The study of plant development relies on techniques for the accurate visualization of plant tissue structure to understand cell patterning and patterns of gene expression. Current techniques for three-dimensional (3D) imaging are limited with respect to the thickness of tissue that may be studied and the resolution that may be achieved. Confocal laser scanning microscopy (CLSM) of living plant tissue works well for thin and semitransparent organs, such as small roots, and for the observation of external tissue layers, such as the epidermis and subepidermal cells (Haseloff, 2003). Optical projection tomography allows optical sectioning and 3D reconstruction of plant organs of up to ∼15 mm thickness, but resolution is limited and small cells and intracellular details are not resolvable (Lee et al., 2006). Truernit et al. (pages 1494–1503) describe a greatly improved propidium iodide plant tissue staining method that is used in combination with CLSM to reveal remarkably detailed 3D cellular organization of plant tissues from deep within multilayer tissues without embedding or sectioning. The method also enables 3D imaging of gene expression in entire tissues and organs as well as single cell resolution of gene expression (see figure).
Figure 1.
3D visualization and GUS protophloem marker gene expression. CLSM image of intact cotyledon of Arabidopsis expressing a GUS marker gene in protophloem cells stained with propidium iodide (white) and GUS (blue). Overview of cotyledon (A); magnified section showing GUS expression in vascular bundle (B); magnification showing GUS expression in elongated protophloem cells (C).
The authors present beautiful figures and supplemental movies online showing optical sections of leaf primordia, developing leaves, roots, and lateral root primordia. Resolution extended to visualization of small cells in meristem tissue, ovules within the silique, and embryos within intact seed coats. The method was then used to identify and visualize details of phloem structure, including sieve elements and sieve plates, and protophloem cells and their differentiation states in developing Arabidopsis seedlings. Finally, the authors investigated phloem development in intact tissues of phloem mutants woodenleg and altered phloem development, providing additional information on the function of the disrupted genes. The method represents a significant step forward in studying 3D architecture of plant tissues during development.
References
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