Comparative transcriptomic analysis reveals the roles of microtubule-related genes and transcription factors in fruit length regulation in cucumber (Cucumis sativus)
ABSTRACT: Cucumber (Cucumis sativus L.) fruit is a type of fleshy fruit that is harvested immaturely. Early fruit development directly determines the final fruit length and diameter, and consequently the fruit yield and quality. Different cucumber varieties display huge variations of fruit length, but how fruit length is determined at the molecular level remains poorly understood. To understand the genes and gene networks that regulate fruit length in cucumber, high throughout RNA-seq data were used to compare the transcriptomes of early fruit from two near isogenic lines with different fruit lengths. 3955 genes were found to be differentially expressed, among which 2368 genes were significantly up-regulated and 1587 down-regulated in the line with long fruit. Microtubule and cell cycle related genes were dramatically activated in the long fruit, and transcription factors were implicated in the fruit length regulation in cucumber. Thus, our results built a foundation to dissect the molecular mechanism of fruit length control in cucumber, a key agricultural trait of significant economic importance. Comparative analysis of fruit from two near-isogenic lines, 408 (long fruit) and 409 (short fruit), was employed to discover genes and networks that regulate the fruit length. Two biological replicates were used from each line.
Project description:To distinguish PsCA-responsive proteins, total proteins extracted from the non-acclimated fruit, the fruit exposed to 3 d of cold acclimation, the control fruit stored at 5oC for 12 d and the fruit exposed to 3d of PsCA treatment followed by 9 d of cold storage were separated by 2-DE, respectively. More than 750 protein spots were detected using PDQuest Advanced 2-D Gel Analysis software (Version 8.0) following the 2-DE gel analysis. Of the 750 spots, 58 that displayed significant and reproducible changes in abundance were isolated, and 56 spots were successfully characterized using MS/MS coupled with database searching, which matched 51 proteins. 38 proteins have scores greater than threshold scores of 61(P < 0.05) and they are regarded as the significant differentially- accumulated proteins (SDAPs).
Project description:Early stages of fruit growth from initial set through exponential growth are critical determinants of size and yield, however, there has been little detailed analysis of this phase of development. In this study we combined morphological analysis with 454 pyrosequencing to study gene expression changes occurring in young cucumber fruit at five ages from anthesis through the end of exponential growth. The resulting 1.13 million ESTs were assembled into 27,859 contigs with a mean length of 834 base pairs and a mean of 67 reads per contig. Principal component analysis separated the fruit ages into three groups corresponding with cell division/pre-exponential growth (0 and 4 days post pollination (dpp)), peak exponential expansion (8dpp), and late/post-exponential expansion stages of growth (12 and 16 dpp). Transcripts predominantly expressed at 0 and 4 dpp included homologs of histones, cyclins, and plastid and photosynthesis related genes. The group of genes with peak expression at 8dpp included cytoskeleton, cell wall, and lipid metabolism related genes and phloem specific proteins. This group also was dominated by genes with unknown function or without Arabidopsis homologs, suggesting unique factors contributing to the rapid growth phase in cucurbits. A second shift in gene expression was observed at 12-16dpp, which was characterized by abiotic and biotic stress related genes and significant enrichment for stress related- and developmental- transcription factor gene homologs. Collectively, the gene expression information coupled with morphological analyses tells a progressive story of cell division, development of photosynthetic capacity, cell expansion and fruit growth, phloem activity, protection of the fruit surface, and finally transition away from fruit growth toward defense and maturation. mRNA profiling of early fruit development using 454 sequencing.
Project description:The timing of flower opening is essential for pollination and thus seed production. Despite flower opening has been a long lasting topic for plant biologists, the underling molecular mechansim remains elusive. Here we used a unique cucumber line ‘6457’ that spontaneously produces super ovary with delayed corolla opening as material, and explored the physiological, cytological, nutritional and transcriptomic reasons for the delayed corolla opening in super ovary. Our data showed that cell division and cell expansion persisited for a longer period of time in the super ovary, especially during the green yellow bud stage and yellow bud stage. Similarly, RNA-seq analyses showed that RNA and protein synthesis related genes were upregulated during these two stages, which may account for the decreased nitrogen and phosphrate content in the super ovary. Further, activation of transcription factors were delayed in the green yellow bud stage, while signalling kinases related genes were upregulated in the yellow bud stage in the super ovary. Photosynthesis related genes were also significantly activated in the super ovary, which corresponds to the increased soluble suble content. Phytohoromones such as cytokinins and gibberllins were elevated in the super ovary. Consistently, cytokinins and gibberllins related genes showed significantly enhanced expression. Therefore, both developmental and nutritional factors regulate the timing of female flower opening in cucumber, and provide a valuable foundation for dissecting the underling regulatory pathways of flower opening in planta. The normal ovary blooms at 4-5 days after labeling (when the ovary is visible) (DAL), with the majority blooms at 5 DAL, while the anthesis of super ovary occurs at 8-9 DALs, with most super ovaries blooms at 9 DAL.In the normal ovary, corolla at 1 DAL (N1), 3 DAL (N3), 4 DAL (N4), and 5 DAL (N5) is the typical stage for green bud, green yellow bud, yellow bud, and flowering, respectively, whereas in the super ovary, corolla at 1 DAL (S1) is the green bud, corolla at 3, 4 and 5 DAL (S3, S4, S5) is the green yellow bud, corolla at 7 and 8 DAL (S7, S8) is the yellow bud, and corolla at 9 DAL (S9) is the typical flowering stage.Transcriptome profiling of 11 stages include N1,N3,N4,N5,S1,S3,S4,S5,S7,S8 and S9 were performed, and each stage had 3 biological replicates.
Project description:Nitrogen is the most important mineral nutrient of plant. As a worldwide and economically important vegetable, cucumber (Cucumis sativus L.) has a strong nitrogen-dependence. We took whole transcriptome sequencing approach to compare the gene expression profiles of cucumber leaves and roots grown under sufficient or insufficient nitrate supply. Analysis of the transcriptome data revealed that the root and leaf adapt different response mechanisms to long-term nitrogen deficiency. Photosynthesis and carbohydrate biosynthetic process were pronouncedly and specifically reduced in leaf, while the ion transport function, cell wall and phosphorus-deficiency response function seem systematically down-regulated in root. Genes in nitrogen uptake and assimilation are decreased in root, but some are increased in leaf under nitrogen deficiency. Several lines of evidence suggest that the altered gene expression networks support the basic cucumber growth and development likely through successful nitrogen remobilization involving in the induced expression of genes in ABA and ethylene pathways. cucumber leaf and root mRNA of 28-day after sowing nitrogen deficiency and sufficiency deep sequencing, using Illumina HiSeq 2000
Project description:Purpose: The goals of this study are using RNA-seq to obtain cucumber and Botrytis cinerea transcriptome changes during infection Methods: mRNA profiles of anti-infection samples and interaction sample were generate by deep sequencing,using Illumina Hiseq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow,In total, 248,908,688 raw reads were generated; after removing low-quality reads and those containing adapter and poly-N, 238,341,648 clean reads remained to map the reference genome. There were 3,512 cucumber (differential expression genes) DEGs and 1,735 B. cinerea DEGs. GO enrichment and KEGG enrichment analysis were performed on these DEGs to study the interaction between cucumber and B. cinerea. To verify the reliability and accuracy of our transcriptome data, 5 cucumber DEGs and 5 B. cinerea DEGs were chosen for RT-PCR verification. Conclusions:To the best of our knowledge, this is the first analysis of large-scale transcriptome changes of cucumber during the infection of Botrytis cinerea. These results will increase our understanding of the molecular mechanisms of the cucumber defense Botrytis cinerea and may be used to protect plants against disasters caused by necrotrophic fungal pathogens. mRNA profiles of infection and anti-infection cucumber were generated by deep sequencing, using Illumina Hiseq 2500 .
Project description:Here we performed a transcriptomic study on the effect of sodium nitroprusside (SNP) on cucumber leaves under alkali stress using Solexa/Illumina's high-throughput digital gene expression (DGE) system. Two DGE libraries (from one NaHCO3-treated sample and one NaHCO3 + SNP-treated sample) were constructed, and the gene expression variations between the two samples were compared. Hundreds of differentially expressed genes were obtained by the comparison, and GO analysis of these genes suggested that many biological processes, molecular function, cellular components were related to SNP’s mitigated effect on alkaline stress. The authenticity of the DGE data was further confirmed by analyzing real-time RT-PCR using several random-selected genes. The experiment had 2 treatments: 30 mM NaHCO3 (alkali stress treatment, indicated as Na) and 30 mM NaHCO3+100 μM sodium nitroprusside (indicated as SNP). Samples from Na- and SNP-treated leaves, used for RNA isolation, were harvested, and the two corresponding tag libraries of samples were constructed in parallel. Illumina sequencing of transcripts from Na- and SNP-treated samples to get gene information for cucumber leaves in different treatments.
Project description:Trichomes are the hair-like structures that are widely present on the surface of aerial organs and function in plant defense against biotic and abiotic stresses. Previous studies focus on the single cell trichomes in Arabidopsis and cotton, or multicellular glandular trichomes in tomato, but the developmental process and molecular mechanisms controlling multicellular non-glandular trichome development are largely neglected. Here, we extensively characterized the fruit trichome (spine) development in wild type cucumber and in a tiny branched hair (tbh) mutant that contains a spontaneous mutation and has hairless foliage and smooth fruit surface. Our data indicated that cucumber trichome was multicellular and non-glandular, with no branches or endoreduplication. Further, the major feature of cucumber trichome development was spine base expansion. Transcriptome profiling through Digital Gene Expression indicated that meristem-related genes and transcription factors were implicated in the fruit spine development, and polarity regulators were upregulated during spine base expansion. qRT-PCR verified the reliability of our RNA-SEQ data, and in situ hybridization confirmed the enriched expression of meristem regulators CUP-SHAPED COTYLEDON3 (CUC3) and STM (SHOOT MERISTEMLESS) , as well as the abaxial identity gene KANADI (KAN) in cucumber fruit spine. Together, our results suggest a distinct regulatory pathway involving meristem genes and polarity regulators in multicellular trichome development in cucumber. Using Digital Gene Expression technology to compare the genome-wide gene expression profiles in the fruit spines of wild type cucumber and the tbh mutant, as well as the fruit spines on fruits of 0.5cm and 1.6cm long, repectively. Two biological repelicates were generated for each tissue.
Project description:We used Bio-Rad laboratories, lnc. PCR assay panel to analyze expression levels of MAPK genes in various tissues or organs. Cucumber plants of the ‘Jinyan No.4’ cultivar were reared in growth chambers at 28 ± 1 °C with a photoperiod of 16 h light/8 h dark and light intensity of 400 μmol m−2 s−1. The roots, leaves, stems, female flower buds (approximately 3 d before anthesis), male flower buds (approximately 1.0 cm in length), and fruits (10 d after pollination) were collected from flowering plants for tissue expression analysis. The experiment was designed to gain insight into the temporal and spatial transcription patterns of 58 MAPK cascade genes in cucumber.
Project description:We compared organ specific chloroplast gene expression in cucumber between fully developed leaves (12th counting from the bottom of plant) and growing tips, female flowers, young leaves, young fruits (3-5 cm long). We have also characterized chloroplast gene transcription in cucumber etiolated seedlings in comparison to mature cucumber leaves.
Project description:Phloem is essential for higher plant development and survival by transporting photosynthetic products and systemic signals from source to sink organs. To date, most phloem studies were performed with the phloem exudate for physiological, transcriptomic or proteomic analyses. However, as yet, no transcriptomic profiling was performed to the phloem tissue itself, probably due to the technique difficulties to access the phloem tissue inside the plant body. In this study, laser microdissection combined with RNA-Seq technology was used to gain phloem-specific transcript profiling in three different organs (pedicle, stalk and fruit) of cucumber, a model plant species for phloem research. We found that transcription factors and biotic/abiotic stress related genes are highly enriched in the sink organs, while cell growth via calcium ion, hormone actions and cell cycle control was largely restricted in the pedicle and stalk, and genes implicated in transporting amino acids and sugars are mostly up-regulated in the pedicle. Further, we found excellent corroboration between phloem-specific gene expression, and physiological characterization with phloem function. In addition, we identified 432 cucumber-unique genes and several phloem-specific markers for future functional studies. This study provides new insights into the molecular genetics of the function of phloem tissue in cucumber, rather than the conventional phloem sap analyses. Overall design: Transcriptome profiling by laser microdissection combined with RNA sequencing analysis was employed to discover critical genes that are expressed and regulated in phloem tissue in three different organs (pedicle, stalk and fruit) of cucumber. Two biological replicates were generated for each sample.