The Plant Cell, Vol. 2, 795-803, August 1990 O 1990 American Society of Plant Physiologists
Light-Mediated Control of Translational lnitiation of
Ribulose-1,5=BisphosphateCarboxylase in Amaranth
Cotyledons
J a m e s O. Berry,"Tb D a v i d E. Breiding," and Daniel F. Klessig".'
aWaksman Institute, Rutgers, The State University of New Jersey, P.O. Box 759, Piscataway, New Jersey 08855
bDepartmentof Biological Sciences, State University of New York, Buffalo, New York 14260
In cotyledons of 6-day-old amaranth seedlings, the large subunit (LSU) and the small subunit (SSU) polypeptides of
ribulose-l,5-bisphosphate carboxylase are not synthesized in the absence of light. When dark-grown seedlings
were transferred into light, synthesis of both polypeptides was induced within the first 3 to 5 hr of illumination
without any significant changes in levels of their mRNAs. In cotyledons of light-grown seedlings and of dark-grown
seedlings transferred into light for 5 hr (where ribulose-1,5-bisphosphatecarboxylase synthesis was readily detected
in vivo), the LSU and SSU mRNAs were associated with polysomes. In cotyledons of dark-grown seedlings, these
two mRNAs were not found on polysomes. In contrast to the SSU message, mRNAs encoding the nonlight-regulated,
nuclear-encoded proteins actin and ubiquitin were associated with polysomes regardless of the light conditions.
Similarly, mRNA from at least one chloroplast-encoded gene (rp12) was found on polysomes in the dark as well as
in the light. These results indicate an absence of translational initiation in cotyledons of dark-grown seedlings which
is specific to a subset of nuclear- and chloroplast-encoded genes including the SSU and LSU, respectively. Upon
illumination, synthesis of both polypeptides, and possibly other proteins involved in light-mediated chloroplast
development, was induced at the level of translational initiation.
INTRODUCTION
In higher plants, light serves both as a primary energy
source and as an environmental stimulus which regulates
development. In particular, the development of photosynthetically active chloroplasts from proplastids or etioplasts
is light dependent. Many, but not all, dark-grown plants
lack thylakoid organization, chlorophyll, and several of the
polypeptides associated with photophosphorylation and
C02 fixation. lllumination of these dark-grown plants
causes the accumulation of chlorophyll and induces the
development of thylakoid components into photosynthetically functional units. Light also induces changes in the
synthesis of a number of nuclear- and plastid-encoded
proteins, including in some species ribulose 1,5-bisphosphate carboxylase (RuBPCase) (Apel, 1979; Schroder et
al., 1979; Santel and Apel, 1981; Sims and Hague, 1981;
Smith and Ellis, 1981; Tobin and Silverthorne, 1985; Berry
et al., 1985).
RuBPCase is found in the chloroplasts of all higher
plants and is a principle enzyme in photosynthetic carbon
fixation. This enzyme is composed of eight large (51 to 58
kD) and eight small (12 to 18 kD) subunits. The large
subunit (LSU) is encoded on the chloroplast genome and
translated on 70s chloroplast ribosomes (Coen et al.,
' To whom correspondence should be addressed.
1977; Ellis, 1981), while the small subunit (SSU) is nuclear
encoded and translated on free cytoplasmic ribosomes as
a 20-kD precursor (Cashmore et al., 1978; Chua and
Schmidt, 1978; Highfield and Ellis, 1978). The precursor is
processed to its final size during transpor? into the chloroplast, where it assembles with LSUs to form the active
holoenzyme.
The control of RuBPCase production and activity in a
number of plant species is very complex with regulation
occurring at many levels. Control at the level of LSU or
SSU mRNA accumulation has been well documented (for
review, see Tobin and Silverthorne, 1985). In several cases
alterations in transcriptional activity have been shown to
be responsible for changes in SSU mRNA levels (Tobin
and Silverthorne, 1985; Fluhr et al., 1986). At the other
end of the spectrum, regulation at the post-translational
level via turnover of the protein (Mishkind and Schmidt,
1983) as well as by activation (Miziorko and Lorimer, 1983;
Portis et al., 1986) or inhibition (Gutteridge et al., 1986) of
the enzyme's activity has also been reported.
We have previously described the effects of environmental (light) (Berry et al., 1985, 1986, 1988) and developmental (Berry et al., 1985; Nikolau and Klessig, 1987)
signals on the expression of LSU and SSU genes in the
C,. dicotyledonous plant Amaranthus hypochondriacus.
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The Plant Cell
The expression of these genes is regulated not only at the
level of mRNA accumulation but also post-transcriptionally.
In amaranth cotyledons, rapid and dramatic alterations in
the synthesis of the LSU and SSU polypeptides occur in
response to changes in illumination without corresponding
changes in mRNA levels. Because the stability of these
polypeptides and the functionality in vitro of their respective mRNAs were not affected by alterations in illumination,
the expression of these genes appears to be regulated, in
part, at the translational level. Light-mediated translational
regulation of this enzyme has also been observed in other
plant species including Lemna (Slovin and Tobin, 1982),
Volvox (Kirk and Kirk, 1985), pea (Inamine et al., 1985),
and barley (Klein and Mullet, 1986; Klein et al., 1988;
Gamble et al., 1989). In addition, translation appears to
play a role in regulating LSU and SSU gene expression
during the latter stages of leaf development in amaranth
(Nikolau and Klessig, 1987).
We have recently shown that when cotyledons of lightgrown amaranth seedlings were transferred to total darkness, both LSU and SSU mRNAs remained bound to
polysomes but were not translated in vivo, suggesting that
under these conditions control was exercised, in part, at
the translational elongation step (Berry et al., 1988). In this
report, we show that the RuBPCase LSU and SSU mRNAs
are not associated with polysomes in dark-grown (etiolated) cotyledons but are rapidly recruited onto polysomes
upon illumination. In contrast, mRNAs encoding constitutive (nonlight-regulated) polypeptides are associated with
polysomes in cotyledons of both dark-grown and lightshifted seedlings. These results demonstrate a selective
absence of translational initiation (and therefore synthesis)
of both RuBPCase subunits when amaranth seedlings are
grown in the absence of light.
SSU polypeptides was dramatically enhanced during the
first 5 hr of illumination, the corresponding mRNA levels
increased only modestly (twofold to threefold) during this
time period (Figure 1 B). Therefore, the marked increase in
synthesis of the RuBPCase proteins was not accompanied
by similar changes in LSU and SSU mRNA levels.
Association of LSU and SSU mRNAs with Polysomes
The above results, as well as our previous studies (Berry
et al., 1986), indicated that the rapid induction of
RuBPCase synthesis, which occurs when dark-grown
B
O)
RESULTS
Effect of Light on LSU and SSU Gene Expression
The light-mediated stimulation of RuBPCase synthesis in
cotyledons was examined by illuminating 6-day-old, darkgrown amaranth seedlings. Similar to our previous studies
using 8-day-old seedlings (Berry et al., 1986), synthesis of
the LSU and SSU in 6-day-old seedlings increased from
nearly undetectable amounts in the dark to levels similar
to that observed in light-grown seedlings within 3 to 5 hr
post-illumination, as shown in Figure 1 A. While total protein
synthesis was enhanced twofold to fivefold by 5 hr postillumination (data not shown), LSU and SSU synthesis was
specifically increased 20-fold or more above general protein synthesis during light induction.
Figure 1B shows that levels of the LSU and SSU mRNAs
were lower in cotyledons grown in darkness than those
maintained in the light. Although synthesis of the LSU and
Figure 1. RuBPCase Synthesis and mRNA Accumulation in Response to Light.
(A) LSU (L) and SSU (S) polypeptide synthesis. Amaranth seedlings were germinated and grown either in complete darkness or
under illumination. Six days after planting, part of the dark-grown
seedlings (D+5hL) were exposed to light for 5 hr. Excised cotyledons were incubated with [35S]methionine for 2 hr. LSU and
SSU were immunoprecipitated from extracts containing equal
amounts of radioactivity incorporated into protein, fractionated by
SDS-PAGE, and fluorographed.
(B) LSU and SSU mRNA accumulation. Total RNA was extracted
in parallel from 6-day-old, light- and dark-grown cotyledons, and
from dark-grown cotyledons exposed to light for 5 hr. Samples (5
^g per lane) of total RNA were fractionated on an agaroseformaldehyde gel, transferred to nitrocellulose, and probed with
32
P-labeled LSU (pAlst) and SSU (pAss!) clones.
Translational Regulation of RuBPCase
seedlings are transferred to light, may be due to regulation
at the translational level. To confirm this, changes in the
association of these mRNAs with polysomes during light
induction were examined. Figure 2A shows 10% to 40%
sucrose gradient profiles of polysomes obtained from cotyledons of light-grown and dark-grown seedlings and from
dark-grown seedlings transferred to light for 5 hr. Comparison of polysome profiles indicates that a smaller proportion of the ribosomes are engaged in translation in
dark-grown versus light-grown seedlings. When the etiolated seedlings were transferred to light, an increasing
proportion of these ribosomes shifted from the monosome
regions to the polysome regions of the gradient and presumably began actively synthesizing proteins. These moderate changes in the polysome profiles were consistent
with the differences in rates of general protein synthesis
observed under the various illumination conditions.
Comparison of these three polysome profiles suggests
that there may be differences in the populations of polysome-associated mRNAs in seedlings subjected to the
various illumination conditions. To test this, the sucrose
gradient profiles were analyzed by dot blotting using specific probes (Figure 2B). Both LSU and SSU mRNAs were
associated with polysome regions of the sucrose gradient
(fractions 1 to 4, Figure 2B) from light-grown seedlings.
However, with polysomes isolated from dark-grown seedlings (when little, if any, RuBPCase synthesis was detected), only small amounts of these two transcripts were
detected in the polysome fractions. Within 5 hr of transfer
into light (after RuBPCase synthesis was initiated), dramatic increases in the levels of both mRNAs were found
in the polysome fractions.
One interpretation of these results is that the LSU and
SSU mRNAs from cotyledons of dark-grown seedlings
were associated with very large polysomes. This might
occur if translational elongation/termination was inhibited
in the dark so that ribosomes continued to accumulate on
the mRNA. Under the sedimentation conditions used here,
the very large polysomes might be pelleted at the bottom
of the gradient and thus escape detection. This was unlikely because the gradients contained a dense 1.75 M
sucrose cushion at the bottom to prevent pelleting. To
further rule out this possibility, polysome extracts were
also sedimented through denser 15% to 60% sucrose
gradients. Under these conditions, again only small
amounts of LSU and SSU mRNA were found in the heavy
portion of the gradient (data not shown), thus confirming
that very few of the RuBPCase mRNAs were associated
with polysomes in cotyledons of dark-grown seedlings.
Very few LSU or SSU mRNAs were detected in any
region of the gradient in extracts from cotyledons of darkgrown seedlings compared with those from light-shifted
seedlings (Figure 2B). This was unexpected because only
very modest changes in the levels of these two mRNAs
were observed by RNA gel blot analysis (Figure 1B). A
likely explanation for this discrepancy is the manner of
797
Light
Dork
Dark+5h Light
1
2 3 4 5
6 7 8 9
B
L
D
LSU
D+5HL
1
2 3 4 5 6 7 8 9
L"
D
SSU
D+ShL
Figure 2. Changes in Association of LSU and SSU mRNAs with
Polysomes in Response to Light.
(A) Sucrose gradient profiles of polysome preparations isolated
from 6-day-old, light- and dark-grown cotyledons and from darkgrown cotyledons transferred to light for 5 hr.
(B) Dot blot analysis of fractions from sucrose gradient profiles.
Polysomes were prepared from seedlings grown under the illumination conditions indicated. Three /4260 units were loaded onto
each gradient. Sedimentation is from right to left. Polysomes
(cytoplasmic and chloroplastic) are located primarily in fractions 1
to 4; monosomes and ribosomal subunits are in fractions 6 and
7. Distribution of the LSU and SSU mRNAs was determined by
dot blot analysis using pAlsl and pAssI as probes.
798
The Plant Cell
preparation of the crude poly some fraction. Poly some
isolation from plant tissue involves centrifugation of the
initial crude extract through a dense sucrose cushion
(Leaver and Dyer, 1974; Larkins and Davies, 1975; Jackson and Larkins, 1976). This serves to concentrate the
poly somes, monosomes, and free mRNPs and to separate
them from debris which can interfere with their subsequent
analysis. A major problem with this sedimentation procedure is that it tends to enrich for polysomes, while leaving
monosomes and free mRNPs underrepresented in the
pellet (Leaver and Dyer, 1974; Larkins and Davies, 1975).
Presumably, the levels of LSU or SSU mRNA in the profiles
for dark-grown cotyledons were underrepresented because the majority of the mRNA was not associated with
polysomes and therefore sedimented through the sucrose
cushion less rapidly and efficiently than the denser, polysome-associated messages. In fact, when polysomes
were purified using a shorter sedimentation time (3 hr as
opposed to the 18 hr used for Figure 2, data not shown),
even lower amounts of the nonpolysome-associated LSU
and SSU transcripts were recovered from the dark-grown
seedlings. Therefore, although recovery of the LSU and
SSU mRNAs in crude polysome pellets from dark-grown
seedlings was not complete, analysis of the crude polysome preparations confirmed that very few of the LSU and
SSU transcripts were associated with polysomes in darkness but upon illumination were recruited onto polysomes.
These results suggest that light-induced LSU and SSU
synthesis in etiolated seedlings is regulated, in part, at the
level of translational initiation.
RNA
L D D+5hL
L
D D+5hl_
I I
I
B
Translational Runoff Analysis of Polysomes In Vitro
The association of LSU and SSU mRNAs with polysomes
was further examined by in vitro polysome runoff analysis.
Crude polysomes were added to either an Escherichia coli
(for LSU) or rabbit reticulocyte (for SSU) cell-free translation
system containing aurin tricarboxylic acid (ATA) or edeine/
7-methylguanosine monophosphate, respectively, to inhibit initiation. As previously described (Berry et al., 1988),
these inhibitors had little or no effect on polysome-directed
protein synthesis but reduced RNA-directed synthesis by
>90% (Figures 3A and 3B), indicating that the drugs were
functioning as specific inhibitors of initiation.
Both LSU and SSU polypeptides were produced in vitro
when polysomes from either cotyledons of light-grown or
light-shifted seedlings were used to program cell-free
translation extracts in the absence (-) or presence (+) of
the initiation inhibitors (Figures 3A and 3B). In contrast,
polysomes isolated from cotyledons of dark-grown seedlings did not direct the synthesis of detectable amounts of
either subunit in runoff translation (Figures 3A and 3B).
These results confirm that in the light, when LSU and SSU
synthesis was readily detected, both RuBPCase transcripts were associated with translationally active polysomes. In dark-grown seedlings, where synthesis of these
proteins was not detected, the LSU and SSU mRNAs were
-SSU-
Figure 3. Translational Runoff of Polysomes in Vitro.
(A) Translational runoff of chloroplast polysomes. Polysomes
isolated from light-grown seedlings (L), dark-grown seedlings (D),
or dark-grown seedlings shifted to light for 5 hr (D+5hL) were
incubated in an Escherichia coli cell-free translation system in the
absence (-) or presence (+) of the initiation inhibitor ATA. In the
controls shown at left, the extracts were programmed with total
amaranth RNA in the absence or presence of the inhibitor. LSU
was immunoprecipitated from equal aliquots and analyzed by
SDS-PAGE.
(B) Translational runoff of cytoplasmic polysomes. Polysomes
were added to a rabbit reticulocyte cell-free system in the absence
(-) or presence (+) of the initiation inhibitors edeine/m7GMP. In
the controls shown at left, the extracts were programmed with
amaranth polyadenylated RNA in the absence or presence of the
inhibitors. SSU precursor was immunoprecipitated from equal
aliquots and analyzed by SDS-PAGE.
Translational Regulation of RuBPCase
not associated with polysomes and therefore were not
translated.
Specificity of Initiation on Cytoplasmic and
Chloroplastic Ribosomes
Is the lack of polysome association and active translation
of the LSU and SSU mRNAs simply a reflection of the
reduction of polysome number and rate of general protein
synthesis in the dark? The greater severity of repression
of their translation in comparison with general protein
synthesis (20-fold or more) suggests that this is not the
case. However, to directly test this, the expression of
several nuclear or chloroplast encoded genes, whose
expression was unlikely to be light regulated, was examined. As controls for cytoplasmic protein synthesis, the
translation and polysome association of mRNAs for actin,
a cytoskeletal protein (DeRosier and Tilney, 1984), and
ubiquitin, a protein involved in the degradation of other
polypeptides (Ciechanover et al., 1984), were analyzed. In
contrast to the SSU, the levels of actin mRNA (Figure 4B)
and its rate of translation (Figure 4A) were similar in the
absence or presence of light. In addition, actin mRNA was
associated with polysomes isolated from cotyledons of
dark-grown seedlings as well as those from light-grown
and light-shifted seedlings (Figure 4C). Like actin, ubiquitin
mRNA was associated with polysomes at similar levels in
darkness as well as in light (data not shown). Thus, the
absence of translational initiation on cytoplasmic ribosomes is not general but is specific to light-regulated genes
such as those encoding the SSU.
To determine the specificity of LSU initiation on chloroplastic ribosomes, the distribution on polysomes of another
chloroplast-encoded mRNA was examined. The mRNA for
the ribosomal protein L2 (which is encoded by the chloroplast gene rp/2) was present in cotyledons of dark-grown,
light-grown, and light-shifted seedlings (Figure 5A). Although similar levels of L2 mRNA were found in darkgrown plants before and after transferring into light, this
mRNA was less abundant (about 10-fold) in light-grown
seedlings.
Similar levels of L2 mRNA were associated with polysomes under all three illumination conditions (Figure 5B).
Because lower levels of this mRNA were present in the
light-grown cotyledons, a higher proportion of the L2 transcripts was bound to polysomes in these seedlings than
in dark-grown or light-shifted seedlings (compare Figures
5A and 5B). However, in contrast to the LSU transcripts,
similar levels of L2 mRNA were found on polysomes in
dark-grown cotyledons before and after transfer into light.
Therefore, the lack of polysome association observed for
the LSU transcripts in dark-grown seedlings is not characteristic of this Chloroplastic mRNA.
DISCUSSION
We have previously shown that whereas RuBPCase synthesis occurs continuously in cotyledons of light-grown
799
amaranth seedlings, in dark-grown (etiolated) seedlings
LSU and SSU synthesis occurs only as a burst between 2
and 5 days after planting (Berry et al., 1985). Although no
RuBPCase synthesis is observed after day 5 in the dark,
mRNAs for both subunits are maintained through day 7.
The results presented here indicate that in 6-day-old, darkgrown amaranth cotyledons, where RuBPCase synthesis
is not observed, the majority of the RuBPCase mRNAs
were not associated with polysomes. When these dark-
B
*Q<u
I.2? ro- "
+
-l
Q
Q
+
Q
t
1 2 3 4 5 6 7 8 9
L
D
D+5HL
Figure 4. Actin Polypeptide Synthesis, mRNA Accumulation, and
mRNA Association with Polysomes in Response to Light.
(A) Actin polypeptide synthesis. Amaranth cotyledons were grown
and labeled as described for Figure 1A. Actin was immunoprecipitated from extracts containing equal amounts of radioactivity
incorporated into protein, fractionated by SDS-PAGE, and fluorographed. As a marker, actin was immunoprecipitated from
extracts of total protein from cultured monkey kidney (CV1) cells
which were labeled for 2 hr with 35S-methionine.
(B) Actin mRNA accumulation. Total RNA was subjected to RNA
gel blot analysis as described for Figure 1B using a cloned actin
gene (pSAc3) as a probe.
(C) Dot blot analysis of actin mRNAs associated with polysomes.
Polysome preparation and analysis was the same as described in
Figures 2A and 2B, except that crude polysome extracts were
purified by 3 hr of sedimentation through a sucrose cushion
instead of the 18 hr used for Figure 2. We have found that this
shorter sedimentation is adequate for the recovery and analysis
of polysome-associated mRNAs, while the longer sedimentation
is useful for the isolation and identification of both polysome- and
nonpolysome-associated messages (Berry et al., 1988). Polysomes purified by both methods show similar distributions of
polysome-associated mRNA. Distribution of actin mRNAs was
determined using pSAcS as a probe.
800
The Plant Cell
.C
D)
05
Q
Q
B
1
2
3
4
5
6
7
8
9
L
D
D+5hL
Figure 5. Accumulation and Polysome Association of Ribosomal
Protein L2 (rp!2) mRNA in Light- and Dark-Grown Cotyledons.
(A) Accumulation of L2 mRNA. Total RNA from light- and dark-
grown cotyledons was subjected to RNA gel blot analysis as
described for Figure 1B using ^P-labeled L2 (pUC8c1) clone as a
probe.
(B) Dot blot analysis of L2 mRNAs associated with polysomes.
Polysome preparation and analysis are the same as those shown
in Figure 4. Distribution of mRNAs was determined using L2
(pUC8c1) clone as a probe.
grown seedlings were transferred into light (light-shifted),
both the LSU and SSU mRNAs were rapidly mobilized
onto polysomes and synthesis of the two polypeptides
was initiated.
Cotyledons from dark-grown seedlings exhibited a lower
polysome/monosome ratio than either light-grown or lightshifted seedlings. This suggests that in addition to the
RuBPCase messages, the association of a number of other
mRNAs with polysomes is affected by light. Increased
polysome/monosome ratios in response to red light illumination has been reported for a number of plant species
(Yamamoto et al., 1975; Smith, 1976; Fourcroy et al.,
1979). In mustard seedlings, this shift is due to the increased synthesis of new mRNAs (Mosinger and Schopfer,
1983), while in bean leaves, there is evidence for the lightinduced mobilization of stored mRNA onto polysomes
(Giles et al., 1977). In amaranth cotyledons, LSU and SSU
mRNA levels increased only slightly following illumination,
suggesting that light induced translational initiation on
mRNAs that were already present in the dark. With regard
to the LSU, these results are consistent with recent studies
which indicate that post-transcriptional mechanisms play
important roles in regulation of plastid gene expression
(Deng and Gruissem, 1987; Mullet and Klein, 1987).
At least two nuclear-encoded mRNAs were associated
with polysomes in dark-grown seedlings when the SSU
transcripts were not found on polysomes. Actin and
ubiquitin mRNAs, both of which are translated on cytoplasmic ribosomes, were on polysomes in both dark- and
light-grown seedlings. Furthermore, actin was translated
at similar levels in darkness as well as in light. Similarly,
the mRNAs encoding the plastid ribosomal protein L2 were
associated with chloroplast polysomes under all three
illumination conditions. While it is interesting that the L2
transcripts levels were lower in light-grown than in either
dark-grown or light-shifted seedlings, the important point
is that these chloroplast-encoded mRNAs were polysome
associated in the dark when the LSU transcripts were not.
Therefore, the absence of translational initiation on both
cytoplasmic and chloroplastic ribosomes is not a general
occurrence in the dark but is selective for the RuBPCase
transcripts, and possibly other mRNAs involved in lightregulated chloroplast development as well.
Although the chloroplast-encoded L2 mRNA was polysome associated in the dark, we do not know whether it
was actually being translated. Klein et al. (1988) found that
chloroplast-encoded mRNAs for psaA-psaB, psbA, and
LSU were associated with polysomes in young (4.5 days
old), dark-grown barley seedlings. In these barley seedlings, the LSU was synthesized in the dark; however, the
synthesis of the psaA-psaB and psbA polypeptides was
either arrested in translation elongation or the newly made
proteins were rapidly degraded. Similarly, we have found
that when light-grown amaranth seedlings are transferred
to darkness, LSU synthesis is depressed without any
corresponding changes in the polysome association of the
LSU mRNAs (Berry et al., 1988). Both of these studies
indicate that chloroplastic protein synthesis can be regulated at the level of translational elongation as well as
initiation, and this may also be the case for L2. Further
characterization of L2 protein synthesis awaits the production of specific antisera.
The distribution on polysomes of the light-regulated and
nonlight-regulated transcripts described here for the differ-
Translational Regulation of RuBPCase
ent light conditions may not be representative of all the
mRNAs in the cotyledons. It is very likely that there are
mRNAs which are intermediate in their degree of association with polysomes. In fact, we have found that two
chloroplast mRNAs, those encoding subunit IV of cytochrome b6f(petD) and the p subunit of chloroplast ATPase
(atpB), were associated with polysomes in dark-grown
cotyledons to a lesser extent than the ribosomal protein
L2 mRNA but to a greater degree than LSU mRNA (data
not shown).
The results presented here demonstrate that during
light-mediated chloroplast development in amaranth the
synthesis of both RuBPCase subunits is rapidly induced
at the level of translational initiation. This induction occurs
coordinately on both cytoplasmic and chloroplastic ribosomes. Similar observations of activation of chloroplastic
translational initiation have recently been reported by Gamble et al. (1989) using 8-day-old, dark-grown barley seedlings. When the dark-grown barley seedlings were illuminated, an overall increase in translational initiation occurred
for the rbcL, psbA, and psaA-psaB gene products. While
Kraus and Spremulli (1986) have demonstrated that the
level of a chloroplast translation initiation factor is enhanced by light, it is not known if or how this factor might
specifically control the translation of light-regulated chloroplastic mRNAs. Alternatively, the difference in polysome
association for the various chloroplastic RNAs may simply
reflect differences in ribosome binding affinities of the
mRNAs (Lodish, 1974). These differences may become
particularly apparent if ribosomes or translation factors are
limiting in the absence of light. For example, if LSU mRNA
has a weak ribosome binding site, discrimination against
it could be relieved as the ribosomes or factors become
less limiting in the presence of light.
Interestingly, the translation of LSU and SSU transcripts
appears t o be regulated by different mechanisms in plants
exposed to different light regimes. During a light-to-dark
transition, the rapid shutdown of LSU and SSU synthesis
appears to be due to regulation of translational elongation,
while during a dark-to-light transition, synthesis of both
RuBPCase subunits appears to be induced at the level of
translational initiation. Translation appears to be only one
level at which LSU and SSU gene expression is controlled
in amaranth. Changes in mRNA levels, due to altered
transcription or mRNA stability, also occur and appear to
be responsible for long-term changes in expression of the
RuBPCase genes (Berry et al., 1986). Furthermore,
changes in the synthesis of the chloroplast-encoded LSU
and the nuclear-encoded SSU occur in parallel, being
coordinately induced by light and depressed by darkness.
Therefore, an extensive pattern of communication must
exist between the biosynthetic machinery of the nucleus,
cytoplasm, and chloroplast which is used to coordinately
regulate the expression of these two genes. Clearly, much
more work is needed to define the intricate pattern of
regulation for this essential gene system.
801
METHODS
Plant Material and Growth Conditions
Amaranthus hypochondriacus var RI 03 was grown as described
previously (Berry et al., 1985).For the light induction experiments,
seeds were germinated and plants were grown in lightproof boxes
which were placed in a darkroom. Extreme care was taken to
avoid any exposure of the dark-grown seedlings to light. After 6
days, these dark-grown seedlings were transferred into an illuminated growth chamber, and cotyledons were harvested at the
appropriate times. Cotyledons from dark-grown seedlings were
harvested under a Kodak No. 7 green safelight.
Analysis of Protein Synthesis
Rates of in vivo protein synthesis were determined by radioactive
labeling with 35S-methionine.
The procedures for labeling, extraction of proteins, immunoprecipitation of LSU and SSU polypeptides, and analysis by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)were described previously (Berry et
al., 1985). Actin was immunoprecipitated with antisera kindly
provided by James Lessard (Childrens Hospital, Medical Center,
Cincinnati, OH) according to the method of Lin (1981).
Probes Used in RNA Gel Blot and Dot Blot Analyses
LSU and SSU mRNAs were detected by using 32P-labeledcloned
amaranth LSU (pAlsl) or SSU (pAssl) genes as probes (Berry et
al., 1985). The clones for actin (pSAc3) and for ubiquitin (pXlg20)
were generously provided by Richard Meagher (University of
Georgia, Athens, GA) and Mark Dworkin (Columbia University,
New York, NY), respectively. Clones for the chloroplast genes
rpsl6 (pXWD14), rp12 (pUC8cl), and petD (pXWD9) were the gift
of Wilhelm Gruissem and David Stern. The probe for the p subunit
of chloroplast ATPase (atbB) was kindly provided by Nam-Hai
Chua.
Analysis of mRNA
Total RNA was isolated from cotyledons as previously described
(Berry et al., 1985). Five micrograms per lane of total RNA was
fractionated on agarose-formaldehyde gels and transferred to
nitrocellulose paper for RNA gel blot analysis by the method of
Maniatis et al. (1982). Hybridization conditions for the actin and
ubiquitin probes were as described by Klessig and Berry (1983),
while those for the other probes were according to Berry et al.
(1985).
Analysis of Polysomes
Polysomes were isolated as previously described (Berry et al.,
1988). Crude polysomes were purified from initial extracts by
pelleting through a 1.75 M sucrose cushion (Jackson and Larkins,
1976) at 60,000 rpm using a Ti80 rotor (Beckman) for either 3 hr
or 18 hr. The crude polysome preparations were resuspended,
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The Plant Cell
clarified by centrifugation for 1 min in a microfuge at 4OC to
remove any remaining debris, and stored at -7OOC. Three A260
units of the purified polysome preparation were loaded onto a
10% to 40% sucrose gradient [containing 40 mM Tris-HCI (pH
8.4) 20 mM KCI, 10 mM MgCl,] which was overlayed on top of a
1.75 M sucrose cushion (0.5 mL). Polysomes were also analyzed
using denser 15% to 60% sucrose gradients. The gradients were
centrifuged at 39,000 rpm in an SW41 rotor (Beckman) for 150
min at 4OC. The gradient profiles were determined by using a
Perkin-Elmer Lambda 3A spectrophotometer with a flow cell.
Gradient fractions were collected and immediatelydiluted with an
equal volume of 50 mM Tris-HCI (pH 8.0), 50mM EDTA, 1% SDS,
then phenolextracted and ethanol precipitated.The RNA fractions
were characterized by dot blot analysis (Bethesda Research
Laboratories, Hybri-Dot manifold) on nitrocellulose or by RNA gel
blot analysis using the previously described clones as probes.
Hybridization conditions were the same as described above for
RNA gel blot analysis.
Translational runoff was performed as described previously
(Berry et al., 1988)except that the total crude polysomes obtained
after 3 hr sedimentation through a sucrose cushion were used to
program the cell-free lysates. Five micrograms or 1O pg of polysomes were incubated in nuclease-treated rabbit reticulocyte
lysates (Pelham and Jackson, 1976) (Green Hectares, Oregon,
WI) or cell-freeextracts of Escherichiacoli (Zubay, 1973), respectively. Reticulocyte lysates (50 pL final volume), containing 25 pg/
mL chloramphenicol to suppress translation by chloroplastic
polysomes, were supplemented with 200 pM 7-methylguanosine
monophosphate (m’GMP) (Darzynkiewicz et al., 1987) and 1O p M
edeine (Kozak and Shatkin, 1978) to block translational initiation,
or with an equivalent volume of water (controls).Similarly, E. coli
lysates (30 pL final volume), containing 50 pg/mL cycloheximide
to block translation by cytoplasmic polysomes, were supplemented with the initiation inhibitor aurin tricarboxylic acid (ATA,
50 pM) (Grollman and Stewart, 1968) or an equivalent volume of
water. 35S-Methionine-labeledtranslation products were analyzed
by SDS-PAGE with or without prior immunoprecipitation.All drugs
were obtained from Sigma except for edeine, which was a generous gift from Dr. P. Walter.
ACKNOWLEDGMENTS
We are grateful to Richard Meagher, Mark Dworkin, Wilhelm
Gruissem,David Stern, and James Lessardfor their gifts of clones
or antisera. This work was supported by Grant DCB-8903578
from the National Science Foundation to D.F.K. and by United
States Department of Agriculture Grant 89-37262-4893to J.O.B.
D.E.B. was supported in part by Public Health Service Fellowship
F32-GM11550 from the National lnstitutes of Health. D.F.K. is
recipient of Faculty Research Award 270 from the American
Cancer Society.
Received March 19, 1990; revised June 1, 1990.
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Light-Mediated Control of Translational Initiation of Ribulose-1,5-Bisphosphate Carboxylase in
Amaranth Cotyledons
J. O. Berry, D. E. Breiding and D. F. Klessig
PLANT CELL 1990;2;795-803
DOI: 10.1105/tpc.2.8.795
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