Digital Single-Cell Analysis of Plant Organ Development Using 3DCellAtlas

Abstract

Diverse molecular networks underlying plant growth and development are rapidly being uncovered. Integrating these data into the spatial and temporal context of dynamic organ growth remains a technical challenge. We developed 3DCellAtlas, an integrative computational pipeline that semiautomatically identifies cell types and quantifies both 3D cellular anisotropy and reporter abundance at single-cell resolution across whole plant organs. Cell identification is no less than 97.8% accurate and does not require transgenic lineage markers or reference atlases. Cell positions within organs are defined using an internal indexing system generating cellular level organ atlases where data from multiple samples can be integrated. Using this approach, we quantified the organ-wide cell-type-specific 3D cellular anisotropy driving Arabidopsis thaliana hypocotyl elongation. The impact ethylene has on hypocotyl 3D cell anisotropy identified the preferential growth of endodermis in response to this hormone. The spatiotemporal dynamics of the endogenous DELLA protein RGA, expansin gene EXPA3, and cell expansion was quantified within distinct cell types of Arabidopsis roots. A significant regulatory relationship between RGA, EXPA3, and growth was present in the epidermis and endodermis. The use of single-cell analyses of plant development enables the dynamics of diverse regulatory networks to be integrated with 3D organ growth.

Temperature variability is integrated by a spatially embedded decision-making center to break dormancy in Arabidopsis seeds

“…The cellular locations underlying this decision-making process remain unknown, however. Using 3D digital single-cell analysis, we localized ABA and GA hormone synthesis, degradation, and response reporter constructs in primary dormant embryos (23,24) to reveal the cellular basis of decision making in seeds ( The activity of the ABA-responsive RAB18 promoter demonstrated the transcriptional response to this hormone to be localized to the outer cell layers of the embryo radicle, principally the root cap and epidermis ( Fig. 2A).…”

The Transcription Factor ATHB5 Affects GA-Mediated Plasticity in Hypocotyl Cell Growth during Seed Germination

“…Whole-organ digital single-cell analysis provides the ability to understand and capture both cellular growth in 3D and the dynamics of genetically encoded components within individual cells (Montenegro-Johnson et al, 2015). We used this approach to characterize a spatially distinct growth module in the midhypocotyl of the embryonic axis whose expansion is responsible for the completion of the seed to seedling transition under optimal conditions.…”

“…The spatial and temporal relationship between the ATHB5 protein and EXPA3 promoter activity was quantified at single-cell resolution across the time course of seed germination using 3DCellAtlas (Barbier de Reuille et al, 2015;Montenegro-Johnson et al, 2015). A strong linear relationship between the detection of mean GUS reporter concentration and staining 20 mm (F).…”

“…1G). Principal directions of 3D cell anisotropy were quantified by calculating elongation factors for each direction (Montenegro-Johnson et al, 2015;Supplemental Fig. S2, A-L).…”

“…The transcription factors (TFs) GNC and GNL (Richter et al, 2010), and SCARECROW-LIKE3 have been shown to act downstream of DELLA; however, mechanistic links between DELLA-mediated growth repression and the gene expression that alters biophysical properties of plant cells represents a key gap in our understanding of plant development. The dynamic regulation of these TFs downstream of DELLA within the context of 3D cell growth that mediates developmental transitions also remains poorly understood (Fernandez et al, 2010;Schiessl et al, 2012;Montenegro-Johnson et al, 2015;Shapiro et al, 2015).…”