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Luteolin-7- O -glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7- O -glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7- O -glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7- O -glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a k cat / K m value of 772 s -1 ·M -1 , which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7- O -glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7- O -glucoside, which holds broad application prospects in the food and pharmaceutical industry.

Melon fly ( Bactrocera cucurbitae ) is the most common pest of cucurbits, and it directly causes damage to cucurbit fruits in the early developmental stage. The infection of fruit tissues induces oxidative damage through increased generation of cellular reactive oxygen species. The effects of melon fly infestation on the production of defensive enzymes and antioxidant capabilities in five cucurbit species, namely, bottle gourd, chayote, cucumber, snake gourd, and bitter gourd, were investigated in this study. The total phenolic and flavonoid content was considerably higher in melon fly infestation tissues compared to healthy and apparently healthy tissues. The chayote and bottle gourd tissues expressed almost 1.5- to 2-fold higher phenolic and flavonoid contents compared to the tissues of bitter gourd, snake gourd, and cucumber upon infestation. Defensive enzymes, such as peroxidase (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), and catalase (CAT), were high in healthy and infected tissues of chayote and bottle gourd compared to bitter gourd, snake gourd, and cucumber. The activity of POD (60-80%), SOD (30-35%), PPO (70-75%), and CAT (40-50%) were high in infected chayote and bottle gourd tissue, representing resistance against infestation, while bitter gourd, snake gourd, and cucumber exhibited comparatively lower activity suggesting susceptibility to melon fly infection. The antioxidant properties were also high in the resistant cucurbits compared to the susceptible cucurbits. The current research has enlightened the importance of redox-regulatory pathways involving ROS neutralization through infection-induced antioxidative enzymes in host cucurbit resistance. The melon fly infestation depicts the possible induction of pathways that upregulate the production of defensive enzymes and antioxidants as a defensive strategy against melon fly infestation in resistant cucurbits.

We investigated changes in starch content and starch metabolic enzyme activities in developing and postharvest squash of distinct species, Cucurbita maxima and Cucurbita moschata, which accumulate high and low levels of starch, respectively. The total activity of starch phosphorylase in developing fruits significantly correlated (r = 0.99) to the amount of starch among Cucurbita species (C. maxima, C. moschata and C. pepo). Separable activity of a plastidial L-form phosphorylase in C. maxima fruit markedly increased corresponding with starch accumulation. We isolated two genes (CmPhoL1 and CmPhoH1) encoding an L-form and a cytosolic H-form phosphorylase from C. maxima fruit. The expression of CmPhoL1 in the fruit dramatically increased at the beginning of starch accumulation. Recombinant CmPhoL1 enzyme showed similar kinetic parameters in both glucan synthesis and phosphorolysis: this enzyme can catalyze the invertible reaction in vitro depending on the concentration of substrates. These results suggest that CmPhoL1 plays a role in the starch accumulation process during squash development, but the aid of other starch synthetic enzymes may be required for in vivo glucan synthesis reaction by CmPhoL1. An importance of plastidial starch phosphorylase in the starch accumulation in the fruit organ was indicated., (© 2018 Scandinavian Plant Physiology Society.)

Photorespiration is required in C 3 plants to metabolize toxic glycolate formed when ribulose-1,5-bisphosphate carboxylase-oxygenase oxygenates rather than carboxylates ribulose-1,5-bisphosphate. Depending on growing temperatures, photorespiration can reduce yields by 20 to 50% in C 3 crops. Inspired by earlier work, we installed into tobacco chloroplasts synthetic glycolate metabolic pathways that are thought to be more efficient than the native pathway. Flux through the synthetic pathways was maximized by inhibiting glycolate export from the chloroplast. The synthetic pathways tested improved photosynthetic quantum yield by 20%. Numerous homozygous transgenic lines increased biomass productivity by >40% in replicated field trials. These results show that engineering alternative glycolate metabolic pathways into crop chloroplasts while inhibiting glycolate export into the native pathway can drive increases in C 3 crop yield under agricultural field conditions., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

The winter squash ( Cucurbita moschata , Cm) superoxide dismutase (SOD ) CmSOD gene and Arabidopsis thaliana ( At ) SOD gene were transferred under a ubiquitin promoter into Arabidopsis via Agrobacterium tumefaciens . The expression and amount of SOD and the SOD activities in the AtSOD and CmSOD transgenic lines were significantly higher than those of non-transgenic (NT) plants exposed to 23 or 4°C treatment for 6 ~ 192-h periods. Furthermore, expressions of the cold-inducible gene ( AtCBF2 ) and desiccation-responsible transcription factors ( AtRD 29A/B) were also activated in all transgenic lines compared to NT plants after chilling treatments. Compared to NT plants under chilling stress, superoxide (•O2 - ) accumulation was significantly lower, and chlorophyll (Chl) contents were significantly higher in all transgenic lines with higher SOD activity. Moreover, Arabidopsis seedlings overexpressing AtSOD and CmSOD also displayed greater resistance to chilling and less oxidative injury than NT plants under chilled conditions, indicating that the overexpression of AtSOD and CmSOD in Arabidopsis enhanced chilling tolerance by eliminating •O2 - . The expression of AtRD29A was strongly up-regulated only in AtSOD transgenic plants treated with abscisic acid (ABA), while it was repressed in other transgenic plants, indicating ABA-sensitive AtCBF2 and AtRD29A/B transcriptional regulation signaling pathways in transgenic Arabidopsis under chilling conditions.

A cantilever nanobiosensor functionalized with vegetable source of peroxidase was developed as an innovative way for glyphosate herbicide detection over a wide concentration range (0.01 to 10 mg L -1 ) using atomic force microscopy (AFM) technique. The extract obtained from zucchini (Cucurbita pepo source of peroxidase), with high enzymatic activity and stability has been used as bio-recognition element to develop a nanobiosensor. The polarization-modulated reflection absorption infrared spectroscopy (PM-RAIRS) demonstrated the deposition of enzyme on cantilever surface using self-assembled monolayers (SAM) by the presence of the amide I and II bands. The detection mechanism of glyphosate was based on the changes in surface tension caused by the analyte adsorption, resulting in a conformational change in the enzyme structure. In this way, the results of nanobiosensor demonstrate the potential of the sensing device for detecting glyphosate with a detection limit of 0.028 mg L -1 .

Perfluorooctane sulfonamide (FOSA) is an important perfluorooctane sulfonate (PFOS) precursor used for commercial applications. In order to investigate the transformation and responses of selected antioxidant and degradation enzymes of FOSA in the plants, in vivo exposure of soybean (Glycine max L. Merrill) and pumpkin (Cucurbita maxima L.) were conducted in the solution-plant microcosms. FOSA was readily taken up by soybean and pumpkin roots and translocated to shoots, and metabolized to PFOS, perfluorohexane sulfonate (PFHxS) and perfluorobutane sulfonate (PFBS). Although morphological and biomass effects were not visible, significant changes in oxidative stress response were observed except for thiobarbituric acid reactive substances (TBARS). Superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased by 19.2-30.8% and 19.2-20.7% in soybean (8-12 d) respectively, and increased by 39.2-92.8% and 21.1-37.6% in pumpkin (3-12 d) respectively, suggesting an activation of the antioxidant defense system in the plants exposed to FOSA. Glutathione-S-transferase (GST) activities were decreased in soybean (2-12 d) with 9.0-36.1% inhibition and increased in pumpkin (3-12 d) with 22.5-47.3% activation respectively; cytochrome P450 (CYP450) activities were increased markedly in soybean and pumpkin with 13.2-53.6% and 26.7-50.2% activation respectively, giving indirect evidences on the involvement of CYP450 and GST in degradation of FOSA in plants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast., (Copyright © 2018. Published by Elsevier Inc.)

Plant salt tolerance can be improved by grafting onto salt-tolerant rootstocks. However, the underlying signaling mechanisms behind this phenomenon remain largely unknown. To address this issue, we used a range of physiological and molecular techniques to study responses of self-grafted and pumpkin-grafted cucumber plants exposed to 75 mM NaCl stress. Pumpkin grafting significantly increased the salt tolerance of cucumber plants, as revealed by higher plant dry weight, chlorophyll content and photochemical efficiency (Fv/Fm), and lower leaf Na+ content. Salinity stress resulted in a sharp increase in H2O2 production, reaching a peak 3 h after salt treatment in the pumpkin-grafted cucumber. This enhancement was accompanied by elevated relative expression of respiratory burst oxidase homologue (RBOH) genes RbohD and RbohF and a higher NADPH oxidase activity. However, this increase was much delayed in the self-grafted plants, and the difference between the two grafting combinations disappeared after 24 h. The decreased leaf Na+ content of pumpkin-grafted plants was achieved by higher Na+ exclusion in roots, which was driven by the Na+/H+ antiporter energized by the plasma membrane H+-ATPase, as evidenced by the higher plasma membrane H+-ATPase activity and higher transcript levels for PMA and SOS1. In addition, early stomatal closure was also observed in the pumpkin-grafted cucumber plants, reducing water loss and maintaining the plant's hydration status. When pumpkin-grafted plants were pretreated with an NADPH oxidase inhibitor, diphenylene iodonium (DPI), the H2O2 level decreased significantly, to the level found in self-grafted plants, resulting in the loss of the salt tolerance. Inhibition of the NADPH oxidase-mediated H2O2 signaling in the root also abolished a rapid stomatal closure in the pumpkin-grafted plants. We concluded that the pumpkin-grafted cucumber plants increase their salt tolerance via a mechanism involving the root-sourced respiratory burst oxidase homologue-dependent H2O2 production, which enhances Na+ exclusion from the root and promotes an early stomatal closure.

Phytoremediation is a low cost technology based on the use of plants to remove a wide range of pollutants from the environment, including the insecticide DDT. However, some pollutants are known to enhance generation of reactive oxygen species (ROS), which can generate toxic effects on plants affecting the phytoremediation efficiency. This study aims to analyze the potential use of antioxidant responses as a measure of tolerance to select plants for phytoremediation purposes. Tomato and zucchini plants were grown for 15 days in soils contaminated with DDTs (DDT + DDE + DDD). Protein content, glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) activities were measured in plant tissues. Exposure to DDTs did not affect protein content or CAT activity in any of the species. GST, GR and GPx activity showed different responses in exposed and control tomato plants. After DDTs exposure, tomato showed increased GR and GPX activity in stems and leaves, respectively, and a decrease in the GST activity in roots. As no effects were observed in zucchini, results suggest different susceptibility and/or defense mechanisms involved after pesticide exposure. Finally, both species differed also in terms of DDTs uptake and translocation. The knowledge about antioxidant responses induced by pesticides exposure could be helpful for planning phytoremediation strategies and for the selection of tolerant species according to particular scenarios., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gibberellins (GAs) are hormones that control many aspects of plant development, including flowering. It is well known that stamen is the source of GAs that regulate male and bisexual flower development. However, little is known about the role of GAs in female flower development. In cucumber, high levels of GA precursors are present in ovaries and high levels of bioactive GA 4 are identified in sepals/petals, reflecting the expression of GA 20-oxidase and 3-oxidase in these organs, respectively. Here, we show that the biologically inactive precursor GA 9 moves from ovaries to sepal/petal tissues where it is converted to the bioactive GA 4 necessary for female flower development. Transient expression of a catabolic GA 2-oxidase from pumpkin in cucumber ovaries decreases GA 9 and GA 4 levels and arrests the development of female flowers, and this can be restored by application of GA 9 to petals thus confirming its function. Given that bioactive GAs can promote sex reversion of female flowers, movement of biologically inactive precursors, instead of the hormone itself, might help to maintain floral organ identity, ensuring fruit and seed production., (© 2016. Published by The Company of Biologists Ltd.)

The effect of dissolved organic matter (DOM) on nanoparticle toxicity to plants is poorly understood. In this study, tannic acid (TA) was selected as a DOM surrogate to explore the mechanisms of neodymium oxide NPs (Nd2O3 NPs) phytotoxicity to pumpkin (Cucurbita maxima). The results from the tested concentrations showed that 100 mg L(-1) Nd2O3 NPs were significantly toxic to pumpkin in term of fresh biomass, and the similar results from the bulk particles and the ionic treatments were also evident. Exposure to 100 mg L(-1) of Nd2O3 NPs and BPs in 1/5 strength Hoagland's solution not only significantly inhibited pumpkin growth, but also decreased the S, Ca, K and Mg levels in plant tissues. However, 60 mg L(-1) TA significantly moderated the observed phytotoxicity, decreased Nd accumulation in the roots, and notably restored S, Ca, K and Mg levels in NPs and BPs treated pumpkin. TA at 60 mg L(-1) increased superoxide dismutase (SOD) activity in both roots (17.5%) and leaves (42.9%), and catalase (CAT) activity (243.1%) in the roots exposed to Nd2O3 NPs. This finding was confirmed by the observed up-regulation of transcript levels of SOD and CAT in Nd2O3 NPs treated pumpkin analyzed by quantitative reverse transcription polymerase chain reaction. These results suggest that TA alleviates Nd2O3 BPs/NPs toxicity through alteration of the particle surface charge, thus reducing the contact and uptake of NPs by pumpkin. In addition, TA promotes antioxidant enzymatic activity by elevating the transcript levels of genes involved in ROS scavenging. Our results shed light on the mechanisms underlying the influence of DOM on the bioavailability and toxicity of NPs to terrestrial plants.

This study is aimed to investigate the activity of urease (EC 3.5.1.5, urea amidohydrolase) that catalyzes the hydrolysis of urea in 5-day-old Cucurbita pepo cotyledons subjected to various concentrations of different growth regulators. The treatment of C. pepo cotyledons with different concentrations (100-600 μmol) of different auxins [indole-3-acetic acid (IAA), indole butyric acid (IBA), indole propionic acid (IPA) and naphthalene acetic acid (NAA)]; or with different concentrations (100-300 μmol) of different cytokinins [kinetin, zeatin and benzyladenine (6-BA)] resulted in a significant increase of urease activity, compared to control. The optimal effects were recorded for each of 500 μmol of IAA and 300 μmol of zeatin treatments. A gradual increase in urease activity was detected in cotyledons treated with various concentrations (0.2-1.0 mM) of 28-homobrassinolide (HBL), in relative to control. A substantial increase in urease activity was observed in cotyledons subjected to different concentrations of triazole (10-60 mg L(-1)), containing either triadimefon (TDM) or hexaconazole (HEX), compared to control. The combination of 300 μmol zeatin with any of protein inhibitors, namely 5-fluorouridine (FUrd), cordycepin and α-amanitin, resulted in the alleviation of their inhibitory effect on the urease activity.

In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

This study demonstrates a new strategy to develop online electrochemical systems (OECSs) for continuously monitoring neurochemicals by efficiently integrating in vivo microdialysis with an oxidase-based electrochemical biosensor with low-potential electron mediators to shuttle the electron transfer of the oxidases. By using thionine and xanthine oxidase (XOD) as examples of low-potential mediators and oxidases, respectively, we demonstrate that the use of low-potential mediators to shuttle the electron transfer of oxidases would offer a new approach to the development of oxidase-based biosensors with theoretical and technical simplicity. To construct the XOD-based biosensor, thionine was adsorbed onto carbon nanotubes and used to shuttle the electron transfer of XOD. The XOD-based biosensor was positioned into an electrochemical cell that was directly coupled with in vivo microdialysis to form an online electrochemical system (OECS) for continuous and selective measurements of the substrate of XOD (with hypoxanthine as an example). The OECS based on the low-potential mediators is highly selective against the species endogenously existing in the brain system, which is attributed to the low operation potential benefited from the low redox potentials of the mediators. Moreover, the OECS demonstrated here is stable and reproducible and could thus be envisaged to find some interesting applications in physiological and pathological investigations. This study essentially offers a new strategy to develop online electrochemical systems, which is of great importance in understanding the molecular basis of physiological and pathological events.

Lipase is one of the more important enzymes used in various industries such as the food, detergent, pharmaceutical, textile, and pulp and paper sectors. A novel aqueous two-phase system composed of surfactant and xylitol was employed for the first time to purify lipase from Cucurbita moschata. The influence of different parameters such as type and concentration of surfactants, and the composition of the surfactant/xylitol mixtures on the partitioning behavior and recovery of lipase was investigated. Moreover, the effect of system pH and crude load on the degree of purification and yield of the purified lipase were studied. The results indicated that the lipase was partitioned into the top surfactant rich phase while the impurities partitioned into the bottom xylitol-rich phase using an aqueous two phase system composed of 24% (w/w) Triton X-100 and 20% (w/w) xylitol, at 56.2% of tie line length (TLL), (TTL is one of the important parameters in this study and it is determined from a bimodal curve in which the tie-line connects two nodes on the bimodal, that represent concentration of phase components in the top and bottom phases) and a crude load of 25% (w/w) at pH 8.0. Recovery and recycling of components was also measured in each successive step process. The enzyme was successfully recovered by the proposed method with a high purification factor of 16.4 and yield of 97.4% while over 97% of the phase components were also recovered and recycled. This study demonstrated that the proposed novel aqueous two phase system method is more efficient and economical than the traditional aqueous two phase system method for the purification and recovery of the valuable enzyme lipase.

An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the present study angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from egg-yolk protein preparation (YP). Enzymatic hydrolysis conducted using unconventional enzyme from Cucurbita ficifolia (dose: 1000 U/mg of hydrolyzed YP (E/S (w/w)=1:7.52)) was employed to obtain protein hydrolysates. The 4-h hydrolysate exhibited a significant (IC₅₀=482.5 μg/mL) ACE inhibitory activity. Moreover, hydrolysate showed no cytotoxic activity on human and animal cell lines which makes it a very useful multifunctional method for peptide preparation. The compiled isolation procedure (ultrafiltration, size-exclusion chromatography and RP-HPLC) of bioactive peptides from YP hydrolysate resulted in obtaining peptides with the strong ACE inhibitory activity. One homogeneous and three heterogeneous peptide fractions were identified. The peptides were composed of 9-18 amino-acid residues, including mainly arginine and leucine at the N-terminal positions. To confirm the selected bioactive peptide sequences their analogs were chemically synthesized and tested. Peptide LAPSLPGKPKPD showed the strongest ACE inhibitory activity, with IC₅₀ value of 1.97 μmol/L., Biological Significance: Peptides with specific biological activity can be used in pharmaceutical, cosmetic or food industries. Because of their potential role as physiological modulators, as well as theirhigh safety profile, they can be used as natural pharmacological compounds or functional food ingredients. The development of biotechnological solutions to obtain peptides with desired biological activity is already in progress. Studies in this area are focused on using unconventional highly specific enzymes and more efficient methods developed to conduct food process technologies. Natural peptides have many advantages. They are mainly toxicologically safe, have wide spectra of therapeutic actions, exhibit less side effects compared to synthetic drugs and are more efficiently absorbed in the intestinal tract. The complexity of operation of large scale technologies and high cost of purification techniques are limiting factors to the commercialization of food-derived bioactive peptides. Research on the isolation of bioactive peptides in order to reduce the processing time and costs is continuously developing. Bioactive peptides can also be released from protein by-products of the food industry, which reduce the substrate expense and production cost as well as provide the added advantage of an efficient waste disposal. Moreover, proteins as precursors of food-derived peptides are well-tolerated by the human body and therefore their application in drug development may reduce costs and duration of toxicological studies during research, development and clinical trials., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In order to investigate the contribution of polyamines and related amino acids in the maintenance of zucchini fruit quality during cold storage, two varieties of Cucurbita pepo with different degrees of chilling tolerance were used, Natura (more tolerant) and Sinatra (moresensitive). After harvest, free putrescine levels decreased during storage at 20 °C, whereas in fruit kept at 4 °C this polyamine accumulated in both varieties, but with higher levels in the sensitive variety (Sinatra). This behavior suggests that putrescine is accumulated as a response to low temperature in zucchini fruit by stress-induced chilling injury, and not due to the postharvest storage itself. ADC activity responds quickly to chilling but sharply decreases after 14 days, whereas its expression remains high in both varieties. ODC activity takes over when the cold stress is relatively severe, as this activity was found to be much higher in Sinatra. ODCexpression also correlated with ODC activity. DAO activity increased in Natura fruit, and conversely decreased in Sinatra fruit during storage at 4 °C, whereas the proline content was higher in Natura and lower in Sinatra. Therefore, we suggest that putrescine degradation and proline accumulation contribute to the acquisition of chilling tolerance in zucchini fruit. GABA content decreased in both varieties, with a greater reduction in Natura fruit and less in Sinatra fruit. In addition, GABA transaminase showed a higher activity in Natura fruit than in Sinatra fruit during cold storage, suggesting that GABA catabolism could be involved in the tolerance to postharvest cold storage in zucchini fruit., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Protein isolates of pumpkin (Cucurbita pepo L) seeds were hydrolyzed by acid protease to prepare antioxidative peptides. The hydrolysis conditions were optimized through Box-Behnken experimental design combined with response surface method (RSM). The second-order model, developed for the DPPH radical scavenging activity of pumpkin seed hydrolysates, showed good fit with the experiment data with a high value of coefficient of determination (0.9918). The optimal hydrolysis conditions were determined as follows: hydrolyzing temperature 50°C, pH 2.5, enzyme amount 6000 U/g, substrate concentration 0.05 g/ml and hydrolyzing time 5 h. Under the above conditions, the scavenging activity of DPPH radical was as high as 92.82%.

Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo., (© 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.)

The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant "C. moschata", thermolabile "C. maxima" and moderately heat-tolerant interspecific inbred line "Maxchata" genotypes were exposed to moderate (37 °C) and severe (42 °C) heat shocks. "C. moschata" exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O2(-)) and malondialdehyde (MDA) contents in the roots compared to stems, followed by "Maxchata". The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among "C. maxima" and "Maxchata", most of these genes were highly induced under heat stress in "Maxchata", which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.

The control of gene expression is a crucial regulatory mechanism in carotenoid accumulation of fruits and flowers. We investigated the role of transcriptional regulation of nine genes involved in the carotenoid biosynthesis pathway in three varieties of Cucurbita pepo with evident differences in fruit color. The transcriptional levels of the key genes involved in the carotenoid biosynthesis were higher in flower-, leaf-, and fruit skin tissues than flesh tissues. This correlated with higher concentration of carotenoid content in these tissues. The differential expression among the colored and white cultivars detected for some genes, such as LCYe, in combination with other regulatory mechanisms, could explain the large differences found in terms of carotenoid content among the three varieties. These results are a first step to elucidate carotenogenesis in C. pepo and demonstrate that, in general, regulation of the pathway genes is a critical factor that determines the accumulation of these compounds.

Enzymatic decolourization of the azo dye, Direct Yellow (DY106) by Cucurbita pepo (courgette) peroxidase (CP) is a complex process, which is greatly affected by pH, temperature, enzyme activity and the concentrations of H2O2 and dye. Courgette peroxidase was extracted and its performance was evaluated by using the free-CP (FCP) and immobilized-CP (ICP) forms in the decolourization of DY106. Immobilization of peroxidase in calcium alginate beads was performed according to a strategy aiming to minimize enzyme leakage and keep its activity at a maximum value by optimizing sodium alginate content, enzyme loading and calcium chloride concentration. The initial conditions at which the highest DY106 decolourization yield was obtained were found at pH 2, temperature 20 degrees C, H2O2 dose 1 mmol/L (FCP) and 100 mmol/L (ICP). The highest decolourization rates were obtained for dye concentrations 50 mg/L (FCP) and 80 mg/L (ICP). Under optimal conditions, the FCP was able to decolorize more than 87% of the dye within 2 min. While with ICP, the decolourization yield was 75% within 15 min. The decolourization and removal of DY106 was proved by UV-Vis analysis. Fourier transform infrared (FT-IR) spectroscopy analysis was also performed on DY106 and enzymatic treatment precipitated byproduct.

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

Ascorbate oxidase (AO), a biologically active macromolecule, was successfully immobilized into a biocompatible sandwich-type composite film for developing the vitamin C (VC) biosensor, and the content of VC in commercial juices was amperometrically determined. The biocompatible and conducting poly(3,4-ethylenedioxythiophene) composite film and highly stable and selective multiwalled carbon nanotubes -Nafion composite film were prepared as inner and outer films of biosensor. AO molecules were immobilized between these two composite films. The as-fabricated biosensor displayed an excellent bioelectrocatalytic performance towards the oxidation of VC, a fast current response, a low working potential, a high sensitivity, a wide linear range, and a low detection limit. Moreover, the working mechanism of the biosensor was proposed, and its kinetics was also discussed. In addition, the specificity, reproducibility, and feasibility of the as-fabricated biosensor were also evaluated. Good results of the VC determination in commercial juices indicated that the as-fabricated biosensor was a potential candidate for the electrochemical determination of VC in agricultural crops. Inner and outer films provided a promising platform for the immobilization of biologically active species.

Gibberellin (GA) signalling during pumpkin male flower development is highly regulated, including biosynthetic, perception, and transduction pathways. GA 20-oxidases, 3-oxidases, and 2-oxidases catalyse the final part of GA synthesis. Additionally, 7-oxidase initiates this part of the pathway in some cucurbits including Cucurbita maxima L. (pumpkin). Expression patterns for these GA-oxidase-encoding genes were examined by competitive reverse transcription-PCR (RT-PCR) and endogenous GA levels were determined during pumpkin male flower development. In young flowers, GA20ox3 transcript levels are high in stamens, followed by high levels of the GA precursor GA(9). Later, just before flower opening, transcript levels for GA3ox3 and GA3ox4 increase in the hypanthium and stamens, respectively. In the stamen, following GA3ox4 expression, bioactive GA(4) levels rise dramatically. Accordingly, catabolic GA2ox2 and GA2ox3 transcript levels are low in developing flowers, and increase in mature flowers. Putative GA receptor GID1b and DELLA repressor GAIPb transcript levels do not change in developing flowers, but increase sharply in mature flowers. Emasculation arrests floral development completely and leads to abscission of premature flowers. Application of GA(4) (but not of its precursors GA(12)-aldehyde or GA(9)) restores normal growth of emasculated flowers. These results indicate that de novo GA(4) synthesis in the stamen is under control of GA20ox3 and GA3ox4 genes just before the rapid flower growth phase. Stamen-derived bioactive GA is essential and sufficient for male flower development, including the petal and the pedicel growth.

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Abstract: An ascorbate oxidase purified from the green fruit of zucchini squash (Cucurbita pepo medullosa) was immobilized photochemically on a polyethylene disc with 85% retention of initial activity of free enzyme. The optimum pH (5.5) was unchanged, while K(m) was decreased. The polyethylene discs were employed for determination of ascorbic acid in serum and foodstuffs. The working linear range was 2.8 μM to 16 μM. The mean value of ascorbic acid in serum as measured by the method was 0.16 mg/dl in males and 0.209 mg/dl in females. The immobilized enzyme was used 100 times over 4 months, when stored at 4°C.

Background and Aims: Nectar is a very complex mixture of substances. Some components (sugars and amino acids) are considered primary alimentary rewards for animals and have been investigated and characterized in numerous species for many years. In contrast, nectar proteins have been the subject of few studies and little is known of their function. Only very recently have detailed studies and characterization of nectar proteins been undertaken, and then for only a very few species. This current work represents a first step in the identification of a protein profile for the floral nectar of Cucurbita pepo. In this regard, the species studied is of particular interest in that it is monoecious with unisexual flowers and, consequently, it is possible that nectar proteins derived from male and female flowers may differ., Methods: Manually excised spots from two-dimensional (2-D) electrophoresis were subjected to in-gel protein digestion. The resulting peptides were sequenced using nanoscale LC-ESI/MS-MS (liquid chromatography-electrospray ionization/tandem mass spectrometry). An MS/MS ions search was carried out in Swiss-Prot and NCBInr databases using MASCOT software., Key Results: Two-dimensional electrophoresis revealed a total of 24 spots and a different protein profile for male and female flower nectar. Four main proteins recognized by 2-D electrophoresis most closely resemble β-d-xylosidases from Arabidopsis thaliana and have some homology to a β-d-xylosidase from Medicago varia. They were present in similar quantities in male and female flowers and had the same molecular weight, but with slightly different isoelectric points., Conclusions: A putative function for xylosidases in floral nectar of C. pepo is proposed, namely that they may be involved in degrading the oligosaccharides released by the nectary cell walls in response to hydrolytic enzymes produced by invading micro-organisms. Several types of oligosaccharides have been reported to increase the pathogenic potential of micro-organisms. Thus, it is possible that such a mechanism may reduce the virulence of pathogens present in nectar.

In the present study, enzyme urease has been immobilized on amine-functionalized gold nanoparticles (AuNPs). AuNPs were synthesized using natural precursor, i.e., clove extract and amine functionalized through 0.004 M L: -cysteine. Enzyme (urease) was extracted and purified from the vegetable waste, i.e., seeds of pumpkin to apparent homogeneity (sp. activity 353 U/mg protein). FTIR spectroscopy and transmission electron microscopy was used to characterize the immobilized enzyme. The immobilized enzyme exhibited enhanced activity as compared with the enzyme in the solution, especially, at lower enzyme concentration. Based on the evaluation of activity assay of the immobilized enzyme, it was found that the immobilized enzyme was quite stable for about a month and could successfully be used even after eight cycles having enzyme activity of about 47%. In addition to this central composite design (CCD) with the help of MINITAB version 15 Software was utilized to optimize the process variables viz., pH and temperature affecting the enzyme activity upon immobilization on AuNPs. The results predicted by the design were found in good agreement (R2 = 96.38%) with the experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed the individual and cumulative effect of pH and temperature on enzyme activity indicating that the activity increased with the increase of pH up to 7.5 and temperature 75 °C. The effects of each variables represented by main effect plot, 3D surface plot, isoresponse contour plot and optimized plot were helpful in predicting results by performing a limited set of experiments.

Background and Aims: Aside from those on Arabidopsis, very few studies have focused on spatial expression of cyclin-dependent kinases (CDKs) in root apical meristems (RAMs), and, indeed, none has been undertaken for open meristems. The extent of interfacing between cell cycle genes and plant growth regulators is also an increasingly important issue in plant cell cycle studies. Here spatial expression/localization of an A-type and B-type CDK, auxin and cytokinins are reported in relation to the hitherto unexplored anatomy of RAMs of Cucurbita maxima., Methods: Median longitudinal sections were cut from 1-cm-long primary root tips of C. maxima. Full-length A-type CDKs and a B-type CDK were cloned from C. maxima using degenerate primers, probes of which were localized on sections of RAMs using in situ hybridization. Isopentenyladenine (iPA), trans-zeatin (t-Z) and indole-3yl-acetic acid (IAA) were identified on sections by immunolocalization., Key Results: The C. cucurbita RAM conformed to an open transverse (OT) meristem typified by an absence of a clear boundary between the eumeristem and root cap columella, but with a distinctive longitudinally thickened epidermis. Cucma;CDKA;1 expression was detected strongly in the longitudinally thickened epidermis, a tissue with mitotic competence that contributes cells radially to the root cap of OT meristems. Cucma;CDKB2 was expressed mainly in proliferative regions of the RAM and in lateral root primordia. iPA and t-Z were mainly distributed in differentiated cells whilst IAA was distributed more uniformly in all tissues of the RAM., Conclusions: Cucma;CDKA;1 was expressed most strongly in cells that have proliferative competence whereas Cucma;CDKB2 was confined mainly to mitotic cells. iPA and t-Z marked differentiated cells in the RAM, consistent with the known effect of cytokinins in promoting differentiation in root systems. iPA/t-Z were distributed in a converse pattern to Cucma;CDKB2 expression whereas IAA was detected in most cells in the RAM regardless of their proliferative potential.

A pumpkin 2S albumin with ribonuclease (RNase) activity was purified from pumpkin seeds (Cucurbita sp.) by liquid chromatographic techniques. It manifested potent RNase activity toward baker's yeast RNA and calf liver RNA, and some polyhomoribonucleotides, including poly(A), poly(U) and poly(C) but not poly(G). Moreover, it was able to hydrolyze total RNA of both animal and plant origins. Ions such as Na(+), Mg(2+), Ca(2+), and Zn(2+) inhibited its RNase activity. Since RNase activity has not been previously reported in 2S albumins, this work may shed further light on the biological importance of this group of proteins., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

An amperometric biosensor based on zucchini (Cucurbita pepo) tissue homogenate is presented. The zucchini tissue homogenate was crosslinked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The zucchini tissue contained the enzyme ascorbate oxidase and this enzyme catalyzed the oxidation of ascorbic acid in the presence of dissolved oxygen. The principle of the measurements was based on the determination of the decrease in the dissolved oxygen level. Determinations were carried out by standard curves, which were obtained by the measurement of the decrease in the oxygen level related to ascorbic acid concentration. Optimization and characterization studies of the biosensor were carried out in detail. First of all, the amounts of zucchini tissue homogenate, gelatin, and glutaraldehyde percentage were optimized. Experimental parameters such as buffer system, pH, buffer concentration, and temperature were also optimized carefully. Thermal stability, storage stability, and repeatability of the biosensor were investigated. A linear response was observed from 5x10(-6) M to 1.2x10(-3) M ascorbic acid. Finally, the results of some plant and drug samples analyzed with the presented biosensor compared with the spectrophotometric method (Tillman reagent) used as a reference.

The ascorbate oxidase is the enzyme used to determine the content of ascorbic acid in the pharmaceutical and food industries and clinics analyses. The techniques currently used for the purification of this enzyme raise its production cost. Thus, the development of alternative processes and with the potential to reduce costs is interesting. The application of aqueous two-phase system is proposed as an alternative to purification because it enables good separation of biomolecules. The objective of this study was to determine the conditions to continuously pre-purify the enzyme ascorbate oxidase by an aqueous two-phase system (PEG/citrate) using rotating column provided with perforated discs. Under the best conditions (20,000 g/mol PEG molar mass, 10% PEG concentration, and 25% citrate concentration), the system showed satisfactory results (partition coefficient, 3.35; separation efficiency, 54.98%; and purification factor, 1.46) and proved suitable for the pre-purification of ascorbate oxidase in continuous process.

Urease immobilized on alginate was utilized to detect and quantify As(3+) in aqueous solution. Urease from the seeds of pumpkin (vegetable waste) was purified to apparent homogeneity by heat treatment and gel filtration (Sephadex G-200). Further enzyme was entrapped in 3.5% alginate beads. Urea hydrolysis by enzyme revealed a clear dependence on the concentration and interaction time of As(3+). The process variables effecting the quantitation of As(3+) was investigated using central composite design with Minitab 15 software. The predicted results were found in good agreement (R(2)=96.71%) with experimental results indicating the applicability of proposed model. The multiple regression analysis and ANOVA showed that enzyme activity decreased with increase of As(3+) concentration and interaction time. 3D plot and contour plot between As(3+) concentration and interaction time was helpful to predict residual activity of enzyme for a particular As(3+) at a particular time.

The high number of peroxidase genes explains the description of numerous physiological functions and the fact that the in planta function of a single isoform has never been characterized yet. We analyzed in transgenic Arabidopsis thaliana the localization of a zucchini isoperoxidase (APRX), previously purified thanks to its pectin binding ability. We confirmed that the protein is localized near the cell wall, mainly produced in the elongation area of the hypocotyls and respond to exogenous auxin. In addition, the ectopic overexpression of APRX induced changes in growth pattern and a significant reduction of endogenous indole-3-acetic acid (IAA) level. In agreement with these observations APRX showed an elevated in vitro auxin oxidase activity. We propose that APRX participates in the negative feedback regulation of auxin level and consequently terminates the hypocotyl elongation process.

The casepase is considered to regulate the process of programmed cell death in the development of organisms. In this study, caspase 3-like protease was detected by immunohistochemistry and immunoelectron microscopy during the development of sieve element and tracheary element of stem in Cucurbita moschata Duch. Antibody with brown color (under light microscopy) and gold particles (under transmission electron microscopy) for detecting caspase 3-like protease was mainly displayed in inner phloem, external phloem and xylem in the region close to procambium. From the results it was considered that caspase 3-like protease did exist in vascular elements and played different roles during the development of sieve and tracheary elements, and different types of programmed cell death might be carried out. The caspase 3-like protease mainly participated in making cytoplasmic streaming cease and in degrading P-protein bodies; however, it rarely participated in the function for signal transferring in the developmental sieve element. However, it might induce calcium accumulation for rupturing the tonoplast in the signal of PCD in the developmental tracheary element.

We have previously demonstrated that the targeting signal of pumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targeting signal 1)-like sequence, QKL, located at -13 to -11 from the C-terminus, which is different from the typical PTS1 SKL motif located in the C-terminus. Here we show that Cat1 import into peroxisome is dependent on the cytosolic PTS receptor, Pex5p, in Arabidopsis, similar to typical PTS1 import, and that other components for transport of peroxisomal matrix proteins such as Pex14p, Pex13p, Pex12p and Pex10p also contribute to the import of Cat1. Interestingly, however, we found that Cat1 interacts with the N-terminal domain of Pex5p, but not the C-terminal domain for interaction with the typical PTS1, revealing that Pex5p recognizes Cat1 in a manner distinct from typical PTS1.

Two of the six esterases identified in Cucurbita pepo cv. "Eskandrani" were purified to homogeneity using two chromatography steps: anion exchange and gel filtration. The molecular weights of C. pepo esterases EIc and EII were 50,000 +/- 1500 and 68,000 +/- 1900 Da from gel filtration and 47,000 and 66,000 Da from SDS/PAGE, respectively, suggesting a monomeric structure for both enzymes. Esterases EIc and EII had K(m) values of 1.22 and 1.56 mM and pH optima at 9.0 and 8.0, respectively. The substrate specificity of C. pepo esterases EIc and EII were determined for a number of p-nitrophenyl esters, where their affinity toward these substrates were decreased as carbon atom number increased. Esterases EIc and EII had the same temperature optima, 40 degrees C. Thermal stability studies of esterases EIc and EII indicated that half maximal activities of EIc and EII esterases were reached at 55 degrees C and 50 degrees C, while they lost 45%, 51% and 70%, 77% of their activities after 30 and 90 min of incubation at 40 degrees C, respectively. The effect of different metal cations and inhibitors were examined. The inhibition studies revealed that the active sites of the two esterases contain serine and cysteine residues. The characteristics of C. pepo esterases are closely similar to those of microbial esterases used in food processing and food industry.

Ascorbic acid oxidase (AAO) is a plant blue-copper protein catalyzing dioxygen reduction to water using ascorbic acid as the electron donor. In spite of extensive molecular characterization the physiological role of AAO is still uncertain. Abundant mRNA, protein and activity of AAO were observed in illuminated leaves of Cucurbita pepo. AAO activity was found to be proportional to light intensity. The light effect was rapidly reversed in dark and activity remained low throughout the dark period. Activity was elicited in dark by increased oxygen concentration. AAO activity increased in the facultative CAM Kalanchoë blossfeldiana upon induction of the CAM cycle and decreased during germination of C. pepo and Zea mays under hypoxic conditions. These results strongly suggest that AAO activity could be part of a dynamic system for oxygen management in plants.

The kinetic and thermodynamic properties of ascorbate oxidase (AO) activity and stability of a Cucurbita maxima extract were investigated. Activity tests performed at 25 degrees C using initial ascorbic acid concentration in the range 50-750 M allowed estimating the Michaelis constant for this substrate (Km = 126 microM) and the maximum initial rate of ascorbic acid oxidation (A0,max = 1.57 mM min-1). The main thermodynamic parameters of the enzyme reaction (DeltaH* = 10.3 kJ mol-1; DeltaG* = 87.2 kJ mol-1; DeltaS* = -258 J mol-1 K-1) were estimated through activity tests performed at 25-48 C. Within such a temperature range, no decrease in the initial reaction rate was detected. The long-term thermostability of the raw extract was then investigated by means of residual activity tests carried out at 10-70 degrees C, which allowed estimating the thermodynamic parameters of the irreversible enzyme inactivation as well (DeltaH*D = 51.7 kJ mol-1; DeltaG*D = 103 kJ mol-1; S*D = -160 J mol-1 K-1). Taking into account the specific rate of AO inactivation determined at different temperatures, we also estimated the enzyme half-life (1047 min at 10 degrees C and 21.2 min at 70 degrees C) and predicted the integral activity of a continuous system using this enzyme preparation. This work should be considered as a preliminary attempt to characterize the AO activity of a C. maxima extract before its concentration by liquid-liquid extraction techniques.

Gibberellins (GAs) form a large family of plant growth substances with distinct functions during the whole life cycle of higher plants. The rate of GA biosynthesis and catabolism determines how the GA hormone pool occurs in plants in a tissue and developmentally regulated manner. With the availability of genes coding for GA biosynthetic enzymes, our understanding has improved dramatically of how GA plant hormones regulate and integrate a wide range of growth and developmental processes. This review focuses on two plant systems, pumpkin and Arabidopsis, which have added significantly to our understanding of GA biosynthesis and its regulation. In addition, we present models for regulation of GA biosynthesis in transgenic plants, and discuss their suitability for altering plant growth and development.

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.

Previous studies show that low temperature strongly induces suberin layers in the roots of chilling-sensitive cucumber plants, while in contrast, low temperature produces a much weaker induction of suberin layers in the roots of the chilling-tolerant figleaf gourd [S.H. Lee, G.C. Chung, S. Steudle, Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and -tolerant figleaf gourd, J. Exp. Bot. 56 (2005) 985-995; S.H. Lee, G.C. Chung, E. Steudle, Low temperature and mechanical stresses differently gate aquaporins of root cortical cells of chilling-sensitive cucumber and figleaf gourd, Plant Cell Environ. (2005) in press; S.J. Ahn, Y.J. Im, G.C. Chung, B.H. Cho, S.R. Suh, Physiological responses of grafted-cucumber leaves and rootstock roots affected by low root temperature, Scientia Hort. 81 (1999) 397-408]. Here, the effect of low temperature on fatty acid unsaturation and lipoxygenase activity was examined in cucumber and figleaf gourd. The double bond index demonstrated that membrane lipid unsaturation shows hyperbolic saturation curve in figleaf gourd roots while a biphasic response in cucumber roots to low temperature. In figleaf gourd, the hyperbolic response in the double bond index was primarily due to accumulation of linolenic acid. Chilling stress also significantly induced lipoxygenase activity in figleaf gourd roots. These results suggest that the degree of unsaturation of root plasma membrane lipids correlates positively with chilling-tolerance. Therefore, studies that compare the effects of chilling on cucumber and figleaf gourd may provide broad insight into stress response mechanisms in chilling-sensitive and chilling-tolerant plants. Furthermore, these studies may provide important information regarding the relationship between lipid unsaturation and lipoxygenase function/activity, and between lipoxygenase activity and water channeling during the response to chilling stress. The possible roles of these processes in chilling tolerance are discussed.

Intramolecular electron transfer within zucchini squash ascorbate oxidase is inhibited in a novel manner in the presence of an equimolar concentration of Ag(+). At pH 5.5 in acetate buffer reduction of the enzyme by laser flash photolytically generated 5-deazariboflavin semiquinone occurs at the Type I Cu with a rate constant of 5 x 10(8) M(-1)s(-1). Subsequent to this initial reduction step, equilibration of the reducing equivalent between the Type I Cu and the trinuclear Type II, III copper cluster (TNC) occurs with rate constant of 430 s(-1). The 41% of the reduced Type I Cu is oxidized by this intramolecular electron transfer reaction. When these reactions are performed in the presence of Ag(+) equimolar to dimeric AO, the bimolecular reduction of the enzyme by the 5-deazariboflavin semiquinone is not affected. As in the case of the native enzyme, intramolecular electron transfer between the Type I Cu and the TNC occurs, which continues until 25% of the reducing equivalent has been transferred. At that point, the reducing equivalent is observed to more slowly return to the Type I Cu, resulting a second reduction phase whose rate constant (100 s(-1)) is protein and Ag(+) concentration independent. The data suggest that partial reduction of the TNC results in Ag(+) binding to the enzyme which causes the apparent midpoint potential of the TNC as a whole to decrease thereby reversing the direction of electron flow. These results are consistent with the inhibitory effect of Ag(+) on the steady-state activity of ascorbate oxidase [S. Maritano, E. Malusa, A. Marchesini, presented at The Meeting on Metalloproteins, SERC Daresbury Laboratory, Warrington, England, 1992; A. Marchesini, XIX Convegno Nazionale SICA, Italian Society of Agricultural Chemistry, Reggio Calabria, Italy, September 2001.].

A biosensor modified with peroxidase from crude extract of zucchini (Cucurbita pepo) was developed for the determination of dopamine in pharmaceutical samples. This enzyme catalyses the oxidation of dopamine to dopaminequinone, in presence of hydrogen peroxide, which the electrochemical reduction can be followed at a peak potential of -0.02 V. The recovery of dopamine from the samples ranged from 94.8% to 106% and a rectilinear analytical curve for dopamine concentration from 5.0 x 10(-4) to 3.0 x 10(-3) mol L-1 (r=0.9982) was obtained. The detection limit was 2.6x10(-5) mol L-1 and the relative standard deviation was less than 1.2% for 7.9 x 10(-4) mol L-1 dopamine in 0.1 mol L-1 phosphate buffer solution at pH 6.0 (n=10).

The expression of three genes that encode proteins involved in peroxisome biogenesis, beta-oxidation and the glyoxylate cycle was studied in Arabidopsis plants by fusing their promoter regions to the reporter gene luciferase. Malate synthase showed an extremely restricted pattern of expression, being detected only in young seedlings and the root tips of older plants. PEX1 and 3-ketoacyl thiolase (PED1) were expressed in roots, mature leaves, stems and flowers. However, only thiolase was up-regulated by starvation. Immunoblotting confirmed that neither malate synthase nor the other unique glyoxylate cycle enzyme isocitrate lyase are expressed in senescent leaves. These results indicate that, in contrast to cucumber, pumpkin and barley, the glyoxylate cycle does not play a role in the recycling of carbon from the turnover of membrane lipids during senescence and starvation in Arabidopsis.

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interest for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase-ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.