Chunsun Gu

Reference Gene Selection for Quantitative Real-Time PCR in Chrysanthemum Subjected to Biotic and Abiotic Stress

Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1α (elongation factor 1α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.

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.

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.

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.

Candidate reference genes for gene expression studies in water lily

The selection of an appropriate reference gene(s) is a prerequisite for the proper interpretation of quantitative Real-Time polymerase chain reaction data. We report the evaluation of eight candidate reference genes across various tissues and treatments in the water lily by the two software packages geNorm and NormFinder. Across all samples, clathrin adaptor complexes medium subunit (AP47) and actin 11 (ACT11) emerged as the most suitable reference genes. Across different tissues, ACT11 and elongation factor 1-alpha (EF1alpha) exhibited a stable expression pattern. ACT11 and AP47 also stably expressed in roots subjected to various treatments, but in the leaves of the same plants the most stably expressed genes were ubiquitin-conjugating enzyme 16 (UBC16) and ACT11.

Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1.

The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter.

Chrysanthemum CmNAR2 interacts with CmNRT2 in the control of nitrate uptake

Nitrate transporters are an important component of plant growth and development. Chrysanthemum morifolium is an important ornamental species, for which a sufficient supply of nitrogenous fertilizer is required to maintain economic yields. In this study, the full-length cDNA of the nitrate transporter genes CmNRT2 and CmNAR2 were isolated. CmNRT2 transcript accumulation was inducible by both nitrate and ammonium, but the latter ion down-regulated the transcript accumulation of CmNAR2. CmNRT2 might be a plasma membrane localized protein, while CmNAR2 was distributed throughout the cell. CmNAR2 was shown to interact with CmNRT2 by in vitro and in vivo assays. Arabidopsis thaliana plants heterologously expressing CmNRT2 showed an increased rate of nitrate influx, while this trait was unaltered in plants expressing CmNAR2. Double transformants (CmNRT2 plus CmNAR2) exhibited an enhanced rate of nitrate influx into the root. Our data indicated that the interaction of CmNAR2 with CmNRT2 contributed to the uptake of nitrate.

Identification and expression of an APETALA2-like gene from Nelumbo nucifera.

Arabidopsis transcription factor APETALA2 (AP2) controls multiple aspects of plant growth and development, including seed development, stem cell maintenance, and specification of floral organ identity. Based on sequence similar of Arabidopsis AP2 and its homologues genes from other plant species, degenerate RT-PCR and rapid amplification of cDNA ends assay were used to clone AP2 genes from lotus (Nelumbo nucifera). A 2,048-bp cDNA fragment was obtained, which contains a 1,536-bp open reading frame encoding a protein of 511 amino acids. The protein contains two AP2 domains that are conserved in AP2 proteins from other plant species, thus was named as N. nucifera APETALA2 (NnAP2). Quantitative RT-PCR revealed that NnAP2 gene was expressed in flowers, roots, leaves, and stems of N. nucifera, with flowers which have the highest transcript levels. Further analysis showed that in all five lotus cultivars examined, including “Zhongguogudailian,” “Yaoniangyujiao,” “Jinxia,” “Hongtailian,” and “Yiliangqianban,” petals always have the highest expression levels when compared with the other four flower organs, though the number of petals in these cultivars ranged from simple to thousands. However, NnAP2 expression level in four nonsimple petal flower cultivars was higher than that in the simple petal flower cultivar Zhongguogudailian, indicating that NnAP2 may play a role in specification of petal identity during the evolutionary process of the ancient species N. nucifera.