Unravelling Glucoraphanin and Glucoerucin Metabolism across Broccoli Sprout Development: Insights from Metabolite and Transcriptome Analysis.

Variations in the concentration of glucoraphanin (GRA) and glucoerucin (GER), as well as the corresponding breakdown products, isothiocyanates (ITCs) and nitriles, were investigated during the growth of broccoli sprouts. The concentrations of GRA and GER decreased sharply from 33.66 µmol/g to 11.48 µmol/g and 12.98 µmol/g to 8.23 µmol/g, respectively, after seed germination. From the third to the seventh day, both GRA and GER were maintained as relatively stable. The highest concentrations of sulforaphane (17.16 µmol/g) and erucin (12.26 µmol/g) were observed on the first day. Hereafter, the concentrations of nitrile hydrolyzed from GRA or GER were higher than those of the corresponding ITCs. Moreover, the ratio of sulforaphane to sulforaphane nitrile decreased from 1.35 to 0.164 from 1 d to 5 d, with a similar trend exhibited for erucin/erucin nitrile after 2 d. RNA-seq analysis showed that BolMYB28 and BolCYP83A1, involved in aliphatic glucosinolate (GSL) biosynthesis, remained largely unexpressed until the third day. In contrast, the genes operating within the GSL-myrosinase hydrolysis pathway were highly expressed right from the beginning, with their expression levels increasing significantly after the third day. Additionally, we identified two BolESPs and six BolNSPs that might play important roles in promoting the production of nitriles during the development of broccoli sprouts.

A mutation in CsABCB19 encoding an ATP-binding cassette auxin transporter leads to erect and compact leaf architecture in cucumber (Cucumis sativus L.).

Leaf architecture, including leaf position and leaf morphology, is a critical component of plant architecture that directly determines plant appearance, photosynthetic utilization, and ultimate productivity. The mechanisms regulating leaf petiole angle and leaf flatness in cucumber remain unclear. In this study, we identified an erect and compact leaf architecture mutant (ecla) from an EMS (ethyl methanesulfonate) -mutagenized cucumber population, which exhibited erect petioles and crinkled leaves. Histological examination revealed significant phenotypic variation in ecla was associated with asymmetric cell expansion. MutMap sequencing combined with genetic mapping revealed that CsaV3_5G037960 is the causative gene for the ecla mutant phenotype. Through protein sequence alignment and Arabidopsis genetic complementation, we identified this gene as a functional direct homolog encoding the ATP-binding cassette transporter AtABCB19, hence named CsABCB19. A nonsynonymous mutation in the eleventh exon of CsABCB19 leads to premature termination of translation. The expression level of CsABCB19 in the ecla mutant was significantly reduced in all tissues compared to the wild type (WT). Transcriptome analysis revealed that auxin and polarity-related genes were significantly differentially expressed in mutant petioles and leaves, compared with those in WT. Auxin assay and exogenous treatment further demonstrated that CsABCB19 regulates leaf architecture by mediating auxin accumulation and transport. Our research is the first report describing the role of the ABCB19 transporter protein in auxin transport controlling cucumber leaf development. Furthermore, this study provides recent insights into the genetic mechanisms conferring morphological diversity and regulation of petiole angle and leaf flattening. DATA AVAILABILITY: The RNA-seq data in this study have been deposited in the NCBI SRA under BioProject accession number PRJNA874548.

Evaluate cities' urban water resources system resilience along a river and identify its critical driving factors.

The emergence of a series of problems, such as serious water environment pollution and flood disasters, leads to an increase in an urban water resources system's vulnerability and pressure. In order to alleviate an urban water resources crisis, calculating the water resources system resilience (WRSR) and identifying its impact factors are essential. This paper constructed a resilience evaluation framework of the "Economy-Social Development-Infrastructure-Water Resources-Ecological Environment" water resources system from three stages, i.e. resistance, restoration and adaptability. Then, taking 21 cities along the Yangtze River Economic Belt's lower reaches (YREB's lower reaches) as an example, we scientifically measured the WRSR by using the Projection Pursuit Evaluation Model of Real Coding based on the Accelerating Genetic Algorithm (RAGA-PP) and analyzed its driving factors with Geographic Detectors (GD). (1) From 2010 to 2018, the overall level of the WRSR along the YREB's lower reaches was low, and it was characterized by attenuation from central cities to marginal cities; Suzhou had the highest average WRSR (0.656), while Chizhou had the lowest (0.207). (2) From stage characteristics, compared with 2010, resistance and restoration of most cities along the YREB's lower reaches showed an upward trend in 2018; however, the adaptability level of some cities was still low. From the perspective of sub-system resilience, resilience levels of different sub-systems in each urban water resources system (UWRS) were different. (3) For impact factors, average factor interpretations of per capita GDP, average wages of employees and affected areas of floods, geological disasters and typhoons were largest. Based on this, this paper provides relevant suggestions for improving the WRSR along the YREB's lower reaches to improve ability of UWRS to resist external risks.

Fine mapping of CscpFtsY, a gene conferring the yellow leaf phenotype in cucumber (Cucumis sativus L.).

BACKGROUND: Leaf color mutants are ideal materials to study pigment metabolism and photosynthesis. Leaf color variations are mainly affected by chlorophylls (Chls) and carotenoid contents and chloroplast development in higher plants. However, the regulation of chlorophyll metabolism remains poorly understood in many plant species. The chloroplast signal-recognition particle system is responsible for the insertion of the light-harvesting chlorophyll a/b proteins (LHCPs) to thylakoid membranes, which controls the chloroplast development as well as the regulation of Chls biosynthesis post-translationally in higher plants. RESULTS: In this study, the yellow leaf cucumber mutant, named yl, was found in an EMS-induced mutant library, which exhibited a significantly reduced chlorophyll content, abnormal chloroplast ultrastructure and decreased photosynthetic capacity. Genetic analysis demonstrated that the phenotype of yl was controlled by a recessive nuclear gene. Using BSA-seq technology combined with the map-based cloning method, we narrowed the locus to a 100 kb interval in chromosome 3. Linkage analysis and allelism test validated the candidate SNP residing in CsaV3_3G009150 encoding one homolog of chloroplast signal-recognition particle (cpSRP) receptor in Arabidopsis, cpFtsY, could be responsible for the yellow leaf phenotype of yl. The relative expression of CscpFtsY was significantly down-regulated in different organs except for the stem, of yl compared with that in the wild type (WT). Subcellular localization result showed that CscpFtsY located in the chloroplasts of mesophyll cells. CONCLUSIONS: The yl mutant displayed Chls-deficient, impaired chloroplast ultrastructure with intermittent grana stacks and significantly decreased photosynthetic capacity. The isolation of CscpFtsY in cucumber could accelerate the progress on chloroplast development by cpSRP-dependant LHCP delivery system and regulation of Chls biosynthesis in a post-translational way.

The mutation of C-24 reductase, a key enzyme involved in brassinolide biosynthesis, confers a novel compact plant architecture phenotype to cucumber.

KEY MESSAGE: A novel compact plant architecture mutant, cpa-2, was identified from EMS-induced mutagenesis. Bulked segregant analysis sequencing and map-based cloning revealed CsDWF1 encoding C-24 reductase enzyme as the candidate gene. The compact architecture is a vital and valuable agronomic trait that helps to reduce the labor of plant management, and improve the fruit yield by increasing planting density in cucumbers. However, the molecular basis underlying the regulation of plant architecture in cucumber is complex and largely unknown. In this study, a novel recessive compact allele, designated as cpa-2 (compact plant architecture-2) was fine mapped in a 109 kb region on chromosome 7 by the strategy of bulked segregant analysis sequencing combined with map-based cloning. Gene annotation of the corresponding region revealed that the CsaV3_7G030530 (CsDWF1) gene encoding C-24 reductase, which acts as the key enzyme in brassinosteroids biosynthesis, functions as the candidate gene for cpa-2. Sequence analysis showed that a single-nucleotide mutation (G to A) in the second exon of CsaV3_7G030530 caused an amino acid substitution from E502 to K502. Compared with wild-type CCMC, CsDWF1 had lower expression levels in the stem, leaf and ovary of cpa-2. In addition, the compact phenotype in cpa-2 could be partially restored by exogenous BR application. Transcriptome analysis revealed that many genes related to plant growth hormones were differentially expressed in cpa-2 plants. This is the first report about the characterization and cloning of the CsDWF1 gene. This work revealed the importance of CsDWF1 in plant development regulation and extended our understanding of the interaction between BRs and other hormones for plant architecture development.

A SNP mutation in the CsCLAVATA1 leads to pleiotropic variation in plant architecture and fruit morphogenesis in cucumber (Cucumis sativus L.).

Plant architectures is predominantly determined by branching pattern, internode elongation, phyllotaxis, shoot determinacy and reproductive organs. Domestication or improvement of this critical agronomic trait played an important role in the breakthrough of crop yield. Here, we identified a mutant with fasciated plant architecture, named fas, from an ethyl methanesulfonate (EMS) induced mutant population in cucumber. The mutant exhibited abnormal phyllotaxy, flattened main stem, increased number of floral organs, and significantly shorter and thicker fruits. However, the molecular mechanism conferring this pleiotropic effect remains unknown. Using a map-based cloning strategy, we isolated the gene CsaV3_3G045960, encoding a leucine-rich repeat receptor-like kinase, a putative direct homolog of the Arabidopsis CLAVATA1 protein referred to as CsCLV1. Endogenous hormone assays showed that IAA and GA3 levels in fas stems and ovaries were significantly reduced. Conformably, RNA-seq analysis showed that CsCLV1 regulates cucumber stem and ovary development by coordinating hormones and transcription factors. Our results contribute to the understanding of the function of CsCLV1 throughout the growth cycle, provide new evidence that the CLV signaling system is functionally conserved in Cucurbitaceae.

Long-distance control of pumpkin rootstock over cucumber scion under drought stress as revealed by transcriptome sequencing and mobile mRNAs identifications.

Grafting with pumpkin rootstock is commonly used not only to improve the quality of cucumber fruits but also to confer biotic or abiotic stress tolerance. However, the molecular mechanism of grafted cucumbers to drought stress and the possible roles of mobile mRNAs to improve stress tolerance have remained obscure. Hence, we conducted transcriptome sequencing and combined it with morpho-physiological experiments to compare the response of homografts (cucumber as scion and rootstock) (C) and heterografts (cucumber as scion and pumpkin as rootstock) (P) to drought stress. After applying drought stress, homografts and heterografts expressed 2960 and 3088 genes in response to drought stress, respectively. The identified DEGs in heterografts under drought stress were categorized into different stress-responsive groups, such as carbohydrate metabolism (involved in osmotic adjustment by sugar accumulation), lipid and cell wall metabolism (involved in cell membrane integrity by a reduction in lipid peroxidation), redox homeostasis (increased antioxidant enzymes activities), phytohormone (increased ABA content), protein kinases and transcription factors (TFs) using MapMan software. Earlier and greater H2O2 accumulation in xylem below the graft union was accompanied by leaf ABA accumulation in heterografts in response to drought stress. Greater leaf ABA helped heterografted cucumbers to sense and respond to drought stress earlier than homografts. The timely response of heterografts to drought stress led to maintain higher water content in the leaves even in the late stage of drought stress. The identified mobile mRNAs (mb-mRNAs) in heterografts were mostly related to photosynthesis which would be the possible reason for improved chlorophyll content and maximum photochemical efficiency of PSII (Fv/Fm). The existence of some stress-responsive pumpkin (rootstock) mRNAs in cucumber (scion), such as heat shock protein (HSP70, a well-known stress-responsive gene), led to the higher proline accumulation than homografts. The expression of the mobile and immobile stress-responsive mRNAs and timely response of heterografts to drought stress could improve drought tolerance in pumpkin-rooted plants.

A SNP Mutation in Homeodomain-DDT (HD-DDT) Transcription Factor Results in Multiple Trichomes (mt) in Cucumber (Cucumis sativus L.).

Trichome is a natural physical barrier protecting plants against environmental stresses, natural infestations, ultraviolet rays and pathogenicity. Trichome also helps plants in maintaining appropriate water content by reducing transpiration rate. The molecular mechanism regulating unicellular trichome development in Arabidopsis has been extensively elucidated, but the molecular mechanism regulating multicellular trichome development remains unclear. In this study, we identified a multiple trichomes (mt) mutant from a cucumber EMS (Ethylmethylsulfone) mutagenesis population. Genetic analysis indicated that an incomplete dominant gene controls the mt trait. Using a combination of map-based cloning and BSA-seq (Bulked Segregant Analysis -Sequencing), we identified the candidate gene, CsaV3_6G050410, responsible for the mt mutation. Sequence alignment revealed one base substitution in gene CsaV3_6G050410, resulting in an amino acid substitution. The deduced amino acid sequence of CsaV3_6G050410 encodes a HD-DDT (homeodomain-DDT) transcriptional regulatory protein containing a conserved homeobox domain and a DDT domain. Gene expression analysis revealed that the expression level of CsaV3_6G050410 in the mt mutant was similar to that in the WT (wild type). Transcriptome analysis indicated that the mt gene may regulate the development of the epidermis by influencing plant hormone signaling pathways or participating in several transcription factor pathways. The results of this study are fundamental for a better understanding of the function of the HD-DDT transcription factor in the trichome development of cucumber.

Identification of a putative candidate gene encoding 7-dehydrocholesterol reductase involved in brassinosteroids biosynthesis for compact plant architecture in Cucumber (Cucumis sativus L.).

KEY MESSAGE: By the strategy of bulked segregant analysis sequencing combined with genetic mapping, CsDWF5, which encodes 7 dehydrocholesterol reductase that involved in brassinosteroids biosynthesis, was identified as the candidate gene for cpa. Dwarf architecture is one of the most important breeding goals in crops. The biosynthesis and signal transduction of brassinosteroids (BRs) have a great impact on plant growth and development including plant architecture. Here, we identified a compact plant architecture (cpa) mutant from an EMS-induced cucumber population. cpa displayed the extremely dwarf phenotype with shortened internode and petiole, darkened and wrinkled leaf. Genetic analysis revealed that cpa was caused by a single recessive gene. By the strategy of bulked segregant analysis sequencing combined with genetic mapping, CsDWF5, encoding a 7-dehydrocholesterol reductase that involved in sterol biosynthesis, was identified as the candidate gene for cpa. One single nucleotide mutation (G→A) in splicing site causing 3-bp insertion (TAG) was found in the first base of the sixth intron of CsDWF5 in cpa, which furtherly resulted in the frameshift mutation and got a premature stop codon. The expression of CsDWF5 gene was significantly down regulated in different tissues of the cpa mutant compared with that in wild type. The phenotype of cpa could be partially recovered by exogenous BR treatment. Transcriptome analysis identified 1096 genes that exhibited differential expression between the cpa mutant and wild type. KEGG enrichment analysis indicated that differentially expressed genes were significantly enriched in BR biosynthesis and plant-pathogen interaction pathways. These results provide perspectives on the molecular mechanisms underlying the dwarfing phenotype in cucumber.

An irregularly striped rind mutant reveals new insight into the function of PG1ß in cucumber (Cucumis sativus L.).

KEY MESSAGE: Via bulked segregant analysis sequencing combined with linkage mapping, the ist gene responsible for the irregularly striped rind mutation was delimited to a 144-kb region in cucumber. Sequencing and expression analysis identified Csa1G005490 as the candidate gene. The rind appearance of cucumber is one of the most important commercial quality traits. Usually, an immature cucumber fruit has a uniform rind that varies from green to yellow to white among different cultivated varieties. In the present paper, we isolated a novel fruit appearance cucumber mutant, ist, that has an irregularly striped rind pattern. The mutant displayed green irregular stripes on a yellow-green background at the immature fruit stage. Genetic analysis revealed that a single recessive gene, ist, is responsible for this mutation. A BSA (bulked segregant analysis) sequencing approach combined with genetic mapping delimited the ist locus to an interval with a length of 144 kb, and 21 predicted genes were annotated in the region. Based on mutation site screening and expression analysis, two single-nucleotide polymorphisms within the candidate gene, Csa1G005490, were identified as constituting the mutation. Csa1G005490 encodes a polygalacturonase-1 noncatalytic subunit beta protein (PG1ß) known to be involved in fruit softening. The expression of Csa1G005490 was significantly lower in the ist mutant than in the wild type. Transcriptome analysis identified 1796 differentially expressed genes (DEGs) between the ist mutant and wild type. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these DEGs were enriched mostly in photosynthesis and chlorophyll metabolism pathways. Decreased expression patterns of several chlorophyll synthesis genes in the mutant suggest that ist plays a key role in chlorophyll biosynthesis. These results will provide new insight into the molecular mechanism underlying rind appearance polymorphisms in cucumber.

A leaf shape mutant provides insight into PINOID Serine/Threonine Kinase function in cucumber (Cucumis sativus L.).

Optimizing leaf shape is a major challenge in efforts to develop an ideal plant type. Cucumber leaf shapes are diverse; however, the molecular regulatory mechanisms underlying leaf shape formation are unknown. In this study, we obtained a round leaf mutant (rl) from an ethyl methanesulfonate-induced mutagenesis population. Genetic analysis revealed that a single recessive gene, rl, is responsible for this mutation. A modified MutMap analysis combined linkage mapping identified a single nucleotide polymorphism within a candidate gene, Csa1M537400, as the mutation underlying the trait. Csa1M537400 encodes a PINOID kinase protein involved in auxin transport. Expression of Csa1M537400 was significantly lower in the rl mutant than in wild type, and it displayed higher levels of IAA (indole-3-acetic acid) in several tissues. Treatment of wild-type plants with an auxin transport inhibitor induced the formation of round leaves, similar to those in the rl mutant. Altered expression patterns of several auxin-related genes in the rl mutant suggest that rl plays a key role in auxin biosynthesis, transport, and response in cucumber. These findings provide insight into the molecular mechanism underlying the regulation of auxin signaling pathways in cucumber, and will be valuable in the development of an ideal plant type.

Fine Mapping of CsVYL, Conferring Virescent Leaf Through the Regulation of Chloroplast Development in Cucumber.

Leaf color mutants in higher plants are ideal materials for investigating the structure and function of photosynthetic system. In this study, we identified a cucumber vyl (virescent-yellow leaf) mutant in the mutant library, which exhibited reduced pigment contents and delayed chloroplast development process. F2 and BC1 populations were constructed from the cross between vyl mutant and cucumber inbred line 'Hazerd' to identify that the vyl trait is controlled by a simply recessive gene designated as CsVYL. The CsVYL gene was mapped to a 3.8 cM interval on chromosome 4 using these 80 F2 individuals and BSA (bulked segregation analysis) approach. Fine genetic map was conducted with 1542 F2 plants and narrowed down the vyl locus to an 86.3 kb genomic region, which contains a total of 11 genes. Sequence alignment between the wild type (WT) and vyl only identified one single nucleotide mutation (C→T) in the first exon of gene Csa4G637110, which encodes a DnaJ-like zinc finger protein. Gene Expression analysis confirmed the differences in transcription level of Csa4G637110 between wild type and mutant plants. Map-based cloning of the CsVYL gene could accelerate the study of chloroplast development and chlorophyll synthesis of cucumber.