A Simplified Wheat Protoplast Transformation System and Guideline for Avoiding Protein Localization Artifacts

The transient protoplast transformation system is a vital tool for studying protein subcellular localization and phase separation in wheat. However, current protocols remain underdeveloped, and the lack of systematic vector design analysis frequently leads to localization artifacts. Here, we established a simplified wheat mesophyll protoplast transformation method featuring a shortened cycle, streamlined handling, and no variety limitations, enabling stable acquisition of high-quality confocal imaging data. Using this method, we systematically examined the effects of the fluorescent tag position (N- vs. C-terminal) and promoter type (native, single CaMV35S and double CaMV35S) on protein localization and phase separation. Tag position proved decisive: improper fusion can affect the recognition of localization signals, leading to inaccurate patterns. Regarding promoters, the native promoter represents the optimal choice for physiological accuracy. Constitutive strong promoters such as CaMV35S boost gene expression and thereby enhance fluorescent signals for easier imaging, but overexpression may compromise localization fidelity and exacerbate molecular crowding effects, resulting in false-positive phase-separated aggregates. Conversely, insufficient expression will lead to false-negative outcomes. This standardized transformation system and the defined vector design principles offer a robust framework for minimizing artifacts in wheat protein localization and phase separation research.