Plant Physiol. (1998) 118: 419-430
Proteasome Inhibitors Prevent Tracheary Element Differentiation
in Zinnia Mesophyll Cell Cultures1
Bonnie J. Woffenden,
Thomas B. Freeman2, and
Eric P. Beers*
Department of Horticulture, Virginia Polytechnic Institute and
State University, Blacksburg, Virginia 24061
 |
ABSTRACT |
To determine whether proteasome
activity is required for tracheary element (TE) differentiation, the
proteasome inhibitors clasto-lactacystin 
-lactone and
carbobenzoxy-leucinyl-leucinyl-leucinal (LLL) were used in a zinnia
(Zinnia elegans) mesophyll cell culture system. The addition of proteasome inhibitors at the time of culture initiation prevented differentiation otherwise detectable at 96 h.
Inhibition of the proteasome at 48 h, after cellular commitment to
differentiation, did not alter the final percentage of TEs compared
with controls. However, proteasome inhibition at 48 h delayed the
differentiation process by approximately 24 h, as indicated by
examination of both morphological markers and the expression of
putative autolytic proteases. These results indicate that proteasome
function is required both for induction of TE differentiation and for
progression of the TE program in committed cells. Treatment at 48 h with LLL but not clasto-lactacystin -lactone resulted in partial uncoupling of autolysis from differentiation. Results from gel analysis of protease activity suggested that the
observed incomplete autolysis was due to the ability of LLL to inhibit
TE cysteine proteases.
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INTRODUCTION |
The role of proteases in regulating PCD is currently an extremely
active area of research. Principal among the proteases shown to be
required for PCD in animal systems is the large and still growing
family of proteases known as caspases (cysteinyl
aspartate-specific proteases; for review, see
Nicholson and Thornberry, 1997
), which appear to be involved in
disabling cell-repair processes essential for maintaining homeostasis,
thereby leading to apoptosis. However, caspases are not the only
proteases demonstrated to play a role in PCD. Animal apoptosis pathways
can also be initiated by several other mechanistically distinct
proteases, including the Ser protease granzyme B (Greenberg, 1996),
members of the cathepsin family (Deiss et al., 1996), and the Cys
protease calpain (Squier and Cohen, 1996).
In plants it is well documented that increases in Ser and Cys proteases
are associated with two developmentally programmed suicide pathways,
organ senescence (for review, see Hadfield and Bennett, 1997) and TE
differentiation (for review, see Beers, 1997; Fukuda, 1997; Pennell and
Lamb, 1997). Although it is generally assumed that these plant enzymes
function in the autolysis of intracellular proteins rather than as
components of regulatory proteolytic cascades, the apparent
participation of multiple proteolytic pathways during animal PCD
indicates that the ability of plant proteases to regulate PCD may be
underestimated.
An additional proteolytic system, the ATP-dependent
ubiquitin-proteasome pathway of proteolysis, is known to regulate
numerous cellular processes via degradation of short-lived regulatory
proteins in mammals, yeast, and plants (Shanklin et al., 1987; Glotzer et al., 1991; Hochstrasser et al., 1991). The importance of the ubiquitin-proteasome pathway in degrading long-lived proteins is
well-established for mammals, but its function in this capacity in
lower eukaryotes is uncertain (for review, see Goldberg, 1997). Ubiquitin is a 76-amino acid protein that becomes covalently ligated to
cellular proteins via isopeptide bonds between the carboxy-terminal Gly
of ubiquitin molecules and the 
-amino group of Lys residues of the
target protein (for review, see Ciechanover and Schwartz, 1994;
Varshavsky, 1997). Attachment of ubiquitin to protein targets requires the activity of multiple enzymes, including an
ATP-dependent ubiquitin-activating enzyme (E1), one of a family of
ubiquitin-conjugating enzymes (E2s), and, in some cases, one of a
number of ubiquitin-protein ligases (E3s). Ubiquitin may be attached to
protein substrates as a monomer or it may be ligated to a Lys residue
of another ubiquitin molecule, forming polyubiquitin chains.
Polyubiquitination of proteins is sufficient to target them for
degradation by a large (26S), ATP-dependent multicatalytic protease,
the proteasome. Additionally, proteasome-mediated degradation of a few
proteins has been shown to occur without ubiquitination (Murakami et
al., 1992; Jariel-Encontre et al., 1995).
Recent evidence indicates that the ubiquitin-proteasome pathway may
regulate PCD in some systems. Levels of ubiquitin-proteasome pathway
components were observed to increase during animal PCD events,
including molt-induced claw muscle atrophy in lobster (Shean and
Mykles, 1995) and intersegmental muscle degeneration during
metamorphosis of the hawkmoth (Haas et al., 1995; Jones et al., 1995).
Depending on the experimental system under investigation, proteasome
activity may promote PCD (Grimm et al., 1996; Sadoul et al., 1996; Cui
et al., 1997) or prevent it (Shinohara et al., 1996; Drexler, 1997;
Monney, 1998). Requirements for proteasome activity during
differentiation events not involving PCD have also been documented from
animal systems, including maturation of starfish oocytes (Sawada et
al., 1997) and differentiation of photoreceptor cells in the
Drosophila melanogaster eye (Li et al., 1997). In plants
up-regulation of components of the ubiquitin pathway has been detected
during diverse developmental PCD events, including leaf senescence
(Garbarino and Belknap, 1994), anther dehiscence (Li et al., 1995), and
fruit ripening (Picton et al., 1993). Thus, the ubiquitin-proteasome
pathway is broadly implicated as a regulator of cell fate.
Using the zinnia (Zinnia elegans) mesophyll cell culture
system for TE differentiation (Fukuda and Komamine, 1980a; Church, 1993; Fukuda, 1997), we investigated the role of proteolysis during cell differentiation and PCD. Mature TEs function in planta in water
and solute transport and are characterized by a patterned deposition of
lignified, cellulosic, secondary cell wall thickenings and the absence
of a protoplast within the cell corpse. The zinnia mesophyll cell
system permits the study of plant cell differentiation, death, and
autolysis during the semi-synchronous TE differentiation of 40% to
60% of the cultured cells. Although no genes or proteins controlling
cell death or the initiation of the autolytic phase in differentiating
TEs have been identified, hydrolytic enzyme activity increases
dramatically late in the differentiation process.
Markers for TE autolysis include an endonuclease (Thelen and Northcote,
1989), a RNase (Ye and Droste, 1996), and proteases (Minami and Fukuda,
1995; Ye and Varner, 1996; Beers and Freeman, 1997). The
ubiquitin-proteasome pathway also appears to be required for proper
vascular tissue development. Transgenic tobacco expressing a mutant
ubiquitin unable to form polyubiquitin chains exhibited aberrant
vasculature (Bachmair et al., 1990), and in Coleus,
regeneration of xylem vessel elements after wounding was accompanied by
increased levels of ubiquitin and/or ubiquitin-protein conjugates
(Stephenson et al., 1996).
Ubiquitin-protein conjugating activity is detectable in zinnia TE
culture extracts (B.J. Woffenden and E.P. Beers, unpublished data).
Using inhibitors of the proteasome, we asked whether the proteasome has
a regulatory role during TE differentiation. Proteasome inhibitors used
included LAC (Dick et al., 1997), which was derived from the microbial
metabolite lactacystin (Omura et al., 1991), and the synthetic
tripeptide-aldehyde inhibitors LLnL, LLM (Sasaki et al., 1990), and LLL
(Tsubuki et al., 1996). LAC irreversibly inhibits the tryptic,
chymotryptic, and peptidylglutamic cleavage activities of the
proteasome by covalently binding to the amino-terminal Thr residue of
the -subunits (Fenteany et al., 1995). Radiographic, crystallographic, and mutagenesis studies have demonstrated that this
residue provides the active-site nucleophile (Löwe et al., 1995;
Seemüller et al., 1995). Specificity of LAC for the proteasome has been established to the exclusion of the Cys proteases calpain, papain, and cathepsin B, and the Ser proteases chymotrypsin and trypsin
(Fenteany et al., 1995). Conversely, peptide aldehydes are reversible,
competitive inhibitors that act as transition-state analogs (Löwe
et al., 1995) and inhibit both calpain and the proteasome (Rock et al.,
1994; Tsubuki et al., 1996).
The data presented here implicate the proteasome as a regulator in both
early and late stages of TE differentiation in zinnia mesophyll cell
cultures but do not support a direct role for the proteasome in TE
autolysis. Rather, autolysis appears to depend at least in part on
LLL-sensitive Cys proteases.
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MATERIALS AND METHODS |
Plant Material and Growth Conditions
Seeds of zinnia (Zinnia elegans cv Envy; Grimes Seeds,
Concord, OH) were sown in 4-inch pots containing Sunshine Mix 1 (Wetsel Seed Co., Harrisonburg, VA). Plants were grown at 27°C under a 16-h
photoperiod at 85 µmol photons m
2
s1 and watered as needed with distilled water.
Mesophyll Cell Isolation and Culture
The first true leaves were harvested from 7- to 9-d-old seedlings,
and mesophyll cells were isolated and cultured in TE inductive medium
according to the method of Roberts et al. (1992), except that cells
were cultured in scintillation vials in 2.4 mL of medium. Cells were
collected from suspension cultures by centrifugation at 50g
for 2 min at the times indicated in the figure legends. Pellets were
stored at 
70°C until extraction (when harvesting cells for RNA
extraction, this step was preceded by freezing in liquid nitrogen).
Proteasome Inhibitor Treatments
The proteasome inhibitors LLnL, LLM (Sigma), LLL (sold as MG132,
Calbiochem), or LAC (Calbiochem) were added to TE cultures as follows:
Because the level of solubility of LLL in aqueous solution is
approximately 50 µM, LLL was added to cultures as 20 µM pulses every 6 h, either between 0 and 18 h
or between 48 and 66 h, to a final concentration as indicated in
the figure legends. LAC was added to a final concentration as indicated
in the figure legends. DMSO solvent controls were evaluated for all treatments. Regardless of the method of inhibitor addition (i.e. pulsed
multiple times or single additions), inhibitor additions begun at the
time of culture initiation and at 48 h are referred to as
t0 and t48,
respectively.
Cell Counts
For counting, an aliquot of cells was mixed with an equal volume
of 1% Evans blue (Sigma), which is excluded from live cells (Roberts
and Haigler, 1989), in culture medium. Values for three populations
were recorded using a hemacytometer: live, nondifferentiated cells,
live TEs, and dead TEs. To score incomplete autolysis, dead TEs were
counted as either autolytically cleared or retaining cellular contents.
Hoechst 33342 Staining of Cultured Cells
Paraformaldehyde-fixed (Planchais et al., 1997) cells stained with
1 µg mL1 Hoechst 33342 in Galbraith buffer
(20 mM Mops, pH 7.0, 45 mM MgCl2, 30 mM sodium citrate, and 1%
[w/v] Triton X-100; Galbraith et al., 1983) were viewed and
photographed using a fluorescence microscope (model MC 63, Zeiss).
Protein Extraction
In each of two independent experiments for each inhibitor, four
replicates (scintillation vials) were pooled at each harvest (72 and
96 h) and scored for TE differentiation. Extracts were prepared by
four freeze-thaw cycles in 100 mM
NaPO4 buffer, pH 7.2, containing 20 µM leupeptin and 14 mM 2-mercaptoethanol.
Lysed cells were pelleted by centrifugation at 12,000g for
10 min at 4°C. The supernatant was concentrated approximately 25-fold
using YM10 concentrators (Millipore) and stored at 70°C for
subsequent use in either activity gels or immunoblots.
Antibody Production and Purification and Immunoblot Analysis of
Ubiquitin-Protein Conjugates
Antibodies to denatured, cross-linked bovine ubiquitin (Sigma)
were prepared in chickens at Cocalico Biologicals (Reamstown, PA). The
immunoglobulin fraction was purified from egg yolk using the caprylic
acid extraction protocol of McLaren et al. (1994) and was then
subjected to affinity purification (Hershko et al., 1982; Haas and
Bright, 1985). After resolution by SDS-PAGE (13.5% [w/v] acrylamide)
using the buffer system of Laemmli (1970), zinnia proteins were
electrophoretically transferred to PVDF membranes (Immobilon-P,
Millipore) using a semidry transfer apparatus (Amersham-Pharmacia Biotech) according to the manufacturer's recommendations. The transfer
buffer was 48 mM Tris, 39 mM Gly, pH 8.4, 1.3 mM SDS, 20% methanol. Blocking and incubation in primary
and secondary antibodies were performed with "Blotto" made
according to the method of Johnson et al. (1984). Blots were incubated
in antibody diluted 1:1000 in Blotto at room temperature, with rotation
for 2 h (primary) or 1 h (secondary). Blots were washed
between steps using 200 mM NaCl buffered with 50 mM Tris-HCl, pH 7.4. Colorimetric detection with the
substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (both from Sigma) was catalyzed by alkaline
phosphatase-conjugated, goat anti-chicken antibody (KPL, Gaithersburg,
MD).
Protease Activity Gels
Protease activity gels were prepared essentially according
to the method of Beers and Freeman (1997). Aliquots from each extract, representing an equal number of cells (1 × 105), were resolved by SDS-PAGE (12% [w/v]
acrylamide). Samples were not boiled prior to electrophoresis.
Hydrolysis of the gelatin substrate (0.5% [w/v]) resulted in
unstained bands in the substrate-impregnated gels, indicating the
position of the proteolytic activity in resolving gels. For in vitro
inhibitor studies, polyacrylamide gel lanes were excised and incubated
at room temperature for 15 min in 4 µM LAC, 20 µM LLL, or 0.1% DMSO as a solvent control prior to exposure to the substrate gels, as described above.
RNA Isolation
Following each of two independent experiments for LLL and one
experiment for LAC, 11 replicates were pooled, an aliquot was removed
and scored for TE differentiation, and the balance was frozen in liquid
nitrogen and stored at 70°C until extraction. Total RNA was
prepared by the method of Chirgwin et al. (1979). Immediately after the
addition of 2 mL of a guanidine thiocyanate stock solution (4 M guanidine thiocyanate, 0.5%
N-lauroylsarcosine, 25 mM sodium citrate, pH
7.0, 100 mM 2-mercaptoethanol, and 0.1% antifoam A), the
sample was homogenized on ice for a total of 3 min and clarified by
centrifugation. RNA was precipitated overnight at 20°C by the
addition of 0.025 volume of 1 N acetic acid and 0.75 volume
of ethanol. The pellet was resuspended at one-half the original volume
in guanidine-HCl solution (7.5 M guanidine-HCl, 25 mM sodium citrate, pH 7.0, and 50 mM
2-mercaptoethanol), and precipitated (0.025 volume of 1 N
acetic acid and 0.5 volume of ethanol) at 20°C for at least 3 h. This was repeated twice, reducing the volume of guanidine-HCl by
one-half each time. The pellet was washed by suspension in absolute
ethanol (20°C), extracted twice in 100 µL of
diethyl-pyrocarbonate-treated water, and precipitated (0.1 volume of 2 M potassium acetate, pH 5.0, and 2 volumes of ethanol) overnight at 20°C. The pellet was washed twice with 95%
ethanol, dried, and resuspended in diethyl-pyrocarbonate-treated water.
RNA Probe Synthesis
For template preparation, 1 µg of plasmid p48h-17 in pBluescript
K/S (Stratagene) was linearized with BglII, purified by
electrophoresis through low-melting-point agarose (FMC, Rockland, ME),
recovered, and rendered free of RNase by phenol extraction and
precipitation. Biotinylated antisense p48h-17 was prepared using T7
polymerase and biotin RNA-labeling reagents (Boehringer Mannheim)
according to the manufacturer's directions. The probe was checked for
integrity by RNA gel electrophoresis as described below.
RNA Gel-Blot Analysis
Samples and biotinylated RNA molecular mass markers (New England
Biolabs) were separated on 1.2% agarose gels containing formaldehyde as described by Sambrook et al. (1989), except that the formaldehyde gel-running buffer contained 5 mM sodium acetate. After
electrophoresis, gels were photographed and washed three times for 10 min each in 2× SSC and transferred to positively charged nylon
membranes (Boehringer Mannheim) by overnight capillary transfer in 2×
SSC. The membranes were UV cross-linked and then dried for 2 h at
80°C.
The membranes were hydrated for 2 min in 5× SSC and prehybridized in
prehybridization/hybridization buffer (NorthernMAX, Ambion, Austin, TX)
in a bag at 68°C for at least 1 h. The bag was drained, refilled
with fresh buffer containing biotinylated antisense probe, and
hybridized overnight at 68°C. Membranes were washed twice for 5 min
each time at room temperature in 2× SSC/0.1% SDS, at 0.2× SSC/0.1%
SDS, and then twice for 15 min at 68°C in 0.1 × SSC/0.1% SDS.
Bands were visualized by chemiluminescent detection (Phototope K6 kit,
New England Biolabs) following the manufacturer's directions except
that one or two additional washes were added following incubation in
streptavidin.
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RESULTS |
Lactacystin and LLL Prevent TE Formation when Applied at Culture
Initiation and Delay Differentiation when Applied after Cell Fate
Determination
Preliminary experiments using the proteasome inhibitors LLnL, LLM
(data not shown), LAC, and LLL revealed that only the latter two
effectively inhibited TE formation when added at culture initiation (t0). Therefore, subsequent experiments
were conducted using only LAC and LLL. Figure
1 shows the effect of a range of
concentrations of LAC (Fig. 1A) and LLL (Fig. 1B) on the inhibition of
TE differentiation. The observed effective doses for nearly complete
inhibition of TE development that were used in subsequent experiments
were 4 µM LAC and 80 µM LLL.
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| Figure 1.
Effects of inhibitor dosage and time of addition
on TE differentiation. Shown are the percentages of TEs in total cells
(live, undifferentiated cells plus TEs;  ) detectable at 96 h
following treatment with the indicated concentrations of LAC (A) or LLL
(B) at t0. Also shown are the percentages of
TE detectable at 96 h following removal of inhibitors at 24 h
of culture ( ). C, Percentages of TEs in total cells detectable at
96 h following addition of 4 µM LAC to TE cultures
at the times indicated. Values are the mean percentages ± SD of a minimum of four replicates.
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The concentrations of LAC and LLL used in these studies are comparable
to the nontoxic levels reported previously from research in animal
systems (Palombella et al., 1994; Grimm et al., 1996), and several
parameters indicate that these concentrations of inhibitors are
nontoxic in the zinnia system. Most significantly, cell growth was not
affected. The mean length of nondifferentiating cells present in
cultures treated at culture initiation with either LAC or LLL increased
1.8-fold (from 48 to 88 µm) over 150 h in culture, as did the
cells of control cultures. Additionally, these levels of proteasome
inhibitors did not increase cell mortality above that seen in the
solvent controls (data not shown), as determined from the ability of
cells to exclude the nonpermeant vital stain Evans blue. Cells in
inhibitor-treated cultures also continued to exhibit obvious
cytoplasmic streaming and secrete characteristic protoplast fragments,
as observed by Groover et al. (1997) to be normal behavior of cells in
healthy zinnia mesophyll cell cultures. Finally, a washout experiment
demonstrated that a high percentage of cells (near control levels for
LAC) scored at 96 h could respond to signals leading to TE
differentiation, even after a 24-h exposure to inhibitor (Fig. 1, A and
B).
In three independent experiments, the addition of either 4 µM LAC (Fig. 1A) or 80 µM LLL (Fig. 1B) at
t0 of culture resulted in virtually
complete inhibition of TE differentiation over the 96-h culture period.
Although new TEs did develop in inhibitor-treated and control cultures
during an additional 2 d of culture beyond 96 h, LAC-treated
cultures reached only 35% of control levels and LLL-treated cultures
attained only 22%.
Delaying addition by as little as 6 h after culture initiation
resulted in decreased efficacy, and inhibition was no longer detected
following additions at and beyond 48 h of culture (Fig. 1C). At or
soon after 48 h in TE cultures, differentiation was evident as
secondary cell wall thickenings became visible. A similar time course
of LLL addition was not conducted because the method of application
(four pulses over 18 h) required to achieve the effective dose of
80 µM in solution precluded single-time-point additions.
To determine whether the proteasome plays a role late in the
differentiation process, for example, during the cell death or autolytic programs, TE cultures were treated with 4 µM
LAC or 80 µM LLL at t48.
Table I shows that following LAC or LLL
addition at t48 the percentage of TEs
visible by 72 h in treated cultures was reduced to 13% and 70%,
respectively, of the levels in control cultures. That these lower
levels of TEs represented a delay and not a prohibition of
differentiation was evident by 96 h, when the percentage of
differentiated cells in inhibitor-treated cultures was nearly identical
to the control levels.
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Table I.
Effect of t48 treatment with
proteasome inhibitors on the percentage of TE differentiation
Numbers within parentheses indicate the total number of replicates from
a minimum of two independent experiments.
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Despite the ultimate attainment of control-level numbers of TEs in
t48 LAC-treated cultures, Table I
illustrates that the LAC-induced delay in TE differentiation apparent
by 72 h was still evident at 96 h of culture, as reflected by
a 5-fold higher level of live TEs in treated cultures compared with
controls. The observed level of live TEs in LAC-treated cultures at
96 h was intermediate between the levels observed in control
cultures at 72 h (39%) and 96 h (4%). The stages of
differentiation represented among live TEs in LAC-treated cultures at
96 h ranged from cells with barely detectable cell wall
thickenings to those with extensive thickenings (data not shown). This
observed higher level of live TEs at 96 h in LAC-treated cultures
was apparently not the result of an uncoupling of secondary cell wall
thickening from cell death, as was revealed by culturing cells an
additional 24 h, by which time equivalent numbers of mature, dead
TEs were detected in inhibitor-treated cultures and controls (data not
shown). In contrast to these results with LAC, no such disparity in the
number of live TEs in control versus treated cultures was evident at
late times in the LLL experiments (Table I).
LLL Prevents Completion of TE Autolysis
Despite equivalent numbers of dead TEs present at 96 h in
t48 LLL-treated and control cultures (Table
I), we observed that LLL treatment induced an approximately 6-fold
increase (from 15% to 85%) in the percentage of TEs that had not yet
completed autolytic clearing by 96 h and retained some portion of
intracellular contents. In contrast, t48
LAC treatment did not result in retention of protoplasmic material by
dead TEs above the levels observed in control cultures (data not
shown). Bright-field micrographs shown in Figure
2, A and B, depict cells harvested at
96 h from control and LLL-treated cultures. All TEs visible in
Figure 2A are mature TEs of the control culture, lacking any detectable
contents (representative of 85% of control TEs). All TEs exhibiting
protoplasmic retention in LLL-treated cultures appeared plasmolyzed,
with the collapsed protoplasm most often localized to one or two tight
masses within the cell (Fig. 2B, arrowhead) but sometimes dispersed
throughout the cell (Fig. 2B, arrow).
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| Figure 2.
Characterization of LLL-induced, incomplete
TE autolysis. For all panels, samples were taken from TE cultures at
96 h for staining and microscopy. C and D show the same field of
view. A, Bright-field microscopy of unstained control culture TEs after
autolysis. B, Bright-field microscopy of unstained TEs from the
t48 LLL-treated culture. The two forms of
protoplasmic retention by dead TEs are indicated: condensed to a single
or few locations (arrowhead) or distributed throughout the cell
(arrow). C, Bright-field microscopy of paraformaldehyde-fixed cells
from the t48 LLL-treated culture. Arrowhead
indicates protoplasm retained by dead TE that failed to complete
autolysis. Live TEs showed diffuse protoplasm similar to that of live,
undifferentiated cells. D, Fluorescence microscopy of cells from the
t48 LLL-treated culture following DNA
staining with Hoechst 33342. Blue fluorescence indicates the presence
of nDNA; red fluorescence is due to chlorophyll autofluorescence. LT,
live TE; DT, dead TE; u, live, undifferentiated cell; d, dead,
undifferentiated cell (likely killed during isolation of mesophyll
cells from leaves). Bar = 10 µm.
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Three other cytological characteristics did not distinguish dead TEs
exhibiting incomplete autolysis in LLL-treated cultures from mature TEs
in control cultures. First, secondary cell wall thickenings of
LLL-treated and control dead TEs appeared equivalent under bright-field
microscopy (Fig. 2, compare A and B). Second, phloroglucinol staining
of cells harvested at 96 h revealed no detectable differences in
the extent of cell wall lignification between dead TEs in LLL-treated
cultures and those in control cultures (data not shown). Finally,
Calcofluor white staining of cells at 96 h did not distinguish TEs
of LLL-treated cultures from those in control cultures with respect to
the degree of cell wall cellulose deposition (data not shown).
It has been reported that degradation of the nucleus and other
organelles occurs late during the autolysis of differentiating TEs,
just before or after tonoplast disruption (Groover et al., 1997). To
characterize the intracellular material retained by TEs in LLL-treated
cultures, we determined whether TEs present at 96 h in LLL-treated
cultures contained a nucleus (or at least dye-binding DNA) by staining
with a fluorescent DNA-binding dye (Hoechst 33342). Figure 2, C and D,
are bright-field and fluorescence micrographs, respectively, of
paraformaldehyde-fixed cells demonstrating two important features of
TEs and cells present at 96 h in LLL-treated cultures. First, live
TEs exhibiting a diffuse and uniformly distributed protoplasm similar
to that observed in live, undifferentiated cells contained intact
nuclei (Fig. 2, C and D). Second, we did not observe TEs exhibiting
both plasmolysis (i.e. incomplete autolysis) and dye-binding DNA. These
results indicate that the apparent ability of LLL to stabilize
intracellular contents against autolysis does not include the
preservation of nDNA.
LAC and LLL Have Different Effects on the Activity and mRNA Levels
of Cys Proteases Associated with Late Stages of TE Differentiation
Data presented thus far consist of a characterization of
morphological markers associated with TE development as indicators of
apparent inhibitor-induced disruptions in the differentiation program.
To evaluate potential LAC- and LLL-induced changes in biochemical and
molecular markers of TE differentiation, the activities and mRNA levels
of Cys proteases putatively involved in autolysis were examined.
TE-specific proteases are well-documented markers for the late stages
of TE differentiation (Minami and Fukuda, 1995; Ye and Varner, 1996;
Beers and Freeman, 1997). Examining the expression and activity levels
of these proteases may therefore indicate the extent to which the
TE-differentiation program is affected by proteasome inhibition and
provide clues as to the apparent inability of LLL-treated cells to
complete autolysis.
Because the level of intracellular components decreases during TE
autolysis, it was decided that a comparison of TE markers on an
equal-cell-number basis would best represent relative effects of
proteasome inhibitors on the progression of differentiation. Proteins
were protected from degradation during and following isolation by
inclusion of leupeptin in the extraction buffer (Beers and Freeman,
1997). Protease activity gels were prepared using extracts from
1.5 × 105 cells harvested at 72 and 96 h to determine the effects of t48 application of proteasome inhibitor on the activity of two Cys proteases (28 and 24 kD) postulated to participate in the autolysis of
TEs (Beers and Freeman, 1997).
The activities of the 28- and 24-kD proteases were detectable at very
low levels in extracts from LAC-treated cells harvested at 72 h
compared with the control (Fig. 3A). By
96 h, however, protease activities in extracts from LAC-treated
cultures had increased to levels similar to those observed in 72-h
control extracts. During the same period in control cultures, activity of the 24-kD protease decreased to a barely detectable level, whereas
activity of the 28-kD enzyme was undetectable by 96 h. In
contrast, LLL treatment resulted in the recovery of a slightly higher
level of the 24-kD protease at 72 h compared with controls (Fig.
3B). By 96 h, as was observed in extracts from LAC experiments, activity of the 24-kD protease decreased to a barely detectable level
in control samples, whereas the level of activity of this enzyme
remained relatively high in extracts from LLL-treated cells. Despite
the apparent overall lower level of protease activity in LLL versus LAC
experiments (Fig. 3, compare A and B, 72-h DMSO controls), identical
results concerning the relative levels of protease activity (treated
versus control) were obtained in a second independent experiment for
each inhibitor. These protease activity profiles indicate that the
addition of proteasome inhibitors results in altered regulation of
expression and/or activity of TE-associated Cys proteases, and in the
case of the LAC experiments, the results are consistent with an
inhibitor-induced delay in the progression of the differentiation
program of approximately 24 h.
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| Figure 3.
LAC or LLL treatment at 48 h alters the
activity of TE Cys proteases. LAC (A) or LLL (B) was applied to TE
cultures at t48, and cells were harvested at
72 and 96 h. Shown are activity gels prepared following SDS-PAGE
of total protein extracted from 1.5 × 105 zinnia
cells per lane (see ``Materials and Methods''). Bands represent
regions of proteolytic activity in gelatin-impregnated substrate gels.
Molecular masses of protein standards are indicated on the left (in
kD).
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Expression of p48h-17, a Cys protease that is up-regulated in the late
stages of TE differentiation (Ye and Varner, 1993, 1996), was examined
following inhibitor treatment at t48.
During our efforts to prepare total RNA from treated and control cells for p48h-17 RNA gel-blot analysis, we discovered that equivalent numbers of cells yielded markedly different quantities of RNA. Cells
harvested from LAC- and LLL-treated cultures yielded 1.7- and 3.4-fold
higher levels of RNA, respectively, compared with controls at 72 h, and less than 0.5 µg of total RNA per 106
cells at 96 h. This low level of RNA recovery from
inhibitor-treated cultures at 96 h occurred despite our efforts to
denature RNases throughout RNA extraction (Chirgwin et al., 1979).
Back-extraction of cell pellets yielded no additional RNA (data not
shown), revealing that the observed differences in RNA yield apparently
were not due to treatment-induced differences in the retention of RNA
by the cells. Since it was not possible to isolate useful quantities of
RNA at 96 h following LAC or LLL treatments, we have presented RNA
gel-blot data (equal cell number and equal RNA comparisons) for the
72-h time point only.
Cells treated with LLL and harvested at 72 h yielded a higher
level of p48h-17 mRNA compared with the control, whether analysis was
conducted on an equal-cell-number or an equal-RNA basis (Fig. 4A). Similarly, but to a lesser extent,
p48h-17 mRNA was more abundant in cultured cells following LAC
treatment than in controls when compared on an equal-cell-number or on
an equal-RNA basis (Fig. 4B).
View larger version (46K):
[in this window]
[in a new window]
| Figure 4.
LLL and LAC treatments at 48 h result in
elevated levels of p48h-17 expression. LLL (A) or LAC (B) was added to
t48 TE cultures, and cells were harvested at
72 h. Shown are RNA gel blots of total RNA performed on both an
equal-cell-number (106) and an equal-RNA (3.5 µg
per lane) basis probed with biotinylated antisense p48h-17.
RNA levels loaded for equal-cell-number analyses are as follows: LLL,
13.8 µg, and corresponding DMSO, 4.1 µg; LAC, 8.1 µg, and
corresponding DMSO, 4.8 µg. Corresponding ethidium bromide-stained
agarose gels are shown below each blot.
|
|
LAC Is More Effective than LLL at Stabilizing Endogenous
Ubiquitinated Proteins in Cultured Zinnia Cells
To confirm that the proteasome inhibitors used in this study were
capable of exerting their effects via inhibition of the proteasome, we
examined the effects of LAC and LLL on levels of endogenous
ubiquitin-protein conjugates. Immunoblots of total zinnia
protein probed with anti-ubiquitin antibody reveal that treatment of
cells with either LAC or LLL resulted in the stabilization of
conjugates extracted from 72-h cultures (Fig. 5). The apparent stabilization by the
reversible inhibitor LLL was no longer evident by 96 h in culture
(data not shown). In contrast, treatment of cells at
t48 with the irreversible inhibitor LAC
resulted in a high degree of ubiquitin-protein conjugate stabilization through 96 h of culture (Fig. 5B).
View larger version (65K):
[in this window]
[in a new window]
| Figure 5.
Endogenous ubiquitin-protein conjugates are
stabilized by LLL and LAC treatments at 48 h. LLL (A) or LAC (B)
was added to t48 TE cultures, cells were
harvested at 72 h (LLL and LAC) and 96 h (LAC only), and
protein was extracted for immunoblot analysis as for Figure 3. Extracts
from 1.5 × 105 cells (LAC) and 2 × 105 cells (LLL) were loaded per lane. Blots were probed
with anti-ubiquitin antibody. Molecular masses of protein standards and
the position of free ubiquitin (Ub) are indicated on the right (in
kD).
|
|
LLL Inhibits the Activity of Cys Proteases of Potential Importance
to TE Autolysis
LLL has been reported to inhibit the Cys protease calpain in
addition to the proteasome (Tsubuki et al., 1996). To address the
possibility that LLL inhibition of Cys proteases putatively involved in
TE autolysis might explain the failure of TEs in LLL-treated cultures
to complete autolysis, protease activity gels were prepared in the
presence of LLL and LAC. LLL treatment resulted in complete inhibition
of the 28- and 24-kD TE-specific proteases (Fig.
6). As expected, LAC treatment had no
effect on the activity of these proteases (Fig. 6).
View larger version (29K):
[in this window]
[in a new window]
| Figure 6.
LLL but not LAC inhibits the activity of TE Cys
proteases. Following SDS-PAGE of total zinnia protein, excised gel
lanes were incubated in 4 µM LAC, 20 µM
LLL, or 0.1% DMSO prior to exposure to gelatin-impregnated substrate
gels. Bands represent regions of proteolytic activity in substrate
gels. Molecular masses of protein standards are indicated on the left
(in kD).
|
|
| |
DISCUSSION |
We have demonstrated that, when added at the time of culture
initiation, inhibitors of the proteasome can prevent TE
differentiation, apparently without significantly affecting the health
of cultured zinnia cells. In addition, proteasome inhibition following
the appearance of cell wall thickenings resulted in an approximately 2-fold increase in the time required to complete differentiation. This
delay was demonstrated by characterization of morphological markers and
by evaluation of the expression of Cys proteases putatively involved in
autolysis. Although LLL is capable of inhibiting both the proteasome
and Cys proteases, the observation that application of the specific
proteasome inhibitor LAC prevents TE differentiation indicates that
proteasome inhibition by LLL is sufficient for prevention of TE
differentiation. In contrast, the ability of LLL to partially uncouple
autolysis from the TE-differentiation program is probably due to its
inhibition of autolytic Cys proteases.
The Proteasome as Mediator of Early Signals Leading to TE
Differentiation
The ubiquitin-proteasome pathway is known to regulate the
cell cycle (for review, see Pagano, 1997), and although cell division can occur in up to 40% of cells undergoing TE differentiation, the
remaining 60% of TEs form without intervening mitosis (Fukuda and
Komamine, 1980b). Therefore, disruption of cell cycling by proteasome
inhibitors is not likely to account for the nearly complete prevention
of TE formation reported here. Rather, our results indicate that TE
differentiation may require the proteolytic removal of an endogenous
differentiation inhibitor(s), perhaps due to a role for the proteasome
in transducing differentiation signals initiated by the phytohormones
auxin and/or cytokinin, which are required for TE development (Church
and Galston, 1988). It has been proposed that the turnover of
short-lived repressor proteins is a requirement for auxin response (for
review, see Abel and Theologis, 1996), and recent work by Estelle and
colleagues (Ruegger et al., 1998) specifically implicates the
ubiquitin-proteasome pathway by showing that two Arabidopsis genes,
TIR1 and AXR1, which encode proteins related to
ubiquitin pathway components, are required for normal auxin signal
transduction.
The Proteasome as a Regulator of the Time Course of TE
Differentiation
That application of LAC after cellular commitment to TE
development causes a strong delay in the overall program is
corroborated by the 24-h delay in the peak of Cys protease activity
noted in extracts from LAC-treated cells compared with controls (Fig.
3A). One interpretation of the apparently contrasting 72-h profile of
protease activity extractable from LLL-treated cultures is that LLL
does not result in a similar delay in peak activity of TE proteases.
However, the higher level of 24-kD protease activity detectable at
72 h following LLL treatment compared with controls (Fig. 3B) may
have resulted from up-regulation of the expression of genes encoding
LLL-sensitive proteases in response to LLL-mediated inhibition of Cys
proteases (Fig. 6). Increases in mRNA levels of enzymes following
application of competitive inhibitors has been documented, as in the
case of 3-hydroxy-3-methylglutaryl CoA reductase (Cohen and Griffioen,
1988). Alternatively, the higher level of protease activity observed at
72 h following LLL treatment may have resulted from inhibition by
LLL of an unknown protease(s) that normally functions to degrade the
24-kD enzyme as part of a posttranslational mechanism for regulating
its activity prior to autolysis. Such a posttranslational mechanism
would not be expected to be affected by LAC treatment, which has no
activity against Cys proteases.
Our inability to harvest useful quantities of RNA from
inhibitor-treated TE cultures at 96 h may indicate that RNA was
degraded prior to or during isolation from inhibitor-treated cells.
RNase activity levels have been shown to increase after 48 h in
normal TE cultures (Thelen and Northcote, 1989; Ye and Droste, 1996), with the highest levels detected at 84 h (Thelen and Northcote, 1989). A proteasome-inhibitor-induced delay in the overall
differentiation program would be expected to include a delay in peak
RNase activity relative to the controls. Thus, recovery of low levels
of RNA at 96 h from proteasome inhibitor-treated cultures, despite
the presence of 40% nondifferentiated cells, may represent indirect evidence of a delay in the differentiation process relative to control
cultures, in which mostly empty, mature TEs contain little or no RNase
to degrade RNA released from nondifferentiating cells during RNA
isolation. Similarly, the recovery at 72 h of higher levels of RNA
from inhibitor-treated cells compared with controls may reflect the
presence of higher levels of early- stage (preautolysis) TEs in treated
cultures at this time.
The apparent higher level of p48h-17 mRNA observed at 72 h in both
LLL- and LAC-treated cultures is also consistent with an inhibitor-induced delay in the TE differentiation process, assuming that p48h-17 expression peaks prior to 72 h in normal TE cultures. Ye and Droste (1996) reported the highest level of p48h-17 mRNA expression at 60 h in TE cultures, although data from later times in culture were not presented. Whether p48h-17 encodes the 24-kD protease detected in this study is not known. However, when expressed in transgenic tobacco, p48h-17 yielded a 20-kD mature Cys protease, close to both its predicted size (22.7 kD; Ye and Varner, 1996) and to
the 24-kD Cys protease detected here and previously (Beers and Freeman,
1997). Therefore, it seems reasonable to speculate on the significance
of p48h-17 expression relative to the activity of the 24-kD Cys
protease.
The inverse relationship evident between 72-h p48h-17 mRNA and 72-h
protease activity levels following LAC treatment may indicate that 72-h
p48h-17 mRNA levels predict the much higher 96-h, 24-kD protease
activity levels. Alternatively, as discussed above, protease levels may
normally be kept low, despite high transcript levels, by some as yet
undescribed posttranslational proteolytic mechanism that is inhibited
by LLL and not by LAC. Thus, uncoupling of this regulatory mechanism
might be expected in LLL-treated cells, revealing an apparent direct
correlation between p48h-17 transcript level and TE protease levels.
Ubiquitin-Protein Conjugate Stabilization by LLL and LAC
It is possible that the endogenous conjugate profiles evident in
cell extracts from inhibitor-treated cultures were not the direct
result of proteasome inhibition but, rather, represent the profile of a
delayed culture relative to that of a normally progressing culture.
However, if this were the case, we would expect the levels of
conjugates extracted at 96 h from LAC-treated cells to appear more
similar to those from 72-h control cells, thereby reflecting the
approximately 24-h delay observed at both the morphological (Table I)
and biochemical (Fig. 3) levels. Instead, conjugate stabilization in
LAC-treated cultures clearly persisted through 96 h.
The Role of Cys Proteases in TE Autolysis
In contrast to LAC treatment, LLL treatment resulted in the
partial uncoupling of autolysis from differentiation of what otherwise appeared to be normal TEs (Fig. 2). We have presented evidence that LLL
but not LAC inhibits TE-associated Cys proteases (Fig. 6), suggesting
that the apparent prohibition of TE autolysis in LLL-treated cultures
is due to inhibition of the activity of the 28- and 24-kD Cys proteases
or other LLL-sensitive proteases not detected by our activity gels and
not caused by inhibition of the proteasome.
Hoechst 33342 staining of cells following LLL treatment demonstrated
that the cellular material retained by 85% of the dead TEs in these
cultures does not include nDNA, indicating that nuclear integrity is no
longer maintained in TEs that exhibit incomplete autolysis. This
absence of DNA is consistent with the proposal that endonucleases
expressed during TE differentiation function to degrade DNA and RNA
during autolysis (Thelen and Northcote, 1989) independently of Cys
proteases. Although tonoplast rupture is known to be associated with
autolysis of intracellular components in developing TEs (Groover et
al., 1997), the results presented here represent the first
demonstration to our knowledge that application of a Cys protease
inhibitor prevents the complete autolysis of zinnia TEs.
Additionally, although the activity of the ubiquitin-proteasome pathway
has been previously implicated as a necessary component of vascular
differentiation (Bachmair et al., 1990; Stephenson et al., 1996), to
our knowledge this is the first demonstration that a specific inhibitor
of the proteasome can reversibly prevent TE differentiation and that
proteasome function is also required for regulating the time course of
TE differentiation in zinnia mesophyll cell cultures. The proteasome,
however, does not appear to participate directly in TE autolysis.
As used in this study, LLL and LAC were not able to uncouple the
differentiation process from cell death. If TE death is dependent on
the proteasome or on LLL-sensitive proteases, perhaps the window of
opportunity to uncouple death from TE differentiation is narrow, occurring at a time not tested in this study. Although it is possible that proteases do not play an important role in the regulation of plant
PCD, further investigation using inhibitors against proteases other than the proteasome and Cys proteases may lead to the
identification of proteases specifically involved in the regulation of
cell death during TE differentiation.
| |
FOOTNOTES |
1
This research was supported by National Science
Foundation grant no. MCB 9418377.
2
Present address: DEKALB Genetics Corporation, 62 Maritime Drive, Mystic, CT 06355-1958.
*
Corresponding author; e-mail ebeers{at}vt.edu; fax
1-540-231-3083.
Received June 23, 1998;
accepted July 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
LAC, clasto-lactacystin
-lactone.
LLL, carbobenzoxy-leucinyl-leucinyl-leucinal.
LLM, acetyl-leucinyl-leucinyl-methional.
LLnL, acetyl-leucinyl-leucinyl-norleucinal.
PCD, programmed cell death.
TE, tracheary element.
| |
ACKNOWLEDGMENTS |
We thank Dr. Zheng-Hua Ye for kindly providing p48h-17, Dr.
Larry Dick for helpful discussions concerning the use of LAC, and Dr.
James Westwood for critical reading of the manuscript.
| |
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Top
Abstract
Introduction