Microscopic gene and cell dynamics underpin macroscopic plant growth and form

Unlike animals, the final body plan of a plant is elaborated after embryogenesis by the activities of meristems, or growing points. The shoot apical meristem is a population of cells located at the tip of the shoot axis. It produces lateral organs, stem tissues and regenerates itself. In most plants little or no shoot tissue results from embryogenesis: essentially the entire shoot system derives from postembryonic development in the meristem.

Plant meristems must regulate organ formation by carefully balancing (i) the maintenance of undifferentiated stem cells with (ii) the commitment of appropriately positioned cells towards differentiation. Genetic mechanisms are in place to maintain the size of the meristem. Genes like Clavata3 and Wuschel form part of a local feedback circuit to regulate stem cell behaviour in the tip of the meristem.

Cellular and genetic interactions at the microscopic scale underpin the growth and ultimate macroscopic form of plants. These local, short-range interactions can be recoded, and are responsible for important traits  in domesticated crops.

  Genetic control of meristem activity at the cellular level . Image: Sonia Boscá, Steffen Knauer and Thomas Laux, Frontiers in Plant Science, https://doi.org/10.3389/fpls.2011.00093 (2011)

Genetic control of meristem activity at the cellular level. Image: Sonia Boscá, Steffen Knauer and Thomas Laux, Frontiers in Plant Science, https://doi.org/10.3389/fpls.2011.00093 (2011)


Domestication of tomato and increased fruit size

Modern tomato varieties emerged from wild Peruvian species (Solanum pimpinellifolium). Two mutant alleles which have played a major role in the breeding of large fruit sizes are locule number (lc) and fasciated (fas). Fas encodes the tomato homologue of Clavata3 (CLV3), and Lc encodes Wuschel (WUS). These genes are crucial parts of the well-characterised CLAVATA3-WUSCHEL negative feedback circuit that controls shoot apical meristem size in tomato, and other plants. Mutant fasciated and locule number alleles result in expanded meristem sizes, which in turn give rise to increased tomato fruit size and locule number. The mutants were identified as agronomically important traits during domestication of tomato. (from Rodriquez-Leal et al. Cell 171, 470-480, 2017).

   Clavata3  ( CLV3 ) and  Wuschel  ( WUS ) genes form part of a feedback loop that regulates the number of proliferating meristematic cells in plants, including tomato.  Mutations in the tomato genes result in increased meristem size, which contributes to increased fruit size and locule number. (Locules are indicated by yellow arrowheads). Image: Rodriquez-Leal  et al . Cell 171, 470-480, 2017.

Clavata3 (CLV3) and Wuschel (WUS) genes form part of a feedback loop that regulates the number of proliferating meristematic cells in plants, including tomato. Mutations in the tomato genes result in increased meristem size, which contributes to increased fruit size and locule number. (Locules are indicated by yellow arrowheads). Image: Rodriquez-Leal et al. Cell 171, 470-480, 2017.


Neo-domestication 

In a series of important experiments, Rodriquez-Leal et al. demonstrated that it was possible to recreate this important set of agronomic trait by using gene-editing techniques. They used CRISPR-mediated gene editing to create an allelic series of tomato clv3 (slclv3 or fas) mutants which showed a range of quantitative effects on floral organ number with a simple relationship between reduced gene expression and increased phenotypic severity. Similar experiments were performed for the tomato Wuschel gene, and other genes that regulate shoot architecture in tomato. (from Rodriquez-Leal et al. Cell 171, 470-480, 2017). These results demonstrate that it is possible, in principle, to reprogram plant growth and engineer stable agronomically important traits in a new plant. However, the engineering of novel traits requires a deeper understanding of existing regulatory networks.

  Generating an agronomic trait by gene-editing . A. Phenotypes of WT,  fas , and gene-edited  clv3  tomato plants. B. The  SlCLV3  promoter was targeted by eight gRNAs (blue arrowheads). C. PCR analysis showing multiple deletion alleles in four T0 plants. Image: Rodriquez-Leal  et al . Cell 171, 470-480, 2017

Generating an agronomic trait by gene-editing. A. Phenotypes of WT, fas, and gene-edited clv3 tomato plants. B. The SlCLV3 promoter was targeted by eight gRNAs (blue arrowheads). C. PCR analysis showing multiple deletion alleles in four T0 plants. Image: Rodriquez-Leal et al. Cell 171, 470-480, 2017


Multi-scale models for plant growth

To model and predict the form of reprogrammed plants, integrated, multiscale models for plant growth are required. These software models need to capture (i) interactions within cells, including gene regulatory networks, cell polarity, cell wall determinants, and reactions to strain or geometry regulating cell division and expansion, (ii) genetic interactions between neighboring cells that can regulate cell behaviour, and (iii) cellular growth that results in physical strains that are transmitted across tissues and constrain cell growth, because physical constraints on cell size and shape regulate timing and orientation of individual cell divisions and guide morphogenesis. Multiscale models provide an essential tool for engineering multicellular systems. Standardised DNA parts facilitate assembly of DNA circuits that may be introduced into plant systems by transformation, and the performance of DNA-based circuits can be measured using quantitative imaging techniques. Although a genetic circuit may regulate or alter the behavior of an individual cell in an easily predictable fashion, the consequences of altered cell interactions, propagation of changes across large cell populations, changes in tissue-wide physical and chemical interactions, and feedback on the properties of individual cells are difficult to predict.  

  Multi-scale view of plant growth.  (i) Interaction between cytoskeletal elements and local cell wall determinants, strain or geometry regulates the polarity of cell division and elongation. (ii) Genetic interactions between neighbouring cells trigger gene expression, cell proliferation and differentiation. (iii) Cellular growth results in physical strains that are transmitted across tissues and constrain cell growth. (iv) Physical constraints on cell size and shape regulate timing and orientation of individual cell divisions and guide morphogenesis.

Multi-scale view of plant growth. (i) Interaction between cytoskeletal elements and local cell wall determinants, strain or geometry regulates the polarity of cell division and elongation. (ii) Genetic interactions between neighbouring cells trigger gene expression, cell proliferation and differentiation. (iii) Cellular growth results in physical strains that are transmitted across tissues and constrain cell growth. (iv) Physical constraints on cell size and shape regulate timing and orientation of individual cell divisions and guide morphogenesis.


Marchantia polymorpha as a new model system for multi-scale modelling

We have adopted Marchantia polymorpha as a simple model system for plant synthetic biology. We have identified and sequenced a Cambridge isolate of Marchantia polymorpha, and are using the annotated genome to compile a novel library of DNA building blocks based on a common syntax for DNA parts and a technique for rapid assembly of DNA circuits. We are building an open system for reprogramming plant metabolism and form in a simple engineering testbed. 

The plants are extraordinarily prolific. A single cross can produce millions of propagules in the form of single-cell spores. Spores can be harvested in huge numbers and stored indefinitely in a cold, desiccated state. Each spore can germinate to produce a new plant, and, unlike higher plants, can undergo the entire developmental sequence to produce an adult plant under direct microscopic observation. Marchantia plants can be easily transformed with fluorescent protein gene markers, and directly visualised using advanced fluorescence microscopy techniques. The early stages of development in Marchantia are open and unobscured by surrounding tissues. This allows easy and direct observation of formative processes during morphogenesis. 

Sequencing efforts have provided a draft of the ~280Mbp genome. Most of the major gene families present in more advanced plants are represented by a single or few orthologues in Marchantia, meaning that there is low genetic redundancy. The apparent simplicity of genetic networks in liverworts, combined with the growing set of techniques for genetic manipulation, culture and microscopy, are set to make this primitive plant a major new system for analysis and engineering. 

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Simple morphology
Marchantia polymorpha is the best- studied species of liverwort. Liverworts form a sister clade to modern owering plants, thought to have emerged around 480M years ago. 

Male and female plants
Marchantia plants are haploid with a simple prostrate forms. Male (right) and female (left) plants produce distinctive gamete-bearing structures. 

Sexual reproduction
Fertilisation results in the production of a short-live diploid phase (sporophyte), which terminates in the production of yellow sporangia. 

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Millions of spores
Each sporangium contains 100,000’s of spores, which can he stored cryogenically and used for propagation. 

Spore germination
Spores germinate rapidly when transferred to suitable media, and the entire process of early development is exposed, and can be visualised directly. 

Tiny plants in culture
Germinating spores give rise to plantlets with recognisable body plans and anatomical features within a few days, 

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Chloroplast development
Chloroplast development can
be sampled and visualised in a synchronised cohort of germinating spores (image: Bernardo Pollak). 

Microscopy
High resolution microscopy techniques allow the non invasive imaging of subcellular features and dynamics in intact plants (image: Fernan Federici). 

Oil cells
Marchantia produces oil cells, which are devoted to the production of secondary compounds. The differentiation of these cells can be directly visualised in situ (image: Nuri Purswani). 

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Modular architecture
Cell proliferation, patterning and differentiation results in the formation of repeated air chamber structures across the surface of the plant. 

Simple root system
Marchantia lacks a proper root system, instead elongated rhizoid cells perform this role. Speci cation of rhizoids shows similarity to that of root hairs in higher plants. 

Clonal propagation
Marchantia form cup-like organs that spontaneously produce clonal propagules called gemmae. 

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Asexual propagules
Gemmae have a regular size and morphology, and can be harvested and germinated for simple observation of engineered growth and development (image: Jim Haseloff) 

Quantitative imaging
The dynamics of cellular growth and development can be quantitatively measured and parameterised using quantiative imaging techniques (image: Nuri Purswani). 

Simple genome
The Marchantia polymorpha genome is relatively small (280 MB) and comprises 8 autosomes and 1 sex chromosome that make up the haploid genome. 


Background material

 Intro2Marchantia

Intro2Marchantia

 Synthetic Botany

Synthetic Botany

1. A short description of the background and properties of Marchantia as an experimental organism. (Click to download: Intro2Marchantia.pdf, 1.7 MB, 8 pages). See also https://www.openplant.org

2. A review article arguing the need for simple model systems and improved theoretical frameworks to tackle engineering of whole plants. (Click to download: Synthetic Botany, 1.1 MB, 19 pages)