Jiafu Jiang

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.

Heterologous Expression of a Nelumbo nucifera Phytochelatin Synthase Gene Enhances Cadmium Tolerance in Arabidopsis thaliana

Phytochelatin synthase (PCS) is a key enzyme involved in the synthesis of phytochelatins, which are thought to play important roles in heavy metal detoxification. The sacred lotus (Nelumbo nucifera), one of the most popular ornamental species, has been shown to be a potential phytoremediator of heavy metal polluted water. However, the phytochelatin synthase gene in N. nucifera has not been identified yet. Here, we report the isolation and function characterization of a N. nucifera homologue of phytochelatin synthase. The sequence obtained shares a high degree of similarity with PCSs from other plant species and was named as Nelumbo nucifera phytochelatin synthase1 (NnPCS1). By using quantitative RT-PCR, we found that the expression of NnPCS1 in leaves of N. nucifera was dramatically increased in response to Cadmium (Cd) treatment. We further showed that, when exposed to Cd stress, Arabidopsis transgenic plants heterologous expressing NnPCS1 accumulated more Cd when compared with wild type. These results suggest that NnPCS1 involved in the response of N. nucifera to Cd stress and may represent a useful target gene for the phytoremediation of Cd-polluted water.

A transcriptomic analysis of Chrysanthemum nankingense provides insights into the basis of low temperature tolerance

BackgroundA major constraint affecting the quality and productivity of chrysanthemum is the unusual period of low temperature occurring during early spring, late autumn, and winter. Yet, there has been no systematic investigation on the genes underlying the response to low temperature in chrysanthemum. Herein, we used RNA-Seq platform to characterize the transcriptomic response to low temperature by comparing different transcriptome of Chrysanthemum nankingense plants and subjecting them to a period of sub-zero temperature, with or without a prior low temperature acclimation.ResultsSix separate RNA-Seq libraries were generated from the RNA samples of leaves and stems from six different temperature treatments, including one cold acclimation (CA), two freezing treatments without prior CA, two freezing treatments with prior CA and the control. At least seven million clean reads were obtained from each library. Over 77% of the reads could be mapped to sets of C. nankingense unigenes established previously. The differentially transcribed genes (DTGs) were identified as low temperature sensing and signalling genes, transcription factors, functional proteins associated with the abiotic response, and low temperature-responsive genes involved in post-transcriptional regulation. The differential transcription of 15 DTGs was validated using quantitative RT-PCR.ConclusionsThe large number of DTGs identified in this study, confirmed the complexity of the regulatory machinery involved in the processes of low temperature acclimation and low temperature/freezing tolerance.

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.

The constitutive expression of Chrysanthemum dichrum ICE1 in Chrysanthemum grandiflorum improves the level of low temperature, salinity and drought tolerance

AbstractThe quality and productivity of chrysanthemum are severely compromised by various abiotic stresses. Here, we describe the isolation of CdICE1 from Chrysanthemum dichrum using RACE PCR, which shared identical nucleotide of ICE1 ORF from Chrysanthemum grandiflorum variety ‘Jinba’. CdICE1 contains a conserved bHLH domain, a nuclear localization domain, a S-rich motif and a ACT domain. The constitutive expression of CdICE1 in C. grandiflorum improved the tolerance of C. grandiflorum to low temperature/freezing, drought and salinity. When the transgene was inserted in the antisense direction, the expression of the endogenous ICE1 gene was down-regulated, and the level of the plant’s sensitivity to abiotic stress increased. The level of expression of CgDREBa and CgDREBb, activities of superoxide dismutase and peroxidase and the proline content were enhanced in the sense transgenic lines, and lowered in the antisense ones under stresses. In conclusion, CdICE1 represents a promising candidate for a biotechnological approach to improve the level of crop abiotic stress tolerance. Key message Overexpression of CdICE1 in C. grandiflorum confers the stress tolerance via its regulation of CgDREB involved in the oxidative and osmotic homeostasis pathways.

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.

Rapid Genetic and Epigenetic Alterations under Intergeneric Genomic Shock in Newly Synthesized Chrysanthemum morifolium × Leucanthemum paludosum Hybrids (Asteraceae)

The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5–50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses.

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.