Two-photon fluorescence and Second-harmonic generation imaging for nondestructive circadian profiling of starch content in fresh intact Arabidopsis leaf

Juo-Nang Liao^1,2, Wei-Liang Chen^1*, Chao-Yuan Lo¹, Man-Hong Lai¹, Huang-Lung Tsai², Yu-Ming Chang¹

¹Center for Condensed Matter Sciences, National Taiwan University, Taipei, Taiwan
²Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan

* Presenter:Wei-Liang Chen, email:wechen@ntu.edu.tw

When plant chloroplasts conduct photosynthesis during the day, a fraction of photosynthetic products is stored in chloroplasts by forming starch granules to continue the provision of carbon energy during the night. Currently, profiling the starch temporal pattern requires either sacrificing the leaves or generating transgenic plants at the risk of changing the metabolisms. In this work, we demonstrated a nondestructive method using two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging to quantify starch granules within chloroplasts of fresh intact leaves across a day-night cycle. We used two Arabidopsis lines having normal and excess starch contents: wild-type (Columbia-0) and starch excess 1 (sex1). The starch granules were visualized by SHG imaging, while the chloroplasts in mesophyll cells were visualized by TPF imaging. Furthermore, the SHG and TPF signals were confirmed using spectral mapping. Our results not only showed starch circadian patterns consistent with those profiled by enzymatic assays, but also provided micron scale spatial resolution of starch distribution within leaves. We demonstrated that TPF-SHG imaging is a potential tool for revealing real-time variations of starch circadian rhythm in leaf cells, without the need for destructive sample preparation.

Keywords: Arabidopsis, starch, circadian rhythm, second harmonic generation, two-photon fluorescence