Periodic Reporting for period 1 - AS-CELLEXPAND (Role of alternative splicing in cell expansion and plant growth)
Período documentado: 2018-09-01 hasta 2020-08-31
Abiotic stresses are known to dramatically affect plant growth by influencing the capacity of cells to expand properly, which substantially limits crop production worldwide. Therefore, a detailed understanding of how cell expansion regulators are modulated in response to environmental cues is crucial in the development of efficient strategies to improve crop production, particularly given the threatening environmental fluctuations imposed by global climate change. Alternative splicing (AS) is a key posttranscriptional mechanism that generates multiple mRNAs from the same gene, thus regulating gene expression and expanding proteomic diversity, which is emerging as crucial in allowing plants to adapt their development to stressful environments. This project aimed at investigating the impact of this understudied regulatory layer on genes modulating cell expansion during plant growth under stress conditions.
SR (serine/arginine-rich) proteins belong to a highly conserved family of splicing factors that plays a crucial role in regulating AS. Research in the host laboratory had contributed to determine that both the expression and function of plant SR proteins are stress regulated at multiple levels, with previous work showing that loss-of-function mutations in plant SR protein genes affect the capacity of seedlings to develop under abiotic stress. Our analyses of publicly available RNA-seq data have also revealed that AS events in cell expansion regulators are differentially regulated by environmental cues that impact plant growth. Together, these results encourage us to investigate a role for SR protein-mediated AS of cell expansion regulator genes in plant growth under abiotic stress.
We proposed to focus on three major tasks. In Task 1, we implemented a computational pipeline to detect and quantify all types of AS events in Arabidopsis. This pipeline was then applied on available RNA-seq data to generate an AS database, thus allowing the identification of AS events in genes involved in cell growth during plant development and in response to environmental stresses. The implementation of these tools was done in collaboration with the group of Manuel Irimia from the Centre for Genomic Regulation (CRG) in Barcelona, who has pioneered this method in several animal species, achieving accurate quantification and high validation rates. In parallel, Task 2 focused on generating mutant and transgenic Arabidopsis lines with altered SR protein levels to analyze their growth response to abiotic stresses. For plant lines with altered growth, we are investigating the SR regulation of specific AS events by means of RNA-seq and RT-qPCR. Finally, in Task 3, and based on the results from the two previous tasks, reverse genetics were planned to be used to functionally validate the predicted role of individual splice variants in plant growth adaptation to environmental stress.
SR (serine/arginine-rich) proteins belong to a highly conserved family of splicing factors that plays a crucial role in regulating AS. Research in the host laboratory had contributed to determine that both the expression and function of plant SR proteins are stress regulated at multiple levels, with previous work showing that loss-of-function mutations in plant SR protein genes affect the capacity of seedlings to develop under abiotic stress. Our analyses of publicly available RNA-seq data have also revealed that AS events in cell expansion regulators are differentially regulated by environmental cues that impact plant growth. Together, these results encourage us to investigate a role for SR protein-mediated AS of cell expansion regulator genes in plant growth under abiotic stress.
We proposed to focus on three major tasks. In Task 1, we implemented a computational pipeline to detect and quantify all types of AS events in Arabidopsis. This pipeline was then applied on available RNA-seq data to generate an AS database, thus allowing the identification of AS events in genes involved in cell growth during plant development and in response to environmental stresses. The implementation of these tools was done in collaboration with the group of Manuel Irimia from the Centre for Genomic Regulation (CRG) in Barcelona, who has pioneered this method in several animal species, achieving accurate quantification and high validation rates. In parallel, Task 2 focused on generating mutant and transgenic Arabidopsis lines with altered SR protein levels to analyze their growth response to abiotic stresses. For plant lines with altered growth, we are investigating the SR regulation of specific AS events by means of RNA-seq and RT-qPCR. Finally, in Task 3, and based on the results from the two previous tasks, reverse genetics were planned to be used to functionally validate the predicted role of individual splice variants in plant growth adaptation to environmental stress.
This project is providing very exciting and unexpected data. First, it has revealed a new role for the stress hormone ABA in controlling cell expansion when seedlings grown in the dark perceive light for the first time. Moreover it suggests that the control that this hormone exerts on growth is, at least in part, mediated by changes in the pattern of AS of growth regulators. Finally, our data also points to a role of two SR protein splicing regulators in this process. We are now focused on understanding the molecular mechanisms by which ABA controls these proteins and characterizing further the changes in AS mediated by these two SR proteins in response to light and ABA stimuli.
During the implementation of this project, I actively participated in the dissemination of my research results. This has been achieved by presenting my research in different scientific events. Moreover, in collaboration with the group of Manuel Irimia at CRG we have created (Plant alternative splicing and transcription Data Base; pastdb.crg.eu) the first web resource displaying comprehensive transcriptome-wide quantitative profiles for GE and AS in A. thaliana for a wide array of samples. In addition to RNA-seq based quantifications, PastDB also provides numerous associated genomic features for each AS event, including relevant sequences, splice site strength estimates, predicted impact on the main protein ORF, and suggestions of primers for RT-PCR validations (Figure 1).
In addition to the dissemination of my research results, the completion of this grant has been key in allowing me to better define the research area in which I aim at developing an independent line of research in the future. Both the experience in the host laboratory and in the secondment have provided me with the skills required to perform research in the AS field. Moreover, the research conducted during these years has broadened my knowledge of the gene regulatory mechanisms controlling plant growth in response to different environmental stimuli and increased my competitiveness in this field.
During the implementation of this project, I actively participated in the dissemination of my research results. This has been achieved by presenting my research in different scientific events. Moreover, in collaboration with the group of Manuel Irimia at CRG we have created (Plant alternative splicing and transcription Data Base; pastdb.crg.eu) the first web resource displaying comprehensive transcriptome-wide quantitative profiles for GE and AS in A. thaliana for a wide array of samples. In addition to RNA-seq based quantifications, PastDB also provides numerous associated genomic features for each AS event, including relevant sequences, splice site strength estimates, predicted impact on the main protein ORF, and suggestions of primers for RT-PCR validations (Figure 1).
In addition to the dissemination of my research results, the completion of this grant has been key in allowing me to better define the research area in which I aim at developing an independent line of research in the future. Both the experience in the host laboratory and in the secondment have provided me with the skills required to perform research in the AS field. Moreover, the research conducted during these years has broadened my knowledge of the gene regulatory mechanisms controlling plant growth in response to different environmental stimuli and increased my competitiveness in this field.
This proposal brings together different plant biology fields, where it addressed key open questions:
- ABA and growth: Does ABA stimulate or inhibit plant growth?
Extensive work has described the ABA functions as a stress hormone and developmental regulator of key processes such as seed germination, but little is known of its role in cell expansion. Due to different interpretations of the few experiments addressing this issue, controversy remains on the extent of ABA contribution to cell growth. Our data point to a role for the hormone in repressing growth.
- Tissue-specific cell expansion regulation: How is it established, does it require alternative splicing?
Because plants germinated under the soil surface elongate their hypocotyl (embryonic stem) at high rates, dark-grown hypocotyls are generally used to study plant cell expansion dynamics. Recent studies have focused on the molecular control of cotyledon cell expansion, which is strongly repressed in the dark and activated rapidly after light exposure. My data show a link between alternative splicing and cotyledons expansion, and may thus shed new molecular insight into cell expansion control in this tissue. Whether other plant organs use alternative splicing to regulate growth will be a key future question.
- Biological relevance of alternative splicing: How does alternative splicing control plant development?
Alternative splicing is emerging as a vital process for implementing developmental responses to external signals such as light or abiotic stress. However, this notion stems mostly from genomewide analyses showing splicing changes in response to these cues, but few studies have addressed how they modulate molecularly the splicing outcome. I have discovered that two splicing factors control cotyledon expansion in response to ABA and light signals. Functional analysis of these genes will provide molecular insight into how alternative splicing allows plants to cope with environmental changes.
- Molecular integration of environmental signals: How are stress and light signals integrated to control growth?
Research efforts are focusing increasingly on understanding how different environmental signals are integrated into the growth program. My preliminary data indicate that light regulates endogenous ABA levels in cotyledons to control their expansion. Understanding the molecular link between light and ABA will contribute significantly to expand knowledge in this important research field.
- ABA and growth: Does ABA stimulate or inhibit plant growth?
Extensive work has described the ABA functions as a stress hormone and developmental regulator of key processes such as seed germination, but little is known of its role in cell expansion. Due to different interpretations of the few experiments addressing this issue, controversy remains on the extent of ABA contribution to cell growth. Our data point to a role for the hormone in repressing growth.
- Tissue-specific cell expansion regulation: How is it established, does it require alternative splicing?
Because plants germinated under the soil surface elongate their hypocotyl (embryonic stem) at high rates, dark-grown hypocotyls are generally used to study plant cell expansion dynamics. Recent studies have focused on the molecular control of cotyledon cell expansion, which is strongly repressed in the dark and activated rapidly after light exposure. My data show a link between alternative splicing and cotyledons expansion, and may thus shed new molecular insight into cell expansion control in this tissue. Whether other plant organs use alternative splicing to regulate growth will be a key future question.
- Biological relevance of alternative splicing: How does alternative splicing control plant development?
Alternative splicing is emerging as a vital process for implementing developmental responses to external signals such as light or abiotic stress. However, this notion stems mostly from genomewide analyses showing splicing changes in response to these cues, but few studies have addressed how they modulate molecularly the splicing outcome. I have discovered that two splicing factors control cotyledon expansion in response to ABA and light signals. Functional analysis of these genes will provide molecular insight into how alternative splicing allows plants to cope with environmental changes.
- Molecular integration of environmental signals: How are stress and light signals integrated to control growth?
Research efforts are focusing increasingly on understanding how different environmental signals are integrated into the growth program. My preliminary data indicate that light regulates endogenous ABA levels in cotyledons to control their expansion. Understanding the molecular link between light and ABA will contribute significantly to expand knowledge in this important research field.