Phosphoregulation of the photorespiratory enzyme, glycolate oxidase, in response to light and CO2 content in Arabidopsis thaliana

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Phosphoregulation of the photorespiratory enzyme, glycolate oxidase, in response to light and CO2 content

in Arabidopsis thaliana

Younes Dellero, Edouard Boex-Fontvieille, Sophie Massot, Valérie Flesch, Françoise Gilard, Marlène Davanture, Michel Zivy, Guillaume Tcherkez,

Michael Hodges, Mathieu Jossier

To cite this version:

Younes Dellero, Edouard Boex-Fontvieille, Sophie Massot, Valérie Flesch, Françoise Gilard, et al..

Phosphoregulation of the photorespiratory enzyme, glycolate oxidase, in response to light and CO2 content in Arabidopsis thaliana. SPS Conference 2013: Plant signalling in a changing environment, Jul 2013, Evry, France. �hal-03128225�

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Phosphoregulation of photorespiration : an unexplored domain

Phosphoregulation of the photorespiratory enzyme, glycolate

oxidase, in response to light and CO ₂ content in Arabidopsis thaliana

Our phosphoproteome analysis revealed that photorespiratory GOX can be differentially phosphorylated with respect to C0₂ content and light/dark transition. GOX phosphorylation-mimic mutants at T4 and T158 have modified enzymatic parameters that indicate that GOX phosphorylation could regulate photorespiratory flux. Further work will be carried out to understand in planta the role of these phosphorylation events and their impact on plant metabolism including the Krebs cycle to limit the carbon and nitrogen loss.

Younes Dellero

¹

, Edouard Boex-Fontvieille

¹

, Sophie Massot

¹

, Valérie Flesch

¹

, Françoise Gilard

^1,2

, Marlène Davanture

³

, Michel Zivy

³

, Guillaume Tcherkez

^1,2

, Michael Hodges

¹

, Mathieu Jossier

¹

*

Conclusions

Log (IsotopicRatio)

100 380 1000 Dark Light

A quantitative phosphoproteome revealed a differentially phosphorylated residue of GOX at T158.

100 380 1000

Dark Light

Km (µM) Chloroplast

Peroxisome

Mitochondrion

SHMT

CO₂ NH₄⁺

2-P-Glycolate

GS2 Fd-GOGAT

Glycolate

GOX

O₂

H₂O₂ H₂O CAT

½ O₂

Glyoxylate

Glycine Serine

Hydroxypyruvate Glycerate 3-P-Glycerate

Calvin cycle

Gln

ATP ADP

Glu 2OG

Glu

Glu 2OG GGT

NH₄⁺

PGLP

Pi

DiT2 DiT1

HPR

NADH NAD⁺

ATP ADP

NAD⁺ NADH

P

P P

P

P P

Cytosol

P

GDH GPD

LPD GDT

P

GDC

P

P P

Glycolate Glycerate

Glycine SGT

P

Serine GLYK

RuBP

O₂

RuBisCO

P

mRNA abundance

Leaf GOX activity

Ab antiAtGOX2 Coomassie blue

Col-0 gox2.1 gox2.2 gox1

Mutation at T4 or T158 modifies GOX kinetic parameters

atgox1 and atgox2 are affected in organic acid content but show no growth phenotype in air

• Photorespiration is an essential process for all oxygenic photosynthetic organisms (Bauwe et al., 2010). It allows the recycling of the toxic metabolite 2-phosphoglycolate produced by Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) oxygenase activity. The photorespiratory cycle has often been described as ‘wasteful’ to plant productivity as it leads to the production of CO₂ and ammonia that must be re- assimilated.

• It is of paramount importance for plants to be capable of modifying their metabolism in response to rapidly changing environmental conditions such as light/dark transitions and changing CO₂ content. Although little is known about photorespiratory cycle regulation, the PhosPhAt database indicates that many photorespiratory enzymes can be phosphorylated, thus suggesting that regulation by protein phosphorylation is a possible mechanism.

• We present results of a quantitative phosphoproteome analysis revealing that amongst the 8 photorespiratory enzymes, the glycolate oxidase (GOX) is differentially phosphorylated in response to light/dark transition and to altered photorespiratory conditions. We examined each GOX phosphorylation site identified in our phosphoproteome analysis (T158 and T360) and in PhoPhAt (T4) using mutated recombinant GOX proteins to evaluate the impact of each phosphorylation events on enzymatic parameters.

• It should be noted that Arabidopsis rosette leaves contains two photorespiratory GOX genes: GOX1 (At3g14420) & GOX2 (At3g14415).

A global quantitative phosphoproteomics analysis was undertaken using leaf extracts from Arabidopsis thaliana plants subjected to a 4 h light-dark transition or a 4 h light either at 100ppm, 380ppm or 1000ppm CO₂ in 21% O₂ to modify the photorespiration rate (oxygenation-to-carboxylation ratio). 264 phosphopeptides showed a significant change in their phosphorylation status between the different conditions (99% confidence level) (a). One of these phosphopeptides corresponded to the photorespiratory enzyme GOX (T158 of AtGOX1 and AtGOX2) (b). Mutation of T158, T360 (a 2nd phosphorylated residue identified in our phosphoproteome) and T4 (present in the PhosPhAt database) (c) either to a valine or an alanine (to suppress phosphorylation) or to an aspartic acid (to mimic a phosphorylation) modified the kinetic parameters of recombinant AtGOX1 and AtGOX2 (d).

To investigate the consequences of a reduced GOX activity on leaf photorespiration and primary metabolism, T-DNA insertion mutants of AtGOX1 and AtGOX2 exhibiting different GOX activities, protein and mRNA levels (a) were subjected to GC-MS metabolomic analyses. Our data show a strong decrease of several organic acid contents in atgox1 and atgox2 rosette leaves (b). The knock-down of both GOX genes in amiR-gox1/2 plants show a "photorespiratory" phenotype in a normal atmosphere (c) although they grow normally in air containing 3000ppm CO₂. This mutant will be used as a tool to study the consequence of GOX phosphorylation in planta by introducing mutated forms of maize GOX1 into the amiRNA line.

Hodges et al, 2013

Phosphorylated enzymes of the phostorespiratory cycle according to the PhosPhAt database.

Hodges et al, Plant Biology, 2013

1 Institut de Biologie des Plantes, Saclay Plant Sciences, Université Paris Sud, 91405 Orsay cedex, France

2 Plateforme Métabolisme Métabolome, IFR87 La Plante et son Environnement, Institut de Biologie des Plantes, Université Paris Sud, 91405 Orsay cedex, France

3 INRA, Plateforme d'Analyse Protéomique de Paris Sud Ouest (PAPPSO), 91190 Gif-sur-Yvette, France

*Corresponding author : mathieu.jossier@u-psud.fr

AtGOX1 / AtGOX2

Threonine 158 T4

Identified in PhosPhAt database

T158

2^nd phosphorylated threonineT360

detected in our phosphoproteome

0 20 40 60 80 100

WT T4V T4D

WT T158V T158D

WT T360A T360D

% Activityof wild-type protein

200 200 2100

Km (µM) ^{232 ± 88} ^{117 ± 34} ^-

Km (µM) ^{241 ± 57} ^{212 ± 38} ^{229 ± 53}

PhosPhAt (http://phosphat.mpimp-golm.mpg.de/)

99% confidence level

(a) (b) (c) (d)

T158 FMN T4 FMN

Active site amino acids

Mimic a phosphorylation (T4D and T158D) or suppress it (T158V) leads to the inhibition of recombinant GOX1 and GOX2 activity in vitro .

% Activityof wild-type protein% Activityof wild-type protein

Vmax

Phosphorylated residues in 3D structure of spinach GOX

Even if atgox1 and atgox2 do not show a developmental phenotype, several

organic acids of the Krebs cycle are less abundant. While amiR-gox1/2 plants with almost no GOX activity exhibit a "photorespiratory" phenotype in air.

Protein abundance

0 20 40 60 80 100 120

go2.1

(nmol/min/mg protein)

* *

*

Col-0 gox2.1 gox2.2 gox1 0

1 2 3 4 5 6

Col-0 go2.1 go2.2 go1

mRNA/ Actine2-8

GO2 GO1

*

* *

Col-0 gox2.1 gox2.2 gox1 GOX1

GOX2 Citrate

Malate

0 0,5 1 1,5 2 2,5 3

Col-0 1 AmirGO C 1

mRNA abundance

GOX1 GOX2

mRNA/ Actine 2-8

Col-0 amiR-gox1/2

(a) (b) (c)

* * *

Arbitraryunit

Bauwe et al, Trends in Plant Science, 2010

GABA

Succinate