Fine mapping and transcriptome profiling reveal CpAPRR2 to modulate immature fruit rind color formation in zucchini (Cucurbita pepo).

KEY MESSAGE: A large fragment deletion of CpAPRR2, encoding a two-component response regulator-like protein, which influences immature white rind color formation in zucchini (Cucurbita pepo). Fruit rind color is an important agronomic trait that affects commodity quality and consumer choice in zucchini (Cucurbita pepo). However, the molecular mechanism controlling rind color is unclear. We characterized two zucchini inbred lines: '19' (dark green rind) and '113' (white rind). Genetic analysis revealed white immature fruit rind color to be controlled by a dominant locus (CpW). Combining bulked segregant analysis sequencing (BSA-seq) and Kompetitive Allele-Specific PCR (KASP) markers, we mapped the CpW locus to a 100.4 kb region on chromosome 5 and then narrow down the candidate region to 37.5 kb using linkage analysis of 532 BC1 and 1613 F2 individuals, including 6 coding genes. Among them, Cp4.1LG05g02070 (CpAPRR2), encoding a two-component response regulator-like protein, was regarded to be a promising candidate gene. The expression level of CpAPRR2 in dark green rind was significantly higher than that in white rind and was induced by light. A deletion of 2227 bp at the 5' end of CpAPRR2 in '113' might explain the white phenotype. Further analysis of allelic diversity in zucchini germplasm resources revealed rind color to be associated with the deletion of CpAPRR2. Subcellular localization analysis indicated that CpAPRR2 was a nuclear protein. Transcriptome analysis using near-isogenic lines with dark green (DG) and white (W) rind indicated that genes involved in photosynthesis and porphyrin metabolism pathways were enriched in DG compared with W. Additionally, chlorophyll synthesis-related genes were upregulated in DG. These results identify mechanisms of zucchini rind color and provide genetic resources for breeding.

Genome-wide identification and role of HSFs in antioxidant response of hot water treated zucchini fruit during cold storage.

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.

Transcriptomic analysis provides an insight into the function of CmGH9B3, a key gene of ß-1, 4-glucanase, during the graft union healing of oriental melon scion grafted onto squash rootstock.

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 ß-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.

Multi-Omics Analysis Reveals the Distinct Features of Metabolism Pathways Supporting the Fruit Size and Color Variation of Giant Pumpkin.

Pumpkin (Cucurbita maxima) is an important vegetable crop of the Cucurbitaceae plant family. The fruits of pumpkin are often used as directly edible food or raw material for a number of processed foods. In nature, mature pumpkin fruits differ in size, shape, and color. The Atlantic Giant (AG) cultivar has the world's largest fruits and is described as the giant pumpkin. AG is well-known for its large and bright-colored fruits with high ornamental and economic value. At present, there are insufficient studies that have focused on the formation factors of the AG cultivar. To address these knowledge gaps, we performed comparative transcriptome, proteome, and metabolome analysis of fruits from the AG cultivar and a pumpkin with relatively small fruit (Hubbard). The results indicate that up-regulation of gene-encoded expansins contributed to fruit cell expansion, and the increased presence of photoassimilates (stachyose and D-glucose) and jasmonic acid (JA) accumulation worked together in terms of the formation of large fruit in the AG cultivar. Notably, perhaps due to the rapid transport of photoassimilates, abundant stachyose that was not converted into glucose in time was detected in giant pumpkin fruits, implying that a unique mode of assimilate unloading is in existence in the AG cultivar. The potential molecular regulatory network of photoassimilate metabolism closely related to pumpkin fruit expansion was also investigated, finding that three MYB transcription factors, namely CmaCh02G015900, CmaCh01G018100, and CmaCh06G011110, may be involved in metabolic regulation. In addition, neoxanthin (a type of carotenoid) exhibited decreased accumulation that was attributed to the down-regulation of carotenoid biosynthesis genes in AG fruits, which may lead to pigmentation differences between the two pumpkin cultivars. Our current work will provide new insights into the potential formation factors of giant pumpkins for further systematic elucidation.

Sequence characteristics, genetic diversity and phylogenetic analysis of the Cucurbita ficifolia (Cucurbitaceae) chloroplasts genome.

BACKGROUND: Curcubita ficifolia Bouché (Cucurbitaceae) has high value as a food crop and medicinal plant, and also has horticultural value as rootstock for other melon species. China is home to many different cultivars, but the genetic diversity of these resources and the evolutionary relationships among them, as well as the differences between C. ficifolia and other Cucurbita species, remain unclear. RESULTS: We investigated the chloroplast (cp) genomes of 160 C. ficifolia individuals from 31 populations in Yunnan, a major C. ficifolia production area in China. We found that the cp genome of C. ficifolia is ~151 kb and contains 128 genes, of which 86 are protein coding genes, 34 encode tRNA, and eight encode rRNAs. We also identified 64 SSRs, mainly AT repeats. The cp genome was found to contain a total of 204 SNP and 57 indels, and a total of 21 haplotypes were found in the 160 study individuals. The reverse repeat (IR) region of C. ficifolia contained a few differences compared with this region in the six other Cucurbita species. Sequence difference analysis demonstrated that most of the variable regions were concentrated in the single copy (SC) region. Moreover, the sequences of the coding regions were found to be more similar among species than those of the non-coding regions. The phylogenies reconstructed from the cp genomes of 61 representative species of Cucurbitaceae reflected the currently accepted classification, in which C. ficifolia is sister to the other Cucurbita species, however, different interspecific relationships were found between Cucurbita species. CONCLUSIONS: These results will be valuable in the classification of C. ficifolia genetic resources and will contribute to our understanding of evolutionary relationships within the genus Cucurbita.

Genome-Wide Identification of the WRKY Gene Family and Functional Characterization of CpWRKY5 in Cucurbita pepo.

The WRKY gene family is crucial for regulating plant growth and development. However, the WRKY gene is rarely studied in naked kernel formation in hull-less Cucurbita pepo L. (HLCP), a natural mutant that lacks the seed coat. In this research, 76 WRKY genes were identified through bioinformatics-based methods in C. pepo, and their phylogenetics, conserved motifs, synteny, collinearity, and temporal expression during seed coat development were analyzed. The results showed that 76 CpWRKYs were identified and categorized into three main groups (I-III), with Group II further divided into five subgroups (IIa-IIe). Moreover, 31 segmental duplication events were identified in 49 CpWRKY genes. A synteny analysis revealed that C. pepo shared more collinear regions with cucumber than with melon. Furthermore, quantitative RT-PCR (qRT-PCR) results indicated the differential expression of CpWRKYs across different varieties, with notable variations in seed coat development between HLCP and CP being attributed to differences in CpWRKY5 expression. To investigate this further, CpWRKY5-overexpression tobacco plants were generated, resulting in increased lignin content and an upregulation of related genes, as confirmed by qRT-PCR. This study offers valuable insights for future functional investigations of CpWRKY genes and presents novel information for understanding the regulation mechanism of lignin synthesis.

Genome­wide analysis of the MYB gene family in pumpkin.

The MYB gene family exerts significant influence over various biological processes and stress responses in plants. Despite this, a comprehensive analysis of this gene family in pumpkin remains absent. In this study, the MYB genes of Cucurbita moschata were identified and clustered into 33 groups (C1-33), with members of each group being highly conserved in terms of their motif composition. Furthermore, the distribution of 175 CmoMYB genes across all 20 chromosomes was found to be non-uniform. Examination of the promoter regions of these genes revealed the presence of cis-acting elements associated with phytohormone responses and abiotic/biotic stress. Utilizing quantitative real-time polymerase chain reaction (qRT-PCR), the expression patterns of 13 selected CmoMYB genes were validated, particularly in response to exogenous phytohormone exposure and various abiotic stressors, including ABA, SA, MeJA, and drought treatments. Expression analysis in different tissues showed that CmoMYB genes are expressed at different levels in different tissues, suggesting that they are functionally divergent in regulating growth and abiotic stresses. These results provide a basis for future studies to characterize the function of the MYB gene family under abiotic stresses in pumpkins.

Unveiling the mechanism of source-sink rebalancing in cucumber-pumpkin heterografts: the buffering roles of rootstock cotyledon.

Grafting onto pumpkin rootstock is widely applied in cucumber production to improve growth and yield, as well as to overcome soil-borne diseases and enhance resistance to abiotic stresses. In this study, we constructed the cucumber-pumpkin heterografts with the one-cotyledon grafting method, and examined the effects of heterografting on biomass allocation and sugar partitioning, with cucumber and pumpkin self-grafts used as control. Compared with cucumber self-grafts, heterografting onto pumpkin rootstock promoted photosynthesis in cucumber scion, and led to higher sucrose contents in the 1st true leaf (source) and newly emerged leaf (sink). Thereby, the scion part of heterografts accumulated more biomass than cucumber self-grafts. In contrast, when compared to pumpkin self-grafts, grafting with cucumber scion reduced root vigor and biomass but promoted cotyledon growth in pumpkin rootstock. The roots (sink) of heterografts contained less sucrose and hexoses, and showed reduced sucrose synthase (SuSy) and hexokinase (HXK) activities. However, the rootstock cotyledon (source) contained more sucrose and starch, and showed higher activities of HXK, cell-wall invertase (CWIN), and enzymes for starch synthesis and degradation. Furthermore, removal or shade of rootstock cotyledon led to reduced growth of root and scion. Silencing of CmoMEX1a gene in rootstock cotyledon inhibited maltose export and reduced root growth of heterografts. These results indicated that rootstock cotyledon, especially its starch content, played a buffering role in the growth regulation of cucumber-pumpkin heterografts. Taken together, our results provided a major contribution to our understanding of source-sink sugar partitioning and scion-rootstock growth balancing in cucumber-pumpkin heterografts.

CmoPIP1-4 confers drought tolerance in pumpkin by altering hydrogen sulfide signaling.

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 (H2S) 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 H2S-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 H2S-mediated signaling, influencing stomatal density and aperture in pumpkin plants, and thereby enhancing their drought tolerance.

Molecular markers associated with resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus in tropical pumpkin (Cucurbita moschata Duchesne ex Poir.) breeding line AVPU1426.

Virus diseases are a major production constraint for pumpkin. Recessive resistance to squash leaf curl China virus and tomato leaf curl New Delhi virus has been mapped in Cucurbita moschata (Duchesne ex Poir.) breeding line AVPU1426 to chromosomes 7 and 8, respectively. Molecular markers tightly associated with the resistance loci have been developed and were able to correctly predict resistance and susceptibility with an accuracy of 99% for squash leaf curl China virus resistance and 94.34% for tomato leaf curl New Delhi virus in F2 and back cross populations derived from the original resistance source AVPU1426. The markers associated with resistance are recommended for use in marker-assisted breeding.

Artificial neural networks optimize the establishment of a Brazilian germplasm core collection of winter squash (Cucurbita moschata D.).

With widespread cultivation, Cucurbita moschata stands out for the carotenoid content of its fruits such as ß and α-carotene, components with pronounced provitamin A function and antioxidant activity. C. moschata seed oil has a high monounsaturated fatty acid content and vitamin E, constituting a lipid source of high chemical-nutritional quality. The present study evaluates the agronomic and chemical-nutritional aspects of 91 accessions of C. moschata kept at the BGH-UFV and propose the establishment of a core collection based on multivariate approaches and on the implementation of Artificial Neural Networks (ANNs). ANNs was more efficient in identifying similarity patterns and in organizing the distance between the genotypes in the groups. The averages and variances of traits in the CC formed using a 15% sampling of accessions, were closer to those of the complete collection, particularly for accumulated degree days for flowering, the mass of seeds per fruit, and seed and oil productivity. Establishing the 15% CC, based on the broad characterization of this germplasm, will be crucial to optimize the evaluation and use of promising accessions from this collection in C. moschata breeding programs, especially for traits of high chemical-nutritional importance such as the carotenoid content and the fatty acid profile.

Structural and functional characterization of genes PYL-PP2C-SnRK2s in the ABA signalling pathway of Cucurbita pepo.

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.

Morphological Analyses and QTL Mapping of Mottled Leaf in Zucchini (Cucurbita pepo L.).

The mottled leaf is one of the agronomic traits of zucchini and can be applied as a marker trait in aggregation breeding. However, the genetic mechanism responsible for mottled leaf has yet to be elucidated. In the present study, we used two inbred lines (line '19': silver mottled leaf; line '113': normal leaf) as parents for the physiological and genetic analysis of mottled leaf. The synthesis and net photosynthetic rate of chlorophyll were not significantly affected in the mottled areas of leaves. However, we detected a large space between the palisade parenchyma in the leaf mottle area of line '19', which may have caused the mottled leaf phenotype. Light also plays an important role in the formation of mottled leaf, and receiving light during the early stages of leaf development is a necessary factor. Genetic analysis has previously demonstrated that mottled leaf is a quantitative trait that is controlled by multiple genes. Based on the strategy of quantitative trait locus sequencing (QTL-seq), two QTLs were identified on chromosomes 1 and 17, named CpML1.1 and CpML17.1, respectively. Two major loci were identified using R/qtl software version 1.66 under greenhouse conditions in April 2019 (2019A) and April 2020 (2020A) and under open cultivation conditions in May 2020 (2020M). The major QTL, CpML1.1, was located in a 925.2-kb interval on chromosome 1 and explained 10.51%-24.15% of the phenotypic variation. The CpML17.1 was located in a 719.7-kb interval on chromosome 17 and explained 16.25%-38.68% of the phenotypic variation. Based on gene annotation, gene sequence alignment, and qRT-PCR analysis, the Cp4.1LG01g23790 at the CpML1.1 locus encoding a protein of the TPX2 family (target protein of Xklp2) may be a candidate gene for mottled leaf in zucchini. Our findings may provide a theoretical basis for the formation of mottled leaf and provide a foundation for the fine mapping of genes associated with mottled leaf. Molecular markers closely linked to mottled leaf can be used in molecular-assisted selection for the zucchini mottled leaf breeding.

A long noncoding RNA functions in pumpkin fruit development through S-adenosyl-L-methionine synthetase.

Long noncoding RNAs (lncRNAs) play important roles in various biological processes. However, the regulatory roles of lncRNAs underlying fruit development have not been extensively studied. The pumpkin (Cucurbita spp.) is a preferred model for understanding the molecular mechanisms regulating fruit development because of its variable shape and size and large inferior ovary. Here, we performed strand-specific transcriptome sequencing on pumpkin (Cucurbita maxima "Rimu") fruits at 6 developmental stages and identified 5,425 reliably expressed lncRNAs. Among the 332 lncRNAs that were differentially expressed during fruit development, the lncRNA MSTRG.44863.1 was identified as a negative regulator of pumpkin fruit development. MSTRG.44863.1 showed a relatively high expression level and an obvious period-specific expression pattern. Transient overexpression and silencing of MSTRG.44863.1 significantly increased and decreased the content of 1-aminocyclopropane carboxylic acid (a precursor of ethylene) and ethylene production, respectively. RNA pull-down and microscale thermophoresis assays further revealed that MSTRG.44863.1 can interact with S-adenosyl-L-methionine synthetase (SAMS), an enzyme in the ethylene synthesis pathway. Considering that ethylene negatively regulates fruit development, these results indicate that MSTRG.44863.1 plays an important role in the regulation of pumpkin fruit development, possibly through interacting with SAMS and affecting ethylene synthesis. Overall, our findings provide a rich resource for further study of fruit-related lncRNAs while offering insights into the regulation of fruit development in plants.

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.

Identification and functional characterization of conserved cis-regulatory elements responsible for early fruit development in cucurbit crops.

A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved noncoding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865 to 43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2, and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

Identification of ARF genes in Cucurbita pepo L and analysis of expression patterns, and functional analysis of CpARF22 under drought, salt stress.

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.

Transcriptome-based analysis of candidate gene markers associated with resistance mechanism to Phytophthora melonis that causes root and crown rot in pumpkin.

Root and crown rot incited by an oomycete, Phytophthora melonis , causes significant yield losses in commercial pumpkin (Cucurbita pepo ) production worldwide. Currently, resistant cultivars and knowledge of molecular mechanism of C. pepo against P. melonis are scarce. Here, we analysed the quantitative gene expression changes of 10 candidate gene markers (bHLH87, ERF014, HSF, MYB, PR-1, WRKY21, CPI, POD, PSK, SGT ) in pumpkin roots and leaves at three time points (h post-inoculation, hpi) following inoculation with P. melonis in two resistant (Ghelyani and Tanbal), and two susceptible (Marmari and Khoreshti) varieties of pumpkin. Gene expression using quantitative real time PCR along a time course revealed the strongest transcriptomic response at 48 and 72hpi in resistant genotypes, 1.1-2.7-fold in roots and leaves, respectively, with a high significant correlation (r =0.857**-0.974**). We also found that CPI , PSK, SGT1 and POD act as a dual regulator that similarly modulate immunity not only against P. melonis , but also against other diseases such as early blight (Alternaria cucumerina) , powdery mildew (Podosphaera xanthii ), downy mildews (Pseudoperonospora cubensis ), and pathogenic plant nematodes (Meloidogyne javanica ). Furthermore, significantly higher activities of the ROS scavenging defence enzymes, catalase (1.6-fold increase) and peroxidase (6-fold increase) were observed in the roots of resistant cultivars at different hpi compared with non-inoculated controls. In addition, the biomass growth parameters including leaf and root length, stem and root diameter, root fresh weight and volume were significantly different among studied genotypes. Cumulatively, the transcriptome data provide novel insights into the response of pumpkins for improving pumpkin breeding to P. melonis .

Production of transgenic periclinal chimeras in pumpkin - a tool for revealing cell fates of L1 meristem.

Genetic engineering is commonly used to improve the agronomic traits of crops. However, genetic transformation in pumpkin remains a challenge. Conducting transformation trials, we accidentally created transgenic L1 periclinal chimeras in pumpkins. Using our modified Agrobacterium-mediated transformation, we generated transgenic L1 periclinal chimeras which have high value in research on development of the meristem. Fluorescence observations of transformed L1 cells enabled us to reveal cell fates. These L1 cells can develop into stomata, epidermal hairs, seed coat, and epidermis of the root, stem, leaf, flower, and fruit. These periclinal chimeras can be propagated vegetatively with minimal risk of transgene flow. This study offers new perspectives on development of the meristem and a promising technique for creating transgenic periclinal chimeras in plants.

Natural variation in the promoter of FLOWERING LOCUS T-LIKE 2 in pumpkin (Cucurbita moschata Duch.) is associated with flowering time under short-day conditions.

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 F2 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.