Plant Physiol. (1908 -v3, 140)-150
Studies on the Role of RNA Synthesis in
Auxin Induction of Cell Enlargement'
Larry D. Nooden
Department of Botany, University of Michigan, Ann Arbor, Michigan2 48104
and Botany Department, University of Edinbuirgh, Edinburgh, Great Britain
Received a 22, 1967.
Abstract. Selective inhibitors were used to study the connection between nucleic acid
synthesis and indoleacetic acid (IAA) induction ot cell enlargement. Actinomycin D (act D)
and azaguanine (azaG) almost completely inhibit IAA-induced growth in aged artichoke tuber
disks when they are added simultaneously with IAA. In contrast, when they are added 24
hours after the hormone, these inhibitors have little or no effeCt Gn the induced growth which
continues for 48 hours or more with little or no inhibition. Inhibitors of protein synthesis
still stop growth when applied 24 hours after the IAA, thus protein synthesis and presumably
supporting metabolism are still essential.
In corn coleoptile sections auxin-induced growth did not show any pronounced tendency
to become less sensitive to act D as the IAA treatment progressed. Act D did not completely
inhibit the response to IAA unless the sections were pretreated with act D for 6 hours. In
contrast to act D, cordycepin produced almost complete inhibition of IAA-induced growth
when added with the IAA.
Although IAA has a very large and very rapid stimulatory effect (within 10 min) on
incorporation of 32P-orthophosphate into RNA in disks, it did not cause a detectable change
in the base composition of the RNA synthesized. Furthermore, the promotive effect could
be accounted for through increased uptake of the 2P. That much of the RNA synthesis in
these tissues is not necessary for auxin action is indicated by the results with fluorouracil
(FU). FU strongly inhibits RNA synthesis, probably acting preferentially on ribosomal
RNA synthesis, without inhibiting auxin-induced growth in the disks or coleoptile sections.
FU also strongly inhibited respiration in auxin-treated disks indicating that the large promotion
of respiration by auxin likewise may not be entirely necessary for growth.
At least in the artichoke disks, RNA synthesis is required for auxin induction of cell
enlargement and not for cell enlargement itself.
The possible relationships of auxin induction of cell enlargement and RNA svnthesis are
discussed.
!lith,oti-h cell enlar'gement is one of the m11ost
hasic processes in planlt development anld imainy
systemis in which celil enlargemenit is regtilated by
atuxin have been studield and described extensively,
the primary mode of aciti,on o,f auxini relmain,s tinIknown.
This paper extends the sttudies on the reqtiiremenat for protein and RNA synthesis for atixininducod growth wvhich wvacs reported in earlier
papers by thi!s author (31-34) 'and manyl others.
The concluhsion that protein and RNA synthesis are
ieces's!arv stems frolm studies with a varietv of
'-elec,t,ive inhiibitors whose vallue, as lpointed out
earlier (32), depends on their specificity. In genleral, however, all the inhibitor stuldies suibstantiate
the involvemenit of proiteini and R\RN A synvthesis inl
'Supported in part by NIH Postdoctoral Fellowslhip
No. F2-GM-12,160 in Botany l)epartmennt of the University of Edinburgh fromii 2/1/64 through 7/1/65.
and in part by a grant from the H. H. Rackham Sclhool
of Graduate Studies at the Uniiversity of AMichigiani.
2 Present address.
atuxini induction of cell enllargemni,ent. 'T'he niext
question is wheither or not auixini acts throulgh Ian
effect on these synthetic processes.
In th,is paper the role o f RNA and protein
synthesis in auxin-induced grouwith is studied furthe,r
with selective inhibitors: some experiments on the
effects o,f these compounds on RNA synthesis and
respi,ra(tion are also described.
Thiese findings have been presented in a short
plaper given at the AIBS meetings (31).
Materials and Methods
Matcrials. Artichoke (Helianthus . tuberosus)
tubers were grown in the Edinburgh Botanical
Garden,s and the University of AMichigan Botanical
Gardens. The tubers were harvested in the fall
aind st,ored in mInoist sand a,t 3° tuntil they we-re used.
from May to Jtuly in the following year.
Disks (1 X 10.5 mm) were prepared froni
timbers jtuSt before and after sprotlting as describe(d
in earlier papers (32, 33). To "age" the (lisks
they were soaked for 24 houirs before uise in sO ItM
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Plant Biologists. All rights reserved.
N-OODEN\-R,A SY'NTHESIS
chloramphenicol. The tissute nearest the budls generallyv seemle(l to give a larger alnd more rep roducible
growith reisponse. Di,sks wZhich floated oni water
w-ere always dliscarded as thly gave a relatively
poor response to auixiini.
The corn seeds uised wvere Zca )Il(avS variety
Barbecue Hvbrid fro,m WV. Atlee Burpee Company
and a White Sotuth African Horsetooth variety.
The seeds were soaked in a 0.25 % (w/w) NaCIO,
x-ashed in ruinnling tap watter for 8 houirs anid
germinate(l at 295 in m-oist vermicuilite in dim red
light. Coleopti,les (3 cm long) were excised and
after remo-al of the leaves wvere floated on water
for about 2 hours. Starting about 4 mm I)elow the
tip, 2.0 cmi secitions (iniltial fr wt 50-60 mg per
section ) were cuit from these coleoptiles. The
coloeoptile sectioiis from the 2 varieties oif corn were
simillar in all characteristics wch ch were compared.
TreatneWts. Tin growth stll(lies the "aged" artichoke tuber diskxs were suIspen(led oIn a plastic screen
julst totIching the treatment so,lutionls, as dlescribe(l
earlier (32,33). Where radioisotopes were uised or
respiration imleasuired, tihe treatmienlt soluitions coIntained 50 ju-x chloramphenicol, which has nio inhbitory effect on plaint growth (33), btut is generally
adequtiate to suppress bacteria comnp'etely (28). Thuts
it is possible to tuse the diffeorence in sensitivities to
chloramphenicol to prevenit bacterial growth selectively. Chlorampheni,col at 30 JLM ha,s been shown
to be very effective in suppressing bacterial growth
in infected disks even when stucrose is added, and
150 Ai chloramphenicol gives essent<ally complete
inhibition (20). Also, the dangers of bacterial
oontamiinaftion were miniimize(d since no sulgars were
added to the medium, special care w-as taken to
prevent bacterial growth in the stock solutionis,
brokein prot-oplasts were wasihed anway, and tubers
showing s:gns of in,fection (brownish spot's near the
vascular bu ndles) were discarded. Stock so4lutions
of IAA (100 mg/l) were adjtisted to pH 5.5 w\ith
NaOH.
Corn coileoptile sections were floated onl 10 ml
of treatment solution containing 2 % (w/w) stucrose
with or without IAA, 32p, etc. and incuibated at 250
on a rotary sh,aker. Artichoke disks being exposed
to 32p wvere similarly incubated with 10 disks per
sample buit without the suicrose; otherwise they were
treated as described above. The 32P-orthophosphate
used wiith the coleoptilIe sections contained 1 poi
KH2PO4 a-s carrier but "c,arrier-free' 32P-P; was
used with the tuiber di;sks. Incorporation of 32P
into RNA in the tuiber disks was increased by IAA
to about the samne extent with or wxithouit added
carrier.
IIncuibatioIis with 3 wPwere ended b- washinig
the tissuies careftulily with distillled wvater except
when uptake o.f 32P_P1 wa s studied: then the disks
were w-ashed with 0.05 M KH,PO. (pH 6.8).
Wheni RNA%-aws to be extractedl from ti'ssues, they
were stored in ethanol at - 17° with mercap,toethanol uttil grinding about 12 houirs later.
AND AUXIN ACTIO1-
141
Determnination of Acid-Soluble 32P* Tulber (lisks
exposed to 32 -%P1were washed wi?th 0.05 Mi KH2PO4
(pIH 6.8) and grouind immed!iately in 0.005 M
KH.,PO, (pH 6.8) with 5 % (v/v) mercaptoethanol at 3°. An equial volume of 10 % (w/xw)
trichloroacetic acid was added. After 30 minutes
at 30, the mixtture was centrifuged until the sutpernataant wa,s clear, and samples of the supernatant
were taken foir dletermination of the 32P. Similar
experiments were done with the corn coleoptile
sectiionlis.
ISOl(Itiot of RAVA. 'I'lle tissues were ground
with anmortar and(l pestle in 0.5 ml of H2O-saturated
phenol (waished several times xwith 10 mtM EDTA,
pH 7.4), 0.5 ml of KH. PO.G butffer (0.1 -u, pH 7.4)
containing 0.2 % (w/w) soldium ( ),decvl sutlfate
(SDS) an(d abouit 0.01 ml mercaptoethanol at 3°.
After washing the mortar and pestile with iniore
phenol and buffer, the mixtuires were combuiied,
stirredl thoroughly several times ancl centrifuged.
T,he interp)hase anid phenol base were extracted
twice more with b)uffer pluis S-DS, onice at 600 for
15 minuiites anld onice at 30.
RNA was precipitated initially by adding 3
volumes oif 100 % ethan,ol ancd sttoring the mixtures
at -170 for aboult 12 hours and subsequently from
3 M gulanidine chloride (see 21) at least 2 times.
This precipitate was wa;shed with 90 % (v/v)
ethanol (lx), 100% ethanol (2x), 3:1 ethanolHCCI: (2x), and 3:1 ethanol-ether (lx).
Determiiinationi of the RNA Baise Comptpositiont.
For determinabion of UV absorp'tion a smal,l portion
of the hydrolysate was diluted wi,th 0.1 N HClO4
and the UV abqiorption measureod. In calcuflatin1g
the amiount of RNA nticleot'des in tihe 0.1 N HC10,
solution, the molar extinction coefficient a;t 260 m/
w as taken as 12,000 (p 393 in 22). The RNA was
hydrolyzed with KOH and( the nucleot'des were
separated ionophoretically in a waay s:milar to that
described bv Loening (21). The 4 ntcleotides
were located on the driefl strip wiith a UV lamp and
were cult out for counnting. Radioactivilty w as determined in a liquid scintillatfon sspectrometer uising
toluene with 1 ,4-bis- [2- ( 5-phenynloxazolyl ) ] -benzene
(POPOP) and 2,5-diphenxloxazole (PPO).
Measutremient of Respiratory Rates. The respiration rate was determined as hefore (33).
Results
Effect of Delayed Addition of Inhibitors of RNA
Synthesis on1 IAA -Induced Growzth. Actinoni vcin
D (act D) has been shown to inhibit auxin-iinduiced
growth in a variety of tissuies inClhidllng articlhoke
tunber disks (25, 34). In most cases act D wxaas
added together wvith or before the IAA. Figuire la
shows that act D (50 mg/l) added together with
IAA suippresses atuxin-indutced growth completely
(ludring the firsit 24 houirs, the inhibition being evident by 12 hoturs, and thereafter only a very small
incremenit of grow%vth duie to the auixin is apparent.
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142
PLANT PHYSIOLOGY
IAA.
50
HELIANTHUS TUBER DISKS
(IAA) -10 mg/I
HELIANTHUS TUBER DISKS
(IAA) = 10 mg/l
(Actinomycin D - 50 mg/I
(Azoguanine) = 0.8mM
~IAA
aIE
(0
IAA+i A;zaG ot
hr
~~~~~~~~~24
40
z
IAA + Act D
2
at24hr
3
~30
Uf)
n
w
LLJ
w
Lz
ii
n
U)
Lii
Ac D
//!~~~~~~~A
z
at/hrW
2°~~~~~~~~~~~~~~~~~
2
IAA AzaG;
20~~~~~~~~~~~~~2
+
10
12
0
TIME ( Hours)
2
t
24
48
36
TIME (Hours)
72
['i ;. h(. (left) Effe,zt o)f d l1avd addkition of aIctinllc)l\inl 1) oll !AA\-indticcf(l -i-cmth iii .(-i-cd alrtichoke tulber
(liSkl.s
FIG. 11). (right) E ffest of delayed adldition of aza-uianine ')I IAA-iioee(l -rowth in a-e(l ar-ticlhokc tuber disks.
A 24-1hour (lelav. Thlec arroxvs shlow whenl the inhibitor N-xas a(ldl-Cl. Before the start of tle e-Nperlillwunt. the (Ii sks
we re soaked for 24 hours iin 50 juMi chloramplicnicol.
o
HELIANTHUS TUBER DISKS
50
(IAA) = 10mg/I
(Azaguanne) 0 8 mM
IAA
/
IAA/
=
40 -HELANTHUS TUBER DISH
(IAA) - 10mg/I
10 mg/ 1
/Azaguanhne)
IAA +AzaG
0.8mM
(?20hr)
@i40
c
30
a.~~~~~~~~~~~~~~~~~~~~~~~~~~a
I~~~~~0a
0
I
330p
,A1
Z
AA+AzG(IOhr)
/
20
-o/
0
H°Ijo
3:~~
ui/
, 20
IAA+AzaG
IRAaGSr
(5hr)
Ll-
VZ
20
~~~~~~~~IAA+AzaG (Ohr)
cr
z
0
_
L
.
C-)~ ~ ~ ~ ~ ~ ~ 0
0
TIME (Hours)
TIME ( Hours)
FIG. 2a. (left) Effect of (delaxed addition of azaguaiiine on IA A-induced grotli in aged artichoke tuber (lisks.
Delays of 5 to 20 hours.
FIG. 21). (righlt) Comparisoio of the effect of delayed addition (of azaguanne on ar-tichoke tuber disks aged 24 and
48 hotirs. The arroNx-s inidicate wheni the azaG or IAA x -as addled. At time equals 0 holtrs thel disks had been soake(d
for 24 holurs in 50 u.xr chloramphenicol.
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Copyright © 1968 American Society of Plant Biologists. All rights reserved.
143
In corn coleoptile sections there could be a sinall
escape from a requirement for RNA synthesis
.NOODE)N-RNA SYNTHESIS AND) AUXIN ACTION
If act D is added to artichoke tuber disks 24 hours
after IAA, at a time when the disks have started
to respond to IAA, the act D fails to inhibit the
IAA-induced growth which continues for another
48 hours.
Very similar results were obtained using the
base analog 8-azagtuanine (azaG) as shown in
figture lb. Additional experiments show that azaG
added between 0 and 24 hoturs after IAA inhibits
incompletely (fig 2a). The escape frrom sensitivity
to azaG occurs even when the addition of IAA is
delaye(d another 24 houtrs, making the total length
of soaking in water 48 hours (fig 2b). As wouild
be expected, the ability of the (lisks to respon(l to
IAA is decreased after 48 houirs o.f soaking.
In other transfer experiments
not
showni
below the limits of resolution of the experiments
in figure 3. Unlike artichoke disks, the auxin
induction of growth in corn coleoptile sections i,s
not completely suppressed by act D added together
with the IAA (figs 3 ancl 4). In fact the sections
eventtually respond to abotut the same extent whether
IAA is added together with or 1 hour after the
act D (fig 4). When the IAA is given 3 hours
after the act D, it has mtuch less effect. Addition
of IAA 6 hoturs after the act D produtces almost no
promotbon of growth. Increasing the concentration
of act D frem 50 mg/liter to 100 mg/lilter or
decreasing it to 25 mg/liter had only a small effect
on the time requcired for act D to inhibit growth
wheni aidded with the IAA (fig 5). In colntrast to
act D, 5 mmi cordycepinl produces very rapId an(l
nearly complete suppression of the effect of IAA
on growth in co,rn ccleoptile sections tunder similar
col1(litions (tab'e I).
Althoulgh glulcose xwas able to overcome some of
the inhib.l tory- effects of act D in Sarcoma 37
ascites cells (13), in the presenit experiment-, ne,ther
58 mNI (2 %) suicrose nor 10 mm glulcose show any
tendency to overcome the inhibitory effect of act D
on IAA-induced growth in corn coleoptile sections.
here,
the disks treated wvith IAA for less than 24 houtrs
do noit grow as much as those exposed to IAA for
24 houtrs or more. After 24 hours of exposuire to
IAA, the diski grow to abo-ut the same extent
whether they are transfer,red to w,ater alone or to
IAA an(d azaG or to IAA alone, and disks transferredl to azaG alone grow- only sligh,t'y less.
Guanosine (0.4 m-I) added Nwvi!th the IA.\ and
azaG (0.4 mM) was able to prexvent completelx the
inhibWtory effect of azaG on the growth of the d'sks.
The results of similar exper ments with corn
co'.eopt:le sections were d fferent. In the first place
auxin-induced growth in this tissuie is not affected
by 0.8 m-iM
azaG.
Fuirthermore, experiments
Table I. Inlhlibitioni of I4A-induced Gr-owth in Cor0n
Colcoptile Sections by, Cordycepin
Burpee's Barbectue Hybrid Corn.
with
from. senisitiv_'i.ty.
Regardless of whether act D (50 mg/l) is aplaed
writh IAA or 3 hours latel-, it appears to inhibit
within 2 to 3 hours (fig 3). Even after 12 houlrs
of treatment with IAA, the growtth rate of the
coleoptiles is inh,ibited between 2 andl 3 houirs after
addition of act D. Curiously, act D ad(led only1
1 h.mur after IAA affects growth after 1 to 2 hours.
As a result the sections given act D s multJtaneouSl1
with the IAA andl 1 hotur later have identical growth
act D failed to reveal
cuirves
any escape
IAA
10 Imlg/liter-
+
I'l!tbatio)n in
hirs
(fig 3).
3.8
5.0
6.2
10.4
16.4
4
as
% intcrease in fr wt
0.9
1.1
1.3
1.4
2.2
1.8
4.1
9.4
14.7
19.4
29.3
38.1
2.0
2.4
3.2
3.2
2.9
2.9
ZEA MAYS COLEOPTILE SECTIONS
(IAA) = 10 mg/I
(Actinomycn D0)
IGAA
50 mg/I
Act
D
ot
3 h,
D, eveni at hi gh concentrathese tissutes tio become flacc.l
even after prolonged expostures, 48 hours for coleoptile sections and more than 72 houirs for the tuiber
disks.
Effect of Delayed Aidditiont of Inihibitors of
Proteini. Syntthesis on IAA-Induced Growoth. Chloramphenicol (6 mM), pulromycin (0.1 mM), aind
At
30
tions,
ID
|ISA
20
A
Act Doat
Act D atO0
,
h
cr
LI
10
0
Gr1owc!$h
2.2
1
2
3
8
I-1
5
5
6
40
+
Cordycepill
;Act O
2
3
4
5
6
7
8
TIME (Hours)
FIG. 3. Effect of delayed addition of actinomycin D
growth in corn coleoptile sections. The
when the act D was added. Burpee's
Barbecue Hybrid Corn.
on IAA-induced
arrows indicate
no
time d-d act
ever cauise
cyclloheximh.ide (50 y.I ) all inhi-'bit IAA-indulced
growth in the ttiber disks completely when added
with IAA (table II). Unlike aza-guanine and
actinomycin D, these sutbstanices inhibit IAA-induiced
growth even if they are added 24 hoturs after IAA.
The exact time when inhibition oiccuirs after the
delayed addition of inhibitors differs in each case.
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144
I'LAN-T PH-YSIOLOG:'
Inhlibitors of Protein Syntlhesis
Tal)le II. Effeet of Delajyed Addition of
.4 rti-hoke Tnbcr D)isk.s
The disks were soaked in 50 /utm chlioramiiphenicol for 24 hoursl)eto:-e
IAA
10 III-/]
Chlor amnplhen icol
6
+
I \ \
II'Mi
Transfer at 24 hours to:
IAA
10 Ig-l Chloran3iphlelnicol
+
+
+
C-cloheximlidlc
.0
tie experiment.
of
12
24
36
48
72
4.7
11.0
3.6
3.4
3.0
10.4
3.6
3.5
23.2
14.7
31.0
16.6
39.8
18.4
5.2
5.6
4.9
6.0
4.6
5.6
2.6
3.4
11.5
13.4
3.2
4.4
20.4
24.2
16.8
0.9
6.0
30.1
15.9
-6.8
7.2
4.7
4.4
2.4
3.0
11.0
11.4
3.2
3.5
23.2
19.4
31.0
21.3
7.2
4.9
39.8
23.6
7.4
6.0
Cx-cloheximide
+
Puron cvin
0.1 hli'M
I \\
thie ta;-t
(;iGowc'th in .-Ifcd
4.9
5.8
/m
T
+~
fI.-1I-j,di d
Grox-th as, % inicrease in fr Nxt at hrs
+v
IAA
o
Puronivcmi
+
+
+
+
17.8
3.6
6.6
5.8
4.9
Irtichokc T1 ber Disks
TJblet I1. I tf..t of Ie?n' .-I(nloys oil Girowth and Respiration IJndued by I.-. lin -Iqed
I
Th., disks xveri treated wvith IAA and )ase analogs for about 20 lhouirs ])-fore tlleir resl)iratory rI.te \\7aS Ill('l-ntre(l. At tinie equals 0 hours, the (lisks had bcen soaked for 24 lhoir.`l ill 50 [L.\I 'hlo(,:tlionhellicol.
+
IAA 10 illg/1
8-Aziag-uanine 0.8 nim
5-Fluorouracil 4 mnm
4-
,[l 0,/hr/g initial fr vt
% Increase in fr wt at 20 hr
1(5
-+-
11
7.1
Chlioramphenlicol seems to in,hib!it L little m)ore
rapidly than the others (table II). Tin n,o case is
there a strong tendency for growth to escape from
requiremen)t for protein synithesis. The disks
exposed to puromycin and chloramphenicol show
Ino tendency to become flaccid even after 72 houirs.
However, cycloheximide causes the disks to lose
sonme water a,ft,er 36 or 48 lionlrs of treaitmenlt.
+1i1
99
7.4
10
153
110 -+
16
9.0
14.2
121 ± 13
20.3
Although pturonycin xxas found to inhibit growth
of the tuiber d,isks here and in an earller study (34),
M\asiidla (24) foun,d that 0.1 mM puron-nyciv ha(d
little effecit on h,is disks. He did, however, find
chloramphenicol and azaG to be effectiv,e inhibittors.
A recent report that sqome preparra,tions of pltIrom'ecini are relatively inactive could providle ani
exl)lanattoll for the differences (43).
Endogenotts and
Effect of Base Analogs
144-Indutced Respiration in Artichtoke Tutber Disks.
As sho'wiv
in table III, a 24-houtr treatment witlh
0.8 mm azaG has no appa)rent effelct on tthe established respirationl of disks sugbgesting that azaG
generaltized toxic effect oni the
does not have
met,abolism of these cells. Of course, this could
on
40
C
ZEA MAYS COLEOPTILE SECTIONS
iAAi) - 10 mg/I
-^Xtnomycin D) 50 ng/l
<
a
already have been inferre(d friom the rellative
cr
20-
2
3
4
TIME (Hours)
5
6
7
I
FIG. 4. Effect of preincubation in actinomvcin D
on IAA-induction of growth in corn coleoptile sections.
The arrows indicate when the IAA was adlded. Burpee's
Barbecue Hx-lbrid Corn.
lack
o,f inhibition of growx tli when azaG is aidded 24
houirs afiter IAA.
Alore interestilig re the effectts of the base
alnalogs onI the TAA1induiced increase in O., uptake,
for xoth azaG alnd 5' -fluorouracil dtronigly interfere
with this increase. Tlhe inlhibitory effec,ts are plrticularlv significanilt whenl cOimparcd to the inlcl'rsCe
induicedc by IAA.
Effect of I44A01 ::'l'- (:) t/ho phlosphltet near-0)
Poration intto RNA. It was consi:dere(l important
to determine whait effect 1AA might have oni in
corporation of MP orthophasphate inito RN.\ in the
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1 45
N-OODEN-RNA SYNTHESIS AND AUXIN ACTION-
IAA
ZEA MAYS COLEOPTILE SECTIONS
(IAA) O mg/l
3
A
C
I01 /
2
/
* ActA
50mq
tion is much more raipid than the effect on growth,
which is evident only after 5 or more houirs.
It was also possible to determine the 32p base
composition, which can give an approximation of
the overall base compo-siti,on of the net RNA synthesized during tihe period of exlpiosuire to 32P-orthophosphate (18). The 32P base co,mpos!iition shown
in table IV is characteristic of ribosomal RNA,
h,av,ing
posures
o0
o
2
4
3
TIME
(Hous)
FIG. 5. Inhibition of IAA-induced growth in corn
cobleoptile sections by different concentrations of actinomy cin D. Burpee's Barbecue Hybrid Corn.
ma terialls used here. As shown in table IV, IAA
inc reases the amotunt of 32p taken up iinto RNA in
art ichoke disks, and the efifect is very rapi:d occutr10 minutes. The increase abservded
rin gwithkin 10
esThe isincrease
afitter 10 minuteis of exposure
quiite l-arge, berved
being
aix )ut 100 %. Tlh,is stimutlation, however, appears
to decrease wiith bime reachiinz 48 0 after 30
minutes. At 30 minutes, this effect is quite variable and often the increase duie to IAA is much
less. Clearly, the effect of TAA On 32p iucorpora-
tg 10tiin
a high GMIP content (37), eveen with exa,s sh,ort as 10 minuties. This suggests that
mosit of the RNA accutmulated in these brief periods
is of the ribo,somal type and, qu,ite likely, stuch
synthesis woulld allso predominate in an instantaneous
cross-section of RNA synthesis. Thtus it appearrs
tihait the 32P pullse-labeling techniquie, which has
been uised successfully to label selectively messenger
RNA in microorgan(isms, does not work well in
these plant tisstues. Of course, some difficulties
couild also result from the multicellultar nature of
the disks. For examiple, the otuter cells wou,ld
probably get a longer expo.sure to 32p thani the
inner cells. IAA does not cautse a detectable change
in the 32p base composition of the RNA synthesized
duigtefrt1Io3
iutso xoueee
during the first 10 to 30 minttes of expostire even
though it increases 32P incorporation.
When the effect of IAA on acid-solutlble 32 in
the disks was studied, it seemeid to stimullate the
uptake of 32P-orthophosphalte (table V).
Table IV. Base Composition of RNA Sviitlicsizcd by Aged Artichoke Tuber Disks during Brief Exposures to IAA
anld 32f' OrthoPhosphate
The disks (10 per sample) were soaked in 50 Am chloramphenicol for 24 hours before the start of the exposure
to 32p
IAA
10 mg/l
Cpm in RNA
nucleotides
ExDosure to
32p-Pi and IAA kmole RNA P
l' -MP
Ratios of 32P cont, ntG\ IP
AMP
mtin
+
10
10
2,890
0.96
6,810
15
15
30
30
10,700
22,680
23,600
35,120
0.92
0.9o
0.95
0.04
0.94
1.21
1.21
1.23
1.25
1.27
1.26
1 09)
1.09
1.00
1.00
1.0J)
1.0)
1.06
1.06
1.00
1.05
1.05
1.02
Table V. Effects of IAA anid Base Analogs on Uptake of 32P-Ortho phosphate inito the Acid-Soluble Fractioni of
Aged Artichoke 7 Tuber Disks durinig a 30-M1inute Incub(atioui
The disks (10 per samlple) were treated with IAA and base analogcs for 5 hlouirs before exposure to 32P-P. with
IAA and base anialogs. Before the start of the experinmnt, the (ciiks were so'uked in 50 utm ehloramphenicol
for 24 hours.
IAA
10 mg/l
Fluorouracil
4 nan
Azaguanine
0.8 mil
+
+
+
I-1
32P soluble in cold 5 % trichloroacetic acid
cp/'n/g originial fr wt
138,000
249.000
161.000
243,000
286,000
286,000
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Copyright © 1968 American Society of Plant Biologists. All rights reserved.
146
PLANT PHYSIOLOGY
Figure 6 shows that IAA also promotes incor-
poraition O,f 32p into RNA in
corn
coleoptile
sec-
30
28
ZEA MAYS
26 -
(IAA)
=
10
COLEOPTILE
mg/l
SE CTIONS
24-
Q.
22-
z
cr
201
18
16
J
c:
z
cx. 12F
0:
4
/7
0 18
tionls; however, this promo,tion appears to follow
the growth promoting effect of IAA (fig 5).
Again IAA stimula,ted uiptake of 3P inlto the sections (table VI).
Effecet of Base Aui(ilogs o7 Grooth a11(n R.NAI
S vlthesis. Althoutgh 5-flutorolracil (FU) ilnhibits
RNA synthesis, it has n1o inhiblitory effect o11, and
perhaps even stimulates, IAA-induiced growth. In
corn coleoXptile sections, 5 mNi FU an,d 5-fltiorodeox\iridine (FUdR) (lo not inhibit growth over
a 20-hotir period with or withotut ad!dled IAA. FIT
(4 mMi) (loes not (lecreatse the growth response of
the (llsks to IAA even after 72 houirs and so,me
stimutlation miay restilt (table III). Similar concentrationls of FUdIR also have no inhibiitory effect
oni the growth of the disks. Thus auxin-induced
growth in these tissuies dliffers fronm GA-induiced
growth in lentil epic,otyls w-hich is inhibite(l by
FUdIR (30).
Aks sho\\wn in tables
andl \II, 4 nval FE'
dramatically inhibits inco-rpo)ratcion of 12p into
RNA in ttiber (lisks withouit affecting its uiptake
into the (Iisks. In corIn coleoptile section!s, EU has
simi'lar th,ouigh less striking effecits (itables VI and
VIII)!.
45
90
60
TIME
180
120
iInutes
FIG. 6. Effect of IAA oni incorporatioin of 32Porthophosphate into RNA in corn coleoptile sections.
White Sotith African Horsetooth Corn.
Sinice FU is known to inhtib:Kt rib-osomnal R.NA
synthesis selectively in a varieity of organiTsms
(16, 17, 19), thi,s change in base composition is
probably dtle to a decrease in ribosomal RNA\ synthesis retlative to some other RNA which is hIIgh
in AMIP.
hffects of [11A (in( Inihibitov-s
oil Uptake of 221)OrthoplosphS1l(lte into Cori (Coleoptile, SeecionnS
dut li1i.J a 3-lIfot LExposuire to 32:'-01 t1loph osphlate
2
Tbl sections (5 per samnple) were pretreated wvith the IAA and inhibitors, for 3 hours l)befire alddition of the "P.
Burpee's Barbecue H-brid Cornl.
Table VI.
IAA
10 m-g/l
Fluorouracil
3.5 mnr
Azaguanine
0.7 nit
Actinomy-cin D
25 mg/l
Coldl 5 % tricliloroacetic acid
32'P SOltble in
c/m
-+
L-
-F
+
q , iginal fr te t
17,000
38.000
14,000
35,000
30.000
16.000
Tlable VII. EIffcts of ! ,I. I (anid Base ._nloj((yS on3Iornolion at 31 into RAJ nl1/e1 .ir tiertlhoke T'Iuber Disks
P
duiriitg a 30-.llinutc Exposurc to '0rthophosphatc
The disks (10 per sample) w ere treated with IAX and inhibitors for 5 bliours before exposre to ':P-P with I AA
andhlasc an'alogs. For 24 hotur-s before the exposure to 32p, tile (liSks were soaked in 50 um chTloramphelicol.
Ratios of
CPM\I in RN.A
IXA
10 rng/I
Fltiorouracil
4
iiu-m
.\Aagtianine
0.8 min
+
+
+
:.2
P contcents
(GAl P
A PIP
128,000
0.92
1.16
291,000
0.93
0.86
0.81
0.91
0.91
1.22
1.00
1.00
13,000)
+
+-
nuticleoti(les per
1umnoe RN \ P L' \1 '
26.200
345,000
356.0()O
(.98
1.01
1.19
1.21)
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1.00
1.00
1.00
1.00
0.97
0.97
0.89
0.88
0.93
0.96
147
NOODEN-R\A SYNTHESIS A-ND AUXIN ACTION1
Table VIII. Effects of Basc A4nialogs oni Inc-orporation of 32P intto RN,4A in Coi-n Coleoptile Sections during a
3-Houtr Exposuire .o 32P-O-t op/o
posphate
The sectiois were treated wxith IAA and bjase an,alogs for 3 holurs before exposure to 32P-Pi with IAA and base
analogs. \Vhite Soutlh African Horsetooth Corn.
Ratios of "2p contents
CP-M in R-NA
10 mg/l
Fluorouracil
3.5 mM
+
+
IAA
Azaguanine
0.7 mM
nucleotides per
Anmole RNA P
+
32,000
52,000
54,000
In the preseince of FU, IAA still stvmutflates
incorporation of 42p into RNA of tuber (tisks and
uiptake of 32p into the acid-soluble fraction. Even
in FU-treated disks where the background of RNA
synithesis has been greatly reedutced, IAA has no
detect-able effect oIn the 32P base compos.:tion.
AzaG
increases
the incorporation o,f 32P illto
the ttuber (l sks diuring, a 30-mintite exposure (table
VII); however, it also increases the amount of
acid-soltuble 32p in sim!ilar experiments ('table V).
Thu,s azaG may not have acttually increased RNA
synthesis buit only tiptake of the precuirsor. Paradoxically, azaG almost completely inhibits IAAinduced growtfh in ttuber disks whlile it stimulates
32p incorporation. On the oither hand, 0.7 milM
azaG does not have
any
significant effect otl 32p
incorporation in coleoptile sections here it also
fails to inhibit growth (table VIII). Tii experiments with colleoptile sections similar to those shown
in table VIII, 25 mg/liter acit D also inhibi:.tedl "''r
incoriporation into RNA by 65 %. 'J'he 32p lbaSe
compo,sit'OI1ons obtained in the acit D-treated sections
were qtu!ite variable but tendled tio be hliglher in
GMP and CMP. The act D a(lso seemed to inter_P in coleoptile sections
fere wilth ilptake (4f
(table VI).
Discussion
These studies explore ftirtiher the possibility thaat
aux,in iniduices cell
enlargement throtigh induction
Mlost
the cell wall.
act
of thle work was done with act(inomvcill D and
azagti,anine, which can interfere with the synthesis
or functioning of RNA.
Act D is welil known as aii inhibitor of RNA
syn,thesi.ls in higher p'ants as wvell as in ani.mals
and bacteria. As has been p)ointe(l ott prev ioiisly
(34), a nuimber of imlportant processes are not
much affected by act D tinder conditions where
RNA syntihesis is or would be expecte(l to be inhibited thereby providing evidence for the drrug's
selectivity. For example, act D doets not seem to
impair gireatly the mtiltiplicatiom of certaini RNA
viruses in higher plants (3,38). So far, all the
data on higher plants suggest that act D acts by
inhibiiting DNA-dependent RN.A synthesis, although
given a suifficiently long treatment it mright wvell
produlce siome seconl(lary effects in sonice tiSsuest.
of
new
enzymes
which
on
UAMP
G-M P
AMIP
0.78
0.74
0.78
0.99
1.00
1.00
1.00
0.86
0.96
CAIP
1.12
1.04
1.09
AzaG, an anallog of gtaninie, may inhibit 1RN..\
(16) and protein synthesis (12,15) in plants, bnlt
it does noit allways seem to inh,ibi,t enzyme formation (5,34). AzaG can be incorporatedl inito RNA
in place of gtu;anine (8,12) and may reduce the
ability of the RNA to serve as a template (8).
Since gtuanine nucleo¢t(des are cofactors in a
variety of biochemical reactions, it is possible that
azaG nucleotides or derivatives may impair these
reactions, btut azaG does not impair established
respiration in the articholke disks and does not
inhibit significantly growth of artichoke disks after
24 hours of expo,sure to IAA. Thuis, the data
suggest that azaG may have some fairly sele,ctive
effect on the operation or synthesi,s of RNA, a,lthotugh it is not necessarily completely specific in
all cases.
Auixin-induced growth of aged artichoke tuber
disks is completely inhibited by 0.8 mM azaG or
50 mg/liter act D when either is added simultaneotusly with the IAA. If the inhtibitors are added
24 hoturs after the atuxin, they have very little or
no inhibitory actbion on auxin-induced. growth and
the disks continue to grow for anot-her 48 hours.
Evidently all the RNA needed for grow,th is synthesized du;ring the first 24 hours of exposure to
IAA, land (th,en, fortuitously, it is stable. Significantly, other experiments have shown that the dicsks
need 'to be exposeed to IAA only during the first
24 hours; diskis tranisferred to waiter after 24 hourrs
oif exposuire to IAA grow about the same ais those
kept in IAA. Thus it appears thalt auxin-induotion
of cell enlargement, not cell enlargement itself,
reqtuires RNA synthesis in the tutber disks.
To determin,e if protein synthesis is still necessary during the growth of the diisks 24 or more
houirs afiter the addiition of aulxin, experiment,s were
performed with other inhibitors.
Chloramphenicol, puromycin and cycloheximide
all inhibited the auxin-indutced growth of the disks
when addedl 24 hoturs after IAA. The exact tiTnecoturse of the appearance of inhibition differedl
slightly among the inhibitors. Some of this d,ifference coutld be due to differing rates of penetration and, therefore, in the time requiired to affect
protein synthesis. There seems little dlouibt that
some protein synithesis, and presuma!bly ancillary
metaabolism, is requlired even 24 houirs after IAA
is added. It also follow-s that azaG and act D do
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14,'-'
PLANT PHYSIOLOGY
not interfere direcitly with pro'teiu syinthesi's and
metia(bollic reactions which are reqtired for growth
by the diisks after the first 24 hours.
The actions of puromycin and chlorampheuicol
oIn pltants have been discussed previously (32, 33)
and little need be added here except to note that
the affTinity of chloramphenicol for ribosomes from
organisms which are relaitively resistant to the
antibiobic seemis to be considerably less (see 39).
Cycloheximide (Ac,tidione) has been shown to
inhilbit protein synthesis in severiall organisms including plants (16, 39), but some ribosomail preparations from plants seem to be insensitive (23, 35).
For some ti,me iit has been known th'at storage
tissule m'ay uindergo some metabolic changes following excision. For example, changes in respiration
rate take pliace in artich'oke tuber disks, and thiis
process i's probalbly complete 24 houlrs afiter excisionl
(42). In order to avoid or minimize possible
effects of interference wvith these (aging processes,
which may be important for subbseqtlenit growth of
the disks (24, 40), these studies were started about
21. hours after excision.
The djisks were 'aged aIn additional 24 houtirs
(making a total of 48 hrs af,ter exciislion) to test the
1)ossibil:i'ty that the esca,pe o'f the growth res-ponse
from a requirement for RNA synthesis which is
observed in articihoke disks is due to aux,in aud not
merely to the completion of some RNA sy nthesisrequiring aging process which 'progresses with or
wiithouit auxin. These disks still respond to auxin:
however, the liaig is longer. Again, azaG radded
simulltaneously wiith IAA completely inh.ibit's growth
induction, and azaG added 24 hauirs after IAA
does not. These experiments tenid to rtule ouit the
possibilihty th'alt the requirement for RNA synthesis
during the first 24 hours of exposure tio aiixiln is
related to soime necessa'ry p)rocess rather than to
the action of auxinl.
Auxin also promotes resp:lrat.iion in soome tissues
where it causes cell enlargement, buit not always
(see 7). The most striking effec'ts of auixini oni
respirabtion hav,e been observed in artichoke tuber
dissks ('see 33, 42) an'd 'tobacco pith (29) where these
effect's seem to precede that on growth. This
raises the po'ssi'bil'iFty that aux'in may act by stimulaiting
respiratory
actbivity
or
related
metiabolic
Aerob.ic respiration is likewise generally
necessiary for auxin action ('see 7) with the possible exception of submerged rice coleoptiles which
may providle ATP through fermenbaition (14).
Since FU strongly suppresses respiraltion in atixintreateed disks wilthout inh'ibiting growth, auix'in
probably does not stiimulate growth by increasing
the activiity of t-he metabolic machinery connected
systems.
with respiration.
The auxin--regulated growith of corn cole-optile
section's is discussed separately here, becau,se it
shows some important (lifferences from that of
artichoke tuber d'isks. The lag l)er.o'd in their
response to auxin is les than an houtr compared
with more than 5 hours for the tuber disks. A's in
sevetral other tissues (,see 34), the growth of corn
coleopti,les i,s not inhibi,ted by azaG. Dike soybean
hypo,coftyl (15) and green pea stem sections (6)
the time required for the inhibi;tory effect of act D
to become apparent in coileopti-les does not increase
siign,ificanltly wvhen act D is add-fedl after the IAA.
A possible ex'p'anaition of the differences betweein
corn coleoptiles and artichoke disks is that the
mRNA which is induced by auxnii is relatively
short-lived in 'the former tissuie and minust be svnthesized continuously in order to support growth.
At 50 mg/lite,r act D completely sup)presses growth
in arbichoke disks, buit this and higlher concentrations do not completely suppress the growth stim-ulation by IAA in the coileoptile sect.ons uinless they
are preincubated in act D for 6 houirs or longer.
Similar delays in the inhibiltory effect oif act D
have been reported for several tisstues. (6, 10,17,
27, 36) w,ith lower concentrations of act D. One
explanation is that act D is merely slow in penetratbing, for it is a large, charged molecule. If
slow penetration of the act D were the sole cauise
of the la,g, then the length of the lag oulght to be
stronglx influlenced by the concentrati.on of act D,
bult above 25 mg/liter, an increased concentration
of ac-t D does not greaitly decrease the time
required for ant act D to inhi'bift. Ful,rthermore, iu
oat coleoptile sectilon's increasing the concentrattion of act D above 25 mg/lfiter produicees little or
no increase in the inhibiltion of auixin-induiced
growlth mea'suredl a't 24 hours (see fig 4 in 32).
Similar results have been obtained with corn coleoptilte secti'on's. Act D ait 25 mg/liter does inhibit
RNA synthesis in the corn coleoptile sections, but
it is dicfficulllt to be sure exacitly how much becautse
of interference wit'h uptake of the labeled precturssor.
Somewhat di fferen't results were obtained wiith
corrdycepin (3'-deoxyadeno'sin'e), whiich terminates
syn-thesis of RNA chains by getting incorporated
thereby leavingf, the end of the chain without the
3'OH group nieeded for further elongation (see 11).
If cordycepin inhiibits growth throutgh an effect oni
nucleic aicid synthesis, then the lag in the action of
act D is not dIuie to the persiistence of mRNA which
mu,st be degraldled before inhibition occurs. The
cause of the dellay observed wiith act D may noit be
simply slowness in penetration butt some yet iinknoxvn factors involved in it,s action.
In this study preliminary experiments were
performed oni the effiect of IAA on incorporation
of 32P-orrth'opho,sph'a'te inito RNA. The promotion
of 32P incorporation by IAA wias very rapid in
tuber dIsks but slow in coleoptile sections. The
distribultion of 32P in the nucleoitides from hyvlrolvze(d RNA was uised as ain approxtimation of the
overa,4ll base composition of tihe RNA synthesized
(see A\e-thods). After incubation with 32P-,orthophosphate, ranging from 10 to 30 mintutes, the
32p base composition of the RNA in the disks
closely resembled that of ribosomall RNA (see 37).
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1493
certain polypeptide chains, the hormone beinig bounid
to the N-terminal of some protein (1). However,
certain svntheti.c compounds such as naphthalene-1nitromethane, indole-3-methane-su-lphoniic acidl an(l
5i-( indole-3-methyl) tetrazole appear to be auixins
(see p 67-69 in 2) btit have acidic grotups which
N-oo)EN-RNA. SYNETHESIS AND AU-XI-N ACTIO-N
Although IAA in some cavseS greatly increases the
amount Of 32p incorporated, it does not significantly change the base composition of the RNA
accumulated. In the artichoke diisks used here promotion of uiptake of 32p by IAA probably accotunts
for some or all of the increased incorporation.
This illustrates a common difficuilty in the uise
of tagged compounds supplied externally to measture
rates of processes inside whole cells or tisstues.
For thlis rea,son iit is important to determine the
effect of trea'tments uised on uiptake of the labeled
prectursors. An additional difficuilty in interpreting
the results of experiments oIn precuirsor tiptake may
arise from the possibillity that there is more thain 1
po,ol of the precursor and that the treatments may
affect the movement or the specific activity of the
precursor inside the cell. Slince these experiments
were carried out, more detailed analyses have been
reported using MAK columns or stucrose grad,lents
to fractionate the RNA (4,10, 25, 44). Ustually,
the auixin promotes 32p incorporation (10, 25, 44)
buit it may inh'ibilt (4). In some cases the different
fractions seemed to be affected eq1allyl (10, 25)
anid in others tunequally (4,44).
As with soybean hypocotyl sections (16,17) and
oat coleoptile secftions (26), FU suppresses RNA
synthesis in artichoke ttuber disks and corn coleoptile sections without interferiing with IAA-induiced
growth. Since FU changes the 32p base compositioln of the RNA synthesized, it apparently acts by
selecitively stluppressing some types of RNA synthesis, probalbly ribosomal anid tranisfer RNA.
FU is known to selectively inhibit riboisomal and
sometimes transfer RNA syinthesis in a variety of
organisms (16, 17, 19). Mloreover, incorporation of
FU into RNA does not necessarily redutce its ability
to function as a temip'"ate for protein synthesis (9).
Even in the presence of 4 ma.r FU, where RNA
synthesis is greatly inhi,Wbtedclai( the backgrouin(d
of RNA synthesis unnecessary for auxi:.n actioll is
greatly reduced, IAA stimullated 32P incorporation
wi,thoutit altering sg-nlificantlv the 32P base conli)os:tion. Again, however, IAA. stimuflated the uiptake
of 32P into the disks.
The data presented in this paper anid the work
of Key and Ingle (17) and, more recently that of
MIasuda et al. (26) support the notion that the
fraction of RNA synithesis which I.mits cell enlargement may he relatively mninor quantitatively.
Qulite likely, the synithesis or supply of ribosomal
or transfer RNA does not limiit growth in the
systems studied here whereas that of mRNA or
some other type of RNA probably is limiting.
The datta presente(d in this paper are conisistent
with the idea that auixin acts by induction of sonme
RNA synthesis, presumably mRNA, whiich resullts
in the synthesis of newF enzymes and ultimately
modifica.tion of the cell wall allowing cell expansion. As has been pointled ouit above, there are
other possible alternatives. For example, it has
been proposed that auxin mav act as an initiator of
seem tunlikely to replace a carboxyl group to form
the necessary peptide linkage. If auxin does act
as a chaiin initiator, then the role of mRNA synthesis might be to maintain an adequate amouint of
the RNA template dutring induction.
A-n objection which can be raised against these
theories is the very rapid effect of IAA on protoplasmic streaming, which occuirs abouit 1 minulte
after treatment (see p) 256 in 7). This effect
seems too fast to represent induction of mRNA,
enzvme synthesis and finalIly changes in the cell
wall. Unfortuinately, it remainis tunclear what bearing, if any, the effect on protoplasm)ic streangi
has on cell enlargement (see 7). In the final
analysis, clear proof of whether or not aulxin cauises
cell expansion by induicing the synthes,is of new
enzymes will requ,ire the identifica,tion of the
chemic,al or phy-sical changes in the cell wall and1
the effect of auixin on the enzymes responsible for
these changes.
Acknowledgment
The auithor is indebted to Merck, Sharpe and Dohme
Laboratories for the actinomycin D and to HofffmannlLaRoche. Inic., for the 5-fluorouracil and 5-fluorodeoxvuridive.
The inlterest, hospitality, andcI advice of Prof. R.
Brownvi and(l Dr. U. Loening of the Botany Department,
University of Edinburglh are gratefully acknowledged.
The autlhor also expresses his appreciation to Dr. Alfred
Sus,sman for reading anid criticizinig this mnanuiscript.
1.
Literature Cited
ARM STRO;NG, D. J. 1966. Hypothesis concerning
the meclhanism of auxiin action. Proc. Natl. Acad.
Sci. U. S. 56: 64-6.
2. AUDUs, L. J. 1959. Planit grrowth substances. Interscience Publishers, New York.
3. BANCROFT, J. B. AND J. L. KEY. 1964. Effect of
actinomycin D anld ethylenediamine tetraacetic
acidl oni the multiplicationl of a plant virus in etiolated soybeani hypocotyls. 'Nature 202: 729-30.
4. CARPENTER, WV. J. G. AND J. H. CHERRY. 1966.
Effects of protein inhibitors and auxin on nucleic
aci(l metabolism in peanut cotyledonls. Plant
Phy-siol. 41: 919-22.
5. CLICK. R. C. AND D. P. HACKETT. 1963. The
role of protein and(l nucleic acid synthesis in the
developmelnt of respiration in potato tuber slices.
Proc. Natl. Acad. Sci. U. S. 50: 243-50.
6. DF HERTOGH, A. A., D. C. M/ICCUNE, J. BROWN.
AND D. ANTOINE. 1965. The effect of ailtagonists of RNA and protein biosynthesis onl IAX
an1d 2,4-D induced growth of green pea steml sections. Contrib. Boyce Thompson Inst. 23: 23-32.
A. WV. AND WV. K. PURVES. 1960. The
7. GALSTOAN
mechanism of action of auxin. Ann. Rev. Plant
Physiol. 11: 239-76.
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1 .750(
PLANT PH YSIOLOGY'
8. GJRUNBERGER, D).. C. O'NEAL, AND M. NIRENBERG.
1966. Stimulation of ami11ino acid incorporation
inlto proteinl bV polyuridylic-8-azaguanm-lic acid.
Bioclhemii. Biophys. Acta 119: 581-85.
9. GTRUNBERG- MIANAGO, MI. AND A. ML. MICHELSON.
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10. HAIMILTON
), T. 1HI., R. J. M\IOORE. A. F. Ru AISEY.
A\. R. \MEANS, AN\) A. R. SCHRANK. 1965. Stimilaltion of svnthesis of ribonucleic acid in sub-
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12.
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19.
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21
22.
23.
atcetic acid. Natuire 208: 1180-83.
H1EIDDELBERGER, C. 1963. Bioclhemiiical mechanisms
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1HEYES, J. K. 1963. TIlhe effects of 8-az'i guanine
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HONIG, (I. R. ANI) M\. RABINOVITZ. 1966. Inihi
hition bh ac.tinomIvxcini 1) of oxidation-dependent
hiosvnthesis in Sarcoma 37 ascites cells. T. Biol.
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KEFFORD, N. P. 1962. Auxin-gibberellin interaction in rice coleoptile elongation. Plant Physiol.
37: 380-86.
KEY, J. L. 1964. Ribonucleic acid and p)rOtein synthesis as essentiall processes for cell elongation.
Ibid. 39: 365-70.
KEY, J. L. 1966. EIffects of puriine and l)yrimi(linie ,nalogues on growthl and RNA iimetabolism
in the soybean hypocotvl-the selective actioni of
5-fluorouracil. Ibid. 41: 1257-64.
K\ItY J. L. ANI) J. INGI.I. 1964. Requtiremlenit for
the synthesis of DNA-like RNA for growth of
excisQd plant tissule. FProc. N\atl. Acad. Sci. U.S.
52: 1382488.
KITAZUM2IE, Y. ANI) M. YCAS. 1963. The Ialculated composition of newly synilthesized yeast riboniucleic acid. Biochem. Biophvs. Acta 76: 391400.
ID KLOET, S. R. A NID P. J. STRIJKERT. 1966. Se
lective inhihition of ribosomal RNA synthesis in
Saccharomy1ccs. carlsbergensis by 5-fluorouracil.
Biocheml. Biophys. Res. Conmmun. 23: 49-55.
LEAVER, C. J. ANI) J. EDELMAN. 1965. AIntibiotics
as a mleanis of control of bacterial contaminiation
of storagc tissuIC disks. Natture 207: 1000-01.
LOENING, U. E. 1965. The synthesis of messeniger
anld( rihosomal RNA in pea seedlings as detected
hy electrophoresis. l1-roc. Rov. Soc. London Ser.
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I\fAGASANIK, B. 1955. Isolation anl(l comiiposition
of th,e pentose nucleic acids and of tlle corresponding niucleoproteiiis. In: The Nuclcic Acids I.
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MfARCUS A. AND J. FEETIEY. 1966. Rihosome actiVation and polvsome formation in vitro: re(uirenmen,t for ATI'. Proc. N at!. X\cad. Sci. 'U.S. 56:
1770-77.
24. A\IASUDA. Y. 1966. Auxin-induced growth of tuber
tissue of Jerusalem artichoke. 11. The relation
to protein and nuclecitc lid mletalolism. Plant
Cell Phvsiol. 7: 75-91.
25. _MASLDA. Y. 1966. -Nil.\m indcedgoxlth of tn(l,e
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41.
42.
43.
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tissue of Jer-ussalemii artichloke. III. Effect of
actinomycin D oni auixin-iii(liced expansioni growth
and RNA synthesis. Ibid. 573-81.
MASUDA, Y., G. SETT1ERFIELD, AND S. T. BAYLEY.
1966. Rihonticleic acldi1ietabolismi and cell expsansion ill Wlat coleoptile. Ilid. 7: 243-62.
MIASUDIA, Y. AN-D S. \V ADA. 1966. Requiremueit of
R'NA for the auxi-inndtced elongation of oat
coleoptile. Physiol. Plantarum 19: 1055-63.
-McLEAN. I. W'., JR., J. L. SCHwN'AB, A. B. HILLEG(AS, ANDDA. S. SCHLINGMAIN. 1949. Susceptibilitv of micro-organisms to chloramiiphenicol
(chloromycetin'. J. Clin. Invest. 28: 953-63.
NEWCO-MB, E. H. 1960. I)issociationi of the effects
of acuxini oni metaholism anid growth of cultured
tobacco pith. Physiol. Plantartilum 13: 459-67.
NITSAN, J. AND A. LA-NG. 1966. DNA synithesis
in the elongatiing niondividing cells of the lentil
epicotv l anld its promotion 1- gibberellin. Plant
Phvsiol. 41: 965-70.
NooD_N., L. 1). 1966. Stud(ies of the role of nucleic acid sy-nlthesis in auxin ini(ductioln of cell
enlar-emiient. Ibid. 41: xlN-i.
NOODkN, L. I). AND 1K. V. THIMANN. 1963. Ev idenlce for a requirement for protein synthesis for
atuxmi-induced cell enlargenili-nit. Proc. Natl. Acad.
Sci. U.S. 50: 194-200.
NOODPN, L. D. AND K. V. THIMANN. 1965. Inlibition of protein sy nthesis and of auxin-induced
growth b-v chloramiphenlicol. Plant Physiol. 40:
193-201.
NOODkN, L. D. \ND K. V. TlM\ANN. 1966.
The
actionI of inhibitors of RNA and p)roteini s!tnthesis
oni cell enlargement. Ibid. 41: 157-64.
PARISI, B. A.ND 0. CIFERRI. 1966. Proteini svnthesis bx- cell-frce extracts from castor bean
seedlings. I. Precparation and chlaracteristics of the
aamino acil incorporating system. Biochem. 5:
1638-45.
PENNY, P. AND) A. WN. GAI.STON. 1966. The kineties of inhibition of (tluxin-inidticed growth in
g-reeni pea stemil segments by actinomn cin D and
other substances. X-\m. J. Botany 53: 1-7.
P'OLLAIRD, C. J. 1964. The slpccificitx of ribosomal
ribonticleic acids of palints. Biochemil. Biophys.
Res. Commiuin. 17: 171-76.
S3.NGER, H. L. ANI) C. A. KNIGHT. 1963. Action
of actinomcCi D otn RNA synthesis in healthy1
and virus-inf ecte( tobacco leav es. Tbid. 13:
455-61.
SCHWEET, R. AND R. HEINTZ. 1966. ProteiIn sv/nthesis. Annii. Rev. Biochemii. 35: 723-58.
SETTERFIELD, G. 1963. Growvth regulationl in excise(l slices of Jerusalem artichoke tuber tissue.
Syml). Soc. Exptl. Biol. 17: 98-126.
SHIGEURA, H. T., G. E. BOXER. M. L. MELONI, AND
S. I). SA'MPSON. 1966. Structure-activitv relationship of some purine 3'-deoxyribonucjleoti(les.
Biochellm. : 994-1004.
SPERLING(; E. AND G. C. LATIES. 1963. The dependenlce of auxin -induced growth on auxin-in(lep)endent metabolic changes in slices of stora -e
tissue. Plant Physiol. 38: 546-50.
STUDZINSI.u G. P. AND R. BASERGA. 1966. Instability of lpuromycin. 'Nature 212: 196-97.
TESTER, C. F. AND L. S. D)URE III. 1967. Nucleic
aicid svrnthesis d(uring the hormone-stimlulated
growth of exciscd oat coleoptiles. BPiocheiii. 6:
2532-38.
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