Characterisation of tomato short RNAs involved in fruit development
Lead Research Organisation:
University of Nottingham
Department Name: Sch of Biosciences
Abstract
Most of the RNA molecules in cells are involved in protein production (ribosomal, transfer or messenger RNAs), however there are RNA molecules with other functions. A very recently discovered class of non-coding RNAs have been called microRNAs (miRNAs) because they are very short (19-24 nucleotides). These miRNAs can recognise specific mRNAs because they have partially complementary sequences to them. As a result of this interaction between miRNAs and mRNAs, the expression of the targeted mRNAs is significantly reduced. Most plant miRNAs regulate the production of transcription factors required for normal development of plants. Many miRNAs have been identified in arabidopsis and rice and several miRNAs found in one species were absent from the other suggesting that there are miRNAs which are present only in a certain group of plants. We hypothesized that miRNAs could regulate the development of different plant characteristics that are missing from arabidopsis and rice. If this was true, novel miRNAs regulating valuable plant traits could be discovered in other species. We tested this hypothesis on fleshy fruit development because of its agronomic importance. Tomato is the model plant for fleshy fruit development and ripening; therefore we sequenced more than half a million short RNAs from tomato fruit and leaf. We have demonstrated that conserved miRNAs are present in fruit and that one of them targeted a known transcription factor required for fruit ripening. We also identified four novel miRNAs, which are absent from arabidopsis and other plant genomes and showed that one of them targeted a CTR family (genes that are involved in fruit ripening) member. In addition we identified novel candidates of another class of short RNA that are produced through a different biogenesis pathway than miRNAs. These are called ta-siRNAs and are produced in a phased manner from precursor non-coding RNAs. Our preliminary work demonstrated that the process of fleshy fruit development is regulated by miRNAs and we would like to establish the biological role of the two miRNAs we showed to target ripening genes through transgenic tomato plants that produce more miRNAs or target genes that are resistant to miRNA targeting. We also propose to validate and characterise the ta-siRNA candidates. Further deep sequencing work will establish the expression profile of short RNAs during fruit development and ripening and also identify short RNAs that are regulated by known transcription factors involved in fruit development. Finally, we developed a novel technique to generate a library of mRNAs that are cleaved by short RNAs and we will apply this protocol for tomato fruit tissue. This tool will help target prediction and will have a big impact for crop research. Most crop species' genome is not known and this tool will enable researchers to investigate short RNA targets in these species. Understanding this layer of regulation in fruit development and ripening may lead to the improvement of key characteristics of fleshy fruits.
Technical Summary
MicroRNAs (miRNAs) are small RNAs, 19-24 nucleotides in length that regulate gene expression by targeting mRNAs. Although many plant miRNAs are phylogenetically conserved, several rice miRNAs are absent from the arabidopsis genome and a few arabidopsis miRNAs are not present in the rice genome. This observation suggests that non-conserved miRNAs could play a role in the development of plant traits, which are missing from arabidopsis and rice. To test whether sRNAs play a role in fleshy fruit development we have sequenced more than half a million sRNAs from tomato fruit. We have demonstrated that one of the conserved miRNAs targeted CNR, a gene required for fruit ripening. We also identified four novel tomato-specific miRNAs and showed that one of them targeted splice variants of CTR4. In addition we identified novel candidates of another class of sRNAs. These are the ta-siRNAs, which are produced in a phased manner from precursors. Our work demonstrated that the process of fleshy fruit development is regulated by miRNAs and we would like to establish the biological role of the two miRNAs we showed to target ripening genes through transgenic tomato plants that over-express miRNAs or target genes that are resistant to miRNAs. We also propose to validate ta-siRNAs using mutant tomato lines that are defective in this pathway and characterise these novel ta-siRNAs. Further high throughput sequencing will establish sRNA expression profile during fruit development and ripening. We will also identify sRNAs that are regulated by known transcription factors involved in fruit development. Finally, we developed a novel technique to generate a library of mRNAs that are cleaved by sRNAs and we will apply this protocol for tomato fruit tissue. It will be used to predict targets of sRNAs identified in the different stages of fruit development. This protocol will be useful to investigate sRNAs in other crop species, since it does not require any genome sequence information.
People |
ORCID iD |
Graham Seymour (Principal Investigator) |
Publications
Tomato Genome Consortium
(2012)
The tomato genome sequence provides insights into fleshy fruit evolution.
in Nature
Description | Nottingham partner was a minor partner in grant (resources for travel and interactions only, about £6K), but working with PI we helped place small RNAs generated during ripening within context of tomato genome assembly and the PI and Seymour wrote a section of the tomato genome paper together on this aspect of the work. The genome paper was published in Nature in 2012. |
Exploitation Route | Science discovery |
Sectors | Agriculture, Food and Drink |
Description | The UK genome project was spearheaded bu the UNOTT PI in collaboration with colleagues at Imperial and James Hutton. The interactions between the UNOTT and UEA research groups helped make an important contribution to tomato genome sequence and this information is being used by multinational biotechnology companies to breed better tomato varieties |
First Year Of Impact | 2011 |
Sector | Agriculture, Food and Drink |
Impact Types | Cultural,Economic |
Description | Interaction on map-based cloning of ripening genes and the mechanistic basis of fruit ripening |
Organisation | Cornell University |
Country | United States |
Sector | Academic/University |
PI Contribution | Sharing of data and ideas relating to the mechanisms controlling fruit ripening |
Collaborator Contribution | Provision of ideas and resources including access to data. This has resulted in a number of important papers and the collaboration has helped to formulate the now accepted model for the biological basis of fruit ripening |
Impact | The Tomato Genome Consortium (including Seymour GB) (2012).The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485:635-641 Seymour GB, Ryder CD, Cevik V, Hammond JP, Popovich A, King GJ, Vrebalov J, Giovannoni JJ and Manning K (2011). A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria × ananassa Duch.) fruit, a non-climacteric tissue. J. Exp. Bot: 62, 1179-1188. Vrebalov, J., Pan, I.L., Matas Arroyo., A.J., McQuinn, R., Chung, M-Y., Poole, M., Rose, J., Seymour, G., Grandillo, S., Giovannoni, J., Irish, V.F. (2009) Freshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1. Plant Cell 21 3041-3062. Manning K., Tor, M., Poole M., Hong, Y.,Thompson, A.J., King G., Giovannoni, J. and Seymour, G.B. (2006). A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nature Genetics, 38: 948-952. |
Description | Presentation to the British Tomato Growers about latest developments in tomato research including genome sequence |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Discussion of latest development on tomato genome sequencing project and UK contribution Tomato Growers showed increased interest and understanding about molecular breeding approaches |
Year(s) Of Engagement Activity | 2011 |
Description | Publicity for the Tomato Genome Project and publication of the project outcomes in Nature |
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 | Media outputs relating to the Genome Sequence and Role of Nottingham University. These included BBSRC and Nottingham University websites for end May and June 2012, BBC website from May 30, 2012. Example URLs are: http://www.nature.com/news/tomato-genome-sequence-bears-fruit-1.10751 http://www.bbsrc.ac.uk/news/food-security/2012/120531-pr-international-consortium-sequences-tomato-genome.aspx http://www.bbc.co.uk/news/science-environment-18253577, http://www.cbc.ca/news/technology/story/2012/06/01/sci-tomato-genome.html http://www.eurobiotechnews.eu/news/news/2012-02/tomato-genome-decoded.html http://news.yahoo.com/tomato-genome-project-bears-fruit-170214265.html http://www.nytimes.com/2012/05/31/science/the-tomato-ripe-juicy-and-bursting-with-genes.html http://www.eurobiotechnews.eu/news/news/2012-02/tomato-genome-decoded.html http://www.thegrocer.co.uk/fmcg/fresh/fruit-and-veg/tomato-genome-set-to-boost-taste-and-health/229610.article Worldwide publicity about new tomato genome sequence and how it will make a step change in breeding improved varieties. Also how tomato is a model for other Solanceous crops, e.g.potato, pepper, eggplant. |
Year(s) Of Engagement Activity | 2012 |
URL | http://www.bbc.co.uk/news/science-environment-18253577 |
Description | Tomato Growers Conference, Coventry, UK |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Presentation on the Tomato Genome Project to the British Tomato Growers Conference to demonstrate prospects for crop improvement using genomics information Demonstrated to growers value of rational approaches to breeding that will allow a step change in crop improvement. Also developed contacts that have been useful to extend industry interactions including funding for a recent TSB project on enhancing tomato shelf life. |
Year(s) Of Engagement Activity | 2011 |
Description | public lecture sponsored to the Society of Biology, East Midlands Branch |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Seymour presented a public lecture sponsored to the Society of Biology, East Midlands Branch entitled 'The Tomato Genome Project' on 17th November 2011 at University of Nottingham. Discussion around use of molecular breeding |
Year(s) Of Engagement Activity | 2011 |