Institut de Biologie des Plantes - UMR 8618

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Composition du groupe :

• Anne-Sophie Bohrer (Docteur)
• Gilles Innocenti (Ingénieur UPS)
• Emmanuelle Issakidis-Bourguet (CR1 CNRS)
• Myroslawa Miginiac-Maslow (Chercheur bénévole)
• Hélène Vanacker (Maître de Conférences)

Redox Signaling Team

OVERVIEW OF RESEARCH THEME

The importance of redox regulation linked to changes in the thiol/disulphide status of proteins in response to perturbations of the cell redox balance is now well-established. Among the key players of this regulation are ubiquitous proteins named thioredoxins (TRX).

TRX are small-sized oxidoreductases catalysing dithiol/disulphide exchanges with their target proteins. Unlike non-photosynthetic organisms, plants possess many isoforms of TRX : more than 20 TRX genes were found in the model higher plant Arabidopsis thaliana (Arabidopsis).

In photosynthetic eukaryotes, TRX are present in the cytosol, mitochondria, nucleus and numerous isoforms are also found in plastids (10 members in Arabidopsis subdivided into 5 types : f, m, x, y and z types). In the chloroplast, biochemical studies initially identified two types of TRX, named the f and the m types according to their respective target enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase. With the completion of plant genomes sequencing additional plastidial TRX isoforms were found and named TRX x, y and z in absence of known related functions. Biochemical studies which we have initiated showed that TRX x and y isoforms are unable to regulate enzymes involved in carbon metabolism but rather have an anti-oxidant function, serving as electron donors (reducing power) for enzymes involved in detoxification of reactive oxygen species (ROS) or in the repair of their deleterious effects. These data strongly suggested that, in plastids, TRX isoforms can have specific roles but this finding remained to be validated in planta.

Further on, searching for new protein targets for the numerous plant TRX, proteomic approaches, to which we have participated, revealed more than 100 plastidial targets implicating TRX in many putative new functions. Most of them remain to be explored.

Aims and Strategies Our main objectives are to : (1) Characterize / validate TRX-dependency of known or suspected new targets and compare the reactivity of plastidial TRXs from Arabidopsis (Biochemical approaches : redox chemistry, recombinant proteins), (2) Validate biochemical results in planta and further evaluate the physiological contribution of plastidial TRXs (T-DNA mutants of Arabidopsis) in optimal and challenging growth conditions.

Recent major results In vitro studies Redox regulation of Glucose-6-Phosphate dehydrogenase. G. Née, PhD 2011 Plants, like all other living organisms, use the reducing power of NADPH in many metabolic processes. In plastids, NADPH is supplied by photosynthesis and the oxidative pentose phosphate pathway which is the major source for reducing power in non-photosynthetic tissues or under non-photosynthetic conditions. The enzyme glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and committed reaction of this pathway. Previous work has shown that G6PDH plastidial isoforms were redox-regulated via disulfide reduction by TRX but all the TRX types were not tested and compared. We tested the regulatory capacities of 7 TRX isoforms, representative of the 5 plastidial types, towards G6PDH1, the main leaf G6PDH isoform in Arabidopsis. The results showed marked specificities of the various TRX isoforms. TRX m and y were efficient, but, most interestingly, Trx f was a very efficient regulator of G6PDH1 activity. Light/dark modulation of G6PDH1 was reproduced in vitro in a reconstituted ferredoxin/TRX system using Trx f allowing to propose a new function for this Trx isoform co-ordinating both reductive (Calvin-Benson cycle) and oxidative pentose phosphate pathways (Née et al., 2009).

TRX dependent-redox regulation of enzymes involved in starch degradation. Collaborations : F. Sparla, univ. Bologna, Italy ansd G. Moorhead, univ. Calgary, Canada. Starch breakdown provides plants with necessary carbon reserves for growth at night. Several proteins are involved in starch degradation including BAM1, a plastid-targeted β-amylase of Arabidopsis, and the glucan phosphatase Starch Excess4 (SEX4) required for proper starch breakdown. In the framework of collaborations with two laboratories respectively studying these enzymes, we could show that TRX f and m can very efficiently modulate their activity in vitro (Valerio et al., 2011 ; Silver et al., 2013).

Biochemical studies of TRXs x, y and z. V. Massot, Ph D 2008 and A-S. Bohrer, Ph D 2012 The tenth plastidial TRX was only recently identified in Arabidopsis. Proteomics studies have found TRX z in a high molecular weight complex as an essential sub-unit of the plastid encoded RNA polymerase. It was known that TRX f and m are reduced in the light by the ferredoxin / TRX reductase (FTR). We could show the FTR dependent reduction of TRX x and y using a physiologically relevant reconstituted light reducing system. We have biochemically characterized Arabidopsis TRX z and found that it has unusual physico-chemical properties, making it unique among plastidial TRXs. TRX z, which is expressed in green tissues in the light, is the first plastidial TRX not reduced by FTR but can be reduced by TRXs f and m, revealing a previously unknown interconnection between plastidial TRX isoforms (Bohrer et al., 2012).

Functional specificities of plastidial TRX as evidenced by in vitro studies are summarized if.

In planta studies

Function of y-type TRXs in the regulation of methionine sulfoxide reductase (MSR). Collaboration P. Rey, CEA Cadarache, France Oxidized methionine in proteins can be reduced back to Met by methionine sulfoxide reductases (MSRs) A and B. In collaboration with the team headed by P. Rey (CEA, Cadarache) we had previously shown that in vitro MSRB2 was supplied with reducing power by TRXs, with y-type isoforms being the most efficient. Three MSR isoforms, MSRA4, MSRB1 and MSRB2 are present in Arabidopsis chloroplasts. Under conditions of high light and long photoperiod, plants knocked-down for each plastidial MSR type or for both of them display reduced growth. In contrast, over-expression of plastidial MSRBs is not associated with beneficial effects suggesting a physiological limitation of an increase in the amount of MSR in the chloroplast. To identify the physiological reductants for plastidial MSRs, mutants deficient for TRXs f, m, x or y were studied. Results show that mutant lines lacking both TRX y1 and y2 or only TRX y2 specifically display a significantly reduced leaf MSR capacity (- 25%) and growth rate under high light, related to those of plants lacking plastidial MSRs. Thus, we could propose that TRX y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs (Laugier et al., 2013).

Function of TRX f1 in starch synthesis. Collaboration P. Geigenberger, LMU, Munich, Germany

Biochemical studies have shown that TRX f is an important regulator of enzymes involved in primary metabolism, however, genetic evidence for its physiological importance was largely lacking. To test the functional significance of Trx f in vivo, two Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in TRX f leaf content almost to the detection limit. While their growth rates and photosynthetic parameters remained unchanged, trx f1 mutant lines showed a decrease in leaf starch accumulation, an increase of sucrose levels and a decrease in starch-to-sucrose ratio during the day. Analysis of metabolite levels indicated carbon shortage in the TRX f1-mutant leaves at the end of night. To investigate the reason for the inhibition of starch synthesis, redox-activation of ADP-glucose pyrophosphorylase (AGPase), the key-enzyme of starch synthesis and a known target of TRX, was studied. Knock-out of TRX f1 led to a strong attenuation in reductive light-activation of AGPase in leaves during the day. Lack of TRX f1 also impaired light-activation of AGPase in isolated chloroplasts, while sucrose-dependent redox-activation of the enzyme in darkened leaves was not affected. Overall, these results provided in planta evidence for the role played by TRX f in the light-activation of AGPase and photosynthetic carbon partitioning in plants (Thormählen et al., 2013).

Recent publications

 Laugier, E., Tarrago, L., Courteille, A., Innocenti, G., Eymery, F., Rumeau, D., Issakidis-Bourguet, E., and Rey, P. (2013) Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light. Plant Cell Environ. 36 : 670-682.

 Thormählen, I., Ruber, J., Von Roepenack-Lahaye, E., Ehrlich, S.M., Massot, V., Hümmer, C., Tezycka, J., Issakidis-Bourguet, E., and Geigenberger, P. (2013) Inactivation of thioredoxin f1 leads to decreased light activation of ADP-glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants. Plant Cell Environ. 36 : 16-29.

 Silver, D.M., Silva, L.P., Issakidis-Bourguet, E., Glaring, M.A., Schriemer, D.C., and Moorhead, G.B. (2013) Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation. FEBS J. 280 : 538-548.

 Bohrer, A.S., Massot, V., Innocenti, G., Reichheld, J.P., Issakidis-Bourguet, E., and Vanacker, H. (2012) New insights into thereduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis. J. of Exp. Botany 63 : 6315-6323.

 Valerio, C., Costa, A., Marri, L., Issakidis-Bourguet, E., Pupillo, P., Trost, P., and Sparla, F. (2011) Thioredoxin-regulated beta-amylase (BAM1) triggers diurnal starch degradation in guard cells, and in mesophyll cells under osmotic stress. J of Exp Botany, 62 : 545-555.

 Marchand, C., Vanacker, H., Collin, V., Issakidis-Bourguet, E., Le Maréchal, P., and Decottignies P. (2010) Thioredoxins targets in Arabidopsis roots. Proteomics 10 : 2418-2428.

 Née, G., Zaffagnini, M., Trost, P., and Issakidis-Bourguet, E. (2009) Redox regulation of chloroplastic glucose-6-phosphate dehydrogenase : a new role for f-type thioredoxin. FEBS Lett. 583 : 2827-2832.

 Marri, L., Zaffagnini, M., Collin, V., Issakidis-Bourguet, E., Lemaire. S.D., Pupillo, P., Sparla, F., Miginiac-Maslow, M., and Trost, P. (2009) Prompt and easy activation by specific thioredoxins of Calvin cycle enzymes of Arabidopsis thaliana associated in the GAPDH/CP12/PRK supramolecular complex. Molecular Plant 2 : 259-269.