Spatial and single-cell expression analyses reveal complex expression domains in early wheat spike development.

backgroundWheat is important for global food security. Understanding the molecular mechanisms driving spike and spikelet development can benefit the development of more productive varieties.

resultsHere we integrate single-molecule fluorescence in situ hybridization (smFISH) and single-cell RNA sequencing (scRNA-seq) to generate an atlas of cell clusters and expression domains during the early stages of wheat spike development. We characterize spatiotemporal expression of 99 genes by smFISH in 48,225 cells at early transition (W1.5), late double ridge (W2.5), and floret primordia stages (W3.5). These cells are grouped into 21 different expression domains, including four in the basal region of the developing spikelets and three different meristematic regions, which are consistent across spikelets and sections. Using induced mutants, we reveal functional roles associated with the specific expression patterns of LFY in intercalary meristems, SPL14 in inflorescence meristems, and FZP in glume axillae. Complementary scRNA-seq profiling of 26,009 cells from W2.5 and W3.5 stages identifies 23 distinct cell clusters. We use the scRNA-seq information to impute the expression of 74,464 genes into the spatially anchored smFISH-labelled cells and generate a public website to visualize them. We then use experimental and imputed expression profiles, together with co-expression studies and correlation matrices, to annotate the scRNA-seq clusters. From co-expression analyses, we identify genes associated with boundary genes TCP24 and FZP, as well as the meristematic genes AGL6 and ULT1.

conclusionsThe smFISH and scRNA-seq studies provide complementary tools for dissecting gene networks that regulate spike development and identifying new co-expressed genes for functional characterization.