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Plant Physiol. (1999) 119: 1457-1464
Cloning and Characterization of a Gibberellin-Induced RNase
Expressed in Barley Aleurone Cells1
Sally W. Rogers* and
John C. Rogers
Institute of Biological Chemistry, Washington State University,
Pullman, Washington 99164-6340
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ABSTRACT |
We cloned a cDNA for a
gibberellin-induced ribonuclease (RNase) expressed in barley
(Hordeum vulgare) aleurone and the gene for a second
barley RNase expressed in leaf tissue. The protein encoded by the cDNA
is unique among RNases described to date in that it contains a novel
23-amino acid insert between the C2 and C3 conserved sequences.
Expression of the recombinant protein in tobacco (Nicotiana
tabacum) suspension-cultured protoplasts gave an active RNase
of the expected size, confirming the enzymatic activity of the protein.
Analyses of hormone regulation of expression of mRNA for the aleurone
RNase revealed that, like the pattern for  -amylase, mRNA levels
increased in the presence of gibberellic acid, and its antagonist
abscisic acid prevented this effect. Quantitative studies at early
times demonstrated that cycloheximide treatment of aleurone layers
increased mRNA levels 4-fold, whereas a combination of gibberellin plus
cycloheximide treatment was required to increase -amylase mRNA
levels to the same extent. These results are consistent with loss of
repression as an initial effect of gibberellic acid on transcription of
those genes, although the regulatory pathways for the two genes may
differ.
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INTRODUCTION |
The plant hormone GA plays a key role in the regulation of
expression of genes involved in many different plant functions (Huttly
and Phillips, 1995 ). In conjunction with its antagonist ABA,
GA3 controls the germination of monocotyledonous
seeds, including the intensively studied events involved in its effects
on the cereal aleurone layer (Jones and Jacobsen, 1991). Although the role of GA3 in inducing expression of the
-amylase gene families has received considerable and detailed
attention in recent years, earlier experiments showed a connection
between the presence of GA3 and the induction of
numerous other hydrolytic enzymes, including RNase (Chrispeels and
Varner, 1967) and DNase (Taiz and Starks, 1977; Brown and Ho, 1986;
Brown et al., 1988). -Amylase gene expression in cereal aleurone has
been a model system for studying the effects of
GA3 and ABA on transcription. To date all models for mechanisms by which GA3 increases and ABA
suppresses transcription in cereal aleurone are derived from studies of
-amylase promoters (Gubler and Jacobsen, 1992; Huttly et al., 1992;
Lanahan et al., 1992; Rogers and Rogers, 1992), although transient
expression assays have demonstrated GA3
transcriptional effects on promoters from protease (Cejudo et
al., 1992) and (1-3,1-4)- -glucanase (Wolf, 1992), genes that are
expressed in aleurone cells during germination.
Although RNases may be present in many different compartments in plant
cells (Green, 1994), our interests are in RNases, as the one described
here, that enter the secretory pathway. These RNases are homologous to
the extracellular RNase T2 from the fungus Aspergillus
oryzae (Kawata et al., 1988). In general, proteins enter the
secretory pathway by cotranslational translocation into the ER lumen
and will follow a default pathway of secretion to the cell exterior
unless they contain structural determinants causing them to be retained
or diverted to another organelle (Okita and Rogers, 1996). RNases
secreted to the cell exterior include those with functions in
self-incompatibility (Matton et al., 1994) and functions somehow
related to wound responses (Ye and Droste, 1996). Alternatively, RNases
could be sorted from the secretory pathway to lytic or protein storage
vacuoles (Neuhaus and Rogers, 1998); RNase MC purified from
gourd (Homordica charantia) seeds presumably would be an
example of the latter (Ide et al., 1991). The function of RNases in
vacuoles is engimatic because they should be separated in those
organelles from potential substrate molecules in the cell cytoplasm or
in the extracellular space. One potential role would be in autophagic
vacuoles that are induced by Suc starvation, where cytoplasm and
cytoplasmic organelles are engulfed and degraded (Aubert et al., 1996;
Moriyasu and Ohsumi, 1996). A similar role might pertain for RNases
that are induced by phosphate starvation or by tissue senescence
(Taylor et al., 1993; Bariola et al., 1994; Köck et al., 1995;
Dodds et al., 1996).
In germinating cereal grains RNases are thought to contribute to
nutrient mobilization during digestion of the dead starchy endosperm
(Green, 1994). Ingle and Hageman (1965) reported two RNase activities
with different pH optima in the germinating seeds of corn and found
that the increase in RNase activity required protein synthesis. Dry
barley (Hordeum vulgare) seeds were the source for two
RNases and a nuclease characterized by Pietrzak et al. (1980), who
found sufficient similarity in the two analyzed RNases to result in
identical reactions in a double immunodiffusion test and to show
similar substrate specificities.
There are two separate classes of enzymes induced by GA treatment of
aleurone layers that degrade RNA. The first is a Zn-containing metalloenzyme that digests both DNA and RNA (Brown and Ho, 1986, 1987); the second class includes at least two separate activities that
appear to be RNA specific (Brown and Ho, 1986). Both activities were
secreted from aleurone cells into the incubation medium, although the
peaks of accumulation occurred after 72 h of hormone treatment
(Brown and Ho, 1986). It is interesting that Chrispeels and Varner
(1967) found that RNase activity induced by GA accumulated in aleurone
layers for 24 h and then was progressively released into the
medium. Release of the enzyme appeared to be an active process because
it could be prevented by treatment with protein and RNA synthesis
inhibitors (Chrispeels and Varner, 1967). It would be reasonable to
expect secretion of the enzyme, where it could contribute to starchy
endosperm degradation, but it is not clear where the enzyme might be
sequestered before the time secretion occurs.
As a second hydrolytic enzyme induced by GA3 in
barley aleurone, the RNase described here provides a means by which to
test the universality of the conclusions based on the -amylase model regarding the effects of GA on transcription. Two general models have
been offered to explain how GA3 could increase
transcription from -amylase gene promoters. The first proposes that
GA3 causes an increase in the abundance of mRNA
for a Myb transcription factor, GAMyb, and synthesis of
increased amounts of the GAMyb protein, which, in turn, specifically
binds to the GARE and is responsible for the increase in transcription.
In this role, GAMyb is proposed to be the "master regulator" of the
system (Gubler et al., 1995, 1997). A second model proposes that a
transcriptional repressor, HRT, interacts with the GARE
sequences and prevents transcription in the absence of
GA3: The primary action of that hormone is to cause loss of transcriptional repression, perhaps by physical displacement of the HRT protein (Raventós et al., 1998). This model would not exclude the possibility that additional transcription factors could then be recruited to the derepressed promoter by GA3 to further increase the level of expression.
One advantage of the second model is that it would explain the
antagonistic effects of ABA by proposing that ABA would stabilize the
repressor on the promoter (Raventós et al., 1998). It will be
important to test the -amylase-derived models by discerning hormonal
regulation of other genes expressed in aleurone cells, and results
presented here represent a step toward that goal.
Here we describe the cloning of a cDNA for a GAR-RNase expressed
in barley aleurone but not in leaf tissue. The predicted protein
sequence of the RNase includes a canonical signal peptide; therefore,
the enzyme should enter the secretory pathway. Its structure, however,
is unique compared with other plant RNases because it contains a novel
23-amino acid insertion. Detailed studies of early times following
treatment with GA3 and with the protein synthesis
inhibitor Chx indicate the existence of two separate mechanisms that
result in increases of mRNA for the GAR-RNase and for high-pI
-amylase.
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MATERIALS AND METHODS |
Cloning and rDNA Techniques
Methods for cloning, DNA sequencing, and blot hybridization were
described previously (Khursheed and Rogers, 1988). A cDNA for a barley
(Hordeum vulgare L.) RNase (pSR360) was isolated from a
library prepared from GA3-treated aleurone layers
of barley var Himalaya using Lambda-Zap (Stratagene). The library (a
gift from Dr. John Mundy, University of Copenhagen, Denmark) was
screened using a protein expression strategy to find proteins
interacting with a multimer of a known -amylase promoter
cis-acting element sequence. This clone appeared to be
positive in the screen, but sequencing revealed its homology to known
RNases. A gene for a different RNase was recovered from a barley
genomic library (var Igri) in a Lambda-Fix II vector (Stratagene) using
the full-length RNase cDNA as a probe. Analysis of the protein sequence
for structural features was performed using the BCM Protein Secondary
Structure Prediction Program (accessible at
http://dot.imgen.bcm.tmc.edu:9331/; Kim C. Worley, Human Genome
Center, Baylor College of Medicine).
Southern-Blot Analysis
Southern-blot hybridization of restriction enzyme digests of DNA
from barley cvs Morex and Steptoe was kindly performed by D. Kudrna and
A. Kleinhofs (Department of Crop and Soil Sciences, Washington State
University, Pullman), as described previously (Kleinhofs et al., 1993).
Northern-Blot Analysis
Preparation of total RNA from aleurone and leaves,
electrophoresis, and blotting were as described previously using 20 µg of RNA per lane (Rogers, 1985). For repeat probes blots were
boiled in 0.1× SSC and 0.1% SDS for 10 min between hybridizations.
Probes included the full-length RNase cDNA, the high-pI -amylase
cDNA-coding sequence (Rogers, 1985), and the coding sequence for a PAPI
present in aleurone, the abundance of which is not affected by either GA3 or ABA (Mundy and Rogers, 1986). In Chx
experiments the inhibitor was added concurrently with hormone addition.
Washing conditions included a final wash in 0.1× SSC and 0.1% SDS at
65°C for 30 min. Blots were analyzed using a phosphor imager (model
445SI, Molecular Dynamics, Sunnyvale, CA) with Imagequant software
(Macintosh). Barley cv Himalaya seeds, 1996 and 1998 crops, were
purchased from the Department of Agronomy, Washington State University.
Transient Expression in Tobacco Suspension-Cultured
Protoplasts
For expression in protoplasts, the aleu-RNase cDNA was placed
between the BamHI and SacI sites in pBI221
(Jefferson et al., 1987). Culture of tobacco (Nicotiana
tabacum cv Xanthi) TxD cells, protoplast preparation, and
transient expression of constructs under control of the cauliflower
mosaic virus 35S promoter after electroporation were described
previously (Holwerda et al., 1992). Control cells were electroporated
with unmodified pBI221. Twenty-four hours after electroporation,
protoplasts (approximately 0.4 mL packed volume) and medium (10 mL)
were separated by centrifugation at 200g. Protoplasts were
dissolved directly in 0.4 mL of 2× sample buffer, and proteins in 0.5 mL of medium were precipitated with acetone and taken up in 50 µL of
sample buffer. The presence of RNase activity in each fraction was
assayed after electrophoresis through an acrylamide gel containing RNA
as described previously (Brown and Ho, 1986), except that the
incubation buffer was 0.1 M sodium succinate, pH
5.5, 0.01 M KCl, and 1 mM
Cys and the incubation temperature was 50°C.
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RESULTS |
Sequence Information
The predicted protein sequence encoded by pSR360 is clearly that
of an RNase, as shown by its homology to other known plant RNases (Fig.
1A; numbering is for reference only and
does not represent residue numbers for any of the sequences).
Accordingly, we have identified the clone as GAR-RNase (accession no.
AF000939). Based on the amino acid sequence, the expected molecular
mass of the mature GAR-RNase protein, minus the signal peptide, is 24.8 kD and the expected pI is 5.14, making it likely to correspond to the
25-kD RNase identified by Brown and Ho (1986). GAR-RNase contains the
five highly conserved RNase regions (Green, 1994; Fig. 1A, C1-C5), as
well as the two pairs of Cys residues (positions 100 and 127 and 207 and 234) that form disulfide linkages characteristic of T2 RNases in
fungi (Kawata et al., 1988). Other highly conserved amino acids include
the third active-site His (position 137) and the Glu (position 140, bold print; Green, 1994). GAR-RNase contains an insert of 23 amino
acids, with a third pair of Cys residues, which is not found in
previously described RNases and has no detectable homology to any
sequences in the current database. The unique region does contain a
central DGA, which is common to other sequences, and a hydrophobic
region, followed by a strongly hydrophilic one (Fig. 1B). The residues
indicated with dots above have a high likelihood of forming an
-helix (data not presented). Its hydrophilic nature indicates that
the helical structure is likely to be on the protein surface. By
homology, the position of the insert corresponds to residue 52 of RNase
Rh (Kurihara et al., 1992), which, according to its crystal structure,
is on the surface of the molecule.
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| Figure 1.
Sequence analysis. A, A comparison is presented
of predicted amino acid sequences of barley aleurone RNase (GAR-RNase)
(accession no. AF000939) and the leaf RNase (Leaf) encoded by the gene
isolated from a barley genomic library (accession no. AF000940) and
known sequences from the database for RNases of zinnia (accession no.
U19924; Zinnia) and Arabidopsis (accession no. U05206; At). Numbering
begins with the initial Met. Asterisks indicate active-site His
residues, and conserved Glu is shown in bold. Five highly conserved
regions are underlined and labeled C1 to C5. A 23-amino acid insert
unique to GAR-RNase is present from residues 73 to 95. Dots above
residues indicate a region that is predicted to form an -helix.
Vertical lines show positions of predicted signal peptide cleavage. B,
Kyte-Doolittle hydropathy plot of the predicted protein sequence of
GAR-RNase cDNA (Kyte and Doolittle, 1982). The area highlighted
in gray is the 23-amino acid insert unique to GAR-RNase. Numbering of
amino acids is the same as in A. S, Hydrophobic region corresponding to
signal peptide.
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We also identified a barley genomic clone encoding a different RNase
(accession no. AF000940). The predicted sequence of the protein encoded
by this gene is also presented in Figure 1A (Leaf). As shown from the
sequence comparison, it lacks the insert found in the cDNA and is more
closely related to the zinnia and Arabidopsis RNases than is GAR-RNase.
When compared with GAR-RNase, there is only 41% sequence identity,
indicating that the cDNA and the gene encode different members of the
RNase family. As shown below, this RNase is expressed in leaf but not
aleurone tissue. The predicted size of the RNase gene product is 22.4 kD, which is somewhat smaller than the 25.9-kD size determined by Lantero and Klosterman (1973) for their purified leaf RNase. In addition to the sequences shown in Figure 1A, the GenBank database contains an expressed sequence tag clone from barley (accession no.
L43983), which represents a partial RNase cDNA containing a large
deletion 5 to the C5 region and an extended 3 region (data not
shown). This presumably represents a third, entirely different type of
barley RNase. The predicted signal peptide cleavage sites for both
GAR-RNase and the leaf RNase, based on the ExPASy program (Heinrik et
al., 1997), are indicated on Figure 1A by vertical lines.
The results of Southern-blot hybridizations using the cDNA as a probe
and washing at high stringency indicate that this RNase belongs to a
small gene family with one or two other genes closely related to that
encoding the cDNA. This is illustrated in Figure 2, where restriction digests of DNA from
barley cvs Morex (lanes M) and Steptoe (lanes S) are presented.
Digestions with EcoRV and HindIII (lanes 3-6),
enzymes that do not cut within the coding sequence of the cDNA, gave
single hybridizing bands for both DNAs. In contrast, digestion with
EcoRI, an enzyme that cuts within the cDNA-coding sequence,
gave three bands with the same pattern in both DNAs: a faint band <5
kb, a strong band of approximately 6 kb, and an intermediate band of
approximately 7 kb (lanes 1 and 2). Since two bands could be explained
by the EcoRI site in the cDNA sequence, the third presumably
is derived from another, homologous gene. This estimate is consistent
with the identification of two RNases expressed in aleurone (Brown and
Ho, 1986).
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| Figure 2.
Southern blot probed with GAR-RNase cDNA. DNA from
barley cvs Morex (lanes M) and Steptoe (lanes S) was digested with
EcoRI (RI; lanes 1 and 2), EcoRV (RV;
lanes 3 and 4), or HindIII (H; lanes 5 and 6),
electrophoresed, transferred to a membrane, and hybridized with the
GAR-RNase cDNA probe. Sizes of molecular mass markers (in kb) are
indicated to the left.
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Enzymatic Activity of GAR-RNase Expressed as a Recombinant Protein
We used transient expression in tobacco suspension-cultured
protoplasts to establish that the GAR-RNase protein, as predicted from
its sequence, was an enzymatically active RNase. An assay was used with
which RNase activity is determined in situ after denaturation of
proteins with SDS and electrophoresis through an acrylamide gel
containing RNA (Brown and Ho, 1986). Activity is shown as cleared bands
when RNA remaining in the gel is stained after incubation. As shown in
Figure 3B, lane 2, control protoplasts transfected with plasmid pBI221 expressing Escherichia coli
GUS contained an RNase activity that migrated at approximately 18 kD
(indicated by a dot), whereas medium from those protoplasts (lane 4)
lacked detectable activity. In contrast, protoplasts transfected with
the GAR-RNase construct (lane 1) not only had the approximately 18-kD
activity (dot) but also a similar amount migrating at approximately 26 kD (arrow). In addition, a lesser amount of this approximately 26-kD
activity was present in the medium from these protoplasts (asterisk).
The fact that a similar proportion of the endogenous, approximately
18-kDa activity was also present in the medium indicates that both may
have resulted from a small amount of protoplast lysis. As shown in
Figure 3A, the amounts of protein in the protoplast extracts (lanes 1 and 2) and in the medium samples (lanes 3 and 4) for the two sets were
similar. Therefore, the presence of the approximately 26-kD RNase
activity was not an artifact of unequal loading but was, instead, due
to specific expression of GAR-RNase in the test cells. Although the
approximately 26-kD size of the RNase activity is an estimate limited
by the fact that intramolecular disulfide bonds were not reduced before
electrophoresis of the samples, it fits well with the size of GAR-RNase
predicted from the cDNA sequence.
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| Figure 3.
Expression of recombinant GAR-RNase in tobacco
suspension-cultured protoplasts. A, Coomassie blue-stained gel from
SDS-PAGE. B, RNase activity gel. Lanes M, Molecular mass markers
with size in kD to the right. Lanes 1, Cell extract from protoplasts
expressing GAR-RNase; lanes 2, extract from protoplasts expressing
E. coli GUS; lanes 3, medium from GAR-RNase-expressing
cells; and lanes 4, medium from control cells. Dots indicate
approximately 18-kD RNase activity in both cell extracts, arrow
indicates approximately 26-kD RNase activity in extract from
GAR-RNase-expressing cells, and asterisk indicates approximately 26-kD
RNase activity in medium from GAR-RNase-expressing cells.
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Analysis of mRNA Expression Patterns
We used aleurone-specific PAPI mRNA as an internal standard,
because its abundance in aleurone is not altered with hormone treatments (Mundy and Rogers, 1986). Blots were sequentially hybridized with an RNase probe and then combined -amylase and PAPI cDNAs; therefore, the expression patterns of RNase and -amylase mRNAs could
be compared directly. Northern blots prepared from barley aleurone
total RNA (Fig. 4A) showed that mRNA for
GAR-RNase was at essentially undetectable levels in untreated layers
(lane 1), as was true for high-pI -amylase mRNA (Rogers, 1985).
Neither probe hybridized to leaf RNA (data not shown). In a manner
similar to high-pI -amylase mRNA, the GAR-RNase mRNA level was
substantially increased after 24 h of exposure to
10 6 M GA3
(lane 2), and incubation with 105 M
ABA reduced the GA3-mediated mRNA increase by
>80% for both (lane 3). The presence of similar amounts of 0.7 kb of
PAPI mRNA in each of the lanes indicates that the differences observed
for GAR-RNase and -amylase hybridizations in the different samples could not be explained by RNA gel-loading variations. The specificity of the pattern observed for GAR-RNase mRNA was confirmed by using the
RNase genomic clone as a control. A second northern blot (Fig. 4B) was
probed with an approximately 600-bp SalI fragment containing a portion of the coding sequence of the leaf RNase gene. These results
demonstrate that this RNase was expressed in leaf (lane 3) but not in
aleurone (lanes 1 and 2) and the results establish the tissue
specificity of expression of each of the gene types.
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| Figure 4.
Hormone effects on mRNA expression. A,
Hybridization with the GAR-RNase probe. Total RNA was isolated from
barley aleurone layers treated for 24 h with no hormone (0; lane
1), with GA3 (G; lane 2), or with GA plus ABA (G + A, lane
3). Following electrophoresis, RNA was transferred to nitrocellulose
and blots were probed sequentially. First, the blot was probed with the
full-length GAR-RNase cDNA. After the signal was collected by phosphor
imager analysis, the blot was stripped and probed with a mixture of
high-pI -amylase cDNA (Amy) and PAPI probes. Sizes (in kb) are
indicated to the right of each panel. B, Hybridization with barley leaf
RNase probe. Total RNA blots were prepared and probed as for Figure 3A,
except that here the RNase probe was an approximately 600-bp
SalI fragment of coding sequence from the RNase gene.
Lane 1, No hormone (0); lane 2, 24 h with GA3 (G); and
lane 3, leaf RNA (L). Sizes of mRNA (in kb) are indicated to the right
of each panel.
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Chx Effects
We wanted to investigate mechanisms that could contribute to the
GA3-induced increase in mRNA documented in Figure
4A. In other hormonally regulated systems, Chx has been used to test for the presence of a transcriptional repressor (Theologis, 1986; Franco et al., 1990; Ballas et al., 1995). If the half-life of a
repressor protein is short enough, inhibition of protein synthesis by
Chx should result in depletion of repressor before other positive transcription factors are depleted. As repressor is depleted, transcription should increase. Therefore, aleurone layers were incubated untreated or treated with GA3, Chx, or
Chx plus GA3, and the relative amounts of
GAR-RNase and high-pI -amylase mRNAs were measured.
Results are presented in Figure 5 for
aleurone layers incubated for 6 h under various conditions. In
this experiment the same blot was hybridized sequentially with
GAR-RNase, PAPI, and high-pI -amylase probes; the blot was not
stripped between hybridizations. Amounts of hybridization for the two
probes were expressed as relative abundance. These numbers were
corrected for variation in loading based on hybridization to the PAPI
cDNA probe, using phosphor imager analysis. Little hybridization for
the approximately 0.9-kb GAR-RNase was present in control RNA (Fig. 5,
lane 1); in contrast, strong hybridization signals were obtained from
Chx-treated (lane 2) and GA3-treated (lane 3)
layers, where the signals represented 4- and 8-fold increases over the
control, respectively. Surprisingly, little or no hybridization was
obtained from the Chx plus GA3-treated layers
(lane 4). The latter result was not an artifact of unequal sample
loading or transfer to the membrane because the signal for the
approximately 0.7-kb PAPI mRNA hybridization, positioned immediately
below the position of GAR-RNase, was essentially equal in all of the
samples (lanes 5-8). For high-pI -amylase, little hybridization was
detected in control (lane 9) and Chx-treated (lane 10) samples.
GA3 treatment (lane 11) caused a 12-fold increase in mRNA over the control level, and mRNA also increased 4-fold over the
control in the Chx plus GA3-treated sample (lane
12). These results were obtained with 1998 harvest seeds, and similar results were also obtained with 1996 harvest seeds.
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| Figure 5.
Effect of Chx on mRNA expression patterns. A blot
carrying 20 µg/lane of total aleurone RNA from de-embryonated
half-seeds not treated (0; lanes 1, 5, and 9), treated with 50 µM Chx (C; lanes 2, 6, and 10), treated with
106 M GA (G; lanes 3, 7, and 11), or treated
with GA plus Chx (GC; lanes 4, 8, and 12) for 6 h was probed
sequentially with cDNAs for GAR-RNase, PAPI, and high-pI -amylase
(Amy). After the blot was washed at high stringency, images were
captured and quantitated with a phosphor imager. The signals for PAPI
in each lane were used to correct for differences in loading. RA,
Relative abundance, where the value obtained with GA was set to 1.0.
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We analyzed the different results obtained with GAR-RNase and high-pI
-amylase as follows. Chx treatment was effective because it resulted
in an increase in GAR-RNase mRNA; therefore, the negative result with
-amylase (lane 10) is a true difference and not the result of
inadequate treatment. Similarly, the Chx treatment was sufficient to
prevent GA3 induction of GAR-RNase (lane 4);
therefore, the presence of a substantial signal in the Chx plus
GA3 lane for -amylase (lane 12) is not simply
due to inadequate Chx treatment, but, instead, means that
GA3 has an effect on -amylase mRNA
accumulation when synthesis of new protein is blocked. These results
are consistent with the conclusion that loss of a short-lived protein
affected mRNA levels for both GAR-RNase and high-pI -amylase but by
different mechanisms. For GAR-RNase, loss of the protein was sufficient to induce mRNA accumulation, and addition of the GA signal blocked that
process. In contrast, loss of a short-lived protein alone was not
sufficient to cause accumulation of -amylase mRNA, but loss of a
protein coupled with the effects of the GA signal transduction pathway
was.
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DISCUSSION |
Here we present the protein sequences for two new barley RNases
and characterize the patterns of expression at the mRNA level for each.
The RNase cDNA and gene from which the protein sequences were derived
provide additional tools with which to increase our understanding of
both the roles of RNases in many phases of plant development and
interaction of the plant hormones GA3 and ABA in
regulating gene expression.
The role of S1-RNases in self-incompatability in various
Nicotiana species has been the subject of intense recent
study (McClure et al., 1989, 1990). The sequences of the two RNases
presented here fall into a class different from S1- RNases (Green,
1994); the enzymes in this class have been termed "S-like RNases"
to indicate their different functions and sequence divergence from S-RNases (Taylor et al., 1993). Phosphate starvation has been shown to
induce expression of a number of different S-like RNases (Taylor et
al., 1993; Bariola et al., 1994; Köck et al., 1995; Dodds et al.,
1996), and it is likely that these enzymes contribute to a phosphate
starvation rescue system in plants (Dodds et al., 1996). Because
GAR-RNase is expressed in aleurone during germination, it may
contribute to digestion of RNA in the dead starchy endosperm cells and
thereby may function to help mobilize Pi for the developing embryo.
Although the protein has a predicted signal peptide and almost
certainly is translocated into the ER during synthesis, we have not yet
obtained antibodies to GAR-RNase and therefore cannot comment about its
possible intracellular or secreted location in aleurone cells. The fact
that most of the GAR-RNase activity expressed in the tobacco
protoplasts was intracellular may indicate that it is sorted to
vacuoles, as well as possibly being secreted.
The characterization of a wound-induced RNase cDNA in zinnia (Ye and
Droste, 1996) suggests an additional, defensive task for which plants
might elicit RNase activity. The leaf RNase encoded by the barley
genomic sequence is closely related to this type of RNase and may have
a similar function. Comparison of the protein sequence encoded by
GAR-RNase cDNA with those for other RNases reveals a 23-amino acid
insert, which, exclusive of the conserved DGA core, appears to be
unique among presently known RNases. The insertion is predicted, using
the crystal structure of the Rhizopus niveus RNase Rh as a
model (Kurihara et al., 1992), to be located on the surface of the
molecule. The insertion contains two Cys residues; because the other
Cys residues in the protein correspond to invariant residues known to
participate in intramolecular disulfide bonds (Green, 1994), we predict
that the two Cys residues in this insertion would be linked together.
This would isolate the insertion in a looped domain with the disulfide
bridge at the base. Analysis of the possible structure of this
insertion indicates that one-half of the insertion would likely form an
-helix, which should protrude from the surface of the molecule. In
the future, it will be of considerable interest to learn how the
insertion affects the mechanism of action of this aleurone-specific
RNase. Similarly, structural analysis of the protein encoded by the
genomic sequence described here may prove useful in understanding what
role RNases play in plant wound response.
As previously observed for most other
GA3-regulated genes (Huttly and Phillips, 1995),
mRNA levels for GAR-RNase were greatly increased in aleurone layers
treated with GA3, and this increase was largely
prevented by the simultaneous presence of ABA. Similar to
GA3/ABA regulation of -amylase gene
expression, it is likely that the primary action of these hormones in
regulating GAR-RNase gene expression is at the transcriptional level.
However, we recently isolated a genomic clone for GAR-RNase and assayed
promoter function in the particle bombardment transient expression
system (Lanahan et al., 1992; Rogers and Rogers, 1992; Rogers et al.,
1994). The promoter has three potential GARE sequences within  500
nucleotides but lacks all other sequences found in amylase
GA-response complexes. Its promoter, assayed as either a 500- or a
3000-nucleotide construct, gives very little transcription above
baseline, and such a small amount of response to
GA3 that assays 6 times longer than usual (Rogers
and Rogers, 1992) are required to detect it (data not presented).
Stepwise truncation of the promoter and mutation of potential GARE
sequences did not change this result. At present we do not know how
GA3 regulates GAR-RNase mRNA abundance.
In this regard, the effects of Chx observed in our studies may help in
understanding mechanisms for transcriptional regulation. Chx treatment
of aleurone layers caused a 4-fold increase in GAR-RNase mRNA over a
6-h incubation, under conditions in which GA3
treatment gave an 8-fold increase. In addition, Chx plus
GA3 caused a 4-fold increase in high-pI
-amylase mRNA abundance under conditions in which
GA3 treatment alone gave a 12-fold increase.
These results in general provide support for a recently proposed model
by which GA3/ABA might regulate transcription
(Raventós et al., 1998), where loss of repression, an early
result of GA3 treatment, would allow a certain
level of transcription, whereas later effects of GA signaling would
increase transcription by recruiting positive factors to the promoter.
Our data indicate that loss of a short-lived protein by inhibiting
protein synthesis with Chx, in either the absence (for GAR-RNase) or
the presence (for high-pI -amylase), results in increased abundance
of both RNase and -amylase mRNA. These results would be
consistent with a model in which loss of a short-lived
transcriptional repressor allowed a certain level of transcription from
the gene promoters, but we cannot exclude other possible mechanisms,
such as effects on mRNA stability.
The effects of protein synthesis inhibitors on hormone-regulated
expression of plant genes was first described and carefully studied
with certain auxin-regulated genes (Theologis, 1986; Franco et al.,
1990; Ballas et al., 1995). That system differs greatly from the one
described here. Auxin induces transcription of those genes within
minutes, and the combination of auxin plus Chx caused "superinduction" of the mRNAs. These observations indicated that all of the factors necessary for maximal transcription were present within the cells before auxin treatment. In contrast, in the aleurone system used here, increased mRNA levels were detected only after several hours of treatment with GA3 (data not
shown), and 6 h were required to define the Chx effect. It is
likely that Chx treatment over this prolonged period, in contrast to
the short times used in the auxin experiments, would also cause
depletion of somewhat longer-lived positive transcription factors. The
effects seen from Chx treatment would reflect the relative half-lives of a repressor versus positive factors. If the half-life of the repressor was only moderately less than that of the positive factors, a
result similar to what we present here would be observed. This may
explain why in previous studies with a less-sensitive hybridization system (Muthukrishnan et al., 1983) an effect with Chx was not observed.
| |
FOOTNOTES |
1
This research was supported by the Department of
Energy (grant no. DE-FG 95ER 20165).
*
Corresponding author; e-mail bcsroger{at}wsu.edu; fax
1-509-335-7643.
Received July 21, 1998;
accepted December 28, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Chx, cycloheximide.
GARE, GA response
element.
GAR-RNase, GA-regulated RNase.
PAPI, lipid transfer protein.
| |
ACKNOWLEDGMENTS |
We thank Kay Walker-Simmons for her gift of biologically active
ABA, Lynn Holappa for valuable assistance with preparation of northern
blots, and Liwen Jiang for transfecting tobacco suspension-cultured protoplasts. Special thanks are due to Dave Kudrna and Andy Kleinhofs for providing the Southern-blot data.
| |
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Abstract
Introduction