Optimization of CRISPR-Cas9 system in Eustoma grandiflorum.

The optimization of the CRISPR-Cas9 system for enhancing editing efficiency holds significant value in scientific research. In this study, we optimized single guide RNA and Cas9 promoters of the CRISPR-Cas9 vector and established an efficient protoplast isolation and transient transformation system in Eustoma grandiflorum, and we successfully applied the modified CRISPR-Cas9 system to detect editing efficiency of the EgPDS gene. The activity of the EgU6-2 promoter in E. grandiflorum protoplasts was approximately three times higher than that of the GmU6 promoter. This promoter, along with the EgUBQ10 promoter, was applied in the CRISPR-Cas9 cassette, the modified CRISPR-Cas9 vectors that pEgU6-2::sgRNA-2/pEgUBQ10::Cas9-2 editing efficiency was 37.7%, which was 30.3% higher than that of the control, and the types of mutation are base substitutions, small fragment deletions and insertions. Finally we obtained an efficient gene editing vector for E. grandiflorum. This project provides an important technical platform for the study of gene function in E. grandiflorum.

Investigating Flood Risks of Rainfall and Storm Tides Affected by the Parameter Estimation Coupling Bivariate Statistics and Hydrodynamic Models in the Coastal City.

The public health risk caused by urban floods is a global concern. Flood risks are amplified by the interaction of rainfall and storm tides in coastal cities. In this study, we investigate the flood risks of rainfall and storm tides coupling statistical and hydrodynamic models and evaluate the influence of different parameter estimation methods and bivariate return periods (RPs) on flood risks in the coastal city. The statistical model is used to obtain the bivariate design of rainfall and storm tides with the integration of copula function, most-likely weight function and Monte Carlo simulation method. The bivariate designs are adopted as the input boundaries for the hydrodynamic model established by Personal Computer Storm Water Management Model (PCSWMM), and the flood risk is evaluated by the hydrodynamic model. Subsequently, the influence of different parameter estimation approaches (that is, parametric and non-parametric) and bivariate RPs (that is, co-occurrence RP, joint RP, and Kendall RP) on bivariate designs and flood risks are investigated. With Haikou coastal city in China as the case study, the results show that: (1) Gumbel copula is the best function to describe the correlation structure between rainfall and storm tides for the parametric and non-parametric approaches, and the non-parametric approach is a better fit for the observed data; (2) when the Kendall RP is large (more than 100 years), the flood risk is underestimated with an average of 17% by the non-parametric estimation, and the parametric estimation approach is recommended as it is considered the most unfavorable scenario; (3) the types of bivariate RP have the important impact on the flood risk. When there is no specific application need, the Kendall RP can be adopted as the bivariate design standard of flooding facilities since it can describe the dangerous areas more accurately for multivariate scenario. The results can provide references for reasonable flood risk assessment and flooding facility design in coastal cities.

Mitotically heritable epigenetic modifications of CmMYB6 control anthocyanin biosynthesis in chrysanthemum.

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.

EgMIXTA1, a MYB-Type Transcription Factor, Promotes Cuticular Wax Formation in Eustoma grandiflorum Leaves.

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.