Validating the utility of <scp>APEX2</scp> for protein localization at nanometre resolution in plant cells

Determining protein localization at nanometre resolution with electron microscopy (EM) is crucial for understanding protein functions and cellular processes. The APEX2 tag, an engineered ascorbate peroxidase, was developed to image mammal proteins with EM (Martell et al., 2012). Derived from the plant ascorbate peroxidase APX1, APEX2 catalyses H2O2-dependent polymerization of DAB into dense precipitates, providing high contrast under EM. The APEX2 tag is exceptional for imaging proteins with EM because it is tolerant to EM fixation, free of disulfide bonds, active in all cell compartments and exists as a monomer (Lam et al., 2015). Moreover, since the APEX2 signal does not diffuse across membranes, fusing APEX2 to the N- or C-terminus of an integral membrane protein allows determination of its topology relative to the lipid bilayer. Although the APEX2 method was introduced for protein localization in mammal cells 12 years ago, its adoption in plant cells has been discouraged by the concern about high background staining from abundant endogenous peroxidases (Martell et al., 2017). While recent studies have begun to explore the utility of APEX2 for protein localization in plant cells (Lampugnani et al., 2024), further validation in diverse plant structures remains essential. In this study, we validated the APEX2 application in plant cells by transiently expressing proteins fused to APEX2 in Nicotiana benthamiana leaves (Figure S1, Table S1). Our results demonstrate that endogenous peroxidase activity is not pervasive in the plant cells when using the APEX2 staining method. The APEX2 tag proves invaluable for achieving high-resolution protein localization across diverse cell compartments and for studying membrane topology in plant cells. APEX2 is especially valuable for imaging proteins within the intricate structures of double-membrane organelles, such as mitochondria and plant-specific chloroplasts. To access the utility of APEX2 in mitochondria, we fused two mitochondria proteins, the ATPase β signal peptide and MICU1, to the N-terminus of APEX2 (Figure S1). ATPase β is sorted into the mitochondria matrix, where we observed a strong contrast of the APEX2 signal (Figure 1a), compared to the weak contrast in 35S::GFP-transformed negative controls (Figure 1c). MICU1 complexes with the transmembrane MCU to transport Ca2+ ion. A previous study in Arabidopsis struggled to localize the MICU1 within the narrow intermembrane space (IMS) of mitochondria using confocal microscopy (Wagner et al., 2015). Here we conclusively showed that MICU1 localizes in the IMS (Figure 1b). We localized four chloroplast proteins, including RBCS, PORB, OEP7 and Tic20II, to distinct structures of the chloroplast. RBCS contributes to carbon fixation within the chloroplast matrix (Vitlin Gruber and Feiz 2018), where we observed a densely distributed APEX2 signal (Figure 1d). PORB, an enzyme involved in chlorophyll biosynthesis (Delfosse et al., 2016), exhibited an APEX2 signal at the stroma lamellae between grana thylakoids (Figure 1g), suggesting specific sites of chlorophyll biosynthesis. Given that PORB lacks transmembrane hydrophobic regions, as predicted by TMHMM-2.0, it is likely a peripheral membrane protein associated with the exterior of stroma lamellae. Tic20II and OEP7 are integral membrane proteins located in the inner and outer membranes of the chloroplast, respectively (Machettira et al., 2011). Cells transfected with Tic20II-APEX2 exhibited a dense signal tightly associated with the inner membrane on the matrix-facing side (Figure 1e,f), indicating its C terminus protrudes into the chloroplast matrix (Figure S6). The OEP7-APEX2 signal was observed at the chloroplast envelope, showing a blurred double-membrane structure (Figure 1h,i), which suggests that the outer membrane may exhibit high permeability to DAB products. Although several ascorbate peroxidases (APXs) are predicted to localize in chloroplasts(Figure S2), we did not observe a signal in the negative control (Figure 1j). To validate the utility of APEX2 in studying the endomembrane system, we first tested an ER-resident protein by fusing a signal peptide (SP) before APEX2 and adding a C-terminus HDEL peptide (Figure S1). APEX2 staining was observed in the ER lumen (Figures 1k and S4). We also imaged CNX1, a type I transmembrane protein and ER marker, characterized by an N-terminal lumenal domain and a short cytosolic tail (Liu et al., 2017). In the canonical secretion pathway, CNX1 is co-translationally translocated across the ER membrane, guided by its N-terminal signal peptide. Here, we constructed an APEX2-CNX1 fusion vector, with APEX2 positioned at the N-terminus (Figure S1). The APEX2 signal, observed on the cytosolic side of the ER rather than within the lumen, indicates that while the CNX1 signal peptide, positioned after APEX2, successfully targets the fusion protein to the ER, it does not facilitate translocation of APEX2 into the ER lumen (Figures 1l and S6). The membrane topology revealed by APEX2 allows us to identify the inward folding of the ER membrane, forming a pocket-like structure (Figures 1l and S7). Furthermore, we imaged a Golgi marker, Man1, fused with APEX2 at its C terminus and observed its signal in the Golgi (Figures 1m and S6). In contrast, no DAB staining was observed in the ER and Golgi of the negative control (Figure 1j,c). CBL1 encodes a plasma membrane-anchored protein that has been widely used as a plasma membrane marker (You et al., 2023). The CBL1n-APEX2 construct, created by fusing the 12-amino acid N-terminus of CBL1 to APEX2, exhibits distinct staining at the plasma membrane (Figures 1n and S6). These results demonstrate that APEX2 is a valuable tool for investigating the protein localization and topology within the endomembrane system (Figure S6). The abundance of endogenous peroxidases has been a primary concern for the utility of APEX2 in plant cells. The Arabidopsis thaliana genome encodes a large number of peroxidases across various classes (Fawal et al., 2013). Nine APXs are predicted to localize in different cellular compartments (Table S2), including in the peroxisome, plastid and cytosol. In Nicotiana benthamiana, a total of 13 APX homologues were identified (Figure S2), with nine genes expressed in the leaves (Table S3). Interestingly, despite this concern, we did not observe pervasive endogenous DAB staining background in mesophyll cells, suggesting that most endogenous plant peroxidases do not withstand EM fixation. However, persistent background staining was observed on the exterior of peroxisomes (Figures 1h and S3a), in the intercellular space (Figure S3b) and within vascular tissues and epidermis cells (Figure S3c,d). Notably, the presence of staining on the exterior of peroxisomes raises intriguing questions about the identity of the responsible enzyme. Vessels in vascular tissues likely express specific peroxidases at elevated levels to catalyse lignin deposition during secondary cell wall formation, potentially contributing to the endogenous DAB staining signal. In addition to potential interference from endogenous peroxidase activity, the DAB reaction product may obscure the native ultrastructure of labelled compartments and their immediate surroundings (e.g., thick staining of the plasma membrane in Figure 1n)—a recognized limitation of the APEX2 system. Thus, localization should be verified by other methods, such as cell fractionation and Western blotting. In conclusion, this study addresses long-standing concerns about the utility of APEX2 in plant cells. Given the advantages of APEX2—including its high resolution, simple and robust labeling procedure and its capability to reveal membrane topology—this method proves invaluable for imaging plant proteins and plant-specific organelles. We thank Chuxiong Zhuang and Gang Hao for equipment support. This work was funded by the National Natural Science Foundation of China (31771342) and Guangdong Basic and Applied Basic Research Foundation (2023A1515010817). Y.H. conceptualized this study. J.H., W.H., P.X., M.Z., Z.F., C.L. and X.W. performed and analysed the experimental data. Y.H. and J.H. interpreted the results and wrote the manuscript. All the data are included in this published article and its supporting information. Figure S1 APEX2 fusion constructs for candidate proteins. Figure S2 Phylogenetic analysis of ascorbate peroxidases (APXs). Figure S3 Endogenous background DAB staining in negative control leaves of 35S::GFP-transformed Nicotiana benthamiana. Figure S4 APEX2 signal densitometry visualization. Figure S5 Quantitative analysis of DAB staining intensity comparing specific targets with negative controls. Figure S6 Topology of six membrane proteins. Figure S7 Serial sections of ER-resident pocket-like structures labelled with APEX2-CNX1. Table S1 Candidate proteins for APEX2 fusion and their predicted cellular locations. Table S2 Arabidopsis peroxidase in RedOxiBase. Table S3 RNA-seq Data of Peroxidase in Nicotiana benthamiana Leaves. File S1 APEX2 vector sequences. File S2 Amino acids of proteins in this study. Data S1 Materials and methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.