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First published online June 12, 2003; 10.1104/pp.103.020453 Plant Physiology 132:1642-1651 (2003) © 2003 American Society of Plant Biologists
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ABSTRACT |
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 TOP ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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-alanine (Ala) betaine, an osmoprotectant suitable under saline and
hypoxic environments, is found in most members of the halophytic plant family
Plumbaginaceae. In Limonium latifolium (Plumbaginaceae), it is
synthesized via methylation of -Ala by the action of a trifunctional
S-adenosyl L-methionine (Ado-Met): -Ala
N-methyltransferase (NMTase). Peptide sequences from purified
-Ala NMTase were used to design primers for reverse transcriptase-PCR,
and several cDNA clones were isolated. The 5' end of the cDNA was cloned
using a 5'-rapid amplification of cDNA ends protocol. A 500-bp cDNA was
used as a probe to screen a 
-gt10 L. latifolium leaf cDNA
library. Partial cDNA clones represented two groups, NMTase A and NMTase B,
differing only in their 3'-untranslated regions. The full-length NMTase
A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp
5'-untranslated region. The deduced amino acid sequence of 375 residues
had motifs known to be involved in the binding of Ado-Met. The NMTase mRNA was
expressed in L. latifolium leaves but was absent in Limonium
sinuatum, a member of the genus that lacks the synthetic pathway for
-Ala betaine. NMTase mRNA expression was high in young and mature leaves
and was enhanced by light. NMTase cDNA was expressed in yeast
(Saccharomyces cerevisiae) under the control of a galactose-inducible
promoter. Protein extracts of galactose-induced recombinant yeast had
Ado-Met-specific NMTase activities that were highly specific to -Ala,
N-methyl -Ala, and N,N-dimethyl -Ala as methyl
acceptors. NMTase activities were not detectable in comparable protein
extracts of yeast, transformed with vector control. The NMTase protein
sequence shared homology with plant caffeic acid O-methyltransferases
and related enzymes. Phylogenetic analyses suggested that -Ala NMTase
represents a novel family of N-methyltransferases that are
evolutionarily related to O-methyltransferases.
).
However, many plants have evolved metabolic adaptations to these abiotic
stress factors. Accumulation of osmoprotectants is a common adaptation found
in stress-tolerant plants, bacteria, and marine algae
(Yancey et al., 1982;
Bohnert and Sheveleva, 1998).
Quaternary ammonium compounds (QACs) represent some of the most effective
osmoprotectants known in biology (Anthoni
et al., 1991; Rhodes and
Hanson, 1993).
Because only certain stress-tolerant plants accumulate the common QAC Gly
betaine and many crops do not, it was suggested that engineering crops for Gly
betaine overproduction could be a way to improve their stress tolerance
(McCue and Hanson, 1990).
Transgenic plants, overexpressing bacterial and plant pathways for Gly betaine
synthesis, accumulated relatively small quantities of the QAC
(Rontein et al., 2002), but
nonetheless exhibited stress-tolerant phenotypes (Sakamoto and Murata,
2000,
2001;
Hibino et al., 2002). However,
reiterative metabolic engineering experiments indicated that availability of
the substrate choline limited Gly betaine synthesis in transgenic plants
(McNeil et al., 2000).
In the evolution of the stress-tolerant plant family Plumbaginaceae,
-Ala betaine replaced Gly betaine (Hanson et al.,
1991,
1994). -Ala betaine
synthesis is not constrained by choline availability, because it is derived by
the methylation of the non-protein amino acid, -Ala. Unlike Gly betaine
synthesis, -Ala betaine synthesis does not require oxygen, and hence it
was suggested to be suitable for osmoprotection under saline and hypoxic
conditions (Hanson et al.,
1994; Rathinasabapathi,
2000). Accordingly, -Ala betaine was distributed among
species of the Plumbaginaceae, adapted to a wide range of adverse stress
environments including saline and hypoxic conditions
(Hanson et al., 1994).
Although -Ala betaine accumulation has been intensely studied for its
role in osmoprotection (Hanson et al.,
1994; Rathinasabapathi et al.,
2000,
2001), early work on this
com-pound in marine algae also suggested it to have a cholesterol-reducing
effect in animal feeding experiments (Abe
and Kaneda, 1973).
We have characterized the -Ala betaine synthetic pathway in
Limonium latifolium, a member of the Plumbaginaceae. -Ala
betaine is synthesized by N-methylation of -Ala via
N-methyl and N,N-dimethyl -Alas
(Rathinasabapathi et al.,
2000). -Ala methylation, catalyzed by a S-adenosyl
L-Met (Ado-Met)-dependent N-methyltransferase (-Ala
N-methyltransferase [NMTase]) was specific to -Ala
betaine-accumulating members of the Plumbaginaceae and was absent in species
lacking this pathway (Rathinasabapathi et
al., 2000). An 86-kD leaf protein, a dimer of 43-kD subunits,
purified from L. latifolium was trifunctional, catalyzing all three
methylations of -Ala betaine synthesis
(Rathinasabapathi et al.,
2001). This suggested that -Ala betaine synthesis could be
engineered in transgenic plants by simply expressing a single gene. We report
here the isolation and functional expression of a full-length cDNA for
-Ala NMTase as a step toward a metabolic engineering approach to
understanding the biological roles of -Ala betaine.
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RESULTS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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Active -Ala NMTase protein was purified from L. latifolium
leaves using a previously described protocol
(Rathinasabapathi et al.,
2001). The 43-kD band was extracted from a polyacrylamide gel and
subjected to degradation by lysylendo-peptidase (lysC), and several peptides
were sequenced (Table I).
Degenerate primers were designed based on the peptides 1, 2, and 3 in both
sense (F) and antisense (R) orientations
(Table II). Under optimum
conditions, primers 2F and 3R amplified a 500-bp cDNA. Primer combinations 2F
and 1R and 1F and 3R produced shorter products of 400 and 100 bp, respectively
(data not shown). The 500-bp PCR product was named clone 23 and was sequenced
(accession no. AY216896).
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PCR primers were designed to amplify cDNA, 5' to clone 23. This was done by using a degenerate sense primer based on peptide 4 and an antisense primer based on the 5' end of clone 23. This resulted in two overlapping products, 423a (accession no. AY216897) and 423b (accession no. AY216898). The longer product, 423b (Fig. 1) was obtained by chance because the 5' end primer had shared a small region of homology in the target sequence.
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A cDNA partially overlapping clone 423b was further amplified employing a 5'-RACE protocol (Fig. 1) using the gene-specific primer 423R 5' to 3'-TCAACTCGTCATTCTTCCCGTAGTAC. We used a high-fidelity enzyme system to reduce potential errors during 5'-RACE PCR. However, to detect possible mutations during PCR amplification, this reaction was done in duplicate in independent reactions, and the products were sequenced. Both 5'-RACE sequences matched exactly with each other (accession no. AY216899).
cDNA Library Screening
To validate the PCR-based cloning strategy, we also screened a
-gt10 L. latifolium cDNA library using clone 23 cDNA as a
probe. Among the many positive clones identified, three clones, gt31, gt41,
and gt61 (accession nos. AY216900, AY216901, and AY216902, respectively), were
sequenced in both strands (Fig.
1). All three were partial cDNAs. The cDNA sequences of these
clones verified the PCR-based cDNA cloning in that the open reading frame
obtained via both methods was the same. The 3'-untranslated region,
however, differed among clones. Clones gt41 and gt61 represented NMTase A. The
3'-untranslated region of clone gt31 differed from the other clones in
several nucleotides (data not shown), representing NMTase B.
Characterization of the Full-Length NMTase cDNA
The full-length (1,414-bp) NMTase A cDNA (accession no. AY216903),
reconstructed by splicing clone gt61 to the 5'-RACE product, had 119 bp
of 5'-untranslated region, an open reading frame of 1,128 bp, and a
3'-untranslated region of 167 bp. The open reading frame had two ATGs
within the first 30 bp. The sequence context near the first Met had matched
well with the sequence context conserved around the initiation codon for plant
genes (Joshi, 1987;
Cavener and Ray, 1991). A
putative polyadenylation signal, AAATAAT
(Heidecker and Messing, 1986),
preceded poly(A+) by 17 bp.
The deduced amino acid sequence had 375 amino acid residues
(Fig. 2). It had all of the
peptides that were sequenced (Table
I) from purified -Ala NMTase
(Fig. 2). All three motifs
implicated in Ado-Met binding (Joshi and
Chiang, 1998) were conserved in -Ala NMTase
(Fig. 2). There is no
recognizable signal sequence (Emanuelsson
et al., 2000) based upon sequence analyses.
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NMTase Expression Is Unique to -Ala Betaine-Accumulating
Species
RNA blots probed with -Ala NMTase cDNA indicated that a 1.4-kb NMTase
mRNA is expressed in L. latifolium leaves but not in the leaves of
Limonium sinuatum (Fig.
3), a species that does not methylate -Ala
(Hanson et al., 1991). RNA
blots from both of these species had nearly equal signal intensities when the
filter was probed with a L. latifolium actin probe
(Fig. 3), and equal loading was
verified by an ethidium bromide-stained gel
(Fig. 3).
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Tissue Specificity and Regulation of NMTase Expression
Young and mature leaves had high levels of -Ala NMTase steady-state
mRNA expression, whereas old leaves, floral stems, flowers, and roots had
relatively lower levels of expression (Fig.
4A). Corroborating expression at the RNA level, crude extracts of
young and mature leaves had detectable NMTase activities (150–200 pmol
h–1 mg–1 protein), and
floral stems, flowers, and roots had no detectable activity. We used excised
mature leaves to test what factors may regulate -Ala NMTase mRNA levels.
Leaves incubated in a nutrient medium were exposed to light or dark conditions
for 24 h. Some of the leaves incubated under light were also exposed to sodium
chloride, indole butyric acid, GA3, and kinetin in the nutrient
medium for 24 h. -Ala NMTase expression was about 10-fold higher under
light than dark (Fig. 4B),
which was also verified by incubating whole plants under dark and light
conditions (data not shown). Under light, exogenous auxin to excised leaves
directly or indirectly down-regulated NMTase, and other treatments including
sodium chloride did not significantly affect NMTase RNA levels
(Fig. 4B).
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-Ala NMTase Is Coded by a Single or Low Copy Number of
Genes
Analysis of L. latifolium genomic DNA by Southern-blot hybridization showed a 5,000-bp band for an EcoRI digest, a 5,000-bp band for a HindIII digest, and a 2,000-bp band for a double digest (Fig. 5). The simple patterns obtained were consistent with a single or a few copies of the gene in L. latifolium genome.
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Heterologous Expression of -Ala NMTase cDNA
-Ala NMTase cDNA was cloned into yeast expression vector pYES-NT/B to
derive pYES-NMTase construct and was introduced into Brewer's yeast
(Saccharomyces cerevisiae) strain InVSc1 (Invitrogen, Carlsbad, CA).
This system allowed Gal-induced expression of the recombinant fusion protein
with a hexa-His tag at the N terminus. After Gal induction for 12 h and
analysis of the protein extracts in immunoblots, a single protein band was
stained with hexa-His-specific antibodies
(Fig. 6, inset). The molecular
mass of the immunostained protein band, 47.5 kD, was comparable with the
expected mass of the recombinant protein, 45.3 kD. Protein extracts of yeast
containing pYES-NMTase construct had detectable NMTase activities with
-Ala, N-methyl -Ala, and N,N-dimethyl -Ala
as methyl acceptors (Fig. 6).
Protein extracts of yeast containing a control pYES vector with
-galactosidase gene cloned under the Gal promoter (pYES-lacZ) had the
expected immunoreactive -galactosidase recombinant protein when probed
with hexa-His-specific antibodies (data not shown) but had no detectable
NMTase activities (Fig. 6).
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-Ala NMTase Has High Substrate Specificity
Protein extracts of yeast containing pYES-NMTase or pYES-lacZ were assayed
for Ado-Met-specific methyltransferase activities with the following potential
substrates as methyl acceptors: -alanyl Gly,
DL--aminoisobutryic acid, L-Ala, L-Pro,
trans-4-hydroxy L-Pro, Gly, putrescine, 
-amino-n-butyric
acid, N-methyl DL-Ala, and N,N-dimethyl Gly
(Table III). The compounds
tested, when used as methyl acceptors, could support only less than 2% of the
activity found with -Ala (Table
III). This suggested that -Ala NMTase has high substrate
specificity. Little (<1.2%) methyltransferase activity was found in protein
extracts of yeast containing pYES-lacZ
(Table III), used as a negative
control.
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-Ala NMTase Is Related to O-Methyltransferases
The deduced amino acid sequence of -Ala NMTase showed sequence
homology to caffeic acid O-methyltransferases and related
O-methyltransferases (Fig.
7). The homology was highest at the carboxyl end of the protein
including the conserved motifs described for Ado-Met-binding proteins. A
phylogenetic analysis indicated that -Ala NMTase was monophyletic,
whereas nodes with caffeic acid O-methyltransferases and related
enzymes were either bifurcating or polytomous in nature
(Fig. 8).
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DISCUSSION |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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-Ala NMTase
(Fig. 1) using peptide sequence
data for the purified -Ala NMTase. The deduced amino acid sequence
(Fig. 2) resulted in a
theoretical mass of 41,286 D and a pI of 5.84, closely resembling experimental
determinations of 43,000 D and 5.15 for the NMTase
(Rathinasabapathi et al.,
2001). -Ala NMTase had all three motifs conserved for
Ado-Met binding (Joshi and Chiang,
1998).
Our results suggest that -Ala NMTase is probably a cytoplasmic enzyme
because no signal sequences can be identified in the deduced amino acid
sequence (Fig. 2). It is
interesting to note that pantothenate synthetase, involved in ligating
D-pantoate and -Ala to make pantothenate, is also a
cytoplasmic enzyme (Genschel et al.,
1999). There are perhaps regulatory features that allow the
-Ala pool to be shared by both pantothenate and -Ala betaine
synthetic pathways in the same compartment.
Our previous radiotracer and enzyme measurements showed that -Ala
betaine synthesis was specific to -Ala betaine-accumulating members of
the Plumbaginaceae (Hanson et al.,
1991; Rathinasabapathi et al.,
2000). Consistent with this, -Ala NMTase mRNA expression was
present in L. latifolium and absent in L. sinuatum
(Fig. 3).
In L. latifolium, -Ala NMTase mRNA was expressed at
relatively high levels in young and mature leaves but at lower levels in
floral stem, flowers, and old leaves (Fig.
4A). NMTase activity measurements in protein extracts from these
tissues also matched these mRNA expression patterns, suggesting that
-Ala betaine synthesis in L. latifolium is developmentally
regulated at the transcriptional level. Previous radiotracer studies showed
that root tissue of L. latifolium had a capacity to synthesize
-Ala betaine (Hanson et al.,
1991). In contrast, RNA blots indicated poor expression of
-Ala NMTase in roots (Fig.
4A), and no detectable NMTase activities could be measured in
crude protein extracts of roots, using the assay routinely employed to
investigate NMTase in leaves. This contradiction suggests the possibility of
another root-specific NMTase isoform diverged both in its sequence and
properties from the leaf isoform.
-Ala NMTase expression was not significantly affected by salinity
stress treatment imposed on excised leaves
(Fig. 4B). This was consistent
with our previous observations that -Ala NMTase activity was not induced
by salinity stress treatment
(Rathinasabapathi et al.,
2000) and that -Ala betaine levels are somewhat constitutive
in L. latifolium leaves (Hanson
et al., 1991).
Light promoted the expression of -Ala NMTase
(Fig. 4B), similar to what was
observed for phosphobase N-methyltransferase in the synthesis of
choline (Weretilnyk et al.,
1995). Exogenous auxin directly or indirectly down-regulated the
NMTase (Fig. 4B). Genes that
are up-regulated by light and down-regulated by auxin are known in other
plants (Datta et al., 1993).
Further studies on the physiological significance of this regulation are in
progress.
When the full-length NMTase cDNA was expressed in yeast, a protein of
expected molecular mass was made upon induction with Gal
(Fig. 6, inset). The difference
between the mass of the band labeled by the immunoblot and the expected mass
is likely due to imprecision of the SDS-PAGE technique, but modifications of
the protein in yeast cannot be ruled out. Protein extracts of Gal-induced
yeast had NMTase activities with all three methyl acceptor substrates
-Ala, N-methyl -Ala, and N,N dimethyl
-Alas (Fig. 6). Such
activities were not observed in cultures of vector control yeast, which were
grown and extracted similarly (Fig.
6). This indicated that the NMTase cDNA cloned was stably
expressed in yeast and coded for the trifunctional NMTase. The specific
activity toward N,N-dimethyl -Ala was less than that found with
-Ala or N-methyl -Ala as the methyl acceptors
(Fig. 6), like what was found
with the enzyme purified from L. latifolium leaves
(Rathinasabapathi et al.,
2001).
When a range of amino acids and methyl amines were tested as potential
methyl acceptors, none of them acted as a substrate. This confirmed that the
recombinant -Ala NMTase expressed in yeast had high substrate
specificity, as observed previously in partially purified L.
latifolium leaf protein
(Rathinasabapathi et al.,
2000). This suggests that metabolic engineering of -Ala
methylation using this enzyme will result in specific and predictable
synthesis of -Ala betaine.
-Ala NMTase protein sequence shared homology to
O-methyltransferases (Fig.
7) but did not have significant homology to
N-methyltransferases. Several O-methyltransferases involved
in important secondary product synthesis pathways have recently been
structurally characterized (Parvathi et
al., 2001; Zubieta et al.,
2001,
2002). Our analysis comparing
-Ala NMTase with those sequences
(Fig. 7) revealed the
structural elements common and unique among them. Like the
O-methyltransferases, -Ala NMTase had a large C-terminal
catalytic domain responsible for Ado-Met binding. The N terminus of -Ala
NMTase had several unique regions, and future work will examine the
biochemical significance of these regions in determining substrate
specificity.
Our phylogenetic analyses (Fig.
8) are consistent with the hypothesis that -Ala NMTase had
evolved from an O-methyltransferase ancestral to caffeic acid
O-methyltransferases and related enzymes. This is similar to what was
known for putrescine N-methyltransferase in tobacco (Nicotiana
tabacum), where some homology to O-methyltransferases was
observed (Hibi et al., 1994).
We suggest that diverse O-methyltransferases can be progenitors to
N-methyltransferases and some sequences annotated as
O-methyltransferases in the genome databases based on sequence
homology may actually be N-methyltransferases.
Although a variety of QAC osmoprotectants are known in plants
(Rhodes and Hanson, 1993),
only the biosynthesis of the most common QAC, Gly betaine, has thoroughly been
investigated. Engineering of Gly betaine synthesis requires installing two
genes for choline oxidation and additional gene(s) to increase the
availability of choline (McNeil et al.,
2001; Rontein et al.,
2002).
It may be possible to avoid the problem of poor availability of choline by
engineering -Ala betaine synthesis instead of Gly betaine synthesis in
transgenic crops. Recent research in our laboratory indicates that
D-pantoate is what limits pantothenate synthesis in plants and not
-Ala (B. Rathinasabapathi, C. Sigua, and S.B. Raman, unpublished data).
This suggests that it should be possible to engineer transgenic plants
synthesizing -Ala betaine without adversely reducing pantothenate
levels. Additionally, because -Ala betaine synthesis will not require
oxygen for its activity, it would be suitable under hypoxic conditions.
Current research in our laboratory therefore focuses on the expression of
-Ala NMTase in model plants and testing whether such manipulation would
result in the synthesis and accumulation of -Ala betaine. Cloning,
characterization, and functional validation of the novel -Ala NMTase
described here has opened up this important opportunity.
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MATERIALS AND METHODS |
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TOPABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSLITERATURE CITED |
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If not otherwise indicated, chemicals used were from Sigma-Aldrich (St.
Louis) and were of the highest purity available. Magna Lift nylon (0.45 µm,
137 mm) circles were from Osmonics Inc. (Minnetonka, MN). dGTP
(
-32P, 800 Ci mmol–1) was
purchased from Amersham Biosciences (Piscataway, NJ). Plasmid purification and
gel extraction kits were from Qiagen (Valencia, CA). RACE protocol kit was
from BD Biosciences Clontech (Palo Alto, CA). Mr markers,
Taq polymerase, dNTPs, and restriction enzymes were from Promega
(Madison, WI). Oligonucleotide primers were synthesized from the custom primer
synthesis unit of Invitrogen.
Plant Material
Limonium latifolium and Limonium sinuatum plants were
grown under controlled conditions in a greenhouse as described previously
(Rathinasabapathi et al.,
2001). For salinity treatment, plants were grown in vermiculite
and irrigated daily with one-half-strength Hoagland medium
(Hoagland and Arnon, 1950).
Sodium chloride was added to the nutrient solution at 50 mM per
every 3 d until reaching 200 mM. The plants were kept in this
salinity for another week. Fully expanded leaves were harvested for RNA
extraction to prepare a cDNA library.
RNA Extraction
Young, mature (i.e. fully expanded), and old (i.e. senescing) leaves,
roots, floral stems, and flowers were harvested from 2-year-old plants grown
in a greenhouse. Total RNA was extracted using a modified hot borate method
(Wan and Wilkins, 1994). In
brief, tissue was ground in a mortar in liquid nitrogen and dithiothreitol
powder (2 mM final concentration) and was further extracted in a
boiling medium containing 0.2 M sodium borate, 30 mM
EDTA, and 1% (w/v) SDS, pH 9. Proteins were digested using proteinase K (5 mg
g–1 fresh weight tissue) at 42°C for 2 h. SDS
was removed by precipitation by the addition of potassium chloride solution to
a final concentration of 145 mM. The extract was then filtered
through four layers of cheesecloth and centrifuged 20,000 g. Barium
chloride at 75 mM was added to the supernatant to remove
carbohydrates. Following this step, lithium chloride at 2 M was
used to precipitate the total RNA. Lithium chloride step was repeated two more
times to achieve high-purity total RNA. The RNA was concentrated by
precipitation in ethanol, redissolved in RNase-free water, quantified using a
UV-visible spectrophotometer, and analyzed by gel electrophoresis.
Poly(A+) RNA was isolated from leaf total RNA using Oligo(dT)-cellulose (GenElute-mRNA miniprep kit, Sigma-Aldrich). First-strand cDNA was synthesized using oligo(dT)20 primers and reverse transcriptase (RT; Thermoscript RT-PCR System, Invitrogen).
Degenerate Primers
Active NMTase protein was purified from leaves of Limonium
latifolium using the procedure described previously
(Rathinasabapathi et al.,
2001). The purified protein (20 µg) was separated on an
SDS-PAGE gel and stained with Coomassie Blue. The 43-kD band was eluted from
the gel and was digested with lysyl endopeptidase C (LysC). Peptide sequencing
was done by Edman degradation (Tempst et
al., 1990). On the basis of the peptide sequences, degenerate
primers were designed for RT-PCR. Each primer included one or two
inosines.
RT-PCR
RT-PCR was performed using a RT-PCR kit (Thermoscript RT-PCR system, Invitrogen) according to manufacturer's instructions. RT-PCR reactions were in a volume of 50 µL in thin-walled amplification tubes. The reactions contained 10 µL of first-strand reaction, 200 µM of each of the four dNTPs, 2 mM magnesium chloride, 4 µM each of the sense and antisense primers, and 5 units of Taq DNA polymerase in 10 mM Tris-HCl, pH 9, 50 mM KCl, and 0.1% (v/v) Triton X-100. Forty cycles each with 93°C for 30 s for denaturation, 60°C for 30 s for annealing, and 72°C for 1.5 min for extension were performed in a thermal cycler (MiniCycler, MJ Research, Watertown, MA). The products were analyzed in an agarose (1%, w/v) gel and stained with ethidium bromide.
cDNA Library Construction
Poly(A+) RNA was isolated from L. latifolium
leaves from plants salinized with 200 mM NaCl. First- and
second-strand cDNAs were made using Moloney murine leukemia virus RT
(Sambrook et al., 1989). cDNAs
size-selected for 1,000 bp were cloned into the EcoRI site of
-vector gt10 (BD Biosciences Clontech). The primary library had a
titer of 1.5 x 106 plaque forming units
mL–1.
Library Screening
Clone 23 was labeled with [32P]dGTP (800 Ci
mmol–1, Amersham BioSciences, Piscataway, NJ)
using a random primer labeling kit (Invitrogen) according to manufacturer's
instructions. Plaque lifts of the library on nylon membranes were screened
using the radiolabeled probe by following the formamide procedure
(Sambrook et al., 1989).
Positive clones were identified after autoradiography and were purified using
standard protocols (Sambrook et al.,
1989). -DNA was extracted using Wizard Lambda prep system
(Promega) according to manufacturer's protocol.
DNA Sequencing and Analysis
DNA sequencing was in both strands using the fluorescent chain-terminating
dideoxynucleotides method. DNA sequences were analyzed by several software
packages including BLAST (Patnaik and
Blumenfeld, 2001).
RNA Blots
Total RNA from L. latifolium and L.
sinuatum was loaded onto a formaldehyde agarose (1.2%, w/v) gel, 10
µg lane–1, and blotted onto nylon membranes
(Sambrook et al., 1989). Equal
loading of RNA in the gels was verified by ethidium bromide staining of the
gel. Completion of RNA transfer was verified by methylene blue staining of the
blots and ethidium bromide staining of the gels. The blots were probed using
clone 23 cDNA probe labeled with [32P]dGTP. Band intensities in
autoradiographs were analyzed using densitometry. The RNA blots were also
probed with a 395-bp L. latifolium actin cDNA (S.B. Raman
and B. Rathinasabapathi, unpublished data).
Treatment of Excised Leaves
For the experiments on -Ala NMTase mRNA expression, mature leaves
were excised and incubated in 50 mL of Hoagland nutrient medium
(Hoagland and Arnon, 1950) for
24 h under light (photosynthetic photon flux density, 46 µmol
m–2 s–1, cool-white
fluorescent lamps) or complete darkness. Some of the leaves under light were
treated with kinetin (1 mg L–1), indole-3-butyric acid (1 mg
L–1), GA3 (1 mg
L–1), or 200 mM NaCl in Hoagland
nutrient medium.
Heterologous Expression
Coding sequences of NMTase cDNA were amplified with primers 5' to
3'-GCGGATCCAATGGCGAACCACTCCTCAGCTG and 5' to
3'-CTCGAGTCACTTCTGGAACTCTACCACGG with BamHI and XhoI
restriction enzyme recognition sites at the primer ends, respectively. The
amplified product was digested with the appropriate restriction enzymes and
subcloned into BamHI and XhoI restriction sites of yeast
expression vector pYES-NTB. The resulting plasmid was verified by sequencing
and was introduced into INVSc1 strain using the lithium chloride method
(Ausubel et al., 1995).
Recombinants were selected and maintained on minimal medium lacking uracil.
Preliminary experiments were carried out to determine the optimum duration of
growth in Glc-free Gal-containing medium for optimal induction of recombinant
protein (data not shown). Total protein was extracted from cells following
breakage of the cells using glass beads
(Ausubel et al., 1995) in
breaking buffer containing 50 mM sodium phosphate buffer (pH 7.4)
with 1 mM EDTA, 5% (v/v) glycerol, and 1 mM
4-(2-aminoethyl) benzenesulfonyl fluoride. Protein extracts were digested with
enterokinase for 1 h at 30°C according to manufacturer's protocol
(Invitrogen) and were assayed for NMTase activity using the radiometric method
described previously (Rathinasabapathi et
al., 2001). Heat-killed controls and cultures without Gal
induction had no detectable NMTase activities. Substrate specificity assays
were done with 10 mM potential methyl acceptors, following the
radiometric method described previously
(Rathinasabapathi et al.,
2000). Total protein was estimated by the method of Peterson
(1977), and bovine serum
albumin was the standard.
Immunoblots
SDS-PAGE was performed according to the method of Laemmli
(1970) in 12% (w/v) separation
gel and 5% (w/v) stacking gel. Proteins were transferred to a polyvinylidene
difluoride membrane (Bio-Rad, Hercules, CA) using an electroblotting device.
The protein blots were probed according to manufacturer's protocol, with
monoclonal antibodies against (His)6Gly conjugated to alkaline
phosphatase (Invitrogen).
DNA Extraction and Southern-Blot Hybridization
Genomic DNA was isolated from leaves of L. latifolium
using a cetyltrimethyl-ammonium bromide method
(Rogers and Bendich, 1994).
DNA was digested with restriction enzymes and separated on an agarose gel. DNA
was blotted onto a nylon membrane following the procedure described by
Sambrook et al. (1989).
Southern-blot hybridization was performed following the formamide procedure as
described (Sambrook et al.,
1989).
Multiple Sequence Comparisons and Phylogeny
BLAST analysis indicated sequences highly homologous to -Ala NMTase.
Multiple sequence comparisons were done using ClustalX
(Thompson et al., 1997). A
phylogenetic tree was drawn using the neighbor-joining method in PAUP
(Phylogenetic Analysis Using Parsimony, v4.0b10, Sinaur Associates,
Sunderland, MA). Bootstrap values were obtained by running the PAUP program,
and the tree was rooted using midpoint rooting. GenBank identification numbers
of sequences used for these analyses are as follows, listed top-down in the
phylogenetic tree: GI:6688808, GI: 3421382, GI:7488967, GI:7271883,
GI:18025321, GI:3913289, GI:2832224, GI: 1169009, GI:231757, GI:642952,
GI:729135, GI:4808524, GI:1170555, GI:19550749, GI:542050, GI:7447884,
GI:13399464, GI:15223364, GI:758580, GI:17366954, and GI:2282586.
|
ACKNOWLEDGMENTS |
|---|
Received January 15, 2003; returned for revision March 25, 2003; accepted April 18, 2003.
|
FOOTNOTES |
|---|
1 This work was supported by the United States Department of Agriculture
National Research Initiative Competitive Grants Program (grant no.
2001–35318–10947 to B.R.) and by the Florida Agricultural
Experiment Station. This is journal series no. R–09042. 
* Corresponding author; e-mail brath{at}mail.ifas.ufl.edu; fax 352–392–5653.
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