Aiping Song

Next-Generation Sequencing of the Chrysanthemum nankingense (Asteraceae) Transcriptome Permits Large-Scale Unigene Assembly and SSR Marker Discovery

Background Simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Chrysanthemum is one of the largest genera in the Asteraceae family. Only few Chrysanthemum expressed sequence tag (EST) sequences have been acquired to date, so the number of available EST-SSR markers is very low. Methodology/Principal Findings Illumina paired-end sequencing technology produced over 53 million sequencing reads from C. nankingense mRNA. The subsequent de novo assembly yielded 70,895 unigenes, of which 45,789 (64.59%) unigenes showed similarity to the sequences in NCBI database. Out of 45,789 sequences, 107 have hits to the Chrysanthemum Nr protein database; 679 and 277 sequences have hits to the database of Helianthus and Lactuca species, respectively. MISA software identified a large number of putative EST-SSRs, allowing 1,788 primer pairs to be designed from the de novo transcriptome sequence and a further 363 from archival EST sequence. Among 100 primer pairs randomly chosen, 81 markers have amplicons and 20 are polymorphic for genotypes analysis in Chrysanthemum. The results showed that most (but not all) of the assays were transferable across species and that they exposed a significant amount of allelic diversity. Conclusions/Significance SSR markers acquired by transcriptome sequencing are potentially useful for marker-assisted breeding and genetic analysis in the genus Chrysanthemum and its related genera.

Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB2 Enhances Drought and Salinity Tolerance, Increases Hypersensitivity to ABA and Delays Flowering in Arabidopsis thaliana

Knowledge on genes related to plant responses to adverse growth conditions and development is essential for germplasm improvement. In this study, a chrysanthemum R2R3-MYB transcription factor gene, designated CmMYB2 (GenBank accession No. JF795918), was cloned and functionally characterized. Expression of CmMYB2 in chrysanthemum leaves was up-regulated in response to drought, salinity and cold stress, as well as by treatment with exogenous abscisic acid (ABA). When the gene was constitutively expressed in Arabidopsisthaliana, it increased plant sensitivity to ABA and reduced stomatal aperture. Plant survival under drought was improved than in the wild type, as was the plants’ salinity tolerance. The level of expression of a number of genes associated with the stress response, including RD22, RD29A, RAB18, COR47, ABA1 and ABA2, was raised in the CmMYB2 transgenic Arabidopsis plants. CmMYB2 transgenic Arabidopsis plants were also delayed in flowering. The expression of CONSTANS (CO), FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY) and APETALA1 (AP1) genes involved in flowering was down-regulated in the CmMYB2 transgenics. Together, these results suggest that CmMYB2 may be a promising gene for the drought and salt tolerance improvement and flowering-time modulation.

A Chrysanthemum Heat Shock Protein Confers Tolerance to Abiotic Stress

Heat shock proteins are associated with protection against various abiotic stresses. Here, the isolation of a chrysanthemum cDNA belonging to the HSP70 family is reported. The cDNA, designated CgHSP70, encodes a 647-residue polypeptide, of estimated molecular mass 70.90 kDa and pI 5.12. A sub-cellular localization assay indicated that the cDNA product is deposited in the cytoplasm and nucleus. The performance of Arabidopsis thaliana plants constitutively expressing CgHSP70 demonstrated that the gene enhances tolerance to heat, drought and salinity. When CgHSP70 was stably over-expressed in chrysanthemum, the plants showed an increased peroxidase (POD) activity, higher proline content and inhibited malondialdehyde (MDA) content. After heat stress, drought or salinity the transgenic plants were better able to recover, demonstrating CgHSP70 positive effect.

Chrysanthemum WRKY gene CmWRKY17 negatively regulates salt stress tolerance in transgenic chrysanthemum and Arabidopsis plants

Key messageCmWRKY17was induced by salinity in chrysanthemum, and it might negatively regulate salt stress in transgenic plants as a transcriptional repressor.AbstractWRKY transcription factors play roles as positive or negative regulators in response to various stresses in plants. In this study, CmWRKY17 was isolated from chrysanthemum (Chrysanthemum morifolium). The gene encodes a 227-amino acid protein and belongs to the group II WRKY family, but has an atypical WRKY domain with the sequence WKKYGEK. Our data indicated that CmWRKY17 was localized to the nucleus in onion epidermal cells. CmWRKY17 showed no transcriptional activation in yeast; furthermore, luminescence assay clearly suggested that CmWRKY17 functions as a transcriptional repressor. DNA-binding assay showed that CmWRKY17 can bind to W-box. The expression of CmWRKY17 was induced by salinity in chrysanthemum, and a higher expression level was observed in the stem and leaf compared with that in the root, disk florets, and ray florets. Overexpression of CmWRKY17 in chrysanthemum and Arabidopsis increased the sensitivity to salinity stress. The activities of superoxide dismutase and peroxidase and proline content in the leaf were significantly lower in transgenic chrysanthemum than those in the wild type under salinity stress, whereas electrical conductivity was increased in transgenic plants. Expression of the stress-related genes AtRD29, AtDREB2B, AtSOS1, AtSOS2, AtSOS3, and AtNHX1 was reduced in the CmWRKY17 transgenic Arabidopsis compared with that in the wild-type Col-0. Collectively, these data suggest that CmWRKY17 may increase the salinity sensitivity in plants as a transcriptional repressor.

Ambient temperature enhanced freezing tolerance of Chrysanthemum dichrum CdICE1 Arabidopsis via miR398

BackgroundICE (Inducer of CBF Expression) family genes play an important role in the regulation of cold tolerance pathways. In an earlier study, we isolated the gene CdICE1 from Chrysanthemum dichrum and demonstrated that freezing tolerance was enhanced by CdICE1 overexpression. Therefore, we sought to determine the mechanism by which ICE1 family genes participate in freezing tolerance.ResultsUsing EMSA (Electrophoretic Mobility Shift Assay) and yeast one-hybrid assays, we confirmed that CdICE1 binds specifically to the MYC element in the CdDREBa promoter and activates transcription. In addition, overexpression of CdICE1 enhanced Arabidopsis freezing tolerance after transition from 23°C to 4°C or 16°C. We found that after acclimation to 4°C, CdICE1, like Arabidopsis AtICE1, promoted expression of CBFs (CRT/DRE Binding Factor) and their genes downstream involved in freezing tolerance, including COR15a (Cold-Regulated 15a), COR6.6, and RD29a (Responsive to Dessication 29a). Interestingly, we observed that CdICE1-overexpressing plants experienced significant reduction in miR398. In addition, its target genes CSD1 (Copper/zinc Superoxide Dismutase 1) and CSD2 showed inducible expression under acclimation at 16°C, indicating that the miR398-CSD pathway was involved in the induction of freezing tolerance.ConclusionsOur data indicate that CdICE1-mediated freezing tolerance occurs via different pathways, involving either CBF or miR398, under acclimation at two different temperatures.

The Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB1 Alters Lignin Composition and Represses Flavonoid Synthesis in Arabidopsis thaliana

Plant R2R3-MYB transcription factor genes are widely distributed in higher plants and play important roles in the regulation of many secondary metabolites at the transcriptional level. In this study, a chrysanthemum subgroup 4 R2R3-MYB transcription factor gene, designated CmMYB1, was isolated through screening chrysanthemum EST (expressed sequence tag) libraries and using rapid application of cDNA ends (RACE) methods and functionally characterized. CmMYB1 is expressed in the root, stem, leaf and flowers, but most strongly in the stem and most weakly in the root. Its heterologous expression in Arabidopsis thaliana reduced the lignin content and altered the lignin composition. The heterologous expression also repressed the flavonoids content in A. thaliana. Together, these results suggested that CmMYB1 is a negative regulator of genes involved in the lignin pathway and flavonoid pathway, it may be a promising gene for controlling lignin and flavonoids profiles in plants.

Phylogenetic and Transcription Analysis of Chrysanthemum WRKY Transcription Factors

WRKY transcription factors are known to function in a number of plant processes. Here we have characterized 15 WRKY family genes of the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of 15 distinct sequences were isolated; initially internal fragments were amplified based on transcriptomic sequence, and then the full length cDNAs were obtained using RACE (rapid amplification of cDNA ends) PCR. The transcription of these 15 genes in response to a variety of phytohormone treatments and both biotic and abiotic stresses was characterized. Some of the genes behaved as would be predicted based on their homology with Arabidopsis thaliana WRKY genes, but others showed divergent behavior.

Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding

Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future.

A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum.

Chrysanthemum plants are subject to serious virus diseases, so detection and identification of virus pathogens is important to prevent the virus spread. A reliable one‐step multiplex RT‐PCR was developed to simultaneously detect two viruses and two viriods: chrysanthemum virus B, tomato Aspermy virus, chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid. In addition, we investigated the detection limit and the efficiency of single and multiplex RT‐PCR assays. The results showed that the multiplex RT‐PCR assay proved to be as sensitive as the single one. In conclusion, this technique is potentially useful in routine diagnosis of chrysanthemum viruses and viroids.

RNA-Seq derived identification of differential transcription in the chrysanthemum leaf following inoculation with Alternaria tenuissima

BackgroundA major production constraint on the important ornamental species chrysanthemum is black spot which is caused by the necrotrophic fungus Alternaria tenuissima. The molecular basis of host resistance to A. tenuissima has not been studied as yet in any detail. Here, high throughput sequencing was taken to characterize the transcriptomic response of the chrysanthemum leaf to A. tenuissima inoculation.ResultsThe transcriptomic data was acquired using RNA-Seq technology, based on the Illumina HiSeq™ 2000 platform. Four different libraries derived from two sets of leaves harvested from either inoculated or mock-inoculated plants were characterized. Over seven million clean reads were generated from each library, each corresponding to a coverage of >350,000 nt. About 70% of the reads could be mapped to a set of chrysanthemum unigenes. Read frequency was used as a measure of transcript abundance and therefore as an identifier of differential transcription in the four libraries. The differentially transcribed genes identified were involved in photosynthesis, pathogen recognition, reactive oxygen species generation, cell wall modification and phytohormone signalling; in addition, a number of varied transcription factors were identified. A selection of 23 of the genes was transcription-profiled using quantitative RT-PCR to validate the RNA-Seq output.ConclusionsA substantial body of chrysanthemum transcriptomic sequence was generated, which led to a number of insights into the molecular basis of the host response to A. tenuissima infection. Although most of the differentially transcribed genes were up-regulated by the presence of the pathogen, those involved in photosynthesis were down-regulated.