The ethylene-responsive transcription factor ERF024 is a novel regulator of climacteric fruit ripening in melon.

Fruit ripening is an essential developmental stage in Angiosperms triggered by hormonal signals such as ethylene, a major player in climacteric ripening. Melon is a unique crop showing both climacteric and non-climacteric cultivars, offering an ideal model for dissecting the genetic mechanisms underpinning this process. The major quantitative trait locus ETHQV8.1 was previously identified as a key regulator of melon fruit ripening. Here, we narrowed down ETHQV8.1 to a precise genomic region containing a single gene, the transcription factor CmERF024. Functional validation using CRISPR/Cas9 knock-out plants unequivocally identified CmERF024 as the causal gene governing ETHQV8.1. The erf024 mutants exhibited suppression of ethylene production, leading to a significant delay and attenuation of fruit ripening. Integrative multi-omic analyses encompassing RNA-seq, DAP-seq, and DNase-seq revealed the association of CmERF024 with chromatin accessibility and gene expression dynamics throughout fruit ripening. Our data suggest CmERF024 as a novel regulator of climacteric fruit ripening in melon.

Genome-wide characterization and functional analysis of the melon TGA gene family in disease resistance through ectopic overexpression in Arabidopsis thaliana.

TGA-binding (TGA) transcription factors, characterized by the basic region/leucine zipper motif (bZIP), have been recognized as pivotal regulators in plant growth, development, and stress responses through their binding to the as-1 element. In this study, the TGA gene families in melon, watermelon, cucumber, pumpkin, and zucchini were comprehensively characterized, encompassing analyses of gene/protein structures, phylogenetic relationships, gene duplication events, and cis-acting elements in gene promoters. Upon transient expression in Nicotiana benthamiana, the melon CmTGAs, with typical bZIP and DOG1 domains, were observed to localize within the nucleus. Biochemical investigation revealed specific interactions between CmTGA2/3/5/8/9 and CmNPR3 or CmNPR4. The CmTGA genes exhibited differential expression patterns in melon plants in response to different hormones like salicylic acid, methyl jasmonate, and ethylene, as well as a fungal pathogen, Stagonosporopsis cucurbitacearum that causes gummy stem blight in melon. The overexpression of CmTGA3, CmTGA8, and CmTGA9 in Arabidopsis plants resulted in the upregulation of AtPR1 and AtPR5 expression, thereby imparting enhanced resistance to Pseudomonas syringae pv. Tomato DC3000. In contrast, the overexpression of CmTGA7 or CmTGA9 resulted in a compromised resistance to Botrytis cinerea, coinciding with a concomitant reduction in the expression levels of AtPDF1.2 and AtMYC2 following infection with B. cinerea. These findings shed light on the important roles of specific CmTGA genes in plant immunity, suggesting that genetic manipulation of these genes could be a promising avenue for enhancing plant immune responses.

Genome-wide identification of SAMS gene family in Cucurbitaceae and the role of ClSAMS1 in watermelon tolerance to abiotic stress.

S-Adenosyl-L-methionine (SAM) is widely involved in plant growth, development, and abiotic stress response. SAM synthetase (SAMS) is the key enzyme that catalyzes the synthesis of SAM from methionine and ATP. However, the SAMS gene family has not been identified and their functions have not been characterized in most Cucurbitaceae plants. Here, a total of 30 SAMS genes were identified in nine Cucurbitaceae species and they were categorized into 3 subfamilies. Physicochemical properties and gene structure analysis showed that the SAMS protein members are tightly conserved. Further analysis of the cis-regulatory elements (CREs) of SAMS genes' promoter implied their potential roles in stress tolerance. To further understand the molecular functions of SAMS genes, watermelon SAMSs (ClSAMSs) were chosen to analyze the expression patterns in different tissues and under various abiotic stress and hormone responses. Among the investigated genes, ClSAMS1 expression was observed in all tissues and found to be up-regulated by abiotic stresses including salt, cold and drought treatments as well as exogenous hormone treatments including ETH, SA, MeJA and ABA. Furthermore, knockdown of ClSAMS1 via virus-induced gene silencing (VIGS) decreased SAM contents in watermelon seedings. The pTRSV2-ClSAMS1 plants showed reduced susceptibility to drought, cold and NaCl stress, indicating a positive role of ClSAMS1 in abiotic stresses tolerance. Those results provided candidate SAMS genes to regulate plant resistance against abiotic stresses in Cucurbitaceae plants.

Ethylene produced in carpel primordia controls CmHB40 expression to inhibit stamen development.

Sex determination evolved to control the development of unisexual flowers. In agriculture, it conditions how plants are cultivated and bred. We investigated how female flowers develop in monoecious cucurbits. We discovered in melon, Cucumis melo, a mechanism in which ethylene produced in the carpel is perceived in the stamen primordia through spatially differentially expressed ethylene receptors. Subsequently, the CmEIN3/CmEIL1 ethylene signalling module, in stamen primordia, activates the expression of CmHB40, a transcription factor that downregulates genes required for stamen development and upregulates genes associated with organ senescence. Investigation of melon genetic biodiversity revealed a haplotype, originating in Africa, altered in EIN3/EIL1 binding to CmHB40 promoter and associated with bisexual flower development. In contrast to other bisexual mutants in cucurbits, CmHB40 mutations do not alter fruit shape. By disentangling fruit shape and sex-determination pathways, our work opens up new avenues in plant breeding.

Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen.

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Chromosome-Level Genome Assembly of Herpetospermum pedunculosum (Cucurbitaceae).

This study presents a chromosome-level reference genome assembly of a traditional Tibetan medicinal plant, Herpetospermum pedunculosum belonging to the Cucurbitaceae family. Following a combined PacBio high-fidelity sequencing and Hi-C analysis, a final H. pedunculosum genome assembly, 804.11 Mb in length was obtained, 90.45% of which was anchored into ten pseudochromosomes with a contig N50 of 24.39 Mb. In addition, 579.55 Mb repetitive sequences and 23,924 high-confidence protein-coding genes were annotated. Phylogenetic analysis revealed that H. pedunculosum was sister to a clade formed by cucumber, zucchini, and wax gourd. Further whole-genome duplication analysis revealed no recent polyploidization event in the H. pedunculosum genome. The high-quality H. pedunculosum genome presented here will be highly useful in investigating the molecular mechanisms underlying the biosynthesis of its active compounds and adaptation strategies to the extreme environment. It will also provide great insights into comparative genomic studies of Cucurbitaceae and flowering plants.

Mining Candidate Genes Related to Heavy Metals in Mature Melon (Cucumis melo L.) Peel and Pulp Using WGCNA.

The content of metal ions in fruits is inseparable from plant intake of trace elements and health effects in the human body. To understand metal ion content in the fruit and pericarp of melon (Cucumis melo L.) and the candidate genes responsible for controlling this process, we analyzed the metal ion content in distinct parts of melon fruit and pericarp and performed RNA-seq. The results showed that the content of metal ions in melon fruit tissue was significantly higher than that in the pericarp. Based on transcriptome expression profiling, we found that the fruit and pericarp contained elevated levels of DEGs. GO functional annotations included cell surface receptor signaling, signal transduction, organic substance metabolism, carbohydrate derivative binding, and hormone-mediated signaling pathways. KEGG pathways included pectate lyase, pentose and glucuronate interconversions, H+-transporting ATPase, oxidative phosphorylation, plant hormone signal transduction, and MAPK signaling pathways. We also analyzed the expression patterns of genes and transcription factors involved in hormone biosynthesis and signal transduction. Using weighted gene co-expression network analysis (WGCNA), a co-expression network was constructed to identify a specific module that was significantly correlated with the content of metal ions in melon, after which the gene expression in the module was measured. Connectivity and qRT-PCR identified five candidate melon genes, LOC103501427, LOC103501539, LOC103503694, LOC103504124, and LOC107990281, associated with metal ion content. This study provides a theoretical basis for further understanding the molecular mechanism of heavy metal ion content in melon fruit and peel and provides new genetic resources for the study of heavy metal ion content in plant tissues.

A common whole-genome paleotetraploidization in Cucurbitales.

Cucurbitales are an important order of flowering plants known for encompassing edible plants of economic and medicinal value and numerous ornamental plants of horticultural value. By reanalyzing the genomes of two representative families (Cucurbitaceae and Begoniaceae) in Cucurbitales, we found that the previously identified Cucurbitaceae common paleotetraploidization that occurred shortly after the core-eudicot-common hexaploidization event is shared by Cucurbitales, including Begoniaceae. We built a multigenome alignment framework for Cucurbitales by identifying orthologs and paralogs and systematically redating key evolutionary events in Cucurbitales. Notably, characterizing the gene retention levels and genomic fractionation patterns between subgenomes generated from different polyploidizations in Cucurbitales suggested the autopolyploid nature of the Begoniaceae common tetraploidization and the allopolyploid nature of the Cucurbitales common tetraploidization and the Cucurbita-specific tetraploidization. Moreover, we constructed the ancestral Cucurbitales karyotype comprising 17 proto-chromosomes, confirming that the most recent common ancestor of Cucurbitaceae contained 15 proto-chromosomes and rejecting the previous hypothesis for an ancestral Cucurbitaceae karyotype with 12 proto-chromosomes. In addition, we found that the polyploidization and tandem duplication events promoted the expansion of gene families involved in the cucurbitacin biosynthesis pathway; however, gene loss and chromosomal rearrangements likely limited the expansion of these gene families.

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides.

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides.

Ethylene plays a dual role in sex determination and fruit shape in cucurbits.

Shapes of vegetables and fruits are the result of adaptive evolution and human selection. Modules controlling organ shape have been identified. However, little is known about signals coordinating organ development and shape. Here, we describe the characterization of a melon mutation rf1, leading to round fruit. Histological analysis of rf1 flower and fruits revealed fruit shape is determined at flower stage 8, after sex determination and before flower fertilization. Using positional cloning, we identified the causal gene as the monoecy sex determination gene CmACS7, and survey of melon germplasms showed strong association between fruit shape and sexual types. We show that CmACS7-mediated ethylene production in carpel primordia enhances cell expansion and represses cell division, leading to elongated fruit. Cell size is known to rise as a result of endoreduplication. At stage 8 and anthesis, we found no variation in ploidy levels between female and hermaphrodite flowers, ruling out endoreduplication as a factor in fruit shape determination. To pinpoint the gene networks controlling elongated versus round fruit phenotype, we analyzed the transcriptomes of laser capture microdissected carpels of wild-type and rf1 mutant. These high-resolution spatiotemporal gene expression dynamics revealed the implication of two regulatory modules. The first module implicates E2F-DP transcription factors, controlling cell elongation versus cell division. The second module implicates OVATE- and TRM5-related proteins, controlling cell division patterns. Our finding highlights the dual role of ethylene in the inhibition of the stamina development and the elongation of ovary and fruit in cucurbits.

CRISPR/Cas9 gene editing uncovers the roles of CONSTITUTIVE TRIPLE RESPONSE 1 and REPRESSOR OF SILENCING 1 in melon fruit ripening and epigenetic regulation.

Melon (Cucumis melo) has emerged as an alternative model to tomato for studying fruit ripening due to the coexistence of climacteric and non-climacteric varieties. Previous characterization of a major quantitative trait locus (QTL), ETHQV8.1, that is able to trigger climacteric ripening in a non-climacteric background resulted in the identification of a negative regulator of ripening CTR1-like (MELO3C024518) and a putative DNA demethylase ROS1 (MELO3C024516) that is the orthologue of DML2, a DNA demethylase that regulates fruit ripening in tomato. To understand the role of these genes in climacteric ripening, in this study we generated homozygous CRISPR knockout mutants of CTR1-like and ROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two growing seasons with an earlier ethylene production profile being observed compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single-cytosine methylome analyses of the ROS1-knockout mutant revealed changes in DNA methylation in the promoter regions of the key ripening genes such as ACS1, ETR1, and ACO1, and in transcription factors associated with ripening including NAC-NOR, RIN, and CNR, suggesting the importance of ROS1-mediated DNA demethylation for triggering fruit ripening in melon.

Regulation of ethylene biosynthesis and signal transduction by nitric oxide leading to resistance against Alternaria alternata in Hami melon.

BACKGROUND: Hami melons are tasty and nutritive, but susceptibility to the fungus Alternaria alternata is one of the main problems leading to the postharvest loss of this fruit. The purpose of this research was to evaluate the effectiveness of nitric oxide (NO) on regulation of ethylene biosynthesis as well as signal transduction against black spot disease caused by A. alternata in the Hami melon. RESULTS: Nitric oxide reduced the growth of lesion diameter and lesion depth in melons inoculated with A. alternata. Ethylene production was significantly inhibited by NO, which was supported by the reduction of 1-aminocyclopropene-1-carboxylate (ACC) synthase (ACS) activity and the deferment of ACC content and ACC oxidase (ACO) activity. Nitric oxide treatment also significantly regulated the expression of four ethylene biosynthesis genes CmACS1, CmACS2, CmACO1, and CmACO2, and eight signal ethylene transduction genes CmETR1, CmETR2, CmCTR1, CmEIN2, CmEIL1, CmEBF1, CmERF1B and CmERF2. The modes of NO regulating these genes can be divided into five categories: promotion (CmEIN2, and CmEIL1), delay (CmACS1, CmETR2, CmCTR1 and CmERF2), up-regulation (CmETR1, CmEBF1 and CmERF1B), down-regulation (CmACS2), and first inhibition and then induction (CmACO1 and CmACO2). CONCLUSION: The NO treatment enhanced the postharvest disease resistance of Hami melon attacked by A. alternata, possibly by postponing ethylene biosynthesis and signal transduction. © 2021 Society of Chemical Industry.

Systematic genome-wide analysis of the ethylene-responsive ACS gene family: Contributions to sex form differentiation and development in melon and watermelon.

Ethylene is an important regulatory phytohormone for sex differentiation and flower development. As the rate-limiting enzyme encoding genes in ethylene biosynthesis, ACS gene family has been well studied in cucumber; however, little is known in other cucurbit crops, such as melon and watermelon, which show diverse sex types in the field. Here, we identified and characterized eight ACS genes each in the genomes of melon and watermelon. According to the conserved serine residues at C-terminal, all the ACS genes could be characterized into three groups, which were supported by the exon-intron organizations and conserved motif distributions. ACS genes displayed diverse tissue-specific expression patterns among four melon and three watermelon sex types. Furthermore, a comparative expression analysis in the shoot apex identified orthologous pairs with potential functions in sex determination, e.g., ACS1s and ACS6s. All ACS orthologs in melon and watermelon exhibited similar expression patterns in monoecious and gynoecious genotypes, except for ACS11s and ACS12s. As expected, the majority of ACS genes were responsive to exogenous ethephon; however, some orthologs exhibited opposite expression patterns, such as ACS1s, ACS9s, and ACS10s. Collectively, our findings provide valuable ACS candidates related to flower development in various sex types of melon and watermelon.

The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism.

Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.

Development of an efficient transient expression system for Siraitia grosvenorii fruit and functional characterization of two NADPH-cytochrome P450 reductases.

Siraitia grosvenorii (Luo hanguo or monk fruit) is a valuable medicinal herb for which the market demand has increased dramatically worldwide. As promising natural sweeteners, mogrosides have received much attention from researchers because of their extremely high sweetness and lack of calories. Nevertheless, owing to the absence of genetic transformation methods, the molecular mechanisms underlying the regulation of mogroside biosynthesis have not yet been fully elucidated. Therefore, an effective method for gene function analysis needs to be developed for S. grosvenorii fruit. As a powerful approach, transient expression has become a versatile method to elucidate the biological functions of genes and proteins in various plant species. In this study, PBI121 with a ß-glucuronidase (GUS) marker and tobacco rattle virus (TRV) were used as vectors for overexpression and silencing, respectively, of the SgCPR1 and SgCPR2 genes in S. grosvenorii fruit. The effectiveness of transient expression was validated by GUS staining in S. grosvenorii fruit, and the expression levels of SgCPR1 and SgCPR2 increased significantly after infiltration for 36 h. In addition, TRV-induced gene silencing suppressed the expression of SgCPR1 and SgCPR2 in S. grosvenorii fruit. More importantly, the production of the major secondary metabolites mogrol, mogroside IIE (MIIE) and mogroside III (MIII) was activated by the overexpression of SgCPR1 and SgCPR2 in S. grosvenorii fruit, with levels 1-2 times those in the control group. Moreover, the accumulation of mogrol, MIIE and MIII was decreased in the SgCPR1 and SgCPR2 gene silencing assays. Therefore, this transient expression approach was available for S. grosvenorii fruit, providing insight into the expression of the SgCPR1 and SgCPR2 genes involved in the mogroside biosynthesis pathway. Our study also suggests that this method has potential applications in the exploration of the molecular mechanisms, biochemical hypotheses and functional characteristics of S. grosvenorii genes.

Bioprospecting of Sechium spp. varieties for the selection of characters with pharmacological activity.

Bioprospecting identifies new sources of compounds with actual or potential economic value that come from biodiversity. An analysis was performed regarding bioprospecting purposes in ten genotypes of Sechium spp., through a meta-analysis of 20 information sources considering different variables: five morphological, 19 biochemical, anti-proliferative activity of extracts on five malignant cell lines, and 188 polymorphic bands of amplified fragment length polymorphisms, were used in order to identify the most relevant variables for the design of genetic interbreeding. Significant relationships between morphological and biochemical characters and anti-proliferative activity in cell lines were obtained, with five principal components for principal component analysis (SAS/ETS); variables were identified with a statistical significance (< 0.7 and Pearson values ≥ 0.7), with 80.81% of the accumulation of genetic variation and 110 genetic bands. Thirty-nine (39) variables were recovered using NTSYSpc software where 30 showed a Pearson correlation (> 0.5) and nine variables (< 0.05), Finally, using a cladistics analysis approach highlighted 65 genetic bands, in addition to color of the fruit, presence of thorns, bitter flavor, piriform and oblong shape, and also content of chlorophylls a and b, presence of cucurbitacins, and the IC50 effect of chayote extracts on the four cell lines.

Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

Chromosome-level assembly of the melon thrips genome yields insights into evolution of a sap-sucking lifestyle and pesticide resistance.

Thrips are tiny insects from the order Thysanoptera (Hexapoda: Condylognatha), including many sap-sucking pests that are causing increasing damage to crops worldwide. In contrast to their closest relatives of Hemiptera (Hexapoda: Condylognatha), including numerous sap-sucking species, there are few genomic resources available for thrips. In this study, we assembled the first thrips genome at the chromosomal level from the melon thrips, Thrips palmi, a notorious pest in agriculture, using PacBio long-read and Illumina short-read sequences. The assembled genome was 237.85 Mb in size, with 1,324 contigs and a contig N50 of 567 kb. All contigs were assembled into 16 linkage groups assisted by the Hi-C technique. In total, 16,333 protein-coding genes were predicted, of which 88.13% were functionally annotated. Among sap-sucking insects, polyphagous species (e.g., T. palmi and Bemisia tabaci) usually possess more detoxification genes than oligophagous species (e.g., Diaphorina citri). The polyphagous thrips genomes characterized so far have relatively more detoxification genes in the GST and CCE families than polyphagous aphids, but they have fewer UGTs. HSP genes, especially from the Hsp70s group, have expanded in thrips compared to other hemipterans. These differences point to different genetic mechanisms associated with detoxification and stress responses in these two groups of sap-sucking insects. The expansion of these gene families may contribute to the rapid development of pesticide resistance in thrips, as supported by a transcriptome comparison of resistant and sensitive populations of T. palmi. The high-quality genome developed here provides an invaluable resource for understanding the ecology, genetics, and evolution of thrips as well as their relatives more generally.

Phylotranscriptomics in Cucurbitaceae Reveal Multiple Whole-Genome Duplications and Key Morphological and Molecular Innovations.

The ability of climbing plants to grow upward along others to reach the canopy for photosynthesis is hypothesized as a key innovation in flowering plants. Most members of the Cucurbitaceae, a family containing ∼1000 species and many important crops, are climbers and have characteristic tendrils and pepo fruits. Here, we present 127 newly sequenced transcriptomes and genomes along with other datasets for a total of 136 cucurbits representing all tribes to establish a robust Cucurbitaceae phylogeny containing eight highly resolved major clades. We analyzed whole-genome duplication, diversification dynamics, and ancestral morphologies, and found that after early genome duplication event(s), a burst of diversification and morphological innovations in flower, fruit, and root characters occurred under the climate optimum in the Early Eocene. Species radiation during the Mid-Eocene Climatic Optimum also coincided with several morphological changes shared by 80% of cucurbits. We found that the cucurbit-specific tendril identity gene TEN originated from a paleo-polyploidization event at the origin of the family. Our results support the hypothesis that cucurbit diversifications were probably driven by increased genetic diversity following polyploidizations and by trait morphological innovations under paleo-climate upheavals. Our study provides a phylogenetic framework and new insights into morphological and genomic changes underlying the adaptive evolution of Cucurbitaceae.