Analysis of barley (Hordeum vulgare) grain development using three-dimensional digital models.

Analysis of gene expression in the developing barley caryopsis requires effective instruments for visualization of the grain and the 3D expression patterns. Digital models of developing barley (Hordeum vulgare) grains were reconstructed from serial sections to visualize the complex three-dimensional (3D) grain anatomy, to generate and analyse 3D expression patterns, and to quantify tissues during growth. The models provide detailed spatial descriptions of developing grains at anthesis, at the syncytial stage of endosperm development and at the onset of starch accumulation, visualizing and quantifying 18 tissues or tissue complexes. Total caryopsis volumes and volume changes of specific tissues between the stages were determined, and proportions of ovule- and non-ovule-tissues and ratios of filial to maternal tissues were calculated from the model data. To generate and analyse 3D expression patterns, data from mRNA localization by in situ hybridizations were integrated into the models. At the onset of starch accumulation, cell-wall invertase (HvCWINV1) mRNA is mainly localized in the transfer cells and to a lesser degree in zones of the starchy endosperm. Using the model, an expression gradient across the grain was visualized. The expression pattern in the upper region of the caryopsis resembles that found in the median region at an earlier stage, indicating the presence of a developmental gradient. At anthesis, mRNA of the protease nucellin was visualized in a distinct zone of the nucellus near the antipodal cells.