Original video: https://www.youtube.com/watch?v=I-OsCqGp4Ws&t=6s
The Arabidopsis thaliana sepals, the outermost green, leaf-like floral organs, serve as an ideal model for studying plant organ development due to their simplicity and accessibility. Their smooth shape helps enclose and protect the developing flower (Roeder, 2021), and anatomically, they resemble leaves with an outer (abaxial) and inner (adaxial) epidermal layer surrounding loosely packed mesophyll cells (Roeder, 2021). Sepals initiate early during floral development, and their small size and robustness to environmental variation allow for high-resolution study of organ development at the cellular level (Roeder, 2021). For live imaging, sepals are easier to access compared to internal floral organs or young leaves because the dissection of surrounding tissue, while necessary, is less complex (Roeder, 2021; Harline & Roeder, 2023). To image sepals from early developmental stages, it is essential to first dissect older flowers from the inflorescence, as these can obscure the younger flowers from view. Arabidopsis flowers emerge from the inflorescence meristem in a continuous spiral phyllotactic pattern, with younger flowers closer to the meristem and older flowers located farther away (Smyth et al., 1990; Bradley et al., 1997; Byrne et al., 2003). The rapid upward elongation of the pedicel causes older flowers to overshadow younger ones, making dissection necessary for live imaging.
Arabidopsis sepals, with their small size, are excellent models for analyzing growth at a cellular resolution under a confocal microscope. However, imaging deep cell layers of sepals poses challenges due to the low signal-to-noise ratio in the deeper tissues, especially in live imaging datasets. To study how differential growth in connected cell layers generates distinct organ morphologies, it is critical to live-image deep into the tissue, despite these difficulties. Here, we provide an optimized methodology for live imaging Arabidopsis sepals and subsequent image processing to overcome some of these challenges.
For optimal dissection, we prefer six- to seven-week-old Arabidopsis plants grown in 21°C long-day conditions, which prolongs vegetative growth phase relative to 24 hours light, resulting in larger meristem sizes and thicker inflorescence axes. Although plants can be grown under short day conditions as well, which leads to larger meristem size compared to long-day/24 hour light conditions (Hamant et al., 2019), plants exhibit significant delay in flowering under short day conditions (Mouradov et al., 2002), which is not necessary for our dissection purposes. Dissection can either be performed while the inflorescence is on the plant or on a clipped inflorescence to allow for easier maneuvering during dissection. However, clipping the inflorescence bears the risk of faster dehydration, and thus requires quicker dissection. The choice of whether to dissect in planta or on a clipped inflorescence depends on the researcher.
Although the dissection techniques vary among researchers, we describe the procedure that works best for us, resulting in minimal tissue damage. For dissection, we grip the inflorescence in one hand, and use the tweezers to press the base of the pedicel and pull the flower backwards in a gentle yet swift manner. We adopt a stage-wise dissection approach by rotating the inflorescence, starting with the oldest flowers first and gradually progressing to younger ones, all the way down to stages 7 to 8 [Figures 1A–C, staging performed according to (Smyth et al., 1990)].
Keywords: growth, 2.5D segmentation, live imaging, image processing, deep tissue imaging, Arabidopsis, MorphoGraphX, sepals
Citation: Singh Yadav A and Roeder AHK (2024) An optimized live imaging and multiple cell layer growth analysis approach using Arabidopsis sepals. Front. Plant Sci. 15:1449195. doi: 10.3389/fpls.2024.1449195
Received: 14 June 2024; Accepted: 29 July 2024;
Published: 03 September 2024.
Attribution 4.0 International — CC BY 4.0 - Creative Commons
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