Transcriptomic analysis on cucumber tendril reveals GLRs play important roles in thigmotropism and thigmomorphogenesis

Thigmotropism and thigmomorphogenesis are two related and pervasive processes that play crucial roles in plant adaptation to the environment.However,there have been few investigations into the molecular regulatory mechanisms of these phenomena.Cucumber(Cucumis sativus L.)tendrils are ideal material for studying thigmotropism and thigmomorphogenesis because they display a combination of the two processes.Here,we generated the transcriptome profiles of cucumber tendrils at the young,stretch,and coiling stages.Genes related to receptor proteins,transmembrane transport,and ion transport were significantly enriched among those differentially expressed between stages.Pharmacological assays illustrated that three GLUTAMATE RECEPTOR(GLR)genes might play a vital function in perceiving or transducing touch stimulation signals.Comparing the transcriptomes of tendrils and roots after touch stimulation,we found that genes related to extracellular stimulus and xyloglucan metabolism might have conserved functions in the regulation of thigmomorphogenesis.The transcriptome atlas of thigmotropism and thigmomorphogenesis of cucumber tendrils constructed in this study will help further elucidate the molecular mechanisms behind these processes.

Natural variation in SlSOS2 promoter hinders salt resistance during tomato domestication

Increasing soil salinization seriously impairs plant growth and development,resulting in crop loss.The Salt-Overly-Sensitive(SOS)pathway is indispensable to the mitigation of Na+toxicity in plants under high salinity.However,whether natural variations of SOS2 contribute to salt tolerance has not been reported.Here a natural variation in the SlSOS2 promoter region was identified to be associated with root Na+/K+ratio and the loss of salt resistance during tomato domestication.This natural variation contains an ABI4-binding cis-element and plays an important role in the repression of SlSOS2 expression.Genetic evidence revealed that SlSOS2 mutations increase root Na+/K+ratio under salt stress conditions and thus attenuate salt resistance in tomato.Together,our findings uncovered a critical but previously unknown natural variation of SOS2 in salt resistance,which provides valuable natural resources for genetic breeding for salt resistance in cultivated tomatoes and other crops.

Genetic dissection of climacteric fruit ripening in a melon population segregating for ripening behavior

Melon is as an alternative model to understand fruit ripening due to the coexistence of climacteric and non-climacteric varieties within the same species,allowing the study of the processes that regulate this complex trait with genetic approaches.We phenotyped a population of recombinant inbred lines(RILs),obtained by crossing a climacteric(Védrantais,cantalupensis type)and a non-climcteric variety(Piel de Sapo T111,inodorus type),for traits related to climacteric maturation and ethylene production.Individuals in the RIL population exhibited various combinations of phenotypes that differed in the amount of ethylene produced,the early onset of ethylene production,and other phenotypes associated with ripening.We characterized a major QTL on chromosome 8,ETHQV8.1,which is sufficient to activate climacteric ripening,and other minor QTLs that may modulate the climacteric response.The ETHQV8.1 allele was validated by using two reciprocal introgression line populations generated by crossing Védrantais and Piel de Sapo and analyzing the ETHQV8.1 region in each of the genetic backgrounds.A Genome-wide association study(GWAS)using 211 accessions of the ssp.melo further identified two regions on chromosome 8 associated with the production of aromas,one of these regions overlapping with the 154.1 kb interval containing ETHQV8.1.The ETHQV8.1 region contains several candidate genes that may be related to fruit ripening.This work sheds light into the regulation mechanisms of a complex trait such as fruit ripening.

Chromosome-scale genome assembly of Cucumis hystrix-a wild species interspecifically cross-compatible with cultivated cucumber

Cucumis hystrix Chakr.(2n=2x=24)is a wild species that can hybridize with cultivated cucumber(C.sativus L.,2n=2x=14),a globally important vegetable crop.However,cucumber breeding is hindered by its narrow genetic base.Therefore,introgression from C.hystrix has been anticipated to bring a breakthrough in cucumber improvement.Here,we report the chromosome-scale assembly of C.hystrix genome(289 Mb).Scaffold N50 reached 14.1 Mb.Over 90%of the sequences were anchored onto 12 chromosomes.A total of 23,864 genes were annotated using a hybrid method.Further,we conducted a comprehensive comparative genomic analysis of cucumber,C.hystrix,and melon(C.melo L.,2n=2x=24).Whole-genome comparisons revealed that C.hystrix is phylogenetically closer to cucumber than to melon,providing a molecular basis for the success of its hybridization with cucumber.Moreover,expanded gene families of C.hystrix were significantly enriched in“defense response,”and C.hystrix harbored 104 nucleotide-binding site-encoding disease resistance gene analogs.Furthermore,121 genes were positively selected,and 12(9.9%)of these were involved in responses to biotic stimuli,which might explain the high disease resistance of C.hystrix.The alignment of whole C.hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C.hystrix chromosomes.Finally,we developed four cucumber-C.hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods.The assembled C.hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.

Enhancing sheepgrass through genomic exploration and targeted editing

The growth of grass industry is essential for meeting human needs for proteins and sustaining grassland ecosystems(Bengtsson et al., 2019). Leymus chinensis, also known as sheepgrass, is a perennial Triticeae forage renowned for its adaptability and top-notch forage quality(Li et al., 2013).

A near-complete cucumber reference genome assembly and Cucumber-DB,a multi-omics database

Dear Editor,Cucumber(Cucumis sativus)is a key vegetable crop and an essential model for genetics and genomics research in Cucurbitaceae,especially in studies on plant sex determination and vascular development.As the first sequenced genome among vegetable crops,the cucumber reference genome has been a crucial genetic resource for comparative genomics and genetic studies.However,the latest version of the cucumber cv.Chinese Long reference genome(CLv3.0),generated using PacBio long-reads data,is incomplete.Despite a predicted genome size of 350-367 Mb,CLv3.0 encompasses only 226.2 Mb,leaving 72 gaps and over 120 Mb of unknown sequences.This low assembly completeness(approximately 62%)is primarily due to the high proportion(~30%)of heterochromatic satellite and rDNA sequences in cucumber,substantially higher than that observed in many other species such as Arabidopsis,rice,maize,and watermelon,where it is less than 5%(Han et al.,2008;Huang et al.,2009;Li et al.,2019).

A commentary of“Mechanism of alkaline tolerance and application in crop improvement”:Top 10 Chinese Scientific Advances of 2023,China

As global warming accelerates,soil degradation,exacerbated by extreme weather,has become a significant environmental issue worldwide.China,in particular,is grappling with a yearly reduction in arable land due to soil degradation,acidification,and salinization,posing potential threats to food security.The country has a vast area of saline-alkaline land,over half of which are high-pH soda saline soils.These lands often experience simultaneous salt and alkali stress,severely inhibiting plant growth and crop yield.Therefore,finding ways to utilize this marginal and saline-alkaline land for food production is a critical scientific challenge.In this context,Chinese scientists have achieved a breakthrough towards harnessing saline-alkaline land.

The gap-free potato genome assembly reveals large tandem gene clusters of agronomical importance in highly repeated genomic regions

Dear Editor,Potato is a vital food security crop and is ranked as the world’s third most important food crop after rice and wheat.In 2011,the first genome assembly of a doubled monoploid potato DM1-3516 R44(DM)was released(Potato Genome Sequencing Consortium,2011),which has been widely used as one of the most popular reference genomes in the last decade and served as a valuable resource in plant genomics and potato genetics community(Leisner et al.,2018;Yang et al.,2020;Zheng et al.,2020).

Vegetable biology and breeding in the genomics era

Vegetable crops provide a rich source of essential nutrients for humanity and represent critical economic values to global rural societies. However, genetic studies of vegetable crops have lagged behind major food crops, such as rice, wheat and maize,thereby limiting the application of molecular breeding. In the past decades, genome sequencing technologies have been increasingly applied in genetic studies and breeding of vegetables. In this review, we recapitulate recent progress on reference genome construction, population genomics and the exploitation of multi-omics datasets in vegetable crops. These advances have enabled an in-depth understanding of their domestication and evolution, and facilitated the genetic dissection of numerous agronomic traits, which jointly expedites the exploitation of state-of-the-art biotechnologies in vegetable breeding. We further provide perspectives of further directions for vegetable genomics and indicate how the ever-increasing omics data could accelerate genetic, biological studies and breeding in vegetable crops.

Haplotype-resolved and chromosome-level genome assembly of Colorado potato beetle

As the most important non-cereal food crop,potatoes are a staple food for 1.3 billion people(Stokstad,2019).However,these cultivated potatoes are challenged with multiple biotic stresses(i.e.,pathogens and pests),some of which can evade host resistance,leading to a serious dampening of potato yield.One of these pests is the Colorado potato beetle(hereafter referred to as CPB,Leptinotarsa decemlineata Say).CPB is a member of the order Coleoptera,and originated in the southwestern United States and Mexico(Alyokhin et al.,2008).

Phytoene synthases 1 modulates tomato fruit quality through influencing the metabolic flux between carotenoid and flavonoid pathways

The deterioration in fruit quality of commercial tomatoes is a major concern of modern tomato breeding.However,the metabolism and genetics of fruit quality are poorly understood.Here,we performed transgenic and molecular biology experiments to reveal that tomato phytoene synthase 1(SlPSY1)is responsible for the accumulation of an important flavor chemical,6-methyl-5-hepten-2-one(MHO).To dissect the function of SlPSY1 in regulating fruit quality,we generated and analyzed a dataset encompassing over 2000 compounds detected by GC-MS and LC-MS/MS along with transcriptomic data.The combined results illustrated that SlPSY1 deficiency imparts novel flavor to yellow tomatoes with 236 volatiles significantly changed and improves fruit firmness,possibly due to accumulation of seven cutins.Further analysis indicated SlPSY1 is essential for carotenoid-derived metabolite biosynthesis by catalyzing prephytoene-PP(PPPP)to 15-cis-phytoene.Notably,we showed that SlPSY1 can influence the metabolic flux between carotenoid and flavonoid pathways,and this metabolic flux was confirmed by silencing SlCHS1.Our study provided insights into the multiple effects of SlPSY1 on tomato fruit metabolome and highlights the potential to produce high-quality fruit by rational design of SlPSY1 expression.

The Genome of Medicinal Plant Macleaya cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism

The overuse of antibiotics in animal agriculture and medicine has caused a series of potential threats to public health. Macleaya cordata is a medicinal plant species from the Papaveraceae family, providing a safe resource for the manufacture of antimicrobial feed additive for livestock. The active constituents from M. cordata are known to include benzylisoquinoline alkaloids （BIAs） such as sanguinarine （SAN） and chelerythrine （CHE）, but their metabolic pathways have yet to be studied in this non-model plant. The active biosynthesis of SAN and CHE in M. cordata was first examined and confirmed by feeding ^13C-labeled tyrosine. To gain further insights, we de novo sequenced the whole genome of M. cordata, the first to be sequenced from the Papaveraceae family. The M. cordata genome covering 378 Mb encodes 22,328 predicted protein-coding genes with 43.5% being transposable elements. As a member of basal eudicot, M. cordata genome lacks the paleohexaploidy event that occurred in almost all eudicots. From the genomics data, a complete set of 16 metabolic genes for SAN and CHE biosynthesis was retrieved, and 14 of their biochemical activities were validated. These genomics and metabolic data show the conserved BIA metabolic pathways in M. cordata and provide the knowledge foundation for future productions of SAN and CHE by crop improvement or microbial pathway reconstruction.

An ACC Oxidase Gene Essential for Cucumber Carpel Development

Sex determination in plants gives rise to unisexual flowers that facilitate outcrossing and enhance genetic diversity. In cucumber and melon, ethylene promotes carpel development and arrests sta- men development. Five sex-determination genes have been identified, including four encoding 1-aminocyclopropane-l-carboxylate （ACC） synthase that catalyzes the rate-limiting step in ethylene biosynthesis, and a transcription factor gene CmWIP1 that corresponds to the Mendelian locus gynoecious in melon and is a negative regulator of femaleness. ACC oxidase （ACO） converts ACC into ethylene; how- ever, it remains elusive which ACO gene in the cucumber genome is critical for sex determination and how CmWIP1 represses development of female flowers. In this study, we discovered that mutation in an ACO gene, CsAC02, confers androecy in cucumber that bears only male flowers. The mutation disrupts the enzymatic activity of CsAC02, resulting in 50% less ethylene emission from shoot tips. CsAC02 was ex- pressed in the carpel primordia and its expression overlapped with that of CsACS11 in female flowers at key stages for sex determination, presumably providing sufficient ethylene required for proper CsACS2 expression. CmAC03, the ortholog of CsACO2, showed a similar expression pattern in the carpel region, suggesting a conserved function of CsACO2/CmACO3. We demonstrated that CsWlP1, the ortholog of CmWlP1, could directly bind the promoter of CsAC02 and repress its expression. Taken together, we propose a presumably conserved regulatory module consisting of WlP1 transcription factor and ACO controls unisexual flower development in cucumber and melon.

Rare SNP Identified a TCP Transcription Factor Essential for Tendril Development in Cucumber

Rare genetic variants are abundant in genomes but less tractable in genome-wide association study. Here we exploit a strategy of rare variation mapping to discover a gene essential for tendril development in cucumber （Cucumis sativus L.）. In a collection of 〉3000 lines, we discovered a unique tendril-less line that forms branches instead of tendrils and, therefore, loses its climbing ability. We hypothesized that this unusual phenotype was caused by a rare variation and subsequently identified the causative single nucleotide poly- morphism. The affected gene TEN encodes a TCP transcription factor conserved within the cucurbits and is expressed specifically in tendrils, representing a new organ identity gene. The variation occurs within a pro- tein motif unique to the cucurbits and impairs its function as a transcriptional activator. Analyses of transcrip- tomes from near-isogenic lines identified downstream genes required for the tendril＇s capability to sense and climb a support. This study provides an example to explore rare functional variants in plant genomes.

An ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 gene mutation confers light green peel in cucumber

The peel color of fruit is an important commercial trait in cucumber, but the underlying molecular basis is largely unknown. A mutant showing light green exocarp was discovered from ethyl methane sulfonate （EMS） mutagenized cucumber line 406 with dark green exocarp. Genetic analysis showed the mutant phenotype is conferred by a single recessive gene, here designated as lgp （light green peel）. By re-sequencing of bulked segregants, we identified the candidate gene Csa7Go51430 encoding ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 （ARCS） that plays a vital role in chloroplast division in Arabidopsis. A single nucleotide polymorphism （SNP） causing amino acid alteration in the conserved GTPase domain of Csa7Go5143o showed co-segregation with the altered phenotype. Furthermore, the transient RNA interference of this gene resulted in reduced number and enlarged size of chloroplasts, which were also observed in the Igp mutant. This evidence supports that the non-synonymous SNP in Csa7G051430 is the causative mutation for the light green peel. This study provides a new allele for cucumber breeding for light green fruits and additional resource for the study of chloroplast development.

A structural and data-driven approach to engineering a plant cytochrome P450 enzyme

Functional manipulation of biosynthetic enzymes such as cytochrome P450 s(or P450 s) has attracted great interest in metabolic engineering of plant natural products.Cucurbitacins and mogrosides are plant triterpenoids that share the same backbone but display contrasting bioactivities.This structural and functional diversity of the two metabolites can be manipulated by engineering P450 s.However,the functional redesign of P450 s through directed evolution(DE) or structure-guided protein engineering is time consuming and challenging,often because of a lack of high-throughput screening methods and crystal structures of P450 s.In this study,we used an integrated approach combining computational protein design,evolutionary information,and experimental data-driven optimization to alter the substrate specificity of a multifunctional P450(CYP87 D20)from cucumber.After three rounds of iterative design and evaluation of 96 protein variants,CYP87 D20,which is involved in the cucurbitacin C biosynthetic pathway,was successfully transformed into a P450 mono-oxygenase that performs a single specific hydroxylation at C11 of cucurbitadienol.This integrated P450-engineering approach can be further applied to create a de novo pathway to produce mogrol,the precursor of the natural sweetener mogroside,or to alter the structural diversity of plant triterpenoids by functionally manipulating other P450 s.

A Truncated F-Box Protein Confers the Dwarfism in Cucumber

Dwarfism is an important plant architecture trait in crop breeding（Peng et al.,1999;Sasaki el al.,2002）.In cucurbits.the compact plant type was proposed to develop new varieties for the once-over mechanical harvest for concentrated fruit set and higher densities（Li et al.,2011;Mondal et al.,2011）.

Genome architecture and tetrasomic inheritance of autotetraploid potato

Potato(Solanum tuberosum)is the most consumed non-cereal food crop.Most commercial potato cultivars are autotetraploids with highly heterozygous genomes,severely hampering genetic analyses and improvement.By leveraging the state-of-the-art sequencing technologies and polyploid graph binning,we achieved a chromosome-scale,haplotype-resolved genome assembly of a cultivated potato,Cooperation-88(C88).lntra-haplotype comparative analyses revealed extensive sequence and expression differences in this tetraploid genome.We identified haplotype-specific pericentromeres on chromosomes,suggesting a distinct evolutionary trajectory of potato homologous centromeres.Furthermore,we detected double reduction events that are unevenly distributed on haplotypes in 1021 of 1034 selfing progeny,a feature of autopolyploid inheritance.By distinguishing maternal and paternal haplotype sets in C88,we simulated the origin of heterosis in cultivated tetraploid with a survey of 3110 tetra-allelic loci with deleterious mutations,which were masked in the heterozygous condition bytwo parents.This study provides insights into the genomic architecture of autopolyploids and will guide their breeding.

Comparison of the distribution of the repetitive DNA sequences in three variants of Cucumis sativus reveals their phylogenetic relationships

Repetitive DNA sequences with variability in copy number or/and sequence polymorphism can be employed as useful molecular markers to study phylogenetics and identify species/chromosomes when combined with fluorescence in situ hybridization （FISH）. Cucumis sativus has three variants, Cucumis sativus L. var. sativus, Cucumis sativus L. var. hardwickii and Cucumis sativus L. var. xishuangbannesis. The phylogenetics among these three variants has not been well explored using cytological landmarks. Here, we concentrate on the organization and distribution of highly repetitive DNA sequences in cucumbers, with emphasis on the differences between cultivar and wild cucumber. The diversity of chromosomal karyotypes in cucumber and its relatives was detected in our study. Thereby, sequential FISH with three sets of multi-probe cocktails （combined repetitive DNA with chromosome-specific fosmid clones as probes） were conducted on the same metaphase cell, which helped us to simultaneously identify each of the 7 metaphase chromosomes of wild cucumber C. sativus var. hardwickii. A standardized karyotype of somatic metaphase chromosomes was constructed. Our data also indicated that the relationship between cultivar cucumber and C. s. var. xishuangbannesis was closer than that of C. s. var. xishuangbannesis and C. s. var. hardwickii.

Mutation in a novel gene SMALL AND CORDATELEAF 1 affects leaf morphology in cucumber

Plant species exhibit substantial variation in leaf morphology.VWe isolated a recessive mutant gene termed small and cordate leaf 1（sclh）that causes alteration in both leaf size and shape of cucumber.Compared to wild type leaves,the sclh mutant had fewer numbers of epidermal pavement cells.A single nucleotide polymorphism was associated with this leaf phenotype,which occurred in a putative nucleoside bisphosphate phosphatase.RNA-seq analysis of the wild type and sclh mutant leaves suggested that SCL;regulation may not involve known hormonal pathways.Our work identified a candidate gene for SCL;that may play a role in leaf development.