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

Lead Research Organisation: John Innes Centre
Department Name: Cell and Develop Biology

Abstract

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.

Technical Summary

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.

Planned Impact

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).

Publications

10 25 50
 
Description Understanding how the plant stem grows is 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 spite of its relevance, stem development is one of the least understood aspects of plant development.

In this project, we took 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. Using Arabidopsis as the model, we found that:

1. Genes that have been used extensively to modify plant height and improve crop yield (called DELLA genes) function mostly in the terminal region of the stem, where cell proliferation occurs. We have identified links between these genes and the molecular machinery that controls cell proliferation. We also revealed an unanticipated role for the DELLA genes in controlling the size of the proliferating, apical region (the shoot meristem), in a way that is genetically separable from their role in regulating plant height. We also found that this role of DELLA genes is mediated by a regulator of cell division. These findings are important because meristem size influences flower number, which is a key factor in crop yield potential. Therefore, separating the effects of DELLA genes on stem growth and meristem size could unlock further yield increases in widely-used semi-dwarf varieties. A manuscript describing the results from this part of the project has recently been submitted.

2. Another regulatory gene that controls stem growth (called RPL) has multiple direct links to other genes previously implicated in stem formation, and therefore seems to function as a general coordinator of stem growth. We found that RPL controls three-dimensional patterns of cell division and growth through repression of organ boundary genes. controls three-dimensional patterns of cell division and growth through repression of organ boundary genes. The work provided insight into the internal cell behaviour that drives development of a 3D structure and opened the way to study and modify a developmental process that influences plant traits with key practical importance. These findings were published in Bencivenga et al., Developmental Cell 39: 198-208, 2016.
Exploitation Route We have discussed some of the findings with representatives from plant breeding industry (including Syngenta, Limagrain, Bayer and KWS). Both our approaches and findings (especially on DELLA genes) attracted interest.
Sectors Agriculture, Food and Drink

URL http://www.bbsrc.ac.uk/news/fundamental-bioscience/2016/160922-pr-new-techniques-uncover-hidden-secrets-plant-stem-development/
 
Description The project is of fundamental nature, although it focuses in an area of practical importance for crop improvement. We have discussed some of the findings with representatives from plant breeding industry (including Syngenta, Limagrain, Bayer and KWS). Both our approaches and findings (especially on DELLA genes) attracted interest.
First Year Of Impact 2014
Sector Agriculture, Food and Drink
 
Title Affymetrix oligonucleotide array dataset - genes regulated by REPLUMLESS 
Description The Arabidopsis thaliana REPLUMLESS gene (RPL), also known as PENNYWISE (PNY) and BELLRINGER (BLR) encodes a BEL1-like TALE homeodomain (BLH) transcription factor that controls multiple aspects of meristem and floral development, including meristem maintenance, phyllotaxis, the transition to flowering, stem development and floral organ patterning [1-3]. As part of a screen for genes that mediate the function of RPL in the processes above, we compared gene expression in inflorescence apices of wild-type (Landsberg-erecta) and the rpl-1 mutant [2] using Affymetrix oligonucleotide arrays. The data have been deposited in the public database Gene Expression Omnibus (GEO) with accession number GSE78511 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE78511) 1. Byrne, M.E., Groover, A.T., Fontana, J.R., and Martienssen, R.A. (2003). Development 130, 3941-3950. 2. Roeder, A.H.K., Ferrandiz, C., and Yanofsky, M.F. (2003). Current Biology 13, 1630-1635. 3. Smith, H.M.S., and Hake, S. (2003). Plant Cell 15, 1717-1727. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Not easy to gauge because I do not have access to information on who may have downloaded and used the data. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE78511
 
Title REPLUMLESS ChIP-seq dataset 
Description The Arabidopsis thaliana REPLUMLESS gene (RPL), also known as PENNYWISE (PNY) and BELLRINGER (BLR) encodes a BEL1-like TALE homeodomain (BLH) transcription factor that controls multiple aspects of meristem and floral development, including meristem maintenance, phyllotaxis, transition to flowering, stem development and floral organ patterning [1-3]. As part of a screen for genes that mediate the function of RPL in the processes above, we performed ChIP-seq to identify genome-wide RPL binding sites within inflorescence apices. The dataset was deposited at the Gene Expression Omnibus public repository (GEO, accession GSE78727) 1. Byrne, M.E., Groover, A.T., Fontana, J.R., and Martienssen, R.A. (2003). Development 130, 3941-3950. 2. Roeder, A.H.K., Ferrandiz, C., and Yanofsky, M.F. (2003). Current Biology 13, 1630-1635. 3. Smith, H.M.S., and Hake, S. (2003). Plant Cell 15, 1717-1727. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Not easy to gauge because I do not have access to information on who may have downloaded and used the data. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE78727
 
Title RGA ChIP-seq data 
Description Dataset of genes that are targeted by a key regulatory gene that controls plant height, related to publication by Serrano-Mislata et al. 2017, Nature Plants, doi: 10.1038/s41477-017-0003-y. The target genes were identified by immunoprecipitation of chromatin bound to the Arabidopsis DELLA protein RGA, followed by high-throughput sequencing of the bound DNA. Raw and analysed data have been deposited in a public database (NCBI Gene Expression Omnibus, accession GSE94926). 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact The data ara available for other researchers to use, but the actual use is not accessible until published. 
URL https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94926
 
Title Raw and analysed images from Serrano-Mislata et al. 2017 
Description Collection of raw confocal images and analysed images (13 Gb total) used to substantiate the publication Serrano-Mislata et al. 2017, Nature Plants, doi: 10.1038/s41477-017-0003-y. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact The dataset had been viewed 117 times and dowloaded 36 times by Febriuary 2017. 
URL https://figshare.com/articles/Serrano-Mislata_et_al_2017_image_analysis/4675801/1
 
Description Collaboration with IGDB, Beijing, China 
Organisation Chinese Academy of Sciences
Department Institute of Genetics & Developmental Biology
Country China 
Sector Academic/University 
PI Contribution Initiated joint project on the genetic control of stem development and inflorescence architecture. I participate in directing the research and I provide expertise and access to research facilities.
Collaborator Contribution Visiting PhD student (paid by IGDB) for one year
Impact None yet, collaboration recent
Start Year 2015
 
Description Collaboration with Utrecht University 
Organisation Utrecht University
Country Netherlands 
Sector Academic/University 
PI Contribution Collaboration with Dr. Marcel Proveniers from Utrecht University, The Netherlands, on the role of the ATH1 gene in controlling stem initiation. We have contributed ChIP-seq data, imges and image analysis.
Collaborator Contribution The Dutch group has contributed genetic resources and gene expression data, and a PhD student (Savani Silva) has visited my lab to perform imaging and image analysis to complete a manuscript currently in preparation.
Impact Manuscript currently in preparation.
Start Year 2017
 
Title Image analysis software 
Description Tracking and analysing quantitatively the growth and division of cells in three dimensions is a significant challenge. To track cell growth and division in time-lapse confocal images of Arabidopsis inflorescence apices, we developed a package of Python scripts and Fiji macros to landmark, segment, locate, track and measure cells in 3D (3D_meristem_analysis). The package with instructions and annotated source code is available as Supplemental Software in a paper by Serrano-Mislata et al. (http://dx.doi.org/10.1016/j.cub.2015.10.008). 
Type Of Technology Software 
Year Produced 2015 
Open Source License? Yes  
Impact The software has significantly changed the way we approach our research and has opened new research directions. For example, it is being used to show how regulatory genes control the rates and orientation of tissue growth during the early stages of stem development. The method is also being used for the quantitative analysis of floral bud growth in cereals, in collaboration with Scott Boden (JIC) - this is expected to impact on research that is directly relevant to crop yield. 
URL http://dx.doi.org/10.1016/j.cub.2015.10.008
 
Title Image analysis software - new functions 
Description Analysing quantitatively the growth and division of cells in three dimensions is a significant challenge. We developed a package of Python scripts and Fiji macros to landmark, segment, locate, track and measure the orientation of cell growth and division in plant tissues in 3D. The package with instructions and annotated source code is available as Supplemental Software in a paper by Bencivenga et al (2016), http://www.cell.com/developmental-cell/abstract/S1534-5807(16)30588-3 
Type Of Technology Software 
Year Produced 2016 
Open Source License? Yes  
Impact The software has significantly changed the way we approach our research and has opened new research directions. For example, it has been used to show how regulatory genes control the rates and orientation of tissue growth during the early stages of stem development and is being used for the quantitative analysis of floral bud growth in cereals, in collaboration with Scott Boden (JIC) - this is expected to impact on research that is relevant to crop yield. 
URL http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S1534580716305883/1-s2.0-S15345807163...
 
Title Image analysis software - new functions 
Description Analysing quantitatively the growth and division of cells in three dimensions is a significant challenge. We developed a package of Python scripts and Fiji macros to landmark, segment, locate, track and measure the orientation of cell growth and division in plant tissues in 3D. We have added additional functions to teh package, which allow better segmentation, automatic detection of recent cell divisions and their orientation, and analysis of genetically marked sectors in 3D. The package with instructions and annotated source code is available as Supplemental Software in a paper by Serrano-Mislata et al., 2017. (https://doi.org/10.6084/ m9.figshare.4675801.v1) 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact The software and associated mages had been viewed 117 times and downloaded 36 times by February 2018. 
 
Description Breeders Event JIC 2014 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Approximately 4 representatives for the plant breeding industry across Europe visited JIC for one day and attended science talks. The PI gave a talk about current work on the regulation of stem development in Arabidopsis and Brassica. Several of the industry representatives subsequently asked questions about the findings and about the approaches used, and more detailed discussions continued over dinner.

The meeting happened recently, so there has not yet been further action apart form additional e-mails form industry delegates asking for more details of approaches and publications.
Year(s) Of Engagement Activity 2014
 
Description GARNET interview 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Interview with Geraint Parry for Arabidopsis Research roundup (GARNET, http://www.garnetcommunity.org.uk/node/773) about the work published in Bencivenga et al. 2016, Dev. Cell 39(2): 198-208. The interview was subsequently placed on YouTube ( https://www.youtube.com/watch?v=Ya0ErZCYOBg).
Year(s) Of Engagement Activity 2016
URL https://www.youtube.com/watch?v=Ya0ErZCYOBg
 
Description Interview on BBC Radio 4 programme Farming Today 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interview on BBC Radio 4 programme Farming Today (http://www.bbc.co.uk/programmes/b006qj8q), broadcast on 22 August 2017. The interview was prompted by a press release related to our Serrano-Mislata et al. 2017 paper in Nature Plants. The aim was to explain to farmers and the general public how geens that control plant height have been used to improve crop productivity, and how our research opened new opportunities for further improvement.
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/programmes/b006qj8q
 
Description YouTube video on meristem development 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A YouTube video was produced by the TV presenter and YouTube Educator Maddie Moate, based on our work on meristem development, and more specifically on the work published in Serrano-Mislata et al (2015), Active control of cell size generates spatial detail during plant organogenesis. Current Biology 25: 2991-2996. The video is part of the series "How does it grow" and attracted over 1300 views within the first two months of being posted.
Year(s) Of Engagement Activity 2015
URL https://www.youtube.com/watch?v=Z0XsH7UzSfo