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.
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.
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).
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).
People |
ORCID iD |
Robert Sablowski (Principal Investigator) |
Publications
D'Ario M
(2019)
Cell Size Control in Plants
in Annual Review of Genetics
Serrano-Mislata A
(2018)
The pillars of land plants: new insights into stem development.
in Current opinion in plant biology
Sablowski R
(2016)
Coordination of plant cell growth and division: collective control or mutual agreement?
in Current opinion in plant biology
Bencivenga S
(2016)
Control of Oriented Tissue Growth through Repression of Organ Boundary Genes Promotes Stem Morphogenesis.
in Developmental cell
Bush M
(2022)
A Phloem-Expressed PECTATE LYASE-LIKE Gene Promotes Cambium and Xylem Development.
in Frontiers in plant science
Serrano-Mislata A
(2017)
DELLA genes restrict inflorescence meristem function independently of plant height.
in Nature plants
Shi B
(2016)
Two-Step Regulation of a Meristematic Cell Population Acting in Shoot Branching in Arabidopsis.
in PLoS genetics
Ejaz M
(2021)
ARABIDOPSIS THALIANA HOMEOBOX GENE 1 controls plant architecture by locally restricting environmental responses.
in Proceedings of the National Academy of Sciences of the United States of America
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 has been viewed 677 times and dowloaded 732 times by March 2022. |
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 for "Science Today" on BNR radio Amsterdam |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Interview on 21/4/2021 for the for "Science Today" programme on BNR radio Amsterdam, following the publication by Ejaz et al. (2021), PNAS. Title: "Waarom het ene plantje lang en het andere kort is" |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.podnl.com/ep/Wetenschap-Vandaag-or-BNR-Waarom-het-ene-plantje-lang-en-het-andere-kort-is |
Description | Interview for 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 Radio 4 Farming Today on 22/8/17. The interview centred on our publication on how a gene that has been widely used to increase crop yields by reducing plant height could actually have a hidden cost by limiting the number of flowers that can be produced, and how this hidden cost could be removed to maximise the benefits on plant yield. |
Year(s) Of Engagement Activity | 2017 |
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 |