Genome-wide identification and expression analysis of the JMJ-C gene family in melon (Cucumis melo L.) reveals their potential role in fruit development.

BACKGROUND: Proteins with the jumonji (JMJ)-C domain belong to the histone demethylase family and contribute to reverse histone methylation. Although JMJ-C family genes have an essential role in regulating plant growth and development, the characterization of the JMJ-C family genes in melon has not been uncovered. RESULTS: In this study, a total of 17 JMJ-C proteins were identified in melon (Cucumis melo L.). CmJMJs were categorized into five subfamilies based on the specific conserved domain: KDM4/JHDM3, KDM5/JARID1, JMJD6, KDM3/JHDM2, and JMJ-C domain-only. The chromosome localization analyses showed that 17 CmJMJs were distributed on nine chromosomes. Cis-acting element analyses of the 17 CmJMJ genes showed numerous hormone, light, and stress response elements distributed in the promoter region. Covariance analysis revealed one pair of replicated fragments (CmJMJ3a and CmJMJ3b) in 17 CmJMJ genes. We investigated the expression profile of 17 CmJMJ genes in different lateral organs and four developmental stages of fruit by RNA-seq transcriptome analysis and RT-qPCR. The results revealed that most CmJMJ genes were prominently expressed in female flowers, ovaries, and developing fruits, suggesting their active role in melon fruit development. Subcellular localization showed that the fruit-related CmJMJ5a protein is specifically localized in the cell nucleus. CONCLUSIONS: This study provides a comprehensive understanding of the gene structure, classification, and evolution of JMJ-C in melon and supports the clarification of the JMJ-C functions in further research.

Pollen tube emergence is mediated by ovary-expressed ALCATRAZ in cucumber.

Pollen tube guidance within female tissues of flowering plants can be divided into preovular guidance, ovular guidance and a connecting stage called pollen tube emergence. As yet, no female factor has been identified to positively regulate this transition process. In this study, we show that an ovary-expressed bHLH transcription factor Cucumis sativus ALCATRAZ (CsALC) functions in pollen tube emergence in cucumber. CsALC knockout mutants showed diminished pollen tube emergence, extremely reduced entry into ovules, and a 95% reduction in female fertility. Further examination showed two rapid alkalinization factors CsRALF4 and CsRALF19 were less expressed in Csalc ovaries compared to WT. Besides the loss of male fertility derived from precocious pollen tube rupture as in Arabidopsis, Csralf4 Csralf19 double mutants exhibited a 60% decrease in female fertility due to reduced pollen tube distribution and decreased ovule targeting efficiency. In brief, CsALC regulates female fertility and promotes CsRALF4/19 expression in the ovary during pollen tube guidance in cucumber.

Natural variation in CRABS CLAW contributes to fruit length divergence in cucumber.

Fruit length is a key domestication trait that affects crop yield and appearance. Cucumber (Cucumis sativus) fruits vary from 5 to 60 cm in length. Despite the identification of several regulators and multiple quantitative trait loci (QTLs) underlying fruit length, the natural variation, and molecular mechanisms underlying differences in fruit length are poorly understood. Through map-based cloning, we identified a nonsynonymous polymorphism (G to A) in CRABS CLAW (CsCRC) as underlying the major-effect fruit size/shape QTL FS5.2 in cucumber. The short-fruit allele CsCRCA is a rare allele that has only been found in round-fruited semi-wild Xishuangbanna cucumbers. A near-isogenic line (NIL) homozygous for CsCRCA exhibited a 34∼39% reduction in fruit length. Introducing CsCRCG into this NIL rescued the short-fruit phenotype, and knockdown of CsCRCG resulted in shorter fruit and smaller cells. In natural cucumber populations, CsCRCG expression was positively correlated with fruit length. Further, CsCRCG, but not CsCRCA, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression. Knockout of CsARP1 produced shorter fruit with smaller cells. Hence, our work suggests that CsCRCG positively regulates fruit elongation through transcriptional activation of CsARP1 and thus enhances cell expansion. Using different CsCRC alleles provides a strategy to manipulate fruit length in cucumber breeding.

Genome-Wide Identification of the SUN Gene Family in Melon (Cucumis melo) and Functional Characterization of Two CmSUN Genes in Regulating Fruit Shape Variation.

Melon (Cucumis melo) is an important economic crop cultivated worldwide. A unique SUN gene family plays a crucial role in regulating plant growth and fruit development, but many SUN family genes and their function have not been well-characterized in melon. In the present study, we performed genome-wide identification and bioinformatics analysis and identified 24 CmSUN family genes that contain integrated and conserved IQ67 domain in the melon genome. Transcriptome data analysis and qRT-PCR results showed that most CmSUNs are specifically enriched in melon reproductive organs, such as young flowers and ovaries. Through genetic transformation in melons, we found that overexpression of CmSUN23-24 and CmSUN25-26-27c led to an increased fruit shape index, suggesting that they act as essential regulators in melon fruit shape variation. Subcellular localization revealed that the CmSUN23-24 protein is located in the cytoplasmic membrane. A direct interaction between CmSUN23-24 and a Calmodulin protein CmCaM5 was found by yeast two-hybrid assay, which indicated their participation in the calcium signal transduction pathway in regulating plant growth. These findings revealed the molecular characteristics, expression profile, and functional pattern of the CmSUN genes, and may provide the theoretical basis for the genetic improvement of melon fruit breeding.

Genome-wide characterization and expression analysis of SAUR gene family in Melon (Cucumis melo L.).

MAIN CONCLUSION: We identified 66 melon SAUR genes by bioinformatic analyses. CmSAUR19, 38, 58, 62 genes are specifically expressed in different stages of fruit growth, suggesting their participation in regulating fruit development. Auxin plays a crucial role in plant growth by regulating the multiple auxin response genes. However, in melon (Cucumis melo L.), the functions of the auxin early response gene family SAUR (Small auxin up RNA) genes in fruit development are still poorly understood. Through genome-wide characterization of CmSAUR family in melon, we identified a total of 66 CmSAUR genes. The open reading frames of the CmSAUR genes ranged from 234 to 525 bp, containing only one exon and lacking introns. Chromosomal position and phylogenetic tree analyses found that the two gene clusters in the melon chromosome are highly homologous in the Cucurbitaceae plants. Among the four conserved motifs in CmSAUR proteins, motif 1, motif 2, and motif 3 located in most of the family protein sequences, and motif 4 showed a close correlation with the two gene clusters. The CmSAUR28 and CmSAUR58 genes have auxin response elements located in the promoters, suggesting they may be involved in the auxin signaling pathway to regulate fruit development. Through transcriptomic profiling in the four developmental stages of fruit and different lateral organs, we selected 16 differentially-expressed SAUR genes for performing further expression analyses. qRT-PCR results showed that five SAUR genes are specifically expressed in flower organs and ovaries. CmSAUR19 and CmSAUR58 were significantly accumulated in the early developmental stage of the fruit. CmSAUR38 and CmAUR62 showed high expression in the climacteric and post-climacteric stages, suggesting their specific role in controlling fruit ripening. This work provides a foundation for further exploring the function of the SAUR gene in fruit development.

Roles of CsBRC1-like in leaf and lateral branch development in cucumber.

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes, as plant-specific transcription factors, play vital roles in flower pattern, leaf development and plant architecture. Our recent study shows that the TCP gene BRANCHED1 (CsBRC1) specifically regulates shoot branching in cucumber. Here, we found CsBRC1 had a closely related paralogous gene CsBRC1-like. The synteny analysis revealed that these two genes originated from a segmental duplication. CsBRC1-like displayed different expression patterns in cucumber compared with CsBRC1, indicating that they may have functional differentiation. Ectopic expression of CsBRC1-like in Arabidopsis brc1-1 mutant resulted in reduced rosette branches and rosette leaves, whereas silencing CsBRC1-like in cucumber only led to a deformed true leaf of seedling rather than affecting the shoot branching. RNA-seq analysis of wild-type and CsBRC1-like-RNAi plants implicated that CsBRC1-like might regulate early leaf development through affecting the transcripts of auxin and cytokinin related genes in cucumber. Moreover, CsBRC1-like directly interacts with CsTCP10a and CsBRC1 in vivo. Our results demonstrated that CsBRC1-like has a specific role in regulating leaf development, and CsBRC1-like and CsBRC1 may have overlapping roles in shoot branching.

Gene regulatory network controlling carpel number variation in cucumber.

The WUSCHEL-CLAVATA3 pathway genes play an essential role in shoot apical meristem maintenance and floral organ development, and under intense selection during crop domestication. The carpel number is an important fruit trait that affects fruit shape, size and internal quality in cucumber, but the molecular mechanism remains elusive. Here, we found that CsCLV3 expression was negatively correlated with carpel number in cucumber cultivars. CsCLV3-RNAi led to increased number of petals and carpels, whereas overexpression of CsWUS resulted in more sepals, petals and carpels, suggesting that CsCLV3 and CsWUS function as a negative and a positive regulator for carpel number variation, respectively. Biochemical analyses indicated that CsWUS directly bound to the promoter of CsCLV3 and activated its expression. Overexpression of CsFUL1A , a FRUITFULL-like MADS-box gene, resulted in more petals and carpels. CsFUL1A can directly bind to the CsWUS promoter to stimulate its expression. Furthermore, we found that auxin participated in carpel number variation in cucumber through interaction of CsARF14 with CsWUS. Therefore, we have identified a gene regulatory pathway involving CsCLV3, CsWUS, CsFUL1A and CsARF14 in determining carpel number variation in an important vegetable crop - cucumber.

CsBRC1 inhibits axillary bud outgrowth by directly repressing the auxin efflux carrier CsPIN3 in cucumber.

Shoot branching is an important agronomic trait that directly determines plant architecture and affects crop productivity. To promote crop yield and quality, axillary branches need to be manually removed during cucumber production for fresh market and thus are undesirable. Auxin is well known as the primary signal imposing for apical dominance and acts as a repressor for lateral bud outgrowth indirectly. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family gene BRANCHED1 (BRC1) has been shown to be the central integrator for multiple environmental and developmental factors that functions locally to inhibit shoot branching. However, the direct molecular link between auxin and BRC1 remains elusive. Here we find that cucumber BRANCHED1 (CsBRC1) is expressed in axillary buds and displays a higher expression level in cultivated cucumber than in its wild ancestor. Knockdown of CsBRC1 by RNAi leads to increased bud outgrowth and reduced auxin accumulation in buds. We further show that CsBRC1 directly binds to the auxin efflux carrier PIN-FORMED (CsPIN3) and negatively regulates its expression in vitro and in vivo. Elevated expression of CsPIN3 driven by the CsBRC1 promoter results in highly branched cucumber with decreased auxin levels in lateral buds. Therefore, our data suggest that CsBRC1 inhibits lateral bud outgrowth by direct suppression of CsPIN3 functioning and thus auxin accumulation in axillary buds in cucumber, providing a strategy to breed for cultivars with varying degrees of shoot branching grown in different cucumber production systems.

A Functional Allele of CsFUL1 Regulates Fruit Length through Repressing CsSUP and Inhibiting Auxin Transport in Cucumber.

Fruit length is a prominent agricultural trait during cucumber (Cucumis sativus) domestication and diversifying selection; however, the regulatory mechanisms of fruit elongation remain elusive. We identified two alleles of the FRUITFULL (FUL)-like MADS-box gene CsFUL1 with 3393C/A Single Nucleotide Polymorphism variation among 150 cucumber lines. Whereas CsFUL1A was specifically enriched in the long-fruited East Asian type cucumbers (China and Japan), the CsFUL1C allele was randomly distributed in cucumber populations, including wild and semiwild cucumbers. CsFUL1A knockdown led to further fruit elongation in cucumber, whereas elevated expression of CsFUL1A resulted in significantly shorter fruits. No effect on fruit elongation was detected when CsFUL1C expression was modulated, suggesting that CsFUL1A is a gain-of-function allele in long-fruited cucumber that acts as a repressor during diversifying selection of East Asian cucumbers. Furthermore, CsFUL1A binds to the CArG-box in the promoter region of SUPERMAN, a regulator of cell division and expansion, to repress its expression. Additionally, CsFUL1A inhibits the expression of auxin transporters PIN-FORMED1 (PIN1) and PIN7, resulting in decreases in auxin accumulation in fruits. Together, our work identifies an agriculturally important allele and suggests a strategy for manipulating fruit length in cucumber breeding that involves modulation of CsFUL1A expression.

The MADS-Box Gene CsSHP Participates in Fruit Maturation and Floral Organ Development in Cucumber.

Cucumber is an important vegetable crop bearing fleshy pepo fruit harvested immature. Fruits left unpicked in time during summer production, as well as unfavorable environmental conditions during post-harvest shelf, will cause cucumber fruits to turn yellow and ripen, and thus impair the market value. Identification of maturity-related genes is of great agricultural and economic importance for cucumber production. Here, we isolated and characterized a MADS-box gene, Cucumis sativus SHATTERPROOF (CsSHP) in cucumber. Expression analysis indicated that CsSHP was specifically enriched in reproductive organs including stamens and carpels. Ectopic expression of CsSHP was unable to rescue the indehiscence silique phenotype of shp1 shp2 mutant plant in Arabidopsis. Instead, overexpression of CsSHP resulted in early flowering, precocious phenotypes, and capelloid organs in wild-type Arabidopsis. Biochemical analysis indicated that CsSHP directly interacted with cucumber SEPALLATA (SEP) proteins. CsSHP expression increased significantly during the yellowing stage of cucumber ripening, and was induced by exogenous application of abscisic acid (ABA). Therefore, CsSHP may participate in fruit maturation through the ABA pathway and floral organ specification via interaction with CsSEPs to form protein complex in cucumber.

Molecular basis of cucumber fruit domestication.

Cucumber (Cucumis sativus L.) is an economically important vegetable crop that is cultivated worldwide. Compared to the wild ancestor bearing small, bitter and seedy fruit, domesticated cucumbers exhibit significant variation in fruit appearance, size and flavor. Understanding the molecular basis of domestication related traits can provide insights into fruit evolution and make crop breeding more efficient. Here we review recent advances in relating to the genetic basis of fruit morphological traits (femaleness, fruit spine, wart, size, color and carpel development) and organoleptic features (bitterness) during cucumber domestication.

CsTFL1b may regulate the flowering time and inflorescence architecture in cucumber (Cucumis sativus L.).

Cucumber is an important vegetable with indeterminate growth habit which is beneficial to its yield. In this study, we cloned the TFL1 homolog CsTFL1b in cucumber. CsTFL1b shares highly sequence similarity to TFL1 from Arabidopsis and has conservative histidine amino acid residue which is necessary for TFL1 function. However, phylogenetic analysis suggested that cucurbits TFL1s (CsTFL1b of cucumber and CmTFL1 of melon) formed a subclade which is far from the AtTFL1 in Arabidopsis or CEN in Antirrhinum. CsTFL1b was highest expressed in male flower but barely expressed in SAM which was different from TFL1 in Arabidopsis with highly transcription accumulation in SAM and CsTFL1b was located in nucleus and cytoplasm. Upon ectopic expression of CsTFL1b in Arabidopsis, the flowering time of transgenic plants was significantly delayed in both wild type and tfl1-11 mutant background but the terminal flower phenotype of tfl1-11 mutant was partially rescued. These results may underlie the discrepant function of CsTFL1b in cucumber from that in Arabidopsis.

CsSPL functions as an adaptor between HD-ZIP III and CsWUS transcription factors regulating anther and ovule development in Cucumis sativus (cucumber).

Anther and ovule genesis preconditions crop fertilization and fruit production; however, coordinative regulation of anther and ovule development and underlying molecular pathways remain largely elusive. Here, we found that SPOROCYTELESS (SPL)/NOZZLE (NZZ) expression was nearly abolished in a Cucumis sativus (cucumber) mutant with severely defective anther and ovule development. CsSPL was expressed specifically in the developing anthers and ovules. Knock-down of CsSPL reduced male and female fertility with malformed pollen and suppressed ovule development. Importantly, CsSPL directly interacted with CsWUS (WUSCHEL) in the nucellus and YABBY family genes in integuments, and positively regulated CsWUS expression, meanwhile the HD-ZIP III gene CsPHB (PHABULOSA), expressed specifically in the nucellus, promoted CsSPL expression by binding to the CsSPL promoter. Thus, CsSPL acts as an adaptor to link CsPHB and CsWUS functioning, underpinning a previously unidentified regulatory pathway orchestrating sex organ development in planta. In addition, auxin accumulation was reduced in the reproductive organs of CsSPL knock-down plants. Biochemical analyses further showed that CsSPL stimulated the expression of AUXIN RESPONSE FACTOR 3 (CsARF3), and was positively regulated by CsARF13 during reproductive organ development, indicating sequential interactions of CsSPL with auxin signaling components in orchestrating anther and ovule development.

CsLFY is required for shoot meristem maintenance via interaction with WUSCHEL in cucumber (Cucumis sativus).

Cucumber (Cucumis sativus) is an agronomically important vegetable with indeterminant growth habit, in which leaves are produced from the shoot apical meristem (SAM), and unisexual flowers are generated from the leaf axils. LEAFY (LFY) and its homologs have been shown to play important roles in promoting flower development and branching. The LFY homolog gene CsLFY was cloned in cucumber. Molecular biology, developmental biology and biochemical tools were combined to explore the biological function of the LFY homologous gene CsLFY in cucumber. CsLFY was expressed in the SAM, floral meristem and floral organ primordia. Ectopic expression of CsLFY rescued the phenotype of the lfy-5 mutant in Arabidopsis. Knockdown of CsLFY by RNA interference (RNAi) led to defective shoot development and premature discontinuance of leaf initiation in cucumber. Transcription of CsWUS and putative CsLFY target genes including CsAP3 and CUM1 were significantly reduced in the CsLFY-RNAi lines. Further biochemical analyses indicated that CsLFY physically interacts with CsWUS in cucumber. These data suggested that CsLFY has a novel function in regulating shoot meristem maintenance through interaction with CsWUS, and promotes flower development via activation of CsAP3 and CUM1 in cucumber.

Optimization of Mycelia Selenium Polysaccharide Extraction from Agrocybe cylindracea SL-02 and Assessment of their Antioxidant and Anti-Ageing Activities.

The aim of the present study was to optimize the purification of mycelia selenium polysaccharides (MSPS) from Agrocybe cylindracea SL-02 and characterize their in vitro antioxidant and in vivo anti-ageing activities. The Box-Behnken experimental design (BBD) was evaluated, which showed that the optimum conditions included an extraction temperature of 94.99°C, a pH of 9 and a precipitation temperature of 12°C, and the predicted yield was 11.036 ± 0.31%. The in vitro antioxidant assay demonstrated that MSPS had potential effects on scavenging and enhanced the reducing power of reactive oxygen species. The in vivo anti-ageing evaluation showed that MSPS significantly reduced the malonaldehyde (MDA) contents and total cholesterol (CHOL) levels, and remarkably improved the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) in mice in response to D-galactose-induced ageing. Furthermore, the characteristic analysis of MSPS indicated a selenium content of 1.76 ± 0.10 mg/g at a concentration of 6 µg/mL in liquid media and a monosaccharide composition of rhamnose, arabinose, mannose, glucose and galactose at a molar ratio of 29:3:1:18.8:2.7. These results suggest that MSPS might be suitable for functional foods and natural drugs on preventing the ageing progress induced by toxic chemicals.

The antihyperlipidemic activities of enzymatic and acidic intracellular polysaccharides by Termitomyces albuminosus.

Two polysaccharides, EIPS and AIPS were obtained by the hydrolysis of IPS from Termitomyces albuminosus, and their pharmacological effects on blood lipid profiles metabolism and oxidative stress were investigated. The results demonstrated that EIPS was superior to IPS and AIPS on reducing hepatic lipid levels and preventing oxidative stress by improving serum enzyme activities (ALT, AST, and ALP), serum lipid levels (TC, TG, HDL-C, LDL-C and VLDL-C), hepatic lipid levels (TC and TG), and antioxidant status (SOD, GSH-Px, CAT, T-AOC, MDA, and LPO). These conclusions indicated that EIPS, AIPS and IPS might be suitable for functional foods and natural drugs on preventing the high-fat emulsion-induced hyperlipidemia. In addition, the monosaccharide compositions of IPS and its hydrolyzate were also processed.

Antioxidant and hepatoprotective activities of intracellular polysaccharide from Pleurotus eryngii SI-04.

In present study, the intracellular polysaccharide (IPS) and its two fractions of IPS-1 and IPS-2 were obtained and purified by DEAE-52 cellulose chromatography from Pleurotus eryngii SI-04 mycelia, and their hepatoprotective effects were also investigated. The results showed that the IPS-2 had superior hepatoprotective effects by increasing the serum enzyme activities and bilirubin (BIL) levels, decreasing the serum albumin (ALB) and triglyceride (TG) levels, improving the hepatic antioxidant status, and ameliorating the hepatic structure damage. Furthermore, the monosaccharide composition and main bond types were also analyzed. These conclusions demonstrated that the both IPS and its fractions might be suitable for functional foods and natural drugs in preventing the acute liver damage.

Different cucumber CsYUC genes regulate response to abiotic stresses and flower development.

The phytohormone auxin is essential for plant growth and development, and YUCCA (YUC) proteins catalyze a rate-limiting step for endogenous auxin biosynthesis. Despite YUC family genes have been isolated from several species, systematic expression analyses of YUCs in response to abiotic stress are lacking, and little is known about the function of YUC homologs in agricultural crops. Cucumber (Cucumis sativus L.) is a world cultivated vegetable crop with great economical and nutritional value. In this study, we isolated 10 YUC family genes (CsYUCs) from cucumber and explored their expression pattern under four types of stress treatments. Our data showed that CsYUC8 and CsYUC9 were specifically upregulated to elevate the auxin level under high temperature. CsYUC10b was dramatically increased but CsYUC4 was repressed in response to low temperature. CsYUC10a and CsYUC11 act against the upregulation of CsYUC10b under salinity stress, suggesting that distinct YUC members participate in different stress response, and may even antagonize each other to maintain the proper auxin levels in cucumber. Further, CsYUC11 was specifically expressed in the male flower in cucumber, and enhanced tolerance to salinity stress and regulated pedicel and stamen development through auxin biosynthesis in Arabidopsis.