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Regulatory mechanisms of the aspartate metabolic pathway in Arabidopsis thaliana: the need for isogenes in amino acid metabolism :

Regulatory mechanisms of the aspartate metabolic pathway in Arabidopsis thaliana: the need for isogenes in amino acid metabolism Pieter Van Bochaute Promotor: Prof. Dr. ir. Geert Angenon

Overview:

Overview Introduction The aspartate metabolic pathway in Arabidopsis thaliana Regulatory mechanisms Isogenes: redundant or not? Results & Discussion Blocking the flux in the lysine or threonine biosynthetic branch Role of AKLYS1 in biotic stress C onclusions and perspectives

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Introduction  Results  Discussion  Conclusions & perspectives Lysine biosynthetic branch

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Introduction  Results  Discussion  Conclusions & perspectives Threonine biosynthetic branch

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Introduction  Results  Discussion  Conclusions & perspectives Aspartate metabolic pathway

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Isoenzymes : redundant? Curien et al. (2009) Differential spatial and temporal expression patterns AKLYS3 Yoshioka et al. (2001) DHDPS1 Vauterin et al. (1999)

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Introduction  Results  Discussion  Conclusions & perspectives Stress Energy Ethylene , polyamines , glucosinolates , methyldonor (salicylate, jasmonate ,…) Increased resistance to Halyoperonospora arabidopsidis Low energy-state TCA TCA cycle exhaustion , Pro & pipecolic acid (osmolytes) , GABA (signaling) , Arg (precursor for PA, NO) Amino acid transferases ASP2 : 2-OG + Asp  Glu B . cinerea AGD2 : 2-OG + Lys  Glu ? ALD1 : , SA induction P . syringae ? MGL BCAT LKR-SDH Induced by salt and osmotic stress Isoenzymes : redundant? I ncreased expression after infection with B. cinerea

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Roles and regulatory mechanisms of the isoenzymes , especially in stress conditions ?

Overview:

Overview Introduction The aspartate metabolic pathway in Arabidopsis thaliana Regulatory mechanisms Isogenes: redundant or not? Results & Discussion Blocking the flux in the lysine or threonine biosynthetic branch Role of AKLYS1 in biotic stress C onclusions and perspectives Introduction  Results  Discussion  Conclusions & perspectives

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Effects of short-term perturbed flux on : enzymatic activities free amino acid levels transcriptional levels Compare to dhdps1 / dhdps2 KO plants Blocking the flux in the lysine or threonine biosynthetic branch DDAB (N ,N - dimethylglycinato b oranyloxycarbonylmethyl)- dimethylamine - borane) Introduction  Results  Discussion  Conclusions & perspectives

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Blocking the flux in the lysine biosynthetic branch Introduction  Results  Discussion  Conclusions & perspectives DHDPS

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Blocking the flux in the lysine biosynthetic branch Introduction  Results  Discussion  Conclusions & perspectives DHDPS

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Introduction  Results  Discussion  Conclusions & perspectives Blocking the flux in the lysine biosynthetic branch Amino acid levels DHDPS

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Effects of short-term perturbed flux on : enzymatic activities free amino acid levels transcriptional levels Compare to hsk KO plants Blocking the flux in the lysine or threonine biosynthetic branch I nducible HSK silencing (dexamethasone) Introduction  Results  Discussion  Conclusions & perspectives

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Blocking the flux in the threonine biosynthetic branch 10 days 20 days after sowing on DEX containing medium WT HSKis line WT HSKis line Introduction  Results  Discussion  Conclusions & perspectives HSK

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Blocking the flux in the threonine biosynthetic branch Introduction  Results  Discussion  Conclusions & perspectives HSK c fr . Van Damme et al. (2009): 5 % - 10 % residual HSK activity Normal amino acid levels

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Introduction  Results  Discussion  Conclusions & perspectives Blocking the flux in the lysine or threonine biosynthetic branch * * HSK

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Blocking the flux in the threonine biosynthetic branch Amino acid levels * Introduction  Results  Discussion  Conclusions & perspectives HSK

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DHDPS2 : main driver of Lys biosynthesis DHDPS1 : rapid counteraction of decreasing lysine levels Introduction  Results  Discussion  Conclusions & perspectives Blocking the flux in the lysine or threonine biosynthetic branch T ranscriptional levels DHDPS * * * DHDPS HSK

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Introduction  Results  Discussion  Conclusions & perspectives DHDPS2 : main driver of Lys biosynthesis DDAB treatment ≈ dhdps2 KO WT dhdps2 KO

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l DHDPS l > l HSDH l K m,DHDPS ≈ 10 K m, HSDH Curien et al. (2009) K 0.5 , DHDPS1 = 10 µM K 0.5 , DHDPS2 = 30 µM Craciun et al. (2000) Introduction  Results  Discussion  Conclusions & perspectives DHDPS2 : stabilizes flux throughout the pathway

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d hdps1 KO DDAB Introduction  Results  Discussion  Conclusions & perspectives DHDPS2 : stabilizes flux throughout the pathway

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d hdps2 KO DDAB K 0.5, HSDH1 = 407 µM K 0.5 , HSDH2 = 8500 µM Curien et al. (2005) l TS l > l CGS l K m, TS > K m, CGS Adomet activates TS/deactivates CGS Curien et al. (1998, 2003) Introduction  Results  Discussion  Conclusions & perspectives DHDPS2 : stabilizes flux throughout the pathway

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DHDPS2 : influences Thr levels  role? MGL Introduction  Results  Discussion  Conclusions & perspectives MGL and BCAT2 are upregulated during salt and osmotic stress:  Ile, Val, Leu increase TCA Acetyl CoA Glu GDH /ASP2 Overexpression : s usceptible to B. cinerea BCAT2 Activated ENERGY met  AMINO ACID met ? AdoMet

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* Introduction  Results  Discussion  Conclusions & perspectives Blocking the flux in the lysine or threonine biosynthetic branch T ranscriptional levels AKLYS DHDPS HSK * Time after adding DDAB * Time after adding DEX

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Introduction  Results  Discussion  Conclusions & perspectives Short-term Long -term Blocking the flux in the lysine or threonine biosynthetic branch T ranscriptional levels AKLYS

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Introduction  Results  Discussion  Conclusions & perspectives Blocking the flux in the lysine or threonine biosynthetic branch AKLYS1 rapidly growing, young plant parts little variation between different tissues and developmental stages M ain supply of Asp-derived amino acids AKLYS3 vascular tissue rosettes, inflorescence stems, hypocotyls nodes Systemic supply and supplementation Expression analysis

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DHDPS2 main driver of Lys biosynthesis Stabilizes flux throughout the pathway Influences Thr levels Hetero-oligomers with DHDPS1 with enhanced activity Introduction  Results  Discussion  Conclusions & perspectives Blocking the flux in the lysine or threonine biosynthetic branch AKLYS AKLYS1: short -term perturbations AKLYS3: long -term perturbations

Overview:

Overview Introduction The aspartate metabolic pathway in Arabidopsis thaliana Regulatory mechanisms Isogenes: redundant or not? Results & Discussion Blocking the flux in the lysine or threonine biosynthetic branch Role of AKLYS1 in biotic stress C onclusions and perspectives

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Role of AKLYS1 in biotic stress Introduction  Results  Discussion  Conclusions & perspectives D ecrease during maturation C arbohydrate -based activation ( Ufaz et al., 2011) (source – sink)

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Characterizing AKLYS1 expression Introduction  Results  Discussion  Conclusions & perspectives Copy number ?

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Introduction  Results  Discussion  Conclusions & perspectives Characterizing AKLYS1 expression B. cinerea

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Introduction  Results  Discussion  Conclusions & perspectives Characterizing AKLYS1 expression B. cinerea

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Introduction  Results  Discussion  Conclusions & perspectives Characterizing AKLYS1 expression B. cinerea GUS activity in 25 -day-old leaves four days post-inoculation of B. cinerea

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Introduction  Results  Discussion  Conclusions & perspectives Characterizing AKLYS1 expression B. cinerea GUS activity in 25 -day-old leaves four days post-inoculation of B. cinerea A lso observed for defence -related enzymes HR, cell wall strengthening (CWS),… > 2000 proteins in CWS e.g. extensins (Lys- & Val-rich) Amino acid metabolites : Thr /Ile & Lys (energy) , AdoMet ( CWS, Met-SA, Met- JA) , Lys (proline) Induced by B. cinerea ? Phytotoxines , pectine ,…

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Introduction  Results  Discussion  Conclusions & perspectives Mutagenesis of pAKLYS1::GUS seeds 1:1000 M1 plants 4 % in M2 generation

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Introduction  Results  Discussion  Conclusions & perspectives Screening of pAKLYS1::GUS mutants mutants in positive regulators: GUS infected M2/M3 ≤ GUS non-infected M0 - σ mutants in other types of regulators: GUS infected M2/M3 ≥ GUS non-infected M0 + σ 3 M2 15 M2 2 M3 B. cinerea

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Introduction  Results  Discussion  Conclusions & perspectives Response of pAKLYS1::GUS M3 plants in biotic stress

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Introduction  Results  Discussion  Conclusions & perspectives AKLYS1 not involved in primary defence -related responses Higher AKLYS1 expression  no increased containment Insufficient increase of AKLYS1 expression AKLYS1 overexpressers or aklys1 knockouts Response of pAKLYS1::GUS M3 plants in biotic stress

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Introduction  Results  Discussion  Conclusions & perspectives Response of pAKLYS1::GUS M3 plants in abiotic stress Met, Lys, Ile – Leu , Val accumulate in drought stress ( Arabidopsis , tomato, pea) C ross-talk between abiotic – biotic stress-induced responses

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Introduction  Results  Discussion  Conclusions & perspectives Response of pAKLYS1::GUS M3 plants in abiotic stress Leaf surface in osmotic stress Leaf surface in salt stress Leaf surface relative to leaf surface on K1 medium * * * M3 (6D) M0

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Introduction  Results  Discussion  Conclusions & perspectives Response of pAKLYS1::GUS M3 plants in abiotic stress Root length in osmotic stress R oot length in salt stress Leaf surface relative to leaf surface on K1 medium M3 (6D) M0 * * * *

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Introduction  Results  Discussion  Conclusions & perspectives Response of pAKLYS1::GUS M3 plants in abiotic stress C orresponds with AKLYS1 expression Lys metabolites : glutamate  Pro & Arg (polyamines) accumulate in salt & osmotic stress conditions  regulate osmotic pressure, stabilize CWS Met metabolites: AdoMet  polyamines Ile & TCA cycle

Overview:

Overview Introduction The aspartate metabolic pathway in Arabidopsis thaliana Regulatory mechanisms Isogenes: redundant or not ? Results & Discussion Blocking the flux in the lysine or threonine biosynthetic branch Role of AKLYS1 in biotic stress C onclusions and perspectives Introduction  Results  Discussion  Conclusions & perspectives

Conclusions and perspectives:

Conclusions and perspectives DHDPS2 main driver of Lys biosynthesis Stabilizes flux throughout the pathway Influences Thr levels hetero-oligomer formation with DHDPS1 with enhanced activity AKLYS AKLYS1: short -term perturbations AKLYS3: long -term perturbations AKLYS1: involved in response to biotic and abiotic stress Introduction  Results  Discussion  Conclusions & perspectives

Conclusions and perspectives:

Conclusions and perspectives Introduction  Results  Discussion  Conclusions & perspectives DHDPS2 Role of Thr? – Ile – TCA cycle DHDPS hetero-oligomer formation in planta (split-GFP) Structure of hetero-oligomer: catalytic and regulatory properties – Lys inhibition AKLYS B iochemical and biomolecular characterization of the 2 pAKLYS1::GUS mutants. Isolation of mutation(s) : map-based cloning Induction of pAKLYS1 in abiotic stress

Conclusions and perspectives:

Conclusions and perspectives Introduction  Results  Discussion  Conclusions & perspectives More extensive transcriptomics , metabolomics , proteomics Other important crops : e.g. leguminous plants ( Medicago truncatula – 4 DHDPS isoforms)

Questions? :

Questions?

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