' DNA SYNTHESIS AND ELONGATION GROWTHNEW ASPECTS
MARTIN BOPP
B_oitmisches Institut der Universitat Heiddberg, 69, Heidelberg,
Hofmeisterweg 4, Germany
SUMMARY
·' · ., The size of plant cells is determined by hormones. The hormones act by means of rapid reactions probably on the cytomembranes' as weir as by a slow reaction on RNA and finally
DNA. Here it is made clear that a regulated elongation in
hypocotyls of Sinapis seedlings is only possible if DNA synthesis
trtkes place. Inhibition of DNA synthesis by 5-FdUrd stops growth.
There' are no ·quantitative relations between growth and DNAsynthesis but there is an absolute requirement for DNA synthesis.
Within the heterochromatin of Sinapis seedlings a late replication can
be found and this may somehow be linked to the elongation proeesses which follow, i.e. the replication of this DNA frees information
which is a requirement for further elongation.
~
' I
INTRODUCTION
There is an evidence for the fact that the differentiation of the
cell depends on whether at a particular time DNA synthesis takes
place or not. During this time the pattern of the active, inactive and
potentially active genes is determined. We call this event the primary
differentiation. From a number of recent publications one can conclude that this dependency i~ also true for cell size. The regulation of
th(enlargemeQt specially through auxins follows three stages :
1. Auxin stimulates the elongation growth immediately probably by changing the nature of the permeability of the plasmalemma
(Cocking, 1962 ; Nagata and Takebe, 1970 ; Power and Cocking,
1970; Pilet, 1971). Intact cells treated with auxin elongate in a few
minutes (Nissl and Zenk, 1969; Zenk, 1970; Rayle eta!., 1970 ;
Pope and Black, 1972). One can, therefore, conclude that there is
an immediate effect on the plasmamembrane.
DNA SYNTHESIS AND ELONGATION GROWTH-NEW ASPECTS
H9
2. There is a good amount of evidence that the elongation
growth can take place for a longer time only when RNA and
proteins are synthesized (Bopp, 1965; Nooden and Thimann, ;l9.66t:
Zenk, 1967; Cleland, 1971).
;.
,__ ,
3. Not all the cells elongate in the same. manner after treat..
ment with indoleacetic acid (IAA). For e;xample, the cells of inos&
protonema do not elongate after IAA treatment, but the-. cells. of the
shoot can do this. One can see that the two types of cells have
different reactions under the same conditions. This depends on
''primary differentiation" of the cells. .
. In a .s~ries of recent repo~ts it has tx:en shown .that u.n~€:f-·. ce~j
tam condthons, DNA synthests could be mduced wtth auxtns. Tbts
is true for Jerusalem artichoke tissue where a 2,4::D'.treatmt:!nf reacted after a lag phase of 3 days (Maheshwari and Nooden, 19Jl). In
this case, the growth begins first if there is an early. synthesis-of
DNA. I intend to deal now with the relationship between the IDNA
1
synthesis and elongation growth.
'-''
We start by the observation that 5-FdUrd inhibits · cell
elongation in the shoots of many plants e.g. plan:tlets of ·BFyophyl/um
(Purohit et al. 1969), leaves of Bryopyllum (Bopp and ;Catarino
1968), hypocotyls of Sinapis alba and lower internodes of Vicia faba,
cucumber, lens and slices of potato tuber (Lang and· Nitsan·, 1967 ·;
Degani et al., 1970; Kamisaka and Masuda, 1970). Very pr,obably
FdUrd inhibits DNA synthesis in higher plants, as in bacteria. Obyiously one can assume that in all cases the cell division is inhibite<l
and, therefore, the hypocotyl is shortened. Some workers,believe th,;1.t
by following measurements of the older internodes (HolJ11 and Key
1969) or the roots (Paranjothy and Rhagavan, 1970) this inhibition
can be measured. We have, therefore, investigatC::d the roots of
Vicia faba, and by marking the outside of the roots with black iiik.we
are able to demonstrate that the inhibition is infact <;mly ih the· area
of cen division, the other zones having a normal elongati6h:: ·we
can verify that: in older shoots and roots FdUrd does ri.J't i~bi61t
the elongation, but in young hypocotyls and internodes th~'ce11
elongation is inhibited.
,., . , ·
MATERIALS AND METHODS
All experiments were conducted on Sinapir alba L. (ScMell,
Stuttgart-Plieningen). Seeds were selected and germinated.for''·36 hr
in total darkness at 25°C. After that the testa was removed <Und~r
dim green light. The plants were then. grown in ... squaFe ;. pb\stfc
140
MARTIN BOPP
dishes (10 em) for certain time either in light or darkness. 5-FdUrd
and thymidine taken from stock solutions were applied either at the
titne of sowing of the seeds or later on. The nucleic acids were extracted, separated on a MAK-e ·lumn, Sepharose 4-B-column or with gelelectrophoresis. All other methods have been described in more
&tails in earlier papers (Bopp and Capesius, 1971 a & b; Capesius
aJ')d Bopp, 1970 a & b).
RESULTS
1. Significance of DNA in elongation process.-There are three
suppositions :
(a) During the time of inhibition we should have DNA synthes#s........Jn Bryop/1)'1/um 36 hr after germination, a high DNA synthesis
in the hypQQotyls a~ well as in the cotyledons was noted. By labeling with thymidine the radioactivity was observed only in the DNA"~tk. However, in Sinapis alba the radio-activity decreased rapidly.
Fortyeight hr after germination, DNA synthesis decreased to about
10% of the value at 36 hr (Capesius and Bopp 1970). The bulk
of the newly-synthesized DNA in both Sinapis and Bryophyllum
w~ localized in the nucleus, as demonstrated by autoradiography.
The synthesis of DNA in the nucleus of the hypocotyls of Sina-
piJ was not homogeneous. Replication of DNA took place first in
the euchromatic and later in the heterochromatic parts. Therefore,
one finds many nuclei which show radioactivity only in the chromocentres as was shown earlier (Bopp and Capesius, 1971).
(b) In the research material FdUrd must inhibit DNA
synthesis.-FdUrd inhibits all thymidine dependent processes. This
is specially true for the synthesis of DNA. With the addition
of exo~enous thymidine we can avoid this block. To avoid the
block of thymidine dependent processes by FdUrd, the Sinapsis plants
were first treated with FdUrd and after this the DNA synthesis
was studied with labelled 14 C-thymidine. It was observed that FdUrd
enhanced incorporation of thymidine by 50 % because through
the inhibition of thymidylatesynthetase no endogenous thymidine was
available and, therefore, the incorporation of exogenous thymiQillO was stimulated. But when 3 H-cytidine was fed, incorporation
in the hypocotyl was only between 37-54%, while in the cotyledons
it was betw~n 21-31% of the untreated control as reported earliar
·
(Ca.pesius alld Bopp, 1970),
DNA SYNTHESIS AND ELONGATION GROWTH-NEW ASPECTS
141
(c) This inhibitory effect is specific, ie only the DNA synthesis
is inhibited and not the RNA ana protein.-Fan and Mclachlan
(1967) showed that in the first two days of their experiments, the
DNA synth~sis was almost fully inhibited with FdUrd. while RNA
and protein syntheses during this time were not affected. The same
was also true for their experiments with the enzyme cellulase.
Similar results Vlere obtained by Seitz and Lang (1968). They found
a remarkably poor inhibition of the enzyme invertase as compared
to DNA.
2. Conditions for causal connection between DNA inhibition and
growth inhibition.-If the DNA inhibition on the one hand and the
elongation inhibition on the other, have a causal connection, then 5
conditions must be fulfilled ;
(a) The growth of tissue without DNA synthesis may not be
inhibited by FdUrd.-It is already known and recently redemonstrated that FdUrd does not inhibit the DNA-induced elongation in oatcoleoptiles. In the oat-coleoptiles, there is no DNA synthesis and it is
also not induced by auxin (Bopp, 1967).
(b) The growth inhibition should be abolished by thymidine.When we gave the FdUrd treatment in the beginning of germination
at as low a concentration as 10-8 M, there was a growth inhibition
of about 50%. This inhibition could not be abolished until the
concentration of thymidine was raised 100 times higher to get a
nolma1 growth. But with 10- 5 and I0- 4 M FdUrd, 10 times higher
concentration of thymidine was needed. Thus, to conteract a
complete inhibition of 10- 5M we need a concentration of thymidine
10-4M. Therefore, this is the thymidine concentration which is
needed to guarantee normal elongation growth.
Substances of the RNA synthesis like uridine or uracil,
however, could not abolish the growth inhibition, neither in Sinapis
nor in Bryophyllum. This means that the elongation and the blocking
of DNA synthesis take the same path.
(c) Inhibition of DNA synthesis must precede the inhibition
of elongation.-To test this the lag phase between the application and
the evidence of first inhibition of DNA synthesis was studied.
From the growth curves of Sinapis alba we can demonstrate that
elongation in the dark was completely linear for a long time. lf we
give FdUrd during this linear growth (7 hr after the addition of
FdUrd) the growth was retarded and stoped gradually. After another
4 hr, the hypocotyls did not grow any more. The lag phase of this
process from the time of addition of FdUrd to the beginning of
inhibition amounted to 7 hr.
142
MARTIN BOPP
From our experiments we can also conclude that longer plants
have a longer lag phase and also a longer phase of delay. They
are less inhibited at the same concentration of FdUrd. At a
certain time, growth is not inhibited at all. For example, when the
plants arc longer than 30 mm then no concentration of FdUrd
can inhibit the elongation (Bopp, 1967).
For the inhibition of DNA synthesis, we get the following results : The incorporation of cytidine without FdUrd in the first~
8 hr after the beginning of the experiment (36-44 hr after sowing)
was almost linear. Two hr after FdUrd treatment, the hypocotyls had
10% activity of the control, and the cotyledons about 50%. It is
assumed that during the first time the incorporation is also linear,'
the lag phase in the cotyledons is about 30 min and in the hypocotyt
it is still shorter Therefore, the lag phase is much shorter than
the elongation growth. The inhibition of DNA synthesis precedes
many-fold the inhibition of elongation.
(d) The restoration of DNA synthesis should restore elongation.-After the beginning of inhibition i.e. after 9 hr, we transferred
Sinapis plants to a solution which contained 10 to 20 times TDR.
After 3 hr, the incorporation of radioactive cytidine in the hypocotyls
was about 75% as compared to the control. With a very short
! ag phase the DNA synthesis was re-established. After 24 hr the
growth was resumed which had fully ceased in the FdUtd-treated
plants. Therefore, we can conclude that inhibition and promotion of
DNA synthesis have a short and corresponding growth processes
have a long lag-phase.
(e) Other DNA inhibitors must likewise inhibit the elongation.For this question we studied the effect of mitomycin and cytosinarabinoside. Tables I shows that in Sinapis and Bryophyllum the elongation growth was inhibited. At present, we can not explain as to why
such a high concentration of cytosinarabinoside is needed. Possibly
it is associated with the mechanism of its uptake. Independent of
this mechanism both the substances inhibit the elongation.
We can now summarize that the inhibition of elongation growth
and the inhibition of DNA synthesis really have a causal connec~ion.
3. Non-quantitative relation between DNA synthesis and
growth.- Now we can ask if the whole DNA or a certain fraction
~fit is responsible for this inhibition. Therefore, we could make two
comparisons: In Bryophyllum, DNA synthesized during the elongation
growth is stable. More than 90% does not take part in the metabolism (Bopp. 1970). When we compare the nuw.ber of l~belled
143
DNA SYNTHESIS AND ELONGAtiON GROWTH-NEW ASPECTS
Table I. Effect of different inhibitors of DNA synthesis on the elongation growth of seedlings a/Sinapis and of plantlets o/Bryophyllum. Sinapis seedlings were treated 36 hr after sowing~
and the plantlets of Bryophyllum at the start of the experiment.
Sinapis alba
after 60 h
Cytosinarabinoside
mm
31.6 ± 0.8
22.0± 1.2
31.3 ±0.9
or
/0
100
70
100
Bryophyllum dia,;remontianum
after 5 days
Mitomycin
mm
K
56
to-•M
w-'M
29
50
to-•M
56
01
!0
100
51,7
89
100
cell nuclei with the total amount of synthesized DNA, we can see
that during the growth of the hypocotyl, the number of labelled
nuClei decreased faster than the percentage of the radioactivity incorporated into DNA. After 48 hr there was about I 0% radioactivity
but only 1-2% labelled nuclei. From this, it follows that either
more than one replication takes place in the labelled nuclei or that
extra karyotic components participate in labellin!. For this, we do
not as yet have any direct evidence but only some indirect indication.
One of the suppositions is that the percentage of the heterochromatic
nuclei in the older tissues is higher than in the younger ones.
This means that in this tissue the DNA synthesis in the heterochromatic regions is prolonged, and this could be the result of higher
am.:mnt of DNA in these particular cells (Bopp and Capesius, 1971).
For this reason, we can not expect that the amount of growth
depends on the amount of DNA synthesized.
DISCUSSION
We have seen that FdUrd inhibits elongation growth specially
in this case where it is induced through the external factors. In our
case it is true for the etiolated hypocotyls. While Degani et a/. (1971)
observed an inhibition of GA 3-induced elongation, Maheshwari
144
MARTIN BOPP
and Nooden (1971) and Mamisaka and Masuda (1970) have shown
that this is also the case during the lag phase in auxin-induced
cell elongation in tissue cultures. It has also been demonstrated in
the etiolated liverwort Marchantia (Ninnemann, 1967) and in the
leaves of Bryophyllum (Bopp and Catarino, 1968).
In a number of cases, the DNA synthesis is reactivated by
external conditions, e.g., in Jerusalem artichoke tissue (Maheshwari
and Nooden 1971), in basal portion of soybean tissue (Holm and
Key 1971), in the internodes of Bryophyllum daigremontianum (Bopp,
1967) and in the leaves of Lobularia maritima (Catarino 1969). In the
last case, it is the salt content of the soil which activates the DNA
synthesis. In other ca5es while DNA synthesis is not activated but
still has to take place so that a reaction can follow, and this is the
case with Sinapis.
In no case it was found that DNA synthesis was necessarily linked with cell division. It can, therefore, be concerned with the postmitotic DNA increase. This can not be decided as yet whether there is
an endomitotic increase or a local gene amplification or an extranuclear DNA increase in the mitochondria and chloroplasts. Our
observations, however, support the idea of gene amplification
(Bopp and Capesis, 1971).
The significance of the DNA increase could be that during
this increase necessary genes for the elongation are activated.
This interpretation also agrees with the results of Holm aild Key
(1971). The activity of extracted chromatins is much Jess when
during a treatment with 2,4-D new synthesis of DNA is inhibited
with FdUrd and then the in vitro experiment is done with the same
quantity of chromatin. So, if there is a Jack of DNA synthesis there
are fewer active gene loci than if the DNA is synthesized. This study
confirms the supposition that gene activity is induced during the
auxin-induced DNA synthesis. In principle, the conditions tor the
primary differentiation are the same as for the elongation growth and
differentiation of other kinds. There is, however, an important difference, the responsible genes must continually renew the potential
active state through every DNA synthesis and that state is evidently
very labile.
After DNA synthesis is concluded, as for example in the tissue
of leaves, the elongation is continued for certain time which is demonstrated by the experiments of Maksymowich and Kettrick {1970).
With increase in age of the tissue, the cell elongation is always more
independent of the DNA synthesis.
-~
DNA.SYNtHESIS AND ELONGAtiON GROWTH-NEW ASPECTS
14$
Therefore, every cell has a phase of elongation growth which is.
DNA-dependent. After this time, FdUrd is no longer able to inhibit
the elongation. When DNA synthesis is induced by external factors
then this phase can be prolonged. This points to the fact that there
is a correlation between DNA contents of the cell and the cell size, as
is found often.
ACKNOWLEDGEMENTS
The research was supported by the Deutsche Forschungsgemeinschaft. Many experiments were carried out first of all by my
coworker Dr. I. Capesius.
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