Periodic Reporting for period 1 - CHORPATH (Study of phenol-like compound, a newly discovered class of plant specialized metabolites.)
Período documentado: 2020-04-01 hasta 2022-03-31
Title: Digging into the synthesis of plant metabolites
Summary:
Plants produce arrays of specialized metabolites that are crucial for plant development, defense and interactions with the ever-changing environment. Discovery of metabolites and metabolic pathways by dissecting the underlying genetic basis is an important challenge, not only to increase our understanding of life, but also to provide indispensable applications for humans such as pharmaceuticals. In this project we have investigated the occurrence and biosynthesis of a novel metabolic class of phenol-like compounds (PLC), that was discovered by the host group in the plant model Arabidopsis (unpublished). Notably, chemically synthesized PLC and analogues are in vitro inhibitors of enzymes at branch points in the biosynthesis of a plethora of essential molecules. This inhibitory activity hints to a potential biological role for PLCs in regulating the flux through these pathways, opening perspectives for applications. The direct objective of my project is to unravel the biosynthetic pathway of PLCs. We therefore aim i) to structurally identify additional pathway intermediates and sinks via LC-MS based metabolite profiling and purification followed by NMR, ii) to feed plants with Stable Isotope Labeled precursor molecules, followed by analyzing the MS fragmentation spectra of the labeled molecules, iii) to investigate the role of an operon-like gene cluster involved in the biosynthesis of PLCs via reversed genetics, and iv) to obtain insight in the in planta role of PLC by feeding experiments followed by comparative metabolic profiling.
Summary:
Plants produce arrays of specialized metabolites that are crucial for plant development, defense and interactions with the ever-changing environment. Discovery of metabolites and metabolic pathways by dissecting the underlying genetic basis is an important challenge, not only to increase our understanding of life, but also to provide indispensable applications for humans such as pharmaceuticals. In this project we have investigated the occurrence and biosynthesis of a novel metabolic class of phenol-like compounds (PLC), that was discovered by the host group in the plant model Arabidopsis (unpublished). Notably, chemically synthesized PLC and analogues are in vitro inhibitors of enzymes at branch points in the biosynthesis of a plethora of essential molecules. This inhibitory activity hints to a potential biological role for PLCs in regulating the flux through these pathways, opening perspectives for applications. The direct objective of my project is to unravel the biosynthetic pathway of PLCs. We therefore aim i) to structurally identify additional pathway intermediates and sinks via LC-MS based metabolite profiling and purification followed by NMR, ii) to feed plants with Stable Isotope Labeled precursor molecules, followed by analyzing the MS fragmentation spectra of the labeled molecules, iii) to investigate the role of an operon-like gene cluster involved in the biosynthesis of PLCs via reversed genetics, and iv) to obtain insight in the in planta role of PLC by feeding experiments followed by comparative metabolic profiling.
During this project period, a series of experiments led to new discoveries on the biosynthesis and biological role of PLCs.
1. Structure elucidation of new PLCs in Arabidopsis. In total, 17 PLCs have been identified in this project, 10 of them have been identified by NMR analysis, and 7 of them have been putatively annotated based on their MS fragmentation information.
2. Characterization of a T-DNA insertion mutant for GENE1 of the operon, and generation of knock-out mutants by the CRISPR/cas9 system for four genes (GENE 1 ~ 4) putatively involved in the biosynthesis of the PLCs. For the T-DNA insertion mutant, a homozygous knock-out mutant was obtained for GENE1 and the strongly reduced expression of the GENE1 gene in this knock-out mutant was confirmed by RT-qPCR. For the CRISPR/cas9 edited mutants, two independent alleles were obtained for each of the four genes (GENE 1 ~ 4) putatively involved in the biosynthesis of PLCs, and all these knock-out mutants have been confirmed by sequencing.
3. Metabolite profiling of knock-out mutants vs wild-type plants for four genes (GENE 1~4). The loss-of-function of any one of these four targeted genes resulted in the absence or severe reduction of all PLCs, indicating all these four genes are involved in the biosynthesis of PLCs in Arabidopsis, as hypothesised.
4. GST-tagged recombinant protein purification and in vitro enzymatic activity for each enzyme (GENE 1~4). All of the four recombinant proteins have been purified by affinity chromatography and used for enzymatic assays. In vitro combinatorial enzyme assays suggest that GENE 1-4 sequentially catalyzed the conversion of chorismite to PLCs by four consecutive steps. These in vitro results are consistent with those obtained from the metabolite profiling of knock-out mutants for each gene, suggesting GENE 1~4 forms a gene cluster for the biosynthesis of PLC.
5. Localization of the enzymes encoded by GENE 1~4. By using two independent approaches, including Arabidopsis stable transgenic lines and a tobacco transient expression system, the protein encoded by GENE 1 was found to be localized in the plastid of plant cells. Curiously, no typical signal peptide was found at the N- or C-terminal of this protein. In contrast, the three other proteins (GENE 2~4) studied are cytosolic, suggested by their localization in the cytosol of transfected tobacco leaf cells.
1. Structure elucidation of new PLCs in Arabidopsis. In total, 17 PLCs have been identified in this project, 10 of them have been identified by NMR analysis, and 7 of them have been putatively annotated based on their MS fragmentation information.
2. Characterization of a T-DNA insertion mutant for GENE1 of the operon, and generation of knock-out mutants by the CRISPR/cas9 system for four genes (GENE 1 ~ 4) putatively involved in the biosynthesis of the PLCs. For the T-DNA insertion mutant, a homozygous knock-out mutant was obtained for GENE1 and the strongly reduced expression of the GENE1 gene in this knock-out mutant was confirmed by RT-qPCR. For the CRISPR/cas9 edited mutants, two independent alleles were obtained for each of the four genes (GENE 1 ~ 4) putatively involved in the biosynthesis of PLCs, and all these knock-out mutants have been confirmed by sequencing.
3. Metabolite profiling of knock-out mutants vs wild-type plants for four genes (GENE 1~4). The loss-of-function of any one of these four targeted genes resulted in the absence or severe reduction of all PLCs, indicating all these four genes are involved in the biosynthesis of PLCs in Arabidopsis, as hypothesised.
4. GST-tagged recombinant protein purification and in vitro enzymatic activity for each enzyme (GENE 1~4). All of the four recombinant proteins have been purified by affinity chromatography and used for enzymatic assays. In vitro combinatorial enzyme assays suggest that GENE 1-4 sequentially catalyzed the conversion of chorismite to PLCs by four consecutive steps. These in vitro results are consistent with those obtained from the metabolite profiling of knock-out mutants for each gene, suggesting GENE 1~4 forms a gene cluster for the biosynthesis of PLC.
5. Localization of the enzymes encoded by GENE 1~4. By using two independent approaches, including Arabidopsis stable transgenic lines and a tobacco transient expression system, the protein encoded by GENE 1 was found to be localized in the plastid of plant cells. Curiously, no typical signal peptide was found at the N- or C-terminal of this protein. In contrast, the three other proteins (GENE 2~4) studied are cytosolic, suggested by their localization in the cytosol of transfected tobacco leaf cells.
1. A transporter encoding gene is physically close to and highly co-expressed with those four biosynthetic genes (GENE 1 ~ 4), suggesting this transporter could be involved in the translocation of PLCs. By using three independent approaches, including Arabidopsis stable transformation, and tobacco transient expression and a yeast inducible system, the transporter was demonstrated to be localized at the tonoplast. This suggests this transporter may transfer PLCs into the vacuole, but this hypothesis remains to be further investigated.
2. Arabidopsis GENE 1 was found to utilize chorismate, which is the precursor of phenylalanine biosynthesis. Phenylalanine is at the entry point in lignin biosynthesis. Overexpression of GENE 1 should redirect the chorismate flux to PLC, resulting in less chorismate to lignin biosynthesis. GENE 1 homologous genes are not found in the poplar genome. Therefore, we hypothesize that overexpressing of the Arabidopsis GENE 1 in poplar stems may reduce lignin amount, which is a major objective of the host group to improve the processing efficiency of wood into fermentable sugars. For this purpose, a xylem-specific promoter-driven GENE 1 construct was transformed into poplar (Populus tremula x alba) via Agrobacterium tumefaciens mediated transformation in cooperation with Dr. Jan Van Doorsselaere (Hogeschool VIVES Campus Roeselare, Belgium) and 19 independent transgenic lines have been obtained at the end of this project.
3. PLC biosynthesis was found to be responsive to nitrogen stress in Arabidopsis. Accordingly, root growth of mutants with altered PLC levels was significantly different than that of wildtype under altered nitrogen conditions, suggesting PLCs play a role in plant-nitrogen interaction. Although the biological role of PLCs has not been investigated yet, it is hypothesized that the flux to phenylpropanoids will increase while that towards downstream products of other post-chorismate pathways (i.e. salicylic acid and auxin) will reduce when PLC levels increase in Arabidopsis under nitrogen deprivation.
Currently, PLCs have only been found in Arabidopsis. It is hypothesized that this pathway allows Arabidopsis to adapt to low nitrogen conditions. Discovery of PLCs in Arabidopsis not only expands our knowledge on plant specialized metabolism and gene clusters, but opens perspectives to develop strategies that allow growing crops under low nitrogen conditions. In addition, elucidation the pathway provides the opportunity for production of PLCs in plants or in microbial fermentors in the future.
2. Arabidopsis GENE 1 was found to utilize chorismate, which is the precursor of phenylalanine biosynthesis. Phenylalanine is at the entry point in lignin biosynthesis. Overexpression of GENE 1 should redirect the chorismate flux to PLC, resulting in less chorismate to lignin biosynthesis. GENE 1 homologous genes are not found in the poplar genome. Therefore, we hypothesize that overexpressing of the Arabidopsis GENE 1 in poplar stems may reduce lignin amount, which is a major objective of the host group to improve the processing efficiency of wood into fermentable sugars. For this purpose, a xylem-specific promoter-driven GENE 1 construct was transformed into poplar (Populus tremula x alba) via Agrobacterium tumefaciens mediated transformation in cooperation with Dr. Jan Van Doorsselaere (Hogeschool VIVES Campus Roeselare, Belgium) and 19 independent transgenic lines have been obtained at the end of this project.
3. PLC biosynthesis was found to be responsive to nitrogen stress in Arabidopsis. Accordingly, root growth of mutants with altered PLC levels was significantly different than that of wildtype under altered nitrogen conditions, suggesting PLCs play a role in plant-nitrogen interaction. Although the biological role of PLCs has not been investigated yet, it is hypothesized that the flux to phenylpropanoids will increase while that towards downstream products of other post-chorismate pathways (i.e. salicylic acid and auxin) will reduce when PLC levels increase in Arabidopsis under nitrogen deprivation.
Currently, PLCs have only been found in Arabidopsis. It is hypothesized that this pathway allows Arabidopsis to adapt to low nitrogen conditions. Discovery of PLCs in Arabidopsis not only expands our knowledge on plant specialized metabolism and gene clusters, but opens perspectives to develop strategies that allow growing crops under low nitrogen conditions. In addition, elucidation the pathway provides the opportunity for production of PLCs in plants or in microbial fermentors in the future.