|
Plant Physiol. (1998) 116: 725-732
Formate Dehydrogenase, an Enzyme of Anaerobic Metabolism, Is
Induced by Iron Deficiency in Barley Roots1
Kazuya Suzuki,
Reiko Itai,
Koichiro Suzuki,
Hiromi Nakanishi,
Naoko-Kishi Nishizawa,
Etsuro Yoshimura, and
Satoshi Mori*
Laboratory of Plant Molecular Physiology, Department of Applied
Biological Chemistry, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku,
113 Tokyo, Japan (R.I., H.N., N.-K.N., E.Y., S.M.); and Core Research
for Evolutional Science and Technology, Japan Science and Technology
Corporation, 2-1-6 Sengen, 305 Tsukuba, Japan (Ka.S., Ko.S.,
S.M.)
 |
ABSTRACT |
To identify the proteins induced by
Fe deficiency, we have compared the proteins of Fe-sufficient and
Fe-deficient barley (Hordeum vulgare L.) roots by
two-dimensional polyacrylamide gel electrophoresis. Peptide sequence
analysis of induced proteins revealed that formate dehydrogenase (FDH),
adenine phosphoribosyltransferase, and the Ids3
gene product (for Fe deficiency-specific) increased in Fe-deficient
roots. FDH enzyme activity was detected in Fe-deficient roots but not
in Fe-sufficient roots. A cDNA encoding FDH (Fdh) was
cloned and sequenced. Fdh expression was induced by Fe
deficiency. Fdh was also expressed under anaerobic
stress and its expression was more rapid than that induced by Fe
deficiency. Thus, the expression of Fdh observed in
Fe-deficient barley roots appeared to be a secondary effect caused by
oxygen deficiency in Fe-deficient plants.
| |
INTRODUCTION |
In Fe-deficient calcareous soils graminaceous plants secrete
mugineic acid family phytosiderophores, which are natural Fe chelators,
from the roots (Takagi, 1976 ) to solubilize Fe required for plant
growth. This Fe-acquisition mechanism in graminaceous plants is called
strategy II and in nongraminaceous plants it is called strategy I
(Takagi et al., 1984; Marschner et al., 1986). The pathway of the
biosynthesis of mugineic acid family phytosiderophores has been
established (Mori and Nishizawa, 1987, 1989; Shojima et al., 1989,
1990; Mori et al., 1990; Ma and Nomoto, 1993). Among the enzymes
involved in this biosynthetic pathway, Higuchi et al. (1994, 1996)
purified nicotianamine synthase and Kanazawa et al. (1994) purified
nicotianamine aminotransferase. Comparison of 2D profiles of proteins
in barley (Hordeum vulgare L.) roots under Fe-sufficient and
Fe-deficient conditions (Suzuki et al., 1995, 1997) allowed us to
identify a 36-kD protein that was specifically induced by Fe
deficiency. In addition, several genes related to the Fe-deficiency
response have been reported: Ids1 (Okumura et al., 1991),
Ids2 (Okumura et al., 1994), and Ids3 (Nakanishi
et al., 1993). In this study, we characterized several other proteins induced by Fe-deficiency stress in barley roots, one of which was
identified as FDH. FDH was induced not only by Fe deficiency but also
by anaerobic stress. The relationship between Fe deficiency and
anaerobic stress in barley roots is discussed.
| |
MATERIALS AND METHODS |
Plant Material and Growth Conditions
Seeds of barley (Hordeum vulgare L. cv Ehimehadaka no.
1) were germinated at room temperature on paper towels soaked with distilled water. Plants were transferred 4 d after germination to
a plastic net floating on tap water at pH 5.5 in a greenhouse under
natural light. On d 10, plants were transferred to a continuously aerated nutrient solution of the following composition: 0.7 mm K2SO4, 0.1 mm KCl, 0.1 mm
KH2PO4, 2.0 mm
Ca(NO3)2, 0.5 mm MgSO4, 10 µm
H3BO3, 0.5 µm
MnSO4, 0.2 µm
CuSO4, 0.5 µm
ZnSO4, 0.01 µm (NH4)6Mo7O24,
and 0.1 mm Fe-EDTA. The pH of the culture solution was
adjusted to 5.5 daily with 1 n HCl. Fe deficiency was
started on d 20 using the same solution, but without Fe-EDTA. The
nutrient solution was changed every 7 d. Plant roots were
harvested 40 d after germination. Anaerobiosis was achieved by
bubbling nitrogen gas through the nutrient solution overnight to purge
oxygen gas in the solution, followed by a continuous flow of nitrogen
gas throughout the anaerobic experiment.
Protein Extraction for 2D PAGE
The procedure for extraction of proteins was as described by
Damerval et al. (1986) with slight modifications. The roots were homogenized in liquid nitrogen with a mortar and pestle, and the powder
was resuspended in a cold solution of 10% (w/v) TCA in acetone with
0.1% (v/v) 2-ME. Proteins were allowed to precipitate for 60 min at
 20°C and were then centrifuged at 16,000g for 30 min at
4°C. The supernatant solution was discarded and the pellet was rinsed
with cold acetone containing 0.1% (v/v) 2-ME for 60 min at 20°C
and then centrifuged at 16,000g for 30 min at 4°C. The
supernatant solution was discarded and the pellet was dried under
reduced pressure, dissolved (50 µL mg 1 dry
weight) in sample buffer (9.5 m urea, 2% [w/v] Triton
X-100, and 5% [v/v] 2-ME), and centrifuged at 16,000g for
10 min at room temperature. The supernatant solution was used for 2D
PAGE. Protein concentrations were estimated by the method of Bradford
(1976).
2D PAGE
2D PAGE was performed following the method of O'Farrell (1975).
Gel length in the column (2.5 × 130 mm) was 100 mm. To cover the
pI range from 5.0 to 8.0, the gel contained 1.6% (v/v) pH 5.0 to 8.0 ampholines and 0.4% (v/v) pH 3.0 to 10.0 ampholines. Protein extracts
(200 µg) were subjected to IEF at 400 V for 15 h and at 800 V
for 1 h, and the gels were equilibrated for 15 min in the SDS-PAGE
sample buffer (2.3% [w/v] SDS, 10% [w/v] glycerol, 5% [v/v]
2-ME, 62.5 mm Tris-HCl, pH 6.8, and 0.1% [w/v]
bromphenol blue), before loading onto slab gels for 12.5% (w/v)
SDS-PAGE in the second dimension. The gel was stained with 0.25% (w/v) Coomassie brilliant blue R-250 in a mixture of 50% (v/v) methanol and
10% (v/v) acetate and destained in a solution of 50% (v/v) methanol
and 10% (v/v) acetate.
Chemical and Enzymatic Digestion of Proteins and Amino Acid
Sequence Analysis
Chemical or enzymatic digestion was used to determine the internal
sequence of the proteins. Chemical digestion with CNBr was according to
the method of Gross (1967) with the following modifications. Isolated
protein spots from 50 2D PAGE gels were pooled by electroblotting onto
a PVDF membrane according to the method of Towbin et al. (1979).
Proteins were eluted from the membrane by soaking in a 10-fold volume
of 70% (v/v) formic acid containing 1% (w/v) CNBr in a 1.5-mL
microtube and incubating overnight at 4°C. The supernatant was
collected, dried under reduced pressure, resuspended in the SDS-PAGE
sample buffer, and incubated overnight at room temperature. Enzymatic
digestion was performed according to the method of Cleveland et al.
(1977) or Aebersold et al. (1987). After digestion of proteins, the
peptides were separated by electrophoresis using Tricine/SDS-PAGE
(Schägger and von Jagow, 1987) in 16.5% (w/v) acrylamide gels.
Peptides were transferred by electroblotting onto a PVDF membrane and
stained with Coomassie brilliant blue. Each band on the PVDF membrane was cut out and the amino acid sequence was determined by automated Edman degradation on a gas-phase sequencer (model 477A protein sequencer and model 120A PTH analyzer, Applied Biosystems).
FDH Assay
For the assay of FDH activity, the roots were homogenized in
liquid nitrogen with a mortar and pestle, and the powder was resuspended in the following buffer: 100 mm Tris-HCl, pH
8.0, 1 mm PMSF, 1 mm EDTA, and 0.2% (w/v)
Triton X-100. FDH activity on nondenaturing polyacrylamide gels was
visualized according to the method of Uotila and Koivusalo (1979) as
follows. A 7% (w/v) native acrylamide gel was used with the
discontinuous buffer system of Laemmli (1970). One-hundred micrograms
of soluble protein from roots or leaves was fractionated at 100 V for
2 h at room temperature and then incubated in darkness for 30 min
at room temperature in the following solution: 100 mm
sodium phosphate buffer, pH 7.0, 50 mm sodium formate, 0.8 mm NAD+, 0.03 mg
mL1 phenazine methosulfate, and 0.4 mg
mL1 nitroblue tetrazolium.
Cloning of cDNA Encoding FDH
A pYH23 cDNA library prepared from poly(A+)
RNA of Fe-deficient barley roots (kindly provided by Hirotaka
Yamaguchi, The University of Tokyo) was screened with an FDH PCR
product corresponding to the partial amino acid sequences of FDH:
GGIGTITTYTAYCARGCIGGIGARTAY and GCRTCIGCIACIGCYTGIGTRTCCAT
(shaded sequences in Fig. 3). The PCR probe was labeled with a
random-primer-labeling kit (version 2, Takara Biomedicals,
Gennevilliers, France) in the presence of
[ -32P]dATP. The cloned cDNA was sequenced
according to the protocol of a sequencing kit (Dye Terminator Cycle
Sequencing Ready Reaction kit, Perkin-Elmer) using a DNA sequencer
(model A373, Applied Biosystems). Hybridization probes for Southern
blotting were prepared by digesting the cloned cDNA corresponding to
the FDH gene (Fdh) with SacI, and the smaller
fragment (Fig. 3, underlined) was radiolabeled as described above. The
labeled DNA was purified on a Nick column (Pharmacia) and used as a
probe for both Southern and northern hybridization analyses.
View larger version (56K):
[in this window]
[in a new window]
| Figure 3.
DNA and deduced amino acid sequence of
barley FDH. Position of primers for PCR are shaded and the
SacI fragment is underlined.
|
|
Genomic Southern Hybridization
Barley genomic DNA was prepared from leaves by the method of
Murray and Thompson (1980) using cetyltrimethylammonium
bromide. The DNA was digested with BamHI, EcoRI,
or HindIII, separated on a 0.8% (w/v) agarose gel (30 µg
per lane), and alkali-transferred onto a nylon membrane
(Hybond-N+, Amersham). The membrane was
hybridized with the labeled SacI fragment of Fdh
with 5× SSPE, 4× Denhardt's solution, and 100 µg
mL1 salmon-sperm DNA at 65°C overnight. The
washing conditions were three times with 2× SSPE plus 0.1% (w/v) SDS
at 65°C for 30 min.
RNA Isolation and Northern Hybridization
Total RNA was isolated from roots or leaves according to the
procedure of Logmann et al. (1987). RNA (10 µg per lane) was separated on 1.2% agarose gels containing 5% (v/v) formaldehyde and
blotted onto nylon membranes (Hybond-N+,
Amersham). The membrane was hybridized with the SacI
fragment of Fdh under the same conditions described above.
The washing conditions were: 6× SSPE at 65°C for 10 min and then
twice with 2× SSPE plus 0.1% (w/v) SDS at 65°C for 10 min. The
radioactivity was detected and quantified using an image analyzer
(BAS-2000, Fuji, Tokyo, Japan).
| |
RESULTS |
2D Electrophoresis of Barley Root Proteins
Proteins prepared from Fe-sufficient and Fe-deficient roots and
analyzed on 2D PAGE gels showed different protein patterns after
Coomassie brilliant blue staining (Fig.
1). In the roots of Fe-deficient plants,
the protein spots named C, D, G, V, W, X, and Y were present at higher
concentrations than in Fe-sufficient plants (Fig. 1). Protein spots C,
D, G, and V had previously been observed to increase during Fe
deficiency (Mori et al., 1988; Suzuki et al., 1995), but the W, X, and
Y spots were newly identified in this study.
View larger version (48K):
[in this window]
[in a new window]
| Figure 1.
2D PAGE gel from barley roots grown under
Fe-sufficient (+Fe) and Fe-deficient (Fe) conditions. Each gel was
loaded with 200 µg of root proteins. Several spots of
proteins that increased under Fe deficiency are indicated with
arrowheads. C, Adenine phosphoribosyltransferase; Y, IDS3; and W, FDH.
Other spots are unknown (Table I). Mr is
reported in thousands.
|
|
Amino Acid Sequences of Each Protein
Among the seven proteins with increased concentrations in
Fe-deficient roots, the D protein has been previously identified as a
36-kD peptide (Suzuki et al., 1995). Three proteins (C, W, and Y) were
successfully sequenced. The N-terminal sequence of protein W was
sequenced by Edman degradation, but the N termini of C and Y appeared
to be blocked. The W protein appeared to be FDH (EC 1.2.1.2) based on
homology of the sequences of internal peptides from CNBr digestion
fragments (WCN-3, -4, -5, -6, and -7 in Table
I) to other FDH sequences (the SwissProt
and GenBank databases were used for the homology search). FDH was
previously reported to be expressed in Fe-deficient roots of tomato and
in the roots of the nicotianamine-free mutant chloronerva by
Herbik et al. (1996). The C protein was transferred onto a PVDF
membrane and digested with CNBr (CCNS-2 and CCNM-1 in Table I) or
lysilendopeptidase (CLP-1 and CLP-2 in Table I). The sequences of
peptides from C obtained by chemical or enzymatic digestion suggested
that it is adenine phosphoribosyltransferase (EC 2.4.2.7). The
sequences of CNBr digestion fragments from the Y protein (YCN-2, -3, and -4 in Table I) completely matched the translated products from the
cDNA sequence of the Ids3 gene (Nakanishi et al., 1993,
accession nos. D37796 and D10058). Thus, FDH, adenine
phosphoribosyltransferase, and the Ids3 gene product showed
increased accumulation in Fe-deficient barley roots.
FDH Assay
An increase in FDH activity (Fig. 2)
was also detected in Fe-deficient roots, confirming the accumulation of
FDH observed by 2D PAGE. FDH activity was not detected in Fe-deficient
leaves, Fe-sufficient roots, or Fe-sufficient leaves at the protein
concentration tested (100 µg).
View larger version (79K):
[in this window]
[in a new window]
| Figure 2.
FDH assay on nondenaturing polyacrylamide gel.
Each lane was loaded with 100 µg of soluble proteins from barley
roots or leaves of Fe-sufficient (+Fe) or Fe-deficient (Fe) plants.
|
|
Isolation of Barley cDNA Encoding FDH
A cDNA library prepared from poly(A+) RNA of
Fe-deficient barley roots was screened with a PCR probe designed to
encode FDH (Fig. 3). Several clones were
thus identified. The cloned cDNA encoded 377 amino acids of an open
reading frame corresponding to a protein with a molecular mass of 41.5 kD and a pI of 6.7. The deduced molecular mass and pI were those of W
in 2D PAGE. A sequence consisting of 21 amino acids at the N terminus
might be a transit peptide targeted to mitochondria, based on
comparison with potato (Solanum tuberosum) tuber FDH (Colas
des Francs-Small et al., 1993). Comparison of barley FDH with FDH from
other plants or bacteria is shown in Figure
4. The NAD+-binding
site from Lys-193 to Asn-228 (Lamzin et al., 1992) and the
formate-binding site from Arg-285 are indicated. The sequences were
highly conserved between barley and potato in contrast to the sequences
at the N terminus, which displayed very low sequence homology.
View larger version (56K):
[in this window]
[in a new window]
| Figure 4.
Comparison of amino acid sequences from barley FDH
with FDH from other organisms. The partial amino acid sequences from
the W protein are underlined. The probable NAD+-binding
site is boxed, and the formate-binding site is shaded (Lamzin et al.,
1992). The homology between barley FDH and other FDHs was 82.7%
(S. tuberosum; Colas des Francs-Small et al., 1993), 53.0% (N. crassa; Chow and RajBhandary, 1993), 50.9%
(Pseudomonas sp.; Tishkov et al., 1991), 50.0%
(H. polymorpha; sequence A06214 was submitted to EMBL
data bank by C.P. Hollenberg and Z. Janowicz in 1989), and 49.6%
(C. methylica; Allen and Holbrook, 1995).
|
|
Southern Hybridization Analysis
The copy number of Fdh in barley was assessed by
Southern hybridization analysis (Fig. 5).
One fragment was observed in the BamHI- and
HindIII-digested DNA. Three fragments were detected in the
EcoRI lane but the largest (10 kb) may represent an
incomplete digestion product, since the sum of the molecular masses of
the other two fragments (5.5 and 4.2 kb) is approximately 10 kb. Since there were no restriction enzyme sites for BamHI,
EcoRI, and HindIII in the cloned Fdh
cDNA, we conclude that the Fdh gene is a single copy in the
barley genome and that an EcoRI site is probably present within an intron.
View larger version (25K):
[in this window]
[in a new window]
| Figure 5.
Southern hybridization analysis of
Fdh. Genomic DNA from barley was digested with
BamHI, EcoRI, and HindIII
and then blotted onto a nylon membrane and hybridized with a
32P-labeled SacI fragment of
Fdh.
|
|
Northern Hybridization Analysis
To investigate the expression of Fdh, northern
hybridization analysis was performed (Fig.
6). In the control (Fe-sufficient) plants, no Fdh mRNA was detected in either the leaves or the
roots (Fig. 6, compare +Fe leaf and +Fe root). In contrast,
Fdh was strongly expressed in the roots of Fe-deficient
plants but not in the leaves (Fig. 6, compare Fe leaf and Fe root).
View larger version (36K):
[in this window]
[in a new window]
| Figure 6.
Northern hybridization analysis of
Fdh. RNA was extracted from Fe-deficient () or
Fe-sufficient (+) roots or leaves. Each lane was loaded with 10 µg of
RNA. Total RNA was extracted after 14 d of Fe deficiency.
|
|
The induction of Fdh expression required 1 d of Fe
deficiency, with the amount of Fdh mRNA increasing gradually
day by day (Fig. 7). After 14 d,
Fdh expression reached a maximum and remained constant for
28 d. When Fe was resupplied in the form of Fe-EDTA to the culture
solution of Fe-deficient plants, Fdh mRNA quickly diminished
and was barely detectable on d 7 after the addition of Fe.
View larger version (41K):
[in this window]
[in a new window]
| Figure 7.
Expression of Fdh during Fe
deficiency in barley roots. Each lane was loaded with 10 µg of RNA.
Total RNA was isolated after 0, 1, 3, 5, 7, 10, and 14 d of Fe
deficiency and 1, 3, 5, and 7 d after Fe resupply.
|
|
In bacteria and unicellular algae, formate is produced in large
quantities under anaerobic conditions (Kreuzberg, 1984; Ferry, 1990).
We therefore examined the expression of barley Fdh under anaerobic conditions. As in prokaryotes, Fdh expression
increased in barley under anaerobic conditions (Fig.
8); the increase in Fdh mRNA
began at 12 h compared with 1 d for Fe deficiency. The amount
of Fdh mRNA present after 48 h of anaerobiosis was
approximately equivalent to 10 to 14 d of Fe deficiency
(quantification by the image analyzer is not shown).
View larger version (39K):
[in this window]
[in a new window]
| Figure 8.
Expression of Fdh under anaerobic
conditions. Each lane was loaded with 10 µg of RNA. Total RNA was
isolated after 0, 6, 12, 24, and 48 h of anaerobic treatment.
|
|
| |
DISCUSSION |
Based on peptide sequencing of protein spot W, FDH was among the
proteins induced by Fe deficiency in barley roots. FDH activity and
mRNA were detected in Fe-deficient barley roots but were undetectable in Fe-deficient barley leaves (Figs. 2 and 6). In
Pseudomonas sp. FDH, Arg-288 has been proposed to
be a formate-binding site (Lamzin et al., 1994; Popov and Lamzin,
1994), and the His-341-Gln-317 pair is necessary for the binding of
formate (Tishkov et al., 1996). These amino acids are conserved in all
plant FDHs, including that of barley (Fig. 4). Yeast (Hansenula
polymorpha and Candida methylica) FDH and
Neurospora crassa FDH have two inserted regions that are
absent in barley, potato, and Pseudomonas sp. (Fig. 3, residues 134-135 and 331-335).
Colas des Francs-Small et al. (1993) reported that FDH in potato tubers
was located in the mitochondria. The N-terminal sequence of barley FDH
shows little homology to potato FDH, but its hydropathy plot is similar
to that of transit peptides for mitochondria targeting. Therefore, FDH
activity detected in barley roots might be derived from mitochondria.
FDH mRNA was detectable after 1 d of Fe deficiency, reaching the
maximal level after 2 weeks. A few days after the addition of Fe into
the Fe-deficient solution, Fdh expression decreased and on d
7 was undetectable. Although this inductive response pattern to Fe
deficiency is very similar to that of Ids3, the response to
the Fe resupply is slower (Nakanishi et al., 1993). Ids3 is
one of the clones we have isolated in barley roots by the differential
hybridization method, and it supposedly encodes a putative mugineic
acid synthase. We observed that the transcript of Ids3 gene
is increased by Fe deficiency and we have confirmed in this experiment
that Ids3 is actually translated and the Ids3 protein is accumulated in Fe-deficient barley roots (Fig. 1; Table I).
In addition to Fe-deficient conditions, the barley Fdh was
also expressed under anaerobic conditions (Fig. 8). Moreover, this inductive response to anaerobic stress began 12 h after treatment and was more rapid than the response to Fe deficiency. Formate is
reported to be produced in large quantities in bacteria (Ferry, 1990)
and unicellular algae (Kreuzberg, 1984) under anaerobic conditions.
Colas des Francs-Small et al. (1993) suggested that a major,
uncharacterized metabolic pathway exists in the mitochondria of
nonphotosynthetic tissues, which produces large quantities of formate.
Therefore, induction of FDH by anaerobic stress in barley roots (Figs.
7 and 8) is conceivable in light of the above-mentioned results in
bacteria and algae.
The more rapid response of Fdh transcript to anaerobic
stress than that to Fe deficiency indicates that the expression of Fdh is primarily induced by anaerobic stress. The expression
of Fdh in Fe-deficient barley roots suggests that Fe
deficiency caused changes similar to the ones caused by anoxia through
changes in heme biosynthesis. For example, Fe regulates the
biosynthesis of  -aminolevulinic acid (by -aminolevulinic acid
synthase; Pushnik and Miller, 1989) and protoporphyrinogen IX (by
co-proporphyrinogen III oxidase). These are the common precursors
of protoporphyrin IX, from which chlorophyll a in the
chloroplasts and heme in the mitochondria are synthesized. Moreover, Fe
regulates the biosynthesis of divinyl protochlorophyllide (by
Mg-protoporphyrin IX monomethylester cyclase), which is the precursor
of chlorophyll in the chloroplasts (von Wettstein et al., 1995).
Fe is also incorporated into protoporphyrin IX to become heme in the
mitochondria. Therefore, Fe deficiency not only lowers the amount of
chlorophyll in the chloroplasts of shoots but also the amount of heme
in the mitochondria of both shoots and roots (Marschner, 1995). Since
large amounts of heme are needed for energy production by the
respiratory chain, the inhibition of energy production by decreasing
respiration in mitochondria could be caused by Fe deficiency. In
addition, we previously reported that Fe deficiency in rice roots
caused morphological malformation of mitochondria and a decrease in the
energy charge from 0.748 to 0.520 (Mori et al., 1991). Therefore,
anaerobiosis-like changes may be produced in mitochondria of
Fe-deficient barley roots.
Anaerobic stress causes acute damage to the plant by reducing available
energy, which may result in its turning to formate metabolism to
produce NADH by FDH. On the other hand, Fe deficiency may cause anoxia
by the depletion of Fe from heme and, secondarily, by the depletion of
heme protein as the result of inhibition of heme biosynthesis. If the
metabolism of formate in plant roots is similar to that of bacteria, as
was suggested by Colas des Francs-Small et al. (1993), the formate
pathway would be induced either by the decrease of heme (Fe deficiency)
or by reduced electron transport in the respiratory chain of
mitochondria (anaerobiosis). In conclusion, FDH induction might be
caused by the anoxia induced by Fe deficiency in spite of the presence
of oxygen.
| |
FOOTNOTES |
1
This work has been supported by Core Research
for Evolutional Science and Technology, Japan Science and Technology
Corporation (Tsukuba, Japan).
*
Corresponding author; e-mail asmori{at}hongo.ecc.u-tokyo.ac.jp;
fax 81-3-5684-4822.
Received August 15, 1997;
accepted November 14, 1997.
| |
ABBREVIATIONS |
Abbreviations:
CNBr, cyanogen bromide.
FDH, formate
dehydrogenase.
2D, two-dimensional.
2-ME, 2-mercaptoethanol.
| |
ACKNOWLEDGMENT |
We thank Professor Elizabeth C. Theil of North Carolina State
University (Raleigh) for editing English usage.
| |
LITERATURE CITED |
Aebersold RH,
Leavitt J,
Saavedra RA,
Hood LE,
Kent SBH
(1987)
Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ digestion on nitrocellulose.
Proc Natl Acad Sci USA
84:
6970-6974
[Abstract/Free Full Text]
Allen SJ,
Holbrook JJ
(1995)
Isolation, sequence and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeast Candida methylica.
Gene
162:
99-104
[CrossRef][Medline]
Bradford MM
(1976)
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254
[CrossRef][ISI][Medline]
Chow CM,
RajBhandary UL
(1993)
Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa.
J Bacteriol
175:
3703-3739
[Abstract/Free Full Text]
Cleveland DW,
Fischer SG,
Kirshner MW,
Laemmli UK
(1977)
Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.
J Biol Chem
252:
1102-1106
[Abstract/Free Full Text]
Colas des Francs-Small C,
Ambard-Bretteville F,
Small ID,
Rémy R
(1993)
Identification of a major soluble protein in mitochondria from nonphotosynthetic tissues as NAD-dependent formate dehydrogenase.
Plant Physiol
102:
1171-1177
[Abstract]
Damerval C,
de Vienne D,
Zivy M,
Thiellement H
(1986)
Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins.
Electrophoresis
7:
52-54
Ferry JG
(1990)
Formate dehydrogenase.
FEMS Microbiol Rev
87:
377-382
[CrossRef]
Gross E
(1976)
The cyanogen bromide reaction.
Methods Enzymol
11:
238-255
Herbik A,
Giritch A,
Horstmann C,
Becker R,
Blazer HJ,
Bäumlein H,
Stephan UW
(1996)
Iron and copper nutrition-dependent changes in protein expression in a tomato wild type and the nicotianamine-free mutant chloronerva.
Plant Physiol
111:
533-540
[Abstract]
Higuchi K,
Kanazawa K,
Nishizawa NK,
Chino M,
Mori S
(1994)
Purification and characterization of nicotianamine synthase from Fe-deficient barley roots.
Plant Soil
165:
173-179
[CrossRef]
Higuchi K,
Kanazawa K,
Nishizawa NK,
Mori S
(1996)
The role of nicotianamine synthase in response to Fe nutrition status in Gramineae.
Plant Soil
178:
171-177
Kanazawa K,
Higuchi K,
Nishizawa NK,
Fushiya S,
Chino M,
Mori S
(1994)
Nicotianamine aminotransferase activities are correlated to the phytosiderophore secretions under Fe-deficient conditions in Gramineae.
J Exp Bot
45:
1903-1906
[Abstract/Free Full Text]
Kreuzberg K
(1984)
Starch fermentation via formate producing pathway Chlamydomonas reinhardii, Chlorogonium elongatum, and Chlorella fusca.
Physiol Plant
61:
87-94
[CrossRef]
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Lamzin VS,
Aleshin AE,
Srtokopytov BV,
Yukhnevich MG,
Popov VO,
Harutyunyan EH,
Wilson KS
(1992)
Crystal structure of NAD-dependent formate dehydrogenase.
Eur J Biochem
206:
441-452
[Medline]
Lamzin VS,
Dauter Z,
Popov VO,
Harutyunyan EH,
Wilson KS
(1994)
Structure of holo and high resolution apo formate dehydrogenase.
J Mol Biol
236:
759-785
[CrossRef][ISI][Medline]
Logmann J,
Schelland J,
Willmitzer L
(1987)
Improved method for the isolation of RNA from plant tissues.
Anal Biochem
163:
16-20
[CrossRef][ISI][Medline]
Ma JF,
Nomoto K
(1993)
Two related biosynthetic pathway of mugineic acid in graminaceous plants.
Plant Physiol
102:
373-378
[Abstract]
Marschner H
(1995)
Functions of mineral nutritions: micronutrients.
In
R Marschner,
eds, Mineral Nutrition of Higher Plants, Ed 2.
Academic Press, London, pp 313-404
Marschner H,
Römheld V,
Kissel M
(1986)
Different strategies in higher plants in mobilization and uptake of iron.
J Plant Nutr
9:
695-713
[ISI]
Mori S,
Hachisuka M,
Kawai S,
Takagi S,
Nishizawa NK
(1988)
Peptides related to phytosiderophore secretion by Fe-deficient barley roots.
J Plant Nutr
11:
653-662
Mori S,
Nishizawa N
(1987)
Methionine as a dominant precursor of phytosiderophores in Gramineae plants.
Plant Cell Physiol
28:
1081-1092
[Abstract/Free Full Text]
Mori S,
Nishizawa N
(1989)
Identification of barley chromosome no. 4, possible encoder of genes of mugineic acid synthesis from 2 -deoxymugineic acid using wheat-barley addition lines.
Plant Cell Physiol
30:
1057-1060
[Abstract/Free Full Text]
Mori S,
Nishizawa NK,
Fushiya S,
Nozoe S,
Irifune T
(1990)
Identification of rye chromosome 5R as a carrier of the genes for mugineic acid synthase and hydroxymugineic acid synthase using wheat-rye addition lines.
Jpn J Genet
65:
343-352
Mori S,
Nishizawa NK,
Hayashi H,
Chino M,
Yoshimura E,
Ishihara J
(1991)
Why are young rice plants highly susceptible to iron deficiency?
Plant Soil
130:
143-156
[CrossRef]
Murray MG,
Thompson WF
(1980)
Rapid isolation of high molecular weight plant DNA.
Nucleic Acids Res
8:
4321-4325
[Abstract/Free Full Text]
Nakanishi H,
Okumura N,
Umehara Y,
Nishizawa NK,
Chino M,
Mori S
(1993)
Expression of a gene specific for iron deficiency (Ids3) in the roots of Hordeum vulgare.
Plant Cell Physiol
34:
401-410
[Abstract/Free Full Text]
O'Farrell PH
(1975)
High resolution two-dimensional electrophoresis of proteins.
J Biol Chem
250:
4007-4021
[Abstract/Free Full Text]
Okumura N,
Nishizawa NK,
Umehara Y,
Mori S
(1991)
An iron deficiency specific cDNA (Ids1) with two homologous cysteine rich domains.
Plant Mol Biol
17:
531-535
[CrossRef][ISI][Medline]
Okumura N,
Nishizawa NK,
Umehara Y,
Ohata T,
Nakanishi H,
Yamaguchi T,
Chino M,
Mori S
(1994)
A dioxygenase (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare.
Plant Mol Biol
25:
705-719
[CrossRef][ISI][Medline]
Popov VO,
Lamzin VS
(1994)
NAD+-dependent formate dehydrogenase.
Biochem J
301:
625-643
Pushnik JC,
Miller GW
(1989)
Iron regulation of chloroplast photosynthetic function: mediation of PSI development.
J Plant Nutr
12:
407-421
Schägger H,
Von Jagow G
(1987)
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 kDa to 100 kDa.
Anal Biochem
166:
368-379
[CrossRef][ISI][Medline]
Shojima S,
Nishizawa NK,
Fushiya S,
Nozoe S,
Irifune T,
Mori S
(1990)
Biosynthesis of phytosiderophores: in vitro biosynthesis of 2-deoxymugineic acid from l-methionine and nicotianamine.
Plant Physiol
93:
1497-1503
[Abstract/Free Full Text]
Shojima S,
Nishizawa NK,
Mori S
(1989)
Establishment of a cell free system for the biosynthesis of nicotianamine.
Plant Cell Physiol
30:
673-677
[Abstract/Free Full Text]
Suzuki K,
Hirano H,
Yamaguchi H,
Irifune T,
Nishizawa NK,
Chino M,
Mori S
(1995)
Partial amino acid sequences of a peptide induced by Fe deficiency in barley roots.
In
J Abadía,
eds, Iron Nutrition in Soils and Plants.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 363-369
Suzuki K,
Kanazawa K,
Higuchi K,
Nishizawa NK,
Mori S
(1997)
Immunological characterization of a 36 kDa Fe-deficiency specific peptide in barley roots.
Biometals
10:
77-84
Takagi S
(1976)
Naturally occurring iron-chelating compounds in oat- and rice-root washings.
Soil Sci Plant Nutr
22:
423-433
Takagi S,
Nomoto K,
Takemoto S
(1984)
Physiological aspect of mugineic acid, a possible phytosiderophore of graminaceous plants.
J Plant Nutr
7:
469-477
[ISI]
Tishkov VI,
Galkin AG,
Egorov AM
(1991)
NAD-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101: cloning, expression and the study of gene structure.
Dokl Acad Nauk USSR
317:
745-748
Tishkov VI,
Matorin AD,
Rojkova AM,
Fedorchuk VV,
Savitsky PA,
Dementieva LA,
Lamzin VS,
Mezentzev AV,
Popov VO
(1996)
Site-directed mutagenesis of the formate dehydrogenase active center: role of the His332-Gln313 pair in enzyme catalysis.
FEBS Lett
390:
104-108
[CrossRef][Medline]
Towbin H,
Staehelin T,
Gordon J
(1979)
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc Natl Acad Sci USA
76:
4350-4354
[Abstract/Free Full Text]
Uotila L,
Koivusalo M
(1979)
Purification of formaldehyde and formate dehydrogenases from pea seeds by affinity chromatography and S-formylglutathione as the intermediate of formaldehyde metabolism.
Arch Biochem Biophys
196:
33-45
[CrossRef][ISI][Medline]
von Wettstein D,
Gough S,
Kannangara G
(1995)
Chlorophyll biosynthesis.
Cell
7:
1039-1057
This article has been cited by other articles:
|
|
|
|
 
Y. Ogo, R. N. Itai, H. Nakanishi, H. Inoue, T. Kobayashi, M. Suzuki, M. Takahashi, S. Mori, and N. K. Nishizawa
Isolation and characterization of IRO2, a novel iron-regulated bHLH transcription factor in graminaceous plants
J. Exp. Bot.,
August 1, 2006;
57(11):
2867 - 2878.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. J. Castro, C. Carapito, N. Zorn, C. Magne, E. Leize, A. Van Dorsselaer, and C. Clement
Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress
J. Exp. Bot.,
November 1, 2005;
56(421):
2783 - 2795.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kobayashi, M. Suzuki, H. Inoue, R. N. Itai, M. Takahashi, H. Nakanishi, S. Mori, and N. K. Nishizawa
Expression of iron-acquisition-related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements
J. Exp. Bot.,
May 1, 2005;
56(415):
1305 - 1316.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. V. Bykova, A. Stensballe, H. Egsgaard, O. N. Jensen, and I. M. Moller
Phosphorylation of Formate Dehydrogenase in Potato Tuber Mitochondria
J. Biol. Chem.,
July 3, 2003;
278(28):
26021 - 26030.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Li, M. Moore, and J. King
Investigating the Regulation of One-carbon Metabolism in Arabidopsis thaliana
Plant Cell Physiol.,
March 15, 2003;
44(3):
233 - 241.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Uhde-Stone, K. E. Zinn, M. Ramirez-Yanez, A. Li, C. P. Vance, and D. L. Allan
Nylon Filter Arrays Reveal Differential Gene Expression in Proteoid Roots of White Lupin in Response to Phosphorus Deficiency
Plant Physiology,
March 1, 2003;
131(3):
1064 - 1079.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Yamaguchi, N.-K. Nishizawa, H. Nakanishi, and S. Mori
IDI7, a new iron-regulated ABC transporter from barley roots, localizes to the tonoplast
J. Exp. Bot.,
April 1, 2002;
53(369):
727 - 735.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Thimm, B. Essigmann, S. Kloska, T. Altmann, and T. J. Buckhout
Response of Arabidopsis to Iron Deficiency Stress as Revealed by Microarray Analysis
Plant Physiology,
November 1, 2001;
127(3):
1030 - 1043.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Yamaguchi, H. Nakanishi, N. K. Nishizawa, and S. Mori
Isolation and characterization of IDI2, a new Fe-deficiency-induced cDNA from barley roots, which encodes a protein related to the {{alpha}} subunit of eukaryotic initiation factor 2B (eIF2B{{alpha}})
J. Exp. Bot.,
December 1, 2000;
51(353):
2001 - 2007.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. F. López-Millán, F. Morales, S. Andaluz, Y. Gogorcena, A. Abadía, J. D. L. Rivas, and J. Abadía
Responses of Sugar Beet Roots to Iron Deficiency. Changes in Carbon Assimilation and Oxygen Use
Plant Physiology,
October 1, 2000;
124(2):
885 - 898.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Itai, K. Suzuki, H. Yamaguchi, H. Nakanishi, N.-K. Nishizawa, E. Yoshimura, and S. Mori
Induced activity of adenine phosphoribosyltransferase (APRT) in iron-deficient barley roots: a possible role for phytosiderophore production
J. Exp. Bot.,
July 1, 2000;
51(348):
1179 - 1188.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Gout, S. Aubert, R. Bligny, F. Rébeillé, A. R. Nonomura, A. A. Benson, and R. Douce
Metabolism of Methanol in Plant Cells. Carbon-13 Nuclear Magnetic Resonance Studies
Plant Physiology,
May 1, 2000;
123(1):
287 - 296.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
E.S. Dennis, R. Dolferus, M. Ellis, M. Rahman, Y. Wu, F.U. Hoeren, A. Grover, K.P. Ismond, A.G. Good, and W.J. Peacock
Molecular strategies for improving waterlogging tolerance in plants
J. Exp. Bot.,
January 1, 2000;
51(342):
89 - 97.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Takahashi, H. Yamaguchi, H. Nakanishi, T. Shioiri, N.-K. Nishizawa, and S. Mori
Cloning Two Genes for Nicotianamine Aminotransferase, a Critical Enzyme in Iron Acquisition (Strategy II) in Graminaceous Plants
Plant Physiology,
November 1, 1999;
121(3):
947 - 956.
[Abstract]
[Full Text]
|
|
|
|
Top
Abstract
Introduction