Xiao Xu

Cold tolerance in plants is a complex trait regulated by a network of transcription factors (TFs) and their downstream genes. While C‐repeat binding factors (CBFs) are well‐known for their role in cold tolerance, other regulatory networks remain largely unexplored. This study utilizes a combined approach of Assay for Transposase‐Accessible Chromatin with Sequencing (ATAC‐seq) and RNA‐seq to identify cold‐responsive TFs in Cynodon dactylon (Bermudagrass), a species widely grown in southern China but limited by cold stress. Early‐stage cold stress was found to induce dynamic changes in 331 differentially accessible regions (DARs), with over 45% located in gene promoter regions. Key TFs, including CAMTA1, CAMTA2, WRKY43, WRKY48, WRKY21, and DREB1G, were associated with gained DARs, highlighting their roles in cold response regulation. In contrast, several Heat Shock Factor (HSF) family members, such as HSFA6B, HSFB2A, HSFC1, and HSFB2B, were linked to lost DARs, underscoring their involvement in cold stress regulation. The correlation between chromatin accessibility and gene expression emphasizes the critical role of TFs in plant cold stress adaptation. This study highlights the potential of ATAC‐seq and RNA‐seq as powerful tools for uncovering novel TFs essential for cold tolerance in plants, offering insights for future research and breeding efforts.

Bermudagrass [ Cynodon dactylon (L.) Pers.] is widely used for soil remediation, livestock forage, and as turfgrass for sports fields, parks, and gardens due to its resilience and adaptability. However, low temperatures are critical factors limiting its geographical distribution and ornamental season, even preventing its safe overwintering. PHYTOCHROME‐INTERACTING FACTOR 4 (PIF4) acts as a hub transcription factor, not only regulating various light responses but also integrating multiple external stimuli to improve plant productivity and architectural adaptation under adverse stress conditions, which makes it potential as a target gene. In this study, we cloned and characterized the CdPIF4 genes in bermudagrass. Expression analysis revealed that it is predominantly expressed in leaves and is regulated by photoperiod and cold stress. Using Agrobacterium ‐mediated genetic modification, we successfully generated CdPIF4a ‐overexpressing bermudagrass lines. Under cold stress at 4°C, these transgenic plants demonstrated enhanced cold tolerance, as indicated by higher relative water content, reduced membrane damage, and lower levels of lipid peroxidation levels. Photosynthetic analysis revealed that CdPIF4a ‐overexpressing plants exhibited higher light energy capture and transfer efficiency at this low temperature, with less energy loss. Additionally, they showed higher antioxidant enzyme activity and lower levels of reactive oxygen species levels. The responsive regulation of cold stress‐related genes further validated the role of the CdPIF4a gene in enhancing cold tolerance. This study elucidates that CdPIF4 enhances cold tolerance in bermudagrass through physiological and molecular mechanisms, offering new insights and valuable genetic resources for advancing cold resistance research in bermudagrass and other grass species.

Bermudagrass is one of the most extensively used warm‐season grasses. It is widely used in landscaping, stadium construction and soil remediation due to its excellent regeneration, trampling and stress tolerances. However, studies on its regulatory mechanism and variety improvement by genetic engineering are still at a standstill, owing to its genetic variability and intrinsic limits linked with some resistance to Agrobacterium infection. In this study, we established a higher efficient Agrobacterium‐mediated transformation via screening for vital embryogenic callus and improving infection efficiency. The superior callus was light yellow, hard granular and compact, determined with a differentiation rate of more than 95%. The optimized infestation courses by gentle shaking, vacuuming and sonicating were used. The infested calluses were co‐cultured for 3 days, followed by desiccation treatments for 1 day to get higher infection efficiency. Then the CdHEMA1 gene, essential for chlorophyll biosynthesis, was cloned and transferred into bermudagrass to validate the aforementioned optimization procedures integrally. Molecular‐level analyses indicated that the CdHEMA1 gene had successfully integrated and was greatly increased in transgenic seedlings. Results of the photosynthetic capacity assessment showed that CdHEMA1 overexpression may considerably enhance the contents of photosynthetic pigments, OJIP curve and reaction center density (RC/CSo) to absorb (ABS/CSo, ABS/CSM) and capture (TRo/CSo) more light energy, hence improve the performance indices PI_ABS and PI_CS compared to the wild type. The successful completion of this project would provide a solid platform for further gene function study and molecular breeding of bermudagrass.

Aim Potassium (K) is a key determinant for plant development and productivity. However, more than 90% of K in the soil exists in an insoluble form. K-solubilizing microbes play an important role in the transformation of insoluble K. Thus, the objective of this study was to evaluate K-dissolving ability of Aspergillus aculeatus (F) and growth-promoting properties in perennial ryegrass. Methods and Results Perennial ryegrass inoculated with A. aculeatus exhibited enhanced soluble K accompanied with higher growth rate and turf quality, compared with the non-inoculated regimen. In addition, A. aculeatus also played a primary role in increasing chlorophyll content and photosynthetic capacity of the plant exposed to LK + F (K-feldspar plus A. aculeatus) treatment, compared with CK (control, no K-feldspar and A. aculeatus), F (only A. aculeatus) and LK (only K-feldspar) groups. Furthermore, the antioxidase activities (CAT and POD) were significantly increased while the oxidative damage (EL and MDA) was dramatically decreased in LK + F group compared to the LK (K-feldspar) group. Finally, in perennial ryegrass leaves, the genes expression levels of HAK8, HAK12 and HKT18 were obviously elevated in LK + F group, compared to CK, F and LK groups. Conclusion We concluded that A. aculeatus could solubilize K from bound form and be considered as K-solubilizing biofertilizer through supplementing K in soil. Significance and Impact of the Study A. aculeatus has potential to be used as biofertilizer in sustainable agriculture.