Genetic and developmental basis for natural variation in plant stem architecture

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

Abstract

The height and shape of plants depends to a large extent on the way the stem grows. Although stem height and shape varies widely in nature and in cultivated plants, the genes and mechanisms behind this variation are still poorly understood. Changes in stem height have been very important to increase crop yields in the last decades, but the mutations responsible for these changes can have undesirable side effects (for example, by reducing seed size). Therefore, knowledge about how the stem forms and novel genetic changes that can be used to modify stem growth have both fundamental and practical interest.

One way to identify genes that affect stem formation is to look for genetic changes responsible for differences seen between plant lines of the same species that have originated from different locations and environments. This has two advantages: naturally selected genetic changes are less likely to cause negative side effects and can be more varied and complex than those induced and selected in the lab. This project aims to identify novel genes responsible for natural variation in stem development, and their mechanisms of action. To achieve this, we will combine two recent technical developments. First, resources and methods of unprecedented power have been developed to analyse natural genetic variation in the model species, Arabidopsis. Second, novel imaging and image analysis methods allow a much more detailed and quantitative analysis of how plant tissues grow and how growth relates to changes in gene activity.

In collaboration with a lab at the Gregor Mendel Institute (Vienna), where many of the resources to analyse natural variation in Arabidopsis have been established, we have recently identified small regions of the Arabidopsis genome associated with natural variation in stem width and length. These regions contain only a few genes, for which the available information suggests how they could affect stem growth. For example, in the case of stem width, one of the three candidate genes is similar to genes that affect the orientation of cell growth, so we hypothesize that changes in this gene may affect radial growth at early stages of stem formation. For stem length, one of the two candidate genes has been proposed to control formation of the stem vasculature, which when fully developed is expected to restrict further elongation of the stem, so we hypothesize that this gene may determine terminal stem length through the timing of vascular development.

We now propose to prove which candidate genes are responsible for changes in stem growth. For this, we will swap each of the candidate genes between Arabidopsis lines with different stem shapes. After the causative genes are identified, we will determine the exact changes in DNA sequence that modified gene function. To fully understand the functions of these genes, we will then study where and when they are expressed, and test the effect of turning these genes on and off in specific tissues and developmental stages during stem formation. To understand in detail how these genes modify the growth of stem tissues, we will measure in three dimensions the differences in cell behavior (cell division, oriented cell elongation) between natural accessions and after artificially manipulating when and where these genes function. Finally, we will extend our analysis from simple, static measurements of stem shape (such as width and length) to more complex but also more informative measurements of the speed and timing of changes in stem shape.

Revealing the genetic changes and mechanisms behind changes in stem shape in Arabidopsis will be an essential first step before equivalent genetic changes and mechanisms can be tested in crop species such as rapeseed or wheat. Ultimately our work will provide knowledge and genetic tools understand how stem architecture can be modified not only in crops, but also during plant evolution.

Technical Summary

Plant architecture depends in a large part on the size and shape of the stem, which vary widely in nature and in crops. The genetic and developmental basis for this variation, however, is mostly unknown. Knowledge about stem ontogenesis and novel genetic variation that modifies stem development is not only of fundamental interest in plant development and evolution, but also has great strategic potential for crop improvement.

An effective approach to reveal the genetic basis of natural variation is genome-wide association studies (GWAS), and in recent years Arabidopsis has emerged as a powerful model for GWAS. Also in the last years, novel imaging and quantitative, 3D image analysis methods have created unprecedented opportunities to study the cellular basis of plant growth. Here, we propose to combine both approaches to reveal the genetic basis for natural variation in stem development and the mechanism of action of the underlying genes.

We initiated GWAS in collaboration with Wolfgang Busch (Gregor Mendel Institute, Vienna) and found two significant association peaks, one for terminal stem length and one for stem width. Neither coincided with peaks for flowering time and both were independently supported by QTL analysis. Each peak spanned 2-3 genes, whose predicted functions suggest hypotheses for how they could control stem growth, for example through the timing of vascular differentiation or through polarized cell growth. We now aim to identify causative loci and alleles, to functionally characterize the genes using localized loss and gain of function, and to use quantitative 3D image analysis to reveal their cellular and developmental mechanisms of action. We also propose to extend our GWAS from static measurements to dynamic aspects of stem development, with a view to predictive modeling of stem growth, and to allow comparison with GWAS carried out by our collaborator on the dynamics of root growth.

Planned Impact

This project will benefit four main non-academic beneficiaries in the following ways:

1. Breeders will benefit from knowledge and novel genetic variation that can be used to change plant architecture precisely and predictably, potentially with fewer undesirable pleiotropic effects. 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 crop varieties with increased yield through reduced lodging and more favorable allocation of resources. Changes in stem growth due to changes in vascular development and lignification also have potential use in the bioenergy industry. The channels to these beneficiaries will be breeders, as mentioned above, and licensing of patented knowledge through JIC's technology transfer office, Plant Bioscience Limited (PBL). The time frame for this type of impact is expected to be 10-20 years.

3. 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 and the Year 10 Science Camp (3-4 years).

4. 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. The long-term impact of the project is also relevant to the BBSRC priority areas of food security and bioenergy (3-10 years).

Publications

10 25 50
 
Description Our main findings are listed in relation to each of the projects main objectives:

Objective 1 (identification of genes that cause natural variation in terminal stem length and thickness) and
Objective 3 (functional analysis of causative genes)

We identified genes associated with variation in stem growth across a large number of natural accessions of the model species, Arabidopsis. For this, we looked for association between differences in genome sequences and differences in how the stem grows. For one the genes associated with natural variation in stem growth, we used CRISPR-Cas9 technology to generate new mutations, which confirmed that the gene controls elongation of the terminal region of the stem. The gene encodes a small, secreted peptide that has not been linked previously to the control of plant height. These results are being prepared for publication.

Objective 2 (Analysis of temporal aspects of stem development)

We established methods to measure changes in stem growth over time, rather than the usual static measurement of stem height and thickness, and used these methods to support both our studies of natural variation (objectives 1 and 3 above) and to study the role of genes already known to affect plant height. This helped to clarify the function of the REPLUMLESS gene in the initiation of stem growth (published in Bencivenga et al., Developmental Cell, 2016).

Objective 4 (Use quantitative imaging to understand the cellular basis for the effect of genes on stem growth).

We created new computational tools to analyse quantitatively three-dimensional microscopy images of growing shoot apices. We used these tools to understand how genes that regulate plant height affect the cell behaviour (grow, division) that underpins stem growth. This helped to elucidate the role of REPLUMLESS in stem initiation (see 2 above) and to reveal a novel role for DELLA genes, which are widely used in agricuture to increase crop yield by reducing plant height. The latter finding showed how further yield gains might potentially be unlocked in crops containing DELLA mutations (Serrano-Mislata et al., Nature Plants 2017).
Exploitation Route In a separate project looking at genetic variation in stem growth in Brassicas (crops that include rapeseed, cabbage and broccoli), we found that the gene identified by GWAS in Arabidopsis corresponds to a genomic region associated with variability of Brassica stem growth. Future work in collaboration with other groups at the John Innes Centre will explore whether the gene identified in Arabidopsis could be useful to improve Brassica architecture.
Sectors Agriculture, Food and Drink

URL https://www.nature.com/articles/s41477-017-0003-y
 
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 - 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 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