhizome.

BACKGROUND AND OBJECTIVE: Flavonols in plants are catalyzed by flavonol synthase (FLS) enzyme. FLS was reported expressed in flowers and fruits, i.e., Dianthus caryophyllus L. (Caryophyllaceae), Petunia hybrida Hort. (Solanaceae), Arabidopsis thaliana L. (Brassicaceae), Citrus unshiu Marc. (Rutaceae). However, none reported about FLS in medicinal plants, particularly those which possess anti-inflammatory activity. This study was aimed to extract and identify FLS in the rhizome of Boesenbergia rotunda (Zingiberaceae) and to determine quercetin in the ethanol extract of the rhizome. MATERIALS AND METHODS: The protein extraction of the rhizome was carried out by employing Laing and Christeller's (2004) and Wang's (2014) methods. The extracted-proteins were separated by using SDS-PAGE, followed by the measurement of FLS intensity by using Gel Analyzer. The FLS-1 of recombinant A. thaliana was employed as the standard. The determination of quercetin in the rhizome was carried out using LC-MS. RESULTS: The FLS occurred as a thick band at 38 kDa with intensity 116-158. The LC chromatogram of the extract indicated a small peak at 7.94 min similar to that of quercetin standard. The MS spectra at 7.94 min indicated that quercetin is present in the B. rotunda rhizome (m/z = 303.0549). The concentration of quercetin in the extract is 0.022% w/v. CONCLUSION: The FLS, an enzyme which plays an important role in producing quercetin, was detected in B. rotunda rhizome planted in Indonesia. As a consequence, quercetin in a small amount, was also quantified in the rhizome of this plant. This report will add a scientific insight of B. rotunda for biological sciences.

Pectate lyases: Their role in plants and importance in fruit ripening.

Plant cell walls are complex structures that are modified throughout development. They are a major contributor to the properties of plant structure and act as barriers against pathogens. The primary cell walls of plants are composed of polysaccharides and proteins. The polysaccharide fraction is divided into components cellulose, hemicelluloses and pectin, are all modified during fruit ripening. Pectin plays an important role in intercellular adhesion and controlling the porosity of the wall. A large number of pectin degrading enzymes have been characterised from plants and they are involved in numerous aspects of plant development. The role of pectate lyases in plant development has received little attention, probably because they are normally associated with the action of plant pathogenic organisms. However their importance in plant development and ripening is now becoming well established and new information about the role of pectate lyases in plant development forms the focus of this review.

Effects of Bacillus Subtilis CF-3 VOCs Combined with Heat Treatment on the Control of Monilinia fructicola in Peaches and Colletotrichum gloeosporioides in Litchi Fruit.

In order to study the effect of volatile organic compounds (VOCs) produced by Bacillus subtilis CF-3 combined with heat treatment on Monilinia fructicola in peach and Colletotrichum gloeosporioides in litchi fruit, fruits were treated with B. subtilis CF-3 VOCs and hot air alone or in combination. The quality indexes of peach and litchi fruit after treatment and the changes in defense-related enzymes were measured. The results showed that the B. subtilis CF-3 VOCs combined with heat treatment could significantly reduce the rot index of peach and litchi fruit, and effectively maintain firmness and soluble solids content, as well as reduce weight loss of fruits. The combined treatment effectively enhanced the activity of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD) than either treatment alone, and enhanced the resistance of fruit to pathogenic fungi by activating disease-resistant enzymes (phenylalanine ammonia-lyase [PAL], chitinase [CHI], ß-1, 3-glucanase [GLU]) activity. In this study, B. subtilis CF-3 VOCs combined with heat treatment maintained the quality and delayed the decline of peach and litchi fruit, providing a theoretical basis for future applications. PRACTICAL APPLICATION: The combination of B. subtilis CF-3 VOCs and heat treatment reduce the extent of M. fructicola and C. gloeosporioides. The combination maintain the quality of peach and litchi better. The combination obviously improve the activity of defense-related enzyme in fruit.

Fractionation and activity profiling of fruit bromelain from pineapples of Phuket variety growing in Thailand.

Bromelain is a type of protease found in both fruits and stems of pineapples. Stem bromelain has been extensively studied and is commercially available for applications in various industries. In contrast, studies of fruit bromelain are quite limited since most of pineapples have been consumed freshly, canned or juiced. Nowadays, the consumption of canned fruits, including canned pineapples has decreased greatly. Fruit bromelain could be a new growth point for pineapple industry. In this study, fruit bromelain was extracted from the pineapple juice of Phuket variety and some of its properties were studied. The enzyme was purified by precipitation using ammonium sulfate fractionation followed by ion-exchange and gel filtration chromatography. Consequently, the protease purification level was increased by 95.2 fold. The final specific activity was getting to 448,590 U/mg on average, dominated by cysteine proteases, with optimal activity at 45°C and pH ranging from 6 to 8. The study facilitates the molecular and application research of fruit bromelain. PRACTICAL APPLICATIONS: The research has been carried out at Funong Food Technology Co., Ltd., Guangdong, China, which produces primarily pineapple chunks and juice. As plenty of by-products, like peels and cores of pineapples, are produced, the techniques are employed to extract bromelain from the by-products. The techniques reported in this work are not new or advanced, however, they are applicable during the manufacturing process and the related equipment is easy to set up and operate. What's more, the practical application of the techniques is cost-effective for the manufactories. Take Funong Food Technology Co., Ltd. as an example, they was using 80% saturation ammonium sulfate to precipitate protein from pineapple juice and obtained a bromelain with activity of approximately 8,000 U/mg and yield of 1.7 kg per ton of juice. With the application of the techniques reported in this work, bromelain was first extracted by ammonium sulfate gradient precipitation, and then purified through ion-exchange and gel filtration chromatography. Each step of precipitation and purification generates a different level of activity and recovery of bromelain, ranging from around 2,506 to 448,590 U/mg, which allows for the production of bromelain according to the requirement of the market and brings more profits.

Juglans regia and Ribes nigrum as potential nutraceuticals: Source of thermostable superoxide dismutase enzyme.

In the present study, superoxide dismutase (SOD) extracted from dry fruits; Juglans regia (Walnut; W) and Ribes nigrum (Munakka; M) was partially purified into 0%-40% and 40%-80% fractions based on ammonium sulfate saturation levels. The partially purified fractions (0%-40%) exhibited purification level of 3.09- (W) and 3.22- (M) fold with specific activity 79.32 Umg-1 (W) and 125.23 Umg-1 (M). SOD from both the sources was found to be thermally stable, that is, 80°C (W) and 70°C (M). Kinetic studies showed Km values to be 3.33 mM (W) and 2.86 mM (M), whereas the activation energy (Ea ) calculated as 24.52 KJ mol-1 (W) and 26.25 KJ mol-1 (M). Na+ , Mn2+ , and Ba2+ ions acted as potential inhibitors, whereas Fe2+ stimulated SOD from both the sources. Among these metal ions, Na+ exhibited uncompetitive inhibition in both cases; with Ki values of 0.7 mM (W) and 0.9 mM (M), suggesting the more prominent binding affinity and effectiveness. PRACTICAL APPLICATIONS: Awareness need to be created among people for multifactorial health benefits of nutraceuticals in day-to-day life. Nutritional consumption from fruits, nuts, and vegetables safeguard against various maladies like cardiovascular diseases, diabetes, and cancers. Superoxide dismutase (EC 1.15.1.1) is a standout among the most critical metal-containing enzymes that act as a main line of defense against oxidative stress. Antioxidant-based drugs and formulations have been developed in the recent years and research is emphasized on its impact on oxidative stress levels. In this study, Juglans regia (W) and Ribes nigrum (M) were found to have thermostable SOD enzyme with excellent antioxidant properties. Thermal stability of an enzyme improves its significance making it industry friendly with therapeutically vital products, alongside their utilization as supplement in numerous therapeutic formulations.

A Neighboring Aromatic-Aromatic Amino Acid Combination Governs Activity Divergence between Tomato Phytoene Synthases.

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue-specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Effect of saline irrigation on physiological traits, fatty acid composition and desaturase genes expression in olive fruit mesocarp.

The effect of salinity on physiological traits, fatty acid composition and desaturase genes expression in fruit mesocarp of olive cultivar Leccino was investigated. Significant reduction of shoot elongation (-12%) during salt treatments (80 mM NaCl) was associated with the translocation of Na in the aerial part. After 75 days of treatment, fruits from each plant were subdivided into four maturation groups (MG0, MG1, MG2, MG3) according to ripening degrees. Na accumulation increased in each MG under salinity, reaching the highest values in MG1 fruits (2654 mg kg-1 DW). Salinity caused an acceleration of the ripening process, increased fruit number and decreased total fatty acids content in MG3. An increase in oleic acid at MG1 (53%) was detected, with consequent increase in the oleic/linoleic (41%) and decrease in the polyunsaturated/monounsaturated ratios (30%). Those variations could be explained by the synergic up-regulation of OeSAD1, together with the down-regulation of OeFAD6 transcript levels.

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.

Complex Tannins Isolated from Jelly Fig Achenes Affect Pectin Gelation through Non-Specific Inhibitory Effect on Pectin Methylesterase.

Jelly fig (Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (SPEI) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated SPEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the SPEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, SPEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.