Improving Sedum plumbizincicola genetic transformation with the SpGRF4-SpGIF1 gene and the self-excision CRE/LoxP system.

MAIN CONCLUSION: S. plumbizincicola genetic transformation was optimized using a self-excision molecular-assisted transformation system by integrating the SpGRF4/SpGIF1 gene with XVE and Cre/loxP. Sedum plumbizincicola, despite being an excellent hyperaccumulator of cadmium and zinc with significant potential for soil pollution phytoremediation on farmland, has nonetheless trailed behind other major model plants in genetic transformation technology. In this study, different explants and SpGRF4-SpGIF1 genes were used to optimize the genetic transformation of S. plumbizincicola. We found that petiole and stem segments had higher genetic transformation efficiency than cluster buds. Overexpression of SpGRF4-SpGIF1 could significantly improve the genetic transformation efficiency and shorten the period of obtaining regenerated buds. However, molecular assistance with overexpression of SpGRF4-SpGIF1 leads to abnormal morphology, resulting in plant tissue enlargement and abnormal growth. Therefore, we combined SpGRF4-SpGIF1 with XVE and Cre/loxP to obtain DNA autocleavage transgenic plants induced by estradiol, thereby ensuring normal growth in transgenic plants. This study optimized the S. plumbizincicola genetic transformation system, improved the efficiency of genetic transformation, and established a self-excision molecular-assisted transformation system. This work also established the basis for studying S. plumbizincicola gene function, and for S. plumbizincicola breeding and germplasm innovation.

Exploring Transcriptional Regulation of Hyperaccumulation in Sedum plumbizincicola through Integrated Transcriptome Analysis and CRISPR/Cas9 Technology.

The cadmium hyperaccumulator Sedum plumbizincicola has remarkable abilities for cadmium (Cd) transport, accumulation and detoxification, but the transcriptional regulation mechanisms responsible for its Cd hyperaccumulation remain unknown. To address this knowledge gap, we conducted a comparative transcriptome study between S. plumbizincicola and the non-hyperaccumulating ecotype (NHE) of Sedum alfredii with or without Cd treatment. Our results revealed many differentially expressed genes involved in heavy metal transport and detoxification that were abundantly expressed in S. plumbizincicola. Additionally, we identified a large number of differentially expressed transcription factor genes, highlighting the complexity of transcriptional regulatory networks. We further screened four transcription factor genes that were highly expressed in the roots of S. plumbizincicola as candidate genes for creating CRISPR/Cas9 knockout mutations. Among these, the SpARR11 and SpMYB84 mutant lines exhibited decreased Cd accumulation in their aboveground parts, suggesting that these two transcription factors may play a role in the regulation of the Cd hyperaccumulation in S. plumbizincicola. Although further research will be required to determine the precise targeted genes of these transcription factors, combined transcriptome analysis and CRISPR/Cas9 technology provides unprecedented opportunities for identifying transcription factors related to Cd hyperaccumulation and contributes to the understanding of the transcriptional regulation mechanism of hyperaccumulation in S. plumbizincicola.