Protoplast isolation and transient gene expression in Suaeda aralocaspica, a halophyte with single-cell C4 anatomy

Suaeda aralocaspica, an annual halophyte belonging to the Amaranthaceae family, is widely distributed in saline-alkali and arid habitats, exhibiting remarkable stress tolerance. This species has evolved a unique single-cell C4 (SCC4) photosynthetic pathway, where both C4 and C3 cycles are compartmentalized within a single cell, and demonstrates superior photosynthetic efficiency. This distinctive physiological adaptation makes S. aralocaspica an intriguing model for investigating the photosynthetic mechanism and its environmental adaptation in plants. However, efficient and rapid transformation systems are still lacking for this species. This gap has significantly hindered the dissection of the molecular regulatory networks underlying the SCC4 photosynthetic structure formation and the stress resistance mechanisms. Through systematic optimization of enzymatic digestion parameters and leaf pretreatment methods, we developed an efficient protoplast isolation and transient transformation system for S. aralocaspica. The highest protoplast yield (1.1 × 105/g FW) and viability (64%) were obtained from young leaves under optimized condition: enzyme solution containing 0.75% cellulase R-10 and 0.25% macerozyme R-10, 1.3 M glucose as osmotic regulator, with transverse/longitudinal leaf sectioning combined with epidermal peeling. Protoplast quality was verified by organelle fluorescence staining using different organelle markers. The highest transfection efficiency (75%) was achieved using protoplasts isolated under optimal conditions and transfected with 2400 ng/µL plasmid (5 µg total). Subcellular localization analysis revealed localization of key photosynthetic enzyme SaPEPC1 in the cytoplasm and chloroplasts, and nuclear localization of transcription factor SabHLH169. In summary, we established a simple and efficient system for isolating and transiently transforming S. aralocaspica chlorenchyma protoplasts. This system is shown to express exogenous proteins with the proper subcellular localization, and it provides a platform for further research on S. aralocaspica, including potential applications in somatic cell fusion, genome editing, and signal transduction and transcriptional regulation studies. The limitations of the current system, such as relatively low protoplast yield and challenges with high-throughput or complex molecular biology experiments, are also discussed. This work can serve as a starting point for developing a similar system in other SCC4 plants or Suaeda halophytes.