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Plant Physiol. (1998) 118: 661-674
Comparative Analysis of the Regulation of Expression and
Structures of Two Evolutionarily Divergent Genes for
 1-Pyrroline-5-Carboxylate Synthetase from
Tomato1
Tomomichi Fujita,
Albino Maggio,
Mario Garcia-Rios2,
Ray A. Bressan, and
Laszlo N. Csonka*
Departments of Biological Sciences (T.F., M.G.-R., L.N.C.) and
Horticulture (A.M., R.A.B.), Purdue University, West Lafayette, Indiana
47907
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ABSTRACT |
We
isolated two tomato (Lycopersicon esculentum) cDNA
clones, tomPRO1 and tomPRO2, specifying
 1-pyrroline-5-carboxylate synthetase (P5CS), the first
enzyme of proline (Pro) biosynthesis. tomPRO1 is unusual
because it resembles prokaryotic polycistronic operons (M.G.
García-Ríos, T. Fujita, P.C. LaRosa, R.D. Locy, J.M.
Clithero, R.A. Bressan, L.N. Csonka [1997] Proc Natl Acad Sci USA 94:
8249-8254), whereas tomPRO2 encodes a full-length P5CS.
We analyzed the accumulation of Pro and the tomPRO1
and tomPRO2 messages in response to NaCl stress and
developmental signals. Treatment with 200 mM NaCl resulted in a >60-fold increase in Pro levels in roots and leaves. However, there was a <3-fold increase in the accumulation of the
tomPRO2 message and no detectable induction in the level
of the tomPRO1 message in response to NaCl stress.
Although pollen contained approximately 100-fold higher levels
of Pro than other plant tissues, there was no detectable increase in
the level of either message in pollen. We conclude that transcriptional
regulation of these genes for P5CS is probably not important for the
osmotic or pollen-specific regulation of Pro synthesis in tomato. Using
restriction fragment-length polymorphism mapping, we determined the
locations of tomPRO1 and tomPRO2 loci in
the tomato nuclear genome. Sequence comparison suggested that
tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS
genes.
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INTRODUCTION |
Water stress can be imposed by high salinity, dehydration, or
freezing, which are environmental conditions that lead to the loss of
water from cells. Water stress triggers the accumulation of Pro in a
wide variety of species in all biological kingdoms (Paleg and Aspinall,
1981 ; Gilles et al., 1987; Csonka and Hanson, 1991). It has been
suggested that the accumulation of Pro contributes to the maintenance
of proper balance between extracellular and intracellular osmolality
under conditions of water stress. Direct evidence supporting this
hypothesis was provided by the fact that mutations that resulted in
high level Pro overproduction conferred increased osmotic stress
tolerance in Salmonella typhimurium (Csonka, 1981).
High-level expression of P5CS, a bifunctional enzyme that catalyzes the
first and second reactions of Pro biosynthesis, has been reported to
result in increased salinity stress tolerance in transgenic tobacco
plants (Kishor et al., 1995). However, the significance of Pro
accumulation is still controversial (Verma and Hong, 1996; Hare and
Cress, 1997), and other functions have been proposed for this response,
such as free radical scavenging, nitrogen storage, or pH regulation
(Stewart and Hanson, 1980; Delauney and Verma, 1993).
Pro is synthesized by the following four reactions: (a)
ATP-dependent phosphorylation of glutamate to  -glutamyl phosphate, catalyzed by GK; (b) reduction of -glutamyl phosphate by NADPH to
-glutamyl semialdehyde, mediated by GPR; (c) spontaneous cyclization of -glutamyl semialdehyde to P5C; and (d) NADPH-dependent reduction of P5C to Pro, carried out by P5C reductase. In addition to this so-called "glutamate pathway" of Pro synthesis, an alternate route to Pro has been suggested, involving the conversion of Orn to P5C by
Orn- -amino transferase. There are contradictory conclusions in the
literature concerning the importance of the latter pathway during
salinity stress. Whereas Delauney et al. (1993) found that the level of
the Orn--amino transferase mRNA was markedly decreased by high
salinity, Roosens et al. (1998) observed that this message was induced
by the same stress in Arabidopsis. Isotope-tracing studies suggested
that the pathway via Orn is not important for Pro synthesis during
osmotic stress in tomato (Lycopersicon esculentum) (Rhodes et al., 1986).
The finding that water stress increases the accumulation of Pro in
numerous plant species, together with the demonstration that it is
possible to enhance osmotic stress tolerance in bacteria by Pro
overproduction provided the motivation for the cloning of genes of the
Pro biosynthetic pathway from plants. Genes specifying GK have been
cloned from moth bean (Vigna aconitifolia), Arabidopsis, rice (Oryza sativa), and tomato (Hu et al., 1992;
Savouré et al., 1995; Yoshiba et al., 1995; Maggio et al., 1996;
García-Ríos et al., 1997; Igarashi et al., 1997;
Strizhov et al., 1997). The genes that were cloned from moth bean,
Arabidopsis, and rice encode a P5CS made up of a hybrid GK and GPR.
High salinity or dehydration results in increased accumulation of Pro
in Arabidopsis, rice, and moth bean roots, and has been shown to be
accompanied by an increase in the P5CS message level (Hu et al., 1992;
Savouré et al., 1995; Yoshiba et al., 1995; Igarashi et al.,
1997).
The induction in the level of P5CS mRNA has been determined to be 7- to
8-fold in Arabidopsis (Savouré et al., 1995; Yoshiba et al.,
1995). Genes for the last enzyme of Pro biosynthesis, P5C reductase,
were cloned from soybean, Arabidopsis, and pea (Delauney and Verma,
1990; Williamson and Slocum, 1992; Verbruggen et al., 1993), and it was
observed that osmotic stress resulted in a similar increase in the P5C
reductase message level as were seen for the P5CS message. The
observation that salinity or dehydration stress stimulated the
accumulation of the transcripts for the Pro-biosynthetic genes has been
interpreted to mean that the transcriptional control of these genes is
important for the regulation of Pro synthesis by osmotic stress.
However, 50-fold overproduction of P5C reductase in transgenic tobacco
plants did not lead to increased Pro accumulation (Szoke et al., 1992),
indicating that the much smaller induction of P5C reductase in
NaCl-stressed plants is not likely to be of significance for the Pro
accumulation.
Plant species exhibit substantial variation both in the relative
increases and final levels of Pro attained in response to osmotic
stress (Delauney and Verma, 1993). Arabidopsis, pea, and rice, which
have been used to probe the importance of transcriptional control for
Pro synthesis, are in fact not the best representatives of Pro
accumulators. These plants accumulate only approximately 2 to 6 µmol
Pro/g fresh weight in response to NaCl stress (Williamson and Slocum,
1992; Savouré et al., 1995; Peng et al., 1996; Igarashi et al.,
1997). Thus, unless it is highly concentrated in specific subcellular
compartments or organelles, Pro at such low overall concentrations
would not be expected to be a substantial determinant of the osmotic
potential of the whole cells (Blum et al., 1996; Sharp et al., 1996).
However, plants in the family Solanaceae have been found to contain
much higher levels of Pro (Treichel et al., 1984; Handa et al., 1986;
Rhodes et al., 1986; Delauney and Verma, 1993). The levels of this
imino acid can be regulated over 300-fold in tomato tissue-culture
cells by osmotic stress (Handa et al., 1986; Rhodes et al., 1986).
15N-isotope-tracing experiments indicated that
this increase in the Pro pool in cultured tomato cells upon osmotic
stress was primarily due to a 10-fold increase in the rate of Pro
synthesis via the glutamate pathway. Therefore, if transcriptional
regulation of P5CS is important for the control of Pro synthesis by
water stress, as has been suggested for Arabidopsis and rice, then one might expect that the Solanaceae, which accumulate much more robust levels of Pro under osmotic stress, would be more suitable for the
study of this effect than the model species studied thus far.
For the above reasons, we cloned the genes that specify the first and
second enzymes of Pro biosynthesis in tomato. We obtained two distinct
clones, tomPRO1 and tomPRO2. The
tomPRO1 clone was isolated from a tomato cDNA library by
complementation of GK (proB) and GPR (proA)
mutations in Escherichia coli (García-Ríos
et al., 1991). Surprisingly, this locus proved to have an unusual structure, in that it contains two open reading frames that encode GK
and GPR, arranged as a dicistronic operon (García-Ríos
et al., 1997). The tomPRO2 locus was cloned by hybridization
to a fragment of the first P5CS gene cloned from Arabidopsis (see
below). Like the P5CS genes from Arabidopsis, moth bean, and rice,
tomPRO2 specifies a hybrid GK-GPR as a single polypeptide.
Because mitochondria and chloroplasts, like prokaryotes, are able to
translate polycistronic messages, we considered the possibility that
the tomPRO1 might be present on a plastid genome. To test this, we carried out RFLP mapping of tomPRO1 and
tomPRO2 loci and demonstrated that both are present in the
tomato nuclear genome. To our knowledge, this is the first example of a
polycistronic locus mapped in plant nuclear genome. We also used these
clones to probe the transcriptional regulation of the corresponding
genes by osmotic stress. Our major finding was that transcriptional induction is not likely to be important for the regulation of Pro
synthesis by osmotic stress in tomato, despite the fact that this plant
accumulates Pro to much higher levels than Arabidopsis, pea, and rice.
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MATERIALS AND METHODS |
Isolation of tomPRO2 cDNA
A 1.6-kb fragment of the AtP5CS1 gene
was generated by García-Ríos (1995) by PCR
amplification of Arabidopsis total DNA with primers designed from
highly conserved sequences in the tomPRO1 clone
(García-Ríos et al., 1997), the moth bean P5CS gene (Hu
et al., 1992), and a partial sequence of the AtP5CS2 gene
(Strizhov et al., 1997; L. Szabados, personal
communication). The two primers were 5 -GATGCTCATTTATGGGCTCC-3
(specifying amino acids corresponding to residues 283-288 of the
tomPRO2 product) and 5-CCATTCTGCTCCAAATCTTT-3
(complementary to sequences specifying amino acids
corresponding to residues 553-558 of the tomPRO2 product). The amplified fragment was radiolabeled and used to probe a tomato (Lycopersicon esculentum cv Ailsa Craig) cDNA library in
 gt10 (kindly provided by Dr. G. Martin; described in Martin et al. [1993]). Hybridization of the plaque blots on Hybond N+
membranes (Amersham) was performed in 6× SSC and 1% SDS at 42°C. The blots were washed with 0.5× SSC and 0.1% SDS at 60°C. Among the
positive clones, the one with the longest insert was subcloned into the EcoRI site of pBluescript SKII( )
(Stratagene) and sequenced using an automated fluorescence sequencer
(Applied Biosystems).
Plant Materials
Tomato seeds were planted on 3M paper immersed with 0.25×
Murashige and Skoog solution (JRH Biosciences, Lenexa, KS) under continuous light at 25°C. About 3 weeks after germination, the seedlings were transplanted to plastic containers filled with one-half-strength Hoagland solution and maintained hydroponically in a
greenhouse under natural light. Leaf and root samples were taken at d
2, 6, 16, and 31 after the initiation of NaCl treatment. Various
tissues were collected from nonstressed hydroponic plants. Tomato
tissue-culture cells were grown in the normal liquid medium (S0 cells)
or in the medium containing 15 g/L NaCl (S15 cells), as described by
Hasegawa et al. (1980).
Measurement of Pro Content in Tissues
Frozen materials were ground with a mortar and pestle in
methanol:chloroform:water (12:5:1, v/v), and Pro content was determined by the acid ninhydrin procedure as described in Troll and Lindsley (1955).
Analyses of RNA
Total RNA was obtained by the LiCl-precipitation method as
described in Nagy et al. (1988). For northern-blot analysis, 20 µg of
total RNA was electrophoresed on a formaldehyde-agarose gel (1.2%
agarose). After electrophoresis, the analysis was carried out by the
standard protocol (Sambrook et al., 1989) on Hybond N+ membrane (Amersham). For use as probes of
northern blots, fragments containing nucleotides 1 to 899 of the
tomPRO1 cDNA clone plus an additional 90 bp derived from the
multicloning site in a vector or the full-length EcoRI
fragment of tomPRO2 cDNA were amplified with PCR and labeled
with [ -32P]dATP and/or dCTP by a
random-primer reaction according to the manufacturer's instructions
(Amersham). After hybridization, filters were washed three times for 20 min with 0.1× SSC and 0.1% SDS at 42°C. RNase protection analyses
were performed as described previously (García-Ríos et
al., 1997) with 80 µg of total RNA, and probed with an antisense
riboprobe from tomPRO1 cDNA using the HybSpeed RPA kit
(Ambion, Austin, TX). Levels of mRNA were quantified by scanning of the
autoradioagrams with a densitometer (Molecular Dynamics, Sunnyvale,
CA). To correct for loading differences in the northern blots (Figs.
1B and 2B),
the blots were also probed with a fragment containing the 18S and 25S
rRNA genes of flax (obtained from Dr. Joel Gaffe, Purdue University),
and the densitometer readings for the tomPRO2 signal for
each sample were normalized to the total 25S and 18S rRNA signal.
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| Figure 1.
Relationship between Pro accumulation and mRNA
levels in unstressed tomato tissues. A, Pro levels in different tissues
from unstressed tomato. Pro values are means ± SE
(n = 3). gfw, Grams fresh weight. B, Relative RNA
levels in different tissues from unstressed tomato. Same materials as
in A were used to extract total RNA. The top panel shows the result of
northern-blot analysis, which was carried out using 20 µg of total
RNA and probed with tomPRO2 cDNA, as described in
``Materials and Methods''. The rDNA probe was used as a control for
sample loading. RNA levels in each sample were quantified by
densitometric scanning of the autoradiograms and normalized to the
respective rRNA signal. The bottom panel shows normalized levels of the
tomPRO2 mRNA in the indicated tissues compared with
leaves, where the value for leaves has been set to 1.0. C,
RNase-protection assay of tomPRO1 in roots, leaves, and
pollen from unstressed tomato. RNase-protection analyses were performed
using 80 µg of total RNA with a riboprobe from tomPRO1
cDNA, as described in ``Materials and Methods''.
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| Figure 2.
Relationship between Pro accumulation and
tomPRO2 mRNA levels in response to NaCl stress in tomato
leaves and roots. A, Pro accumulation in leaves and roots of hydroponic
tomato after 2, 6, 16, and 31 d of treatment with 0, 100, and 200 mM NaCl. Pro values are means ± SE
(n = 3). gfw, Grams fresh weight. B, Relative RNA
levels in leaves and roots of hydroponic tomato. The conditions for
treatment were the same as in A. The top panel represents the result of
northern-blot analysis, which was performed using total RNA (20 µg)
probed with tomPRO2 cDNA. The rDNA probe was used as a
control for sample loading, and the results were measured, as described
in the Figure 1B legend. The relative tomPRO2 mRNA
levels are shown in the bottom panel, with the message set to 1.0 for
the 31-d samples in the untreated leaves and roots, respectively.
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Computer Analyses
Analyses of nucleotide and amino acid sequences were carried out
with programs in the Genetics Computer Group (GCG) package of the
University of Wisconsin, Madison, through a UNIX system. Comparisons
against sequences in GenBank and amino acid sequence alignments were
performed using the GAP and PILEUP programs, respectively. The
codon usage table was derived by the CODONFREQUENCY program, and
the codon usage tables for low- and high-expression genes in
Escherichia coli and for genes of tomato were also supplied by the GCG package. For constructing phylogenetic trees, the
neighbor-joining method was performed on the amino acid-composition
data using the SEQBOOT, PROTDIST, NEIGHBOR, and CONSENSE tools from the
PHYLIP program (Phylogeny Inference Package, version 3.5c, 1993; J. Felsenstein, Department of Genetics, University of Washington,
Seattle). Bootstrapping was performed with 100 replicates. Distances
were calculated using the Dayhoff PAM matrix option of PROTDIST.
Abbreviations and accession numbers are: tomPRO2, tomato
P5CS, U60267; Arabid, Arabidopsis P5CS, D32138; ArabidB, Arabidopsis
P5CS2, X86778; Vigna, V. aconitifolia P5CS, M92276;
Medicago, Medicago sativa P5CS, X98421; Rice, Oryza
sativa P5CS, D49714; Homos, Homo sapiens P5CS, X94453;
Cele, Caenorhabditis elegans P5CS, Z50797; Yeast,
Saccharomyces cerevisiae GK and GPR, P32264 and X90565; Coryne, Corynebacterium glutamicum GK and GPR, U31230 and
X82929; Bacsub, Bacillus subtilis GK and GPR, P39820 and
P39821; Tthermo, Thermus thermophilus GK and GPR, D29973;
Trepone, Treponema pallidum GK and GPR, U61535; Haein,
Hemophilus influenzae GK and GPR, P43763 and U32804; Serma,
Serratia marcescens GK and GPR, P17856 and P17857; Ecoli,
E. coli GK and GPR, P07005 and P07004; Synecho,
Synechocystis sp. GK and GPR, D90903 and D64001; Strept,
Streptococcus thermophilus GK and GPR, X92418; tomPRO1,
tomato GK and GPR, U27454.
Complementation of Pro Auxotrophy in E. coli
For the construction of a Pro auxotrophic derivative (KC1325) of
E. coli strain BL21(DL3)pLysS (Novagen, Madison, WI), the proB1658::Tn10 insertion, which is
polar on proA (Mahan and Csonka, 1983), was
transduced with P1 phage (Miller, 1972) into BL21(DE3)pLysS, selecting
Tetr progeny. Strain KC1325 was not able to grow
without Pro, but the parental strain was, confirming that KC1325 is a
Pro auxotroph. A tomPRO2 fragment (nucleotides
44-2197) containing a complete open reading frame was amplified by PCR
with the primers 5-TTCCATGGAGACAGTTGATTCAACTCG-3 and
5-TTGGATCCATCACCCTTGCTGAGTAAGGT-3 (which contain NcoI and BamHI restriction enzyme sites, respectively),
and the fragment was cloned between the NcoI and
BamHI sites of pET32a vector (Novagen) to yield pET32PRO2,
resulting in a fusion protein of tomPRO2 with an N-terminal
extension from Trx-, His-, and S-tag sequences. Construction of pPRO1,
which carries the tomPRO1 cDNA in the EcoRI site
of pBluescript KSII(+) (pKS; Stratagene), has been described by
García-Ríos et al. (1997). Plasmids pPRO1, pET32PRO2,
pKS, and pET32a were electroporated into KC1325, respectively.
Complementation tests were carried out at 37°C on solid medium 63 (Cohen and Rickenberg, 1956) containing 10 mM Glc, 0.05 mM thiamine-HCl, and 1 mM IPTG, with or without
1 mM Pro.
Expression of Recombinant Proteins
For tomPRO1 expression, E. coli strain HB101
(proBA leu thi-1) was transformed with pPRO1. The
transformants were grown in Luria-Bertani broth with ampicillin (100 µg/mL) at 37°C for 10 h. pET32PRO2 was used for the
transformation of the strain KC1325. Production of a
recombinant protein for tomPRO2 was induced by 1 mM IPTG at 25°C for 17 h, based on the
manufacturer's instructions (Novagen). Cells were collected and
resuspended in a 125 mM Tris-HCl (pH 6.8), 4% SDS, 5%
 -mercapthoethanol, and 20% glycerol. Total crude extracts were
separated by 12% or 10% SDS-PAGE and then visualized with Coomassie
brilliant blue R250 as in Sambrook et al. (1989).
RFLP Mapping
RFLP linkage analyses were performed utilizing
F2 progeny from the cross between L. esculentum and Lycopersicon pennellii. DNA samples from
67 of the F2 progeny had been digested by various restriction enzymes, separated by electrophoresis, and transferred to
Hybond N+ membranes. These membranes, which had
been used previously for the mapping of numerous other markers
(Tanksley et al., 1992), were generously provided by Dr. G. Martin
(Purdue University). RFLP markers were also collected and supplied by
Dr. G. Martin. P-labeled probe preparation and
DNA gel-blot analyses were basically the same as for the RNA gel-blot
analyses, except that they were washed with 0.2× SSC, 0.1% SDS at
25°C or with 0.1× SSC and 0.1% SDS at 42°C. Multipoint linkage
analyses were performed using the MapMaker program (version 2.0, Lander
et al., 1987). Recombination frequencies from multipoint analysis were
converted into map distances (in centiMorgans [cM]) using the mapping
function of Kosambi (1944).
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RESULTS |
Isofunctional Enzymes Catalyzing the First Step of Pro Biosynthesis
Are Specified by Two Distinct Genes, tomPRO1 and
tomPRO2, in Tomato
We isolated a polycistronic tomPRO1 locus in tomato,
which specifies GK and GPR divided by an internal stop codon in a
single gene (García-Ríos et al., 1997). Because the
accumulated sequence information indicated that the first step of Pro
biosynthesis is mediated by a bifunctional P5CS in other higher
eukaryotes, we screened a tomato cDNA library with a genomic DNA
fragment from Arabidopsis encoding P5CS. We identified several inserts that strongly hybridized to this probe, the longest of which was selected for further analysis. Nucleotide sequencing of this clone, which was designated tomPRO2, revealed that it contained a
single, long open reading frame, flanked by 43- and 42-bp untranslated regions at the 5 and 3 ends, respectively. The predicted amino acid
sequence of tomPRO2 indicated that it consists of a GK-GPR hybrid as a monocistron, having an overall 76% identity at the amino
acid level to Arabidopsis P5CS. However, tomPRO2 shows
only 35% identity to tomPRO1 products, suggesting
that tomPRO2 represents a homolog of the
Arabidopsis P5CS gene.
The Levels of Pro and of the P5CS Message in Various Tissues in
Unstressed Tomato Plants
In tomato plants grown under nonstressed conditions, Pro was
present in the range of 1 to 7 µmol/g fresh weight in roots, leaves,
and fruits of various stages (Fig. 1A). However, in accord with
previous reports that pollen are rich in free Pro (Khoo and Stinson,
1957; Hong-qi et al., 1982), we found that the level of this imino acid
was 200 µmol/g fresh weight in tomato pollen. We examined the
accumulation of tomPRO1 and tomPRO2 mRNAs in the different tissues in the unstressed plants using northern-blot analyses
with the tomPRO1 and tomPRO2 cDNA inserts as the
probes. The tomPRO2 message was readily observable in all
tissues, but the tomPRO1 message was not detectable by
northern blots (data not shown), indicating that tomPRO1 is
not expressed at all or at a substantially lower level than the
tomPRO2 gene in the tissues analyzed. As shown in Figure 1B,
the level of the tomPRO2 mRNA, normalized to the rRNA level
to correct for loading errors, was nearly the same in all of the
tissues, including pollen. Whereas the tomPRO1 message was
not observable with northern blots, using the more sensitive
RNase-protection assay, we were able to observe it in leaves (Fig. 1C).
The tomPRO1 transcript was undetectable in roots and pollen
even with this assay. Thus, our data indicate that the high-level
accumulation of Pro in pollen was not correlated with a detectable
induction in the levels of the tomPRO1 and
tomPRO2 transcripts.
Pro Is Accumulated to High Levels in NaCl-Stressed Plants
We examined the effect of NaCl stress on Pro levels in
hydroponically grown tomato plants (Fig.
2A). Treatment with 100 mM NaCl elicited an approximately 15-fold increase in the level of Pro
accumulation in both leaves and roots, and treatment with 200 mM NaCl resulted in 60- and 80-fold increases in these
tissues, respectively. In leaves the highest level of Pro was reached
after 6 d, and was maintained until 31 d after treatment; in
roots, Pro decreased to about one-half of the highest level by this
time. The more rapid disappearance of Pro in roots compared with leaves in NaCl-treated plants could reflect a more severe osmotic stress in
leaves because of transpiration and/or slower osmotic adjustment than
in roots.
The same plants that were used for Pro measurement were also subjected
to northern analysis to determine the effect of osmotic stress on the
accumulation of the P5CS transcripts. As before, the tomPRO2
mRNA was much more abundant than the tomPRO1 mRNA, which was
not detectable with northern analysis in any of the tissues in the
NaCl-stressed plants (data not shown). NaCl stress resulted in some
increase in the accumulation of the tomPRO2 transcript. However, the tomPRO2 message level, normalized to the rRNA
signal, increased only about 2-fold after 2 d of treatment with
100 and 200 mM NaCl in both leaves and roots, and remained
at almost the same level for 31 d after the treatment (Fig. 2B).
Although Pro accumulation in leaves and roots treated with 200 mM NaCl was 3- to 7-fold higher throughout the entire
course of the treatment than in the same tissues treated with 100 mM NaCl (Fig. 2A), we have no evidence that the level of
the tomPRO2 mRNA is altered substantially by either the
severity or duration of osmotic stress (Fig. 2B). Thus, our results
show that the tomPRO2 transcript level was induced much less
by NaCl in tomato than in Arabidopsis, rice, and moth bean (Hu et al.,
1992; Savouré et al., 1995; Yoshiba et al., 1995; Igarashi et
al., 1997; Strizhov et al., 1997), even though tomato accumulated
>15-fold higher levels of Pro than those other plants. Thus, our
results suggest that control of the accumulation of the
tomPRO2 message level is probably not important for the regulation of Pro synthesis by NaCl stress.
Effect of NaCl Stress on the Pro Levels and the Accumulation of the
P5CS Transcripts in Tissue-Culture Cells
We also measured the Pro levels in normal and NaCl-adapted
tissue-culture cells. As shown in Figure
3A, cells grown in normal medium (S0) had
a very low level of Pro, whereas cells grown in medium containing 15 g/L NaCl (S15) had an approximately 30-fold higher level of this imino
acid. Despite this difference in the Pro content, the level of the
tomPRO2 message was essentially the same in the two types of
cells, as detected by northern blotting (Fig. 3B), indicating that the
30-fold increase in the Pro levels seen in tissue-culture cells adapted
to 15 g/L NaCl occurred without any notable change in the accumulation
of the tomPRO2 message. The tomPRO1 mRNA was not
detectable by northern-blot analysis in either of the cells, but as
reported earlier (García-Ríos et al., 1997), the more
sensitive RNase-protection assays demonstrated that the S15 cells had a
4-fold higher level of the tomPRO1 message than the S0
cells. However, because of the low abundance of the tomPRO1
message even in the NaCl-adapted cells, the induction of this message
probably is not sufficient to account for the elevation in the Pro
pools size in the tissue-culture cells.
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| Figure 3.
Relationship between Pro accumulation and mRNA
levels in tomato tissue-culture cells. A, Comparison of Pro levels in
tissue-culture cells grown in normal medium (S0) and in medium
containing 15 g/L NaCl (S15). Pro values are means ± SE (n = 3). gfw, Grams fresh weight. B,
Relative RNA levels in the tissue-culture cells S0 and S15. Same
materials as in A were used to extract total RNA. The analysis was
carried out using 20 µg of total RNA and probed with
tomPRO2 cDNA. The rDNA probe was used as a control for
sample loading.
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The tomPRO1 and tomPRO2 Loci Are
Structurally Different and May Have Evolved from Separate Ancestral
Genes
In addition to the different levels of expression of
tomPRO1 and tomPRO2 described above, the
two cDNAs have remarkable structural differences. The
tomPRO1 has a dicistronic structure (García-Ríos et al., 1997), which is unusual in
eukaryotes (Kozak, 1986). Thus far, all genes encoding GK
and GPR in bacteria and yeast have been reported as separate genes,
organized as an operon in most bacteria. In contrast,
tomPRO2 encodes a bifunctional enzyme formed by a hybrid of
GK and GPR without an intervening stop codon, similar to genes encoding
a hybrid GK and GPR, or P5CS, that have been cloned from several higher
eukaryotes, such as C. elegans, H. sapiens, and higher
plants (Hu et al., 1992; Savouré et al., 1995; Yoshiba et al.,
1995; Liu et al., 1996; Igarashi et al., 1997).
Table I shows that the predicted amino
acid sequence of tomPRO2 has greater than 76% identity with
other plant P5CS, but shows a much lower identity (35%-44%) with the
tomPRO1 product and the bacterial and yeast GK and GPR
enzymes. In contrast, the predicted tomPRO1 product has a
low amino acid identity (32%-47%) with GK and GPR from yeast and
prokaryotes and with P5CS from all other eukaryotes. Although the
tomPRO1 product has a similar homology to the corresponding
proteins from different organisms, sequence alignment shown in Figure
4 revealed that tomPRO1 is closer to bacterial GK and GPR than to P5CS of plants. Some regions that are highly conserved in P5CS proteins from plants are either missing, divergent, or carry insertions in tomPRO1 and in
bacterial GK and GPR (e.g. amino acids 71-83, 151-161, and 194-219
in GK, and amino acids 426-451, 476-497, and 571-577 in GPR, where
the numbers of amino acid residues are based on tomPRO1
sequences). However, the GK part of tomPRO1 also shows a
striking difference from most bacterial GKs, because it lacks an
approximately 100-amino acid C-terminal tail that is conserved in most
other prokaryotic GKs (represented by E. coli GK, amino
acids 259-367, and B. subtilis GK, amino acids 256-354
[García-Ríos et al., 1997]). At present, S. thermophilus is the only exception among bacteria that also lacks this C-terminal tail in GK. The tomPRO1 product has
the closest sequence similarity to the GK and GPR from the latter organism (Table I).
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| Figure 4.
Amino acid sequence comparison of
tomPRO1, tomPRO2, and other related
genes. Predicted amino acid sequences of proB,
proA, and P5CS genes were aligned using
the multiple alignment program PILEUP, and the results were highlighted
with the BOXSHADE program. Letters in the black and gray backgrounds
indicate identical and similar residues, respectively. Representative
regions that are highly conserved in P5CS proteins in plants but are
either missing, divergent, or carry insertions in GK and GPR for
tomPRO1 and in bacterial GK and GPR are underlined.
Extended C-terminal tails of GK, which are conserved in most of
prokaryotic GK, are shown by a dashed line. Abbreviations and accession
numbers are provided in ``Materials and Methods''.
|
|
We compared codon usage of tomPRO1 and tomPRO2
with the average codon usage of tomato genes and of genes expressed at
low or high levels in E. coli. The tomPRO2 codon
usage agrees well with the preference of average codon usage from
tomato genes, whereas tomPRO1 codon usage deviates from the
usage of tomato genes (Table II), and in
fact, appears to be between the preference in genes in tomato and in
E. coli (results not shown).
View this table:
[in this window]
[in a new window]
|
Table II.
Comparison of codon frequency usage in tomPRO1,
tomPRO2, and general tomato genes
Values are given of each codon in each amino acid. Trp, Met, and stop
codons are not included.
|
|
Because of the intriguing differences in nucleotide and predicted amino
acid sequences of tomPRO1 and tomPRO2, the
relationship of these two loci to other GK, GPR, and P5CS from various
species was examined further by a phylogenetic analysis. A phylogenetic tree was constructed by the neighbor-joining method from a highly conserved region of GK (amino acids 84-149 of tomPRO1).
Figure 5A revealed that P5CS proteins
from higher eukaryotes are clearly monophyletic, and that
tomPRO2 was tightly clustered as a member of plant P5CS
within this group. On the other hand, tomPRO1 appeared together with T. pallidum and S. thermophilus
within a bacterial group. This trend is also true for the
phylogenetic tree constructed from a highly conserved region of GPR
(361-436 amino acids of tomPRO1) as shown in Figure 5B.
Whereas the GPR domain of tomPRO2 grouped with the
corresponding regions of other eukaryotes, the tomPRO1-encoded GPR clustered with bacterial ones. These
results suggest that tomPRO1 and tomPRO2 were
probably incorporated into the tomato genome separately during
evolution.
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| Figure 5.
Possible evolutionary relationship among the GK
(A) and the GPR (B) proteins. The phylogenetic tree was generated using
the PHYLIP program (Felsenstein, 1993). Numbers are bootstrap values
given as percentages, and only 50% or greater values are indicated at
a node. Abbreviations and accession numbers are as described in
``Materials and Methods''.
|
|
tomPRO1 and tomPRO2 Encode Functional
Enzymes Catalyzing GK and GPR Activities
We demonstrated that the tomPRO2 gene encodes a
functional P5CS by the fact that it can complement both a GK and a GPR
defect in E. coli. As shown in Figure
6B, the plasmid carrying the
tomPRO2 fragment could complement the polar
proB1658::Tn10 mutation, whereas the
vector by itself was unable to do so. In accord with our earlier
observations (García-Ríos et al., 1997), the
tomPRO1 cDNA clone inserted pBluescript KSII(+) could
likewise complement the Pro auxotrophic mutation in KC1325 (Fig. 6B).
All strains could grow on the medium containing Pro (Fig. 6A).
These results demonstrate that although both tomPRO1 and
tomPRO2 show only 35% amino acid sequence identity,
they both specify functional GK and GPR.
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| Figure 6.
Complementation of a proBA mutation
by tomPRO1 and tomPRO2, and their
products expressed in E. coli. A and B, Expression
vectors containing the tomPRO1 and
tomPRO2 cDNA clones were introduced into strain KC1325
(a derivative of BL21[DL3]pLysS carrying the
proB1658::Tn10 insertion, which
is polar on proA). a, KC1325 harboring the vector, pKS
only. b, KC1325 harboring pPRO1. c, KC1325 harboring pET32a only. d,
KC1325 harboring pET32PRO2. Strain KC1325 containing each plasmid was
streaked on minimal M63 medium containing Glc, thiamine, and IPTG with
(A) and without (B) Pro, and incubated for 2 d at 37°C. All
strains could grow on the media supplemented with Pro (A). C, Total
cell extracts from either E. coli strain HB101,
containing pKS (lane 1) and pPRO1 (lane 2), or strain KC1325,
containing pET32a (lane 3) and pET32PRO2 (lane 4), were analyzed by
SDS-PAGE. The gels were stained with Coomasie brilliant blue.
tomPRO1 products are indicated as GK and GPR, and the
tomPRO2 product as P5CS. Numbers at left refer to size
standards (in kD).
|
|
We verified with SDS-PAGE analysis that the tomPRO2 gene
directs the synthesis of a single polypeptide of the expected mass of
98 kD in E. coli (Fig. 6C), whereas tomPRO1
directs the synthesis of two polypeptides: the approximately 33-kD GK
and the approximately 44-kD GPR, in accord with our previous report
(García-Ríos et al., 1997) that tomPRO1 is
recognized as a polycistronic locus in E. coli.
tomPRO1 and tomPRO2 Are Located at
Different Loci within the Tomato Nuclear Genome
Restriction fragments of total tomato DNA were probed with
sequences from the GK region of tomPRO1 and with
full-length tomPRO2. Probes made from both clones hybridized
with an efficiency of less than 10 copies per haploid genome (data not
shown). This result suggested that each locus is present in the nuclear
genome but not in an organelle genome, which is present at a much
higher copy number (>50 copies).
We mapped both genes using a segregating population of progeny from the
cross L. esculentum × L. pennellii. As
shown in Figure 7, the tomPRO1
locus was mapped to a region of 2.6 cM adjacent to the TG33 locus on
chromosome 2. Mapping of the tomPRO1 locus to chromosome 2 supports unambiguously our conclusion that a polycistronic locus is
present in the tomato nuclear genome, as opposed to being in a
chloroplast or mitochondrial genome, which would not only be in a much
higher copy, but would also be maternally inherited. The
tomPRO2 locus proved to be present in a region of
approximately 1 cM between the TG228 and CT92 markers on chromosome 8 (Fig. 7B, left panel). Southern analysis with a tomPRO2
probe at a milder stringency (Fig. 7B, right panel) revealed additional
bands that appeared homologous to tomPRO2, which did not
hybridize to a tomPRO1 probe under these conditions. These
tomPRO2-related bands were mapped to chromosome 6.
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| Figure 7.
Mapping of tomPRO1,
tomPRO2, and tomPRO2 homolog in tomato
nuclear genome. A and B, Southern-blot analyses of total DNA from the
F2 population of crosses between L. esculentum and L. pennellii. Hybridizations were
performed with the GK part of the tomPRO1 cDNA fragment
(A) and the full length of tomPRO2 cDNA as the probes
(B) at a high-stringency wash condition, 0.1× SSC, 0.1% SDS, at
42°C (High), and at a low-stringency wash condition, 0.2× SSC, 0.1%
SDS, at 25°C (Low). Two bands that appeared at a low-stringency
condition are depicted by arrows. This figure shows a representative
portion of the blots, in which a total of 67 of the F2
populations were used for the RFLP mapping. Shown at the top of each
lane is the RFLP pattern representative for the L. esculentum homozygote (e), the L. pennellii
homozygote (p), or their heterozygote (e/p). C, Map position of
tomPRO1, tomPRO2, and
tomPRO2 homologs on the tomato chromosome. The maps were
drawn by segregation analysis of RFLPs based on data by Tanksley et al.
(1992). The map distances (in cM) are indicated on the left. Maps are
not drawn to scale. tomPRO1, tomPRO2, and
tomPRO2 homologs (tomPRO2homo) were located on
chromosomes 2, 8, and 6, respectively.
|
|
| |
DISCUSSION |
Pro Accumulation Is Not Correlated with the tomPRO1
and tomPRO2 Message Levels in Tomato
It has been proposed that transcriptional control of the P5CS
gene, which encodes a bifunctional enzyme catalyzing the first and
second reactions of Pro synthesis, is important for the regulation of
accumulation of this imino acid during osmotic stress in plants. This
conclusion was based on the observation that NaCl stress increased the
P5CS transcript level in moth bean, Arabidopsis, and rice (Hu et al.,
1992; Savouré et al., 1995; Yoshiba et al., 1995; Igarashi et
al., 1997). In rice and Arabidopsis, the increases in the Pro levels
were accompanied by coordinate increases in the P5CS transcript levels.
(The accumulation of Pro was not monitored in moth bean during the
course of induction of the P5CS message [Hu et al., 1992].)
Arabidopsis has two P5CS isoenzymes, encoded in the AtP5CS1
and AtP5CS2 genes (Savouré et al., 1995; Yoshiba et
al., 1995; Strizhov et al., 1997; Zhang et al., 1997).
AtP5CS1, which was estimated to synthesize about 60% to
80% of the total P5CS mRNA (Strizhov et al., 1997), exhibited up to an
8-fold induction upon osmotic stress (Savouré et al., 1995;
Yoshiba et al., 1995; Strizhov et al., 1997), whereas
AtP5CS2 exhibited a <4-fold regulation (Strizhov et al.,
1997; Zhang et al., 1997). Because tomato accumulates much more Pro
than Arabidopsis or rice, our initial hypothesis had been that tomato
might show an even more sensitive regulation of P5CS transcript
accumulation than the other two plants. To test whether this is the
case, we determined the effect of NaCl stress on the accumulation of
the tomPRO1 and tomPRO2 transcripts. We also
determined whether there is a special transcriptional regulation of
these two loci in pollen, which contain very high levels of Pro.
Although NaCl stress was shown to cause some Pro accumulation in rice
and Arabidopsis, this metabolite reached only a maximum of 2 and 6 µmol Pro/g fresh weight in these two species (Savouré et al.,
1995; Igarashi et al., 1997; Zhang et al., 1997). In contrast, we found
that tomato accumulated to 90 and 105 µmol Pro/g fresh weight in
leaves and roots, respectively, after 6 d of treatment with 200 mM NaCl (Fig. 2), representing a 60- to 80-fold increase over the level in unstressed plants. Surprisingly, in view of the
results reported for Arabidopsis and rice, there was only about a 2- to
3-fold change in the tomPRO2 transcript level throughout the
entire time course of NaCl treatment. In roots the accumulation of Pro
was maximal at d 6 of NaCl treatment, after which it declined gradually, but this was not reflected by a decrease in the
tomPRO2 message level (Fig. 2). The Pro pool size was
30-fold higher in the NaCl-adapted S15 tomato tissue-culture cells than
in the control, unadapted S0 cells (Fig. 3). Despite this large
difference in Pro content, the tomPRO2 message was present
at similar levels in the two types of cells.
Previously, we showed that the level of the tomPRO1 message
in the S15 cells was approximately 4-fold higher than in the S0 cells
(García-Ríos et al., 1997). However, because of the low level of this transcript even in the NaCl-stressed cell line, this
induction of the transcript probably is not sufficient to account for
the increase in the Pro content. The highest level of Pro in all
tissues tested was found in pollen of unstressed plants. (We did not
measure the Pro content in pollen of NaCl-stressed plants.) The
tomPRO2 message level, however, was unchanged compared with
other tissues, and the tomPRO1 message was undetectable in pollen. These results indicate that in tomato, the large increases in
Pro levels in response to NaCl stress or pollen-specific developmental signals are brought about without substantial increases in the levels
of the tomPRO1 and tomPRO2 messages.
Because we only determined the steady-state accumulation of these
messages, we cannot infer that tomPRO1 and
tomPRO2 are transcribed constitutively. In principle, it is
conceivable that changes in the rates of synthesis of these transcripts
could be compensated for by comparable changes in their turnover. Zhang
et al. (1997) demonstrated with a GUS reporter fusion that the 2- to
4-fold increase in the level of the AtP5CS2 message after
dehydration or NaCl stress in transgenic Arabidopsis and tobacco plants
was the result of transcriptional induction. However, all of the other studies on the regulation of the accumulation of the P5CS messages in Arabidopsis and rice (Savouré et al., 1995; Yoshiba et al., 1995; Igarashi et al., 1997; Strizhov et al., 1997) involved only measurements of the steady-state levels of these messages, and, therefore, direct evidence is lacking that the increases in the accumulation of these transcripts upon water stress are necessarily brought about by induction of transcription initiation.
Aside from control of the accumulation of P5CS message (at the level of
synthesis or turnover), there are several other possible mechanisms for
the control of Pro biosynthesis. The enzyme for the first step, GK, is
sensitive to feedback regulation by Pro (Hu et al., 1992;
García-Ríos et al., 1997). However, there may be
important differences in the allosteric properties of the enzymes in
tomato and other plants, as indicated by the observations that the
activity of the tomPRO1-encoded P5CS was inhibited 50% by
0.07 mM Pro (García-Ríos et al., 1997),
whereas 5 mM Pro was required to elicit 50% inhibition of
the GK activity of moth bean P5CS (Zhang et al., 1995). We have not
been successful in measuring the kinetic properties of
tomPRO2 product because of difficulties in obtaining this
enzyme in a soluble form. However, we have preliminary evidence that
this enzyme, which is more similar in its amino acid sequence to the
moth bean P5CS than to the tomPRO1 product, is also
sensitive to feedback inhibition by Pro. It is possible that the
regulation of synthesis of Pro in tomato is effected by relief of
allosteric inhibition of the activities of the tomPRO1 and
tomPRO2 products under NaCl or dehydration stress. Tomato
may have an additional gene related to tomPRO2 (Fig. 7), for
which we have no sequence information. If it proves to be related to
P5CS, it could participate in the regulation of Pro synthesis.
The accumulation of Pro could also be regulated by changes in the rate
of its catabolism to glutamate by the combined action of Pro
dehydrogenase and P5C dehydrogenase. However, because Pro dehydrogenase
is a mitochondrial enzyme (Kiyosue et al., 1996), effective catabolism
of Pro would presumably require transport of Pro from the cytosol to
the mitochondria. In Arabidopsis NaCl stress or dehydration
down-regulates the accumulation of the message for Pro dehydrogenase
(Kiyosue et al., 1996; Peng et al., 1996; Verbruggen et al., 1996).
Although repression of the synthesis of Pro dehydrogenase could have a
role in the long-term regulation of Pro accumulation in response to
water stress, the effect of water stress on the activity or stability
of Pro dehydrogenase itself has not been determined in Arabidopsis.
Repression of transcription of the gene for Pro dehydrogenase would be
an efficient mechanism for increasing the Pro pools size only if this
response is accompanied by a simultaneous inactivation or turnover of
preexisting Pro dehydrogenase molecules. Direct evidence on the
relative contributions of the biosynthetic and catabolic pathways for
the regulation of Pro pool size was provided in cultured tomato cells
by the N-isotope-tracing experiments of Rhodes
et al. (1986). These studies indicated that the 300-fold increase in
the Pro accumulation resulting from 25% PEG stress was mainly due to a
10-fold increase in the rate of biosynthesis and provided no evidence
that the rate of Pro catabolism was inhibited under these conditions.
Changes in the intracellular Pro levels could also be accomplished by
translocation of this metabolite between different tissues or cell
compartments. Two genes, ProT1 and ProT2, which
encode closely related Pro-transport proteins, have been cloned from Arabidopsis (Rentsch et al., 1996). Accumulation of the
ProT2 message was strongly elevated by NaCl stress,
indicating that control of the synthesis of Pro-transport proteins also
could be involved in the regulation of the cellular Pro pool sizes.
Two Evolutionarily Distinct Genes Are Present in the Tomato Nuclear
Genome
We showed that the tomPRO1 and tomPRO2 loci
are present in the tomato nuclear genome. Comparison of protein
sequence, codon usage, and phylogenetic analysis suggested that
tomPRO2 is in a tight group containing the P5CS proteins
from plants and other higher eukaryotes. In contrast,
tomPRO1 has several unique features that distinguish it from
the eukaryotic P5CS group. First, tomPRO1 did not show a
high identity to eukaryotic P5CS (32%-35% at the amino acid level).
A comparison of codon usage of tomPRO1 with genes from
E. coli or tomato suggested that the codon usage of tomPRO1 is not typical for either E. coli or
tomato genes, but something in between. Second, tomPRO1
has a dicistronic structure, similar to polycistronic operons found in
bacteria, and in fact, tomPRO1 is recognized as a
dicistronic operon in E. coli (Fig. 6C). Third, phylogenetic
analysis placed tomPRO1 within the same clade of
prokaryotes, separate from the other eukaryotic P5CS genes. These
characteristics suggest that tomPRO1 and tomPRO2, which are both nuclear loci, might have different origins. We present
alternative hypotheses for the possible origin of the tomPRO1 locus, but we would like to emphasize that at this
stage we do not have sufficient data to distinguish among these
speculative alternatives.
There are several reports in which isozymes are encoded by multigene
families in nuclear genomes, as exemplified by the existence of dual
genes for P5CS in Arabidopsis (Strizhov et al., 1997). Multigene
families may be derived by gene duplication or by gene conversion from
a single gene. It is, however, unlikely that the tomPRO1 and
tomPRO2 loci arose in tomato by such mechanisms, because of the difference in their coding regions. The prokaryotic features of
tomPRO1 are consistent with the notion that it may have been acquired by organelle-to-nucleus gene transfer, or by uptake of DNA of
prokaryotic origin into the nuclear genome. According to the theory of
endosymbiosis, mitochondria and chloroplasts originated from once
free-living eubacteria (Gray, 1989), followed by the loss of genes from
the organellar genomes or transfer to the nucleus (Weeden, 1981;
Palmer, 1985). The tufA gene, encoding the chloroplast protein synthesis elongation factor Tu in Arabidopsis, and the rpl22 gene, encoding chloroplast ribosomal protein CL22, are
examples of genes that were transferred from the chloroplast genome to the nucleus (Baldauf and Palmer, 1990; Gantt et al., 1991). There are
two isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in tobacco
and maize, one found in the chloroplasts and the other in the cytosol.
Although both of these isoenzymes are encoded in nuclear genome, they
display sequence divergence corresponding to the prokaryotic/eukaryotic
separation (Shih et al., 1986; Brinkmann et al., 1987). These examples
support the endosymbiotic theory of chloroplast evolution, with
subsequent transfer of genes from the endosymbiont to the host nucleus.
An alternative explanation for the origin of tomPRO1 is that
it may have been acquired from a bacterium or virus by a horizontal gene transfer. This mechanism has been invoked to explain the close
homology between vertebrate hemoglobin genes and the leghaemoglobin gene from legume (Lewin, 1981). Some soil bacteria (e.g. the genus Rhizobium) have two forms of Gln synthetase, a prokaryotic
type and a eukaryotic type. It has been proposed that the
eukaryotic-type genes may have been incorporated by a horizontal
transfer from a host plant to symbiont bacteria (Carlson and Chelm,
1986; Smith et al., 1992).
It is unlikely that tomPRO1 has been translocated from an
organelle to the nucleus, because if tomPRO1 originated from
organelles, then there should be tomPRO1 homologs in other
plants. However, we did not find any homologs in organellar genomes
using a computer search. This is also supported by the results of
Southern-blot analysis that sequences homologous to tomPRO1
are present in species of the Solanaceae family, such as tobacco,
potato, and two wild species of tomato (L. pennellii and
Lycopersicon cheesmanii), but could not be detected in rice
and maize (García-Ríos, 1995). These results suggest
that horizontal gene transfer may have been responsible for the
integration of the tomPRO1 gene into the nuclear genome
after the divergence of dicots and monocots, but before divergence of
the family Solanaceae. An examination of the subcellular localization
of the tomPRO1 product and a more detailed search of
tomPRO1 homologs in other plants may lead to clues as to the origin of tomPRO1, as well as to the mechanism of its
transfer. There is little evidence about the possibility that bacteria
or viruses could be responsible for the introduction of the
tomPRO1 gene into the tomato genome. However, because of its
close sequence similarity to the S. thermophilus proBA and
the common lack of the C-terminal 100-amino acid tail in the GK region,
the tomPRO1 locus may have been derived from a bacterium
related to S. thermophilus.
Although we found evidence for tomPRO1-like genes in some
other Solanaceae (see above), it is not clear whether this locus is
present in other plants. The tomPRO1 clone and the P5CS
clone from moth bean (Hu et al., 1992) were isolated by complementation of a proB point mutation in E. coli, but all
subsequent plant P5CS clones, including tomPRO2, were
isolated on the basis of sequence homology with the P5CS gene family.
It is possible that homologs of tomPRO1 might be present in
other plants, but because of the sequence divergence between
tomPRO1 and the other plant P5CS clones, it is unlikely that
the former type of gene could be cloned by sequence hybridization with
P5CS clones.
There are few reports of the coexistence of prokaryotic and eukaryotic
forms of a gene in a single genome. The coexistence of dual genes
specifying isoforms of enzymes in one organism may serve two functions.
Multiple copies of genes could satisfy a need for high amounts of a
particular gene product, or they could provide an efficient means for
differential regulation of gene-expression development in response to
different factors (Long and Dawid, 1980). It seems likely that
tomPRO1 and tomPRO2 will fit into the latter type
of gene family, because of their distinct pattern of expression.
Because the tomPRO2 message was much more abundant than the
tomPRO1 in all tissues under the conditions we tested, it is
likely that tomPRO2 may have the predominant responsibility for Pro production in these situations, and it is possible that the
expression of the tomPRO1 gene might be restricted to very specific cell types or developmental stages. The significance of the
existence of tomPRO1 and the coexistence of
tomPRO1 and tomPRO2 at this time remains elusive.
| |
FOOTNOTES |
1
This work was funded by the U.S. Department of
Agriculture (grant no. 93-37100-8871).
2
Present address: Department of Natural Sciences,
Texas A&M International University, Laredo, TX 78041.
*
Corresponding author; e-mail lcsonka{at}bilbo.bio.purdue.edu; fax
1-765-496-1496.
Received March 4, 1998;
accepted July 9, 1998.
| |
ABBREVIATIONS |
Abbreviations:
GK, -glutamyl kinase.
GPR, -glutamyl
phosphate reductase.
IPTG, isopropyl--D-thiogalactopyranoside.
P5C, 1-pyrroline-5-carboxylate.
P5CS, 1-pyrroline-5-carboxylate synthetase.
RFLP, restriction
fragment-length polymorphism.
| |
ACKNOWLEDGMENTS |
We thank S. Fletcher for technical support, Dr. M. Hasebe for
help with the construction of the phylogenetic tree, Dr. G. Martin for
materials and assistance with RFLP mapping, Dr. D. Rhodes for helpful
discussions, and Dr. L. Szabados for the sequence of portions of the
ATP5CS2 gene prior to its publication.
| |
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33:
857-865
[CrossRef][ISI][Medline]
Khoo U,
Stinson HT Jr
(1957)
Free amino acid differences between cytoplasmic male sterile and normal fertile anthers.
Proc Natl Acad Sci USA
43:
603-607
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Abstract
Introduction
