High levels of auxin signalling define the stem-cell organizer of the vascular cambium.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium1. Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown2. Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium3. Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.

Cella: 3D data visualization for plant single-cell transcriptomics in Blender.

AIMS: Recent advancements in single-cell transcriptomics have facilitated the possibility of acquiring vast amounts of data at single-cell resolution. This development has provided a broader and more comprehensive understanding of complex biological processes. The growing datasets require a visualization tool that transforms complex data into an intuitive representation. To address this challenge, we have utilized an open-source 3D software Blender to design Cella, a cell atlas visualization tool, which transforms data into 3D heatmaps that can be rendered into image libraries. Our tool is designed to support especially research on plant development. DATA RESOURCES GENERATED: To validate our method, we have created a 3D model representing the Arabidopsis thaliana root meristem and mapped an existing single-cell RNA-seq dataset into the 3D model. This provided a user-friendly visual representation of the expression profiles of 21,489 genes from two perspectives (42,978 images). UTILITY OF THE RESOURCE: This approach is not limited to single-cell RNA-seq data of the Arabidopsis root meristem. We provide detailed step-by-step instructions to generate 3D models and a script that can be customized to project data onto different tissues. KEY RESULTS: Our tool provides a proof-of-concept method for how increasingly complex single-cell RNA-seq datasets can be visualized in a simple and cohesive manner.

Gibberellins promote polar auxin transport to regulate stem cell fate decisions in cambium.

Vascular cambium contains bifacial stem cells, which produce secondary xylem to one side and secondary phloem to the other. However, how these fate decisions are regulated is unknown. Here we show that the positioning of an auxin signalling maximum within the cambium determines the fate of stem cell daughters. The position is modulated by gibberellin-regulated, PIN1-dependent polar auxin transport. Gibberellin treatment broadens auxin maximum from the xylem side of the cambium towards the phloem. As a result, xylem-side stem cell daughter preferentially differentiates into xylem, while phloem-side daughter retains stem cell identity. Occasionally, this broadening leads to direct specification of both daughters as xylem, and consequently, adjacent phloem-identity cell reverts to being stem cell. Conversely, reduced gibberellin levels favour specification of phloem-side stem cell daughter as phloem. Together, our data provide a mechanism by which gibberellin regulates the ratio of xylem and phloem production.

Cytokinins initiate secondary growth in the Arabidopsis root through a set of LBD genes.

During primary growth, plant tissues increase their length, and as these tissues mature, they initiate secondary growth to increase thickness.1 It is not known what activates this transition to secondary growth. Cytokinins are key plant hormones regulating vascular development during both primary and secondary growth. During primary growth of Arabidopsis roots, cytokinins promote procambial cell proliferation2,3 and vascular patterning together with the hormone auxin.4-7 In the absence of cytokinins, secondary growth fails to initiate.8 Enhanced cytokinin levels, in turn, promote secondary growth.8,9 Despite the importance of cytokinins, little is known about the downstream signaling events in this process. Here, we show that cytokinins and a few downstream LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors are rate-limiting components in activating and further promoting secondary growth in Arabidopsis roots. Cytokinins directly activate transcription of two homologous LBD genes, LBD3 and LBD4. Two other homologous LBDs, LBD1 and LBD11, are induced only after prolonged cytokinin treatment. Our genetic studies revealed a two-stage mechanism downstream of cytokinin signaling: while LBD3 and LBD4 regulate activation of secondary growth, LBD1, LBD3, LBD4, and LBD11 together promote further radial growth and maintenance of cambial stem cells. LBD overexpression promoted rapid cell growth followed by accelerated cell divisions, thus leading to enhanced secondary growth. Finally, we show that LBDs rapidly inhibit cytokinin signaling. Together, our data suggest that the cambium-promoting LBDs negatively feed back into cytokinin signaling to keep root secondary growth in balance.

An inducible genome editing system for plants.

Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type-specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a new tool with which target genes can efficiently and conditionally be knocked out by genome editing at any developmental stage. Target genes can also be knocked out in a cell-type-specific manner. Our tool is easy to construct and will be particularly useful for studying genes having null alleles that are non-viable or show pleiotropic developmental defects.