Plant growth originates from the meristems. In the core of each meristem, stem cells coordinate growth and maintain the undifferentiated state of the cells in the meristems. Apical meristems are located at the tip of shoots and roots, and they provide length to plant organs. Lateral meristems, cork and vascular cambia, are formed later in the development, and aligned along the main axis of organs to provide thickness (i.e. lateral or radial growth). Lateral meristems are cylindrical in shape: vascular cambium is nested inside the cork cambium. Vascular cambium produces two types of conductive tissue, xylem (wood) and phloem; and cork cambium forms phellem (cork), a tough protective layer at the surface.
Our group has been studying the development of vascular cambium by using Arabidopsis thaliana root as a model. Despite being a small weed, Arabidopsis undergoes secondary thickening, similar to the process in tree trunk.
Figure 1. Left: Simplified fate map of the root lateral growth. Xylem pole pericycle (XPP) cells produce CC and VC. Yellow arrows indicate the growth direction of the two nested meristems. Top right: Lineage tracing revealed that XPP cells contribute both to VC and CC (blue GUS clone). Bottom right: Clonal induction of auxin accumulation (blue cell) leads to xylem formation (characteristic secondary cell wall, yellow arrowhead), and induction of periclinal cell division (red arrows), the hallmark of cambial activity, in the adjacent cells.
In order to understand the growth dynamics in cellular detail, we developed a novel cell lineage tracing tool (Smetana et al. 2019) to allow us to monitor morphogenesis in course of time. We combined lineage tracing with molecular genetics and showed for the first time a molecular mechanism positioning and specifying the stem cells of vascular cambium in Arabidopsis thaliana root (Smetana et al. 2019). First, the lineage tracing was initiated at the onset of vascular cambium formation, and the resultant fate map revealed that only those procambial and pericycle cells, that are in a physical contact with primary xylem vessels, contribute to the formation of vascular cambium, xylem and phloem. Thus these cells in contact with xylem act as cambial stem cells at the onset of cambium. Subsequent lineage tracing and molecular marker analysis in mature cambium revealed that cells with xylem identity define an organizer of the vascular cambium by positioning the stem cells in the adjacent cambial cells. The identity of the organizer is defined by the local maximum of plant hormone auxin and consecutive expression of class III homeodomain-leucine zipper (HD-ZIP III) family transcription factors. Conditional removal of auxin signalling or HD-Zip IIIs lead to reduced secondary xylem and phloem production. Since lineage tracing revealed a common, bipolar stem cell for xylem and phloem, cell non-autonomous requirement of auxin and HD-ZIP IIIs for phloem identity confirms their role as factors maintaining the organizer of vascular cambium. The ectopic clonal activation of high auxin signalling results in the ectopic expression of the cambial markers, WOX4 and PXY, and subsequently formation of ectopic xylem vessels and induction of periclinal cell divisions in the adjacent cells. These data suggests that local auxin maximum in mature root tissue is sufficient for the formation of cambial stem organizer.
Figure 2. A model highlighting the dynamic nature of the organizer: differentiation of the organizer to a xylem vessel element leads to auxin accumulation in the adjacent cambial cell and thus formation of a new organizer.
After identifying the regulatory mechanism that defines the organizer of vascular cambium (Smetana et al. 2019), we have initiated several projects to link other known cambium regulators to this regulatory mechanism. We have also carried out transcript profilings to identify novel regulators of vascular cambium.
Cork cambium and coordination of growth
In the project (CORKtheCAMBIA) funded by ERC Consolidator grant we are investigating the function of the stem cells of cork cambium, and how the two lateral meristems, cork and vascular cambia, together increase the thickness in plants. To identify the origin of stem cells of cork cambium, we are combining cell lineage tracing and cell ablation approach with detailed molecular marker analysis. In order to discover the molecular factors regulating the stem cell specification of cork cambium, we are utilizing molecular genetics and a novel method (inducible CRISPR/Cas9 mutant targeting) developed in our lab. Since the lateral growth is orchestrated by two adjacent, nested meristems, cork and vascular cambia, the growth process must be tightly co-regulated. Thus, we are in process of developing in silico model to simulate the intertwined growth process conducted by the two nested meristems. By combining modelling with experimentation, we aim to uncover mechanistically how cork and vascular cambium coordinate lateral growth.
Long term goal
Our long term goal is to provide detailed understanding of the regulatory mechanisms specifying the stem cells of lateral meristems. We hope that this will lay the foundation for studies on radial thickening and facilitate rational manipulation of lateral meristems of crop plants and trees.