Watermelon Genome Research Articles

Editing eIF4E in the Watermelon Genome Using CRISPR/Cas9 Technology Confers Resistance to ZYMV

Watermelon is one of the most important cucurbit crops, but its production is seriously affected by viral infections. Although eIF4E proteins have emerged as the major mediators of the resistance to viral infections, the mechanism underlying the contributions of eIF4E to watermelon disease resistance remains unclear. In this study, three CleIF4E genes and one CleIF(iso)4E gene were identified in the watermelon genome. Among these genes, CleIF4E1 was most similar to other known eIF4E genes. To investigate the role of CleIF4E1, CRISPR/Cas9 technology was used to knock out CleIF4E1 in watermelon. One selected mutant line had an 86 bp deletion that resulted in a frame-shift and the expression of a truncated protein. The homozygous mutant exhibits developmental defects in plant growth, leaf morphology and reduced yield. Furthermore, the mutant was protected against the zucchini yellow mosaic virus, but not the cucumber green mottled mosaic virus. In summary, this study preliminarily clarified the functions of eIF4E proteins in watermelon. The generated data will be useful for elucidating eIF4E-related disease resistance mechanisms in watermelon. The tissue-specific editing of CleIF4E1 in future studies may help to prevent adverse changes to watermelon fertility.

Nanopore ultra-long sequencing and adaptive sampling spur plant complete telomere-to-telomere genome assembly

The pursuit of complete telomere-to-telomere (T2T) genome assembly in plants, challenged by genomic complexity, has been advanced by Oxford Nanopore Technologies (ONT), which offers ultra-long, real-time sequencing. Despite its promise, sequencing length and gap filling remain significant challenges. This study optimized DNA extraction and library preparation, achieving DNA lengths exceeding 485 kb; average N50 read lengths of 80.57 kb, reaching up to 440 kb; and maximum reads of 5.83 Mb. Importantly, we demonstrated that combining ultra-long sequencing and adaptive sampling can effectively fill gaps during assembly, evidenced by successfully filling the remaining gaps of a near-complete Arabidopsis genome assembly and resolving the sequence of an unknown telomeric region in watermelon genome. Collectively, our strategies improve the feasibility of complete T2T genomic assemblies across various plant species, enhancing genome-based research in diverse fields.

Genome-wide identification and expression analysis of watermelon (Citrullus lanatus) SSR2 gene during fruit development

BackgroundSterol side-chain reductase 2 (SSR2) is a key enzyme in the synthesis of plant cholesterol pathway. Despite the importance of watermelon as a horticultural cash crop, the SSR2 gene in watermelon has not been previously studied or reported.ResultsIn this study, 28 SSR2 genes were identified in the watermelon genome. The physicochemical properties of 28 ClaSSR proteins were predicted by bioinformatics methods, and the gene structure, conserved motif, chromosome localization, phylogenetic analysis, cis-acting elements, expression patterns, promoter activity analysis and subcellular localization of ClaSSRs were studied. The 28 ClaSSRs were unevenly distributed on 11 chromosomes, and phylogenetic analysis showed that they could be grouped into 4 groups with other related Cucurbitaceae homologous genes. Analysis of gene structure and motifs revealed similarities in exons/introns and motifs between members of the same group, further supporting phylogenetic results. The RT–qPCR results showed variations in ClaSSRs expression during watermelon fruit development. The analysis of promoter activity for ClaSSR25 showed strong activity. Subcellular localization studies confirmed that ClaSSR25 is mainly located in the cytoplasm, which aligns with the predicted outcomes. We additionally estimated the network of protein–protein interactions for ClaSSR25 and analyzed proteins that could potentially interact with ClaSSR25 in melon and Arabidopsis thaliana.ConclusionsWe conducted bioinformatics analysis and expression analysis of members of the watermelon SSR2 gene family in this work, and the outcomes set the stage for further investigations into the watermelon SSR2 gene.Graphical

Telomere-to-telomere Citrullus super-pangenome provides direction for watermelon breeding

To decipher the genetic diversity within the cucurbit genus Citrullus, we generated telomere-to-telomere (T2T) assemblies of 27 distinct genotypes, encompassing all seven Citrullus species. This T2T super-pangenome has expanded the previously published reference genome, T2T-G42, by adding 399.2 Mb and 11,225 genes. Comparative analysis has unveiled gene variants and structural variations (SVs), shedding light on watermelon evolution and domestication processes that enhanced attributes such as bitterness and sugar content while compromising disease resistance. Multidisease-resistant loci from Citrullus amarus and Citrullus mucosospermus were successfully introduced into cultivated Citrullus lanatus. The SVs identified in C. lanatus have not only been inherited from cordophanus but also from C. mucosospermus, suggesting additional ancestors beyond cordophanus in the lineage of cultivated watermelon. Our investigation substantially improves the comprehension of watermelon genome diversity, furnishing comprehensive reference genomes for all Citrullus species. This advancement aids in the exploration and genetic enhancement of watermelon using its wild relatives.

Identification of watermelon genes involved in the ZYMV interaction through a miRNA bio-informatics analysis and characterization of ATRIP and RBOHB

Our objective was to identify differentially expressed watermelon genes in its interaction with ZYMV through a bioinformatics analysis of differentially expressed miRNAs. Small RNAseq data analysis of healthy and ZYMV-infected watermelon (21 dpi) identified 353 miRNAs from which 22 known and 331 new miRNAs. Important information about their precursors, their length, the loci of which they originated on watermelon genome are provided. The ZYMV genome could be a target for mir396a-3p, miR8706a, and miR1886i-5p from the miRbase, but none of them was identified in the watermelon miRNAome. Furthermore, watermelon miRNAome does not contain a miRNA targeting ZYMV genome with an expectation score ≤3.5. In healthy watermelon bioinformatically predicted targets of 32 miRNAs were 34 resistance genes, as CC-NBS-LRR, TIR-NBS-LRR, TIR-NBS. For nine differentially expressed miRNAs the respective target genes (10 in total) were bioinformatically predicted. For cla-new_miR307 (upregulated upon ZYMV infection) and cla-miR166h-3p (downregulated upon ZYMV infection) the targets were predicted to be ClaATRIP and ClaRBOHB, respectively, with ClaATRIP downregulated and ClaRBOHB upregulated upon ZYMV infection. ALSV-mediated VIGS of ClaATRIP rendered watermelon plants more resistant to ZYMV, whereas VIGS of ClaRBOHB resulted in higher levels of ZYMV titer in watermelon. These data suggest that ClaATRIP and ClaRBOHB are a susceptibility and resistant gene, respectively. Our results provide new insights in watermelon miRNAome and could propose new strategies for generating resistant watermelon to ZYMV.

Genome-Wide Identification of AUX/IAA Genes in Watermelon Reveals a Crucial Role for ClIAA16 during Fruit Ripening

The auxin/indole-3-acetic acid (Aux/IAA) gene family plays a critical role in auxin-mediated responses and fruit development. However, studies on its role in watermelon are limited. In this study, 29 ClIAA gene members were identified in the watermelon genome and classified into eleven groups. Of note, ClIAA16, which was found to be up-regulated during fruit ripening, was targeted using CRISPR/Cas9 gene editing. Knockout mutants of ClIAA16 exhibited a 3–4 day delay in ripening compared to the wild type, highlighting the regulatory importance of ClIAA16. Our findings shed light on the importance of ClIAA genes in watermelon fruit ripening and pave the way for further functional studies.

Recent Advances and Challenges in Management of Colletotrichum orbiculare, the Causal Agent of Watermelon Anthracnose

The fungus Colletotrichum orbiculare causes watermelon anthracnose and is an important pathogen of watermelon in the United States, causing a significant impact on yield and quality of the produce. The application of fungicides as preventative and post-occurrence control measures is currently being deployed by growers. Further study of the genetic and molecular basis of anthracnose resistance will help in guiding future watermelon breeding strategies. Several conserved virulence factors (effectors) in C. orbiculare have been reported to interact with the host, at times impairing the host immune machinery. A single dominant gene conferring race 1 anthracnose resistance was reported independently on two watermelon germplasm. The recent advances in genomics, transcriptomics, proteomics, and metabolomics could facilitate a better understanding of the interaction between C. orbiculare effectors and host resistance genes in the already sequenced watermelon genome. In this review, we encompass and discuss (i) the history of watermelon anthracnose, taxonomy, morphology, and diversity in races of C. orbiculare; (ii) the epidemiology of the anthracnose disease and host resistance; (iii) the genetics behind the pathogenesis; and (iv) the current advances in breeding and molecular efforts to elucidate anthracnose resistance.

Identification of watermelon H3K4 and H3K27 genes and their expression profiles during watermelon fruit development.

The ClaH3K4s and ClaH3K27s gene families are subfamilies of the SET family, each with a highly conserved SET structure domain and a PHD structural domain. Both participate in histone protein methylation, which affects the chromosome structure and gene expression, and is essential for fruit growth and development. In order to demonstrate the structure and expression characteristics of ClaH3K4s and ClaH3K27s in watermelon, members of the watermelon H3K4 and H3K27 gene families were identified, and their chromosomal localization, gene structure, and protein structural domains were analyzed. The phylogeny and covariance of the gene families with other species were subsequently determined, and the expression profiles were obtained by performing RNA-Seq and qRT-PCR. The watermelon genome had five H3K4 genes with 3207-8043bp nucleotide sequence lengths and four H3K27 genes with a 1107-5499bp nucleotide sequence. Synteny analysis revealed the close relationship between watermelon and cucumber, with the majority of members displaying a one-to-one covariance. Approximately half of the 'Hua-Jing 13 watermelon' ClaH3K4s and ClaH3K27s genes were expressed more in the late fruit development stages, while the changes were minimal for the remaining half. H3K4-2 expression was observed to be slightly greater on day 21 compared to other periods. Moreover, ClaH3K27-1 and ClaH3K27-2 were hardly expressed throughout the developing period, and ClaH3K27-4 exhibited the highest expression. These results serve as a basis for further functional characterization of the H3K4 and H3K27 genes in the fruit development of watermelon.

Genome-wide characterization of the PLATZ gene family in watermelon (Citrullus lanatus L.) with putative functions in biotic and abiotic stress response

Plant AT-rich sequence and zinc-binding (PLATZ) proteins are plant-specific transcription factors involved in growth, development, and stress responses. Here, we conducted a genome-wide characterization of the watermelon ClPLATZ family and examined its expression responsiveness to defense hormones and pathogen infection along with putative functions in biotic and abiotic stress responses. The watermelon genome contains 12 putative ClPLATZ genes, encoding proteins with a characteristic PLATZ domain, and their promoters contain various cis-elements related to plant growth, development, phytohormones and stress response. The ClPLATZ genes, except ClPLATZ6, are differentially expressed in response to defense hormones (e.g., salicylic acid and methyl jasmonate) and fungal infections caused by Fusarium oxysporum f. sp. niveum and Stagonosporopsis cucurbitacearum. Most ClPLATZ proteins interact with other proteins (viz., ClDP, ClRPT2a, and ClRPC53). Among ClPLATZ proteins, ClPLATZ8, 9, 10, and 11 are predominately localized in the nucleus. ClPLATZ3 and 8 positively, but ClPLATZ11 negatively regulate resistance against Pseudomonas syringe pv. tomato DC3000 in transgenic Arabidopsis lines. ClPLATZ8 and 11 positively regulate stress tolerance to NaCl and mannitol during seed germination in transgenic Arabidopsis. In conclusion, the characterization of the ClPLATZ family provides insights into the biological functions of ClPLATZ genes in growth, development, and stress response in watermelon. Further, the involvement of certain ClPLATZ genes in biotic and abiotic stress response in transgenic Arabidopsis suggests their potential application in engineering stress-tolerant crops.

Production of double haploid watermelon via maternal haploid induction.

Production of double haploid watermelon via maternal haploid induction.

Grafting onto pumpkin alters the evolution of fruit sugar profile and increases fruit weight through invertase and sugar transporters in watermelon

Sweetness is an essential quality trait of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), and it depends on both the concentration and composition of sugars. Grafting is a routine technique for watermelon production globally. However, it remains unknown whether rootstock alter the profiles of sugars in watermelon. To address this issue, we grafted watermelon seedlings onto a Cucurbita maxima × Cucurbita moschata rootstock, photosynthesis in leaves, contents of main carbohydrates, activities of invertase and sucrose synthase and expressions of invertase genes and sugar transporters in watermelon fruits were examined. Results showed that grafting could have delayed fruit ripening as fruits from grafted plants had a higher hexose/sucrose ratio (increased concentration of glucose and fructose and decreased concentration of sucrose) compared to fruits from self-grafted plants. These changes were associated with increased vacuolar invertase (VIN) activity. Meanwhile, a watermelon VIN gene (ClVIN2) and two CWIN genes (ClCWIN1 and ClCWIN4) were identified in updated watermelon genome, and ClVIN2 was upregulated accompanied by the elevated activity of VIN. In addition, rootstock grafted plants displayed improved net photosynthesis rate, increased expression of ClAGA2 (coding alkaline alpha-galactosidase), ClVST1 (coding vacuolar sugar transporter) and ClCWIN4 (coding cell wall invertase), and higher activity of cell wall invertase when compared with self-grafted plants, contributing to increased fruit weight. In summary, our results imply that invertase and related sugar transporters might play a role in sugar profiles and fruit weight differentiation observed during fruit maturity evolution.

Genomic Survey and Expression Analysis of GLKs in Watermelon (Citrullus lanatus)

Golden2-like (GLK) genes positively regulate chloroplast development, increase crop yields, and improve fruit quality. However, there has been no comprehensive identification and characterization of GLKs in watermelon. In this study, a total of 48 ClGLKs were identified in the watermelon genome. Based on phylogenetic analysis, they were divided into five groups. ClGLKs within the same group showed a similar gene structure and conserved motif compositions. Promoter analysis indicated that cis-elements responsive to light were the most abundant, though cis-elements associated with hormones, stress, and developmental regulation were also identified in ClGLKs promoters. Expression analysis indicated significant responses of some ClGLKs to drought and CGMMV stress, suggesting that these genes may participate in responses to biotic and abiotic stresses. Phenotypic analyses revealed enhanced chloroplast development and increased thylakoid density and chlorophyll content in the pericarp of a “dark green” watermelon cultivar. ClGLK8 was identified as the homolog of GLK1-2, the genes that promote chloroplast development and chlorophyll biosynthesis in fruits, and showed significantly increased expression in accordance with chloroplast development and chlorophyll accumulation. Our results provide detailed knowledge of the ClGLKs, which will enhance efforts to further improve the fruit quality of watermelon.

The NF-Y Transcription Factor Family in Watermelon: Re-Characterization, Assembly of ClNF-Y Complexes, Hormone- and Pathogen-Inducible Expression and Putative Functions in Disease Resistance.

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that binds to the CCAAT cis-element in the promoters of target genes and plays critical roles in plant growth, development, and stress responses. In the present study, we aimed to re-characterize the ClNF-Y family in watermelon, examine the assembly of ClNF-Y complexes, and explore their possible involvement in disease resistance. A total of 25 ClNF-Y genes (7 ClNF-YAs, 10 ClNF-YBs, and 8 ClNF-YCs) were identified in the watermelon genome. The ClNF-Y family was comprehensively characterized in terms of gene and protein structures, phylogenetic relationships, and evolution events. Different types of cis-elements responsible for plant growth and development, phytohormones, and/or stress responses were identified in the promoters of the ClNF-Y genes. ClNF-YAs and ClNF-YCs were mainly localized in the nucleus, while most of the ClNF-YBs were localized in the cytoplasm of cells. ClNF-YB5, -YB6, -YB7, -YB8, -YB9, and -YB10 interacted with ClNF-YC2, -YC3, -YC4, -YC5, -YC6, -YC7, and -YC8, while ClNF-YB1 and -YB3 interacted with ClNF-YC1. A total of 37 putative ClNF-Y complexes were identified, e.g., ClNF-YA1, -YA2, -YA3, and -YA7 assembled into 13, 8, 8, and 8 ClNF-Y complexes with different ClNF-YB/-YC heterodimers. Most of the ClNF-Y genes responded with distinct expression patterns to defense hormones such as salicylic acid, methyl jasmonate, abscisic acid, and ethylene precursor 1-aminocyclopropane-1-carboxylate, and to infection by the vascular infecting fungus Fusarium oxysporum f. sp. niveum. Overexpression of ClNF-YB1, -YB8, -YB9, ClNF-YC2, and -YC7 in transgenic Arabidopsis resulted in an earlier flowering phenotype. Overexpression of ClNF-YB8 in Arabidopsis led to enhanced resistance while overexpression of ClNF-YA2 and -YC2 resulted in decreased resistance against Botrytis cinerea. Similarly, overexpression of ClNF-YA3, -YB1, and -YC4 strengthened resistance while overexpression of ClNF-YA2 and -YB8 attenuated resistance against Pseudomonas syringae pv. tomato DC3000. The re-characterization of the ClNF-Y family provides a basis from which to investigate the biological functions of ClNF-Y genes in respect of growth, development, and stress response in watermelon, and the identification of the functions of some ClNF-Y genes in disease resistance enables further exploration of the molecular mechanism of ClNF-Ys in the regulation of watermelon immunity against diverse pathogens.

The HD-ZIP Gene Family in Watermelon: Genome-Wide Identification and Expression Analysis under Abiotic Stresses.

Homeodomain-leucine zipper (HD-ZIP) transcription factors are one of the plant-specific gene families involved in plant growth and response to adverse environmental conditions. However, little information is available on the HD-ZIP gene family in watermelon. In this study, forty ClHDZs were systemically identified in the watermelon genome, which were subsequently divided into four distinctive subfamilies (I-IV) based on the phylogenetic topology. HD-ZIP members in the same subfamily generally shared similar gene structures and conserved motifs. Syntenic analyses revealed that segmental duplications mainly contributed to the expansion of the watermelon HD-ZIP family, especially in subfamilies I and IV. HD-ZIP III was considered the most conserved subfamily during the evolutionary history. Moreover, expression profiling together with stress-related cis-elements in the promoter region unfolded the divergent transcriptional accumulation patterns under abiotic stresses. The majority (13/23) of ClHDZs in subfamilies I and II were downregulated under the drought condition, e.g., ClHDZ4, ClHDZ13, ClHDZ18, ClHDZ19, ClHDZ20, and ClHDZ35. On the contrary, most HD-ZIP genes were induced by cold and salt stimuli with few exceptions, such as ClHDZ3 and ClHDZ23 under cold stress and ClHDZ14 and ClHDZ15 under the salt condition. Notably, the gene ClHDZ14 was predominantly downregulated by three stresses whereas ClHDZ1 was upregulated, suggesting their possible core roles in response to these abiotic stimuli. Collectively, our findings provide promising candidates for the further genetic improvement of abiotic stress tolerance in watermelon.

Thioredoxin h2 inhibits the MPKK5-MPK3 cascade to regulate the CBF-COR signaling pathway in Citrullus lanatus suffering chilling stress.

Thioredoxins (TRXs) are ubiquitous oxidoreductases and present as a multigenic family. TRXs determine the thiol redox balance, which is crucial for plants in the response to cold stress. However, limited knowledge is available about the role of TRXs in watermelon (Citrullus lanatus), which is highly sensitive to chilling stress in agricultural practice. Here, we identified 18 genes encoding 14 typical and 4 atypical TRXs from the watermelon genome, and found that ClTRXh2 localized at the plasma membrane was largely induced by chilling. Virus-induced gene silencing of ClTRXh2 resulted in watermelon plants that were more sensitive to chilling stress. We further found that ClTRXh2 physically interacted with mitogen-activated protein kinase kinase 5 (ClMPKK5), which was confirmed to phosphorylate and activate ClMPK3 in vitro, and the activation of ClMPK3 by ClMPKK5 was blocked by a point mutation of the Cys-229 residue to Ser in ClMPKK5. Additionally, ClTRXh2 inhibited the chilling-induced activation of ClMPK3, suggesting that the ClMPKK5-ClMPK3 cascade is regulated in a redox-dependent manner. We showed that ClMPK3-silenced plants had increased tolerance to chilling, as well as enhanced transcript abundances of the C-repeat/DREB binding factor (ClCBF) and cold-responsive (ClCOR) genes. Taken together, our results indicate that redox status mediated by ClTRXh2 inhibits ClMPK3 phosphorylation through the interaction between ClTRXh2 and ClMPKK5, which subsequently regulates the CBF-COR signaling pathway when submitted to chilling stress. Hence, our results provide a link between thiol redox balance and MAPK cascade signaling, revealing a conceptual framework to understand how TRX regulates chilling stress tolerance in watermelon.

Genome-wide analysis of IQD proteins and ectopic expression of watermelon ClIQD24 in tomato suggests its important role in regulating fruit shape.

The plant-specific IQ67 domain (IQD) is the largest class of calmodulin targets found in plants, and plays an important role in many biological processes, especially fruit development processes. However, the functional role of IQD proteins in the development of watermelon (Citrullus lanatus) shape remains unknown, as the IQD protein family in watermelon has not been systematically characterized. Herein, we elucidated the gene structures, chromosomal locations, evolutionary divergence, and functions of 35 IQD genes in the watermelon genome. The transcript profiles and quantitative real-time PCR analysis at different stages of fruit development showed that the ClIQD24 gene was highly expressed on 0 days after pollination. Furthermore, we found that the ectopic overexpression of ClIQD24 promoted tomato fruit elongation, thereby revealing the significance of ClIQD24 in the progression of watermelon shape. Our study will serve as a reference for further investigations on the molecular mechanisms underlying watermelon fruit shape formation.

Chilling-induced H2O2 signaling activates the antioxidant enzymes in alleviating the photooxidative damage caused by loss of function of 2-Cys peroxiredoxin in watermelon

Cold stress signal transduction refers to a process that involves various physiological and molecular changes. Hydrogen peroxide (H2O2) signaling is stimulated by plants in adapting to various environmental stresses. Here, nine respiratory burst oxidase homolog (ClRboh) genes were identified from the watermelon genome. Chilling stress induced increased gene expression of ClRbohD and H2O2 accumulation in watermelon plants. By using virus-induced gene silencing (VIGS), we found that H2O2-induced chilling tolerance is dependent on ClRbohD by increased electrolyte leakage, membrane lipid peroxidation, and reduced photosystem II activity in ClRbohD-silenced plants compared with control. In addition, silencing of 2-Cys peroxiredoxin (Cl2-CP) increased the chilling sensitivity, while exogenous H2O2 treatment compromised the silencing of Cl2-CP-induced photooxidative damage in watermelon plants. The gene expression and enzyme activities of Cu/Zn-SOD and GR were significantly increased in Cl2-CP-silenced plants rather than pV190 control plants upon H2O2 treatment under chilling stress, suggesting that the activation of antioxidant enzymes by H2O2 serves as an alternative pathway to compensate for the deficiency of Cl2-CP during chilling stress response in watermelon.

Identification and Characterization Roles of Phytoene Synthase (PSY) Genes in Watermelon Development.

Phytoene synthase (PSY) plays an essential role in carotenoid biosynthesis. In this study, three ClPSY genes were identified through the watermelon genome, and their full-length cDNA sequences were cloned. The deduced proteins of the three ClPSY genes were ranged from 355 to 421 amino acid residues. Phylogenetic analysis suggested that the ClPSYs are highly conserved with bottle gourd compared to other cucurbit crops PSY proteins. Variation in ClPSY1 expression in watermelon with different flesh colors was observed; ClPSY1 was most highly expressed in fruit flesh and associated with the flesh color formation. ClPSY1 expression was much lower in the white-fleshed variety than the colored fruits. Gene expression analysis of ClPSY genes in root, stem, leaf, flower, ovary and flesh of watermelon plants showed that the levels of ClPSY2 transcripts found in leaves was higher than other tissues; ClPSY3 was dominantly expressed in roots. Functional complementation assays of the three ClPSY genes suggested that all of them could encode functional enzymes to synthesize the phytoene from Geranylgeranyl Pyrophosphate (GGPP). Some of the homologous genes clustered together in the phylogenetic tree and located in the synteny chromosome region seemed to have similar expression profiles among different cucurbit crops. The findings provide a foundation for watermelon flesh color breeding with regard to carotenoid synthesis and also provide an insight for the further research of watermelon flesh color formation.

Plant hormone signals regulate trehalose accumulation against osmotic stress in watermelon cells.

Trehalose, one of the most chemically stable sugars, can effectively improve the tolerance of various plants against abiotic stress by protecting and stabilizing protein and cell membranes. However, the signaling pathway in trehalose biosynthesis triggered by abiotic stresses is still unclear. In the study, it can be shown that exogenous trehalose can alleviate the inhibitory effect of osmotic stress on cell growth, suppress extracellular alkalization, ROS burst, and maintain the integrity of the microtubular cytoskeleton. Trehalose-6-phosphate synthase (TPS) is the key limiting enzyme for trehalose synthesis and is encoded by 7 ClTPS genes, located in 7 different chromosomes of the watermelon genome. Expression analysis by qRT-PCR indicated that osmotic stress could upregulate the expression of all the family members of ClTPS and promote the accumulation of trehalose in watermelon cells accordingly. Exogenous methyl jasmonate (MeJA), ethephon (ETH), abscisic acid (ABA), or salicylic acid (SA) induced trehalose accumulation, with MeJA being the most effective treatment. When fluridone (FL), an ABA biosynthesis inhibitor, was pre-perfused into the cells before osmotic stress, trehalose accumulation and packed cell volume were suppressed significantly, whereas inhibition of ethylene biosynthesis could even restore cell growth. Moreover, inhibition of trehalose hydrolysis could also increase the tolerance against osmotic stress. This study shows that trehalose biosynthesis is phytohormone-dependent and the hydrolysis of trehalose is involved in osmotic tolerance regulation.

Elevated expression of ribosome-inactivating protein (RIP) genes in potyvirus-resistant watermelon in response to viral infection

Watermelon (Citrullus lanatus) is an important vegetable fruit crop, widely grown in many countries in the tropical and subtropical regions around the world. The narrow genetic base of cultivated watermelon makes it susceptible to many pests and diseases, especially viruses. However, genetic sources of resistance are available in wild relatives that could potentially be used to incorporate the resistance genes into watermelon cultivars. Ribosome-inactivating proteins (RIPs) are prevalent in many plant species and are known to have an important role in defence against bacteria, fungi, and viruses. The objective of this study was to identify RIP-like sequences in the watermelon genome and to conduct comparative measurement of RIP gene expression between the potyvirus-resistant Citrullus amarus ‘PI 244019ʹ versus the susceptible watermelon (C. lanatus) ‘Charleston Gray’ in response to infection by papaya ringspot virus (PRSV). Taking advantage of the recently available genome sequences of ‘PI 244019ʹ and ‘Charleston Gray’, in this study we mined out watermelon RIP genes and conducted comparative sequence and expression analyses of RIP-I and RIP-II between the genotypes. The RIP gene expression analysis revealed elevated RIP gene expression in resistant PI 244019 plants, but not in susceptible ‘Charleston Gray’ plants, in response to inoculation with PRSV, indicating a potential association of RIP gene expression and resistance to potyviruses in watermelon. This elevated expression of RIP genes in association with potyvirus resistance opens a new avenue to investigate the underlying mechanism of potyvirus resistance in watermelon.