Subcellular Localization and Vesicular Structures of Anthocyanin Pigmentation by Fluorescence Imaging of Black Rice (<i>Oryza sativa</i> L.) Stigma Protoplast

Anthocyanins belong to the group of flavonoid compounds broadly distributed in plant species responsible for attractive colors. In black rice (<i>Oryza sativa</i> L.), they are present in the stems, leaves, stigmas, and caryopsis. However, there is still no scientific evidence supporting the existence of compartmentalization and trafficking of anthocyanin inside the cells. In the current study, we took advantage of autofluorescence with anthocyanin’s unique excitation/emission properties to elucidate the subcellular localization of anthocyanin and report on the in planta characterization of anthocyanin prevacuolar vesicles (APV) and anthocyanic vacuolar inclusion (AVI) structure. Protoplasts were isolated from the stigma of black and brown rice and imaging using a confocal microscope. Our result showed the fluorescence displaying magenta color in purple stigma and no fluorescence in white stigma when excitation was provided by a helium–neon 552 nm and emission long pass 610–670 nm laser. The fluorescence was distributed throughout the cell, mainly in the central vacuole. Fluorescent images revealed two pools of anthocyanin inside the cells. The diffuse pools were largely found inside the vacuole lumen, while the body structures could be observed mostly inside the cytoplasm (APV) and slightly inside the vacuole (AVI) with different shapes, sizes, and color intensity. Based on their sizes, AVI could be grouped into small (Ф < 0.5 um), middle (Ф between 0.5 and 1 um), and large size (Ф > 1 um). Together, these results provided evidence about the sequestration and trafficking of anthocyanin from the cytoplasm to the central vacuole and the existence of different transport mechanisms of anthocyanin. Our results suggest that stigma cells are an excellent system for in vivo studying of anthocyanin in rice and provide a good foundation for understanding anthocyanin metabolism in plants, sequestration, and trafficking in black rice.