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Background: Pumpkin (Cucurbita moschata) is an important vegetable crop that often suffers from low-temperature stress during growth. However, the molecular mechanism involved in its response to chilling stress remains unknown. In this study, we comprehensively investigated the effect of chilling stress in pumpkin seedlings by conducting physiological, transcriptomic, and metabolomic analyses., Results: Under chilling stress, there was an overall increase in relative electrical conductivity, along with malondialdehyde, soluble sugar, and soluble protein contents, but decreased superoxide dismutase and peroxidase activities and chlorophyll contents in seedling leaves compared with controls. Overall, 5,780 differentially expressed genes (DEGs) and 178 differentially expressed metabolites (DEMs) were identified under chilling stress. Most DEGs were involved in plant hormone signal transduction and the phenylpropanoid biosynthesis pathway, and ERF, bHLH, WRKY, MYB, and HSF transcription factors were induced. Metabolomic analysis revealed that the contents of salicylic acid (SA), phenylalanine, and tyrosine increased in response to chilling stress. The findings indicated that the SA signaling and phenylpropanoid biosynthesis pathways are key to regulating the responses to chilling stress in pumpkins., Conclusion: Overall, our study provides valuable insights into the comprehensive response of C. moschata to chilling stress, enriching the theoretical basis of this mechanism and facilitating the development of molecular breeding strategies for pumpkin tolerance to chilling stress., (© 2024. The Author(s).)

Cucumber is a widely consumed vegetable crop known for its rich nutrient composition and distinctive flavor, influenced by both volatile and non-volatile compounds. Grafting and delayed harvesting are crucial strategies for increasing cucumber yield. The present study investigates the impact of delayed harvesting at different developmental stages and grafting on the metabolic profile, flavor, and overall quality of cucumber fruits Yuxiu 2 (YX) using UPLC-MS/MS and GC-MS/MS techniques. The results indicate that delayed harvesting of YX led to significant increases in length, diameter, and weight from 12 to 24 days after pollination (DAP), with minimal growth beyond 24 DAP. However, grafting did not affect these physical parameters compared to self-rooted plants. Furthermore, metabolic profiling reveals that delayed harvesting enhances the concentration of certain non-volatile metabolites, including alkaloids, organic acids, and phenolic acids, while leading to a reduction in flavonoid contents. Overall, 140 non-volatile and 26 volatile differential metabolites were screened at three developmental stages. Notably, four new organic acids (6-amino hexanoic acid, 5-amino valeric acid, 1-hydroxy-2-naphthoic acid, and succinic semialdehyde) and three novel alkaloids (3-indole acetonitrile, epinephrine, and serotonin) were identified. Volatile compounds, such as aldehydes, esters, terpenes, alcohols, and ketones, exhibit a peak in concentration at 24 DAP, followed by a decline. The characteristic cucumber flavor compound, (E,Z)-2,6-nonadienal, remains consistent across all developmental stages. In grafted cucumber fruits, a total of 113 non-volatile and 11 volatile differential metabolites were screened, and among them, ten unique non-volatile metabolites were detected in grafted fruits, contributing to the sour and bitter taste of cucumbers. Moreover, some of the metabolites like (1S,4S,4aR)-1-isopropyl-4-methyl-7-methylene-1,2,3,4,4a,5,6,7-octahydronaphthalene with pentylenetetrazol contribute to an undesirable camphor-like odor. The study concludes that while delayed harvesting and grafting practices can increase cucumber yield, they also significantly alter the fruit's metabolic profile, impacting taste and flavor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Long-distance signals play a vital role in plant stress response. γ-aminobutyric acid (GABA) has been proposed to be a signal and protects crops against diverse stresses. However, whether GABA acts as a long-distance signal to plant response to stresses remains unknown. Here, we found that the GABA content in cucurbita rootstocks, especially figleaf gourd, was significantly higher than that in cucumber. Figleaf gourd rootstock obviously enhanced cold tolerance and GABA accumulation in roots, xylem sap and leaves of grafting cucumber seedlings. Conversely, GABA synthesis inhibitor 3-mercaptopropionic acid (3-MPA) irrigation was more effective than its foliar application in inhibiting grafting-induced cold tolerance. Moreover, fluorescence microscopy confirmed that GABA can be transported from root to shoot through the xylem when the roots of grafted seedlings were fed with fluorescein isothiocyatate-labeled GABA under normal and cold stress conditions. Importantly, 3-MPA irrigation attenuated grafting-induced cold tolerance, as revealed by a decline in the GABA accumulation, the transcripts of ICE1, CBF1 and COR47, the activities of the antioxidant enzymes, and an increase in stomatal aperture. Collectively, our findings strongly support that GABA functions as a novel long-distance signal in figleaf gourd rootstock-induced cold tolerance of grafted cucumber seedlings by modulating CBF-signalling pathways, antioxidant system and stomatal aperture, providing new evidence for long-distance signaling-mediated cold response of plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Plant domestication often alters plant traits, including chemical and physical defenses against herbivores. In squash, domestication leads to reduced levels of cucurbitacins and leaf trichomes, influencing interactions with insects. However, the impact of domestication on inducible defenses in squash remains poorly understood. Here, we investigated the chemical and physical defensive traits of wild and domesticated squash (Cucurbita argyrosperma), and compared their responses to belowground and aboveground infestation by the root-feeding larvae and the leaf-chewing adults of the banded cucumber beetle Diabrotica balteata (Coleoptera: Chrysomelidae). Wild populations contained cucurbitacins in roots and cotyledons but not in leaves, whereas domesticated varieties lacked cucurbitacins in all tissues. Belowground infestation by D. balteata larvae did not increase cucurbitacin levels in the roots but triggered the expression of cucurbitacin biosynthetic genes, irrespective of domestication status, although the response varied among different varieties. Conversely, whereas wild squash had more leaf trichomes than domesticated varieties, the induction of leaf trichomes in response to herbivory was greater in domesticated plants. Leaf herbivory varied among varieties but there was a trend of higher leaf damage on wild squash than domesticated varieties. Overall, squash plants responded to both belowground and aboveground herbivory by activating chemical defense-associated gene expression in roots and upregulating their physical defense in leaves, respectively. While domestication suppressed both chemical and physical defenses, our findings suggest that it may enhance inducible defense mechanisms by increasing trichome induction in response to herbivory., (© 2024. The Author(s).)

The study investigated the lipid oxidation of pumpkin seed kernels (PSK) under different storage conditions (room temperature, vacuum-room temperature, refrigeration, and vacuum-refrigeration) using HPLC-MS and GC-MS. Experimental results found the vacuum-refrigeration group showed the lowest PV (0.24 g/100 g), diene (8.68), hexanal (356.64 ± 16.06 ng/g), and nonanal (132.05 ± 8.38 ng/g) after a 9-month storage. A total of 586 lipids, including 6 classes and 27 subclasses, were detected, 46 of which showed significant differences. Refrigeration samples had the highest diacylglycerol content, while room temperature samples demonstrated the highest triacylglycerol and phosphatidylcholine content. Differential lipid metabolite analyses indicated that storage conditions mainly affected glycerolipid metabolism, glycerophospholipid metabolism, and sphingolipid metabolism pathways in PSK, while glycerolipid and glycerophospholipid metabolism were still dominant. It revealed that refrigeration was more effective than vacuum in inhibiting the oxidation of PSK. These findings could offer valuable references for the storage, transportation, preservation, and the development and utilization of PSK., Competing Interests: Declaration of competing interest These authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Pumpkin is a highly nutritious plant, rich in valuable nutrients that benefit human health. Due to the high perishability of this fruit, the production of pumpkin juice is a practical way to use it effectively. Recently, fermented vegetable juices have been used as a dairy alternative due to their nutritional and potential probiotic properties. This study investigated the fermentation of pumpkin juice using different strains of lactic acid bacteria (LAB), with and without heat treatment. The effects of fermentation on microbial growth, pH, acidity, extract, sugars, carotenoids, polyphenols, and antioxidant properties were analyzed. The heat-treatment process did not greatly impact the dry matter content, pH, acidity, extract, or sugar content. However, it led to a reduction in carotenoid and polyphenol levels. During fermentation, there was a consistent decrease in pH and an increase in total acidity, with no noticeable differences between bacterial strains regarding their influence on these parameters. The study revealed that there were no distinctions between LAB strains in their effects on pH, acidity, and carotenoid content in fermented pumpkin juice. Nonetheless, both L . sakei and L . plantarum proved to be effective in the fermentation process, with L. sakei demonstrating greater adaptability. The expected pH, acidity, and sugar content changes were consistently observed throughout the fermentation process. Overall, results confirm the efficacy of the used Lactobacillus strains in fermenting pumpkin juice and highlight the potential impact of heat treatment on the nutritional composition of the juice. The purpose of thermal processing of pumpkin juice, which is conducted with lactic acid fermentation, is crucial for the food industry. It extends the product's shelf life, improves its nutritional and taste profiles, and guarantees its microbiological safety.

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.

The crosstalk of phytohormones in the regulation of growth and development and the response of plants to environmental stresses is a cutting-edge research topic, especially in crop species. In this paper, we study the role and crosstalk between abscisic acid (ABA), ethylene (ET), and jasmonate (JA) in the control of germination and seedling growth in water or in standard nutrient solution and under salt stress (supplemented with 100-200 mM NaCl). The roles of ET and JA were studied using squash ET- and JA-deficient mutants aco1a and lox3a , respectively, while the crosstalk between ET, JA, and ABA was determined by comparing the expression of the key ABA, JA, and ET genes in wild-type (WT) and mutant genotypes under standard conditions and salt stress. Data showed that ET and JA are positive regulators of squash germination, a function that was found to be mediated by downregulating the ABA biosynthesis and signaling pathways. Under salt stress, aco1a germinated earlier than WT, while lox3a showed the same germination rate as WT, indicating that ET, but not JA, restricts squash germination under unfavorable salinity conditions, a function that was also mediated by upregulation of ABA. ET and JA were found to be negative regulators of plant growth during seedling establishment, although ET inhibits both the aerial part and the root, while JA inhibits only the root. Both aco1a and lox3a mutant roots showed increased tolerance to salt stress, a phenotype that was found to be mainly mediated by JA, although we cannot exclude that it is also mediated by ABA.

Zucchini squashes are cold-sensitive and vulnerable to chilling injury (CI) resulting from reactive oxygen species (ROS) and hot water (HW) immersing effectively reduce CI symptoms during cold storage. However, mechanism involved in reduced ROS due to HW treatment has not been characterized well. In this study, tender green zucchini fruit were treated with HW for 15 min at 45 ± 1 °C and stored for 15 d at 4 ± 1 °C and above 90 % relative humidity. Results showed substantial reduction in CI index, electrolyte leakage, malonaldehyde (MDA) contents and ROS accumulation along with increased activity of ROS-scavenging enzymes due to HW treatment. To gain insight into the molecular mechanism involved in antioxidant defense system, transcriptomic analysis revealed that heat shock factors (HSF) accumulated due to HW treatment regulated the ROS pathway during cold stress. CpHSFA4a was one of the highly expressed transcription factors (TF) due to HW treatment that regulated the transcription of ROS enzymes related genes. CpHSFA4a bind actively with heat shock element (HSE) in promoter regions of CpSOD, CpCAT, CpAPX1, CpAPX2, and CpAPX3, activated and increased the expression of these genes. In conclusion, HW treatment alleviated the CI by maintaining ROS homeostasis through CpHSFA4a mediated ROS pathway in zucchini squashes during cold storage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Dieldrin, an organochlorine pesticide (OCP) widely used for crop protection in the second half of the 20th century till the 70's, is worldwide still present in arable soils. It can be transferred to crops, notably cucurbits, depending on plant species and cultivars. Finding strategies to decrease OCP bioavailability in soil is therefore a main concern. Phytomanagement strategies could provide (i) ready-to-use short term solution for maintaining the production of edible plant parts with dieldrin concentrations below the Maximum Residue Limits (MRL) and (ii) long-term solution for dieldrin phytoextraction reducing progressively its bioavailability in the soil. This field study aimed at determining dieldrin accumulation capacities and allocation pattern in 17 non-Cucurbitaceae species and 10 Cucurbita pepo varieties, and assessing the dieldrin phytoextraction potential of these plant species when grown to maturity in a historically dieldrin-contaminated soil. Out of the non-Cucurbitaceae species, vetiver was the only one able to accumulate significant amounts of dieldrin, which mainly remained in its roots. All C. pepo varieties were able to uptake and translocate high dieldrin amounts into the shoots, leading to the highest phytoextraction potential. Despite the intraspecific variability in dieldrin concentration in zucchini plant parts, mainly in the reproductive organs, the phytoextraction capacity for shoots and fruits was high for all tested varieties (147 to 275 μg dieldrin plant -1 , corresponding to 5.6 % of the n-heptane extractable soil dieldrin), even for the one with low fruit dieldrin concentration. Both food safety and phytoextraction could be achieved by selecting productive zucchini varieties displaying low dieldrin concentration in fruits and high one in shoots., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chia (Salvia hispanica L.) seed (CS) and Pumpkin (Cucurbita moschata) seed (PS) are used in ruminant diets as energy sources. The current experiment studied the impact of dietary inclusion of CS and PS on nutrient intake and digestibility, milk yield, and milk composition of dairy sheep. Twelve primiparous Texel × Suffolk ewes [70 ± 5 days in milk (DIM); 0.320 ± 0.029 kg milk yield] were distributed in a 4 × 3 Latin square design and fed either a butter-based control diet [CON; 13 g/kg dry matter] or two diets with 61 g/kg DM of either CS or PS. Dietary inclusion of CS and PS did not alter live weight (p >0.1) and DM intake (p >0.1). However, compared to the CON, dietary inclusion of both CS and PS increased the digestibility of neutral detergent fiber (p <0.001) and acid detergent lignin (p < 0.001). Milk production (p = 0.001), fat-corrected milk (p < 0.001), and feed efficiency (p < 0.001) were enhanced with PS, while the highest milk protein yield (p < 0.05) and lactose yield (p < 0.001) were for CS-fed ewes. Compared to the CON diet, the ingestion of either CS and/or PS decreased (p < 0.001) the C16:0 in milk. Moreover, both CS and PS tended to enhance the content of C18:3n6 (p > 0.05) and C18:3n3 (p > 0.05). Overall short-term feeding of CS and/or PS (up to 6.1% DM of diet) not only maintains the production performance and digestibility of nutrients but also positively modifies the milk FA composition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Robles Jimenez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The melon (Cucumis melo L.) is a globally cherished and economically significant crop. The grafting technique has been widely used in the vegetative propagation of melon to promote environmental tolerance and disease resistance. However, mechanisms governing graft healing and potential incompatibilities in melons following the grafting process remain unknown. To uncover the molecular mechanism of healing of grafted melon seedlings, melon wild type (Control) and TRV-CmGH9B3 lines were obtained and grafted onto the squash rootstocks (C. moschata). Anatomical differences indicated that the healing process of the TRV-CmGH9B3 plants was slower than that of the control. A total of 335 significantly differentially expressed genes (DEGs) were detected between two transcriptomes. Most of these DEGs were down-regulated in TRV-CmGH9B3 grafted seedlings. GO and KEGG analysis showed that many metabolic, physiological, and hormonal responses were involved in graft healing, including metabolic processes, plant hormone signaling, plant MAPK pathway, and sucrose starch pathway. During the healing process of TRV-CmGH9B3 grafted seedlings, gene synthesis related to hormone signal transduction (auxin, cytokinin, gibberellin, brassinolide) was delayed. At the same time, it was found that most of the DEGs related to the sucrose pathway were down-regulated in TRV-CmGH9B3 grafted seedlings. The results showed that sugar was also involved in the healing process of melon grafted onto squash. These results deepened our understanding of the molecular mechanism of GH9B3, a key gene of β-1, 4-glucanase. It also provided a reference for elucidating the gene mechanism and function analysis of CmGH9B3 in the process of graft union healing., (© 2024 Wiley‐VCH GmbH.)

Background: The core regulation of the abscisic acid (ABA) signalling pathway comprises the multigenic families PYL, PP2C, and SnRK2. In this work, we conducted a genome-wide study of the components of these families in Cucurbita pepo., Results: The bioinformatic analysis of the C. pepo genome resulted in the identification of 19 CpPYL, 102 CpPP2C and 10 CpSnRK2 genes. The investigation of gene structure and protein motifs allowed to define 4 PYL, 13 PP2C and 3 SnRK2 subfamilies. RNA-seq analysis was used to determine the expression of these gene families in different plant organs, as well as to detect their differential gene expression during germination, and in response to ABA and cold stress in leaves. The specific tissue expression of some gene members indicated the relevant role of some ABA signalling genes in plant development. Moreover, their differential expression under ABA treatment or cold stress revealed those ABA signalling genes that responded to ABA, and those that were up- or down-regulated in response to cold stress. A reduced number of genes responded to both treatments. Specific PYL-PP2C-SnRK2 genes that had potential roles in germination were also detected, including those regulated early during the imbibition phase, those regulated later during the embryo extension and radicle emergence phase, and those induced or repressed during the whole germination process., Conclusions: The outcomes of this research open new research lines for agriculture and for assessing gene function in future studies., (© 2024. The Author(s).)

Drought is a major limiting factor for the growth and development of pumpkins. Plasma membrane intrinsic proteins (PIPs) are major water channels that play a crucial role in the regulation of cellular water status and solute trafficking during drought conditions. CmoPIP1-4 is a plasma membrane-localized protein that is significantly upregulated in roots and leaves under drought-stress conditions. In this study, the overexpression of CmoPIP1-4 enhances drought resistance in yeast. In contrast, CRISPR-mediated CmoPIP1-4 knockout in pumpkin roots increased drought sensitivity. This increased drought sensitivity of CmoPIP1-4 knockout plants is associated with a decline in the levels of hydrogen sulfide (H 2 S) and abscisic acid (ABA), accompanied by an increase in water loss caused by greater levels of transpiration and stomatal conductance. In addition, the sensitivity of CmoPIP1-4 CRISPR plants is further aggravated by reduced antioxidative enzyme activity, decreased proline and sugar contents, and extensive root damage. Furthermore, expression profiles of genes such as CmoHSP70s, CmoNCED3, CmoNCED4, and others involved in metabolic activities were markedly reduced in CmoPIP1-4 CRISPR plants. Moreover, we also discovered an interaction between the drought-responsive gene CmoDCD and CmoPIP1-4, indicating their potential role in activating H 2 S-mediated signaling in pumpkin, which could confer drought tolerance. The findings of our study collectively demonstrate CmoPIP1-4 plays a crucial role in the regulation of H 2 S-mediated signaling, influencing stomatal density and aperture in pumpkin plants, and thereby enhancing their drought tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests., (Copyright © 2024. Published by Elsevier Masson SAS.)

This study aimed to investigate the characterization of zinc oxide nanoparticles (ZnONPs) produced from Cucurbita pepo L. (pumpkin seeds) and their selective cytotoxic effectiveness on human colon cancer cells (HCT 116) and African Green Monkey Kidney, Vero cells. The study also investigated the antioxidant activity of ZnONPs. The study also examined ZnONPs' antioxidant properties. This was motivated by the limited research on the comparative cytotoxic effects of ZnO NPs on normal and HCT116 cells. The ZnO NPs were characterized using Fourier-transform infrared spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Transmission Electron Microscope/Selected Area Electron Diffraction (TEM/SAED), and Scanning Electron Microscope-Energy Dispersive X-ray (SEM-EDX) for determination of chemical fingerprinting, heat stability, size, and morphology of the elements, respectively. Based on the results, ZnO NPs from pumpkins were found to be less than 5 μm and agglomerates in nature. Furthermore, the ZnO NPs fingerprinting and SEM-EDX element analysis were similar to previous literature, suggesting the sample was proven as ZnO NPs. The ZnO NPs also stable at a temperature of 380°C indicating that the green material is quite robust at 60-400°C. The cell viability of Vero cells and HCT 116 cell line were measured at two different time points (24 and 48 h) to assess the cytotoxicity effects of ZnO NP on these cells using AlamarBlue assay. Cytotoxic results have shown that ZnO NPs did not inhibit Vero cells but were slightly toxic to cancer cells, with a dose-response curve IC50 = ~409.7 μg/mL. This green synthesis of ZnO NPs was found to be non-toxic to normal cells but has a slight cytotoxicity effect on HCT 116 cells. A theoretical study used molecular docking to investigate nanoparticle interaction with cyclin-dependent kinase 2 (CDK2), exploring its mechanism in inhibiting CDK2's role in cancer. Further study should be carried out to determine suitable concentrations for cytotoxicity studies. Additionally, DPPH has a significant antioxidant capacity, with an IC50 of 142.857 μg/mL. RESEARCH HIGHLIGHTS: Pumpkin seed extracts facilitated a rapid, high-yielding, and environmentally friendly synthesis of ZnO nanoparticles. Spectrophotometric analysis was used to investigate the optical properties, scalability, size, shape, dispersity, and stability of ZnO NPs. The cytotoxicity of ZnO NPs on Vero and HCT 116 cells was assessed, showing no inhibition of Vero cells and cytotoxicity of cancer cells. The DPPH assay was also used to investigate the antioxidant potential of biogenic nanoparticles. A molecular docking study was performed to investigate the interaction of ZnO NPs with CDK2 and to explore the mechanism by which they inhibit CDK2's role in cancer., (© 2023 Wiley Periodicals LLC.)

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, CaCo 2 , 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.

Late flowering is a serious bottleneck in pumpkin (Cucurbita moschata Duch.) agriculture production. Although key genes governing flowering time have been reported in many species, the regulatory network of flowering in pumpkin remains largely obscure, thereby impeding the resolution of industry-wide challenges associated with delayed fruit ripening in pumpkin cultivation. Here, we report an early flowering pumpkin germplasm accession (LXX-4). Using LXX-4 and a late flowering germplasm accession (HYM-9), we constructed an F 2 segregation population. A significant difference in FLOWERING LOCUS T-LIKE 2 (FTL2) expression level was identified to be the causal factor of the flowering time trait discrepancy in LXX-4 and HYM-9. Moreover, we have shown that a 21 bp InDel in the FTL2 promoter was the key reason for the waxing and waning of its transcript level. The 21 bp deletion excluded a repressor-AGL19 and recruited activators-BBX7, WRKY40 and SVP to the FTL2 promoter in LXX-4. Together, our data add a useful element to our knowledge which could be used to simplify breeding efforts for early-maturing pumpkin., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Background: Auxin transcription factor (ARF) is an important transcription factor that transmits auxin signals and is involved in plant growth and development as well as stress response. However, genome-wide identification and responses to abiotic and pathogen stresses of the ARF gene family in Cucurbita pepo L, especially pathogen stresses, have not been reported., Results: Finally, 33 ARF genes (CpARF01 to CpARF33) were identified in C.pepo from the Cucurbitaceae genome database using bioinformatics methods. The putative protein contains 438 to 1071 amino acids, the isoelectric point is 4.99 to 8.54, and the molecular weight is 47759.36 to 117813.27 Da, the instability index ranged from 40.74 to 68.94, and the liposoluble index ranged from 62.56 to 76.18. The 33 genes were mainly localized in the nucleus and cytoplasm, and distributed on 16 chromosomes unevenly. Phylogenetic analysis showed that 33 CpARF proteins were divided into 6 groups. According to the amino acid sequence of CpARF proteins, 10 motifs were identified, and 1,3,6,8,10 motifs were highly conserved in most of the CpARF proteins. At the same time, it was found that genes in the same subfamily have similar gene structures. Cis-elements and protein interaction networks predicted that CpARF may be involved in abiotic factors related to the stress response. QRT-PCR analysis showed that most of the CpARF genes were upregulated under NaCl, PEG, and pathogen treatment compared to the control. Subcellular localization showed that CpARF22 was localized in the nucleus. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt and drought stress., Conclusion: In this study, we systematically analyzed the CpARF gene family and its expression patterns under drought, salt, and pathogen stress, which improved our understanding of the ARF protein of zucchini, and laid a solid foundation for functional analysis of the CpARF gene., (© 2024. The Author(s).)

Although 6:2 fluorotelomer carboxylic acid (6:2 FTCA), which is one of the most popular substitutes for perfluorooctanoic acid (PFOA), has been widely distributed in environments, little is known about its biotransformation mechanism and phytotoxic effects in plants. Here, we showed that 6:2 FTCA could be taken up by pumpkin (Cucurbita maxima L.) roots from exposure solution and acropetally translocated to shoots. Biotransformation of 6:2 FTCA to different carbon chain perfluorocarboxylic acid (PFCA) metabolites (C2-C7) via α-and β-oxidation in pumpkin was observed, and perfluorohexanoic acid (PFHxA) was the major transformation product. The results of enzyme assays, enzyme inhibition experiments and gene expression analysis indicated that cytochrome P450 (CYP450), glutathione-S-transferase (GST) and ATP-binding cassette (ABC) transporters were involved in the metabolism of 6:2 FTCA in pumpkin. Plant-associated rhizobacteria and endophyte also contributed to 6:2 FTCA degradation through β-oxidation. The chlorophyll (Chl) content and genes involved in photosynthesis were significantly improved by 6:2 FTCA. The reductions of antioxidant and metabolic enzyme activities reflected the antioxidant defense system and detoxification system of pumpkin were both damaged, which were further confirmed by the down-regulating associated genes encoding phenylpropanoid biosynthesis, endoplasmic reticulum-related proteins, ascorbate-glutathione cycle and ABC transporters. This study is helpful to understand the environmental behaviors and toxicological molecular mechanisms of 6:2 FTCA in plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The aim of this study was to conduct transcriptomic, proteomic, and metabolomic analysis to provide a comprehensive view of plant response to tetracycline stress. Pumpkin seeds were cultured for 7 days without or with tetracycline at 10 mg/L. Pumpkin roots showed excessive growth inhibition, but not yet strong growth restraining in cotyledons. Tetracycline affected the abundance of metabolites related to flavonoid biosynthesis and amino acid metabolism. Main changes of metabolites in flavonoid biosynthesis were consistent with mRNA changes. Amino acid changes are mainly mediated by proteins or mRNAs. To be specific, tetracycline treatment increased the levels of rutin, caffeate, cinnamaldehyde, 4-hydroxycinnamic acid, ferulic acid, naringenin, apigenin, luteolin, (-)-epigallocatechin, astragalin, L-serine, and glutathione and the transcript levels related to these compounds; and decreased the levels of indole pyruvate, indole acetaldehyde, L-arginine, S-adenosylhomocysteine, L-glutamine, and gamma-glutamylcysteine and the transcript levels related to these compounds. Tetracycline treatment also increased the levels of oxoglutaric acid, L-glutamic acid, gamma-aminobutyric acid, and gamma-glutamylalanine and enzymes involved in their production; and decreased the levels of L-isoleucine, L-valine, and L-leucine and enzymes involved in their production. We elucidated several biological processes (e.g. phenylpropanoid/flavonoid biosynthesis pathways, amino acid metabolic pathways) that were altered by tetracycline, and provided a multi-omic perspective on the mechanisms underlying the response to tetracycline stress in pumpkin roots. We provide a useful reference for the development of environmental quality management methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Atlantic giant (AG, Cucurbita maxima ) is a type of giant pumpkin in the Cucurbitaceae family and has the world's largest fruit. AG possesses excellent ornamental and economic value due to its well-known large fruit. However, giant pumpkins are usually thrown away after viewing, thus generating a waste of resources. To explore the additional value of giant pumpkins, a metabolome assay was performed between AG and Hubbard (a small fruit pumpkin) fruits. We found that bioactive compounds, especially flavonoids (including 8-prenylnaringenin, tetrahydrocurcumin, galangin, and acacetin) and coumarins (including coumarin, umbelliferone, 4-coumaryl alcohol, and coumaryl acetate), with extensive antioxidant and pharmacological functions, showed higher accumulation in AG fruit than in Hubbard fruits. Comparative transcriptomics of the two pumpkin fruits indicated that the differentially expressed genes (DEGs) encoding PAL, C4H, 4CL, CSE, HCT, CAD, and CCoAOMT were relatively highly expressed, which promoted an increased accumulation of the identified flavonoids and coumarins in giant pumpkins. In addition, the construction of a co-expression network and cis -element analysis of the promoter demonstrated that differentially expressed MYB, bHLH, AP2, and WRKY transcription factors might play vital roles in regulating the expression of DEGs involved in the biosynthesis of several flavonoids and coumarins. Our current results provide new insights into the accumulation of active compounds in giant pumpkins.

Pumpkin is often used as a rootstock for other Cucurbitaceae crops due to its resistance to soil-borne diseases and abiotic stress. Pumpkin rootstocks use a sodium transporter (CmHKT1;1) to promote the transport of Na + from the shoot to the root effectively and improve the salt tolerance of the scion. However, the molecular regulatory mechanisms that influence the activity of CmHKT1;1 during salt stress response remain unknown. In this study, CmCNIH1, a cornichon homolog, was identified as a potential cargo receptor for CmHKT1;1. Yeast two-hybrid, biomolecular fluorescence complementation and luciferase complementary assays demonstrated that CmCNIH1 and CmHKT1;1 could interact. CmCNIH1 was a key component of the cellular vesicle transport machinery located in the endoplasmic reticulum (ER), ER export site and Golgi apparatus. A CmCNIH1 knockout mutant was more sensitive to salt stress than the wild-type (WT). In addition, ion homeostasis was disrupted in cmcnih1 mutants, which had higher Na + and lower K + content in shoots and roots than the WT. Two-electrode voltage-clamp experiment displayed that CmCNIH1 could not influence the Na + current that passed through the plasma membrane (PM) in CmHKT1;1-expressing Xenopus laevis oocytes. Data from co-localization assays indicated that intact CmCNIH1 protein could alter the subcellular localization of CmHKT1;1 in tobacco leaf, pumpkin root and yeast. In summary, CmCNIH1 may function as a cargo receptor that regulates the localization of CmHKT1;1 to the PM to improve salt tolerance in pumpkin., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Heat stress is the most major environmental element contributing to rabbit health problems and reduced production. It is proposed that essential oils be applied to alleviate heat stress-induced oxidative damage in rabbits. The purpose of this feeding trial was to determine the protective impact of pumpkin seed essential oil (PSO)-supplemented diets in reducing the threat of unambient temperature on growing rabbits. Five groups of 5-week-old rabbits were allocated randomly into separated galvanized wire battery. The first group was raised under normal conditions (18 ± 2 °C) and fed a control diet (control group; CNT), whereas the other four groups were exposed to high ambient temperature (38 ± 2 °C) and fed a control diet supplemented with 0 (PSO 0.0 ), 0.5 (PSO 0.05 ), 1.0 (PSO 1.0 ), and 2.0 (PSO 2.0 ) mL PSO/kg diet. Results indicated that all supplemented groups and the positive control have higher live body weight compared with the heat stress group (PSO 0.0 ) at 9 weeks of age. Supplementing of PSO resulted in significant improvement in weight gain at 5-9 weeks and 9-13 weeks compared with PSO 0.0 group. The highest feed intake was detected in PSO 0.05 group compared with that in other groups. Both PSO 2.0 and PSO 2.0 groups showed the lowest feed conversion ration compared with other groups. Heat-stressed rabbits given a high dose of PSO (1 to 2 mL) had higher hemoglobin concentrations and lower white blood cell counts throughout the experiment than those given a control diet and subjected to heat stress. All hepatic and renal function parameters improved significantly in the rabbits fed a high dose of PSO as compared to the heat-stressed control group, while protein constituents were significantly higher in experimental groups fed 2 mL PSO compared with other groups. Heat-stressed rabbits administered graded amounts of PSO had the lowest plasma glucose, cortisol, thyroid, and corticosterone concentrations and were noticed to be equivalent to the control group fed unsupplemented diet and reared under normal conditions. The immunohistochemistry analysis demonstrated that rabbit groups reared under heat stress and given 2 mL PSO supplemented diets had negative caspase-3 immunoreactivity surrounding portal tract and normal structure. In conclusion, adding pumpkin seed oil up to 2 mL/kg diet for growing rabbits is indorsed to promote growth as well as antioxidant and immunological status under heat stress conditions., (© 2023. The Author(s).)

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.

It is generally recognized that the root uptake capacity of grafted plants strongly depends on the rootstocks' well-developed root system. However, we found that grafted plants showed different nitrate uptake capacities when different varieties of oriental melon scion were grafted onto the same squash rootstock, suggesting that the scion regulated the nitrate uptake capacity of the rootstock root. In this study, we estimated the nitrate uptake capacity of grafted plants with the different oriental melon varieties' seedlings grafted onto the same squash rootstocks. The results indicated a significant difference in the nitrate uptake rate and activity of two heterologous grafting plants. We also showed a significant difference in CmoNRT2.1 expression in the roots of two grafting combinations and verified the positive regulation of nitrate uptake by CmoNRT2.1 expression. In addition, the two varieties of oriental melon scion had highly significant differences in CmHY5 expression, which was transported to the rootstock and positively induced CmoHY5-1 and CmoHY5-2 expression in the rootstock roots. Meanwhile, CmHY5 could positively regulate CmoNRT2.1 expression in the rootstock roots. Furthermore, CmoHY5-1 and CmoHY5-2 also positively regulated CmoNRT2.1 expression, respectively, and CmoHY5-1 dominated the positive regulation of CmoNRT2.1 , while CmHY5 could interact with CmoHY5-1 and CmoHY5-2, respectively, to jointly regulate CmoNRT2.1 expression. The oriental melon scion regulated the nitrate uptake capacity of the melon/squash grafting plant roots, and the higher expression of CmHY5 in the oriental melon scion leaves, the more substantial the nitrate uptake capacity of squash rootstock roots.

The present study was designed to evaluate the protective effect of fluted pumpkin seeds (FPS) against caffeine (CAFF) induced testicular toxicity in rats. Thirty young healthy male Wistar rats (196 ± 12 g) were randomly organized into five sets of six animals per each group: control, caffeine (CAFF; 50 mg kg -1 bw) and FPS co-treatment groups (CAFF + 50 mg FPS, CAFF + 100 mg FPS and CAFF + 200 mg FPS kg -1  bw). CAFF and FPS were administered daily and twice per week respectively by oral gavage for 40 days. CAFF treatment decreased testicular lactate dehydrogenase enzyme activity level, which was attenuated on co-administration with FPS at 50 and 100 mg kg -1  bw. Furthermore, CAFF decreased seminiferous epithelia thickness and spermatogenesis score index and increased the number of tubules with abnormal histological features, which were attenuated on co-administration with FPS at 50 mg kg -1  bw much more than at the higher doses (p < 0.05). CAFF did not affect malondialdehyde and glutathione concentrations and glutathione peroxidase (GSH-Px) activity in the testes whereas FPS co-treatment at the higher doses elevated glutathione level and GSH-Px activity and did not affect spermatogenesis score index at the highest dose (200 mg kg -1  bw). Testicular malondialdehyde concentrations remained unaffected in all FPS co-treatment groups. Overall, FPS is able to minimize the CAFF-induced testicular injury at lower than at the higher tested doses., (© 2022 Wiley-VCH GmbH.)

The Cucurbitaceae family accumulates dioxin-like compounds in its fruits. We previously showed that A20/AN1 zinc finger protein (ZFP) genes were highly expressed in the zucchini (Cucurbita pepo) subspecies pepo, which accumulates dioxin-like compounds at high concentrations. Transgenic tobacco (Nicotiana tabacum) plants overexpressing A20/AN1 ZFP genes show accumulation of dioxin-like compounds in their upper parts. However, the mechanisms underlying the accumulation of dioxin-like compounds regulated by the A20/AN1 ZFPs remain unclear. Here, we show that A20/AN1 ZFPs positively regulate the expression of the major latex-like protein (MLP) and its homolog genes in N. tabacum and C. pepo. MLPs are involved in the transport of dioxin-like compounds from the roots to the upper parts of C. pepo. Overexpression of A20/AN1 ZFP genes in N. tabacum leads to the upregulation of pathogenesis-related protein class-10 genes with the binding ability toward dioxin-like compounds. Our results demonstrated that A20/AN1 ZFPs upregulate MLP and its homolog genes in N. tabacum and C. pepo, resulting in the accumulation of dioxin-like compounds., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ethylene biosynthesis and signal transduction play critical roles in plant sex differentiation. ACS (1-aminocyclopropane-1-carboxylic acid synthase) is a rate-limiting enzyme in ethylene biosynthesis. However, the understanding of the ACS gene family in Cucurbita maxima is limited. Here, we identified and characterized 13 ACS genes in the C. maxima genome. All ACS genes could be divided into three groups according to a conserved serine residue at the C-terminus. Thirteen CmaACS genes were found to be randomly distributed on 10 of the 20 chromosomes of C. maxima . The ACS gene exhibits different tissue-specific expression patterns in pumpkin, and four ACS genes ( CmaACS1 , CmaACS4 , CmaACS7 , and CmaACS9 ) were expressed specifically in both the female and male flowers of C. maxima . In addition, the expression levels of CmaACS4 and CmaACS7 were upregulated after ethephon and IAA treatments, which ultimately increased the number of female flowers, decreased the position of the first female flower and decreased the number of bisexual flowers per plant. These results provide relevant information for determining the function of the ACS genes in C. maxima , especially for regulating the function of ethylene in sex determination.

Background: Fusarium oxysporum f. sp. cucumerinum (FOC) is the causal agent of cucumber Fusarium wilt, which can cause extensive damages and productivity losses. Cucurbita ficifolia Bouché (Cucurbitaceae) is usually used as rootstock for cucumber because of its excellent resistance to Fusarium wilt. Our previous study found that C.ficifolia has high FOC resistance, the underlying mechanism of which is unclear., Results: Transcriptome and proteome profiling was performed on the basis of RNA-Seq and isobaric tag for relative and absolute quantitation technology to explore the molecular mechanisms of the response of Cucurbita ficifolia Bouché to Fusarium oxysporum f. sp. cucumerium infection. Comparative analyses revealed that 1850 genes and 356 protein species were differentially regulated at 2d and 4d after FOC inoculation. However, correlation analysis revealed that only 11 and 39 genes were differentially regulated at both the transcriptome and proteome levels after FOC inoculation at 2d and 4d, respectively. After FOC inoculation, plant hormones signal transduction, transcription factors were stimulated, whereas wax biosynthesis and photosynthesis were suppressed. Increased synthesis of oxidative-redox proteins is involved in resistance to FOC., Conclusions: This study is the first to reveal the response of C. ficifolia leaf to FOC infection at the transcriptome and proteome levels, and to show that FOC infection activates plant hormone signaling and transcription factors while suppressing wax biosynthesis and photosynthesis. The accumulation of oxidative-redox proteins also plays an important role in the resistance of C. ficifolia to FOC. Results provide new information regarding the processes of C. ficifolia leaf resistance to FOC and will contribute to the breeding of cucumber rootstock with FOC resistance., (© 2022. The Author(s).)

In this study, pumpkin seedlings were subjected to cadmium stress (100 mg/L cadmium ion solution, 10 days) without or with wheat straw biochar at different concentrations (0%, 0.5%, 1%, and 2% w/v). As the biochar concentration increased, the amount of cadmium accumulated in the root and stem of pumpkin seedlings decreased and the fresh weight of root, stem and leaf increased. The highest cadmium concentration was in the root, followed by the stem and then the leaf. 1% and 2% biochar treatments reduced the oxidative stress of cadmium to seedlings, and added the contents of fatty acid, carbohydrate, amino acid and indoleacetic acid in the root. With the increase of biochar concentration, the metabolites promoting root growth increased. These results provide new information about how biochar alleviates cadmium stress by affecting the metabolic response., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cucurbits have been used as phloem research models for many decades because their exudates can be accessed with ease. However, cucurbit plants possess two distinct phloem systems known as the fascicular phloem (FP) and extrafascicular phloem (EFP). Therefore, the molecular composition and function of certain exudates can be misinterpreted due to their unclear origin. To characterize the anatomy and function of the different phloem systems more clearly, we generated specific antibodies against marker proteins (PP1 homologs) allowing the clear identification of the EFP at the organ, tissue and cellular levels by immunological staining. We also used detailed microscopy to determine common and unique anatomical features of the FP and EFP sieve elements (SEs) in cucumber (Cucumis sativus). The comparison of exudation rates and the dynamic viscosity, density and sugar content of the exudates from plants grown in the light and dark revealed the consistent composition and behavior of the EFP exudate even when photosynthesis was prevented, thus differing from the properties of the FP exudate. Furthermore, the analysis of phloem transport using a fluorescein disodium salt showed only wound-induced exudation of dye from the EFP, indicating the absence of transport in this tissue. Our results show that it is important to distinguish between the EFP and FP in cucurbits, particularly their differing behaviors in response to wounding., (Copyright © 2022 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objectives: Inflammation, insulin resistance, hyperinsulinemia and cell damage are the major patho-physiological reasons behind type 2 diabetes (T2DM), which is one of the most prevalent non communicable metabolic disorders in the world. Oral hypoglycemic drugs and insulin shots are usually exercised to treat the diabetic patients but it produces many side effects. Thereby paving the way for natural hypoglycemic agents; a Himalayan herb and alternative nutritional therapy; low glycaemic indexed pumpkin seed, are used in combination for a better management of the disease. The aim of the study was to explore the combined efficacy of Gymnadenia orchidis Lindl root Salep and low-glycemic indexed-pumpkin seeds in better management of T2DM and associated complications., Methods: Balb/c mice were randomly allocated to six different groups (n=5). Streptozotocin along with high-fat-diet was used to induce T2DM. The experimental animals were supplemented with low-glycemic food or root Salep (200 mg/kg body weight) or combination of both according to their groups for 21 days, post which various biochemical tests were performed., Results: T2DM augmented the IL-6, IFN-γ, TNF-α, BAX, Insulin levels, and HOMA-IR with concurrent reduction of IL-4, QUICKI, Bcl-2, estradiol and progesterone levels. FACS revealed augmented cellular damage in T2DM mice. Interestingly, root Salep and pumpkin seeds normalized those parameters in T2DM animals suggesting significant (p<0.001) improvement of immunity of the diseased animals and ameliorated associated complications., Conclusions: Root Salep and pumpkin seed display synergism among binomial set of herbal agents which may be safely used for T2DM management., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

The native pumpkin 2S albumin, a multifunctional protein, possess a variety of potential biotechnologically exploitable properties. The present study reports the characterization of recombinant pumpkin 2S albumin (rP2SA) and unraveling of its potential DNA/RNA binding site. The purification and characterization of the rP2SA established that it retains the characteristic α-helical structure and exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. In vitro studies revealed that rP2SA exhibits potent antiviral activity against chikungunya virus (CHIKV) at a non-toxic concentration with an IC 50 of 114.5 μg/mL. In silico studies and site-directed mutagenesis were employed to unravel the potential DNA/RNA binding site. A strong positive charge distribution due to presence of many arginine residues in proximity of helix 5 was identified as a potential site. The two of the arginine residues, conserved in some 2S albumins, were selected for the mutation studies. The mutated forms of recombinant protein (R84A and R91A) showed a drastic reduction in DNase and RNase activities suggesting their presence at binding site and involvement in the nuclease activity. A metal binding site was also identified adjacent to DNA/RNA binding site. The present study demonstrated the structural and functional integrity of the rP2SA and reports potential antiviral activity against CHIKV. Further, potential DNA/RNA binding site was unraveled through mutation studies and bioinformatics analysis., Competing Interests: Declaration of competing interest The authors have no conflict of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Serpins constitute a large family of protease inhibitors with regulatory functions found in all living organisms. Most plant serpins have not been functionally characterized, with the exception of Arabidopsis thaliana AtSerpin1, an inhibitor of pro-apoptotic proteases, which is involved in the regulation of the programmed cell death induction, and Cucurbita maxima CmPS1, a phloem protein, which presumably inhibits insect digestive proteases and binds RNA. CmPS1 interacts most efficiently with highly structured RNA; in particular, it forms a specific complex with tRNA. Here, we demonstrated that AtSerpin1 also forms a complex with tRNA. Analysis of tRNA species bound by AtSerpin1 and CmPS1 in the presence of tRNA excess revealed that both proteins have no strict selectivity for individual tRNAs, suggesting specific interaction of AtSerpin1 and CmPS1 proteins with elements of the secondary/tertiary structure universal for all tRNAs. Analysis of CmPS1 binding of the microRNA precursor pre-miR390 and its mutants demonstrated that the pre-miR390 mutant with a perfect duplex in the hairpin stem lost the ability to form a discrete complex with CmPS1, whereas another variant of pre-miR390 with the native unpaired nucleotide residues in the stem retained this ability. These data indicate that specific interactions of plant serpins with structured RNA are based on the recognition of structurally unique spatial motifs formed with the participation of unpaired nucleotide residues in the RNA duplexes.

Main Conclusion: Genome-wide annotation reveals that the gene birth-death process of the Cucurbita R family is associated with a species-specific diversification of TNL and CNL protein classes. The Cucurbitaceae family includes nearly 1000 plant species known universally as cucurbits. Cucurbita genus includes many economically important worldwide crops vulnerable to more than 200 pathogens. Therefore, the identification of pathogen-recognition genes is of utmost importance for this genus. The major class of plant-resistance (R) genes encodes nucleotide-binding site and leucine-rich repeat (NLR) proteins, and is divided into three sub-classes namely, TIR-NB-LRR (TNL), CC-NB-LRR (CNL) and RPW8-NB-LRR (RNL). Although the characterization of the NLR gene family has been carried out in important Cucurbita species, this information is still linked to the availability of sequenced genomes. In this study, we analyzed 40 de novo transcriptomes and 5 genome assemblies, which were explored to investigate the Cucurbita expressed-NLR (eNLR) and NLR repertoires using an ad hoc gene annotation approach. Over 1850 NLR-encoding genes were identified, finely characterized and compared to 96 well-characterized plant R-genes. The maximum likelihood analyses revealed an unusual diversification of CNL/TNL genes and a strong RNL conservation. Indeed, several gene gain and loss events have shaped the Cucurbita NLR family. Finally, to provide a first validation step Cucurbita, eNLRs were explored by real-time PCR analysis. The NLR repertories of the 12 Cucurbita species presented in this paper will be useful to discover novel R-genes., (© 2021. The Author(s).)

Background: Postprandial hyperglycemia and decreased insulin secretion are relevant to risk factors in the development of type 2 diabetes and its complications. Plant foods with antidiabetic properties could be an affordable alternative in the prevention and treatment of this disease. In the present study, the antihyperglycemic and hypoglycemic activity of Bixa orellana, Psidium guajava L., Cucurbita moschata, Raphanus sativus L. and Brassica oleracea var. capitata - Mayan plant foods - were evaluated at doses of 5 and 10 mg kg -1 . Antihyperglycemic activity was measured in healthy Wistar rats and those with obesity induced by high-sucrose diet (group HSD) (20%). The hypoglycemic activity was measure in healthy CD1 mice., Results: Fasting glucose, Lee index and the body weight of HSD rats increased significantly (P ≤ 0.05) after 12 weeks of induction compared to healthy rats. In healthy rats, P. guajava and Bixa orellana (10 mg kg -1 ) demonstrated higher and statistically different (P ≤ 0.05) antihyperglycemic activity compared to control acarbose (0.5 mg kg -1 ). In the HSD rat group, all Mayan plant foods (10 mg kg -1 ) demonstrated antihyperglycemic activity statistically equal (P ≤ 0.05) to control acarbose. However, Brassica oleracea and R. sativus registered the highest antihyperglycemic activity. Bixa orellana and P. guajava (5 mg kg -1 ) showed similar hypoglycemic activity (P ≤ 0.05) to glibenclamide (0.5 mg kg -1 ) but was not significant (P ≤ 0.05) compared to insulin (5 UI kg -1 )., Conclusion: The present study provides valuable evidence on the possible health benefits of Mayan plant foods. These foods could contribute to the development of therapeutic diet strategies for the prevention and treatment of diabetes. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Cucurbita pepo is used as a vegetable in Pakistan and its seeds are also rich in tocopherol. Data showed the pivotal role of tocopherol in the treatment of Parkinson's disease (PD). The current study was designed to probe into the antiparkinson activity of methanolic extract of C. pepo (MECP) seeds in the haloperidol-induced Parkinson rat model. Behavioral studies showed improvement in motor functions. The increase in catalase, superoxide dismutase, glutathione levels whereas the decreases in the malondialdehyde and nitrite levels were noted in a dose-dependent manner. Acetylcholine-esterase (AchE) activity was increased. Molecular docking results revealed significant binding interaction of selected phytoconstituents within an active site of target protein AchE (PDB ID: 4EY7). Furthermore, α-synuclein was up regulated with down regulation of TNF-α and IL-1β in the qRT-PCR study. Subsequently, ADMET results on the basis of structure to activity predictions in terms of pharmacokinetics and toxicity estimations show that selected phytochemicals exhibited moderately acceptable properties. These properties add knowledge towards the structural features which could improve the bioavailability of selected phytochemicals before moving towards the initial phase of the drug development. Our integrated drug discovery scheme concluded that C. pepo seeds could ameliorate symptoms of PD and may prove a lead remedy for the treatment of PD., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The finely tuned balance between sources and sinks determines plant resource partitioning and regulates growth and development. Understanding and measuring metabolic indicators of source or sink limitation forms a vital part of global efforts to increase crop yield for future food security. We measured metabolic profiles of Cucurbita pepo (zucchini) grown in the field under carbon sink limitation and control conditions. We demonstrate that these profiles can be measured non-destructively using hyperspectral reflectance at both leaf and canopy scales. Total non-structural carbohydrates (TNC) increased 82% in sink-limited plants; leaf mass per unit area (LMA) increased 38% and free amino acids increased 22%. Partial least-squares regression (PLSR) models link these measured functional traits with reflectance data, enabling high-throughput estimation of traits comprising the sink limitation response. Leaf- and canopy-scale models for TNC had R 2 values of 0.93 and 0.64 and %RMSE of 13 and 38%, respectively. For LMA, R 2 values were 0.91 and 0.60 and %RMSE 7 and 14%; for free amino acids, R 2 was 0.53 and 0.21 with %RMSE 20 and 26%. Remote sensing can enable accurate, rapid detection of sink limitation in the field at the leaf and canopy scale, greatly expanding our ability to understand and measure metabolic responses to stress., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

A novel β-glucosidase was purified from pumpkin (Cucurbita moschata) seed by anion exchange chromatography and gel permeation chromatography, and its molecular mass was determined to be 42.8 kDa by gel permeation chromatography. The heterodimeric structure consisting of two subunits, free from disulfide bonds, was determined by native-PAGE analysis followed by zymography. The enzyme was maximally active at pH 4.0 and 70°C, and V max , K m , and k cat values were 0.078 units mg -1 protein, 2.22 mM, and 13.29 min -1 , respectively, employing p-nitrophenyl-β-d-glucopyranoside as the substrate. The high content of glycine determined by amino acid analysis implies that the enzyme possesses flexible conformations and interacts with cell membranes and walls in nature. Circular dichroism studies revealed that the high stability of the enzyme within the pH range of 2.0-10.0 is due to its reversible pH-responsive characteristics for α-helix-antiparallel β-sheet interconversion., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

To investigate the effect of relative humidity (RH) on chilling injury (CI), zucchini fruit were stored in cold rooms (4 ± 0.4 ℃) with different RHs (near-saturated RH [NSH] with 96-100% and normal RH with 72-76% served as control). Storage in NSH delayed weight loss and CI, maintained firmness and skin color. Higher antioxidant enzyme activities and greater scavenging capacities of free radicals were found in NSH-fruit than in the control fruit. The decrease of the unsaturated fatty acids was delayed in NSH-fruit due to lower activities of related membrane lipid degrading enzymes as compared to the control fruit. NSH-fruit also maintained higher activities of energy metabolism-associated enzymes than control fruit, leading to high levels of adenosine triphosphate (ATP). Taken together, we attributed the alleviation of CI by NSH storage to its enhancement of antioxidant capacities and its effect on maintaining higher energy status in zucchini fruit., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 .

Encapsulation systems promote targeted delivery to the gastrointestinal tract. An oil-in-water (O/W) nanoemulsion was covered using new delivery system composition based on zein and sodium alginate. The impact of aqueous phase (distilled water and cooked pumpkin puree), pH (2-4), and zein-alginate concentration solution (0.05-0.20% w/v) was investigated on particle size, zeta potential, incorporation efficiency (IE), stability, viscosity, and glucose release from single-layer (SLN) and double-layer nanoemulsion (DLN). DLNs showed a larger droplet size and zeta potential. The slow gradual release of glucose proved effective application of zein/alginate as delivery system for nanoemulsion. Moreover, cooked pumpkin and 0.12% of zein exhibited more delayed release of glucose than distilled water as an aqueous phase of DLN and as a delivery system respectively. Up-to-49% IE, up-to-50% stability in a period of 7-day storage, and controlled-release glucose for 240 min under in vitro gastrointestinal conditions were obtained in DLN. The results of the current study revealed that SLN covered by zein at 0.12% of concentration can be an ideal delivery system composition for patients with hypoglycemia and clinical problems., (Copyright © 2021. Published by Elsevier Masson SAS.)

AINTEGUMENTA-like (AIL) proteins are members of the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain family of transcription factors involved in plant growth, development, and abiotic stress responses. However, the biological functions of AIL members in pumpkin (Cucurbita moschata Duch.) remain unknown. In this study, we identified 12 AIL genes in the pumpkin genome encoding proteins predicted to be localized in the nucleus. Phylogenetic analysis showed that the AIL gene family could be classified into six major subfamilies, with each member encoding two AP2/ERF domains separated by a linker region. CmoAIL genes were expressed at varying levels in the examined tissues, and CmoANT genes showed different expression patterns under auxin (IAA), 1-naphthylphthalamic acid (NPA), and abscisic acid (ABA) treatments. Ectopic overexpression of CmoANT1.2 in Arabidopsis increased organ size and promoted growth of grafted plants by accelerating graft union formation. However, there was no significant difference at the graft junction for WT/WT and WT/ANT under IAA or NPA treatments. Taken together, the results of this study provide critical information about CmoAIL genes and their encoded proteins, and suggest future work should investigate the functions of CmoANT1.2 in the grafting process in pumpkin., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Dwarfism is an important agronomic trait in pumpkin that can increase yield. In this study, the dwarf Cucurbita pepo L. line X10 exhibited significantly longitudinally shorter cell length in the stem than did the normal-vine line JIN234. The dwarf stature of X10 was recovered with exogenous gibberellin (GA 3 ) application, suggesting that X10 might be sensitive to GA biosynthesis. Genetic analysis revealed that this dwarf trait is controlled by a single completely dominant locus: CpDw (Cucurbita pepo L. Dwarf). Using 1,300 F 2 individuals derived from a cross between X10 and JIN234, we mapped the CpDw locus to a region of approximately 24.6 kb on chromosome 10 that contain 5 annotated genes. The high expression level of Cp4.1LG10g05910.1 and high GA2ox enzyme activity in X10 revealed that the GA 2-oxidase gene Cp4.1LG10g05910.1 is a candidate gene for CpDw. Alignment of the Cp4.1LG10g05910.1 gene revealed two nonsynonymous single nucleotide polymorphism (SNP) mutations in the two exons, as well as several SNPs and InDels in the important functional elements of promoter between parental lines. Further allelic diversity analysis of the Cucurbita spp. germplasm resources indicated that Cp4.1LG10g05910.1 may be involved in vine growth during the early developmental stage in C. pepo but not in C. maxima or C. moschata. This study provides an important theoretical basis for the genetic regulation of vine length and crop breeding in pumpkin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Pumpkin seed cake (PSC), a byproduct of pumpkin seed oil processing, is used in ruminant feed as a beneficial protein source. Experiments were conducted to evaluate PSC as a substitute for soybean meal in the diets of lactating cows based on performance, rumen fermentation, antioxidant function and nitrogen partitioning. Six multiparous lactating cows were used in a replicated 3 × 3 Latin square experiment with 27-day periods. The cows were randomly divided into three treatment groups: group (1) was fed a diet containing no PSC (0PSC), and groups (2) and (3) were fed diets in which soybean meal was replaced with PSC and dried distillers grains with solubles (DDGS) at levels of 50% (50PSC) and 100% (100PSC), respectively. The diets were isonitrogenous and contained identical roughage but different proportions of PSC and DDGS. Replacement of soybean meal with PSC and DDGS did not influence rumen degradation, milk performance, rumen fermentation, DM intake or apparent total tract digestibility, and nitrogen partitioning between milk, feces and urine did not differ in the animals fed the three diets. However, compared with a diet containing no PSC, the total antioxidant capacity (P < 0.05) and antioxidant enzymes (total superoxide dismutase, glutathione peroxidase and catalase) activities (P < 0.05) were increased in the animals that received the 50PSC and 100PSC diets. In contrast, addition of PSC significantly reduced concentrations of aspartate transaminase (P < 0.05), alkaline phosphatase (P < 0.05) and malondialdehyde (P < 0.05) in the plasma. These results demonstrate that PSC can be completely substituted for soybean meal in the diet of dairy cows without any negative impact on milk performance, rumen fermentation or apparent digestibility and that this dietary change improves antioxidant functions and blood parameters in dairy cows, indicating that PSC has the potential for use as a feed source for dairy cows., (Copyright © 2020. Published by Elsevier B.V.)

Floral nectar is a sugary solution produced by nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in nectaries and nectar. Hypoxia-related processes are induced in nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo nectaries thus plays an important role in the synthesis and secretion of nectar sugar., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cyclocephalini beetles of the genus Cyclocephala (Coleoptera: Melolonthidae: Dynastinae) use flowers of some plants as food, shelter, and mating sites. However, little is known about floral scent chemistry involved in this interaction. Here we show that a sesquiterpene alcohol mediates attraction of Cyclocephala paraguayensis Arrow, on bottle gourd flowers, Lagenaria siceraria (Cucurbitaceae). Both males and females started to aggregate on the flowers at twilight; after that, mating began and remained for the entire night. GC-FID/EAD analysis of the L. siceraria floral scent collected in the field revealed that only the major constituent of the airborne volatiles elicited electroantennographic responses on male and female antennae of C. paraguayensis. This compound was identified as (3S,6E)-nerolidol, which was tested in two field trapping trials in Brazil. In the first bioassay, traps baited with nerolidol (mix of isomers) captured significantly more adult C. paraguayensis than control traps. In the second field trial, catches in traps baited with a mixture of isomers or enantiopure nerolidol were significantly higher than captures in control traps, but the treatments did not differ significantly. Analysis from the gut content of adult C. paraguayensis showed the presence of pollen, suggesting that they also use bottle gourd flowers for their nourishment. Taken together, these results suggest that (3S,6E)-nerolidol plays an essential role in the reproductive behavior of C. paraguayensis by eliciting aggregation, mating, and feeding., Competing Interests: The authors declare no conflict of interest.

Background: The fruit of Cucurbita ficifolia Bouché is known in Mexico as 'chilacayote'. The scientific interest that C. ficifolia Bouché has acquired is due to its important hypoglycemic effect. The present research aimed (i) to discover whether this hypoglycemic property is present at different stages of development of this fruit, and (ii) to characterize some bioactive compounds with antioxidant or anti-inflammatory properties. Ethylene production, respiration rate, and maturity indices were determined during fruit development. The chemical characterization of the aqueous extracts of each stage of maturity studied was determined and their hypoglycemic effects were bioassayed using groups of normal mice with diabetes induced by streptozotocin at a dose of 500 mg -1 kg -1 body weight., Results: Respiration rate and ethylene production showed a typical pattern for non-climacteric fruit and the quality parameters did not show significant changes. Phenolic compounds such as gallic acid and chlorogenic acid were found to have the highest concentration at 15 days of development. Extracts at 15 days showed a hypoglycemic effect that was 11% greater than that of glibenclamide in diabetized mice., Conclusion: All stages of development of C. ficifolia fruit had a hypoglycemic effect; however, the aqueous extract from the fruit at 15 days of development showed a better effect than glibenclamide. This finding highlights the potential of this maturity stage, and shows that it is appropriate for inclusion in treatments of type 2 diabetes mellitus. The results also indicate that phenolic compounds are mainly responsible for this effect and not d-chiro-inositol as previously thought. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Crop domestication and improvement often concurrently affect plant resistance to pests and production of secondary metabolites, creating challenges for isolating the ecological implications of selection for specific metabolites. Cucurbitacins are bitter triterpenoids with extreme phenotypic differences between Cucurbitaceae lineages, yet we lack integrated models of herbivore preference, cucurbitacin accumulation, and underlying genetic mechanisms. In Cucurbita pepo, we dissected the effect of cotyledon cucurbitacins on preference of a specialist insect pest (Acalymma vittatum) for multiple tissues, assessed genetic loci underlying cucurbitacin accumulation in diverse germplasm and a biparental F 2 population (from a cross between two independent domesticates), and characterized quantitative associations between gene expression and metabolites during seedling development. Acalymma vittatum affinity for cotyledons is mediated by cucurbitacins, but other traits contribute to whole-plant resistance. Cotyledon cucurbitacin accumulation was associated with population structure, and our genetic mapping identified a single locus, Bi-4, containing genes relevant to transport and regulation - not biosynthesis - that diverged between lineages. These candidate genes were expressed during seedling development, most prominently a putative secondary metabolite transporter. Taken together, these findings support the testable hypothesis that breeding for plant resistance to insects involves targeting genes for regulation and transport of defensive metabolites, in addition to core biosynthesis genes., (© 2020 John Wiley & Sons Ltd.)

: Grafting has been widely used to improve plant growth and tolerance in crop production, as well as for clarifying systemic mRNA signaling from donor to recipient tissues in organ-to-organ communication. In this study, we investigated graft partner interaction mechanisms of Cucumis sativus (Csa) and Cucurbita moschata (Cmo) using a large-scale endogenous mRNA transport. The results indicated that most mobile transcripts followed an allocation pathway from source to sink. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that mRNA mobility functions are universally common and individually specific. Identification of mRNA mobility between distant tissues in heterografts with RT-PCR (reverse transcription PCR), RT-qPCR (reverse transcriptional quantitative real time PCR), and clone sequencing were used to estimate 78.75% of selected mobile transcripts. Integration of bioinformatic analysis and RT-qPCR identification allowed us to hypothesize a scion-to-rootstock-to-scion feedback signal loop of Csa move-down and Cmo move-up mRNAs, where Csa scion move-down mRNAs were involved in carbon fixation and biosynthesis of amino acid pathways, and Cmo root received Csa move-down mRNA and then delivered the corresponding Cmo upward mRNA to scion to improve photosynthesis of cucumber scion. This formed a feedback signal loop of scion-to-rootstock-to scion to explain why pumpkin rootstock enhanced cucumber production in the industry, which was utilized for organ communication and mediates photosynthesis processes in heterograft cucurbit crops., Competing Interests: Funding: This work was supported by the National Key Research and Development Program of China (2018YFD1000800 and 2019YFD1000300), National Natural Science Foundation of China (31772358 and 31872158), and Earmarked Fund for China Agriculture Research System (CAS-23).