Sweeter than SWEET: a single-cell leaf vasculature transcriptome atlas.

The year 2020 was full of challenges and setbacks for countless researchers around the globe. It is doubly heartening, then, to report on a landmark study of single-cell RNA sequencing (scRNA-seq) of the Arabidopsis leaf vasculature. scRNA-seq enables thestudy of cell-to-cell variation in the transcriptome, allowing an assessment of transcriptional stochasticity that can lead to a deeper understanding of cell function, reveal new cell types, and uncover unknown pathways in development. The new work by Kim et al. (2021) presents a single-cell transcriptome atlas of the Arabidopsis leaf vasculature, delivering new information about cell types and function in this tissue and providing an excellent resource for further research. The study represents a major step forward in our understanding of the transcriptional profile of vascular cells in Arabidopsis leaves and provides new insights into specific cell types such as the phloem parenchyma (PP) and bundle sheath. In addition, it provides a valuable resource for data mining and design of future experiments investigating leaf cell functions. The author first optimized protoplast isolation protocols to enrich for leaf vascular cells and verified that relative abundances of leaf cells were unaltered, such that the scRNA-seq data derived from the protoplasts should cover essentially all cell types in a mature leaf. The modified protoplasting procedure uses the confocal imaging technique of Cayla et al. (2019) with a “tape-sandwich” described by Wu et al. (2009)—essentially stripping off the abaxial epidermis with double-sided sticky tape to remove trichomes, guard cells, and epidermal cells and enrich for cell types of the vasculature, especially PP cells. They obtained 5,230 single-cell transcriptomes from two biological replicates, and a median 3,342 genes from 27,159 unique transcript signatures per cell. From these data, they identified 19 distinct cell type clusters covering all known major leaf cell types. They chose to focus on leaf vasculature in this study and assigned clusters to the known leaf vascular cell types, including xylem parenchyma, PP, companion cell (CC), and procambium (see figure). The authors report new information on the little understood PP, which plays an important role in active phloem loading in Arabidopsis. First, PP cells were separated into two distinct clusters associated with SWEET genes encoding efflux transporters; one cluster enriched with genes associated with callose deposition and the other over-represented by genes associated with photosynthetic activity. Future work will seek to determine if these clusters represent spatially distinct cell types or developmental trajectories. Secondly, expression of UmamiT gene family members, known to encode amino acid exporters, were enriched in the PP clusters and coexpressed with SWEET genes, leading the authors to speculate that UmamiTs may function in the efflux of amino acids from PP, as the SWEETs do for sucrose. Finally, members of the bZIP family were identified as putative coregulators of functionally related membrane transporters in the PP as well as other cell types. This appears to be an area ripe for further investigation, as bZIP family members are known to act as dimers or heteromers. Kim et al. also provide evidence that PP cells and the sieve element CC complex cells have distinct functions and group into clearly distinct clusters. PP cells appear to be more closely related to procambium and xylem. In addition, expression of APL, encoding a transcription factor known to promote phloem differentiation, was enriched in CC but not PP clusters. Moreover, apl mutants show normal expression of PP markers, indicating that APL may not be involved in PP formation. Another interesting set of observations came from a comparison of leaf vascular cells to those of root vascular cells. The authors compared markers from leaf phloem clusters In B ri ef