This review discusses the advancements and applications of single-cell RNA sequencing (scRNA-seq) in plant systems and synthetic biology. ScRNA-seq has emerged as a powerful tool for understanding the molecular underpinnings of complex plant systems by revealing high-resolution gene expression patterns at the cellular level and investigating cell-type heterogeneity. The review highlights the advantages of scRNA-seq over traditional bulk RNA sequencing, including the ability to identify and characterize distinct cell types, capture temporal changes in gene expression, and provide unbiased gene expression profiles. The technical advancements in scRNA-seq for plant studies are discussed, including sample preparation, library construction, and data analysis. Sample preparation methods such as protoplast isolation and single-nucleus RNA sequencing (snRNA-seq) are compared, with protoplasts offering a more comprehensive view of the transcriptome but facing challenges due to cell wall digestion. snRNA-seq, on the other hand, can isolate complex cell types but may miss information from cytoplasmic RNAs. The review also covers the computational aspects of scRNA-seq data analysis, including pre-processing, alignment, quality control, normalization, imputation, dimensionality reduction, integration, clustering, differential gene expression, and functional analysis. Various tools and methods are highlighted for each step of the analysis process. The applications of scRNA-seq in plant systems biology are explored, including understanding plant development, responses to abiotic and biotic stresses, epigenetic regulation, and cell fate determination. ScRNA-seq has been used to reveal detailed maps of cell types, investigate the role of transcription factors in development, and understand how specific cell types respond to environmental cues. In synthetic biology, scRNA-seq is used to discover and design cell-type-specific promoters, identify regulators for cell-type-specific gene expression, engineer metabolic pathways, and design symbiotic relationships between plants and microbes. The review emphasizes the potential of scRNA-seq in advancing plant biodesign and improving crop yield and quality. Challenges and potential solutions for the application of scRNA-seq in plants are discussed, including the need for optimized sample preparation methods, the development of standardized protocols, and the integration of scRNA-seq with other omics technologies. The review concludes by highlighting the future opportunities for scRNA-seq in plant systems biology and synthetic biology, emphasizing the importance of standardization, multi-omics integration, and expanding the application to a broader range of plant species.This review discusses the advancements and applications of single-cell RNA sequencing (scRNA-seq) in plant systems and synthetic biology. ScRNA-seq has emerged as a powerful tool for understanding the molecular underpinnings of complex plant systems by revealing high-resolution gene expression patterns at the cellular level and investigating cell-type heterogeneity. The review highlights the advantages of scRNA-seq over traditional bulk RNA sequencing, including the ability to identify and characterize distinct cell types, capture temporal changes in gene expression, and provide unbiased gene expression profiles. The technical advancements in scRNA-seq for plant studies are discussed, including sample preparation, library construction, and data analysis. Sample preparation methods such as protoplast isolation and single-nucleus RNA sequencing (snRNA-seq) are compared, with protoplasts offering a more comprehensive view of the transcriptome but facing challenges due to cell wall digestion. snRNA-seq, on the other hand, can isolate complex cell types but may miss information from cytoplasmic RNAs. The review also covers the computational aspects of scRNA-seq data analysis, including pre-processing, alignment, quality control, normalization, imputation, dimensionality reduction, integration, clustering, differential gene expression, and functional analysis. Various tools and methods are highlighted for each step of the analysis process. The applications of scRNA-seq in plant systems biology are explored, including understanding plant development, responses to abiotic and biotic stresses, epigenetic regulation, and cell fate determination. ScRNA-seq has been used to reveal detailed maps of cell types, investigate the role of transcription factors in development, and understand how specific cell types respond to environmental cues. In synthetic biology, scRNA-seq is used to discover and design cell-type-specific promoters, identify regulators for cell-type-specific gene expression, engineer metabolic pathways, and design symbiotic relationships between plants and microbes. The review emphasizes the potential of scRNA-seq in advancing plant biodesign and improving crop yield and quality. Challenges and potential solutions for the application of scRNA-seq in plants are discussed, including the need for optimized sample preparation methods, the development of standardized protocols, and the integration of scRNA-seq with other omics technologies. The review concludes by highlighting the future opportunities for scRNA-seq in plant systems biology and synthetic biology, emphasizing the importance of standardization, multi-omics integration, and expanding the application to a broader range of plant species.