Cellular and regulatory basis of the early stages of stem development.

Virtually all of the aerial parts of the plant originate from specialised structures called shoot apical meristems, where reserves of actively dividing cells are maintained. Decades of study have revealed much about how new leaves or flowers are initiated in the periphery of the meristems. In contrast, little is known about how new stem tissues are produced by the basal region of the SAM, called the rib meristem. This has been in part due to the inaccessibility of the rib meristem to imaging methods that had a key role in revealing other aspects of meristem function. The origin of the stem is not only a major plant developmental process that has been relatively neglected, but is also of great importance in crop improvement: the height and sturdiness of the stem affect the likelihood that plants fall over in bad weather, how much of the plant's resources can be directed to making fruits and seeds, and how easy it is to harvest them. In some plants, including wheat, the stem also stores starch that provides energy reserves for grain filling. Here, we propose to take advantage of recent developments in imaging techniques to reveal the cell division and growth patterns that underpin stem development and how this cell behaviour is influenced by regulatory genes that function in the rib meristem. Using Arabidopsis as the model, we will focus on genes that also control stem development even in distantly related crop plants, such as rice. Specifically, we aim to answer the following questions: 1. What patterns of cell division and growth underpin the early stages of stem formation? We will use high-resolution imaging and computer-based 3D reconstruction to obtain quantitative data on cell growth and division in the developing stem. We will also use methods that genetically mark cells and allow us to follow how their descendants contribute to the formation of new tissues. 2. How do regulatory genes influence the cell behaviour studied in 1? We will use the same methods to compare normal plants and plants in which stem development is altered by mutation of regulatory genes. 3. How is growth and tissue formation co-ordinated across the developing stem? This question will be addressed using plants in which regulatory genes are activated in only a subset of the cells of the developing stem; this will allow us to determine whether specific cells and tissues produce signals that control the behaviour of adjacent cells and tissues. 4. What changes in gene expression underlie the effects of regulatory genes on cell behaviour and signalling during stem development? Regulatory genes typically activate or repress whole sets of other genes to cause changes in cell behaviour. We will compare changes in gene activity caused by activation of different stem regulatory to reveal what genes mediate their effects on cell division, growth and signalling. In addition to giving us insight into a major but poorly understood aspect of how plants grow, the knowledge and methods produced are expected to aid future crop breeding. By knowing the cellular basis for early stem development, it will be possible to design more precise ways to manipulate stem architecture during crop breeding.

Stem development is sustained by a specific region of the shoot apical meristem called the rib meristem. In spite of the strategic importance of stem architecture for crop breeding, the cellular and regulatory basis of RM function and stem development are poorly understood. Here, we will take advantage of recent developments in quantitative imaging and cell tracking to reveal the cell behaviour that underpins the early stages of stem development, and how this behaviour is controlled by widely conserved regulators of stem growth (DELLA proteins and the homeodomain proteins BP/ATH1/RPL). To achieve this, we will: 1. Use quantitative imaging and clonal analysis to reveal the patterns of cell division and growth in the RM that underpin wild-type stem growth; 2. Use inducible gain- and loss-of-function of DELLA and BP/ATH1/RPL to reveal how these regulatory genes change the growth parameters above; 3. Use genetic mosaics for the regulators above to study intercellular communication during early stem development; 4. Use transcriptome analysis to reveal genes that mediate the cell division, growth and signalling events studied in 1-3, and to test whether the DELLA and BP/ATH1/RPL pathways converge on common downstream targets to regulate stem growth. In addition to clarifying an important but relatively neglected aspect of shoot meristem function, this work is expected to generate knowledge and methods to design more precise ways to manipulate stem architecture in crops, e.g. by separating stem height QTL in functional categories based on cellular effects or by identifying novel target genes for breeding.

This project will benefit five main non-academic beneficiaries: 1. Breeders will benefit from knowledge required to change plant architecture precisely and predictably. The fact that at least two of the key regulators studied here (GA and BP) have similar effects on stem growth in Arabidopsis and rice indicates that knowledge originating from this project will be widely applicable. Examples of how the project could benefit breeders include: 1) knowing the key cellular parameters for the early stages of stem growth may allow breeders to recognise different functional categories of QTL affecting stem height; 2) if we find that specific tissues have a key role in controlling stem growth, these tissues could be targeted in future transgenic approaches; 3) downstream targets shared by multiple regulators of stem development could reveal genes with specialised roles in stem development, which would be important targets for breeding. The expected time frame for this beneficial impact will be 5-10 years after the start of the project. 2. Agricultural businesses will benefit from our work indirectly, through future use of the resources and knowledge made available to academic peers and to breeders. The most obvious potential benefit will be increased yield by using new varieties with reduced lodging, easier harvesting or improved harvest index. The channels to these beneficiaries will be breeders, as mentioned above, and licensing of patented knowledge through PBL Technologies (http://www.pbltechnology.com/). The time frame for this type of impact is expected to be 10-20 years. 3. Industry: depending on the career path of the person working on the project, the industrial sector may also benefit from personnel with unique training. This will include theoretical and technical knowledge ranging from molecular genetics to advanced biological imaging and modelling, communication skills including the clarity and rigour required to write papers and talk at scientific meetings, and a network of contacts spanning academia and breeders. The time frame for this type of impact is 3-4 years 4. The general public will benefit from interacting with researchers working in areas of public concern, such as food security and genetic modification. The channels for interaction with the public include the Teacher-Scientist Network (http://www.tsn.org.uk/) and presentations at the Friends of John Innes Society (http://www.jic.ac.uk/corporate/friends/index.htm). This type of impact is expected to happen during the lifetime of the grant (3-4 years). 5. BBSRC will benefit because the project is relevant to two of the current research priorities: using quantitative methods to understand biological processes, and bridging the gap between model and crop species. Because our basic understanding of stem development is very limited, it is necessary to take advantage of a model species to advance the field, but the biological process and the conserved regulatory pathways have been chosen to maximise subsequent application to crop improvement. This type of impact is expected to happen during the lifetime of the grant (3-4 years).