Periodic Reporting for period 1 - EpiAGPs (Breaking frontiers in plant reproduction: understanding the roles of AGPs and epigenetics regulating male-female crosstalk towards fertilization)
Berichtszeitraum: 2017-09-01 bis 2019-08-31
Human life depends on plants, which feeds us, our livestock or fulfils our biofuel needs. Thus to feed this growing population, it will be necessary to double agricultural yields without increasing the amount of arable land.
A fine knowledge of the molecular processes that finely regulate plant reproduction, will allow increasing crop productivity and high quality seed production in a sustainable manner by agricultural engineering, safeguarding the increase of arable land usage.
EpiAGPs is a research project dedicated to plant reproduction study (Fig 1) in the model plant Arabidopsis. Plant reproduction is the ultimate goal of all flowering plants, leading to the formation of seeds and the perpetuation of the next generation of plants.
EpiAGPs was achieved using a combination of genetic, proteomic and transcriptomic approaches in Arabidopsis, an excellent model to study the reproductive process, as it shares a conserved developmental program with major seed-producing crop plants. The main goal was to elucidate in which pathways AGPs act and how they modulate the epigenetic activity during reproduction, in order to make possible, as a long-term goal, to improve seed production and the generation of novel interspecific hybrids.
Overall, EpiAGPs has shed light into the involvement of AGPs in Arabidopsis reproductive process. Dr Pereira has found evidences, which reinforce the prominence of this family of glycoproteins not only in plant reproduction, but also in the plant developmental process. EpiAGPs revealed that the transcription factors NTT and HEC3 are involved in AGPs regulation, and that the epigenetic control involves HDACs.
A fine knowledge of the molecular processes that finely regulate plant reproduction, will allow increasing crop productivity and high quality seed production in a sustainable manner by agricultural engineering, safeguarding the increase of arable land usage.
EpiAGPs is a research project dedicated to plant reproduction study (Fig 1) in the model plant Arabidopsis. Plant reproduction is the ultimate goal of all flowering plants, leading to the formation of seeds and the perpetuation of the next generation of plants.
EpiAGPs was achieved using a combination of genetic, proteomic and transcriptomic approaches in Arabidopsis, an excellent model to study the reproductive process, as it shares a conserved developmental program with major seed-producing crop plants. The main goal was to elucidate in which pathways AGPs act and how they modulate the epigenetic activity during reproduction, in order to make possible, as a long-term goal, to improve seed production and the generation of novel interspecific hybrids.
Overall, EpiAGPs has shed light into the involvement of AGPs in Arabidopsis reproductive process. Dr Pereira has found evidences, which reinforce the prominence of this family of glycoproteins not only in plant reproduction, but also in the plant developmental process. EpiAGPs revealed that the transcription factors NTT and HEC3 are involved in AGPs regulation, and that the epigenetic control involves HDACs.
>Characterization of the putative JAGGER interactors (WP 1)
The first objective was to validate the candidate AGPs receptors gene expression in jagger pistils WP1. Dr Pereira performed Real Time experiments as a mean to validate the RNA-seq data. The experiments were performed using pistils containing fertilized ovules. All the selected genes were validated and we could proceed with confidence to WP1.2 (D1.1). After this validation we performed the characterization of the potential candidates’ mutants. Reproductive defects were analysed in detail in these mutant lines. Dr Pereira contributed to investigate the role of a Cysteine-rich Receptor Like Kinase (CRK) in the control flower development. The characterization of the mutants included morphological analysis using different types of microscopy techniques to compare the phenotypes regarding pollen tube growth and flower development in the mutants with respect to wild type (D1.2 & Milestone1). This revealed important defects in crk mutant flowers’ development. It was shown that this CRK is acting upstream JAGGER. Most probably, JAGGER is acting as a ligand for this specific CRK (D1.3).
> Define transmitting tract (TT) AGPs expression and their role in PT growth along the TT (WP2)
To better understand AGPs functions in reproduction several mutant lines were analysed: agp1, agp12, agp7, agp27, the double mutant jagger agp7, new RNA interference lines generated by Dr Pereira for agp25, agp26 and agp15 were analysed, as well as the double mutant agp25 agp27. All these lines were characterized using several microscopy techniques to check pollen tube growth; pollen and ovule development; and seed set (D2.1). Dr Pereira has created a set of mutant lines by RNAi and CRISPR/Cas9 for several AGPs. Due to the high redundancy found among AGPs family members, Dr Pereira generated several high order mutants. One of the triple mutants displayed a strong phenotype in its development and in the reproductive process; we demonstrated that this 3 AGPs are essential for this processes (D2.1 & Milestone2).
>Determine HDACs activity modulation by AGP (WP3)
Two HDAC mutant lines were analysed regarding defects in pollen tube growth and fertilization. The two lines were characterized using several microscopy techniques to check pollen tube growth; fertilization; and seed set. One of the lines revealed defects in pollen tube growth and ovule development (D3.1). Following this we aimed at identifying expression profiles changes for AGPs in the HDAC mutant lines, to verify if there is any evidence of epigenetic regulation. Our results show that the increase in AGPs’ gene expression in the absence of one of these HDACs reveals that in wild type inflorescences it may be responsible for repressing AGPs’ genes expression, therefore regulating their mode of action (D3.2 & Milestone3). A ChIP seq experiment using anti H3K9Ac antibodies was performed using young seedlings and inflorescences. We verified that most of the AGPs genes do not show differences in the pattern of H3 lysine acetylation (D3.3).
> Define the regulation of AGPs by NTT and HEC TF’s (WP 4)
hec3 mutant line was used to verify how the expression pattern of transmitting tract (TT) specific AGPs changed or not in the absence of this transcription factor. A Real Time PCR was performed. Few AGPs revealed a misregulation in its expression pattern and only 4 of these were TT specific, allowing us to conclude that most probably these AGPs are regulated by the transcription factor HEC3 in the pistil tissues (D4.1). Immunolocalization of AGPs was performed on ntt mutant pistils. The data allowed us to conclude that there is an increase in specific AGPs presence in ntt pistils’ TT and that NTT is regulating their expression (D4.1 & D4.2 & Milestone4).
Overview of the results & their exploitation and dissemination
The overall objectives of the project were achieved. We have highlighted and reinforced the involvement of AGPs in the reproductive process; at the same time, generating a wide range of tools that will benefit future research in the field. EpiAGPs has also shed light into AGPs transcriptional and epigenetic regulation, a theme approached for the first time in the study of AGPs, and essential to better understand its mode of action. Two manuscripts are now in preparation to be published soon, one review on the topic of EpiAGPs and one research paper (D1.3 and D3.4). Most of the project and results have been presented at International Workshops and Conferences. Results obtained along EpiAGPs will continue to be exploited in future projects and disseminated in Conferences.
The first objective was to validate the candidate AGPs receptors gene expression in jagger pistils WP1. Dr Pereira performed Real Time experiments as a mean to validate the RNA-seq data. The experiments were performed using pistils containing fertilized ovules. All the selected genes were validated and we could proceed with confidence to WP1.2 (D1.1). After this validation we performed the characterization of the potential candidates’ mutants. Reproductive defects were analysed in detail in these mutant lines. Dr Pereira contributed to investigate the role of a Cysteine-rich Receptor Like Kinase (CRK) in the control flower development. The characterization of the mutants included morphological analysis using different types of microscopy techniques to compare the phenotypes regarding pollen tube growth and flower development in the mutants with respect to wild type (D1.2 & Milestone1). This revealed important defects in crk mutant flowers’ development. It was shown that this CRK is acting upstream JAGGER. Most probably, JAGGER is acting as a ligand for this specific CRK (D1.3).
> Define transmitting tract (TT) AGPs expression and their role in PT growth along the TT (WP2)
To better understand AGPs functions in reproduction several mutant lines were analysed: agp1, agp12, agp7, agp27, the double mutant jagger agp7, new RNA interference lines generated by Dr Pereira for agp25, agp26 and agp15 were analysed, as well as the double mutant agp25 agp27. All these lines were characterized using several microscopy techniques to check pollen tube growth; pollen and ovule development; and seed set (D2.1). Dr Pereira has created a set of mutant lines by RNAi and CRISPR/Cas9 for several AGPs. Due to the high redundancy found among AGPs family members, Dr Pereira generated several high order mutants. One of the triple mutants displayed a strong phenotype in its development and in the reproductive process; we demonstrated that this 3 AGPs are essential for this processes (D2.1 & Milestone2).
>Determine HDACs activity modulation by AGP (WP3)
Two HDAC mutant lines were analysed regarding defects in pollen tube growth and fertilization. The two lines were characterized using several microscopy techniques to check pollen tube growth; fertilization; and seed set. One of the lines revealed defects in pollen tube growth and ovule development (D3.1). Following this we aimed at identifying expression profiles changes for AGPs in the HDAC mutant lines, to verify if there is any evidence of epigenetic regulation. Our results show that the increase in AGPs’ gene expression in the absence of one of these HDACs reveals that in wild type inflorescences it may be responsible for repressing AGPs’ genes expression, therefore regulating their mode of action (D3.2 & Milestone3). A ChIP seq experiment using anti H3K9Ac antibodies was performed using young seedlings and inflorescences. We verified that most of the AGPs genes do not show differences in the pattern of H3 lysine acetylation (D3.3).
> Define the regulation of AGPs by NTT and HEC TF’s (WP 4)
hec3 mutant line was used to verify how the expression pattern of transmitting tract (TT) specific AGPs changed or not in the absence of this transcription factor. A Real Time PCR was performed. Few AGPs revealed a misregulation in its expression pattern and only 4 of these were TT specific, allowing us to conclude that most probably these AGPs are regulated by the transcription factor HEC3 in the pistil tissues (D4.1). Immunolocalization of AGPs was performed on ntt mutant pistils. The data allowed us to conclude that there is an increase in specific AGPs presence in ntt pistils’ TT and that NTT is regulating their expression (D4.1 & D4.2 & Milestone4).
Overview of the results & their exploitation and dissemination
The overall objectives of the project were achieved. We have highlighted and reinforced the involvement of AGPs in the reproductive process; at the same time, generating a wide range of tools that will benefit future research in the field. EpiAGPs has also shed light into AGPs transcriptional and epigenetic regulation, a theme approached for the first time in the study of AGPs, and essential to better understand its mode of action. Two manuscripts are now in preparation to be published soon, one review on the topic of EpiAGPs and one research paper (D1.3 and D3.4). Most of the project and results have been presented at International Workshops and Conferences. Results obtained along EpiAGPs will continue to be exploited in future projects and disseminated in Conferences.
EpiAGPs provided a complete and precise view about AGPs functions and regulation in the reproductive process, reinforcing their prominent role in this step. Plant reproduction is socially and commercially relevant. All data obtained was generated in Arabidopsis, a model plant sharing a conserved developmental program with major seed-producing crop plants (Fig 2). Therefore this knowledge may easily be transferred into some of these species. The study of AGPs function, interactions and its regulation generated useful information to improve seed and fruit formation and might have positive effects on the production of seedless fruits. It will also facilitate the generation of agronomical interesting interspecific hybrids.
Besides all of this, EpiAGPs has contributed greatly to improve the career development of Dr Pereira, boosting her experience in the field. She has now already applied for two grants to return to her home country, one for in the frame of the Scientific Employment Stimulus (FCT – Science & Technology Foundation), and another one for the Call for SR&TD Project Grants (FCT) and she is now preparing an ERC Starting grant Application.
Besides all of this, EpiAGPs has contributed greatly to improve the career development of Dr Pereira, boosting her experience in the field. She has now already applied for two grants to return to her home country, one for in the frame of the Scientific Employment Stimulus (FCT – Science & Technology Foundation), and another one for the Call for SR&TD Project Grants (FCT) and she is now preparing an ERC Starting grant Application.