chapter i early protein synthesis during the...
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CHAPTER I
EARLY PROTEIN SYNTHESIS DURING THE GERMINATION OF
BARLEY EMBRYOS AND ITS RELATIONSHIP TO RNA SYNTHESIS
SUMMARY
In barley embryo, protein synthesis as judged from
the incorporation of labelled precursors, starts at about
15 min after the commencement of germination. Evidence
suggests that these early proteins are essential for
germination and are programmed by a conserved poly(A)
containing rnRNA preserved in dry embryos. Although low
DNA-dependent RNA polymerase activity is present in dry
barley embryos, RNA synthesis does not commence immediately
after water imbibition. On the other hand, it is initiated
only after 2 hr of germination and its synthesis requires
the essential presence of earJy proteins. Furthermore,
there is a progressive increase in the activity of RNA
polymerase with increase in germination time and after
48 hr of germination, the activity of RNA polymerase is
about 4-fold higher than in dry embryo. However, cyclo
heximide blocks completely the enhanced activity of RNA
polymerase, suggesting a role of early proteins in the
initiation of new RNA synthesis in this developmental
system.
INTRODUCTION
The mature seeds of most flowering plants have a
low moisture content and consequently their metabolic
activity is severely restricted. The development of the
plant which began with the fertilization of the ovum and
continued during seed form8tion is hence temporarily
curtailed. The period of inactivity can last for months,
years or even decades, depending on the species (15) and
continues until the seeds germinate. Growth of the embryo
is then resumed but its pattern of development changes.
During seed formation .tha embryo is produced and root,
stem and leaf primordia develop but usually no further
differentiation occurs. At the same time large quantities
of food reserves are brought in from the maternal parent
and stored in specialized organs of the seed. At the
beginning of germination, in contrast, the embryo expands
rapidly at the expense of the stored reserves, root, stems
and leaves develop so that the seedling becomes autotrophic
as quickly as possible.
The switch in the developmental pattern of the plant,
from seed formation to germination must be affected in
part by a change in gene expression. At some stage, the
genes coding for enzymes which degrade the nutrient reserves
must be derepressed; other genes, coding for storage proteins
and some, at least, of the enzymes which catalysed the
formation of reserves in seed development beame repressed.
In addition, enzymes required for general metabolism and
structural proteins of the protoplast must be replenished
continually during early germination. Is the DNA capable
of allowing transcription early in germination after a
long period of dehydration and inactivity ? Can additional
segments of DNA be depressed and new mRNA species not found
in the ripening seed be transcribed? Or do mRNA species
have to be made during seed formation and stored away so
that they can satisfy the demand for protein synthesis
during early germination? It was with these questions
in mind that the search began in the mid and late 1960s
for the presence of mRNA in seeds; this work is still
continuing. A large amount of evidence has accumulated
suggesting the presence of preformed mRNA in dry seeds.
If it can be demonstrated unequivocally that protein
synthesis begins in germinating seeds before RNA synthesis,
then the protein formed must have been synthesized by the
translation of long-lived RNA. Thus, in addition to
providing proof that seeds contain mRNA, such evidence
would also show that it is actually used in early germination.
Evidence that protein synthesis precedes RNA synthesis
in bean, peas and rice has been p~ovided by Walbot, 1971 (24);
Sielwanowicz and Chmielewoka, 1973, {25) and Bhat and
Padayatty 1974, {26); respectively. In all the three
cases, the presence and participation of long lived mRNA
has been indicated. Autoradiographic studies by Maher
chandani and Naylor, 1973 (27), show that protein synthesis
in aleurone tissue of wild oat grain also is initiated
before RNA synthesis. However, our knowledge of the
properties and functions of long lived mRNA is still very
poor, and we do not know whether or not it plays an
essential role in germination. Stored mRNA seems to
initiate protein synthesis as soon as the seed imbibes.
It has been suggested that some stored mRNA is immature
and requires.polyadenylation during early germination
before being translated (10). On the other hand, poly(A)
RNA has been isolated from a variety of dry seeds, indicating
that atleast part of stored mRNA is translatable (11-13).
Moreover, it has been reported that stored mRNA rapidly
disappears following soaking (14).
Considering these contradictory results, attempts
were made to establish the sequence of events during barley
embryo germination. In this chapter, we show that stable
poly{A)-mRNA is present in the dormant embryo and that the
protein synthesis which starts 15 min after the onset of
germination is absolutely necessary for new RNA synthesis
and for the germination of barley embryo.
RESULTS
Protein Synthesis during Germination:
The change from dormancy t6 germination of seeds
requires water and an appropriate temperature. Dehydration
initiates the transition from the dormant state to a
metabolically active state, in which synthesis of nucleic
acids and proteins are resumed, however, the sequence of
onset of synthesis is under debate. It has been shown by
many workers that during early germination protein synthesis
occurs in absence of concomitant RNA synthesis, however,
according to other reports RNA synthesis occurs simul-
taneously with protein synthesis.
In barley embryo, protein synthesis during the early
phase of germination was studied by measuring the rate of
incorporation of 3H-leucine into the embryos. As shown
in Fig. 1, there is progressive increase in the incorpora
tion of ~-leucine into protein during the first 5 hr of
germination. The lag period of 15 min in the initiation
of protein synthesis, was probably the time required for
the uptake of radioactive precursor into the cells as
observed in other systems. Cycloheximide, an inhibitor of
protein synthesis at a concentration of 20 pg/ml completely
stops the incorporation of ~-leucine into acid-precipitable
fraction. Commencement of protein synthesis almost immediately
3000
1000
500
Fig. 1:
o---o CON'TROL
~ACTINOMYCIN D. o---o CYCLOHEXIMIDE
• • CORDYCEPIN
1 2 3 4
GERMINATION (hrs)
5
B:ffe£:t oLrrr;:q~ and RNA..§.Y.Dthesi§ inhl.bitQ.ts on .1nco.roorat1on Q! 3H-leuci~. At the indicated times ten embtyos were ground, homogenized and precipitated with 10% TCA according to the procecbre described in Materials and Methods. The TCA-insoluble material was collected on Whatrnan GF/c fU ters and washed with 5o ml of 5% TGA. The filters were dried and the radioactivity of the fil tars was detennined in 10 ml of a toluene-based scintillation mixture in a Packard Tri -carb liquid-scintUlation spectrometer.
after imbibition of water, therefore, clearly indicates
the presence of conserved mRNA as well as an active
translational machinery in dry barley embryos. Further
more, actinomycin D (20 pg/ml) and cordycepin (20 pg/ml)
known inhibitors of RNA synthesis had no significant effect
on 3H-leucine incorporation (Fig. 1), suggesting that
protein synthesis during the early phase of germination
could occur in the absence of concomitant synthesis of
RNA. Table 1 shows that cordycepin preferentially blocks
polyadenylation and to some extent new mRNA synthesis.
However, actinomycin D inhibited new RNA synthesis but
was less effective to polyadenylation. Lack of any
appreciable effect on protein synthesis by cordycepin,
an inhibitor of sequential polyadenylation •of mRNA,
therefore, suggests that the stored mRNA of barley embryo
is already processed and active for translation.
Poly(A)sequences are known to be covalently linked
to most of the mRNAs of eukaryotic cells. As in the case
of wheat embryo (8) dormant barley embryo contains about
2% poly(A)-mRNA as determined by cellulose column chromato
graphy (Fig. 2). The fact that the RNA species isolated
is poly(A) rich has been confirmed by poly(U)-sepharose
chromatography and by poly(U)-filter binding techniques.
In vitro translation of this poly(A)-mRNA shows that it is
120
6 0
E" o s c
0 ID N ........ >-1-
lf)
z w 0 _. <C u 1-a.. 0
0 4
0 2
2 4 6 8 10 12 .14 16 18 20 22 24 26
FRACTIONS
Fig. 2: Isolation of PoJ.ri.j.) -mnNA f_rga_dly emb_cr.Q. RNA was extracted frcrn barley embryos ( 250 mg) and precipitated qy alcohol. The precipitate was centrifUged and then washed according to the procedure described in the text. RNA ( rv aJ o. D. Units), was then dissolved in o. 8 ml of l:uffer containing 10mM Tris-Hcl, pH '1.6 and 20·lnM HgC1 2 , incubated with Iliase ( 100 pg/ml) 1 for 30 min at 37°C; lt. 7 ml of extraction buffer and· o. 3 ml of 20% SDS was added to stop the reaction. RNA was then extracted, and precipitated qy alcohol, RNA was collected qy centrifugation, dissolved in buffer H and ·chromatographed on Signa Cell type 38 cellulose according to Materials and Hethods.
TABLE 1
EFFECT OF ACTINOMYCIN-D AND CORDYCEPIN ON POLYADENYLATION
AND mRNA SYNTHESIS DURING FIRST 6 HR OF GERMINATION
Poly(A)-mRNA
Control
a CPM/mg RNA
17,551
+Actinomycin D (20 ug/ml)
5,821
+Cordyce,Pin { 20 ugjml)
14,571
% of control
100
33
83
Poly{A) present in Poly(A)-mRNA
b CPM/mg RNA
2,507
742
487
% of control
12.7
Embryos (50 mg) were incubated with and without inhibitors
for 6 hr in the germination medium containing ~-adenine (20)UCi/
ml) and .RNA was·then extracted.
aAn aliquot of RNA was passed through poly{U)-filter to determine the content of labelled poly{A)-mRNA.
bAnother aliquot of RNA was first digested with RNase T1 {10 units/rnl) and RNaseA(5 pg/ml) for 30 min at 37°C
and then filtered through poly(U)-filter. The filters were dried and counted in a Packard scintillation counter.
biologically active (Fig, J), and therefore, it must be
utilized for protein synthesis during the early phase
of germination.
RNA Synthesis during Germination:
The synthesis of RNA started after a lag period of
about 2 hr (Fig, 4). Using~ -amanitin 5 pg/ml, an
inhibitor of mRNA synthesis, incorporation of ~-uridine
into mRNA ( o< -amani tin sensitive RNA) was detectable after
8 hr of germination and the percentage of o< -amani tin
sensitive RNA synthesis to the total RNA synthesis is
about 24% at the 16 hr (Fig. 4). However, by using high
concentration of JH-adenosine (20 pCi/ml, specific activity,
22 Ci/mmole), we could detect the synthesis of new mRNA
during the first 6 hr of germination (Table 1). Furthermore,
a significant part of the radioactivity incorporated in
the period of 12 to 16 hr was infact in mRNA was confirmed
by the ability to bind ~o poly(U)-filters (Fig. 4, inset).
RNA synthesis during the period of 2 to 8 hr after the
commencement of germination was almost insensitive to
~-amanitin, a characteristic of ribosomal RNA synthesis.
This was further corroborated by the sucrose gradient
analysis of RNA isolated from ribosomes (data not shown).
From these results, it appears that protein synthesis
between 0 to 8 hr is mainly due to the presence of stored
mRNA (early phase of germination) whereas after 8 hr probably
-M IO ~
X
:E a.. u -z 0 h. <{ a::: 0 0.. 0::: 0 u z UJ z u ::> w ....J -u
-.:r --
20
8
o--o +mRNA tr--6. -mRNA . .
15' 30 45 60 MINUTES
Rl
Fig. 3·: In vitro protein synthesis of Poly( A) -mRNA in ·vheat genn extracts.. Poly( A) -mRNA was isolated according to the procedUre described in the legend to figure 2.
-
(tl) r) (._,
0~~~==1---~--~--~--~--~ 0 4 8 12 16 20
GERMINATION (hr.)
Fig. 4: j[fec~of~~anitin on 3H-uridine incorporation into .BNA dJ.ring gennina!ion of ~rley ~br.v:os. J:i,or each point ten embryos were ground, hQnogenized and precipitated with 10% TCA. ~ -Ananitin was added at zero hr along with 3H-uridine, o<'-a.rnanitin sensitive fraction was calculated by subtracting the resistant values from the total counts. (-o-o- ) o< -amanitin resistant; ( .. -....) o<. -amauitin sensitive. Figure 4 inset: Incor-oora tion of JH-uridine in Poly( A) -mRNA during gennination. For each point, 250 mg embryos were used. At the indicated times, embryos were homogenized, RNA was extracted and content of Poly( A)mRNA was detenn ined by Poly( U)- fil tar technique.
both stored and newly synthesized mRNAs are utilized
for translation and this period, therefore, can be
denoted as the "late phase of germination".
An interesting observation was that ~-uridine
incorporation was reduced markedly by cycloheximide,
at a concentration of 20pg/ml (Fig. 5). To examine
further the role of early proteins which could be
critical to RNA synthesis, cycloheximide was added to
the germination medium and incubated for 6 hr. The
cycloheximide was then washed out and the incorporation
of 3H-uridine into RNA was followed. As shown in Fig. 6,
cycloheximide completely inhibited RNA synthesis when
added during the first 6 hr of germination. All these
results suggest that translation of the stored mRNA is
indispensable for new RNA synthesis.
Recently, RNA polymerases have been demonstrated
in dry embryos of wheat (134) and rye (135). We have
confirmed the presence of RNA polymerase in dry barley
embryo and found that the activity of RNA polymerase
increases progressively as the germination time increases
and after 48 hr of germination, the activity of RNA
polymerase is about 4-fold higher as compared to that
in dry embryos (Table 2). As shown in Fig. 7, cycloheximide
(20 pg/ml) could block the appearance of new activity of
M.
16
12 o--o CONTROL
e e +CYCLOHEXIMIDE AT 0 hr
·~ 8 X
~ 0.. u
4
~L_-=~4~~~8t:::~1~2==~1~6====~==~~~ GE-RMINATION (hrs}
Fig. 5: ~ffect of eyclohexJ.mi.d.§LQD_ 3H-YI],dine i11corpora tion into RNA. •ren embryos were used for each point. Cyclobeximide ( 20 )lg/ml) was added at zero hr along with 3H-uridine. .itnbryos were ground with homogenizing medium and precipitated with 10% TCA as described in Materials and Methods.
16
12
M
I~ 8 X
~ 0.. u
4
A I. +eve LOHEX IMI 0 EAT 0-6 hrs.
4 8 . 12 16 GERMINATION (hrs)
20 24
Fig. 6: Effect of GX£J.ohex1m1...~ added 1!!£ing the first 6 hr of gennination on 1a te RNA ~_thesi.§.. Bnbryos incubated for 6 hr in presence or cycloheximide ( 5o~g/ml) were washed several times with water and then ransferred to a fresh gennination medium containing H-uridine. At indicated times, embryos were homogenized and precipitated with 10% TCA according to the procedure described in l4aterials and Nethods.
- -.--
120 ~ ,.....- -
~ -
... -90
> ...... ~ -->
...... r--
u ~ -<( 60
u u. -
.... -u w a. t/)
30 .... ........ -·
- -
~ 0 10 20 40
GERMINATION (hr.)
Fig. 7: .iffect of cl.£lohex1mide on~...JUU>ear~ce of RNA ..P.QJ.xtn§.m.~.JlQ . .t.i vi ty during ge nn ina tiQ!L of ba rm embryo~. Snbryos ( 250 mg) were ground with homogenizing medium and centrifuged at 15,000 r~ for 1 hr. Slpematant was assayed for RNA polymerase activi~. Open bar, control; close bar, with cycloheximide.
TABLE 2
SPECIFIC ACTIVITY OF RNA POLYMERASE AT VARIOUS GERMINATION
TIMES
Dry embryo
Germinated ( 10 hr)
Germinated ( 24 hr)
Germinated ( 48 hr)
Time of incubation
(in hr)
1
2
1
2
1
2
1
2
Specific Activity (pmoles of GTP incorporation per mg protein)
27.1
42,3
50.4
68.5
119.3
139.2
138.7
179.3
Embryos (250 mg) were homogenized in 3 ml homogenizing buffer, centrifuged and RNA polymerase activity in the supernatant was assayed according to Materials and Methods.
RNA polymerases, supporting the view that the early
proteins are essential for RNA synthesis. Furthermore,
cycloheximide inhibited the appearance of new RNA polymerase
activity when added during the first 6 hr of incubation,
but was less effective when added after 8 hr (data not
shown).
Action of Cordycepin and Cycloheximide on Germination:
Barley embryos were germinated in presence of 20 p~l
of cordycepin and after 8 hr, the embryos were washed and
germination was continued in fresh incubation medium,
upto 48 hr. Germination was uneffected (Fig. 8d), however,
a similar treatment with cycloheximide (20pg/ml) stoped
the germination completely (Fig. Be). All these results
suggest that early protein synthesis is indispensable
for late phase of germination. Moreover, if germination
was allowed to proceed upto 8 hr and then the translational
machinery was blocked by cycloheximide (20 yg/ml) from
8 to 10 hr, it was observed that there was no preceptible
change in the development of embryos as compared to control
(Fig. 8a). As shown in Figs. 8b and 8c, continuous presence
of these inhibitors during germination; stopped the germination
completely, suggesting that both early and late phase of
protein synthesis are essential for germination. Furthermore,
results with cordycepin also suggest that stored rnRNA of
barley embryo is already processed whereas new rnRNA synthesis
requires processing before programming.
Fig. 8 Appea_mnce of Wl bryo.§...f!.f.ter !ta._~.!lil.ina tion that have been t,ll!.ll§.fer~ to so1u tion of .:l.WJ.1bi tors at the t~§ indicated. A, control; B, cycloheximide added at zero hr; c, cordycepin added at zero boor; D, corqycepin added at zero hr and removed at 8 br; R, cycloheximide added at zero hr and removed at 8 hr.