Periodic Reporting for period 3 - CORKtheCAMBIA (Thickening of plant organs by nested stem cells)
Período documentado: 2022-09-01 hasta 2024-02-29
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.
We have now carried out lineage tracing and detailed marker analysis during cork cambium development. We have also carried out single cell transcriptome analysis to identify novel cork and vascular cambium factors. The candidate genes will be knocked out with genome editing. A few candidate genes knock outs turned out to be lethal, thus we are utilizing a tissue specific inducible genome editing system to target the candidates conditionally. The tissue specific genome editing system itself is now published (Wang et al Nature Plants 2020). 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).
We developed a tissue-specific, inducible, CRISPR/Cas9-based genome editing system for plants (Wang et al Nature Plants 2020). The system can knock out target gene in desired tissue-type and at desired time. Since the publication of the work, the system has been successfully used in several experiments in my lab and other labs.