Methylation-sensitive amplification polymorphism analysis reveals the DNA methylation dynamics during capitulum development in Chrysanthemum lavandulifolium

Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino is a diploid plant belonging to the Asteraceae family, with typical capitula composed of female ray florets and bisexual disc florets. The differentiation and development of these two types of florets have long been important research focuses; however, the potential epigenetic mechanisms governing these processes have not been elucidated. In the present study, methylation-sensitive amplification polymorphism method was used to trace the dynamic changes of DNA methylation during capitulum development in C. lavandulifolium. DNA methylation patterns and levels were detected in the whole capitula during seven developmental stages, and the obtained results revealed that DNA demethylation was dominant during this process. In addition, DNA methylation patterns and levels showed significant differences between ray and disc florets. Moreover, the expression patterns of candidate genes potentially involved in the development processes of two types of florets were analyzed by real-time quantitative reverse transcription polymerase chain reaction, and correlation analysis indicated that the expression levels of ClPI, ClAG2, ClSEP1, ClCYC2c, ClCYC2d, and ClCYC2e were highly correlated with DNA methylation levels. These results indicate that DNA methylation may be involved in the differentiation and development of ray and disc florets. This study provides epigenetic insights into the capitulum development in C. lavandulifolium.

Transcriptome Analysis of Chrysanthemum lavandulifolium Response to Salt Stress and Overexpression a K+ Transport ClAKT Gene-enhanced Salt Tolerance in Transgenic Arabidopsis

Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum (C. ×morifolium). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 mm NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium. Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT, an important K+ transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K+ and efflux Na+ under salt stress and to absorb K+ under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.

Cloning and function analysis of ClNAC9 from Chrysanthemum lavandulifolium

NAC transcription factors have been found to play an important role in several plant development programs and stress responses. In this study, a NAC gene, ClNAC9 (Chrysanthemum lavandulifolium NAC gene), was isolated from a cDNA library constructed according to the known expressed sequence tag sequence. The cDNA full-length sequence of ClNAC9 is comprised of 881 bp, encoding a putative protein of 217 amino acids. ClNAC9 has a conserved NAC domain in the N-terminus — the NAM domain. ClNAC9 is highly similar to other NACs, especially SENU5 subgroup members. Transgenic Arabidopsis overexpressing ClNAC9 controlled by the CaMV-35S promoter was generated and subjected to saline, alkaline, and drought stresses for morphological and physiological assays. Morphological analyses showed that transgenic plants had enhanced tolerance to saline, alkaline, and drought stresses, as indicated by improved physiological traits, including higher superoxide dismutase and peroxidase activities, and reduced malondialdehyde accumulation. Moreover, overexpression of ClNAC9 enhanced up-regulation of RD29A, RD26, MYB2, and MYB96 expression, and reduced HAB1 expression under saline, alkaline, and drought treatments. Taken together, our results demonstrate that ClNAC9 is likely related to saline, alkaline, and drought resistances and overexpression of ClNAC9 increases the saline, alkaline, and drought resistance of transgenic Arabidopsis.