Plant Alternative Splicing and Abiotic Stress (PASAS)

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

Genetic variation is an important basis for biodiversity and phenotypic variation. How plants respond to external stimuli such as attack by pathogens/pests or stress conditions depends on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control. There are many different mechanisms of post-transcriptional control and alternative splicing (AS) is one of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Thus, AS increases the proteome complexity of an organism and can regulate mRNA levels. Alternative splicing affects all aspects of plant development, viability and adaptability to external conditions including abiotic stress. Nevertheless, AS in plants has been largely over-looked as a major contributor to global gene expression control despite its influence on transcript levels and on generation of differential protein functions. Current estimates suggest that a significant proportion of plant genes (35%) undergo alternative splicing. We have shown that this is clearly underestimated such that substantially more genes undergo AS and many genes have more AS events than have been described to date. In addition, alternative splicing patterns of many genes are regulated by different stress conditions and by altered levels of proteins which are involved in splicing. It is, therefore, essential to have a concerted effort to discover and identify AS events and to develop methods to accurately measure changes in AS at global and gene-specific levels. We will approach this by applying genomics and bioinformatics tools: 454 sequencing, whole genome tiling arrays, an RT-PCR system to quantify AS transcript levels and algorithms for comparing ESTs, to plants experiencing different abiotic stresses and with altered expression of splicing factors. We will identify new, un-annotated AS events, assess the technologies for their potential to provide quantitative analysis of AS and use these tools to study changes in AS under abiotic stress. We will also use these technologies to extend our examinations of the roles of specific trans-acting alternative splicing factors in regulating AS. The proposal is directly relevant to the themes of the ERA-PG call and brings together four research groups already involved in plant alternative splicing research and abiotic stress. The measurable outcomes will be novel, previously undescribed AS events deposited in appropriate databases; techniques for studying changes in AS; a database of AS events with their frequency and consequences; and information on the relationship between transcript levels and AS for many Arabidopsis genes. An overall outcome of this research will be a significantly increased awareness of the importance of AS and the need to consider AS and its consequences in post-transcriptional control of plant gene expression. Knowledge of the complexity and subtlety of all aspects of gene regulation is important in understanding how plants grow and survive and in the prediction of responses to changing environments and such awareness is the cornerstone of future plant breeding policies.

Technical Summary

Alternative splicing (AS) affects all aspects of plant growth, development and responses to abiotic and biotic stress. For example, AS in plants occurs in flowering time control, regulation of the circadian clock, disease resistance, various metabolic enzymes as well as in both transcription factors and splicing factors. AS can affect mRNA stability and protein expression, activity, localization and ability to interact with other proteins. In addition, AS is dynamic and patterns of many genes are regulated by different stress conditions and by altered levels of proteins which are involved in splicing. Although current estimates of the number of plant genes which undergo alternative splicing (35%) represents a substantial fraction of plant genes, we have shown that this figure is an underestimate and have detected numerous previously un-annotated alternative splicing events. Key goals in plant alternative splicing are therefore to identify as many AS events as possible (discovery) and develop methods to accurately measure changes in alternative splicing under different conditions at global and gene-specific levels (quantitation). The main partners in the project will take a co-ordinated approach in the application of genomics and bioinformatic approaches to AS discovery and quantitation. These include 454 (the most appropriate new generation sequencing platform for AS discovery) (Barta, Brown), whole genome tiling arrays (Fluhr), an RT-PCR system to quantify AS transcript levels (Brown) and algorithms for comparing ESTs, handling 454 sequences and extracting AS data from tiling arrays (Fluhr, Barta). The same plant material will be used in the different platforms to allow direct comparison. We will identify novel AS events, assess the technologies for their ability to quantify AS and use these tools to study changes in AS in plants grown under abiotic stress and in mutants of specific trans-acting alternative splicing factors.

Publications

10 25 50
 
Description Plants have around 30,000 genes which are expressed at different times and under different conditions. Expression is where a gene is turned on or off or up or down. When a gene is on, messenger RNA transcripts are made which are translated into proteins which can be structural or enzymatic etc resulting in a particular cellular state and ultimately the phenotype of the plant. Plants are exposed to all kinds of stresses throughout the day and their lifetime and therefore have evolved ways of tolerating changes in their environment - e.g. temperature, drought, UV, ozone, shade etc. Any such adaptation to changes in their environment require changes in the expression of sets of genes which can modify for example, biochemical pathways, membranes and cell behaviour to allow the plant to survive stress. One mechanism by which plants can modify gene expression is alternative splicing. This is where different parts of precursor mRNA transcripts are joined in different ways to make mRNA transcripts which can be targeted for degradation or can encode functionally different proteins. We still know very little about alternative splicing and how it modifies gene expression and how it is regulated. We also do not really know yet to what extent alternative splicing is found in plants and how it reacts to changes in conditions. In this research we have addressed a number of questions on alternative splicing, mechanisms of regulation of expression and how alternative splicing modulates expression in response to stress. 1) By applying a high throughput technology (RNA-seq) to examine what genes are expressed in the model plant Arabidopsis, we were able to show that there is extensive alternative splicing with over 60% of genes showing AS. This is much higher than previously thought and is still likely to be an underestimate. We were also able to use another technology that we developed to validate the RNA-seq results. This method (high resolution AS RT-PCR) showed 92% validation of transcripts predicted from RNA-seq and demonstrated that the analysis "pipeline" developed specifically to report on AS was very good and could be applied to further analysis. 2) We have demonstrated that AS of nearly 20% of Arabidopsis genes can lead to degradation of specific transcripts by nonsense-mediated decay and that some genes regulated in this way are important in stress responses. 3) We have therefore expanded the high resolution panel to include genes which respond to stress at the AS level so we can begin to examine when and how such changes occur (e.g. different conditions) and what the outcome is. One example is in the cold response pathway - a gene signalling pathway which controls the plant's response to low temperature. We know that some cold-responsive genes undergo AS but did not know whether AS was involved in the genes in the signalling pathway - we analysed these genes and found little evidence of AS in these genes. 4) A second example is the circadian clock which determines the timed expression of many genes in plants to optimise expression at different times of the day to optimise the fitness of the plant. It is known that the clock keeps running even when the plant is experiencing low temperatures. Using the RT-PCR we showed that many clock genes undergo AS at low temperatures and thereby modulate expression of key genes in the clock. We have identified temperature-sensitive AS which impacted on the levels of mRNAs able to encode functional proteins. 5) Small temperature changes can affect AS - this may be important for constant modification of expression in an ever-changing daily environment. 6) We have also identified splicing factors which affect AS of particular clock genes and thereby the period of the clock establishing a clear link between AS and the clock. 7) We are currently analysing data on how AS is regulated using lines where splicing factors are expressed at different levels again by RNA-seq and RT-PCR. These lines show developmental and morphological phenotypes as well as sensitivity to for example, ABA and salt stress. Our research has greatly added to knowledge of AS in plants and will demonstrate key roles for AS in response to stress. It will also generate new data on regulation of AS. This work will give a greater appreciation of the importance of AS.
Exploitation Route Much of the research output is in high quality journals. The publication of a body of papers in the area of alternative splicing and regulation in response to different conditions has had impact on the plant community. Direct exploitation is unlikely but raising awareness and understanding how AS gives plasticity to expression are important. The same considerations are being applied to crop plants as both genome sequencing and RNA-seq transcriptomics grows.
Sectors Agriculture/ Food and Drink,Environment

 
Title High resolution RT-PCR for alternative splicing 
Description The method allows us to examine alternative splicing in plant genes; it gives qualitative (discovery of new events) and quantitative data; it has been established for Arabidopsis but we have translated this to barley; it is a system to validate RNA-seq analysis. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact The method has been used by collaborators from around the world and has been established elsewhere by at least three other labs; it has led to many high quality pubications; 
 
Description Regulation of alternative splicing in plants 
Organisation Adam Mickiewicz University in Poznan
Country Poland 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation Bielefeld University
Country Germany 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation Leloir Institute
Country Argentina 
Sector Charity/Non Profit 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation Medical University of Vienna
Country Austria 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Institute of Plant Sciences (ISV)
Country France 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation University of Buenos Aires
Country Argentina 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation University of California, Davis
Country United States 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Alternative splicing and the cold transcriptome of Arabidopsis (John Brown/4th International Post-EURASNET meeting - Poznan, Poland/September 2016) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The meeting is a follow-on from the very successful European Alternative Splicing Network of Excellence (EURASNET). The post-EURASNET meeting series brings together research groups working on alternative splicing in model species, plants, animals and humans. The most up-to-date research on AS is presented from molecular structures to gene therapy. The plant groups continue to be active in this meeting and to collaborate extensively with one another. The active collaboration among plant groups working on AS in the EU is a significant outcome.
Year(s) Of Engagement Activity 2016
 
Description Family Fun Day and Fascination of Plants Day - Dundee Botanic Gardens 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Activities designed to provide information/interest in DNA, genetics, diversity

Excitement from children and interest from parents about DNA when children extracted DNA from raspberries.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
URL http://www.lifesci.dundee.ac.uk/research/ps/engagement-plant-sciences
 
Description Invited talk at 60th Brazilian Genetics Congress, Guaraja, Brazil 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited seminar at the Congress to present results about alternative splicing in the circadian clock
Year(s) Of Engagement Activity 2014
 
Description Invited talk at Danforth Centre in St. Louis, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Seminar about RNA-seq analysis and alternative splicing in the circadian clock to academics including students, PIs etc.
Year(s) Of Engagement Activity 2015