Periodic Reporting for period 1 - EpiShade (Deciphering the epigenetic components of the Shade Avoidance Syndrome in Arabidopsis)
Reporting period: 2019-05-01 to 2021-04-30
In agricultural settings, plants are often growing in high density to optimize land use. In that situation, however, competition for resources such as light occurs and this can lead to lower crop yield. Plants are able to detect high plant density, even at the seedling stage, and activate a series of developmental changes to compete for resources. However, this set of responses often impacts negatively on production, as relocation of resources in the plant is detrimental on food production. To understand the regulation of this response is very valuable as cultivable land is becoming a limiting factor.
When plants sense vegetation proximity they change the expression of hundreds of genes. The aim of this project has been to know which factors are involved in the activation or repression of these genes (experimental approach summary in Image 1). Studying mutants defective in factors that control gene expression and/or shade detection (see an example of the aspect of different mutant plants exposed to shade at Image 2), combined with other experimental approaches (for example microscopic analysis, such at Image 3), we have identified novel players and new mechanisms (called epigenetic) that regulate gene expression when plants detect vegetation proximity.
When plants sense vegetation proximity they change the expression of hundreds of genes. The aim of this project has been to know which factors are involved in the activation or repression of these genes (experimental approach summary in Image 1). Studying mutants defective in factors that control gene expression and/or shade detection (see an example of the aspect of different mutant plants exposed to shade at Image 2), combined with other experimental approaches (for example microscopic analysis, such at Image 3), we have identified novel players and new mechanisms (called epigenetic) that regulate gene expression when plants detect vegetation proximity.
The epigenetic control of gene expression under environmental changes is a field still not well developed but of high interest in a global changing world. The aim of this project is to reveal which epigenetic mechanisms are involved in the response of plants to vegetation proximity (also referred as shade). We used the Arabidopsis model plant to study two main epigenetic mechanisms: histone acetylation and Polycomb activity.
By screening of the multiple histone acetyltransferases and histone deacetylases (involved in the deposition and removal of the acetylation mark respectively) we could detect that altering the levels of histone acetylation, Arabidopsis seedlings have a milder response to shade, as the hypocotyl growth was impaired. However, studying short-term changes of gene expression of well-known shade-induced genes, mild changes were detected. This could be explained by the diversity and redundancy of the acetylation-related enzymes.
When Polycomb activity-related mutants were analysed, strong defective phenotypes were found, especially in the mutant of the histone remodeller LHP1 (LIKE HETEROCHROMATIN PROTEIN 1). The lhp1 mutant moreover, shows important changes on gene expression after shade treatment when compared with the wild-type lines. For that reason, our work was mainly focused on the role of LHP1 in shade signalling. By diverse experimental approaches (genetic analysis, western blot, gene expression, hormone quantification, etc.) we could relate LHP1 activity to multiple levels of the shade signalling cascade such the regulation of phyA stability, auxin synthesis and signalling, repression or activation of gene expression by different mechanisms, etc. The analysis of RNA sequencing results in wild-type and lhp1 mutant at different hours after shade will give a more complete picture of the effect of LHP1 activity on shade-regulated genes.
Furthermore, we could relate the Polycomb associated marks, particularly H3K27me3, with gene expression. We found subsets of shade-regulated genes differently affected by Polycomb-related activity. With the analysis of the ChIP sequencing results, we aim to give a better picture of the regulation.
The results generated during the progression of the project have been presented at scientific conferences, both national and international. The project has contributed to some scientific papers already published, and some are being currently written. The project has been also explained to the general public and students at different stages (from primary to master studies) with special attention on transmitting the scientific critical thinking and data analysis.
By screening of the multiple histone acetyltransferases and histone deacetylases (involved in the deposition and removal of the acetylation mark respectively) we could detect that altering the levels of histone acetylation, Arabidopsis seedlings have a milder response to shade, as the hypocotyl growth was impaired. However, studying short-term changes of gene expression of well-known shade-induced genes, mild changes were detected. This could be explained by the diversity and redundancy of the acetylation-related enzymes.
When Polycomb activity-related mutants were analysed, strong defective phenotypes were found, especially in the mutant of the histone remodeller LHP1 (LIKE HETEROCHROMATIN PROTEIN 1). The lhp1 mutant moreover, shows important changes on gene expression after shade treatment when compared with the wild-type lines. For that reason, our work was mainly focused on the role of LHP1 in shade signalling. By diverse experimental approaches (genetic analysis, western blot, gene expression, hormone quantification, etc.) we could relate LHP1 activity to multiple levels of the shade signalling cascade such the regulation of phyA stability, auxin synthesis and signalling, repression or activation of gene expression by different mechanisms, etc. The analysis of RNA sequencing results in wild-type and lhp1 mutant at different hours after shade will give a more complete picture of the effect of LHP1 activity on shade-regulated genes.
Furthermore, we could relate the Polycomb associated marks, particularly H3K27me3, with gene expression. We found subsets of shade-regulated genes differently affected by Polycomb-related activity. With the analysis of the ChIP sequencing results, we aim to give a better picture of the regulation.
The results generated during the progression of the project have been presented at scientific conferences, both national and international. The project has contributed to some scientific papers already published, and some are being currently written. The project has been also explained to the general public and students at different stages (from primary to master studies) with special attention on transmitting the scientific critical thinking and data analysis.
The proposal has explored the role of epigenetic regulation in plants exposed to vegetation proximity (referred as shade), with special focus on the role of Polycomb activity. Traditionally, Polycomb mediated regulation has been related to genes involved in development, but its role regulating gene expression under environmental changes has been understudied. With this proposal, we find that Polycomb activity modulates the response to environmental changes, particularly changes in light quality (shade). This means a significant advancement in both the light and epigenetics field.
The first mechanism that regulates key genes in the shade signalling response, like HFR1, involves Polycomb activity through the canonical regulatory mechanism of H3K27me3 as a repressive histone mark. In this case, LHP1 plays the well-known role on stabilizing and spreading the repressive H3K27me3 mark. Accordingly, the overexpression of those genes after shade is accompanied by a decrease of the levels of H3K27me3 and LHP1 removal. This is a direct evidence of the role of Polycomb activity regulating genes in response to environmental changes.
A second mechanism relates to the action of LHP1 in activating gene expression, contrarily to the canonical repressive (negative) role. Interestingly, these shade-response genes activated by LHP1 are also auxin-responsive genes. We found that LHP1 activity promotes the degradation of Aux/IAA proteins (involved in the repression of auxin-responsive genes). That way, we have deciphered how LHP1 can have a positive role regulating gene expression and we have found a link between hormone signalling and epigenetic mechanisms.
This work brings knowledge on the fine-tuned regulation of shade responses, an aspect of special interest as plants growing in high density is common in agricultural environments. To understand the regulation of a response that impacts on crop yield as shade will be important especially because of the limiting cultivable land space. Moreover, shade response on plants has been reported to negatively impact on plant defence of biotic and abiotic stresses, which will be of importance in a global changing world. With this work, a new level of understanding, the chromatin and epigenetic regulation, can become an important target for breeders to improve crops.
The first mechanism that regulates key genes in the shade signalling response, like HFR1, involves Polycomb activity through the canonical regulatory mechanism of H3K27me3 as a repressive histone mark. In this case, LHP1 plays the well-known role on stabilizing and spreading the repressive H3K27me3 mark. Accordingly, the overexpression of those genes after shade is accompanied by a decrease of the levels of H3K27me3 and LHP1 removal. This is a direct evidence of the role of Polycomb activity regulating genes in response to environmental changes.
A second mechanism relates to the action of LHP1 in activating gene expression, contrarily to the canonical repressive (negative) role. Interestingly, these shade-response genes activated by LHP1 are also auxin-responsive genes. We found that LHP1 activity promotes the degradation of Aux/IAA proteins (involved in the repression of auxin-responsive genes). That way, we have deciphered how LHP1 can have a positive role regulating gene expression and we have found a link between hormone signalling and epigenetic mechanisms.
This work brings knowledge on the fine-tuned regulation of shade responses, an aspect of special interest as plants growing in high density is common in agricultural environments. To understand the regulation of a response that impacts on crop yield as shade will be important especially because of the limiting cultivable land space. Moreover, shade response on plants has been reported to negatively impact on plant defence of biotic and abiotic stresses, which will be of importance in a global changing world. With this work, a new level of understanding, the chromatin and epigenetic regulation, can become an important target for breeders to improve crops.