Illuminating the cells: transient transformation of citrus to study gene functions and organelle activities related to fruit quality

Xiuxin Deng,Zhen Tian,Qiunan Meng,Pengwei Wang,Yajie Guan,Ping Liu,Wenwu Guo,Yunjiang Cheng,Chuanwu Chen,Jinli Gong,Xiaolu Qu

doi:10.1038/s41438-021-00611-1

Abstract

Although multiple microscopic techniques have been applied to horticultural research, few studies of individual organelles in living fruit cells have been reported to date. In this paper, we established an efficient system for the transient transformation of citrus fruits using an Agrobacterium-mediated method. Kumquat (Fortunella crassifolia Swingle) was used; it exhibits higher transformation efficiency than all citrus fruits that have been tested and a prolonged-expression window. Fruits were transformed with fluorescent reporters, and confocal microscopy and live-cell imaging were used to study their localization and dynamics. Moreover, various pH sensors targeting different subcellular compartments were expressed, and the local pH environments in cells from different plant tissues were compared. The results indicated that vacuoles are most likely the main organelles that contribute to the low pH of citrus fruits. In summary, our method is effective for studying various membrane trafficking events, protein localization, and cell physiology in fruit and can provide new insight into fruit biology research.

Highlights

Citrus is one of the most important and highest-yielding fruits in the world, and identifying genes associated with desirable traits is important for the sustainable development of citrus production
Conclusions are inevitably obtained from observations of fixed plant tissue, which sometimes does not reflect what truly occurs in planta
Agrobacterium-mediated transformation of citrus fruits The transformation of citrus fruits was carried out in kumquat (Fig. 1a), which is one of the major citrus cultivars in southern China[16]

Summary

Introduction

Citrus is one of the most important and highest-yielding fruits in the world, and identifying genes associated with desirable traits is important for the sustainable development of citrus production. The Agrobacterium-mediated infiltration of tobacco leaves is a commonly used method to test gene function in vivo or study protein subcellular localization[2]. The feasibility of this method was demonstrated by expressing different organelle markers to study protein localization, as well as by expressing pHluorin-derived reporters to measure the pH of specific compartments of citrus cells.

Results

Conclusion