Download Dark induction and subcellular localization of the pathogenesis

Plant Molecular Biology 28: 537-547, 1995.
© 1995 Kluwer Academic Publishers. Printed in Belgium.
537
Dark induction and subcellular localization of the pathogenesis-related
PRB-lb protein
Guido Sessa, Xiao-Qing Yang, Vered Raz, Yoram Eyal I and Robert Fluhr*
Department of Plant Genetics, P.O. Box 26, Weizmann Institute of Science, Rehovot 76100, Israel
(* author for correspondence); 1present address: Plant Gene Expression Center, USDA/ARS-UC-Berkeley,
800 Buchanan St, Albany, CA 94710, USA
Received 8 December 1994; accepted in revised form 13 April 1995
Key words: circadian clock, ethylene, pathogenesis-related proteins, post-transcriptional regulation,
subcellular localization
Abstract
The PRB-lb gene codes for a basic-type pathogenesis-related protein of the PR-1 family of tobacco.
PRB-lb mRNA accumulation is induced in response to biotic and abiotic elicitors, such as TMV,
ethylene, salicylic acid, a-amino butyric acid and darkness. In order to determine the location of elements that control dark-regulated PRB-lb gene expression, we tested promoter, transcribed regions and
3'-downstream regions of the gene for their ability to respond to dark induction in transgenic tobacco
plants. An ethylene-inducible promoter region of 863 bp was not able to confer dark induction to a
/~-glucuronidase reporter gene, while a construct containing the transcribed region of the gene and
3'-downstream sequences, driven by the cauliflower mosaic virus 35S promoter, was correctly darkregulated. The results indicate that dark-induction of the PRB-lb gene can be controlled by
3' -downstream elements at the transcriptional level or by transcribed sequences at the post-transcriptional
level. A circadian clock regulation of the PRB-lb gene was excluded, as fluctuations of PRB-lb transcript levels were not observed in plants placed in constant light or darkness. Subcellular localization
of the PRB-lb protein was also determined, in tobacco protoplasts preparations and in cell cultures. The
PRB-lb polypeptide was predominantly detected in protoplast vacuoles and was not secreted to the
media in cell cultures. These results support an intracellular localization for the PRB-lb protein, as
reported for other basic-type components of the pathogenesis-related proteins family.
Introduction
In a variety of plant species, the development of
necrosis in response to pathogenic infections is
accompanied by the de novo synthesis of a large
number of proteins. Pathogenesis-related (PR)
proteins are a subset of proteins synthesized by
The first two authors contributed equally to this paper.
plants during pathogen infection or other stressrelated responses [4, 26]. They have been grouped
into five families (PR-1 to PR-5) on the basis of
function, sequence similarity and immunological
relationship. According to the protein type, PR
proteins accumulate in the extracellular spaces of
plant tissues or in intracellular locations. It has
538
been shown for several PR proteins, such as
fl-(1,3)-glucanase, chitinase and PR-5 proteins,
that generally acidic counterparts are secreted extracellulary, while basic PR proteins accumulate
in the cell vacuoles [30, 31, 49]. In tobacco, subcellular localization studies concerning the PR-1
group are limited to the acidic components, which
are secreted in most cell types [8, 14, 21]. However, in specialized cells, known as crystal idioblasts, three acidic PR-1 proteins have been found
to accumulate in central vacuoles [ 12]. Localization of the basic PR-1 components in tobacco has
yet to be determined. Sequence analysis reveals
that basic PR-1 proteins contain an extra domain
absent in acidic-type isoforms [10, 16, 40].
C-terminal extension in basic-type PR proteins
represent a sorting signal necessary for vacuolar
targeting [31, 36]. Similarly, a vacuolar signal
necessary for proper sorting is found in the
C-terminal propeptide of the barley lectin [3].
Nevertheless, no sequence similarity can be found
in propeptides of different proteins [9].
Inducers of PR genes act generally at the transcriptional level of control [15, 47]. However,
post-transcriptional regulation in plant has been
shown to be an important mechanism in modulating expression of nuclear-encoded and plastid
genes [42, 48]. For instance, light increases transcript stability of the rbcS gene in soybeen seedlings, and of the Fed-1 gene in pea [45, 11 ]. On
the other hand, a fungal elicitor determines the
decrease of proline-rich PvPRP1 transcript despite no change in its transcription rate [51].
We have previously characterized in tobacco
plants the induction pathway of the PRB-lb gene,
which encodes a basic component of the PR-1
protein group [16]. The PRB-lb transcript accumulates in the light in response to ethylene,
~-aminobutyric acid and salicylic acid induction,
and upon TMV inoculation. In addition, PRB-lb
transcript accumulation is induced by exposure of
plants to complete darkness in an elicitorindependent manner [ 16]. The hormone ethylene
has been shown to activate the PRB-lb gene at
the transcriptional level, since it induces expression of a fl-glucurodinase reporter gene driven by
the PRB-lb promoter [ 17, 44]. In this study, using
specific antibodies against the PRB-lb protein,
we characterize subcellular localization and expression pattern in the dark of a 16 kDa protein,
which represents the direct product of the basic
PRB-lb gene. We found the PRB-lb protein to
accumulate in vacuoles of ethylene treated tobacco plants and its expression to be darkregulated either by 3'-downstream elements at
the transcriptional level or by transcribed sequences at the post-transcriptional level.
Materials and methods
Plant material and cell culture
Nicotiana tabacum cv. Samsun N N plants were
grown in greenhouse, in 16 h day and 8 h night
cycles. Experiments were performed on young
potted plants with four to five leaves of at least
10 cm length. Nicotiana sylvestris cv. 2SH cell
suspension cultures were kindly provided by
D. Aviv (The Weizmann Institute of Science,
Rehovot, Israel). The cell cultures were grown in
MS medium [34], containing 3 #g/ml ~t-naphthaleneacetic acid and 1 #g/ml 6-benzylaminopurine,
at 24 °C with moderate shaking.
Preparation of tobacco mesophyll protoplasts and
vacuoles
Fully expanded primary leaves were sterilized and
sliced into strips of about 0.2 m m width. The
strips were then immersed in an enzyme solution
containing 1~o (w/v) cellulase (Worthington
Diagnostic), 1~o (w/v) Rhozyme H P 150 (Genencor), 0.4~o (w/v) Macerase (Behring Diagnostics), in 0.45 M mannitol and 3 m M MES
pH 5.7. The preparation was incubated overnight
at 24 °C. Protoplasts were then separated from
undigested metarial, purified by floatation as described [6] and then counted in a haemacytometer. Vacuoles were isolated by floatation of protoplasts through a discontinuous Ficoll gradient
as described [7]. The purity of vacuolar preparation was checked under the light microscope
and by enzymatic assay of the vacuolar marker
539
g-mannosidase, as previously described [5]. Viability of tobacco cultured suspension cells was
determined as described [50]. After staining with
1 mM fluorescein diacetate, cells were examined
under phase contrast and fluorescence microscopy.
Induction of PR proteins and transcripts in leaves
For ethylene treatment, a constant stream of air
containing 20 ppm ethylene was applied to potted plants in a sealed glass box. Light was provided by a mixture of 'cool white' and Grolux
fluorescent lamps (25-30/IE m-: s-t). Extended
dark treatments were in dark chambers with constant air exchange.
RNA isolation and northern blot hybridization
Total RNA was isolated from tobacco leaves as
described [28]. Total RNA was fractionated in a
formaldehyde denaturing gel and transferred to
nylon membranes. Blots were hybridized to
probes at 50 ° C, for at least 6 h, in hybridization
solution (5 × Denhardt's solution, 5 × SSPE, 1~o
SDS, 50~/o formamide and 100 /~g/ml salmon
sperm DNA). Blots were washed twice for
15 min at room temperature with 1 × SSC, 0.1~o
SDS, and then exposed to X ray film.
Antibody preparation and immunoblotting
Wageningen, Netherlands). Antibodies against
acidic chitinases were prepared as previously
described [29].
Constructs preparation and plant transformation
A schematic representation of the constructs used
in this study is shown in Fig. 1. A construct containing an 863 bp fragment of the PRB-lb promoter region fused to the fl-glucuronidase (GU S)
reporter gene was prepared as previously described [32]. The plasmid pBI121 [22] was used
to express the fl-glucuronidase reporter gene
driven by the cauliflower mosaic virus (CaMV)
35S promoter. An additional construct was created by fusing the CaMV 35S promoter (Hind IIIBam HI fragment) upstream of the PRB-lbcoding region and 3 kb of 3'-downstream
sequences (Barn HI-Xba I fragment). To distinguish the engineered PRB-lb transcript from the
endogenous one, a 76 bp Hind III fragment was
inserted downstream to the PRB-lb-coding region at position 573 (Hind III site) [16]. Overexpression of the PRB-lb gene was obtained
by fusing the PRB-lb-coding region (Bam HIHind III filled-in fragment) downstream of the
nos
PRB-1b promoter
Sa
S't
35S
promoter
SDS-PAGE of plant extracts and immunoblotting procedures were as described [41].
Anti PRB-lb antibodies were raised against proteins overexpressed by cloning a filled-in Ace IHind III fragment of the PRB-lb gene coding
region (from position 114 to 573 [ 16]) at the Sma I
site of the pGEX-3X expression vector [46]. A
43 kDa PRB-lb-glutathione S-transferase fused
protein was isolated from bacteria using glutathione-agarose beads [46], and utilized for
inoculation. Antibodies against the basic-type
(1-3)-fl-glucanase from tomato [23] were a gift
from P.J.G.M. de Wit (Agricultural University,
GUS coding region
NB
S RI
PRB-l b coding
region
l - -
B
PRB-lb
H~H
76bp insertion
35S
promoter
IJI
downstream sequences
//
"
nos
terminator
GUS coding region
J
B
S RI
PRB-lb coding
35S
promo~r
500bp
terminator
~
regionl
nos
/terminat°r
S RI
Fig. 1. Schematic representation and partial restriction map
of gene constructions used in this study. Sa, SalI; St, Stu I;
N, Nde I; B, Barn HI; S, Sst I; RI, Eco RI; H, Hind III;
X, Xba I, co, co enhancer of translation
540
CaMV 35S promoter and the f~ enhancer of
translation [19]. Chimeric genes were cloned in
the plant transformation vector pMON200 [43]
or pGA492 [ 1]. Triparental mating and Agrobacterium-mediated transformation of Nicotiana
tabacum cv. Samsun NN leaves were performed
as previously described [20].
Analysis of GUS activity
For the fluorometric assay, plant tissues were
ground in 200 #1 of GUS lysis buffer, containing
50 mM sodium phosphate pH 7.0, 10 mM EDTA
and 10 mM fl-mercaptoethanol, and the extracts
were tested using the substrate 4-methylumbelliferyl glucuronide (MUG), as described by
Jefferson et al. [22].
Fig. 2. PRB-lb transgene expression. A. Immunoblot reacted
with anti-PRB-lb antibody. B. Immunoblot reacted with antiglucanase antibody. Lanes T, 60 #g protein extract of leaves
from transgenic plants containing the PRB-lb gene controlled
by the 35S CaMV promoter. Lanes N, 20 #g protein extract
of ethylene-induced tobacco NN leaves. Molecular weights of
the polypeptides detected are indicated. The 16 kDa polypeptide detected in panel B is a result of residual signal from the
experiment in panel A.
Results
the transgenic plants (Fig. 2A). To determine that
the detection of the 16 kDa PRB-lb in the transgenic plants resulted from the transformation and
not from cryptic stress conditions induced in the
plant, the same blot was probed with antibody
raised against the basic fl-(1,3)-glucanase, which
is induced in concert with the PRB-lb protein
upon elicitation by different agents. As expected,
the fl-(1,3)-glucanase was detected as a 35 kDa
band only in ethylene-treated plants, and not in
the transgenic plants (fig. 2B). Taken together,
these results suggest that the 16 kDa protein is the
direct product of the PRB-lb gene, while the
20 kDa polypeptide may represent the product of
another related gene.
The PRB-lb gene encodes a 16 kDa protein
Antibodies raised against a PRB-lb-glutathione
S-transferase fused protein cross-react with two
proteins of 16 kDa and 20 kDa molecular mass,
which are present in total extracts of ethyleneinduced tobacco leaves (Fig. 2A) [ 17]. These two
proteins, identified by anti-PRB-lb antibodies,
may result from differential processing of one gene
product or from the expression of two independent homologous genes. In order to differentiate
between these possibilities, the PRB-lb gene was
overexpressed in transgenic tobacco plants using
a construct which contained the PRB-lb-coding
region fused downstream of the CaMV 35S promoter and the f~ enhancer of translation [19].
The transgenic plants constitutively expressed the
PRB-lb polypeptide, which was detected in unstressed conditions as a 16 kDa band (Fig. 2A).
This molecular weight is in agreement with that
calculated according to the PRB-lb deduced
amino acid sequence [ 16], which is 19 kDa for a
preprotein and 16 kDa for the mature protein. In
contrast, the 20 kDa bands observed in ethylenetreated plants, was not detected constitutively in
PRB-Ib protein accumulation is induced by darkness
Daily accumulation of the PRB-lb transcript in
the darkness in greenhouse grown tobacco plants
and in continuously dark-treated plants has been
reported [ 16]. In order to determine if the PRB-lb
polypeptide also accumulates in the dark and its
accumulation is correlated with transcript level,
tobacco plants were transferred to complete
541
darkness for 72 h and were then returned to the
light for an additional 48 h. R N A and proteins
were extracted from leaves sampled at 24 h intervals. A Northern blot was hybridized with a
radioactively labelled probe from the PRB-lbcoding region (Fig. 3A), while a western blot was
reacted with anti-PRB-lb antibodies (Fig. 3B).
As previously observed [16], upon exposure to
darkness, accumulation of PRB-lb transcript was
detectable after 24 h and remained constant in the
following 48 h (Fig. 3A). In the same plants
P R B - l b protein accumulation was observed after
48 h in the darkness, and increased about 2-fold
during an additional 24 h treatment (Fig. 3B). As
observed in the case of PRB-lb induction by
ethylene, protein was detected about 24 h after
maximum transcript accumulation [16]. Rapid
decrease of both the transcript and protein levels
occurred upon the return of the plants to the light
(Fig. 3A and 3B). These results provide evidence
for dark-induced accumulation of both PRB-lb
transcript and protein.
PRB-lb protein expression is not the result of a
circadian rhythm
The daily fluctuating level of PRB-lb transcript
may be a subset of broader circadian regulation,
which has been observed in different plant genes,
such as a tobacco wound-inducible cysteine proteinase gene [27], or the Arabidopsis and wheat
chlorophyll a/b-binding protein genes [2, 35]. To
test whether the accumulation of PRB-lb transcript reflects fluctuating gene expression as a result of an endogenous circadian rhythm, tobacco
plants were kept in constant darkness or exposed
to constant light. The experiment started at noon
and leaves were sampled at intervals of 5 - 7 h for
43 h. PRB-lb transcript and protein expression
were determined by northern and western analysis, respectively. In constant darkness, PRB-lb
transcript accumulation was detectable after the
first 6 h (sampled at 19:00) with a slight increase
in the subsequent hours, but did not show any
decrease in the next day (8:00-19:00; Fig. 4A).
Protein accumulation, as expected, was observed
only after at least 43 h of darkness (Fig. 4B). On
the other hand, neither transcript accumulation
nor protein expression were detectable over
2 days in constant light (Fig. 4C and 4D). Since
the levels of PRB-lb m R N A and protein did not
display any obvious oscillation in constant darkness not in constant light, we conclude that the
expression of the PRB-lb gene is dark-induced
and not circadian rhythm-regulated.
Location of cis-regulatory regions that control dark
induction of the PRB-lb gene
Fig. 3. Dark-induced accumulation of P R B - l b mRNA and
protein in tobacco plants. Tobacco plants were exposed to
dark (D) for 72 h and then returned to light (L) for an additional 48 h. Leaves were sampled at the indicated times.
A. Dark-induced accumulation of basic PRB-lb transcript
analyzed by northern blot. Each lane contains 20/zg of total
RNA, hybridized to a radiolabelled P R B - l b probe B. Darkinduced accumulation of PRB-lb protein analyzed by immunoblot. Each lane contains 20 #g protein extract. The blots
were reacted with anti-PRB-lb antibodies.
To determine where c/s-acting elements responsible to dark responsiveness reside, a fragment of
863 bp upstream to the PRB-lb translation start
site was fused to a G U S reporter gene (Fig. 1).
This construct was previously shown to be
ethylene-induced, indicating that ethylene determines PRB-lb m R N A accumulation at the transcriptional level [17]. Three independent transformant plants were exposed to darkness for
72 h or, as a control, maintained in the light, or
542
light or treated with ethylene, the engineered
PRB-lb mRNA was barely detected (Fig. 5A).
However, when they were exposed to darkness,
a high accumulation of transcript was observed
(Fig. 5A). To exclude the possibility that the dark
induction was mediated by the CaMV 35S promoter, we tested accumulation of GUS mRNA
transcribed by the same 35 S promoter in the light,
in the dark and upon ethylene induction. GUS
mRNA accumulated to high levels in transgenic
plants independently of dark or ethylene treatment (Fig. 5B). We conclude that cis-acting elements responsive to darkness reside in the transcribed regions or in the 3'-downstream
sequences of the PRB-lb gene.
Fig. 4. Circadian survey of PRB-lb expression. Tobacco
plants were maintained constantly in darkness (panel A and
B) or in light (panel C and D) for two days. Times of sampling
are indicated (24 h clock). Total RNA and proteins were extracted from leaves and northern (A and C) or western (B and
D) analysis were performed. In A and C, 20/~g of total RNA
were loaded and hybridized to a radiolabelled PRB-lb probe.
In B and D, 20/~g total protein extract were loaded and reacted with anti-PRB-lb antibodies.
treated with 20 #l/kg ethylene for 48 h. In leaves
of transgenic plants exposed to light or darkness
no significant increase in GUS activity was
observed, while in plants treated by ethylene a
15-fold increase of GUS activity was detected
(Fig. 5C). Thus, the 863 bp PRB-lb promoter
responds to ethylene induction, but not to
darkness. The responsiveness to darkness of
other regions of the gene was tested in four independent transgenic plants containing the PRB-lb
coding region and 3 kb of downstream sequences,
driven by the constitutive CaMV 35S promoter
(Fig. 1). This construct contained a 76 bp tag
insertion which allowed to distinguish the engineered PRB-lb transcript from the endogenous
one. The 76 bp tag was previously shown to
have no effect on PRB-lb expression level [18].
When such transgenic plants were grown in the
The PRB-lb protein is localized in vacuoles of
ethylene-treated leaves and not secreted into the
medium in cell suspension cultures
The subcellular localization of the PRB-lb protein was examined in vacuoles fractionated from
protoplasts of ethylene-treated tobacco leaves
and in cell suspension cultures. Protoplasts were
isolated from leaves exposed to ethylene for
72 h. Vacuoles were then released from protoplasts and purified by centrifugation on a discontinuous Ficoll gradient. The purity of the vacuolar
preparation was evaluated by microscopic examination, and by determination of the enzymatic
activity of the vacuolar marker enzyme a-mannosidase (Fig. 6A). In the vacuole enriched fraction ~-mannosidase activity was about 5-fold
higher than in the protoplast fraction. To test the
localization of the PRB-lb protein, total proteins
from protoplasts and vacuolar fractions were
reacted, by quantitative immunoblot analysis,
with anti-PRB-lb antibodies, the PRB-lb polypeptide was enriched about 5-fold in the vacuolar
fraction relatively to the protoplasts fraction
(Fig. 6B). This ratio was similar to the one observed for ~-mannosidase activity (Fig. 6A and
6B), suggesting that the PRB-lb protein is mainly
localized within the vacuole of induced tobacco
plant cells. As an additional system for studying
PR protein subcellular localization, we utilized
543
Fig. 5. Analysis of transgene activity in the light, in the dark and upon ethylene treatment of constructs containing different portions of the PRB-lb gene. A. Accumulation of PRB-lb mRNA transcribed by a construct containing the PRB-lb-coding region
and 3'-downstream sequences driven by the 35 S CaMV promoter. Each lane contains 20/~g of total RNA extracted from transgenic
tobacco leaves exposed to light (L), or treated with 20/~l/kg ethylene (E), or exposed to dark for 72 h (D). The blot shown is
representative of the four independent transformants tested. B. Accumulation in transgenic plants of GUS mRNA transcribed by
a transgene containing the GUS-coding region driven by the 35S CaMV promoter. Amounts of RNA loaded and treatments as
in panel A. C. GUS activity of a transgene containing 863 bp of the PRB-lb promoter fused to a GUS reporter gene in transgenic
tobacco plants exposed to light (L), or treated with 20 #l/kg ethylene (E), or exposed to dark for 72 h (D). The values reported
are of one transformant representative of the three tested. GUS activity is expressed in pmol 4-methylumbelliferone (4-MU)
produced in 1 h assay by 1 #g total protein extract. Standard deviations in units ofpmol per/~g protein per hour are: light treatment,
+ 5; ethylene treatment, -L-_20; dark treatment, + 3.
A
° 1'°t@
1.0"
OS
immunobloted. PRB-lb antibodies detected the
16 kDa band, which correspond to the PRB-lb
protein, in the cell fraction but not in the culture
medium (Fig. 7A). An additional 20 kDa band,
B
e.* o.,i
•
.>
0- x
.-_:04IN
~0.4"
~
"tNm
0.2"
0
0.0"
_v_a~
pro
.
0
vac
~
pro
Fig. 6. Activity of a ~-mannosidase and PRB-lb expression
in vacuoles and in protoplasts isolated from ethylene-treated
tobacco plants. A. Relative activity of ~-mannosidase in vacuoles and protoplasts fractions. The enzyme activity of the
vacuolar fraction was normalized to 1. B. Normalized densitometric scan of an immunoblot containing fractionated total
protein extracts (4 /~g) from vacuoles and protoplasts from
ethylene-treated leaves and reacted with anti-PRB-lb antibodies. The average of two experiments is presented.
2SH cell suspension cultures, which express PR
proteins in a constitutive manner [33]. Total proteins, extracted from cells and collected from the
medium, were fractionated by SDS-PAGE and
Fig. 7. Immunoblot analysis of PRB-lb protein in tobacco
cells and in the medium. Protein extracts (40/~g) from cells
and medium of tobacco cell culture. A. Blot reacted with
anti-PRB-lb antibodies. The 20 kDa crossreacting polypeptide is not a product of the PRB-Ib gene (Fig. 1). B. Blot reacted with anti-chitinase antibodies.
544
which does not represent a direct product of the
PRB-lb gene (see above), was detected in both
fractions (Fig.7A). As a control, we tested the
localization of secreted proteins by reacting the
immunoblots with an anti-acidic chitinase antibody. The acidic chitinase protein was detected
only in the extracellular medium as previously
observed (Fig. 7B) [29, 30], confirming the integrity of our system. Cell viability was checked by
using fluorescein diacetate staining and estimated
to be 95 %. Our findings in the cell cultures system are consistent with an intracellular location
of the PRB-lb protein.
Discussion
In common with other PR genes, the basic
PRB-lb is induced by elicitors, such as ethylene,
0~-amino butyric acid, salicylic acid and TMV.
However, in a unique fashion, PRB-lb mRNA
also accumulates in response to darkness to levels comparable with those obtained with other
elicitors [16]. The product of the PRB-lb gene, a
16 kDa polypeptide, is detected immunologically
in leaves of tobacco plants exposed for at least
43 h to complete darkness (Figs. 3 and 4). The
appearance of the protein is preceded by transcript accumulation which is first observed after
6 h treatment and reaches its maximal level after
about 24 h treatment (Figs. 3 and 4). The PRB-lb
protein is so far the only component of the PR
proteins family which has been shown to be
inducible by darkness. This characteristic may
suggest a role for the PRB-lb protein also in
physiological processes not directly related to
pathogenesis, as it has been proposed for other
members of the PR protein family [29, 39].
Expression of the PRB-lb gene is differentially
regulated. The plant hormone ethylene stimulates
PRB-lb mRNA accumulation at the transcriptional level, as the 863 bp promoter of the PRB-lb
gene is sufficient to confer ethylene responsiveness to a fl-glucuronidase reporter gene in transgenic plants [17, 32]. However, the same promoter region was insensitive to dark treatment.
Enhanced accumulation of transcript in response
to darkness was detected in transgenic tobacco
plants containing the 35S CaMV promoter fused
to the PRB-Ib transcribed region and downstream sequences. Thus, PRB-lb gene expression
shows a complex regulation pattern which combines different elements of control for ethylene
and dark induction. Similarly, the parsley
4-coumerate:CoA ligase gene promoter sequences
direct cell-type-specific expression while exonic
sequences direct light- and elicitor-induced accumulation [ 13].
The experiments presented here do not permit
us to distinguish whether the dark-responsive
elements act as enhancers at the transcriptional
level or by effecting mRNA stability. The latter
type of regulation plays an important role in the
control of nuclear-encoded and plastid genes [48,
42]. Exogenous signals have been found to affect
mMNA stability of several plant genes [11, 24,
25, 45, 51]. For instance, treatment of cultured
bean cells with a fungal elicitor causes a decrease
in the accumulation of the proline-rich PvPRP1
transcript without changing its transcription rate
[51 ]. On the other hand, light increases message
abundance of a reporter gene fused to the 5' portion of the Ferredoxin 1 transcript driven by the
35S CaMV promoter [ 11 ]. Two elements responsible of mRNA instability have been recognized
in plants; one of them (DST element) is a conserved motif present in the 3'-untranslated region
of the SAUR (small auxin up RNA) gene family
[37]. The other element consists of overlapping
repeats of the sequence AUUUA, that are found
in mammalian in rapidly degraded mRNAs [38].
Sequence comparison between these elements
and the 3' PRB-lb untranslated sequence revealed the presence of one copy of the A U U U A
motif at position 679 [16], but its relevance in
terms of PRB-lb transcript stability in the light
remains to be tested.
We also examined the subcellular localization
of the basic PRB-lb protein by a combination of
vacuole isolation and cell suspension cultures
examination. Our results suggest that the PRB-lb
protein accumulates intracellularly in the vacuole,
similarly to other basic-type PR proteins [6, 30,
31 ]. Protein sorting to plant vacuoles requires a
545
positive signal, as opposed to secretion, which is
thought to be part of a default pathway [9]. In
plants, vacuolar sorting signals may be localized
in N-terminal propeptides, in C-terminal extensions, or in exposed regions of the mature protein
[9]. Amino acid sequence comparison of the
PRB-lb protein with extracellular-located acidic
PR-1 indicates that the PRB-lb contains an 18
amino acid long C-terminus extension [10, 40,
16]. Similar extra domains of different lengths are
found in all the basic PR proteins characterized
so far [26]. In basic chitinase, (1,3)-fl-glucanase
and osmotin, these sequences are necessary for
proper targeting to the vacuoles and are processed
during or after sorting [31, 36]. Thus, the
C-terminal end of basic PRB-lb is a potential
candidate as vacuolar targeting signal sequence;
however, experiments should be performed to
characterize its functional properties. Comparison of the C-terminal extension of PRB-lb and
other vacuolar proteins shows no sequence similarity. However, some of these C-terminal extensions have an overall negative charge, because of
the presence of acidic amino acids, and most of
them are rich in hydrophobic amino acids [9].
These features may be important in forming particular secondary structures or a hydrophobic domain, which may be recognized during the sorting process. The absence of any obvious amino
acid sequence similarity in the vacuolar signals
suggests that the vacuolar targeting signals may
be structural rather than sequence specific, as
previously proposed by Chrispeel and Raikhel
[9].
Acknowledgment
This work was supported by grants from the Israel
Academy of Science, Jerusalem, the Ministry of
Science and Technology, Jerusalem (MOST), the
German Bundesministerium far Forschung und
Technologie, Braunschweig (BMFT), and the Leo
and Julia Forchheimer Center for Molecular Genetics. R. F. is a recipient of the Jack and Florence Goodman Career Development Chair.
G. S. is a recipient of a Scholarship from the
Feinberg Graduate School.
References
1. An G: Binary Ti vectors for plant transformation and
promoter analysis. Meth Enzymol 153:292-305 (1987).
2. Anderson SL, Teakle GR, Martino-Catt SJ, Kay SA:
Circadian clock- and phytochrome-regulated transcription is conferred by a 78 bp cis-acting domain of the
Arabidopsis CAB2 promoter. Plant J 6:457-470 (1994).
3. Bednarek SY, Raikhel NV: The barley lectin carboxylterminal propeptide is a vacuolar protein sorting determinant in plants. Plant Cell 3:1195-1206 (1991).
4. Bol JF, Linthorst HJM, Comelissen BJC: Plant pathogenesis-related proteins induced by virus infection. Annu
Rev Phytopath 28:113-138 (1990).
5. Boiler T, Kende H: Hydrolytic enzymes in the central
vacuole of plant cells. Plant Physio163:1123-1132 (1979).
6. Boiler T, V0geli U: Vacuolar localization of ethyleneinduced chitinase in bean leaves. Plant Physiol 74: 442444 (1984).
7. Boudet AM, Alibert G: Isolation of vacuoles and tonoplast from protoplasts. Meth Enzymol 148: 74- 81 (1987).
8. Cart JP, Dixon DC, Nikolan BJ, Voelkerding KV, Klessig DF: Synthesis and localization ofpathogenesis-related
proteins in tobacco. Mol Cell Biol 7:1580-1583 (1987).
9. Crispeels MJ, Raikhel NV: Short peptide domains target
proteins to plant vacuoles. Cell 68:613-616 (1992).
10. Cornelissen BJC, Horowitz J, van Kan JAL, Goldberg
RB, Bol JF: Structure of tobacco genes encoding
pathogenesis-related proteins from the PR-1 group. Nucl
Acids Res 15:6799-6811 (1987).
11. Dickey LF, Gallo-Meagher M, Thompson WF: Light
regulatory sequences are located within the 5' portion of
the Fed-1 message sequence. EMBO J 11:2311-2317
(1992).
12. Dixon DC, Cutt JR, Klessig DF: Differential targeting of
the tobacco PR-1 pathogenesis-related proteins to the
extracellular space and vacuoles of crystal idioblasts.
EMBO J 10:1317-1324 (1991).
13. Douglas CJ, Hauffe KD, Ites-Morales M-E, Ellard M,
Paszkowski U, Hahlbrock K, Dangl JL: Exonic sequences are required for elicitor and light activation of a
plant defence gene, but promoter sequences are sufficient
for tissue specific expression. EMBO J 10:1767-1775
(1991).
14. Dumas E, Lherminier J, Gianinazzi S, White RF, Antoniw JF: Immunocytochemical location ofpathogenesisrelated b 1 protein induced in tobacco mosaic virusinfected or polyacrylic acid-treated tobacco plants. J Gen
Virol 69:2687-2694 (1988).
15. Eyal Y, Fluhr R: Cellular and molecular biology of
pathogen-related proteins. Oxf Surv Plant Mol Cell Biol
7:223-254 (1991).
16. Eyal Y, Sagee O, Fluhr R: Dark-induced accumulation of
a basic pathogenesis-related (PR-1) transcript and a light
requirement for its induction by ethylene. Plant Mol Biol
19:589-599 (1992).
546
17. Eyal Y, Meller Y, Lev-Yadun S, Fluhr R: A basic-type
PR-1 promoter directs ethylene responsiveness, vascular
and abscission zone-specific expression. Plant J 4: 225234 (1993).
18. Fluhr R, Eyal Y, Meller Y, Raz V, Yang X-Q, Sessa G:
Signal transduction pathways in plant pathogenesis response. In: Bowles DJ, Gilmartin PM, Knox JP, Lunt
GG (eds) Molecular Botany: Signals and Environment,
pp. 131-141, London, Biochemical Society Symposia 60
(1994).
19. Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson
TMA: The 5'-leader sequence of tobacco mosaic virus
RNA enhances the expression of foreign gene transcripts
in vitro and in vivo. Nucl Acids Res 15:3257-3273 (1987).
20. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers
SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227:1229-1231 (1985).
21. Hosokawa D, Ohashi Y: Immunochemical localization of
pathogenesis-related proteins secreted into the intercellular spaces of salycilate-treated tobacco leaves. Plant Cell
Physiol 29:1035-1040 (1988).
22. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions:
fl-glucuronidase as a sensitive and versatile gene fusion
marker in higher plants. EMBO J 6:3901-3907 (1987).
23. Joosten MHAJ, Bergmans CJB, Meulenhoff EJS, Cornelissen BJC, de Wit PGJM: Purification and serological
characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato. Plant Physio194: 585591 (1990).
24. Li Y, Strabala TJ, Hagen G, Guilfoyle TJ: The soybean
SAUR open reading frame contains a cis element responsible for cycloheximide-induced mRNA accumulation.
Plant Mol Biol 24:715-723 (1994).
25. Lincoln JE, Fisher RL: Diverse mechanisms for the regulation of ethylene-inducible gene expression. Mol Gen
Genet 212:71-75 (1988).
26. Linthorst HJM: Pathogenesis-related proteins of plants.
Crit Rev Plant Sci 10:123-150 (1991).
27. Linthorst HJM, van der Does C, Brederode FT, Bol JF:
Circadian expression and induction by wounding of tobacco genes for cysteine proteinase. Plant Mol Biol 21:
685-694 (1993).
28. Logemann J, Schell J, Willmitzer L: Improved method for
the isolation of RNA from plant tissues. Anal Biochem
163:16-20 (1987).
29. Lotan T, Ori N, Fluhr R: Pathogenesis-related proteins
are developmentally regulated in tobacco flowers. Plant
Cell 1:881-887 (1989).
30. Mauch F, Staehelin LA: Functional implications of the
subceUularlocalization of ethylene-induced chitinase and
fl-l,3-glucanase in bean leaves. Plant Cell 1:447-457
(1989).
31. Melchers LS, Sela-Buurlage MB, Vloemans SA,
Woloshuk CP, van Roekel JSC, Pen J, van den Elzen
PJM, Cornelissen BJC: Extracellular targeting of the
vacuolar tobacco proteins AP24, chitinase and fl-l,3-
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
glucanase in transgenic plants. Plant Pol Biol 21: 583593 (1993).
Meller Y, Sessa G, Eyal Y, Fluhr R: DNA-protein interactions on a cis-DNA element essential for ethylene regulation. Plant Mol Biol 23:453-463 (1993).
Mohnen K, Shinshi H, Felix G, Meins JR: Hormonal
regulation of fl-l,3-glucanase messenger RNA levels in
cultured tobacco tissues. EMBO J 4:1631-1635 (1985).
Murasgige T, Skoog F: A revised medium for rapid growth
and bioassays with tobacco tissue culture. Physiol Plant
15:473-497 (1962).
Nagy F, Kay SA, Chua N-H: A circadian clock regulates
transcription of the wheat Cab-1 gene. Genes Devel 2:
376-382 (1988).
Neuhaus J-M, Sticher L, Meins FJr, Boiler T: A short
C-terminal sequence is necessary and sufficient for the
targeting of chitinases to the plant vacuole. Proc Natl
Acad Sci USA 88:10362-10366 (1991).
Newman TC, Ohme-Takagi M, Taylor CB, Green PJ:
DST sequences, highly conserved among plant SAUR
genes, target reporter transcripts for rapid decay in tobacco. Plant Cell 5:701-714 (1993).
Ohme-Takagi M, Taylor CB, Newman TC, Green PJ:
The effect of sequences with high AU content on mRNA
stability in tobacco. Proc Nail Acad Sci USA 90:1181111815 (1993).
Ori N, Sessa G, Lotan T, Himmeloch S, Fluhr R: A
major stylar matrix polypeptide (Sp41) is a member of the
pathogenesis-related proteins superclass. EMBO J 9:
3429-3436 (1990).
Payne G, Middlesteadt W, Desai N, Williams S, Dincher
S, Carnes M, Ryals J: Isolation and sequence of a genomic clone encoding the basic form of a pathogenesisrelated protein 1 from Nicotiana tabacum. Plant Mol Biol
12:595-596 (1989).
Raz V, Fluhr R: Ethylene responsiveness is transduced
via phosphorylation events in the plants. Plant Cell 5:
523-530 (1993).
Rochaix J-D: Post-transcriptional steps in the expression
ofchloroplastic genes. Annu Rev Cell Biol 8:1-28 (1992).
Rogers SG, Klee HJ, Horsch RB, Fraley RT: Improved
vectors for plant transformation; expression cassette vectors and new selectable markers. Meth Enzymol 163:
251-277 (1987).
Sessa G, Meller Y, Fluhr R: A GCC element and a G-box
motif participate in ethylene induced expression of the
PRB-lb gene. Plant Mol Biol (in press).
Shirley BW, Meagher RB: A potential role for RNA turnover in the light regulation of plant gene expression:
ribulose-l,5-biphosphate carboxylase small subunit in
soybean. Nuct Acids Res 18:3377-3385 (1990).
Smith DB, Johnson KS: Single-step purification of
polypeptides expressed in Escherichia coil as fusions with
glutathione S-transferase. Gene 67:31-40 (1988).
Somssich IM: Regulatory elements governing PR gene
expression. In: Nover L (ed) Plant Promoters and Tran-
547
scription Factors, pp. 163-179. Springer-Verlag, Berlin,
Heidelberg, New York (1994).
48. Sullivan ML, Green PJ: Post-transcriptional regulation
of nuclear-encoded genes in higher plants: the roles of
mRNA stability and translation. Plant Mol Biol 23:
1091-1104 (1993).
49. Van den Bulcke M, Bauw G, Castresana C, Van Montagu
M, Vandekerckhove J: Characterization of vacuolar and
extracellular fl-(1,3)-glucanases of tobacco: Evidence for
a strictly compartmentalized plant defense system. Proc
Natl Acad Sci USA 86:2673-2677 (1989).
50. Widholm JM: The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant
cells. Stain Technol 47:189-194 (1972).
51. Zhang S, Sheng J, Liu Y, Mehdy MC: Fungal elicitorinduced bean proline-rich protein mRNA down-regulation is due to destabilization that is transcription and
translation dependent. Plant Cerll 5:1089-1099 (1993).