NTRC new ways of using NADPH in the chloroplast

Physiologia Plantarum 133: 516–524. 2008 Copyright ª Physiologia Plantarum 2008, ISSN 0031-9317

REVIEW

NTRC new ways of using NADPH in the chloroplastMarıa C. Spınola, Juan M. Perez-Ruiz†, Pablo Pulido, Kerstin Kirchsteiger, Manuel Guinea,Maricruz Gonzalez and Francisco J. Cejudo*

Instituto de Bioquımica Vegetal y Fotosıntesis, Universidad de Sevilla y CSIC, Avda Americo Vespucio 49, 41092 Sevilla, Spain

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 23 November 2007;

revised 5 February 2008

doi: 10.1111/j.1399-3054.2008.01088.x

Despite being the primary source of energy in the biosphere, photosynthesis is

a process that inevitably produces reactive oxygen species. Chloroplasts are

a major source of hydrogen peroxide production in plant cells; therefore,

different systems for peroxide reduction, such as ascorbate peroxidase and

peroxiredoxins (Prxs), are found in this organelle. Most of the reducing power

required for hydrogen peroxide reduction by these systems is provided by Fd

reduced by the photosynthetic electron transport chain; hence, the function ofthese systems is highly dependent on light. Recently, it was described a novel

plastidial enzyme, stated NTRC, formed by a thioredoxin reductase (NTR)

domain at the N-terminus and a thioredoxin (Trx) domain at the C-terminus.

NTRC is able to conjugate both NTR and Trx activities to efficiently reduce

2-Cys Prx using NADPH as a source of reducing power. Based on these results,

it was proposed that NTRC is a new pathway to transfer reducing power to

the chloroplast detoxification system, allowing the use of NADPH, besides

reduced Fd, for such function. In this article, the most important features ofNTRC are summarized and the implications of this novel activity in the context

of chloroplast protection against oxidative damage are discussed.

Signaling and toxic effect of reactiveoxygen species

Photosynthesis, the source of organic material for almost

all living organisms (Nelson and Ben-Shem 2004),

produces reactive oxygen species (ROS), which may be

harmful for the cell. ROS production is increased by

environmental factors that include high light intensity,temperature and other abiotic stresses. In photosystem I

(PSI), the production of superoxide anion (O22 ), which is

disproportionated to hydrogen peroxide (H2O2), is well

documented (Asada 1999), whereas in PSII, several

abiotic stresses promote the formation of singlet oxygen

(1O2) (Ledford and Niyogi 2005). ROS accumulation has

a toxic effect because of the high reactivity of these

species, which may produce lipid peroxidation as well as

oxidation of proteins and nucleic acids, thus causing

damage on cell structures (Apel and Hirt 2004).

Besides the toxic effect of ROS, during the past years,

increasing evidence has been reported on the impor-

tant function of ROS as signaling molecules. In plants,

ROS are involved in the control of several processes ofgrowth and development as well as in response to environ-

mental changes. As examples, these processes include

cell growth (Foreman et al. 2003), ABA-dependent signal-

ing in guard cells (Kwak et al. 2003) or plant defense

mechanisms and cell death (Liu et al. 2007). Moreover,

the redox status of the chloroplast is important to

control chloroplast-related processes such as protein

Abbreviations – APX, ascorbate peroxidase; DHA, dehydroascorbate; FTR, ferredoxin thioredoxin reductase; MDA, mono-

dehydroascorbate; NTR, NADPH thioredoxin reductase; Prx, peroxiredoxin; PS, photosystem; ROS, reactive oxygen species; SOD,

superoxide dismutase; Trx, thioredoxin.

†Present address: Plant Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA

516 Physiol. Plant. 133, 2008

import into this organelle (Kuchler et al. 2002) or

signaling from the chloroplast to the nucleus, the so-

called retrograde signaling (Nott et al. 2006).

Of the different ROS produced in the chloroplast,

singlet oxygen and hydrogen peroxide show the highest

signaling capacity. Both act through specific transductionpathways (Op den Camp et al. 2003), although cross talk

between singlet oxygen and hydrogen peroxide signal-

ing has been described in response to stress in Arabidop-

sis (Laloi et al. 2007). During the past years, significant

progress has been made on the signaling activity of

singlet oxygen that, despite its short diffusion capacity,

triggers a signal transduction cascade able to influence

the expression of a large number of genes (Laloi et al.2006). By contrast, hydrogen peroxide is a molecule with

a lower toxic effect and permeable through biological

membranes, hence showing a higher diffusion rate than

singlet oxygen. The function of hydrogen peroxide as

an important signaling molecule has been shown in

different organisms like yeast or mammals (D’Autreaux

and Toledano 2007, Wood et al. 2003); indeed, in yeast,

hydrogen peroxide is able to activate the expression ofantioxidant genes through the activation of a bZIP trans-

cription factor (Vivancos et al. 2005). In plants, global

expression analysis has revealed that a large number of

genes respond to hydrogen peroxide (Vandenabeele et al.

2003, Vanderauwera et al. 2005). The DNA-binding

activity of a R2R3-Myb factor from maize is enhanced

under reducing conditions (Heine et al. 2004), hence

showing that the redox status of transcription factorsmay be important for transcription regulation in plants

(Wormuth et al. 2007).

Chloroplasts contain different systems forhydrogen peroxide reduction

Given the dual effect of ROS and its high production in

chloroplasts, the existence of different mechanisms to

reduce the level of ROS in this organelle, and to balance

their toxic and signaling effect, is not surprising. In the

case of singlet oxygen, because of its high reactivity and

low diffusion rate, it is essentially quenched by non-enzymatic methods including carotenoids, tocopherols,

glutathione or ascorbate (Apel and Hirt 2004). By con-

trast, reduction of hydrogen peroxide is carried out by

several enzymatic mechanisms. Hydrogen peroxide is

produced by the disproportionation of superoxide anion

catalyzed by superoxide dismutase (SOD). Plants con-

tain several isoforms of SOD with different metals at

their active site and different localizations. Copper/zinc(Cu/ZnSOD) has been described to have cytosolic,

peroxisome, plastidial and extracellular localization,

whereas iron (FeSOD) is plastidial and manganese

(MnSOD) is localized in mitochondria (Kliebestein et al.

1998). Superoxide anion produced at PSI is converted to

hydrogen peroxide by a thylakoid-bound CuZnSOD and

then hydrogen peroxide is reduced to water in a reaction

catalyzed by ascorbate peroxidase (APX), which uses

ascorbate as reducing power. In this process, termed thewater–water cycle (Asada 1999), ascorbate is oxidized

to monodehydroascorbate (MDA). The function of this

system requires the regeneration of ascorbate from MDA,

which is reduced to ascorbate by Fd reduced by the

photosynthetic electron transport chain (Asada 2006).

Part of the O22 produced by PSI escapes to the

chloroplast stroma where it is converted to hydrogen

peroxide (by a stromal CuZnSOD), which is reduced bythe stromal isoform of APX (Asada 1999). In this case, the

oxidized forms of ascorbate, MDA and dehydroascorbate

(DHA), are reduced by the corresponding reductases,

which depend on NADPH (MDA reductase) or reduced

glutathione (DHA reductase). Oxidized glutathione is

reduced by an NADPH-dependent glutathione reductase,

an enzyme encoded by a nuclear gene with a dual transit

peptide so that it is imported to mitochondria andchloroplast (Chew et al. 2003). Therefore, the source of

reducing power to reduce MDA and DHA in the chloro-

plast stroma is provided by NADPH. So, the thylakoid-

bound system formed by CuZnSOD and APX constitutes

the initial detoxification barrier to avoid ROS diffusion in

the chloroplast. The reducing power necessary for this

initial system is obtained from Fd reduced by the

photosynthetic electron transport chain (Fig. 1). Althoughthe APX-dependent reduction of hydrogen peroxide is

considered as an essential mechanism to protect the

photosynthetic machinery under conditions causing

2-CysPrx

H2O

Trx

H2O2 ASC

MDA

APX

Fdred

NADPH

NADP+

DHA

Asc GSH

GSSG

FTR

MDAR

GRDHAR

Fig. 1. Reduced Fd is a major source of reducing power for hydrogen

peroxide reduction in the chloroplast. Hydrogen peroxide produced at PSI

is reduced by thylakoid-bound APX using ascorbate as reducing power

(blue) or 2-Cys Prx (green). The source of reducing power for both systems

is Fd reduced by the photosynthetic electron transport chain (yellow)

either directly reducing MDA to ascorbate (ASC) or through the FTR/Trx

pathway to the 2-Cys Prx. Reduction of hydrogen peroxide also occurs in

the stroma (gray); in this case, MDA is reduced by an NADPH-dependent

MDA reductase (MDAR) and DHA by DHA reductase (DHAR) using GSH.

GSSG is reduced by the NADPH-dependent glutathione reductase (GR).

Physiol. Plant. 133, 2008 517

photooxidative damage, genetic evidence has shown that

this system is as well important in the absence of stress

(Rizhsky et al. 2003).

In addition to the APX-based detoxification system,

plants contain another peroxide detoxification system

based on peroxiredoxins (Prxs). In Arabidopsis, the genefamily for Prx is composed of 9 or 10 genes, 4 of which,

encoding 2-Cys PrxA, 2-Cys PrxB, PrxQ and PrxIIE, are

targeted to chloroplasts (Dietz 2003, Dietz et al. 2002).

The presence of 2-Cys Prx in the chloroplast was initially

reported by Baier and Dietz (1997). 2-Cys Prxs are

dimeric enzymes that reduce hydrogen peroxide by

a reaction mechanism that involves the participation of

two Cys residues, located in each subunit of the enzyme(Dietz 2003, Konig et al. 2003). The first Cys residue,

termed peroxidatic, attacks the peroxide and becomes

oxidized to sulfenic acid, which then reacts with the

second, resolving Cys, with the loss of a molecule of

water. Both Cys residues form a disulfide bridge upon

catalysis. For a new catalytic cycle, this disulfide bridge

has to be reduced with the participation of thioredoxins

(Trxs), of which different types exist in the chloroplast(Lemaire et al. 2007). Because chloroplast Trxs use

reducing power from reduced Fd, in a reaction catalyzed

by ferredoxin thioredoxin reductase (FTR), it is assumed

that Prx-dependent peroxide reduction depends on Fd

reduced by the photosynthetic electron transport chain

(Fig. 2A). Therefore, reduced Fd is a major source of

reducing power for hydrogen peroxide reduction in

chloroplasts.

Although the catalytic efficiency of 2-Cys Prx is low,

these proteins are very abundant in chloroplasts. So, it has

been proposed that the participation of these enzymes in

chloroplast protects against oxidative damage (Konig

et al. 2002). However, the relatively poor effect of re-

duced level of 2-Cys Prxs in transgenic lines of Arabidopsisexpressing an antisense gene (Baier and Dietz 1999), as

well as the lack of phenotype under standard growth

conditions of a double mutant of Arabidopsis with severely

reduced levels of 2-Cys Prxs (P. Pulido and F. J. Cejudo,

unpublished results), suggests that these peroxidases are

not essential for plant protection against oxidative dam-

age probably because of the redundancy of function of

the different Prxs present in chloroplasts. Regarding theAPX-dependent system, wheat mutants with reduced

level of thylakoid-bound APX are viable, but exhibit a

clear phenotype of impaired photosynthetic activity (Danna

et al. 2003), and transgenic tobacco plants expressing

antisense thylakoid-bound APX were not viable (Yabu-

ta et al. 2002). However, a null mutant in stromal and

thylakoid APX of Arabidopsis showed necrosis in re-

sponse to excess light only when the level of ascorbateis low (Giacomelli et al. 2007). Altogether, these

results suggest that there is compensation effect among

the different detoxification systems in plant cells.

NTRC, a novel enzyme conjugating NTR andTrx activity

In contrast to other organisms like yeast or mammals,plants contain a large gene family encoding Trxs (Meyer

et al. 2005). While reduction of chloroplast Trxs requires

reduced Fd in a reaction catalyzed by FTR (Schurmann

and Jacquot 2000), cytosolic and mitochondrial Trxs are

reduced by an NADPH-dependent thioredoxin reductase

(NTR) (Fig. 2A) (Laloi et al. 2001). NTRs contain FAD as

co-factor so that these enzymes are able to transfer

reducing power from NADPH to FAD and then to thedisulfide forming its active site, which is able to reduce

the disulfide formed by the active site Cys residues of

oxidized Trx (Fig. 2A). This NADP–Trx system is univer-

sally found in all types of organisms. However, evolu-

tion has produced two types of NTRs (Williams et al.

2000): prokaryotes, lower eukaryotes and plants contain

a low molecular mass NTR formed by two identical

subunits of about 35 kDa, whereas mammal NTR isformed by two identical subunits of larger molecular mass

(approximately 55 kDa) and with a characteristic con-

served selenocysteine residue, which plays an important

role in catalysis, at the C-terminus (Gladyshev et al.

1996).

NTRs from plants are of the low molecular mass type

and are phylogenetically related to the enzyme from

S

SPrx

SH

SHPrx

ROOH

ROHS

STrx

SH

SHTrx

NADPH NTR

Fdred FTRA

NADPH

NADP+ FAD

FADH2SHSH

SS

SHSH

SS

B

NTR domain Trx domain

Fig. 2. NTRC is an NADP–Trx system in a single polypeptide. (A)

Hydrogen peroxide reduction by 2-Cys Prx requires reducing power

supplied by reduced Trx. Trxs are reduced by two pathways: from reduced

Fd in a reaction catalyzed by FTR or by NTR, the NADP–Trx system (in the

square). (B) NTRC is a bifunctional enzyme formed by an NTR domain at

the N-terminus and a Trx domain at the C-terminus.


bacteria and lower eukaryotes (Serrato et al. 2002).

Despite the large gene family for Trx in plants, only two

genes, termed ntra and ntrb, encode NTR in Arabidopsis

and rice (Serrato et al. 2004). In Arabidopsis, both gene

products, NTRA and NTRB, are targeted to the cytosol

and the mitochondria, although NTRB is the predominantenzyme in the mitochondria (Reichheld et al. 2005).

Surprisingly, an ntra-ntrb double mutant of Arabidopsis is

viable (Reichheld et al. 2007), suggesting that additional

reductants of non-chloroplast Trx may exist in plants.

The search of genes encoding NTR, or NTR-like, in

Arabidopsis and rice identified a new gene, stated ntrc,

whose deduced polypeptide, NTRC, showed a high

level of identity with NTRA and NTRB (Serrato et al.2002). The characteristic features of these enzymes,

NADPH- and FAD-binding motifs as well as the double

Cys forming the active site, were conserved in NTRC.

However, the deduced NTRC polypeptide showed two

peculiar features: an extension at the N-terminus with

a high probability to serve as signal peptide to target the

enzyme to chloroplast or mitochondria and an extension

at the C-terminus (of approximately 14 kDa) containingthe motif -CGPC-, a putative active site of Trxs (Serrato

et al. 2001). Truncated polypeptides containing either the

NTR or the Trx domains of NTRC were expressed in

Escherichia coli. Both truncated polypeptides showed the

expected activity (Serrato et al. 2004), hence demon-

strating that NTRC contains the activities of an NADP–Trx

system in a single polypeptide (Fig. 2B).

The search of this novel gene in different organismsrevealed its presence in plants, algae and some, not all,

cyanobacteria, that is, NTRC is exclusive of organisms

able to perform oxygenic photosynthesis. This finding

suggested that the putative transit peptide found in the

deduced polypeptide might target the protein to the

chloroplast. That this is the case was shown with an anti-

NTRC antibody and purified chloroplasts from rice and

Arabidopsis (Serrato et al. 2004) and was confirmed bythe expression of an NTRC::GFP fusion protein in Ara-

bidopsis (Moon et al. 2006). Initial phylogenetic analysis

showed that plant and cyanobacterial NTRC form a group

separated from NTRs from plants and lower eukaryotes

(Serrato et al. 2004), clearly indicating the endosymbiotic

origin of the plant gene. A similar protein composed of

NTR and Trx domains was described in a strain of

Mycobacterium leprae, which is able to use NADPH toreduce insulin, that is, functions as a NADP–Trx system in

a single polypeptide (Wieles et al. 1995a, 1995b).

However, other strains of M. leprae contain contiguous

genes encoding NTR and Trx, suggesting that this single

gene was produced after the mutation of a stop codon

separating both genes in the M. leprae strain. In support

of this view, the M. leprae NTR is related to NTRs

from other prokaryotes but not with NTRC (Serrato et al.

2004).

The phylogenetic relationship of each of the domains of

NTRC with NTRs and Trxs from different organisms was

analyzed. The NTR domain of NTRC is more related to

NTRCs from cyanobacteria than to NTRs from plants,lower eukaryotes or bacteria (Fig. 3). Similarly, the Trx

domain of NTRC shows closer phylogenetic proximity to

cyanobacterial NTRCs than to any of the plant Trxs.

Therefore, the phylogenetic analysis on NTRC and their

domains suggests that the plant gene has an endosymbi-

otic origin, not related with other NTRs or Trxs previously

described in plants.

NTRC is a high-efficiency system forreduction of 2-Cys Prxs

Taking into account that NTRC is a bifunctional enzyme,

with NTR and Trx activities and its localization in the

chloroplast, the enzyme might function either as NTR or

as Trx. In the case that it acts as NTR, it might be able to

reduce plastidial Trxs. The NTR activity of NTRC wastested in vitro in the presence of several plastidial Trxs

including f, m, x and CDSP32. No activity was obtained

with any of these Trxs either in terms of NADPH oxidation

or insulin reduction (Serrato et al. 2004). The other

possibility is that NTRC might act as NTR–Trx system, in

which Trx domain serves as reductant of Prxs or other

redox-regulated enzymes of the chloroplast. This possi-

bility was tested by the incubation of purified recombi-nant NTRC in the presence of 2-Cys Prx, hydrogen

peroxide and NADPH, which resulted in the reconstitu-

tion of a hydrogen peroxide reduction system in vitro. The

activity of this system could be determined either by

NADPH oxidation or by peroxide reduction (Perez-Ruiz

et al. 2006). Moreover, 4-acetamido-4#-maleimidylstil-

bene labeling of thiol groups of the 2-Cys Prx revealed

that NTRC was able to reduce the two disulfides formedby the active site Cys residues of the dimeric Prx.

Therefore, these results showed that NTRC is actually

a novel enzyme conjugating NTR and TRX activities to

efficiently reduce 2-Cys Prx (Fig. 2B). To our knowledge,

this is the only enzyme described in eukaryotes able to

perform such activity, which therefore seems to be

restricted to the chloroplast and to photosynthesis-related

processes. So far, no indication has been reported of theactivity of the cyanobacterial NTRC; so, it is not yet

known whether NTRC has the same function in cyano-

bacteria and plants.

Although not present in eukaryotes, bacteria have an

enzyme, termed AhpF, which actually shows the same

activity described for NTRC; that is, the reduction of

a bacterial 2-Cys Prx, termed AhpC (Poole et al. 2000a).


However, important differences exist between NTRC

and AhpF. First, NTRC is NADPH dependent, whereas

AhpF shows preference for NADH as an electron donor.

Second, NTRC contains a unique Trx domain localizedat the C-terminus of the enzyme, whereas AhpF con-

tains two Trx (or Trx-like) folds, only one of them with

the characteristic active site of Trxs (Poole et al. 2000b,

Wood et al. 2001). It is considered that AhpF evolved

from bacterial Trx reductase based on sequence

similarity (Poole et al. 2000a); however, as discussed

above, NTRC seems to have its origin in a cyanobacte-

rial gene and therefore it is not phylogenetically relatedto AhpF, as shown by the phylogenetic tree of the NTR

domains of NTRC and AhpF, and NTRs from different

sources (Fig. 3).

NTRC allows the use of NADPH forperoxide reduction

As stated before (Fig. 1), two major systems have been

described for peroxide reduction in the chloroplast, onebased on APX and the other on 2-Cys Prx. Both systems

obtain most of the required reducing power from Fd

reduced by the photosynthetic electron transport chain,

which is used to directly reduce MDA to ascorbate (Asada

2006) in the APX-dependent system or through FTR

and Trx in the 2-Cys Prx-dependent system. Among the

different Trxs present in the chloroplast, x-type Trx is the

most efficient electron donor to 2-Cys Prxs (Collin et al.

2003). CDSP32, a protein containing two Trx folds, only

one of them with the characteristic Trx active site, has

been shown to interact in vitro and in vivo with 2-Cys Prxs(Rey et al. 2005) and was proposed as its electron donor

(Broin et al. 2002). However, the rate of peroxide

reduction by CDSP32 is rather low (Perez-Ruiz et al.

2006) so that it is unlikely that the actual function of this

peculiar Trx is to serve as reductant of 2-Cys Prxs.

So far, the proposal of the involvement of the FTR/Trx

(Trx x or CDSP32) pathway as reductant of 2-Cys Prxs

is based on in vitro results, but no deficient mutants ineither of these Trxs have been reported. The character-

ization of such mutants will be required before a conclu-

sion on the involvement of any of these Trxs in the

mechanism of protection against oxidative damage of

the chloroplast can be drawn. By contrast, an Arabidopsis

mutant deficient in FTR has been described, which is

hypersensitive to abiotic stress (Keryer et al. 2004). The

phenotype of this mutant points to the involvement ofthe FTR/Trx pathway in the mechanism of peroxide

detoxification; however, it should be taken into account

that FTR is the reductant of different chloroplast Trxs,

most of them involved in the redox regulation of

many different aspects of the chloroplast metabolism,

as shown by the large number of Trx targets identified

so far (Buchanan and Balmer 2005). Therefore, it is not

clear that the abiotic stress hypersensitivity phenotype

Fig. 3. Phylogenetic tree of NTRs and the NTR domain of NTRCs (or AhpF) from different sources. The phylogenetic tree was constructed using the

neighbor-joining method of the program CLUSTALX, version 1.8, with full-length NTR amino acid sequences and the NTR domain from AhpF or NTRC from

Arabidopsis (AtNtrC), rice (OsNtrC) and different cyanobacteria (Nostoc1, Nostoc2, Synechococcus and P. marinus). Bootstrap analysis was computed

with 1000 replicates. Accession numbers are AtNtrA (NP_179334.3), AtNtrB (NP_195271.1), OsNTRA (BAD33510), OsNTRB (BAD07786), AtNtrC

(NP_565954.1),OsNtrC (CAE46765), Schizosaccharomyces pombe (CAA17692), Saccharomyces cerevisiae (1, P29509; 2, AAA64747.1), Yersinia pestis

(NP_404967), Escherichia coli (P09625), Homo sapiens (1, XP_049211), Nostoc sp. 7120 (1, alr2204; 2, NP_484780), Synechococcus sp. WH 8102

(NP_896780), Prochlorococcus marinus (NP 893267) and Salmonella typhimurium AhpF (P19480).


shown by the FTR-deficient mutant is exclusively because

of the impairment of transfer of reducing power to the

2-Cys Prxs.

While the FTR/Trx system depends on Fd reduced by

the photosynthetic electron transport chain, the NTRC-

dependent pathway uses NADPH as source of reduc-ing power (Perez-Ruiz et al. 2006, Serrato et al. 2004).

This property opens the possibility to use NADPH as

an alternative source of electrons for the 2-Cys Prx-

dependent peroxide reduction system. The fact that

both the ascorbate and the Trx-dependent pathways use

reduced Fd as the major source of reducing power im-

plies that these systems are fully functional under condi-

tions of illumination when the photosynthetic electronchain is highly operative. However, during darkness, the

amount of reduced Fd by the photosynthetic electron flow

drops. Although under these conditions ROS production

is lower than under illumination, it has been reported that

growth in long nights causes oxidative stress because of

ROS produced during respiration (Bechtold et al. 2004)

and therefore reducing power may still be required to

keep the antioxidant systems operative. The kineticfeatures of NTRC, high affinity for NADPH and high

catalytic efficiency for Prx reduction, allow the use of

NADPH as source of reducing power for the Prx-

dependent hydrogen peroxide reduction. In chloroplasts,

NADPH is produced from Fd reduced in the photosyn-

thetic electron flow. However, under darkness, NADPH

may be also produced by the initial reactions of the

oxidative pentose phosphate pathway, catalyzed byglucose-6P dehydrogenase and 6P-gluconate dehydro-

genase (Fig. 4). If this is the case, the function of NTRC

might be more important under darkness. In support of

this view, the phenotype of the NTRC-deficient mutant of

Arabidopsis is more evident when plants are grown under

short-day conditions. Similarly, treatments of prolonged

darkness have a more drastic effect on mutant than onwild-type plants (Perez-Ruiz et al. 2006).

Concluding remarks and future prospects

The two most important hydrogen peroxide reduction

systems described in chloroplasts, APX and 2-Cys Prxs,

rely essentially on Fd reduced by the photosynthetic

electron transport chain as the initial source of reducing

power. The properties of the recently described enzyme

NTRC, containing both NTR and Trx domains in a single

polypeptide, allow the use of an alternative source ofreducing power, NADPH, for 2-Cys Prx-dependent re-

duction of hydrogen peroxide. Because NADPH can be

produced in chloroplasts during darkness in the reactions

catalyzed by dehydrogenases of the oxidative pentose

phosphate pathway, NTRC may be important under these

conditions. Some of the phenotypic features of the

Arabidopsis knockout mutant for NTRC are more evident

when plants are grown under short-day conditions,suggesting that NTRC function becomes more important

under these conditions. However, NTRC-deficient plants

show a strong phenotype including retarded growth,

irregular morphology of mesophyll cells, alteration of

chloroplast structure with lower content of photosyn-

thetic pigments and lower rate of CO2 fixation. This

phenotype suggests additional functions of NTRC. A

possibility is that NTRC might serve as a link between the2-Cys Prx and the APX-dependent detoxification sys-

tems if it is able to reduce MDA (or DHA) to ascorbate

(Fig. 4). Although NTRC shows DHA reductase activity (J.

M. Perez-Ruiz and F. J. Cejudo, unpublished results), the

low rate of reduction obtained suggests that this is not an

important function of NTRC. Additionally, NTRC may be

involved in the use of NADPH for different functions

related not only with the mechanism of peroxidedetoxification but also with chloroplast metabolism or

even with signaling. The finding of these new functions

of NTRC will require the identification of novel

NTRC targets as well as an in-depth characterization

of redox imbalance in the chloroplast of the NTRC

knockout mutant subjected to different abiotic stress

treatments.

Acknowledgements – Work in our laboratory was supported

by grant BIO2004-02023 from Ministerio de Educacion y

Ciencia (Spain) and grant CVI-182 from Junta de Andalucıa

(Spain).

2-Cys Prx

H2O

FTR/TrxH2O2 Asc

MDA

APX

Fd

NADPH

NADP+

DHA

Asc GSH

GSSG

NTRC

NADPH

¿?

Glc6P Ru5P

LIGHT

Fig. 4. NTRC is an alternative pathway to transfer reducing power, from

NADPH, to 2-Cys Prx. NTRC is a stromal enzyme showing high affinity for

NADPH and a high catalytic efficiency for 2-Cys Prx reduction. These

kinetic properties allow the proposal that NTRC is an alternative

pathway for the 2-Cys Prx-dependent reduction of hydrogen peroxide.

NADPH is produced not only from reduced Fd by the photosynthetic

electron transport chain (yellow) but also from the conversion of

glucose-6-phosphate in ribulose-5-phosphate, in the oxidative pentose

phosphate pathway (black), which allows the function of the system

under darkness.


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Edited by K.-J. Dietz


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NTRC new ways of using NADPH in the chloroplast