In vivo imaging of Ca²⁺ accumulation during cotton fiber initiation using fluorescent indicator YC3.60

Non-tip-focused Ca ²⁺ gradient indicated by genetically expressing a FRET-based calcium sensor YC3.60 was established in spherical expanding cotton fibers, which is vital for cotton fiber initiation. Cotton fiber is a single cell elongated from ovule epidermis. It is not only the most important natural fiber used in the textile industry but also an ideal model for studying cell differentiation and elongation. Before linear cell growth, cotton fibers undergo spherical expansion at the beginning of initiation. Ca²⁺, as an important secondary messenger, plays a central role in polarized cell growth including cotton fiber elongation. However, the role of Ca²⁺ in fiber initiation is far from well understood. In this paper, through ovule culture we demonstrate that Ca²⁺ is crucial for fiber initiation. Using transgenic cotton expressing the fluorescent Ca²⁺ indicator YC3.60, we show cellular and intracellular distribution of Ca²⁺ in cotton ovule epidermis and fiber cells. In the initiating fiber cell, Ca²⁺ accumulated mainly at the base of the cell, while in the fast elongating cell, the Ca²⁺ was enriched in the tip region. This cellular distribution of Ca²⁺ reported by YC3.60 was confirmed by the staining with a Ca²⁺-sensitive dye fluo-3/AM. Compared to the fluorescent dye staining, the YC3.60 system can reveal more detailed information on the intracellular distribution without photobleaching. Taken together, our data suggest that Ca²⁺ plays an important role in spherical expansion of cotton fiber initials.