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Plant Physiol. (1998) 116: 859-865
Osmotic Stress Induces Expression of Choline Monooxygenase in
Sugar Beet and Amaranth1
Brenda L. Russell,
Bala Rathinasabapathi, and
Andrew D. Hanson*
Horticultural Sciences Department, University of Florida,
Gainesville, Florida 32611
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ABSTRACT |
Choline monooxygenase (CMO) catalyzes
the committing step in the synthesis of glycine betaine, an
osmoprotectant accumulated by many plants in response to salinity and
drought. To investigate how these stresses affect CMO expression, a
spinach (Spinacia oleracea L., Chenopodiaceae) probe was
used to isolate CMO cDNAs from sugar beet (Beta vulgaris
L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced
beet CMO amino acid sequence comprised a transit peptide and a
381-residue mature peptide that was 84% identical (97% similar) to
that of spinach and that showed the same consensus motif for
coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding
motif was also present. When water was withheld, leaf relative water
content declined to 59% and the levels of CMO mRNA, protein, and
enzyme activity rose 3- to 5-fold; rewatering reversed these changes.
After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol),
CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold
at 400 mm salt, and returned to uninduced levels when salt
was removed. Beet roots also expressed CMO, most strongly when
salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were
readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a
mononuclear Fe center, is inducibly expressed in roots as well as in
leaves of Chenopodiaceae, and is not unique to this family.
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INTRODUCTION |
Like other organisms, many plants accumulate betaines, polyols, or
Pro in response to dry or saline conditions (Yancey, 1994 ; Bohnert et
al., 1995). These compounds, termed compatible solutes or
osmoprotectants, serve as nontoxic solutes for cytoplasmic osmoregulation and can also partly reverse the damaging effects of
salts on proteins and membranes (Yancey, 1994). The metabolic engineering of osmoprotectant accumulation has therefore attracted interest as a way to improve crop stress resistance (McCue and Hanson,
1990; Bartels and Nelson, 1994; Bohnert and Jensen, 1996). One target
for such engineeering is Gly betaine, a potent osmoprotectant that
occurs widely among flowering plants, including the economically important families Chenopodiaceae, Amaranthaceae, Malvaceae,
Compositae, and Gramineae (Gorham, 1995).
Plants synthesize Gly betaine via a two-step oxidation of choline:
choline  betaine aldehyde Gly betaine (Rhodes and Hanson, 1993).
Bacteria use the same route, which has led several groups to express
bacterial genes for choline oxidation (choline dehydrogenase or choline
oxidase) in plants that lack Gly betaine, in some cases targeting the
enzyme to chloroplasts because this is the site of Gly betaine
synthesis in Chenopodiaceae (Rhodes and Hanson, 1993). The
transformants made small amounts of Gly betaine and were significantly
more salt tolerant (e.g. Lilius et al., 1996; Hayashi et al., 1997).
It is now possible to use plant genes for such engineering experiments,
since cDNAs for both enzymes of Gly betaine synthesis have been cloned
from Chenopodiaceae (McCue and Hanson, 1992a; Rathinasabapathi et al.,
1997). The enzyme mediating the second reaction is BADH, an NAD-linked
dehydrogenase that is known also from Amaranthaceae and Gramineae
(Ishitani et al., 1993; Valenzuela-Soto and Muñoz-Clares, 1994).
In Chenopodiaceae, BADH is predominantly ( 90%) located in the
chloroplast stroma (Rathinasabapathi et al., 1994). Salinity and water
deficit raise BADH mRNA and enzyme levels in leaves and roots,
coincident with the accumulation of Gly betaine (McCue and Hanson,
1992a; Ishitani et al., 1995; Wood et al., 1996). BADH cDNAs from
Chenopodiaceae and Gramineae have recently been functionally expressed
in tobacco (Rathinasabapathi et al., 1994; Ishitani et al., 1995).
The enzyme catalyzing the first and committing step of Gly betaine
synthesis is not as well known, having so far been found only in
spinach (Chenopodiaceae). The spinach enzyme (CMO) is a stromal,
Fd-dependent monooxygenase with a Rieske-type [2Fe-2S] center, and it
is completely unrelated to the bacterial choline dehydrogenase and
oxidase enzymes (Burnet et al., 1995; Rathinasabapathi et al., 1997).
Little is known about CMO expression except that it increases in
salinized spinach leaves (Brouquisse et al., 1989; Rathinasabapathi et
al., 1997).
We cannot yet answer three questions about the natural expression of
CMO that bear on its use in metabolic engineering. First, is it induced
by drought as well as by salinity? Second, is it expressed in roots as
well as in leaves? Third, is it unique to Chenopodiaceae or is it found
in other Gly betaine-accumulating plants? To address the first two
questions we chose sugar beet (Chenopodiaceae) because it can
accumulate high levels of Gly betaine in both roots and shoots ( 300 µmol g 1 dry weight), it is a halophyte, and
it withstands drought well (Hanson and Wyse, 1982; Dunham, 1993). This
led us to isolate beet CMO cDNAs for use as homologous probes; analysis
of their deduced amino acid sequences revealed a mononuclear Fe-binding motif.
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MATERIALS AND METHODS |
Plants and Growing Conditions
Sugar beet (Beta vulgaris L., cv Great Western D-2)
seed was obtained from Hilleshög Mono-hy, Inc. (Longmont, CO).
Beet plants from which mRNA was isolated for library construction were
grown in a growth chamber (12-h day, 25°C, 350 µE
m2 s1 PPFD/12-h night,
22°C) and salinized with 400 mm NaCl as described previously (McCue and Hanson, 1992a). For salinity experiments, beet
plants were grown hydroponically (four per 18-L tank) in aerated
one-half-strength Hoagland nutrient solution in a growth chamber (8-h
day, 25°C, 350 µE m2
s1 PPFD/16-h night, 20°C). Salinization began
when plants were 4 weeks old, using an NaCl:CaCl2
mixture (5.7:1 mol/mol, hereafter termed salt), and the concentration
was raised by 50 mm every third day to final values of 100, 250, or 400 mm salt. Water was added daily to replace
evapotranspiration and the medium was replaced every 2 weeks. Plants
were harvested 3 d after the final concentration was reached in
the 400 mm salt treatment, collecting four young leaves
(blade length about 5-20 cm) and the taproot from each. At the same
time, one batch of four plants was transferred from 250 mm
salt to nutrient solution alone, and harvested 3 d later. Unsalinized controls were harvested at the same time as the 400 mm salt treatment or at a similar stage of development to
the plants in this treatment; since the data were similar, only the latter are presented.
For drought experiments, beet plants were grown (one per 4-L pot) in
heavy potting soil (Southland Importers, Greensboro, NC) supplemented
with 5 g per pot of Osmocote 14:14:14 (N:P:K) (Scotts-Sierra,
Maysville, OH) in a greenhouse in natural daylight during November and
December, 1996; minimum temperature was 18°C. Irrigation was with
one-half-strength Hoagland nutrient solution. Seven weeks after
planting, irrigation was withheld for 7 d, by which time the
expanded leaves had been wilted continuously for 3 d. One-half of
the plants was then harvested, taking young leaves as above; the others
were rewatered and harvested 3 d later. Control leaves were
harvested just before irrigation was withheld. In all experiments,
samples from individual plants were frozen in liquid
N2 and stored at  80°C.
Amaranth (Amaranthus caudatus L. cv RRC 1036) seeds were
obtained from U.S. Department of Agriculture, North Central Regional Plant Introduction Station (Ames, IA). Plants were grown (two per 2.5-L
pot) in Metro-Mix 300 (Grace Sierra, Milpitas, CA), supplemented with
Osmocote, in the 8-h day/16-h night conditions given above.
Salinization was with NaCl:CaCl2 in nutrient
solution as above, starting at 5 weeks, applying 1.5 L per pot daily,
and raising the salt level by 50 mm every third day to a
final value of 300 mm. Controls were irrigated with
nutrient solution. After 3 d at 300 mm salt, young,
fully expanded, and expanding leaves ( 50% final size) were
harvested, frozen, and stored as above.
Measurement of  s and RWC
The fourth youngest leaf was sampled for beet, and the first fully
expanded leaf was sampled for amaranth.
s was determined on frozen-thawed
0.8-cm diameter leaf discs using a thermocouple psychrometer (HR-33T,
Wescor, Logan, UT) equipped with C-52 sample chambers. RWC was measured
on sets of nine 1.6-cm discs from the same leaves, as described by
Grumet and Hanson (1986).
Gly Betaine Analysis
Gly betaine was extracted from 50-mg dry weight leaf samples with
a methanol/chloroform/water procedure and isolated by ion-exchange chromatography (Hanson et al., 1991).
[Methyl-2H3]Gly
betaine (448 nmol/sample) was added at the start of the extraction as
internal standard. Gly betaine was converted to its n-butyl
ester and determined by fast atom bombardment MS as described by Rhodes
et al. (1987).
RNA Isolation and Analysis
For beet cDNA library construction, total RNA was isolated as
described previously (Rathinasabapathi et al., 1997) and
poly(A+) RNA was prepared using poly(U) Sephadex
(Hondred et al., 1987). For expression studies, total RNA was isolated
by a single-step method (Puissant and Houdebine, 1990).
Poly(A+) RNA for expression studies was purified
using polyA Spin Kits (New England Biolabs). RNA was separated on 1.2%
formaldehyde gels and blotted to Duralon membranes (Stratagene);
molecular size standards (0.24- to 9.5-kb RNA ladder, Gibco-BRL) were
included. Probes were labeled (> 109 cpm
µg1) by the random primer method using
[ -32P]dCTP. Autoradiographs of northern
blots were scanned using a digital imaging system (IS-1000, Alpha
Innotech, San Leandro, CA) to quantify signals. Signal strengths were
normalized with respect to the amount of rRNA in each track, determined
by reprobing blots with an 18S rRNA probe (Nairn and Ferl, 1988). The
quality of amaranth poly(A+) RNA preparations was
checked by probing blots with the 1.6-kb XhoI fragment of
amaranth PEP carboxylase (Rydzik and Berry, 1996).
cDNA Cloning and DNA Sequence Analysis
A cDNA library (3.1 × 106
plaque-forming units) was constructed in the UniZap XR vector
(Stratagene). Screening and in vivo excision were carried out according
to the supplier's protocols. Filters were hybridized at 42°C and
washed at 55°C in 2× SSC containing 0.1% (v/v) SDS. The amplified
library was screened with a 1150-bp AccI-EcoRV
fragment of spinach CMO cDNA clone pRS3 (Rathinasabapathi et al., 1997)
labeled as described above. This yielded a near-full-length CMO cDNA
(SB2), of which the 5 -terminal 265-bp EcoRI fragment was
used to isolate clone SB30 from the unamplified library. Clones were
sequenced in both strands using the fluorescent chain-terminating dideoxynucleotides method. Sequences were analyzed using the Wisconsin GCG Sequence Analysis Package (Version 8.0, Genetics Computer Group,
Madison, WI).
Protein Isolation and Analysis
Samples (2 g fresh weight) were pulverized in liquid
N2 and extracted with 8 mL of ice-cold buffer
containing 100 mm Tris, 1 mm
Na2EDTA, 10 mm DTT, and 4% (w/v)
PVP, adjusted to pH 8.0 with HCl; for all samples except the
drought-stressed beet leaves, 20 mm Na borate, 50 mm ascorbic acid, 1 mm PMSF, 1 µg
mL1 leupeptin, and 1 µg
mL1 antipain were also included. After
centrifuging at 12,000g (10 min, 4°C), proteins were
precipitated from the supernatant by adding 1 volume of ice-cold 50%
(w/v) PEG 8000 in 25 mm Tris-HCl, pH 8.0 (without or with
borate, ascorbate, leupeptin, and antipain as above), holding on ice
for 2 h, and centrifuging at 9,000g (15 min, 4°C).
This procedure was shown to precipitate 80% of the protein present in
beet leaf extracts. The pellet was redissolved in a buffer (adjusted to
pH 8.0 with HCl) containing Tris 50 mm, 0.5 mm
Na2EDTA, 2 mm DTT, 10% (v/v)
glycerol, and, except for drought-stressed beet leaves, 1 µg
mL1 leupeptin and 1 µg
mL1 antipain. The solution was then desalted on
Sephadex G-25 equilibrated in the same buffer and used for CMO assays
and western analyses. CMO activity was measured by the coupled
radiometric assay described previously (Burnet et al., 1995) using an
incubation time of 10 or 20 min, and adjusting the amount of extract so
that product formation was proportional to time and protein
concentration. Proteins were separated by SDS-PAGE and transferred to
nitrocellulose as described previously (Tokuhisa et al., 1985). Blots
were probed with mouse polyclonal antibodies to spinach CMO
(Rathinasabapathi et al., 1997).
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RESULTS |
Isolation and Characterization of Beet CMO cDNAs
A cDNA library was prepared using mRNA from salinized beet leaves,
and screened with a spinach CMO cDNA (Rathinasabapathi et al., 1997).
This yielded a 5-truncated CMO clone, SB2 (1709 bp). A 5 fragment of
SB2 was then used to isolate SB30, a 3-truncated clone that included
36 bp of 5-untranslated region and 468 bp of coding sequence, of which
462 bp overlapped with SB2. Together, SB2 and SB30 represent a
full-length cDNA of 1751 bp that encodes a polypeptide of 446 amino
acids and has a 377-bp 3-untranslated region. The composite amino acid
sequence is shown in Figure 1, aligned
with that of spinach CMO. Comparison of the beet and spinach N-terminal
regions suggests that beet CMO has a 65-residue stromal targeting
peptide, similar in length to that of spinach CMO (60 residues) and of
typical amino acid composition (Cline and Henry, 1996). The deduced
amino acid sequence of the mature beet CMO polypeptide has 84%
identity and 97% similarity to that of spinach, which are comparable
values to those for their BADHs (McCue and Hanson, 1992a). Beet CMO
contains the consensus sequence
(Cys-X-His-X15-17-Cys-X2-His) for coordinating a Rieske-type [2Fe-2S] cluster, as found in spinach CMO (Rathinasabapathi et al., 1997).
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| Figure 1.
Comparison of the deduced amino acid sequences for
sugar beet and spinach CMO cDNA clones. The beet sequence is a
composite of two overlapping clones (SB2 and SB30, encoding amino acids 3 to 446 and 1 to 156, respectively). Identical amino acid residues are
boxed and shaded; conservative substitutions are shaded. The asterisk
shows the N-terminal residue of the mature spinach CMO polypeptide
(Rathinasabapathi et al., 1997). Solid arrows mark the conserved
Cys-His pairs of the Rieske-type [2Fe-2S] cluster-binding region, and
open arrows indicate the conserved residues of the mononuclear
Fe-binding motif. The accession number for the composite beet CMO
nucleotide sequence is AF 023132.
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Positioned 94 residues along the protein from the Rieske
cluster-binding motif, beet CMO has a sequence very like the recently proposed consensus for coordination sites for mononuclear nonheme Fe:
Glu/Asp-X3-4-Asp-X2-His-X4-5-His
(Jiang et al., 1996; Gray et al., 1997). Exactly the same motif is
present in spinach CMO (Fig. 1); the only departure from the consensus
is that the central Asp and His are three residues apart, not two. Otherwise, the amino acids flanking the Asp and His residues in CMO are
similar or identical to those in other mononuclear Fe-binding sites,
and the location of these conserved residues relative to the Rieske
cluster-binding motif is the same as in other oxygenases (Mason and
Cammack, 1992; Jiang et al., 1996). Together, these observations
strongly imply that beet and spinach CMOs contain mononuclear Fe.
CMO Expression in Beet Leaves during and after Water Deficit
Prolonged withholding of irrigation resulted in marked declines in
RWC and s, which were fully reversed upon
rewatering (Table I). As expected, this
drought treatment promoted Gly betaine accumulation, although it did
not lead to measurable overall osmotic adjustment (i.e. a net increase
in solutes), since s values corrected to 100%
RWC did not fall. Consistent with the accumulation of Gly betaine,
during the stress period the level of CMO mRNA rose 5-fold (Fig.
2A), CMO protein accumulated (Fig. 2B),
and CMO enzyme activity tripled (Fig. 2C). Within 3 d of
rewatering, CMO mRNA, protein, and enzyme activity had all fallen to or
below their prestress levels (Fig. 2). The size of the CMO mRNA
observed in all treatments (1.9 kb) was slightly above that of spinach
(1.7 kb; Rathinasabapathi et al., 1997).
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Table I.
Responses of sugar beet leaves to a cycle of drought
stress and relief
Irrigation was withheld for 7 d, by which time the mature leaves
had been wilted for 3 d, and then restored for 3 d. Control plants were irrigated daily. The s and RWC
measurements were made between 10:00 am and 1:00
pm; data are means of four replicates and were subjected to
analysis of variance. The s values shown are
not corrected to 100% RWC. Gly betaine was determined using leaf discs
pooled from four plants and is expressed on the basis of fresh weight
at harvest.
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| Figure 2.
Expression of CMO in sugar beet leaves during a
cycle of drought stress and relief. Irrigation was withheld until
mature leaves had been continuously wilted for 3 d (Drought), and
then restored for 3 d (Rewater). Controls were irrigated daily. A,
Northern analysis. Lanes contained 10 µg of total RNA; the blot was
probed with two EcoRI fragments comprising the
5-terminal 815 bp from beet clone SB2. The numbers beneath each lane
show the CMO mRNA abundance relative to the control (1.0), normalized
for loading using 18S rRNA as a benchmark. B, Western analysis. Lanes
contained 130 µg of protein (precipitated with 25% PEG); the blot
was probed with mouse antibodies raised against spinach CMO. C, CMO
activity in 25% PEG-precipitated protein (means and se for
three replicates). The experiment was repeated with similar results.
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CMO Expression in Salinized Beet Leaves and Roots
Adding salt to the hydroponic medium lowered its
s by about 0.55 MPa at 100 mm and
1.9 MPa at 400 mm, as predicted (Wyn Jones and Gorham,
1983). The declines in leaf s in salinized
plants were close to these values, and resulted from both osmotic
adjustment and, to a lesser extent, a drop in RWC (Table
II). In leaves the steady-state levels
of CMO mRNA, protein, and enzyme activity all rose as salinity was
increased, and at 400 mm salt they were 4- to 7-fold higher
than in unsalinized controls (Fig. 3).
Three days after relief of salt stress, RWC and
s had both risen markedly (Table II), and CMO
mRNA, protein, and enzyme activity had fallen back to levels no higher
than those in control leaves (Fig. 3).
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Table II.
RWC and s of leaves from
salinized beet plants
Plants were salinized gradually and then maintained at the various
salinity levels for at least 3 d. One batch of plants was salinized to a salt level of 250 mm, then transferred to
nutrient solution for 3 d before harvest. Controls received
nutrient solution alone. The s and RWC
measurements were made 3 to 6 h after the start of the light
period; s values were not corrected to 100%
RWC. Data are means of four or five replicates and were subjected to
analysis of variance.
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| Figure 3.
CMO expression in sugar beet leaves after
long-term salinization. Plants were salinized gradually and then
maintained at the salinity levels shown for at least 3 d. One
batch of plants (250/0) was salinized to an intermediate salt level
(250 mm), then transferred to nutrient solution for 3 d before harvest; a prior experiment had demonstrated that CMO
induction at 250 mm salt was the same as at 400 mm. Controls received nutrient solution alone. A, Northern analysis; numbers below the lanes show the CMO mRNA abundance relative
to the unsalinized control (1.0), normalized for RNA loading. B,
Western analysis; lanes contained 50 µg of protein. C, CMO activity
(means and se for three replicates). Other experimental conditions were as in Figure 2. The experiment was repeated with similar results.
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In taproots CMO mRNA levels were undetectable in the control and low in
the 100 mm salt treatment, but at 400 mm salt
they were comparable to those in leaves (Fig.
4A). CMO protein level and activity (Fig.
4, B and C) were also far higher at 400 mm salt, but were
readily detectable in both other treatments. Expressed per unit
protein, CMO activities in roots were higher than in leaves (Figs. 3C
and 4C). SDS-PAGE analysis of the PEG-precipitated fractions used to
assay CMO indicated that this reflected the high proportion of Rubisco
in leaf samples.
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| Figure 4.
CMO expression in sugar beet taproots after
long-term salinization. The plants were the same as those used for the
leaf data of Figure 3. A, Northern analysis; numbers beneath the lanes
show the CMO mRNA abundance relative to that in unsalinized leaves (1.0; Fig. 3), normalized for RNA loading. B, Western analysis; lanes
contained 100 µg of protein (precipitated with 14% PEG). C, CMO
activity (means and se for three replicates). Other
experimental conditions were as in Figure 2. The experiment was
repeated with similar results.
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CMO Expression in Amaranth
The families Amaranthaceae and Chenopodiaceae belong to the order
Caryophyllales and are considered to stand close to each other within
it (Takhtajan, 1969). Consistent with such a relationship, the beet CMO
cDNA hybridized to a salt-inducible 1.9-kb CMO mRNA from amaranth
leaves (Fig. 5A), and antibodies to
spinach CMO recognized an amaranth CMO polypeptide (Fig. 5B) of the
same mass (about 45 kD) as the CMO monomer from spinach and beet. CMO
enzyme activity was also found in control and salinized amaranth leaves (Fig. 5C), at levels comparable to those in leaves of beet (Fig. 3C)
and spinach (Rathinasabapathi et al., 1997).
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| Figure 5.
Evidence for salinity-inducible CMO in amaranth
leaves. Plants were grown without salinization (Control) or gradually
salinized to a final level of 300 mm (Salt). A, Northern
analysis; lanes contained 3 µg of poly(A+) RNA; the blot
was probed with the two 5 EcoRI fragments from beet
clone SB2 and washed at 30°C in 2× SSC containing 0.1% SDS (v/v).
B, Western analysis; lanes contained 150 µg of protein (precipitated
with 14% PEG). C, CMO activity (means and se for three
replicates). Other experimental conditions were as in Figure 2.
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DISCUSSION |
We have isolated and characterized cDNAs encoding CMO from a
second plant species, with spinach being the first. The deduced amino
sequence of beet CMO contained the Rieske-type [2Fe-2S] cluster-binding motif found in the spinach sequence (Rathinasabapathi et al., 1997), as well as a newly recognized consensus motif for a
mononuclear Fe-binding site (Jiang et al., 1996; Gray et al., 1997).
The presence of a mononuclear Fe center in CMO fits with findings for
other oxygenases. Thus, various bacterial oxygenases with Rieske
[2Fe-2S] clusters are known also to have mononuclear Fe centers
(whence the consensus motif) that are thought to be the site of
dioxygen activation and catalysis (Mason and Cammack, 1992; Jiang et
al., 1996).
If CMO contains mononuclear Fe, this could help to explain why CMO
loses activity during purification (Burnet et al., 1995), since
mononuclear Fe is essential for catalysis but is sometimes readily lost
(Suen and Gibson, 1993; Jiang et al., 1996). Consistent with this
possibility, purified CMO had an Fe:S ratio of 1.06:1 (Rathinasabapathi
et al., 1997), well below the expected value of 1.5:1 for a protein
with a Rieske-type [2Fe-2S] cluster and a mononuclear Fe center. A
systematic study of the effects of Fe2+ treatment
on the activity of purified CMO would consequently be of interest,
although it could be complicated by the effect of
Fe2+ on superoxide-driven Fenton chemistry
(hydroxyl radical formation) in the CMO assay itself (Asada, 1996).
Subjecting beet to a cycle of water stress and relief showed that
drought leads to Gly betaine accumulation in leaves and that this is
associated with up-regulated CMO gene expression manifest in higher
levels of CMO mRNA, protein, and enzyme activity. The latter increases
were completely reversed within 3 d of stress relief, indicating
that both CMO mRNA and protein can be turned over quite rapidly. In
this experiment, as well as in those involving salinity, the increases
and decreases in CMO protein as judged from western blots were roughly
comparable to those in enzyme activity. This suggests that stress does
not affect CMO activity by posttranslational mechanisms, which fits
with mass spectral evidence against CMO having covalent
posttranslational modifications (Rathinasabapathi et al., 1997).
The data for salinized beet confirm observations made with spinach that
CMO is salt inducible in leaves (Rathinasabapathi et al., 1997) and
extend them in two ways. First, the beet results show that the
induction is fully reversed within 3 d of salt removal at both the
mRNA and enzyme levels. This contrasts with the response of BADH to
relief of salt stress: beet BADH mRNA levels decay rapidly but enzyme
activity does not (McCue and Hanson, 1992b). This difference in
poststress behavior between CMO and BADH is consistent with CMO being
more important in controlling flux through the Gly betaine synthesis
pathway. The second novel facet of the beet data is that they show that
CMO is expressed in roots. Since beet roots also express BADH (McCue
and Hanson, 1992a) and produce choline (Hanson and Wyse, 1982), they
are most probably able to synthesize Gly betaine. The reduced Fd
requirement for CMO could presumably be met by electron transfer from
NADPH to nonphotosynthetic Fd, as it is for the glutamate synthase and
nitrite reductase of root plastids (Bowsher et al., 1989, 1992).
Consistent with this, nongreen cell cultures of Atriplex
spp. (Chenopodiaceae) have been shown to produce Gly betaine (Koheil et
al., 1992). Although little conversion of
[14C]ethanolamine or
[14C]formate to Gly betaine was seen in beet
root tissues (Hanson and Wyse, 1982), 14C
accumulated in choline and phosphorylcholine and it is possible that
the endogenous pools of these compounds were large enough to act as
traps for the label.
That amaranth has CMO mRNA, protein, and enzyme is, to our knowledge,
the first evidence that CMO occurs outside the family Chenopodiaceae.
However, since the Amaranthaceae are phylogenetically close to the
Chenopodiaceae, it would be premature to assume that CMO also occurs in
more distant families such as Malvaceae, Compositae, and Gramineae. No
work has yet been done on the biochemistry of choline oxidation in
these groups, but it was recently reported that BADH in Gramineae may
be located mainly in peroxisomes rather than chloroplasts (Nakamura et
al., 1997). If Gly betaine synthesis in Gramineae occurs in the
peroxisome, then CMO is unlikely to be the choline-oxidizing enzyme,
because its Fd requirement would not be met in this compartment. It may
also be significant that we have been unable to detect CMO mRNA or
activity in cotton (Malvaceae), although reprobing the RNA blots with a
cotton -tubulin cDNA gave satisfactory signals, and when cotton and
beet tissues were mixed and extracted together, the beet CMO was not
inactivated (B.L. Russell and A.D. Hanson, unpublished data).
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FOOTNOTES |
1
This work was supported in part by a grant from
the U.S. Department of Agriculture National Research Initiative
Competitive Grants Program (95-37100-1596) and by an endowment from the
C.V. Griffin, Sr. Foundation. This is Florida Agricultural Experiment Station journal series no. R-06188.
*
Corresponding author; e-mail adha{at}gnv.ifas.ufl.edu; fax
1-352-392-6479.
Received September 8, 1997;
accepted October 22, 1997.
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ABBREVIATIONS |
Abbreviations:
BADH, betaine aldehyde dehydrogenase.
CMO, choline monooxygenase.
s, solute potential.
RWC, relative water content.
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ACKNOWLEDGMENTS |
We thank J.O. Berry, R.J. Ferl, and T.A. Wilkins, respectively,
for the gifts of the PEP carboxylase, 18S rRNA, and cotton -tubulin
cDNA clones; D.A. Gage for carrying out the mass spectral analyses of
Gly betaine; and K.D. Nolte for help in preparing figures.
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LITERATURE CITED |
Asada K
(1996)
Radical production and scavenging in the chloroplasts.
In
NR Baker,
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Abstract
Introduction