Final Report Summary - METMADS (MADS box transcription factors: interactions between metabolic pathways and seed development.)
Clearly significant results
> Morphological characterization of stk, abs and goa mutants (WP 1.1)
The first objective was to analyze in detail developmental defects in these stk, abs, goa mutants. In order to perform this objective Dr Ezquer contributed to investigate the role of STK, GOA and ABS in the control of seed development. The characterization of the mutants included morphological analysis using different types of microscopy techniques and histology to compare the accumulation of the various metabolites in wild type respect to the mutants (Deliverable 1.1). This included the analysis on sections of developing seeds and revealed important defects in stk mutant integuments.
> Determination of the expression pattern of STK, ABS and GOA (WP 1.1)
In order to study the pattern of expression of these transcription factors we have performed Real time PCR as well as in situ using specific probes for STK, ABS and GOA. The experiments were performed using pistil containing unfertilized ovules and the siliques containing seed at different stages of development (deliverable 1.2).
>Transcriptome analysis of stk (WP2). In order to determine which genes are down-stream these three important transcription factors we performed RNA seq analysis of stk, goa and abs mutants to be compared with wild type (Deliverable 2). This step has been crucial in order to elucidate the transcriptional network that governs seed formation. This has provided a detailed list of key genes that are deregulated in the stk background. We have selected several metabolic targets and validated the data coming from RNAseq with several qPCRs focusing on three different stages; prefertilized pistils, and siliques from 1-3 and 3-6. DEGs of both stk and goa mutants were compared, and determined common DEGs.
>Production of a STK::STK-GFP complementing line and CHIP-seq analysis. (WP3)
In order, to identify which genes are direct targets of STK, we have performed ChIP sequencing experiments (Deliverable 3). For this analyses we produced and tested a pSTK::STK-GFP stk complementing line that has been successfully validated to carry out this experiments using the appropriate controls to test the expected enrichment efficiency. The obtained enriched target gene fractions were produced and sequenced using Illumina based deep sequencing in collaboration with Prof. Pavesi, from bioinformatics group in the university. In order to determine targets of STK.
>Determination of the role of STK in the regulation of PAs synthesis and mucilage release related genes (WP3)
In order to determine targets of STK. We have combined both Chip-seq deregulated genes and RNA-seq data to obtain a list of deregulated genes. Many of the genes involved in the biosynthesis, regulation and compartimentation of the flavonoids were found to be up-regulated in stk mutant background. We have demonstrated the control of STK over this metabolic process in two steps; First, controlling core enzymes of the PAs production. Second, by controlling transcriptional regulators of key enzymes.
> Metabolic characterization of the Arabidopsis mutant seeds (Wp4)
Ignacio has characterized the metabolic pathways that are under the control of STK, with special emphasis on those involved in mucilage and anthocyanin biosynthesis (Deliverable 4). Biochemical LC-MS analysis supported the morphological observations showing that stk mutant seeds present alterate PAs levels than the wild-type seeds implying that STK is predominantly involved in the repression of genes involved in the regulatory control of PAs accumulation. Focusing on the mucilage biosynthetic pathway, Dr Ezquer performed a deep biochemical characterization of the mucilage composition that lead us to explain the mechanical problem characteristic related to the ability of mucilage to be released in the mutant stk. This experiment in coordination with Rouen University consisted in a GC-MS analysis that provided a detailed comparative monosaccharide profiles in the different mucilage layers and cell wall. This data were also validated with immunolabeling analysis of different CW polymers. With the overall data, we have proposed the first model in which transcription factors involved in differentiation and development of the seed coat are connected to the regulation of key enzymes and factors to activate/disactivate metabolic processes, to add more complexity, we provide clues regarding the biochemical machinery involved in this fine regulation.
Highlight clearly significant results
> Morphological characterization of stk, abs and goa mutants (WP 1.1 Deliverable 1.1) We have deeply characterized defects on seed development in the single mutants. This analysis in STK determined an altered accumulation of pro-anthocyanidins in the seed coat and several defects regarding mucilage extrusion under water imbibition. stk displayed smaller phenotype of the seed, in the case of GOA, it displayed a bigger seed phenotype and was found to be determined maternally.
> Determination of the expression pattern of STK, ABS and GOA (WP 1.1 Deliverable 1.2) In order to determine the possible co-expression between the three factors, qPCR and in situ experiments were performed. This analysis lead us to confirm that GOA and STK are both expressed in the external layer of the seed coat. On the other side, we determined that STK could be expressed in the endothelium layer, where ABS is expressed.
>Transcriptome analysis of stk and goa (WP2, Deliverable 2). Comparative transcriptome analysis of wild-type, stk and goa mutants has been performed by RNA seq. For this, RNA extracted from wild-type and mutants Arabidopsis siliques till 6 DAP were used. We have identified interesting STK and GOA target genes that are involved in Pas synthetic and mucilage pathways in first term. This analysis has provided also with an interesting list of genes which expression is affected by STK that are specific for seed coat and which are involved in key pathways like i) PAs pathways ii) mucilage cell wall-related genes. We have performed the validation of the data coming from the RNAseq and the further analysis of these putative targets provided clues about novel roles of STK controlling seed development.
>Production of a STK::STK-GFP complementing line and CHIP-seq analysis. (WP3, Deliverable 3)
We have performed ChIP sequencing experiments in collaboration with Bioinformatic groups at Milano University. For this analysis we have produced and tested a GFP tagged version (STK::STK-GFP). The obtained enriched target gene fractions were sequenced using Illumina based deep sequencing. With this data we have described several genome wide putative target genes for STK that are involved in seed development focusing on the PAs and mucilage metabolic pathways.
>Determination of the role of STK in the regulation of PAs synthesis and mucilage release related
genes (WP3, Deliverable 3)
In order to determine targets of STK. We have combined both Chip-seq deregulated genes and RNA-seq data to obtain a list of deregulated genes. Many of the genes involved in the biosynthesis, regulation and compartimentation of the flavonoids were found to be up-regulated in stk mutant background. We have investigated the control of STK over this metabolic process in two steps; First, controlling core enzymes of the PAs production. Second, by controlling transcriptional regulators of key enzymes.
> Metabolic characterization of the Arabidopsis mutant seeds (Wp4, Deliverable 4)
To gain insight into the regulation of flavonoid biosynthesis during seed development, we have measured by HPLC in immature seeds of the mutants the content of the PA precursors, catechins and leucoanthocyanidins. To quantify mucilage content, the mucilage isolated from Arabidopsis seeds have been extracted, precipitated and fractionated. The host lab has established a collaboration with the group of Prof. Aharoni (Weizmann institute, Israel) and Prof. Azeddine Driouich in Rouen University to perform both types of characterizations.
Socio –economic impacts of the project
The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
This project provided a complete and precise view about the regulation of flavonoid and CW biosynthetic regulation and formation occurring in the seed coat. Flavonoids importance in several aspects of plant protection and their significance for flavour and astringency of foods and beverages was already known some years ago (for review see Dixon et al., 2005). In particular, it was demonstrated that fruits of avocado that contain higher levels of epicatechin have a stronger resistance to fungal attack (Ardi et al., 1998). In this direction could be interesting to study the resistance of stk mutant seeds to fungi and STK mutagenesis could become a tools to improve plant resistance. Also, the discovery of STK as a master regulator of genes involved in the anthocyanin biosynthetic pathway could be essential to increase the levels of bioactive natural products or modify pigments in plant tissues. Microbial production of plant natural products (PNPs), such as flavonoids could also offer sustainable and economically attractive alternatives to EU. Rapid development of metabolic engineering and synthetic biology of microorganisms expressing altered metabolic pathways based on our candidate genes could show many advantages to replace the current extraction of these useful high price chemicals from plants. Although few of them were actually applied on a large scale for PNPs production, continuous research on these high-price chemicals and the rapid growing global market of them, show the promising future for the production of these PNPs by microorganisms with a more economic and environmental friendly way. The description of new players, target genes involved and proposal of interacting complexes controlling the Pas seed metabolic pathways during seed development constitutes a novel research in the field. The overall project could create the base for the introduction of novel pathways and optimization of the native cellular processes by metabolic engineering in agronomical interest plants or in microorganisms for PNPs production.
Ardi R, Kobiler I, Jacoby B, Keen NT, Prusky D (1998) Involvement of epicatechin biosynthesis in the activation of the mechanism of resistance of avocado fruits to Colletotrichum gloeosporioides. Physiological and Molecular Plant Pathology 53: 269–285
De Folter S, Busscher J, Colombo L, Losa A, Angenent GC (2004) Transcript profiling of transcription factor genes during silique development in Arabidopsis. Plant Molecular Biology 56:351-366
Dixon R a, Xie D-Y, Sharma SB (2005) Proanthocyanidins--a final frontier in flavonoid research? The New phytologist 165: 9–28
> Morphological characterization of stk, abs and goa mutants (WP 1.1)
The first objective was to analyze in detail developmental defects in these stk, abs, goa mutants. In order to perform this objective Dr Ezquer contributed to investigate the role of STK, GOA and ABS in the control of seed development. The characterization of the mutants included morphological analysis using different types of microscopy techniques and histology to compare the accumulation of the various metabolites in wild type respect to the mutants (Deliverable 1.1). This included the analysis on sections of developing seeds and revealed important defects in stk mutant integuments.
> Determination of the expression pattern of STK, ABS and GOA (WP 1.1)
In order to study the pattern of expression of these transcription factors we have performed Real time PCR as well as in situ using specific probes for STK, ABS and GOA. The experiments were performed using pistil containing unfertilized ovules and the siliques containing seed at different stages of development (deliverable 1.2).
>Transcriptome analysis of stk (WP2). In order to determine which genes are down-stream these three important transcription factors we performed RNA seq analysis of stk, goa and abs mutants to be compared with wild type (Deliverable 2). This step has been crucial in order to elucidate the transcriptional network that governs seed formation. This has provided a detailed list of key genes that are deregulated in the stk background. We have selected several metabolic targets and validated the data coming from RNAseq with several qPCRs focusing on three different stages; prefertilized pistils, and siliques from 1-3 and 3-6. DEGs of both stk and goa mutants were compared, and determined common DEGs.
>Production of a STK::STK-GFP complementing line and CHIP-seq analysis. (WP3)
In order, to identify which genes are direct targets of STK, we have performed ChIP sequencing experiments (Deliverable 3). For this analyses we produced and tested a pSTK::STK-GFP stk complementing line that has been successfully validated to carry out this experiments using the appropriate controls to test the expected enrichment efficiency. The obtained enriched target gene fractions were produced and sequenced using Illumina based deep sequencing in collaboration with Prof. Pavesi, from bioinformatics group in the university. In order to determine targets of STK.
>Determination of the role of STK in the regulation of PAs synthesis and mucilage release related genes (WP3)
In order to determine targets of STK. We have combined both Chip-seq deregulated genes and RNA-seq data to obtain a list of deregulated genes. Many of the genes involved in the biosynthesis, regulation and compartimentation of the flavonoids were found to be up-regulated in stk mutant background. We have demonstrated the control of STK over this metabolic process in two steps; First, controlling core enzymes of the PAs production. Second, by controlling transcriptional regulators of key enzymes.
> Metabolic characterization of the Arabidopsis mutant seeds (Wp4)
Ignacio has characterized the metabolic pathways that are under the control of STK, with special emphasis on those involved in mucilage and anthocyanin biosynthesis (Deliverable 4). Biochemical LC-MS analysis supported the morphological observations showing that stk mutant seeds present alterate PAs levels than the wild-type seeds implying that STK is predominantly involved in the repression of genes involved in the regulatory control of PAs accumulation. Focusing on the mucilage biosynthetic pathway, Dr Ezquer performed a deep biochemical characterization of the mucilage composition that lead us to explain the mechanical problem characteristic related to the ability of mucilage to be released in the mutant stk. This experiment in coordination with Rouen University consisted in a GC-MS analysis that provided a detailed comparative monosaccharide profiles in the different mucilage layers and cell wall. This data were also validated with immunolabeling analysis of different CW polymers. With the overall data, we have proposed the first model in which transcription factors involved in differentiation and development of the seed coat are connected to the regulation of key enzymes and factors to activate/disactivate metabolic processes, to add more complexity, we provide clues regarding the biochemical machinery involved in this fine regulation.
Highlight clearly significant results
> Morphological characterization of stk, abs and goa mutants (WP 1.1 Deliverable 1.1) We have deeply characterized defects on seed development in the single mutants. This analysis in STK determined an altered accumulation of pro-anthocyanidins in the seed coat and several defects regarding mucilage extrusion under water imbibition. stk displayed smaller phenotype of the seed, in the case of GOA, it displayed a bigger seed phenotype and was found to be determined maternally.
> Determination of the expression pattern of STK, ABS and GOA (WP 1.1 Deliverable 1.2) In order to determine the possible co-expression between the three factors, qPCR and in situ experiments were performed. This analysis lead us to confirm that GOA and STK are both expressed in the external layer of the seed coat. On the other side, we determined that STK could be expressed in the endothelium layer, where ABS is expressed.
>Transcriptome analysis of stk and goa (WP2, Deliverable 2). Comparative transcriptome analysis of wild-type, stk and goa mutants has been performed by RNA seq. For this, RNA extracted from wild-type and mutants Arabidopsis siliques till 6 DAP were used. We have identified interesting STK and GOA target genes that are involved in Pas synthetic and mucilage pathways in first term. This analysis has provided also with an interesting list of genes which expression is affected by STK that are specific for seed coat and which are involved in key pathways like i) PAs pathways ii) mucilage cell wall-related genes. We have performed the validation of the data coming from the RNAseq and the further analysis of these putative targets provided clues about novel roles of STK controlling seed development.
>Production of a STK::STK-GFP complementing line and CHIP-seq analysis. (WP3, Deliverable 3)
We have performed ChIP sequencing experiments in collaboration with Bioinformatic groups at Milano University. For this analysis we have produced and tested a GFP tagged version (STK::STK-GFP). The obtained enriched target gene fractions were sequenced using Illumina based deep sequencing. With this data we have described several genome wide putative target genes for STK that are involved in seed development focusing on the PAs and mucilage metabolic pathways.
>Determination of the role of STK in the regulation of PAs synthesis and mucilage release related
genes (WP3, Deliverable 3)
In order to determine targets of STK. We have combined both Chip-seq deregulated genes and RNA-seq data to obtain a list of deregulated genes. Many of the genes involved in the biosynthesis, regulation and compartimentation of the flavonoids were found to be up-regulated in stk mutant background. We have investigated the control of STK over this metabolic process in two steps; First, controlling core enzymes of the PAs production. Second, by controlling transcriptional regulators of key enzymes.
> Metabolic characterization of the Arabidopsis mutant seeds (Wp4, Deliverable 4)
To gain insight into the regulation of flavonoid biosynthesis during seed development, we have measured by HPLC in immature seeds of the mutants the content of the PA precursors, catechins and leucoanthocyanidins. To quantify mucilage content, the mucilage isolated from Arabidopsis seeds have been extracted, precipitated and fractionated. The host lab has established a collaboration with the group of Prof. Aharoni (Weizmann institute, Israel) and Prof. Azeddine Driouich in Rouen University to perform both types of characterizations.
Socio –economic impacts of the project
The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
This project provided a complete and precise view about the regulation of flavonoid and CW biosynthetic regulation and formation occurring in the seed coat. Flavonoids importance in several aspects of plant protection and their significance for flavour and astringency of foods and beverages was already known some years ago (for review see Dixon et al., 2005). In particular, it was demonstrated that fruits of avocado that contain higher levels of epicatechin have a stronger resistance to fungal attack (Ardi et al., 1998). In this direction could be interesting to study the resistance of stk mutant seeds to fungi and STK mutagenesis could become a tools to improve plant resistance. Also, the discovery of STK as a master regulator of genes involved in the anthocyanin biosynthetic pathway could be essential to increase the levels of bioactive natural products or modify pigments in plant tissues. Microbial production of plant natural products (PNPs), such as flavonoids could also offer sustainable and economically attractive alternatives to EU. Rapid development of metabolic engineering and synthetic biology of microorganisms expressing altered metabolic pathways based on our candidate genes could show many advantages to replace the current extraction of these useful high price chemicals from plants. Although few of them were actually applied on a large scale for PNPs production, continuous research on these high-price chemicals and the rapid growing global market of them, show the promising future for the production of these PNPs by microorganisms with a more economic and environmental friendly way. The description of new players, target genes involved and proposal of interacting complexes controlling the Pas seed metabolic pathways during seed development constitutes a novel research in the field. The overall project could create the base for the introduction of novel pathways and optimization of the native cellular processes by metabolic engineering in agronomical interest plants or in microorganisms for PNPs production.
Ardi R, Kobiler I, Jacoby B, Keen NT, Prusky D (1998) Involvement of epicatechin biosynthesis in the activation of the mechanism of resistance of avocado fruits to Colletotrichum gloeosporioides. Physiological and Molecular Plant Pathology 53: 269–285
De Folter S, Busscher J, Colombo L, Losa A, Angenent GC (2004) Transcript profiling of transcription factor genes during silique development in Arabidopsis. Plant Molecular Biology 56:351-366
Dixon R a, Xie D-Y, Sharma SB (2005) Proanthocyanidins--a final frontier in flavonoid research? The New phytologist 165: 9–28