Transcriptomic analysis reveals the molecular basis of photoperiod-regulated sex differentiation in tropical pumpkins (Cucurbita moschata Duch.).

BACKGROUND: Photoperiod, or the length of the day, has a significant impact on the flowering and sex differentiation of photoperiod-sensitive crops. The "miben" pumpkin (the main type of Cucurbita moschata Duch.) is well-known for its high yield and strong disease resistance. However, its cultivation has been limited due to its sensitivity to photoperiod. This sensitivity imposes challenges on its widespread cultivation and may result in suboptimal yields in regions with specific daylength conditions. As a consequence, efforts are being made to explore potential strategies or breeding techniques to enhance its adaptability to a broader range of photoperiods, thus unlocking its full cultivation potential and further promoting its valuable traits in agriculture. RESULTS: This study aimed to identify photoperiod-insensitive germplasm exhibiting no difference in sex differentiation under different day-length conditions. The investigation involved a phenotypic analysis of photoperiod-sensitive (PPS) and photoperiod-insensitive (PPIS) pumpkin materials exposed to different day lengths, including long days (LDs) and short days (SDs). The results revealed that female flower differentiation was significantly inhibited in PPS_LD, while no differences were observed in the other three groups (PPS_SD, PPIS_LD, and PPIS_SD). Transcriptome analysis was carried out for these four groups to explore the main-effect genes of sex differentiation responsive to photoperiod. The main-effect gene subclusters were identified based on the principal component and hierarchical cluster analyses. Further, functional annotations and enrichment analysis revealed significant upregulation of photoreceptors (CmCRY1, F-box/kelch-repeat protein), circadian rhythm-related genes (CmGI, CmPRR9, etc.), and CONSTANS (CO) in PPS_LD. Conversely, a significant downregulation was observed in most Nuclear Factor Y (NF-Y) transcription factors. Regarding the gibberellic acid (GA) signal transduction pathway, positive regulators of GA signaling (CmSCL3, CmSCL13, and so forth) displayed higher expression levels, while the negative regulators of GA signaling, CmGAI, exhibited lower expression levels in PPS_LD. Notably, this effect was not observed in the synthetic pathway genes. Furthermore, genes associated with ethylene synthesis and signal transduction (CmACO3, CmACO1, CmERF118, CmERF118-like1,2, CmWIN1-like, and CmRAP2-7-like) showed significant downregulation. CONCLUSIONS: This study offered a crucial theoretical and genetic basis for understanding how photoperiod influences the mechanism of female flower differentiation in pumpkins.

Biosynthesis, Characterization, and Biomedical Applications of Gold Nanoparticles with Cucurbita moschata Duchesne Ex Poiret Peel Aqueous Extracts.

In this study, AuNPs were biosynthesized from Cucurbita moschata fruit peel extracts. Biosynthesized AuNPs exhibited maximum absorbance at a 555 nm wavelength, and XRD analysis indicated that the CM-AuNPs had a particle size of less than 100 nm and a cubic crystal structure. TEM scans revealed that the gold particles exhibited a spherical morphology, with an average size of 18.10 nm. FTIR analysis revealed strong peaks indicating the presence of functional groups involved in the reduction reactions. The surface charge of the biosynthesized AuNPs was determined to be -19.7 mV. The antibacterial and antifungal activities of AuNPs against pathogen strains were assessed by the minimum inhibitory concentration (MIC) method. The cytotoxic effects of CM-AuNPs on cancer cell lines (Sk-Ov-3, CaCo2, and A549) and healthy cell lines (HUVEC) were investigated using the MTT method. The findings indicated that AuNPs biosynthesized by the green synthesis method using C. moschata peel aqueous extract had high inhibition on the growth of pathogenic microorganisms and effective cytotoxic activity against cancerous cell lines at low doses. As a result, it can be concluded that CM-AuNPs will be eminently effective in the production of antibacterial and/or anticancer drugs in the pharmaceutical, food, and cosmetic industries.

Identification of a Major-Effect Quantitative Trait Loci Associated with Begomovirus Resistance in Cucurbita moschata.

Begomoviruses, viz. squash leaf curl China virus and tomato leaf curl New Delhi virus causative diseases are major concerns of quantitative and qualitative losses in pumpkin (Cucurbita moschata) worldwide. Punjab Agricultural University (PAU) in India has identified a resistant source (PVR-1343) against mixed infection (MI-Sq/To) of these begomoviruses. Introgression of resistance in diverse genetic backgrounds requires the identification of quantitative trait loci (QTLs) associated with MI-Sq/To resistance. Phenotyping of 229 F2:3 progenies derived from the PVR-1343 × P-135 cross revealed digenic recessive inheritance against MI-Sq/To resistance in PVR-1343. To identify the genomic region, resistant and susceptible bulks were subjected to whole-genome resequencing along with their parents. The whole-genome resequence analysis of parents and bulks using QTLseq/QTLseqr approaches identified an overlapping 1.52 Mb region on chromosome 7 (qMI-Sq/To7.1), while chromosomal region spanning 0.87 Mb on chromosome17 (qMI-Sq/To17.1) was additionally identified by QTLseqr. However, the highest peak value on chromosome 7 with three algorithms {G', ∆(SNP-index) and -log10 (P value)} highlighted the major contribution of qMI-Sq/To7.1 in MI-Sq/To resistance. Nine polymorphic SNPs identified within the highly significant qMI-Sq/To7.1 region were converted into KASP markers. KASP genotyping of F2 individuals narrowed down the qMI-Sq/To7.1 interval to 103 kb region flanked by two markers, Cmo3914729 and Cmo4018182, which contained 16 annotated genes and accounted for 59.84% of phenotypic variation. The Cmo4018182 KASP marker accurately predicted disease reaction in 91% of diverse Cucurbita genotypes and showed nonsynonym substitutions in the coding region of putative candidate SYNTAXIN-121 gene. These findings pave the way for marker-assisted breeding and elucidating the underlying mechanism of begomovirus resistance in C. moschata.

Genome-Wide Identification of BES1 Gene Family in Six Cucurbitaceae Species and Its Expression Analysis in Cucurbita moschata.

The BES1 (BRI1-EMSSUPPRESSOR1) gene family play a vital role in the BR (brassinosteroid) signaling pathway, which is involved in the growth and development, biotic, abiotic, and hormone stress response in many plants. However, there are few reports of BES1 in Cucurbita moschata. In this study, 50 BES1 genes were identified in six Cucurbitaceae species by genome-wide analysis, which could be classified into 3 groups according to their gene structural features and motif compositions, and 13 CmoBES1 genes in Cucurbita moschata were mapped on 10 chromosomes. Quantitative real-time PCR analysis showed that the CmoBES1 genes displayed differential expression under different abiotic stress and hormone treatments. Subcellular localization showed that the most of CmoBES1 proteins localized in nucleus and cytoplasm, and transactivation assay indicated 9 CmoBES1 proteins played roles as transcription factors. Our analysis of BES1s diversity, localization, and expression in Curcubitaceae contributes to the better understanding of the essential roles of these transcription factors in plants.

In Vivo, In Vitro and In Silico Study of Cucurbita moschata Flower Extract: A Promising Source of Natural Analgesic, Anti-Inflammatory, and Antibacterial Agents.

For thousands of years, medicinal plants have played a pivotal role in maintaining human health and improving the quality of human life. This study was designed to analyze the analgesic, anti-inflammatory, and antibacterial potentials of a hydro-methanolic extract of Cucurbita moschata flowers, along with qualitative and quantitative phytochemical screening. The anti-inflammatory effect was tested using the in vitro membrane stabilizing method for human red blood cells (HRBC), the analgesic effect was tested using the in vivo acetic acid-induced writing method, and the antibacterial effect was tested using the disc diffusion method. In silico ADME/T and molecular docking studies were performed to assess the potential of the stated phytochemicals against Cyclooxygenase-II enzyme. Phytochemical screening confirmed the presence of flavonoids, alkaloids, glycosides, tannins, and carbohydrates. The flower extract demonstrated the maximum protection of human red blood cells at 1000 µg/mL, with a 65.73% reduction in hemolysis in a hypotonic solution. The extract also showed significant (p < 0.05) and dose-dependent analgesic effects at oral doses of 200 and 400 mg/kg on the tested animals. Furthermore, the flower extract exhibited potent antibacterial activity due to the disc diffusion method, which was compared with standard ciprofloxacin. In silico testing revealed that 42 phytochemicals exhibited notable pharmacokinetic properties and passed drug likeness screening tests. Among the six best-selected compounds, 3,4-dihydro-2H-pyran-2-yl)methanamine showed the highest binding affinity (-10.1) with significant non-bonding interactions with the target enzyme. In conclusion, the hydro-methanolic extract of Cucurbita moschata was found to be rich in various phytochemicals that may be associated with therapeutic potential, and this study supports the traditional use of Cucurbita moschata flowers in the management of inflammation and painful conditions.

GWAS Reveals a Novel Candidate Gene CmoAP2/ERF in Pumpkin (Cucurbita moschata) Involved in Resistance to Powdery Mildew.

Pumpkin (Cucurbita moschata Duchesne ex Poir.) is a multipurpose cash crop rich in antioxidants, minerals, and vitamins; the seeds are also a good source of quality oils. However, pumpkin is susceptible to the fungus Podosphaera xanthii, an obligate biotrophic pathogen, which usually causes powdery mildew (PM) on both sides of the leaves and reduces photosynthesis. The fruits of infected plants are often smaller than usual and unpalatable. This study identified a novel gene that involves PM resistance in pumpkins through a genome-wide association study (GWAS). The allelic variation identified in the CmoCh3G009850 gene encoding for AP2-like ethylene-responsive transcription factor (CmoAP2/ERF) was proven to be involved in PM resistance. Validation of the GWAS data revealed six single nucleotide polymorphism (SNP) variations in the CmoAP2/ERF coding sequence between the resistant (IT 274039 [PMR]) and the susceptible (IT 278592 [PMS]). A polymorphic marker (dCAPS) was developed based on the allelic diversity to differentiate these two haplotypes. Genetic analysis in the segregating population derived from PMS and PMR parents provided evidence for an incomplete dominant gene-mediated PM resistance. Further, the qRT-PCR assay validated the elevated expression of CmoAP2/ERF during PM infection in the PMR compared with PMS. These results highlighted the pivotal role of CmoAP2/ERF in conferring resistance to PM and identifies it as a valuable molecular entity for breeding resistant pumpkin cultivars.

The Influence of Ripeness on the Phenolic Content, Antioxidant and Antimicrobial Activities of Pumpkins (Cucurbita moschata Duchesne).

Cucurbita moschata Duchesne (Cucurbitaceae) is a plant food highly appreciated for the content of nutrients and bioactive compounds, including polyphenols and carotenoids, which contribute to its antioxidant and antimicrobial capacities. The purpose of this study was to identify phenolic acids and flavonoids of Cucurbita moschata Duchesne using high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS) at different ripening stages (young, mature, ripened) and determine its antioxidant and antimicrobial activities. According to the results, phenolic acids and flavonoids were dependent on the maturity stage. The mature fruits contain the highest total phenolic and flavonoids contents (97.4 mg GAE. 100 g-1 and 28.6 mg QE. 100 g-1).A total of 33 compounds were identified. Syringic acid was the most abundant compound (37%), followed by cinnamic acid (12%) and protocatechuic acid (11%). Polyphenol extract of the mature fruits showed the highest antioxidant activity when measured by DPPH (0.065 µmol TE/g) and ABTS (0.074 µmol TE/g) assays. In the antimicrobial assay, the second stage of ripening had the highest antibacterial activity. Staphylococcus aureus was the most sensitive strain with an inhibition zone of 12 mm and a MIC of 0.75 mg L-1. The lowest inhibition zone was obtained with Salmonella typhimurium (5 mm), and the MIC value was 10 mg L-1.

Application of the nutrient profile model in the development of food bars with functional potential.

In the present study, the nutrient profile model is used to develop functionally potential food bars prepared with pumpkin pulp flour (PuPF) and pequi peel flour (PePF). The control (0% PuPF, 0% PePF), and treatments T1 (0% PuPF, 2.5% PePF), T2 (5% PuPF, 2.5% PePF), T3 (10% PuPF, 2.5% PePF), and T4 (17.5% PuPF, 2.5% PePF) were defined using the nutrient profile model and subsequently evaluated for antioxidant activity, sensory acceptance, and nutrient profile. All treatments were nutritionally balanced but T3 and T4 were the best, containing, respectively, 35.11 and 59.85 µg g-1 of carotenoids, 230.60 and 261.14 mg gallic acid equivalents 100 g-1 of phenolics, and high antioxidant activity, as determined using ABTS (28.60 and 34.86 µM Trolox g-1), FRAP (67.13 and 80.09 µM ferrous sulfate g-1), and ß-carotene/linoleic acid system (79.08 and 84.83% protection) methods. The nutrient profile model minimized time and resource expenditures throughout the development process.

Metabolic and transcriptomic analysis of two Cucurbita moschata germplasms throughout fruit development.

BACKGROUND: Pumpkins (Cucurbita moschata; Cucurbitaceae) are valued for their fruits and seeds and are rich in nutrients. Carotenoids and sugar contents, as main feature of pumpkin pulp, are used to determine the fruit quality. RESULTS: Two pumpkin germplasms, CMO-X and CMO-E, were analyzed regarding the essential quality traits such as dry weight, soluble solids, organic acids, carotenoids and sugar contents. For the comparison of fruit development in these two germplasms, fruit transcriptome was analyzed at 5 different developmental stages from 0 d to 40 d in a time course manner. Putative pathways for carotenoids biosynthesis and sucrose metabolism were developed in C. moschata fruit and homologs were identified for each key gene involved in the pathways. Gene expression data was found consistent with the accumulation of metabolites across developmental stages and also between two germplasms. PSY, PDS, ZEP, CRTISO and SUS, SPS, HK, FK were found highly correlated with the accumulation of carotenoids and sucrose metabolites, respectively, at different growth stages of C. moschata as shown by whole transcriptomic analysis. The results of qRT-PCR analysis further confirmed the association of these genes. CONCLUSION: Developmental regulation of the genes associated with the metabolite accumulation can be considered as an important factor for the determination of C. moschata fruit quality. This research will facilitate the investigation of metabolic profiles in other cultivars.

Genome-wide characterization and expression analysis of the heat shock transcription factor family in pumpkin (Cucurbita moschata).

BACKGROUND: Crop quality and yield are affected by abiotic and biotic stresses, and heat shock transcription factors (Hsfs) are considered to play important roles in regulating plant tolerance under various stresses. To investigate the response of Cucurbita moschata to abiotic stress, we analyzed the genome of C. moschata. RESULTS: In this research, a total of 36 C. moschata Hsf (CmHsf) members were identified and classified into three subfamilies (I, II, and III) according to their amino acid sequence identity. The Hsfs of the same subfamily usually exhibit a similar gene structure (intron-exon distribution) and conserved domains (DNA-binding and other functional domains). Chromosome localization analysis showed that the 36 CmHsfs were unevenly distributed on 18 of the 21 chromosomes (except for Cm_Chr00, Cm_Chr08 and Cm_Chr20), among which 18 genes formed 9 duplicated gene pairs that have undergone segmental duplication events. The Ka/Ks ratio showed that the duplicated CmHsfs have mainly experienced strong purifying selection. High-level synteny was observed between C. moschata and other Cucurbitaceae species. CONCLUSIONS: The expression profile of CmHsfs in the roots, stems, cotyledons and true leaves revealed that the CmHsfs exhibit tissue specificity. The analysis of cis-acting elements and quantitative real-time polymerase chain reaction (qRT-PCR) revealed that some key CmHsfs were activated by cold stress, heat stress, hormones and salicylic acid. This study lays the foundation for revealing the role of CmHsfs in resistance to various stresses, which is of great significance for the selection of stress-tolerant C. moschata.

Water extract of tendril of Cucurbita Moschata Duch. suppresses RANKL-induced osteoclastogenesis by down-regulating p38 and ERK signaling.

Background: Pumpkin (Curcubita sp.) is a natural product that is commonly used in folk medicine. However, the inhibitory effect and molecular mechanisms of tendril of Cucurbita Moschata Duch. (TCMD) on osteoclast differentiation have yet to be clearly elucidated. Thus, the present study aimed to investigate the effect and underlying mechanism of water extract of TCMD on osteoclast differentiation. Methods: Bone marrow-derived macrophages (BMDMs), osteoclast precursors, were cultured with macrophage colony stimulating factor (M-CSF) 30 ng/ml and receptor activator of nuclear factor-kappa B ligand (RANKL) 100 ng/ml for four days. We investigated the effect of TCMD on RANKL-induced osteoclast differentiation, tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation, and bone resorption assay. RANKL signaling pathways were determined through Western blotting, and osteoclast differentiation marker genes were confirmed by Real-time PCR. Results: TCMD inhibited the RANKL-induced osteoclast differentiation in a dose-dependent manner without cytotoxicity. Further, F-actin ring formation and bone resorption were reduced by TCMD in RANKL-treated BMDMs. In addition, TCMD decreased the phosphorylation of p38 and ERK as well as the expression of osteoclast-related genes in BMDMs treated with RANKL. Conclusion: These findings suggest that TCMD may have preventive and therapeutic effects for destructive bone diseases.

Tendril extract of Cucurbita moschata suppresses NLRP3 inflammasome activation in murine macrophages and human trophoblast cells.

Inflammation is the root cause of many diseases that pose a serious threat to human health. Excessive inflammation can also result in preterm birth or miscarriage in pregnant women. Pumpkin (Cucurbita moschata Duchesne, CMD) is a well-known traditional health food and medicinal herb used in many countries to treat diabetes, obesity, osteoporosis, cancer and other diseases. In this study, we investigated the effects of hot water extract derived from the tendrils of C. moschata Duchesne (TCMD) on NLRP3 inflammasome activation in murine macrophages and human trophoblast cells. The TCMD treatment of LPS-primed bone marrow-derived macrophages (BMDMs) and human trophoblast cells attenuated NLRP3 inflammasome activation induced by inflammasome activators such as ATP, nigericin, and monosodium urate (MSU). TCMD treatment suppressed IL-1ß secretion in a dose-dependent manner, without affecting IL-6 secretion. In addition, TCMD inhibited NLRP3-dependent pyroptosis in BMDMs. TCMD also suppressed the release of mature IL-1ß and activation of cleaved-caspase-1 via limited ASC oligomerization. Furthermore, TCMD significantly inhibited IL-1ß secretion and pyroptotic cell death in human trophoblast cells. These results suggest that TCMD exhibits anti-inflammatory effects mediated via inhibition of NLRP3 inflammasome activation suggesting therapeutic potential against inflammatory diseases, preterm birth, and miscarriage.

New 8-C-p-Hydroxylbenzylflavonol Glycosides from Pumpkin (Cucurbita moschata Duch.) Tendril and Their Osteoclast Differentiation Inhibitory Activities.

Six new 8-C-p-hydroxybenzylflavonol glycosides were isolated from a hot water extract of pumpkin (Cucurbita moschata Duch.) tendril and elucidated as 8-C-p-hydroxybenzylquercetin 3-O-rutinoside, 8-C-p-hydroxybenzoylquercetin 3-O-ß-D-glucopyranoside, 8-C-p-hydroxybenzylkaempferol 3-O-(α-L-rhamnopyranosyl(1→6)-ß-D-galactopyranoside, 8-C-p-hydroxybenzoylkaempferol 3-O-rutinoside, 8-C-p-hydroxybenzylisorhamnetin 3-O-rutinoside, and 8-C-p-hydroxybenzylisorhamnetin 3-O-(α-L-rhamnopyranosyl(1→6)-ß-D-galactopyranoside. Their chemical structures were determined using nuclear magnetic resonance (NMR) and electrospray ionization-mass spectrometer (ESIMS) analyses. The 8-C-p-hydroxybenzylflavonol glycosides were found to inhibit the receptor activator of nuclear factor-κB (RANKL)-induced osteoclast differentiation of bone marrow derived macrophage (BMDM), an osteoclast progenitor. Additionally, 8-C-p-hydroxybenzylflavonol glycosides effectively reduced the expression of osteoclast-related genes, such as tartrate-resistant acid phosphatase, cathepsin K, nuclear factor activated T-cell cytoplasmic 1, and dendritic cell specific transmembrane protein in RANKL-treated BMDMs. These results indicate that the 8-C-p-hydroxybenzylflavonol glycosides may be the main components responsible for the osteoclast differentiation inhibitory effect of pumpkin tendril.

The Profile of Secondary Metabolites and Other Bioactive Compounds in Cucurbita pepo L. and Cucurbita moschata Pumpkin Cultivars.

Plants and animals are sources of various bioactive compounds that exhibit a broad spectrum of health-promoting effects. Scientists continue studies on the chemical composition of many products in search of foods with high nutritional value. The pumpkin (Cucurbita sp.) is unquestionably a source of valuable nutrients. This vegetable is well-known all over the world and it is appreciated due to its high content of carotenoids, but it is still not much used in the processing industry. The aim of present study was to compare the flesh of 15 pumpkin varieties belonging to the Cucurbita pepo and C. moschata species in terms of the bioactive compound content (carotenoids, phenolic acids, flavonols, minerals and vitamins) and to demonstrate whether the variety has an effect on the chemical composition. To date, no such extensive research has been carried out in this area. The research revealed that the pumpkin pulp had high content of carotenoids. In nearly all cases lutein was the most abundant carotenoid. Numerous phenolic acids and flavonols were also identified. All the cultivars contained gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, chlorogenic acid, caffeic acid, and rutin. The pumpkin pulp also contained alpha- and gamma-tocopherol. No beta- or delta-tocopherol was found. Potassium, calcium, and sodium were the most abundant minerals. The research also proved that the profile of bioactive compounds in the pumpkin pulp was considerably diversified and depended on the species and cultivar.

Screening of Cucurbita maxima and Cucurbita moschata Genotypes for Resistance Against Meloidogyne arenaria, M. incognita, M. javanica, and M. luci.

The host response of fifteen winter squash (Cucurbita maxima) and five pumpkin (Cucurbita moschata) dihaploid genotypes to Meloidogyne arenaria, M. incognita, M. javanica, and M. luci was screened in pot experiments. Root galling and nematode reproduction were detected in all combinations of plant genotype and nematode species. Ten genotypes of C. maxima and three genotypes of C. moschata were considered highly resistant (<10% of the susceptible genotype) or moderately resistant (<50% of the susceptible genotype) to one or more Meloidogyne species based on nematode reproduction as a percentage of the most susceptible genotype. Genotypes 55CA15-A3 and G14-IP1 of C. maxima were highly resistant to M. luci and M. arenaria, respectively. Both 14BO01-O2 and G9-A4 genotypes of C. moschata were considered highly resistant to M. arenaria. However, these genotypes still allowed significant nematode reproduction because egg number per plant was higher than initial number of eggs used as inoculum, indicating that all genotypes were hosts.

Biodegradation of γ-hexachlorocyclohexane by transgenic hairy root cultures of Cucurbita moschata that accumulate recombinant bacterial LinA.

KEY MESSAGE: γ-HCH was successfully degraded using LinA-expressed transgenic hairy root cultures of Cucurbita moschata . Fusing an endoplasmic reticulum-targeting signal peptide to LinA was essential for stable accumulation in the hairy roots. The pesticide γ-hexachlorocyclohexane (γ-HCH) is a persistent organic pollutant (POP) that raises public health and environmental pollution concerns worldwide. Although several isolates of γ-HCH-degrading bacteria are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the bacterial survival rate. Cucurbita species incorporate significant amounts of POPs from soils compared with other plant species. Here, we describe a novel bioremediation strategy that combines the bacterial degradation of γ-HCH and the efficient uptake of γ-HCH by Cucurbita species. We produced transgenic hairy root cultures of Cucurbita moschata that expressed recombinant bacterial linA, isolated from the bacterium Sphingobium japonicum UT26. The LinA protein was accumulated stably in the hairy root cultures by fusing an endoplasmic reticulum (ER)-targeting signal peptide to LinA. Then, we demonstrated that the cultures degraded more than 90 % of γ-HCH (1 ppm) overnight and produced the γ-HCH metabolite 1,2,4-trichlorobenzene, indicating that LinA degraded γ-HCH. These results indicate that the gene linA has high potential for phytoremediation of environmental γ-HCH.

Cucurbita moschata Duch. and its active component, ß-carotene effectively promote the immune responses through the activation of splenocytes and macrophages.

Cucurbita moschata Duch. has long been used for traditional health food in many countries. However, to enhance the immune system of Cucurbita moschata Duch. and its major component, ß-carotene is not clear. Here, we determined the immune enhancement effect of Cucurbita moschata Duch. and ß-carotene in mouse splenocytes and RAW 264.7 macrophage cell line. We prepared baked Cucurbita moschata Duch. (Sweetme Sweet Pumpkin(TM), SSP) and steamed Cucurbita moschata Duch. (SC). Splenocytes isolated from the spleen of BALB/c mice were treated with SSP, SC, and ß-carotene for 24 h. RAW 264.7 cells were stimulated with recombinant interferon-γ (rIFN-γ) for 6 h before treatment with SSP, SC, or ß-carotene. SSP, SC and ß-carotene significantly up-regulated the proliferation of splenocyte and mRNA expression of KI-67. The levels of interleukin-2 and IFN-γ were up-regulated by SSP, SC, or ß-carotene in the splenocytes. SC and ß-carotene also increased the levels of tumor necrosis factor-α (TNF-α) in the splenocytes. In addition, SSP, SC, or ß-carotene significantly increased the levels of TNF-α through the nuclear translocation of the nuclear factor-κB and phosphorylation of IκBα in the rIFN-γ-primed RAW 264.7 cells. These data indicate that Cucurbita moschata Duch. and ß-carotene may have an immune-enhancing effect through the production of Th1 cytokines by activation of splenocytes and macrophages.

A Novel Aqueous Two Phase System Composed of Surfactant and Xylitol for the Purification of Lipase from Pumpkin (Cucurbita moschata) Seeds and Recycling of Phase Components.

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.

Phloem long-distance delivery of FLOWERING LOCUS T (FT) to the apex.

Cucurbita moschata FLOWERING LOCUS T-LIKE 2 (hereafter FTL2) and Arabidopsis thaliana (Arabidopsis) FLOWERING LOCUS T (FT), components of the plant florigenic signaling system, move long-distance through the phloem from source leaves to the vegetative apex where they mediate floral induction. The mechanisms involved in long-distance trafficking of FT/FTL2 remain to be elucidated. In this study, we identified the critical motifs on both FT and FTL2 required for cell-to-cell trafficking through mutant analyses using a zucchini yellow mosaic virus expression vector. Western blot analysis, performed on phloem sap collected from just beneath the vegetative apex of C. moschata plants, established that all mutant proteins tested retained the ability to enter the phloem translocation stream. However, immunolocalization studies revealed that a number of these FTL2/FT mutants were defective in the post-phloem zone, suggesting that a regulation mechanism for FT trafficking exists in the post-phloem unloading step. The selective movements of FT/FTL2 were further observed by microinjection and trichome rescue studies, which revealed that FT/FTL2 has the ability to dilate plasmodesmata microchannels during the process of cell-to-cell trafficking, and various mutants were compromised in their capacity to traffic through plasmodesmata. Based on these findings, a model is presented to account for the mechanism by which FT/FTL2 enters the phloem translocation stream and subsequently exits the phloem and enters the apical tissue, where it initiates the vegetative to floral transition.