Summary Flower color, which is determined by various chemical pigments, is a vital trait for ornamental plants, in which anthocyanin is a major component. However, the epigenetic regulation of anthocyanin biosynthesis remains poorly understood. During chrysanthemum cultivation, we found a heterochromatic chrysanthemum accession (YP) whose progeny generated by asexual reproduction contained both yellow‐flowered (YP‐Y) and pink‐flowered (YP‐P) plants. In this study, we aimed to elucidate the epigenetic mechanisms of different flower colors in the YP plant progeny. Metabolome and transcriptome analyses revealed that the difference in flower color between YP‐Y and YP‐P was caused by expression variation of the anthocyanin biosynthesis gene CmMYB6. Bisulfite sequencing revealed that methylation at the CmMYB6 promoter, especially in the CHH context, was higher in YP‐Y than YP‐P. After demethylation of the CmMYB6 promoter using the dCas9‐TET1cd system, the flower color returned from yellow to pink. Furthermore, the methylation status of the CmMYB6 promoter was higher in YP‐Y over three consecutive generations, indicating that this methylation status was heritable mitotically. Finally, investigation of other chrysanthemum cultivars showed that the methylation of CmMYB6 decreased gradually with the increase in anthocyanin content. These results lay an epigenetic foundation for the improvement of flower color in horticultural plants.

In the aerial plant organs, cuticular wax forms a hydrophobic layer that can protect cells from dehydration, repel pathogen attacks, and prevent organ fusion during development. The MIXTA gene encodes an MYB-like transcription factor, which is associated with epicuticular wax biosynthesis to increase the wax load on the surface of leaves. In this study, the AmMIXTA-homologous gene EgMIXTA1 was functionally characterized in the Eustoma grandiflorum. EgMIXTA1 was ubiquitously, but highly, expressed in leaves and buds. We identified the Eustoma MIXTA homolog and developed the plants for overexpression. EgMIXTA1-overexpressing plants had more wax crystal deposition on the leaf surface compared to wild-type and considerably more overall cuticular wax. In the leaves of the overexpression line, the cuticular transpiration occurred more slowly than in those of non-transgenic plants. Analysis of gene expression indicated that several genes, such as EgCER3, EgCER6, EgCER10, EgKCS1, EgKCR1, and EgCYP77A6, which are known to be involved in wax biosynthesis, were induced by EgMIXTA1-overexpression lines. Expression of another gene, WAX INDUCER1/SHINE1, encoding a transcription factor that stimulates the production of cutin, was also significantly higher in the overexpressors than in wild-type. However, the expression of a lipid-related gene, EgABCG12, did not change relative to the wild-type. These results suggest that EgMIXTA1 is involved in the biosynthesis of cuticular waxes.

Eustoma grandiflorum, commonly known as prairie gentian or Texas bluebells, is among the most popular agriculturally propagated species of cut flowers. Due to its widespread appeal, there is increasing interest in understanding the molecular genetic factors underlying floral development and resistance to abiotic stresses. We analyzed 18 potential reference genes in different organs, at different floral developmental stages and under drought- and salt-stress treatments, for use in RT-qPCR analysis. A total of four analytical tool packages, including geNorm, NormFinder, BestKeeper, and RefFinder were employed to determine the most appropriate reference genes under each treatment condition. The results demonstrate that different reference genes should be used for normalization under different experimental treatments. EgPP and EgPP2A2 were the most stable internal control genes across different organ types, EgPP and Eg18S were the most stable under salt-stress, EgPP and EgACT1 were the most stable across different floral development stages, and EgEF1A and EgTUA were the most stable reference genes under drought-stress. Additional gene expression analyses of EgMIXTA1, EgTOE1, and EgP5CS1 further confirmed the applicability of these reference genes. The results represent a significant contribution to future studies of reference gene selection for the normalization of gene expression in Eustoma grandiflorum.