Periodic Reporting for period 4 - CORKtheCAMBIA (Thickening of plant organs by nested stem cells)
Reporting period: 2024-03-01 to 2024-08-31
In plants, growth originates from meristems and the stem cells therein. Lateral meristems, which provide thickness to tree stems and other plant organs, include vascular cambium; and cork cambium. Vascular cambium produces xylem (wood) and phloem. Cork cambium forms cork, a tough protective layer. We recently identified the molecular mechanism that specifies stem cells of vascular cambium. Unexpectedly, this same set of experiments revealed also novel aspects of the regulation of cork cambium, a meristem whose development has remained unknown. CORKtheCAMBIA aims to identify the stem cells of cork cambium and reveal how they mechanistically regulate plant organ thickening. Additionally, we aim to discover the molecular mechanism underlying specification of stem cells of cork cambium.
To identify the origin of stem cells of cork cambium, we will first combine lineage tracing with a detailed molecular marker analysis. To deduce the cell dynamics of cork cambium, we will secondly follow regeneration of the stem cells after ablation of this meristem. These ablation experiments will reveal the role of each tissue types in stem cell maintenance. To discover the molecular factors regulating the stem cell specification of cork cambium, in the third approach, we will utilize molecular genetics and a novel method (inducible CRISPR/Cas9 mutant targeting) developed in my lab to conditionally knockout target genes in tissue specific manner. Since the lateral growth is orchestrated by two adjacent, nested meristems, cork and vascular cambia, the growth process must be tightly co-regulated. Thus, in the final approach, we will develop in silico model of the intertwined growth process. By combining modelling with experimentation, we will uncover mechanistically how cork and vascular cambium coordinate lateral growth. Understanding the coordination of growth by the two meristems will be critical to comprehend the lateral growth as whole, and tissue mechanics during growth in general. Storage organs, such as carrot or sweet potato roots, are produced by a variant of vascular cambium.
CORKtheCAMBIA will thus provide long-awaited insight into the regulatory mechanisms specifying the stem cells of lateral meristem as whole, lay the foundation for studies on radial thickening and facilitate rational manipulation of lateral meristems of crop plants and trees.
To identify the origin of stem cells of cork cambium, we will first combine lineage tracing with a detailed molecular marker analysis. To deduce the cell dynamics of cork cambium, we will secondly follow regeneration of the stem cells after ablation of this meristem. These ablation experiments will reveal the role of each tissue types in stem cell maintenance. To discover the molecular factors regulating the stem cell specification of cork cambium, in the third approach, we will utilize molecular genetics and a novel method (inducible CRISPR/Cas9 mutant targeting) developed in my lab to conditionally knockout target genes in tissue specific manner. Since the lateral growth is orchestrated by two adjacent, nested meristems, cork and vascular cambia, the growth process must be tightly co-regulated. Thus, in the final approach, we will develop in silico model of the intertwined growth process. By combining modelling with experimentation, we will uncover mechanistically how cork and vascular cambium coordinate lateral growth. Understanding the coordination of growth by the two meristems will be critical to comprehend the lateral growth as whole, and tissue mechanics during growth in general. Storage organs, such as carrot or sweet potato roots, are produced by a variant of vascular cambium.
CORKtheCAMBIA will thus provide long-awaited insight into the regulatory mechanisms specifying the stem cells of lateral meristem as whole, lay the foundation for studies on radial thickening and facilitate rational manipulation of lateral meristems of crop plants and trees.
In an attempt to understand how cork and vascular cambium coordinate lateral growth, we identified a set of LOB DOMAIN transcription factors, LBD1, LBD3, LBD4 and LBD11, which regulate radial growth by specifically controlling cellular growth (Ye et al. Current Biology 2021). Tissue specific manipulation of LBDs is thus our prime approach to assess how the two lateral meristem coordinate mechanistically radial growth. In addition to this, we are also studying the downstream action of LBDs. We discovered that LBDs regulate large number of genes encoding primary cell wall modifying enzymes. Among them we identified four PECTIN LYASE LIKE (PLL) genes. Quadruple knockout led to reduced secondary growth, altered pectin content in cell wall and stiffer cells wall, similar to the lbd mutants. Thus, LBDs promote radial growth at least in part by upregulating four PLL genes, which regulated pectin composition in the primary cell wall, and this in turn soften the wall, which then enables accelerated cell growth. We are finalizing this work and submitting it in December 2024.
As planned in the project proposal, we carried out lineage tracing and detailed marker analysis during cork cambium development. We discovered that phloem parenchyma cells initially originated from vascular cambium cells gradually transforms to become periderm cells. This transformation was confirmed with single cell RNA sequencing analysis (scRNAseq). This transition can be accelerated wit jasmonic acid treatment or wounding. Additionally, scRNAseq analysis enabled us to identify novel cork and vascular cambium factors. We generated more than 100 transgenic YFP reporter lines to verify all the clusters in the scRNAseq data, and this way we were able to generate detailed cell types atlas of Arabidopsis root cambium. This paper is now under revision.
As a follow up of the Smetana et al paper (Nature 2019), in which we identified plant hormone auxin defining the stem cell organizer of the cambium, we developed methods to manipulate auxin distribution within the cambium. We discovered that another plant hormone, gibberellic acid, promote polar auxin transport and thus distribution of auxin within the cambium. We showed that broadness of auxin within the cambium determines whether cambium stem cell preferentially produce xylem or phloem (Mäkilä et al. Nature Plants 2023).
Finally, we showed that TDIF ligand-activated PXY receptors promote the expression of CAMBIUM-EXPRESSED AINTEGUMENTA-LIKE (CAIL) transcription factors to define cambium stem cell identity in the Arabidopsis root. By sequestrating the phloem-originated TDIF, xylem-expressed and auxin-induced PXY confines the TDIF signaling front, resulting in the activation of CAIL expression and stem cell identity in only a narrow domain. This was studied by combining experimentation and computational modelling (ten Tusscher lab). Thus, our findings show how signals emanating from cells on opposing sides ensure robust yet dynamically adjustable positioning of a bifacial stem cell layer. This work was published very recently in Science (2024).
As planned in the project proposal, we carried out lineage tracing and detailed marker analysis during cork cambium development. We discovered that phloem parenchyma cells initially originated from vascular cambium cells gradually transforms to become periderm cells. This transformation was confirmed with single cell RNA sequencing analysis (scRNAseq). This transition can be accelerated wit jasmonic acid treatment or wounding. Additionally, scRNAseq analysis enabled us to identify novel cork and vascular cambium factors. We generated more than 100 transgenic YFP reporter lines to verify all the clusters in the scRNAseq data, and this way we were able to generate detailed cell types atlas of Arabidopsis root cambium. This paper is now under revision.
As a follow up of the Smetana et al paper (Nature 2019), in which we identified plant hormone auxin defining the stem cell organizer of the cambium, we developed methods to manipulate auxin distribution within the cambium. We discovered that another plant hormone, gibberellic acid, promote polar auxin transport and thus distribution of auxin within the cambium. We showed that broadness of auxin within the cambium determines whether cambium stem cell preferentially produce xylem or phloem (Mäkilä et al. Nature Plants 2023).
Finally, we showed that TDIF ligand-activated PXY receptors promote the expression of CAMBIUM-EXPRESSED AINTEGUMENTA-LIKE (CAIL) transcription factors to define cambium stem cell identity in the Arabidopsis root. By sequestrating the phloem-originated TDIF, xylem-expressed and auxin-induced PXY confines the TDIF signaling front, resulting in the activation of CAIL expression and stem cell identity in only a narrow domain. This was studied by combining experimentation and computational modelling (ten Tusscher lab). Thus, our findings show how signals emanating from cells on opposing sides ensure robust yet dynamically adjustable positioning of a bifacial stem cell layer. This work was published very recently in Science (2024).
Developing the tissue-specific, inducible gene editing system (Wang et al Nature Plants 2020) went beyond the state of the art, since such a system did not exist before our publication. The system has now been adopted by many labs in their own studies. The fact that the system worked so efficiently was somewhat unexpected.
Also, the identification of a ligand sequestration system in defining the cambium stem cells in narrow domain is beyond state of art (Eswaran et al Science 2024). Additionally, developing the imaging methods to be able to visualize gene expression within the very thin and long cambial cells was instrumental for the Mäkilä et al paper and all the subsequent papers in the future. We studied also the regeneration of periderm (and cork cambium in it). We discovered that periderm integrity in Arabidopsis roots is sensed by diffusion of two gases, a finding that, we believe, change the way scientists understand regeneration in plants.
Also, the identification of a ligand sequestration system in defining the cambium stem cells in narrow domain is beyond state of art (Eswaran et al Science 2024). Additionally, developing the imaging methods to be able to visualize gene expression within the very thin and long cambial cells was instrumental for the Mäkilä et al paper and all the subsequent papers in the future. We studied also the regeneration of periderm (and cork cambium in it). We discovered that periderm integrity in Arabidopsis roots is sensed by diffusion of two gases, a finding that, we believe, change the way scientists understand regeneration in plants.