From the Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
Received for publication, September 19, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Full-length cDNA clones encoding
deoxyhypusine synthase (DHS) and eucaryotic initiation factor 5A
(eIF-5A) have been isolated from a cDNA expression library prepared
from tomato leaves (Lycopersicon esculentum, cv. Match)
exposed to environmental stress. DHS mediates the first of two
enzymatic reactions that activate eIF-5A by converting a conserved
lysine to the unusual amino acid, deoxyhypusine. Recombinant protein
obtained by expressing tomato DHS cDNA in Escherichia coli proved capable of carrying out the deoxyhypusine synthase reaction in vitro in the presence of eIF-5A. Of particular
interest is the finding that DHS mRNA and eIF-5A mRNA show a
parallel increase in abundance in senescing tomato flowers, senescing
tomato fruit, and environmentally stressed tomato leaves exhibiting
programmed cell death. Western blot analyses indicated that DHS protein
also increases at the onset of senescence. It is apparent from previous studies with yeast and mammalian cells that hypusine-modified eIF-5A
facilitates the translation of a subset of mRNAs mediating cell
division. The present study provides evidence for senescence-induced DHS and eIF-5A in tomato tissues that may facilitate the translation of
mRNA species required for programmed cell death.
Eucaryotic translation initiation factor 5A
(eIF-5A)1 is deemed to be
present in all eucaryotic cells and, following post-translational modification to an activated form, appears to play a role in the initiation of protein synthesis (1). This modification entails the
addition of a butylamine residue derived from spermidine to a highly
conserved lysine of inactive eIF-5A, resulting in the formation of the
unusual amino acid, hypusine, and activated eIF-5A (2). The first
reaction leading to the formation of hypusine is catalyzed by
deoxyhypusine synthase (DHS; EC 1.1.1.249), an enzyme that adds
butylamine to the conserved lysine of eIF-5A to form deoxyhypusine. A
subsequent reaction in which deoxyhypusine is converted to hypusine is
catalyzed by deoxyhypusine hydroxylase (EC 1.14.99.29) (2, 3).
Hypusine modification apparently ensues immediately following
translation of eIF-5A inasmuch as there is no accumulation of either
eIF-5A precursor (lysine form) or eIF-5A intermediate (deoxyhypusine
form) unless cells are treated with inhibitors of either deoxyhypusine
synthase or deoxyhypusine hydroxylase (1).
Hypusine-containing eIF-5A is not required for global protein
synthesis. Indeed, in eIF-5A-deficient yeast, protein synthesis is only
inhibited by ~30%, and this is accompanied by only a slight change
in the polysome profile (1, 4, 5). Rather, activated eIF-5A appears to
facilitate the translation of specific subsets of mRNA. For
example, yeast cells in which DHS has been inactivated are incapable of
dividing and simply enlarge (1, 6). These observations have prompted
the proposal that hypusine-containing eIF-5A facilitates translation of
the subset of mRNAs required for cell division and hence is
necessary for cell proliferation (1, 7). Similarly, inhibitors of the
synthesis of spermidine, which is required for DHS activity, have been
shown to decrease cell division (1, 7, 8).
Although it seems clear that hypusine-containing eIF-5A facilitates
protein synthesis, the way in which it does so is not fully understood.
The protein was initially identified as a putative translation
initiation factor based on its ability to stimulate methionyl-puromycin
synthesis under in vitro conditions (9), but this has since
been questioned in light of the fact that a similar effect on
translation is not observed in situ (10, 11). More recent
evidence suggests that eIF-5A facilitates protein synthesis by
promoting nuclear export of specific mRNAs (10). It has also been
proposed that eIF-5A may be involved in mRNA turnover, acting
downstream of decapping (11).
Isoforms of the gene encoding eIF-5A have also been isolated from plant
tissue. For example, two isoforms of eIF-5A exhibiting tissue-specific
expression have been isolated from tobacco (GenBankTM
accession numbers X635411 and X635412) (12), and recently DHS was also
cloned from tobacco (GenBankTM accession number AJ242017)
(13). The eIF-5A gene has also been cloned from humans
(GenBankTM accession number I53801), fungi
(GenBankTM accession numbers P19211 and P23301), yeast
(GenBankTM accession number D83166), Zea mays
(GenBankTM accession number Y07920), Solanum
tuberosum (GenBankTM accession numbers AB004823 to
AB004827), and alfalfa (GenBankTM accession number X59441).
However, apart from an expected involvement in translation, no specific
function has yet been ascribed to plant DHS and hypusine-containing
eIF-5A. The finding that there are isoforms of the plant eIF-5A gene
has prompted the proposal that the different isoforms facilitate the
translation of subsets of mRNA required for specific physiological
functions, including photosynthesis (12). In the present study, we
describe the isolation and characterization of cDNA clones from
tomato that encode DHS and eIF-5A. Of particular interest is the
finding that these genes are both up-regulated at the onset of natural senescence and in the event of cell death attributable to environmental stress, for this indicates that hypusine-modified eIF-5A may facilitate translation of the suite of mRNAs required for programmed cell death.
Plant Material--
Tomato seedlings (Lycopersicon
esculentum, cv. Match) were grown in 8-inch pots for 8 weeks under
greenhouse conditions. By this stage, the seedlings had developed three
pairs of leaves. Cotyledons for Western blot analysis were harvested at
5, 7, 11, 15, and 20 days after planting. Flowers for Northern blot
analysis were harvested at tight bud and open plus senescing stages of development. Fruit for Northern analysis was harvested at breaker (one-tenth of the surface area beginning to turn color), pink, red
firm, and red soft stages of development.
RNA Isolation and Construction of a cDNA Library--
For
preparation of a cDNA library, total RNA was isolated from
drought-stressed leaves of 8-week-old tomato seedlings according to
Davis et al. (14). Drought stress was induced by removing the roots from the seedlings and placing the cut stems in 2 M sorbitol for 6 h. Poly(A)+-RNA was
purified using a Poly(A)tract mRNA Isolation System (Promega). Double-stranded cDNA was prepared using the ZAP express cDNA
synthesis kit (Stratagene).
Library Screening--
Approximately 5 × 105
clones were screened with a DHS probe obtained by reverse
transcriptase-polymerase chain reaction (RT-PCR; PerkinElmer Life
Sciences GeneAmp DNA Thermal Cycler, model 2400). Template RNA for
RT-PCR was isolated from leaves of 8-week-old tomato plants that had
been chill-injured for 2 days at 5 °C in a growth chamber (16-h
light/8-h dark photoperiod) and subsequently rewarmed for 6 h in
the greenhouse. Reverse transcription was performed by adding 500 ng of
total RNA, 50 units of murine leukemia virus reverse
transcriptase (PerkinElmer Life Sciences), 2.5 mM MgCl2, 1 mM of each dNTP, 1 unit of RNase
inhibitor, and 2.5 µM oligo(dT) primer
(5'-GACTGCAGTCGACATCGATTTTTTTTTTTTTTTTT-3') containing ClaI,
PstI, and SalI restriction enzyme sites to 20 µl of 1× RT-PCR buffer (PerkinElmer Life Sciences). The reaction
mixture was incubated at room temperature for 5 min, at 42 °C for 50 min, and then heated to 99 °C for 5 min to inactivate the reverse
transcriptase. The cDNA product was amplified with 2.5 units of
Taq polymerase (Roche Molecular Biochemicals) in 100 µl of
reaction mixture containing 10 µl of reverse transcription
product, 1× PCR buffer plus MgCl2 (Roche Molecular
Biochemicals), 1 mM of each dNTP, 1 µM
upstream primer (5'-AGTCTAGAAGGTGCTCGTCCTGAT-3'), and 1 µM oligo(dT) primer. The PCR parameters were 1 min of
template denaturation at 95 °C, 1 min of primer annealing at
58 °C, and 2 min of primer extension at 72 °C for 35 cycles.
The PCR products were analyzed on 1% agarose gels, excised with
SalI and XbaI, and ligated into SalI-
and XbaI-excised pBluescript KS(+) plasmid (Stratagene).
Positive clones obtained by screening the cDNA library with this
DHS probe were recovered using the ExAssist Helper Phage/SOLR strain
system and recircularized in pBluescript SK( DNA Isolation and Sequencing--
Plasmid DNA was isolated by
alkaline lysis (15) and sequenced at MOBIX (McMaster University,
Hamilton, Ontario, Canada). The open reading frame of tomato DHS
cDNA was compiled and analyzed using the BLAST Search
(GenBankTM, Bethesda, MD), and sequence alignments were
achieved using the BCM Search Launcher (available on the World Wide Web).
Cloning of Eucaryotic Initiation Factor 5A--
One full-length
Arabidopsis and four full-length tomato cDNA clones
encoding eIF-5A were obtained by PCR using an Arabidopsis senescing leaf cDNA library and the environmental stressed tomato leaf cDNA library as templates, respectively. For
Arabidopsis eIF-5A, partial-length cDNA clones were
obtained using the internal upstream primer
(5'-AAARRYCGMCCYTGCAAGGT-3') with T7 (5'-AATACGACTCACTATAG-3') as a
downstream primer and the internal downstream primer
(5'-TCYTTNCCYTCMKCTAAHCC-3') with T3 (5'-ATTAACCCTCACTAAAG-3') as an
upstream primer. For tomato eIF-5As, partial-length cDNA clones
were obtained using the internal upstream primer
(5'-AAARRYCGMCCYTGCAAGGT-3') with T7 as a downstream primer and the
internal downstream primer (5'-ACYTCMACHACCTTGCARGG-3') with T3 as an
upstream primer. Internal primers were designed from the tobacco eIF-5A
sequences (12). The cDNA products were amplified with 2.5 units of
Expand High Fidelity polymerase (Roche Molecular Biochemicals) in 50 µl of reaction mixture containing 1-5 µl of template cDNA, 1×
PCR buffer plus MgCl2 (Roche Molecular Biochemicals), 1 mM of each dNTP, 1 µM upstream primer, and 1 µM downstream primer. The PCR parameters were 1 min of
template denaturation at 95 °C, 1 min of primer annealing at 48 or
52 °C, and 2 min of primer extension at 72 °C for 35 cycles. The
PCR products were subcloned into pBluescript KS(+) plasmid and
sequenced. Full-length cDNAs were obtained by PCR using specific
primers designed from the sequences of the partial-length cDNA PCR
products and Arabidopsis and tomato leaf cDNA libraries
as templates.
Overexpression of Tomato Deoxyhypusine Synthase and Arabidopsis
Eucaryotic Initiation Factor 5A in Escherichia coli--
Tomato DHS
and Arabidopsis eIF-5A were subcloned into the GST fusion
vector, pGEX-5X-3 (Amersham Pharmacia Biotech), using BamHI
and SalI restriction sites and overexpressed in E. coli DH5
Polyclonal antibodies against DHS were raised in rabbits using tomato
recombinant protein eluted from SDS-polyacrylamide gels as antigen.
Deoxyhypusine Synthase Assay--
The enzyme activity of
recombinant tomato DHS was assayed according to Park and Wolff (2) and
Bevec et al. (16) with slight modification. GST-DHS and
GST-eIF-5A fusion proteins were digested with factor Xa in order to
release GST, and the products of this digestion, GST plus DHS and GST
plus eIF-5A, were used for the assay. The standard reaction mixture
contained 5 µg of GST plus DHS, 20 µg of GST plus eIF-5A, 0.5 µCi
of [3H]spermidine-HCl ([terminal
methylene-3H]spermidine; 15 Ci/mmol; PerkinElmer Life Sciences), 1 mM NAD+,
and 1 mM dithiothreitol in 0.3 M glycine-NaOH
(pH 9.0) buffer in a total volume of 200 µl. In control reaction
mixtures, GST plus DHS and GST plus eIF-5A were replaced by
corresponding amounts of GST. The reaction mixture was incubated at
37 °C for 4 h, and 30 µl was then withdrawn and fractionated
by SDS-polyacrylamide gel electrophoresis (15%). Radiolabeled bands
were visualized by soaking the gel in a fix solution (isopropyl
alcohol/water/acetic acid, 25:65:10) for 30 min and for a further 30 min in Amplify (Amersham Pharmacia Biotech). The gel was then dried and
exposed for 6 days using a preflashed film (Amersham Pharmacia Biotech).
Northern and Western Blot Analysis--
For Northern blot
analysis, total RNA (10 µg) was fractionated on 1.0% denatured
formaldehyde-agarose gels and immobilized on Hybon-N+ nylon
membrane (Amersham Pharmacia Biotech). Hybridization conditions were as
described by Wang and Arteca (17). For Western analysis, homogenate
protein (20 µg) was fractionated on 10% SDS-polyacrylamide gels, and
the separated proteins were transferred to polyvinylidene difluoride
membrane (Bio-Rad). Immunoblotting was carried out according to Wang
et al. (18) using antiserum from rabbit as the primary
antibody and alkaline phosphatase-conjugated secondary antibody (Roche
Molecular Biochemicals).
Southern Analysis--
Genomic DNA was isolated from
tomato leaves and digested with the restriction endonucleases,
EcoRI, EcoRV, HindIII, and
XbaI. The digested products (10 µg of DNA) were
fractionated on a 1.0% agarose gel, immobilized on a nylon membrane,
and hybridized with DHS cDNA as for Northern analysis.
Assays--
Chlorophyll was assayed as described by Porra
et al. (19). Protein was measured according to Bradford
(20). Leakage of electrolytes from chill-injured leaves was measured as
a change in conductivity of leaf diffusates (21) using a Conductivity Bridge model 31 (Yellow Springs Instruments, Inc.).
Isolation of a cDNA Clone Encoding Deoxyhypusine
Synthase--
A full-length cDNA clone encoding tomato DHS
(GenBankTM accession number AF296077) was isolated by
screening a cDNA library prepared from sorbitol-treated leaves with
a probe obtained by RT-PCR. The cDNA contains 1610 base pairs,
including a 53-base pair 5'-noncoding sequence and a 414-base pair
3'-noncoding sequence, and encodes a 381-amino acid polypeptide with a
calculated molecular mass of 42.1 kDa (Fig.
1). The derived amino acid sequence of tomato DHS cDNA has high homology with the amino acid sequences of
human DHS (GenBankTM accession number 1352267), yeast DHS
(Saccharomyces cerevisiae, GenBankTM accession
number 731670), fungal DHS (Neurospora crassa,
GenBankTM accession number 1352268), Archaeobacteria DHS
(Methanococcus janaschii, GenBankTM
accession number 2498303), and tobacco DHS (GenBankTM
accession number AJ242017) (Fig. 2). The
tomato DHS sequence also has high homology with an
Arabidopsis genomic clone (GenBankTM accession
number AB017060) that has not been functionally annotated. Using
primers designed from this sequence and the tomato DHS sequence, PCR
products encoding Arabidopsis DHS, carnation DHS, and banana
DHS were cloned using senescing Arabidopsis leaf, carnation
flower, and ripening banana fruit cDNA libraries as templates.
Full-length cDNA clones encoding Arabidopsis DHS
(GenBankTM accession number AF296078), carnation DHS
(GenBankTM accession number AF296079), and banana DHS
(GenBankTM accession number AF296080) were subsequently
obtained by screening these libraries with the PCR products, and the
derived amino acid sequences proved to have high homology with the
tomato DHS amino acid sequence (Fig. 2).
Up-regulation of Deoxyhypusine Synthase Expression during
Environmental Stress--
Northern blot analyses indicated that
expression of the tomato DHS gene is up-regulated in osmotically
stressed leaves. In these experiments, 8-week-old plants bearing 3-4
pairs of leaves were derooted by cutting the stem at the soil surface,
and the aerial part of the plant was placed in 2 M
sorbitol, a treatment known to induce osmotic stress (17). The
abundance of DHS transcript in preparations of total RNA increased
substantially within 6 h of treatment with sorbitol but showed no
increase over the same period in leaf RNA preparations from control
derooted plants placed in water (Fig.
3A). Indeed, no increase in
DHS transcript level was apparent even after 18 h for the water
control plants, indicating that the up-regulation of DHS expression
observed for leaves of sorbitol-treated plants reflected osmotic stress
rather than a wound effect arising from the derooting procedure. This
was further confirmed by simply withholding water from derooted plants.
Within 6 h, the water-deprived plants were wilted, and there was
an increase in DHS transcript level comparable with that induced by
treatment with sorbitol (data not shown). In addition, treatment of the plants for up to 20 h with 1 ppm ethylene had no effect on DHS transcript level, indicating that the up-regulation of DHS in response
to sorbitol treatment or withholding water is not induced by the
increase in ethylene production that normally accompanies drought
stress (17).
Expression of the tomato leaf DHS gene is also up-regulated in response
to chilling injury. In these experiments, total RNA for Northern blots
was isolated from the second leaf pairs of 8-week-old potted plants
that had been exposed to 5 °C in a growth chamber for varying
periods of time and subsequently allowed to rewarm at ambient
temperature in the greenhouse. The degree of chilling injury was
assessed by determining the conductivity of leaf diffusates, which is a
measure of membrane leakiness (21). For plants chilled for 2 days at
5 °C with no subsequent rewarming (C2W0 plants), there was no
up-regulation of DHS expression and no change in leaf diffusate
conductivity (Fig. 4, lanes
1 and 2). However, in keeping with the fact that
chilling injury is more strongly manifested during subsequent rewarming
(22), there was a substantial increase in DHS transcript abundance
coincident with a slight increase in membrane leakage within 6 h
of rewarming (C2W6 plants) (Fig. 4, lane 3). The
C2W6 plants also exhibited slight wilting symptoms. Within 24 h of
rewarming (C2W24 plants), wilting symptoms had completely disappeared,
diffusate conductivity had returned to normal, and DHS transcript
abundance was at background levels, indicating that expression of the
gene had been down-regulated (Fig. 4, lane 4).
Thus, DHS expression was up-regulated coincident with the onset of
symptoms of chilling injury and down-regulated during recovery from the
low temperature episode.
In order to confirm that changes in expression of DHS are correlated
with the onset of chilling injury rather than recovery, tomato plants
were subjected to more severe chilling injury by exposing them to
5 °C for 2 days followed by rewarming at ambient temperature for 1 day and a further 3-day 5 °C chilling episode with no subsequent
rewarming (C3W0 plants). This resulted in stronger up-regulation of DHS
expression and extensive membrane leakage reflecting chilling injury
(Fig. 4, lane 5). The C3W0 plants also exhibited
strong wilting symptoms. After a 6-h rewarming period (C3W6 plants),
there was still high expression of DHS and extensive membrane damage
(Fig. 4, lane 6), and the plants were still
visibly wilted. However, after a 24-h period of rewarming (C3W24), the plants were beginning to regain turgor; diffusate conductivity, although still high by comparison with background levels, was lower,
reflecting repair of membrane damage; and DHS expression was only
barely detectable (Fig. 4, lane 7). Indeed,
within 48 h of returning the C3W0 plants to ambient temperature,
the plants regained full turgor. These observations collectively
indicate that DHS expression is up-regulated coincident with the onset of chilling injury but is not required for the recovery phase.
In a final set of experiments, tomato plants were exposed to 5 °C
continuously for 6 days with no subsequent rewarming (C6W0 plants).
These plants were severely wilted and showed high levels of DHS
expression as well as extensive membrane damage as reflected by high
leaf diffusate conductivity (Fig. 4, lane 8).
During a subsequent 6-h rewarming period (C6W6 plants), DHS expression was further up-regulated, and there was a slight further increase in
leakage (Fig. 4, lane 9). After 24 h of
rewarming (C6W24 plants), DHS expression had begun to abate, membrane
damage was still extensive, and the leaves showed visual symptoms of
dying (Fig. 4, lane 10), and after 48 h at
ambient temperature, the leaves were crinkly and dead. Thus, 6 days of
continuous exposure to 5 °C with no intermittent opportunity for
recovery proved to be a lethal episode of chilling during which there
was strong expression of DHS.
Up-regulation of Deoxyhypusine Synthase Expression during Natural
Senescence--
DHS expression is also up-regulated during natural
senescence. This was demonstrated by probing Northern blots of total
RNA from tomato flowers and fruit at different stages of development with radiolabeled DHS cDNA. Levels of DHS transcript were barely detectable in flower buds and greatly enhanced in preparations of total
RNA isolated from a mixture of open and senescing flowers (Fig.
5A). The abundance of DHS
transcript also proved to be very low in breaker tomato fruit, pink
fruit, and fully ripe red firm fruit but increased substantially as
fully ripe fruit began to soften coincident with the onset of
senescence (Fig. 5B). These observations indicate that DHS
expression is not required for fruit ripening and is only up-regulated
when the fully ripened fruit begins to senesce.
In order to confirm that up-regulated expression of the DHS gene
reflected increased expression of its cognate protein, Western blots
were probed with polyclonal antibodies raised against recombinant DHS
protein. This is illustrated for developing tomato cotyledons in Fig.
6. Food reserves are stored in the
cotyledonary tissue of seeds, and during germination the cotyledons
senesce and their food reserves are mobilized to support growth. DHS
protein was not detectable in cotyledon tissue after 5 days of
germination but was minimally present by day 7, reached a peak by day
15 coincident with maximal levels of chlorophyll in the tissue, and
stayed high between days 15 and 20 as chlorophyll levels began to
decline and the cotyledons became visibly senescent (Fig. 6,
A and B). DHS mRNA levels increased between
days 7 and 11 after germination and remained high through to day 20 coincident with the onset of cotyledon senescence (Figs. 6,
A and B). Thus, mRNA and protein levels for
DHS began to increase well before the onset of a decline in chlorophyll
levels, one of the first manifestations of senescence execution,
indicating that the enzyme may be involved in the upstream regulatory
control of senescence.
Coexpression of DHS and Eucaryotic Translation Initiation Factor
5A--
Full-length cDNA clones for four isoforms of tomato eIF-5A
(eIF-5A1, GenBankTM accession number AF296083; eIF-5A2,
GenBankTM accession number AF296084; eIF-5A3,
GenBankTM accession number AF296085; eIF-5A4,
GenBankTM accession number AF296086) were isolated by PCR
using the cDNA library prepared from sorbitol-treated leaves as a
template and primers designed from tobacco eIF-5A
(GenBankTM accession numbers X635411 and X635412). The
tomato eIF-5A sequences are 89-92% identical at the amino acid level
and 70-80% identical at the nucleotide level, and they are also
closely similar (80-97% identity at the amino acid level to other
plant eIF-5A sequences (Table I). The
extent of sequence identity is lower (50-60% at the amino acid level)
when the tomato eIF-5A sequences are compared with those of human,
fungi, and yeast (Table I).
Full-length cDNA for one of the tomato isoforms, eIF-5A1, was used
to probe Northern blots of total RNA isolated from environmentally stressed tissue and naturally senescing tissue. In keeping with the
fact that DHS activates eIF-5A, the genes encoding these proteins were
found to be up-regulated in parallel in drought-stressed leaf tissue
(Fig. 3, A and B), chill-injured leaf tissue
(Fig. 4), senescing tomato blossoms (Fig. 5A), and senescing
(red soft) tomato fruit (Fig. 5B). It is noteworthy,
however, that DHS expression exhibits more plasticity than eIF-5A
expression during cycles of sublethal chilling stress with intervening
periods of recovery applied to tomato leaves (Fig. 4).
Enzyme Activity of Recombinant Deoxyhypusine Synthase
Protein--
In order to confirm the identity of the isolated tomato
DHS gene, the enzymatic activity of the recombinant protein obtained by
overexpression of DHS cDNA in E. coli was assayed. This
involved measuring the ability of recombinant tomato DHS protein to
transfer butylamine from radiolabeled spermidine to recombinant
Arabidopsis eIF-5A (GenBankTM accession number
AF296082) protein. Recombinant DHS and eIF-5A were both made as GST
fusion proteins. GST was released from the fusion proteins by
proteolytic cleavage with factor Xa, and the resultant protein mixtures
(GST plus DHS and GST plus eIF-5A) were used for the DHS activity
assays. Thus, the standard reaction mixture contained GST plus DHS and
GST plus eIF-5A protein mixtures (Fig. 7,
lane D). In addition, three control reactions
were run: GST alone (Fig. 7A), GST plus DHS (B),
and GST plus eIF-5A (C), and in each case sufficient free
GST was added to make the protein concentrations of the control
reaction mixtures equivalent to that of the standard reaction mixture
(Fig. 7, A-C). Catalytic activity was discerned by
autoradiography after SDS-polyacrylamide gel electrophoresis of the
reaction mixtures. The standard reaction mixture yielded a radioactive
band at 18 kDa (Fig. 7D). This corresponds to the
radiolabeled intermediate form of eIF-5A, which is formed when DHS
transfers butylamine from radiolabeled spermidine to a conserved lysine
residue of inactive eIF-5A (2). No 18-kDa proteins were detectable in
the control reaction mixture containing radiolabeled spermidine and GST
alone (Fig. 7A) or radiolabeled spermidine and GST plus
eIF-5A alone (Fig. 7C). Unused radiolabeled spermidine ran
off the gel (Fig. 7, A and C). These observations collectively indicate that recombinant DHS exhibits its expected catalytic activity and, further, that recombinant eIF-5A is capable of
being deoxyhypusine-modified.
Southern Analysis of Genomic Deoxyhypusine Synthase--
The
Northern blot data illustrated in Figs. 3-6 were obtained using
full-length tomato DHS cDNA as a radiolabeled probe. The same
patterns of DHS expression in chill-injured and drought-stressed leaves
and in naturally senescing flowers and fruit were obtained when
Northern blots of total RNA were probed with cDNA corresponding to
the 3'-noncoding region of tomato DHS (data not shown). This could mean
that there is a single isoform of DHS in tomato. In an attempt to
clarify this, Southern blots of tomato genomic DNA digested with
restriction enzymes were probed with full-length DHS cDNA. Genomic
DNA was digested with XbaI and EcoRI, which cut
in the open reading frame of DHS, and with EcoRV and
HindIII, for which there are no open reading frame cut
sites. In each case, several restriction fragments were detected,
suggesting the presence of multiple DHS isoforms (Fig.
8). However, these data are not conclusive because the tomato DHS genomic sequence(s) is not known, and
it is possible that the restriction enzymes are cutting within one
or more introns.
DHS catalyzes the first reaction in a two-step conversion of
inactive eIF-5A to its activated form. The DHS-mediated reaction entails the transfer of a butylamine residue from spermidine to a
conserved lysine of inactive eIF-5A, forming the unusual amino acid,
deoxyhypusine, which is then converted to hypusine by deoxyhypusine hydroxylase (1). Hypusine-modified eIF-5A, the active form of the
protein, is the only cellular protein known to contain hypusine. It is
present in all eucaryotic cells, and although its precise function has
not been elucidated, it appears to facilitate mRNA translation (1).
It was initially designated as a translation initiation factor based on
in vitro experiments indicating that it stimulates the
formation of methionyl-puromycin, a dipeptide analogue (9). However,
more recent experiments have demonstrated that eIF-5A is not involved
in the initiation of global protein synthesis. For example, mutating
the single isoform of DHS or both isoforms of eIF-5A in yeast results
in only marginal changes in total protein synthesis (1, 4, 5). The DHS
mutants are, however, incapable of cell division, yet they remain alive and simply enlarge (1, 6). Inactivation of both isoforms of yeast
eIF-5A produces the same phenotype. Indeed, the isoforms appear to be
functionally redundant in that inactivation of either alone does not
produce the phenotype (6). A correlation between growth arrest and a
reduction in hypusine formation has also been observed in
spermidine-depleted mammalian cells (23).
These observations have been interpreted as indicating that eIF-5A
facilitates translation of the suite of mRNAs required for cell
division (1) and have prompted the more general view that the various
isoforms of eIF-5A promote translation of specific subsets of mRNA
required for selected cell functions (4). Moreover, recent data
indicate that eIF-5A functions as a nucleocytoplasmic shuttle protein
that facilitates translation by mediating the translocation of specific
mRNAs from the nucleus to the cytoplasm. For example,
immunofluorescence and immunogold labeling studies with mammalian cells
have demonstrated that eIF-5A is localized in both the cytoplasmic and
nuclear compartments, interacts with the general nuclear export
receptor, CRM1, and is transported from the nucleus to the cytoplasm
(24). Also, expression of eIF-5A mutant protein has been shown to
inhibit HIV-1 replication in human T-cells (16, 25). The selective
nature of eIF-5A involvement in mRNA translation is supported by
the finding that inhibitors of the hypusine modification of eIF-5A
cause the disappearance of only certain mRNAs from polysomes
(26). Also, intracellular depletion of eIF-5A in yeast has been shown
to cause a marked accumulation of specific mRNAs in the nuclear
compartment (27).
Two isoforms of eIF-5A have been isolated from tobacco (12), and the
sequences for several other eIF-5A genes including those from Z. mays (GenBankTM accession number Y07920), S. tuberosum (GenBankTM accession numbers AB004823 to
AB004827), and alfalfa (GenBankTM accession number X59441)
are known. In the present study, cDNAs encoding four isoforms of
tomato eIF-5A (GenBankTM accession numbers AF29683 to
AF29686), eIF-5A from Arabidopsis (GenBankTM
accession number AF296082), and eIF-5A from carnation
(GenBankTM accession number AF296081) were isolated. It is
clear from a comparison of eIF-5A sequences that, in keeping with the
fact that it is involved in regulation, this gene is highly conserved among plant species (80-97% identity at the amino acid level) but is
less conserved across the plant and animal kingdoms (50-60% identity
at the amino acid level). DHS genes from tomato (GenBankTM
accession number AF296077), Arabidopsis DHS
(GenBankTM accession number AF296078), carnation DHS
(GenBankTM accession number AF296079), and banana DHS
(GenBankTM accession number AF296080) were also cloned in
the present study, and the sequence for DHS from tobacco has also
recently been reported (28). DHS also appears to be highly conserved among plant species (70-90% identity at the amino acid level) but is
less well conserved across the plant and animal kingdoms (40-60%
identity at the amino acid level). It has been proposed that plant DHS,
like its mammalian and yeast counterparts, mediates the first
step in the hypusine modification of eIF-5A required for its activation
(1), but to date no specific function has been ascribed to plant
eIF-5A.
It is clear from the present study that tomato DHS and eIF-5A are
up-regulated in parallel in response to drought and chilling stress and
coincident with the onset of flower and fruit senescence. Plant DHS has
been shown to have high sequence homology with the plant gene encoding
homospermidine synthase, an enzyme that converts spermidine to
homospermidine in a key reaction of alkaloid biosynthesis (28, 29).
However, two lines of evidence in addition to sequence similarity with
mammalian, yeast, and tobacco DHS clones indicate that the tomato gene
encodes DHS. First, overexpression of tomato DHS cDNA in E. coli produced a recombinant protein that proved capable of
mediating the transfer of a butylamine residue to eIF-5A, the reaction
catalyzed by DHS in situ. Second, tomato DHS and eIF-5A
expression are up-regulated in parallel at the onset of senescence and
in response to environmental stress, which is consistent with the
contention that the cognate protein of the DHS gene is activating
eIF-5A. Indeed, these observations suggest that tomato DHS activates an
isoform of eIF-5A that facilitates the nucleocytoplasmic transport of
mRNAs required for natural senescence as well as premature
senescence induced by environmental stress. That up-regulation of DHS
expression was observed by probing Northern blots with either
full-length or 3'-noncoding cDNA suggests that a single isoform of
DHS may be involved in regulating natural senescence as well as
stress-induced senescence. However, the possibility that additional
isoforms of tomato DHS are involved in the regulation of other
physiological events is not precluded.
Inasmuch as senescence is an active process requiring gene expression
and synthesis of new proteins, it is classified as a form of programmed
cell death. A number of senescence-induced genes have been identified
for leaves and flowers. These include genes encoding ferritin and
metallothionein as well as lipase, a cysteine protease, aspartate
protease, and a vacuolar processing protease (30-32). Indeed, the
translation products of senescence-induced genes invariably include
hydrolytic enzymes that mediate the breakdown of cell structure,
especially membranes, as well as catabolism of macromolecules, and this
results in a progressive decline in protein and lipid (33). The
expression of genes required for growth and development but not
programmed cell death, such as that encoding the small subunit of
ribulose-bisphosphate carboxylase in leaves, is suppressed at the onset
of senescence, whereas housekeeping genes required for cell viability,
such as those supporting respiration, show continued expression
throughout development and senescence until the cells actually die (31,
34). There are also tissue-specific differences in senescence.
Paramount among these is the fact that natural senescence of leaves and
flowers is accompanied by mobilization of nutrients out of the
senescing tissue to other parts of the plant, whereas this is not the
case for fruit senescence. In a similar vein, rapidly induced cell
death in leaves caused by pathogen ingression or severe environmental
stress is normally not accompanied by nutrient mobilization. This type
of programmed cell death in leaves is sometimes referred to as necrosis
rather than senescence, because chlorosis, which defines the
progressive yellowing of leaves reflecting chlorophyll degradation
during natural leaf senescence, is not observed. It is clear from the
present study that DHS and eIF-5A are up-regulated at the onset of
natural senescence, whether or not there is mobilization of nutrients
out of the senescing tissue, and also in the event of necrosis.
Specifically, both genes showed increased expression in senescing
tomato flowers from which nutrients are mobilized and in senescing
tomato fruit from which nutrients are not remobilized. In addition, DHS
and eIF-5A were up-regulated in parallel in tomato leaves under
conditions in which rapid cell death in the absence of chlorosis was
induced by a continuous 6-day exposure to 5 °C, a temperature that
induces chilling injury in tomato. It seems likely, therefore, that
plant DHS and eIF-5A facilitate the translation of mRNAs required
for programmed cell death regardless of how it is initiated, but they may not be involved in the regulation of the mobilization of nutrients that accompanies some types of programmed cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) phagemid (Stratagene).
. Overnight cultures were transferred to 10 times
volume of fresh 2× YT. After 2 h of initial growth in 2× YT at
37 °C, 0.3 mM
isopropyl-
-D-thiogalactoside was added to the cultures,
and growth was continued for an additional 4 h at 30 °C before
the cells were harvested. The GST fusion proteins were purified by glutathione-Sepharose 4B chromatography according to the vector manufacturer's instructions (Amersham Pharmacia Biotech) and treated for 16 h with the protease, factor Xa (Roche Molecular
Biochemicals) (1 µg of protease per 100 µg of GST fusion protein)
to release recombinant DHS and eIF-5A protein. The purity of the
recombinant proteins was confirmed by SDS-polyacrylamide gel
electrophoresis (15% gels).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (60K):
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Fig. 1.
Nucleotide and inferred amino acid sequences
of the cDNA for senescence-induced tomato DHS. Nucleotide
sequence is shown as follows. Lowercase letters,
5'- and 3'-noncoding cDNA sequence; uppercase
letters, open reading frame; underline,
oligonucleotides used for RT-PCR; shaded letters,
mismatched nucleotides; boldface letters,
restriction enzyme sites for subcloning PCR fragments;
asterisk, stop codon. Numbers of nucleotides and amino acids
are indicated.
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Fig. 2.
Alignment of the amino acid sequences of DHS
from tomato, tobacco, carnation, banana, Arabidopsis,
human, fungi (N. crassa), yeast (S. cerevisiae), and Archaeobacteria (M. janaschii). Gaps (-) were introduced to optimize
alignment. Numbering begins with the first amino acid of DHS from
Archaeobacteria. Sequences identical to tomato DHS are
shaded.
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[in a new window]
Fig. 3.
Northern blot analysis of total RNA isolated
from leaves of tomato seedlings. C, control leaves from
intact seedlings; WC, leaves from 6-h water-treated derooted
tomato seedlings; S, leaves from 6-h sorbitol-treated
derooted tomato seedlings. The blot was probed with tomato DHS
(A) and eIF-5A1 (B) cDNAs. Each lane
contained 10 µg of RNA. The corresponding ethidium bromide-stained
agarose gels of fractionated RNA are illustrated.
View larger version (75K):
[in a new window]
Fig. 4.
Northern blot analysis of total RNA isolated
from chilling-treated tomato leaves. The blot was probed with
tomato DHS and eIF-5A1 cDNAs. Each lane contained 10 µg of RNA. The corresponding ethidium bromide-stained agarose gel of
fractionated RNA is illustrated. Measurements (means ± S.E. for
n = 3) of leaf diffusate conductivity are also
indicated. Lane 1, before chilling
(BC); lanes 2-4, chilling for 2 days
followed by rewarming for 0 (C2W0), 6 (C2W6), and
24 (C2W24) h, respectively; lanes
5-7, chilling for 3 days, rewarming for 1 day, chilling for
2 days followed by rewarming for 0 (C3W0), 6 (C3W6), and 24 (C3W24) h, respectively;
lanes 8-10, chilling for 6 days followed by
rewarming for 0 (C6W0), 6 (C6W6), and 24 (C6W24) h, respectively.
View larger version (39K):
[in a new window]
Fig. 5.
Northern blot analysis of total RNA isolated
from developing and senescing tomato tissues. A,
flowers; B, fruit. The stages of fruit development are
breaker (BK), pink (PK), red firm
(RF), and red soft (RS). Each lane
contained 10 µg of RNA. The blots were probed with tomato DHS and
eIF-5A1 cDNAs. The corresponding ethidium bromide-stained agarose
gels of fractionated RNA are illustrated.
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[in a new window]
Fig. 6.
Changes in DHS protein and mRNA levels
and in chlorophyll levels of developing tomato cotyledons.
A, Western blot of total protein probed with polyclonal
antibodies raised against tomato DHS recombinant protein; each
lane contained 20 µg of protein. Northern blot of total
RNA probed with tomato DHS cDNA; each lane
contained 10 µg of RNA. B, chlorophyll levels.
Amino acid (upper value) and nucleotide (lower value) eIF-5A sequence
identities
View larger version (52K):
[in a new window]
Fig. 7.
SDS-polyacrylamide gel electrophoresis (15%)
of the reaction products of the DHS enzyme assay. Each
lane contained 30 µl of reaction mixture
(A-D), and the bands were resolved by autoradiography.
Molecular mass markers (kD) and amounts of GST, GST plus DHS,
and GST plus eIF-5A are indicated.
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[in a new window]
Fig. 8.
Southern analysis of tomato genomic DNA.
Genomic DNA was digested with EcoRI, EcoRV,
HindIII, and XbaI, and the Southern blot was
probed with tomato DHS cDNA. Each lane contained 10 µg
of DNA.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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ACKNOWLEDGEMENT |
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We are grateful to Lynn Hoyles for expert technical assistance.
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FOOTNOTES |
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* This work was supported by the Natural Sciences and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF296077 (tomato DHS), AF296078 (Arabidopsis DHS), AF296079 (banana DHS), AF296080 (carnation DHS), AF296081 (carnation eIF-5A), AF296082 (Arabidopsis eIF-5A), AF296083 (tomato eIF-5A1), AF296084 (tomato eIF-5A2), AF296085 (tomato eIF-5A3), and AF296086 (tomato eIF-5A4).
To whom correspondence should be addressed: Dept. of Biology,
University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. Tel.: 519-888-4465; Fax: 519-746-2543; E-mail: jet@sciborg.uwaterloo.ca.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M008544200
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ABBREVIATIONS |
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The abbreviations used are: eIF-5A, eucaryotic translation initiation factor 5A; DHS, deoxyhypusine synthase; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR.
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