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Article|02 Dec 2022|OPEN
Protein subcellular localization and functional studies in horticultural research: problems, solutions, and new approaches 
Ye Guo1,2 ,† , Zhiru Bao1,2 ,† and Yaling Deng1,2 , Yuhui Li1,2 , , Pengwei Wang,1,2
1Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, Hubei Province 430070, China
2Hubei Hongshan Laboratory, Wuhan 430070, China
*Corresponding author. E-mail:
Both authors contributed equally to the study.

Horticulture Research 10,
Article number: uhac271 (2023)
Views: 221

Received: 06 Sep 2022
Accepted: 01 Dec 2022
Published online: 02 Dec 2022


To ensure different biochemical reactions take place simultaneously, eukaryotic cells have evolved several membrane-bounded organelles, which coordinate their functions through proteins involved in signal transduction, vesicle trafficking, and membrane interactions [1]. Therefore, determining the subcellular localization of a protein is an essential step toward understanding its functions and activities. To do this, people normally co-express genes of interest with a fluorescent organelle marker using the N. Benthamiana (Nicotiana benthamiana) transient expression system and image it with confocal microscopy[2]. This method is commonly used in horticulture research, where generating stable transgenic plants is still challenging for most species. Although there are a few common standards for this operation, such as signals should not be over-saturated, controls should be performed to eliminate the possibility of signal cross-talk between multiple channels (bleed-through), and the microscope settings (e.g. laser output, pinhole) should be kept identical when comparing two independent experiments, things may still go wrong in some circumstances. Here, we have highlighted a few examples of inappropriate image acquisition that happened in various previous studies and provide our reasonings and solutions to these issues.