Plant Cell Physiol. 38(12): 1382-1388 (1997)
JSPP © 1997
Expression of the Plastid ndhF Gene Product in Photosynthetic and NonPhotosynthetic Tissues of Developing Barley Seedlings
Rafael Catala, Bartolome Sabater and Alfredo Guera
Departamento de Biologia Vegetal, Universidad de Alcald de Henares, Alcald de Henares, 28871-Madrid, Spain
A fragment of the NDH-F subunit of the plastid
NAD(P)H dehydrogenase complex (NAD(P)H-plastoquinone-oxidoreductase) from barley was expressed as a fusion protein in Escherichia coli and an antibody to the fusion protein was prepared. Western blot analysis using the
anti-NDH-F antibody showed specificity towards a plastid
poiypeptide of approximately 70 kDa present in both photosynthetic and non-photosynthetic barley tissue. The poiypeptide was found in thylakoid membranes of green leaves
whereas in etiolated leaves it was shown to be associated
with the membrane fraction of etioplasts. NDH-F levels
were higher in roots and etiolated tissue than in greening or
young leaves. During leaf ontogeny, NDH-F levels decreased from young to mature tissue but increased during
senescence. The accumulation of NDH-F in thylakoids of
young leaves was stimulated by photooxidative treatment.
The results indicate a high degree of expression of plastid
ndh genes (which encode NAD(P)H dehydrogenase subunits) in non-photosynthetic plastids and under conditions
which impair the photosynthetic activity of chloroplasts. In
addition to its putative implication in photosynthetic electron transport, a non-photosynthetic role, such as chlororespiration, is proposed for the plastid NAD(P)H dehydrogenase complex.
Key words: Barley (Hordeum vulgare) — Chloroplasts —
Etioplasts — ndh Genes — Photooxidative stress — Senescence.
The presence of ndh genes in the plastid genome of
higher plants was first reported after sequencing the chloroplast genomes of Marchantia polymorpha (Ohyama et al.
1986) and tobacco (Shinozaki et al. 1986). These genes encode polypeptides homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase complex. Further
investigation has shown that the ndh genes are present in
most of the studied plastid genomes of vascular plants
(Hiratsuka et al. 1989, Freyer et al. 1995, Maier et al. 1995)
although they have not been identified in the chloroplast
genomes of some Gymnosperms (Wakasugi et al. 1994) or
Abbreviations: LSU, large subunit of ribulose bisphosphate
carboxylase/oxygenase; FNR, ferredoxin-NADP + reductase;
NDH, plastid NAD(P)H dehydrogenase complex.
parasitic plants (Wolfe et al. 1992, Haberhausen and
Zetsche 1994). ndh Genes are transcribed in different plants
(Matsubayashi et al. 1987, Schantz and Bogorad 1988,
Kanno and Hirai 1993, Maier et al. 1995) but, to date, only
the products corresponding to five of the eleven ndh genes
have been identified (Steinmetz et al. 1986, Wu et al. 1989,
Berger et al. 1993, Guedeney et al. 1996, Martin et al.
1996).
Cuello et al. (1995a, b) described NAD(P)H dehydrogenase activity associated with a large protein complex in
barley thylakoids and suggested that this complex may correspond to the products of ndh genes. Results obtained in
a study of potato thylakoid membranes showed that an
NAD(P)H dehydrogenase protein complex similar to that
reported by Cuello et al. (1995a, b) contained polypeptides
encoded by ndhB and ndhJ (Guedeney et al. 1996).
The function of a putative NAD(P)H dehydrogenase
complex in plastids of higher plants remains unknown. It
has been proposed that the plastid NAD(P)H dehydrogenase complex could be involved in a chlororespiration process or in photosynthetic cyclic electron transport. Evidence
supporting the former hypothesis includes studies carried
out in green algae (Scherer 1990) although several lines of
investigation propose that chlororespiratory activity also
occurs in higher plants (Bennoun 1982, Garab et al. 1989).
In contrast, some authors suggest a role for the complex in
photosynthetic cyclic electron transport since, in Ct plants,
there is a greater abundance of NDH polypeptides in the
bundle sheath (which present high cyclic and low linear
electron transport activity) than in mesophyll chloroplasts
(Kubicki et al. 1996). In addition, it has been shown in
Synechocystis that the NAD(P)H dehydrogenase complex
is involved in both cyclic electron flow and respiratory flow
(Mi et al. 1995). Whichever the role of plastid NAD(P)H dehydrogenase, most authors consider that it might play a
part in the regulation of NAD(P)H/NAD(P) + and ATP/
ADP ratios in chloroplasts. Finally, it is postulated that the
plastid NAD(P)H dehydrogenase complex may be involved
in protection against photooxidative stress (Martin et al.
1996).
It is evident that valuable insight into the function of
the plastid NAD(P)H dehydrogenase complex would be
gained by determining its location and its time of expression in the developing plant. The present investigation
describes the identification of the NDH-F poiypeptide (the
ndhF gene product) in barley plastids and its expression in
1382
Expression of NDH-F
different tissues under different developmental conditions.
The results show that NDH-F levels were higher in non-photosynthetic tissues or in leaves with declining photosynthetic activity (senescent or under strong photooxidative stress).
This finding suggests a non-photosynthesis-dependent role
for the NAD(P)H dehydrogenase complex.
Materials and Methods
Growth of plants—Barley (Hordeum vulgare L. cv. Hassan)
was grown on vermiculite at 23 °C under a 16-h photoperiod of 20
W m~2 white light as described previously (Martin et al. 1996).
Etiolated leaves were obtained by growing plants for 6 d under
similar conditions in the dark. Greening leaves were obtained by
exposure of etiolated plants to continuous light of 20 Wra~2(low
irradiance) or 550 W m'1 (high irradiance) for 18 h. Photooxidative stress was induced by spraying leaves grown under conditions
of low irradiance with a solution of 50 or 500//M paraquat.
Preparation of antibodies—The anti-NDH-F antibody was
prepared by amplification of a 762 bp fragment of the ndhF gene
of barley from cDNA using the primers 5'GGGGATCCGCGTTTTATATGTTTCGG3' and 5'CCCTGCAGATTCCATCAATGACACC3' as described previously (Martin et al. 1996). This fragment is homologous to the region comprised between positions
101565-102312 of the rice chloroplast genome (Hiratsuka et al.
1989). The amplified sequence was inserted into the polylinker
cloning region of the expression vector pUEXl (Amersham,
U.K.). The resulting plasmid pUEF contained the barley ndhV
fragment and encoded the aminoacid sequence H2N-AFYMFRIYLLTFGGYLRVHFQNYSSTKESSLYSISLWGKRIPKGVNRDFVLSTTKSGVSFFSQNIPKIQGNTRNRIGSFTTSFGAKNTFAYPHETGNTMLFPLLILLLFTLFIGFIGISFDNGGMDNGIAELTILSVTLAIFGLFIAYIFYGSAYSFFQNLDLINSFVKRNPKKEFLDQVKKNIYSWSYNRGYIDIFYTRVFTLGIRGLTELTEFFDKGVIDGI-COOH (GenBank, Ace. no. U220O3) fused in frame
with the carboxy-terminal region of the lacZ. pUEF was cloned in
E. coli using standard cloning techniques performed according to
Sambrook et al. (1989). Nucleotide and protein sequences were analyzed using PC/GENE software (Genofit, Germany). The production and purification of the fusion protein was performed
as described in Martin et al. (1996). The polyclonal antibody
anti-NDH-F was prepared by Cambridge Research Biochemicals
(U.K.) using the purified fusion protein as antigen. Antibody to
the LSU was as described by Martin et al. (1996). An antibody
which recognizes the Dl protein of maize (Barkan, 1993) was provided by Dr. Alice Barkan (Univ. of Oregon, U.S.A.).
Preparation of plant extracts and subcellular fractions—To
prepare whole protein extracts from roots or leaf segments, samples were frozen in liquid N2 and homogenized in a mortar until a
fine powder was obtained. This powder was resuspended in a medium containing 0.05 M Tris-HCl pH 6.8, 0.1% SDS and 1 mM
Na2EDTA. The suspension was then mixed in an Omni-mixer (Sorvall, U.S.A.), filtered through 6 layers of muslin and centrifuged
at 20,000 x g for 10 min. The resulting supernatant was considered
the whole protein extract. Etioplasts and their membranes were obtained basically as described by El Amrani et al. (1994). The detailed procedure was as follows: Approximately 50 g of etiolated
leaves were excised and immediately placed in 200 ml of icecold grinding buffer (50 mM HEPES-NaOH pH 7.6, 0.33 M sorbitol, 2 mM ascorbic acid, 1 mM MgCl2, 1 mM MnCl2, 2 mM
Na2EDTA, 0.1% BSA) and homogenized in an Omni-mixer. The
homogenate was then filtered through 6 layers of muslin and cen-
1383
trifuged at 500 x g for 5 min. The supernatant was centrifuged at
2,500 x g for 10 min and the resulting pellet was gently resuspended in a small volume of grinding buffer and centrifuged on a
preformed 5 to 80% (v/v) Percoll gradient (Pharmacia, Uppsala,
Sweden) in grinding buffer. The yellow band of intact etioplasts
was diluted with 10 volumes of washing medium (50 mM HEPESNaOH pH 7.6, 0.33 M sorbitol, 2 mM Na2EDTA) and sedimented
at 2,500 xg for 10 min. The resulting pellet was washed again in
washing buffer to obtain intact etioplasts or resuspended in hypotonic buffer (50 mM HEPES-NaOH, pH 7.6) and centrifuged
at 6,000 x g to obtain etioplast membranes. Each step was performed at a temperature between 0 and 4°C. Mitochondria were
obtained from etiolated leaves as described by Moreau and
Romani (1982) using a self-generated Percoll gradient. Intact chloroplasts were obtained as described previously (Guera et al. 1993).
Thylakoids and other subchloroplastic fractions were obtained
from intact chloroplasts following osmotic shock. In detail, intact
chloroplasts were pelleted and resuspended in 10 mM TricineNaOH pH 7.6, 4mM Mg2Cl for a few seconds. After this treatment, isotonic conditions were restored by adding 1 M sucrose in
10 mM Tricine-NaOH pH 7.6, 4 mM MgClj to produce a final sucrose concentration of 0.3 M. The burst chloroplasts were centrifuged at 6,000 x g for 15 min. The thylakoid pellet was washed in
10 mM Tricine-NaOH, 4mM MgCl2, 0.3 M sucrose and the
supernatant was centrifuged in a discontinuous sucrose gradient
to obtain stroma and chloroplast envelopes as described previously (Guera et al. 1993).
Other procedures—SDS-PAGE was performed as described
by O'Farrel (1975). Western blotting was performed according to
the procedure described by Towbin et al. (1979). Immunodetection was performed using goat anti-rabbit IgG antiserum linked to
horseradish peroxidase (Bio-Rad) as the secondary antibody. Color was developed using tetramethyl-bencidine and H2O2 as substrates. Protein was determined by the method of Lowry et al.
(1951) or Bradford (1976). Chi quantification was carried out
according to Lichtenthaler (1987). Semiquantitative analysis of
NDH-F and LSU was performed by densitometry of immunolabelled membranes using an UVP Easy digital image analyzer.
Results
Identification and localization of NDH-F in barley
leaves—A 762 bp barley cDNA fragment encoding several
putative antigenic determinants of barley NDH-F was cloned into the expression vector pUEX. The resulting fusion
protein was purified and antibody to the protein was generated as described in the Materials and Methods section.
Western blotting confirmed the specificity of the antibody
for a protein extract from barley chloroplasts. The results
showed that serum from the rabbit immunized against the
fusion protein reacted with a 70 kDa polypeptide found in
barley chloroplasts (Fig. 1A). Similar results were obtained
with protein extracts from wheat or oat chloroplasts (data
not shown). The molecular mass calculated for the polypeptide detected using the anti-NDH-F antibody was approximately 10% lower than expected for the ndhF gene product (approximately 80 kDa deduced from the nucleotide
sequence). Similar differences have been reported previously for other ndh gene products (Berger et al. 1993, Guedeney
et al. 1996, Martin et al. 1996) suggesting that these gene
1384
Expression of NDH-F
B
i
kDa
chl
P-I
S E T
kDa
86-
86-
51-
51-
34-
34-
28-
Fig. 1 Characterization of the anti-NDH-F antibody. A: 8/ig
total chloroplast protein from 7 d-old barley plants were loaded
per lane. After SDS-PAGE and transference to PVDF membranes, each lane was incubated with pre-immune (P-I) or immune (I) rabbit anti-NDHF sera. B: Immunodetection of NDH-F
in barley chloroplasts. 8 n% protein from whole chloroplast (chl),
stroma (S), envelope (E) and thylakoid (T) fractions were separated by SDS-PAGE, transferred to PVDF membranes and immunolabelled with anti-NDH-F antibody. The migration of molecular
weight standards is shown in the left margin.
products may be post-translationally processed. In several
experiments, an additional band of slightly lower molecular mass than that of the 70-kDa polypeptide was detected.
This secondary band probably corresponded to a degrada-
tion product of NDH-F as previously described for NDHA (Martin et al. 1996). Once the chloroplasts were fractionated, the NDH-F antibody was able to recognize the
70-kDa polypeptide in thylakoid preparations but not in
the stromal or envelope fractions (Fig. IB). The antibody
did not react with mitochondria (Fig. 2B) or other subcellular fractions. It may, thus, be concluded that the NDH-F
antibody showed specificity for a 70 kDa polypeptide of
the thylakoidal membrane This is in agreement with the
subcellular localization reported for other NDH polypeptides (Berger et al. 1993, Guedenay et al. 1996, Martin et al.
1996).
Identification and localization of NDH-F in non-photosynthetic tissues of barley—Information with respect to
the localization of NDH proteins in non-photosynthetic tissues is scarce. Berger et al. (1993) reported that appreciable
levels of NDH-H were present in etioplasts of sorghum and
maize. In order to test whether NDH-F was also expressed
in etiolated leaves of barley, etioplasts (Fig. 2A, lane 1) and
etioplast membranes (Fig. 3A, lane 1) were isolated from
B
1 2 3
kDa » « • * m>
1
2
3
kDa
86-*•
51-
B
kDa
1
3428-
2
70kDa
m5134- »
28-
i
i:
32kDa
Fig. 2 Subcellular localization of NDH-F in etiolated leaves. A:
SDS-PAGE and Coomassie staining of etioplasts (lane 1) and mitochondria (lane 2) isolated from barley seedlings grown for 6 d in
the dark. B: As in A but after SDS-PAGE, proteins were transferred to a PVDF membrane and the presence of NDH-F was detected using the corresponding antibody. The migration of the molecular weight standards (panel A) or calculation of the molecular
weight of the polypeptide detected by the anti-NDH-F antibody
(panel B) are shown in the left margin. 10/ig total protein were
loaded per lane.
Fig. 3 Expression of NDH-F and Dl protein in etioplasts and
greening plastids. A: SDS-PAGE and Coomassie staining of etioplast membranes isolated from leaves grown for 7 d in the dark
(lane 1). Thylakoids from leaves grown for 6 d in the dark and for
24 additional hours in continuous light of 20 W m~2 (lane 2) or
550 W m~2 (lane 3). 10/ig total protein were loaded per lane. B:
As in A, but after electrophoresis proteins were transferred to a
PVDF membrane and immunodetected using anti-NDH-F antibody. C: As in B but immunodetection was carried out with an
antibody against the D 1 protein.
Expression of NDH-F
seedlings grown for seven d in the dark and the levels
of NDH-F, detected by Western blotting, compared with
those present in thylaloids of greening leaves. It was found
that NDH-F was, in fact, present in the barley etioplasts
(Fig.2B, lane 1). In addition, results showed that levels of
NDH-F were higher in etioplast membranes than in thylakoids of plants grown for six d in the dark followed by 18 h
of illumination (Fig. 3B, lanes 1 and 2 respectively). Lowest
NDH-F levels were detected when etiolated plants were exposed to higher fluence light (Fig. 3B, lane 3). As a control,
the amount of D 1 protein was estimated. This protein is
not detectable in etioplasts and may only be appreciated
after illumination (Fig. 3C). As a further control, mitochondria were isolated from etiolated leaves (Fig.2A, lane 2)
and shown not to react with the NDH-F antibody (Fig. 2B,
lane 2).
To investigate the presence of NDH-F in other nonphotosynthetic tissues, the total protein contents of root
and leaf segments of seven-d barley seedlings were extracted. NDH-F levels in roots were compared with those
along the leaf gradient. Highest levels of NDH-F were
found in roots (r) and in the most basal segment of the
leaves which included basal meristematic tissue (1) (Fig. 4).
NDH-F
1 2
3
4
1385
Levels were lower in the two medial segments (2 and 3) and
in the apical section (4). Similar results were obtained for
NDH-A (unpublished data). These findings confirmed the
presence of NDH-F in non photosynthetic tissues and suggest its differential accumulation during the ontogeny of
the leaf.
Variation of NDH-F levels during leaf ontogeny—
Variation of NDH-F levels in the primary leaf of barley
plants from the 7th to the 21st d of growth was determined.
Levels of chl, protein and LSU were also determined for
comparative purposes. The levels of NDH-F and LSU were
determined after Western blotting of whole protein extracts of leaves including the same amount of total protein
per sample (10 ^g). After immunodetection, membranes
were scanned and bands quantified as described in the
Materials and Methods section. Maximum values obtained
for total protein and chl per g of leaf, LSU and NDH-F
were taken to represent 100%. Figure 5 shows that the
levels of NDH-F fell from the 7th to the 14th d of growth,
i.e. during the period of maximal expansion of the leaves.
After the 14th d of growth the development of the first
symptoms of leaf senescence was indicated by the loss of
chl and LSU, but the amount of NDH-F increased with respect to total protein and reached maximum values in the
most advanced stages of leaf senescence (25th d).
Expression of NDH-F under conditions ofphotooxidative stress—In a recent report (Martin et al. 1996), the
70kDa
LSU
1
2 3
4020--
55kDa
Fig. 4 Expression of NDH-F in different sections of barley seedlings. Whole protein extracts were obtained as described in
Materials and Methods, from roots (r) or different sections (1 to 4
of approx. 2 cm length) of 7 d-barley seedlings. Section 1 corresponds to the most basal region of the primary barley leaf including the basal meristem. Sections 2 and 3 correspond to the medial-basal and medial-apical segments of the primary barley leaf
and section 4 corresponds to the most apical segment. After SDSPAGE, proteins were transferred to PVDF membranes and levels
of NDH-F (upper panel) or LSU (lower panel) were immunodetected with the corresponding antibodies. 10 /ig total protein were
loaded per lane. Corresponding molecular weights are shown on
the left.
Days after sowing
| Belli Dprotein DLSU • NDH-F|
Fig. 5 Expression of NDH-F during leaf ontogeny. Chl (black
bars), proteins (white bars), LSU (light grey bars) and NDH-F
(dark grey bars) were determined as described in Materials and
Methods in the primary leaf of barley seedlings at the times indicated. Results represent the mean of at least three independent
experiments and are expressed as the percentage relative to the
maximum value obtained for each parameter. Standard errors exceeding 5% are shown. See text for details.
Expression of NDH-F
1388
125
0
100 200 300 400 500
Paraquat (|iM)
70kDa
Fig. 6 Expression of NDH-F under conditions of induced photooxidative stress. A: Induction of NDH-F levels after 24 h-treatment of barley seedlings with the indicated amounts of paraquat.
B: Thylakoids were isolated from untreated seedlings (lane 1) or
seedlings treated for 48 h with 50 uM paraquat (lane 2). Western
blotting was performed after SDS-PAGE and NDH-F was detected using the corresponding antibody.
authors described an increase in NDH-A levels when detached leaves were subjected to photooxidative treatment
(100 W m~ 2 +100% O J . In the present study, whole barley
plants were treated with paraquat under low irradiance
conditions to induce photooxidative stress. As shown in
Fig. 6A, treatment of 6-d old plants for 24 h with increasing amounts of paraquat produced a corresponding rise in
NDH-F levels. The increment was greater when treatment
with paraquat was maintained for 48 h (Fig. 6B).
Discussion
Recent reports have confirmed the existence of several
plastid polypeptides with homology to those of the mitochondrial complex I (Berger et al. 1993, Guedeney et al.
1996, Kubicki et al. 1996, Martin et al. 1996). NAD(P)H dehydrogenase activity, probably associated with the putative
plastid complex formed by these polypeptides, has also
been proposed (Cuello et al. 1995a, b, Guedeney et al.
1996). The present investigation extends previous studies
and demonstrates the expression of the barley NDH-F polypeptide. The results show that, as described for other NDH
polypeptides, NDH-F is located in the thylakoid membranes of barley chloroplasts.
Although Berger et al. (1993) described the presence of
NDH-H in etioplasts of sorghum and maize, information
concerning the expression of NDH polypeptides in nonphotosynthetic tissues is scarce. This investigation confirmed that the NDH-F polypeptide of barley was present in
etioplasts. Furthermore, NDH-F was also shown to be expressed in barley roots. These results cannot be due to a
product of cross-reaction with mitochondrial proteins since
the antibody did not react with isolated mitochondria. In
addition, the results indicate that the amount of NDH-F diminishes during greening which contrasts with the accumulation of plastid encoded polypeptides clearly implicated in
photosynthesis (Dl, LSU) after illumination of etiolated
leaves. It is, therefore, concluded that the function of
NDH polypeptides may not be limited to photosynthetic
cyclic electron transport. Interestingly, several isoforms of
ferredoxin NADP + reductase (FNR) and ferredoxin are
present in etiolated leaves or roots (Green et al. 1991). An
association between the plastid NAD(P)H dehydrogenase
complex and FNR has recently been described in potato
(Guedeney et al. 1996) and may suggest that an electron
chain including ferredoxin, FNR and the NAD(P)H dehydrogenase complex could be functional in non-photosynthetic tissues.
The results also suggest that the expression of NDH-F
was lower in mature, fully photosynthetically functional
leaf tissues (where the relative levels of chlorophyll and
LSU are highest) than in young or senescent tissues. Further, high levels of NDH-F were induced by photooxidative
treatment which damage the photosynthetic machinery of
plastids. Similar results were recently described for NDHA levels in detached leaves subjected to dark-induced senescence or photooxidative treatment (Martin et al. 1996). Previous results showed that the transcript levels of ndhB
increased during natural senescence (Martinez et al. 1997)
and that the transcript levels of ndhY also increased under
conditions of photooxidative stress (unpublished data).
This suggests that the expression of ndh genes may be regulated during transcription or post-transcriptional processing. Such data are also more consistent with a non-photosynthetic role (such as chlororespiration) for the NAD(P)H
dehydrogenase complex than with the putative photosynthesis-related function of the complex. It has also been pro-
Expression of NDH-F
posed that the increase in NADH dehydrogenase activity
may reflect the dissipation of the NADH produced during
the break down of excess carbon produced by the degradation of aminoacids in senescent leaf chloroplasts (Cuello et
al. 1995b) or represent a protection mechanism against increased photooxidative stress (Martin et al. 1996). The enhancement of activated oxygen metabolism during natural
leaf senescence has been previously described (Pastori and
del Rio 1997). The present data may be consistent with
mechanisms leading to increased NDH-F levels in senescent
leaves triggered by the accumulation of active-oxygen species. Moreover, an increase in chlororespiratory activity under situations of environmental stress has been previously
described (Scherer 1990). It is postulated that a loss of photosynthetic activity (mediated by photooxidative stress or
other stress) may be accompanied by an increase in chlororespiratory activity although this hypothesis requires further evidence.
In conclusion, the results presented in this paper show
that high levels of NDH-F are found in non-photosynthetic
tissues or leaves which show declining photosynthetic activity. This finding is consistent with a respiratory function
for the plastid NAD(P)H dehydrogenase complex in higher
plants in addition to its putative implication in photosynthetic electron transport.
The authors wish to thank Drs. A. Barkan and R. Maier for
providing the anti-D 1 antibody, Dr. J. Salinas for critical revision
of the manuscript and Dr. M. Martin for technical assistance and
critical discussion. The project was financed by a grant from the
Spanish DGICYT (PB93-0479-C02-01).
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(Received May 20, 1997; Accepted October 8, 1997)