Genome-wide identification, characterization, and expression analysis related to autotoxicity of the GST gene family in Cucumis melo L.

Glutathione S-transferase (GST) plays an important role in plant resistance to biotic and abiotic stresses. In this paper, the characteristics of melon GST gene family members were analyzed from a genome-wide perspective. Forty-nine GSTs were identified in melon genome, belonging to eight classes. Through the phylogenetic analysis of GST proteins in melon and other plants, it was found that members from the same subfamily in different species clustered together, indicating that the subfamilies of GST have diversified before the divergence within these species. The results of chromosome mapping showed that GSTs were present in all chromosomes except for chromosome 5. Gene replication events played an important role in the expansion and evolution of melon GST gene family. Ten GSTs with significant differential expression were screened in the transcriptome database related to melon autotoxicity stress. The differential expression of these 10 GSTs was detected in roots and leaves of melon seedlings treated with cinnamic acid. The relative expression level of CmGSTU7, CmGSTU10, CmGSTU18, CmGSTF2 and CmGSTL1 in roots of melon seedlings was significantly higher than that in control group. It suggested that the five GSTs might play an important role in cinnamic acid mediated autotoxicity stress in melon. The results of this paper were helpful to reveal the evolution and functional succession of GST family and further understand the response of GST to autotoxicity stress in melon.

Genome-wide identification of mitogen-activated protein kinase (MAPK) cascade and expression profiling of CmMAPKs in melon (Cucumis melo L.).

Mitogen-activated protein kinase (MAPK) is a form of serine/threonine protein kinase that activated by extracellular stimulation acting through the MAPK cascade (MAPKKK-MAPKK-MAPK). The MAPK cascade gene family, an important family of protein kinases, plays a vital role in responding to various stresses and hormone signal transduction processes in plants. In this study, we identified 14 CmMAPKs, 6 CmMAPKKs and 64 CmMAPKKKs in melon genome. Based on structural characteristics and a comparison of phylogenetic relationships of MAPK gene families from Arabidopsis, cucumber and watermelon, CmMAPKs and CmMAPKKs were categorized into 4 groups, and CmMAPKKKs were categorized into 3 groups. Furthermore, chromosome location revealed an unevenly distribution on chromosomes of MAPK cascade genes in melon, respectively. Eventually, qRT-PCR analysis showed that all 14 CmMAPKs had different expression patterns under drought, salt, salicylic acid (SA), methyl jasmonate (MeJA), red light (RL), and Podosphaera xanthii (P. xanthii) treatments. Overall, the expression levels of CmMAPK3 and CmMAPK7 under different treatments were higher than those in control. Our study provides an important basis for future functional verification of MAPK genes in regulating responses to stress and signal substance in melon.

Early Defense Responses Involved in Mitochondrial Energy Metabolism and Reactive Oxygen Species Accumulation in Harvested Muskmelons Infected by Trichothecium roseum.

Mitochondria play an essential part in fighting against pathogen infection in the defense responses of fruits. In this study, we investigated the reactive oxygen species (ROS) production, energy metabolism, and changes of mitochondrial proteins in harvested muskmelon fruits ( Cucumis melo cv. Yujinxiang) inoculated with Trichothecium roseum. The results indicated that the fungal infection obviously induced the H2O2 accumulation in mitochondria. Enzyme activities were inhibited in the first 6 h postinoculation (hpi), including succinic dehydrogenase, cytochrome c oxidase, H+-ATPase, and Ca2+-ATPase. However, the activities of Ca2+-ATPase and H+-ATPase and the contents of intracellular adenosine triphosphate (ATP) were improved to a higher level at 12 hpi. A total of 42 differentially expressed proteins were identified through tandem mass tags-based proteomic analyses, which are mainly involved in energy metabolism, stress responses and redox homeostasis, glycolysis and tricarboxylic acid cycle, and transporter and mitochondria dysfunction. Taken together, our results suggest that mitochondria play crucial roles in the early defense responses of muskmelons against T. roseum infection through regulation of ROS production and energy metabolism.

PDC1, a pyruvate/α-ketoacid decarboxylase, is involved in acetaldehyde, propanal and pentanal biosynthesis in melon (Cucumis melo L.) fruit.

Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.

Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var. reticulatus.

Cucumisin [EC 3.4.21.25] was first purified from Cucumis melo var. reticulatus juice by three-phase partitioning (TPP). Optimum purification parameters of the TPP system were determined as 60% ammonium sulfate saturation with 1.0:1.25 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 20°C, respectively. Cucumisin was purified with 4.61 purification fold and 156% activity recovery. The molecular weight of the recovered cucumisin was determined as 68.4kDa and its isoelectric point is 8.7. Optimum pH and temperature of cucumisin were pH 9.0 and 60-70°C, respectively. The protease was very stable at 20-70°C and a pH range of 2.0-12.0. Km and Vmax constants were 2.24±0.22mgmL-1 and 1048±25µ Mmin-1, respectively. The enzyme was stable against numerous metal ions and its activity was highly enhanced by Ca2+, Mg2+, and Mn+2. Cucumisin activity was 2.35-folds increased in the presence of 5mM of CaCl2. It was inactivated by Co2+, Cd2+, Zn2+ and Fe2+ and dramatically by PMSF. Cucumisin milk-clotting activity was highly stable when stored under freezing (-20°C) compared at 4°C and 25°C. Finally, TPP revealed to be a useful strategy to concentrate and purify cucumisin for its use as a milk-clotting enzyme for cheese-making.

Effect of antagonistic yeast XL-1 on resistance-associated enzyme activities in postharvest cantaloupe.

The effect of the antagonistic yeast XL-1 on resistance-associated enzyme activities in postharvest cantaloupe was studied by inoculating the antagonistic yeast XL-1. Cantaloupes were sterilized, dried in air, and soaked in antagonistic yeast treatment liquid for 30 s. After drying in air, the cantaloupe was stored at room temperature (2°-5°C). The activities of resistance-associated enzymes in cantaloupe like polyphenol oxidase, ß-1,3-glucanase, peroxidase, and superoxide dismutase were measured every 7 days. Our results indicated that the antagonistic yeast XL-1 significantly improved the activity of ß-1,3-glucanase and chitinase to promote the disease resistance of postharvest cantaloupe.

Floral primordia-targeted ACS (1-aminocyclopropane-1-carboxylate synthase) expression in transgenic Cucumis melo implicates fine tuning of ethylene production mediating unisexual flower development.

MAIN CONCLUSION: Floral primordia-targeted expression of the ethylene biosynthetic gene, ACS , in melon suggests that differential timing and ethylene response thresholds combine to promote carpels, inhibit stamens, and prevent asexual bud formation. Typical angiosperm flowers produce both male and female reproductive organs. However, numerous species have evolved unisexuality. Melons (Cucumis melo L.) can produce varying combinations of male, female or bisexual flowers. Regardless of final sex, floral development begins with sequential initiation of all four floral whorls; unisexuality results from carpel or stamen primordia arrest regulated by the G and A loci, respectively. Ethylene, which promotes femaleness, is a key factor regulating sex expression. We sought to further understand the location, timing, level, and relationship to sex gene expression required for ethylene to promote carpel development or inhibit stamen development. Andromonoecious melons (GGaa) were transformed with the ethylene biosynthetic enzyme gene, ACS (1-aminocyclopropane-1-carboxylate synthase), targeted for expression in stamen and petal, or carpel and nectary, primordia using Arabidopsis APETALA3 (AP3) or CRABSCLAW (CRC) promoters, respectively. CRC::ACS plants did not exhibit altered sex phenotype. AP3::ACS melons showed increased femaleness manifested by gain of a bisexual-only phase not seen in wild type, decreased male buds and flowers, and loss of the initial male-only phase. In extreme cases, plants became phenotypically hermaphrodite, rather than andromonoecious. A reduced portion of buds progressed beyond initial whorl formation. Both the ACS transgene and exogenous ethylene reduced the expression of the native carpel-suppressing gene, G, while elevating expression of the stamen-suppressing gene, A. These results show ethylene-mediated regulation of key sex expression genes and suggest a mechanism by which temporally regulated ethylene production and differential ethylene response thresholds can promote carpels, inhibit stamens, and prevent the formation of asexual buds.

The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with ß-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for ß-carotene production in orange-fleshed melon varieties.

The cinnamyl alcohol dehydrogenase gene family in melon (Cucumis melo L.): bioinformatic analysis and expression patterns.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development.

Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk.

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu-regulated genes and stimulated Cu accumulation. The C940-fe (fefe) Fe-uptake mutant of melon (Cucumis melo) was characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild-type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe-regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu-deficient plants was independent of the normal Fe-uptake system. One of the four FRO genes in the melon and cucumber (Cucumis sativus) genomes was Fe-regulated, and one was Cu-regulated. Simultaneous Fe and Cu deficiency synergistically up-regulated Fe-uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe-Cu crosstalk in metal homeostasis. The fefe gene is not orthologous to FIT, and thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants.

Effect of abiotic stress stimuli on S-nitrosoglutathione reductase in plants.

S-nitrosylation of protein cysteine thiol groups has recently emerged as a widespread and important reversible post-translational protein modification, involved in redox signalling pathways of nitric oxide and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR), member of class III alcohol dehydrogenase family (EC 1.1.1.1), is considered the key enzyme in the catabolism of major low molecular S-nitrosothiol, S-nitrosoglutathione, and hence to control the level of protein S-nitrosylation. Changes of GSNOR activity after exposure to different abiotic stress conditions, including low and high temperature, continuous dark and de-etiolation, and mechanical injury, were investigated in important agricultural plants. Significantly higher GSNOR activity was found under normal conditions in leaves of Cucumis spp. genotype sensitive to biotrophic pathogen Golovinomyces cichoracearum. GSNOR activity was generally increased in all studied plants by all types of stress conditions. Strong down-regulation of GSNOR was observed in hypocotyls of etiolated pea plants, which did not recover to values of green plants even 168 h after the transfer of etiolated plants to normal light regime. These results point to important role of GSNOR during normal plant development and in plant responses to several types of abiotic stress conditions.

Molecular characterization, 3D model analysis, and expression pattern of the CmUBC gene encoding the melon ubiquitin-conjugating enzyme under drought and salt stress conditions.

Ubiquitin-conjugating (UBC) enzyme is a key enzyme in ubiquitination. Here, we describe the cloning, characterization, and expression pattern of a novel gene, CmUBC, from a melon. Comparison of the deduced amino acid sequences allowed the identification of highly conserved motifs. Synteny analysis between Cucumis sativus L. and Arabidopsis demonstrated that homologs of several Cucumis UBC genes were found in corresponding syntenic blocks of Arabidopsis. The homology structure model of the CmUBC protein was constructed. UBCs from melon, yeast, and Arabidopsis were highly conserved in their three-dimensional folding. CmUBC was ubiquitously expressed in all melon tissues. Increased transcript levels of CmUBC were observed during drought and salinity stresses, which suggested that the expression of the CmUBC gene in melon plants is responsive to physiological water stress. These results suggested that the CmUBC gene might play an important role in the modulation of the ubiquitination pathway.

Synthesis of 9-oxononanoic acid, a precursor for biopolymers.

Polymers based on renewable resources have become increasingly important. The natural functionalization of fats and oils enables an easy access to interesting monomeric building blocks, which in turn transform the derivative biopolymers into high-performance materials. Unfortunately, interesting building blocks of medium-chain length are difficult to obtain by traditional chemical means. Herein, a biotechnological pathway is established that could provide an environmentally suitable and sustainable alternative. A multiple enzyme two-step one-pot process efficiently catalyzed by a coupled 9S-lipoxygenase (St-LOX1, Solanum tuberosum) and 9/13-hydroperoxide lyase (Cm-9/13HPL, Cucumis melo) cascade reaction is proposed as a potential route for the conversion of linoleic acid into 9-oxononanoic acid, which is a precursor for biopolymers. Lipoxygenase catalyzes the insertion of oxygen into linoleic acid through a radical mechanism to give 9S-hydroperoxy-octadecadienoic acid (9S-HPODE) as a cascade intermediate, which is subsequently cleaved by the action of Cm-9/13HPL. This one-pot process afforded a yield of 73 % combined with high selectivity. The best reaction performance was achieved when lipoxygenase and hydroperoxide lyase were applied in a successive rather than a simultaneous manner. Green leaf volatiles, which are desired flavor and fragrance products, are formed as by-products in this reaction cascade. Furthermore, we have investigated the enantioselectivity of 9/13-HPLs, which exhibited a strong preference for 9S-HPODE over 9R-HPODE.

Catabolism of L-methionine in the formation of sulfur and other volatiles in melon (Cucumis melo L.) fruit.

Sulfur-containing aroma volatiles are important contributors to the distinctive aroma of melon and other fruits. Melon cultivars and accessions differ in the content of sulfur-containing and other volatiles. L-methionine has been postulated to serve as a precursor of these volatiles. Incubation of melon fruit cubes with ¹³C- and ²H-labeled L-methionine revealed two distinct catabolic routes into volatiles. One route apparently involves the action of an L-methionine aminotransferase and preserves the main carbon skeleton of L-methionine. The second route apparently involves the action of an L-methionine-γ-lyase activity, releasing methanethiol, a backbone for formation of thiol-derived aroma volatiles. Exogenous L-methionine also generated non-sulfur volatiles by further metabolism of α-ketobutyrate, a product of L-methionine-γ-lyase activity. α-Ketobutyrate was further metabolized into L-isoleucine and other important melon volatiles, including non-sulfur branched and straight-chain esters. Cell-free extracts derived from ripe melon fruit exhibited L-methionine-γ-lyase enzymatic activity. A melon gene (CmMGL) ectopically expressed in Escherichia coli, was shown to encode a protein possessing L-methionine-γ-lyase enzymatic activity. Expression of CmMGL was relatively low in early stages of melon fruit development, but increased in the flesh of ripe fruits, depending on the cultivar tested. Moreover, the levels of expression of CmMGL in recombinant inbred lines co-segregated with the levels of sulfur-containing aroma volatiles enriched with +1 m/z unit and postulated to be produced via this route. Our results indicate that L-methionine is a precursor of both sulfur and non-sulfur aroma volatiles in melon fruit.

Structural analysis of the alcohol acyltransferase protein family from Cucumis melo shows that enzyme activity depends on an essential solvent channel.

Alcohol acyltransferases (AAT) play a key role in ester biosynthesis. In Cucumis melo var. cantalupensis, AATs are encoded by a gene family of four members (CmAAT1-4). CmAAT1, CmAAT3 and CmAAT4 are capable of synthesizing esters, with CmAAT1 the most active. CmAAT2 is inactive and has an Ala268 residue instead of a threonine which is present in all other active AATs, although the role of this residue is still unclear. The present work aims to understand the molecular mechanism involved in ester biosynthesis in melon fruit and to clarify the importance of the Ala268 residue. First, structural models for each protein were built by comparative modelling methodology. Afterwards, conformational interaction between the protein and several ligands, alcohols and acyl-CoAs was explored by molecular docking and molecular dynamics simulation. Structural analysis showed that CmAATs share a similar structure. Also, well-defined solvent channels were described in the CmAATs except for CmAAT2 which does not have a proper channel and instead has a small pocket around Ala268. Residues of the catalytic HxxxD motif interact with substrates within the solvent channel, with Ser363 also important. Strong binding interaction energies were described for the best substrate couple of each CmAAT (hexyl-, benzyl- and cinnamyl-acetate for CmAAT1, 3 and 4 respectively). CmAAT1 and CmAAT2 protein surfaces share similar electrostatic potentials; nevertheless the entrance channels for the substrates differ in location and electrostatic character, suggesting that Ala268 might be responsible for that. This could partly explain the major differences in activity reported for these two enzymes.

Crystal structure of cucumisin, a subtilisin-like endoprotease from Cucumis melo L.

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin-III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.

Isolation and characterization of a muskmelon cDNA encoding Lycopene Beta-cyclase.

Lycopene Beta-cyclase (LCY-B) is thought to play a critical role in Beta-carotene synthesis in fruit. A full-length cDNA clone encoding Lycopene Beta-cyclase was isolated from muskmelon (Cucumis melo L.) by RT-PCR and RACE. The clone, designated CmLcyb1, contains 1871 nucleotides, with an open reading frame of 1512 nucleotides. The deduced 504-amino-acid sequence showed high identities with other plant Lycopene Beta-cyclases. Real time quantitative RT-PCR analysis indicated that CmLcyb1 was expressed in all tissues and organs of muskmelon inbred M01-3 with white mesocarp and, 'Homoka', an orange mesocarp cultivar. The expression levels of CmLcyb1 in roots, stems, leaves and flowers in the two genotypes differed little. The expression level was highest in mature fruit of 'Homoka' and was much higher than that in mature fruit of M01-3. Moreover, the mRNA level of CmLcyb1 was very low in fruits before fruit-size fixation and increased dramatically in the size-fixed fruits of these two genotypes. The mRNA levels of CmLcyb1 during fruit development of 'Homoka' were all higher than those of M01-3. Interestingly, Beta-carotene content showed almost the same change trend as mRNA levels during fruit development in these two genotypes, suggesting that Beta-carotene accumulation may be linked to the CmLcyb1 transcript level in muskmelon fruit.

Intestinal cell targeting of a stable recombinant Cu-Zn SOD from Cucumis melo fused to a gliadin peptide.

The mRNA encoding full length chloroplastic Cu-Zn SOD (superoxide dismutase) of Cucumis melo (Cantaloupe melon) was cloned. This sequence was then used to generate a mature recombinant SOD by deleting the first 64 codons expected to encode a chloroplastic peptide signal. A second hybrid SOD was created by inserting ten codons to encode a gliadin peptide at the N-terminal end of the mature SOD. Taking account of codon bias, both recombinant proteins were successfully expressed and produced in Escherichia coli. Both recombinant SODs display an enzymatic activity of ~5000U mg(-1) and were shown to be stable for at least 4h at 37°C in biological fluids mimicking the conditions of intestinal transit. These recombinant proteins were capable in vitro, albeit at different levels, of reducing ROS-induced-apoptosis of human epithelial cells. They also stimulated production and release in a time-dependent manner of an autologous SOD activity from cells located into jejunum biopsies. Nevertheless, the fused gliadin peptide enable the recombinant Cu-Zn SOD to maintain a sufficiently sustained interaction with the intestinal cells membrane in vivo rather than being eliminated with the flow. According to these observations, the new hybrid Cu-Zn SOD should show promise in applications for managing inflammatory bowel diseases.

1-MCP suppresses ethylene biosynthesis and delays softening of 'Hami' melon during storage at ambient temperature.

BACKGROUND: 'Hami' melon is susceptible to softening during postharvest storage at ambient temperature, which enhances postharvest deterioration and limits transportation and storage. To look for a method of softening control, the effect of 1-methylcyclopropene (1-MCP) on regulating postharvest softening of 'Hami' melon fruit was investigated. RESULTS: 1-MCP treatment at 1 µL L(-1) significantly reduced ethylene production and maintained higher levels of fruit firmness. It also markedly inhibited the accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) and maintained lower activities of ACC synthase and ACC oxidase. In addition, 1-MCP treatment reduced the activities of fruit-softening enzymes such as pectin methyl esterase, polygalacturonase, endo-1,4-ß-glucanase and ß-galactosidase. CONCLUSION: 1-MCP was effective in suppressing ethylene production and delaying fruit softening in 'Hami' melon by decreasing the activities of ethylene biosynthesis enzymes and inhibiting fruit-softening enzymes.

Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.).

Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.