STABY

STABY - OSU
;:Ji.i-1m-im. I'hvsi.,1. I'flaii/iMi 1(58, s. -_'!i!i :((n; (197r»)
On the Fading of an Ephemeral Flower
fc
BllUNO BaUMOAUTNBH1), JaKOIJ IllJKTKRaild I'llll.l.Ti: Mat,,.,;
Department ol General Hotnny, Swiss Federal uistitiitc of Tecl.nol.."V Zi.,,,1,
and Section of Biochemistry, Swiss Federal Research Station for Arboriculture'
\ iticultnrc and Horticulture, Wiidcnswil, Switzerland
;.;,v Term linl.-x: curling reaction, ethylene, protein synthesis, tumor,
ifOBWCU tricolor.
cell wall modification;
Sun unary
The mccharmm in the ribs which leads to the curling „,, of the corolla „f I,
,,,,„,„, w,s
.vfst.gatcd. I seems that two processes are involved in .Ins movement. Dne |„ differential hiw
t*. he nbs start to rol up. At the same time extracellular enay scause „r,,,ucti rth
0m in the structure of the „U walls. This modificationenables the ribs , ,, „1P ,.,„.„„!
7
• nrori'ss.
[ittroductioii
Professor Hotiibs celebrating his jubilee may Find ii nol too [awful (|,.,| 0lir eon•rihun,,. deals with the fading of n Howcr. Unworn, Ibe mornim> -l.nv ,-,,, ;i|s« |„•cnwl as ameaning.nl symbol: lading, seneseouee and abscission of'flowers is ala\$ the prelude to anthesis of a new scl or blossoms.
Ephemeral flowers nro rewarding objects for ll'ic siudv of drvelo. ,l!;1| ,mwvviv*
.hicli normally are rather slow. They are parlicularly suitable lor invesligaiimMhe
J,rioiis phenomena associated vvilb railing and senescence.
The fading of the coroltaof fpomora tricolor starts in the earlv a!-,-,,..,, •l(l(. (|.lY
lllott'crmg and is largely completed in Ibe late evening. Kxtemallv visible svmpfon.s
(fading ...Hud, the curling up of the corolla (Fig, I) and color changes from bine hi
,rple. 1» term* ol physiological proeossos associated will, ,h, Slm-llv lin.ed ,.,,,•„.,
| ... Ipomoca Iricohj: Kvidentl,,, the ribs „,r,,-,, >•/«,• (,„ „ir ,.„,,,„,, uf „„ ;.,.. ^^^
„Present address: University of California, San Diego, Depl. „i m„|„,x ,,„: „,, , , ,„„ ,
. :•:'-'
'.: .
• • -: ..•»....
•.......
• -:•
!'.. I! \iMc.\nT.\i:it. .!. 111< rtk it ami P. Matilk
:ioo
the degradation of biopolymers proson1 in the cytoplasm and in the cell walls as wo
as the cxporl of the breakdown products from (he corolla have been studied (Matiie
and WiNKKM'.Acn 1070, \Vii:mki:n-(;i:iii:i<; el al. 1974). These processes appear to l>o
highly organized, as is convincingly demonstrated by Ihe fact, thai certain hydrolase
such as RNase, /J-glncosidnsc and /?-galaetosidasc arc synthesized dc novo whilst tnf
total protein content nf the corolla is rapidly declining! Wik.mkkx and Wii-:mki:\ I!'1,1'
Ii.\rmcauim:i; el al. 1975). This illustrates the importance of cellular compartiHC"'
latino of lytic processes.
I
tacl
if tr
to e.
This
(Ke
The surprisingly strict timing of the development of the fptimucH corolla points'"
the existence of a controlling mechanism. In fact, Kkxpk and li.\r.M(s.vitT.\Kit (197I1
were able to demonstrate that the curling up of the ageing corolla is induced by exogoi'O"
mis clhvlcnc: moreover, the curling was shown to coincide with a burst of endogenic"*
ethylene. The data available up In now indicates a rather complex effcel of ethvlciifNot only the curling np <>|" the corolla bill also metabolic processes such as the synthesis
of liNase are controlled by ethylene.
1
a, 3-'.;
Detailed work on the physiology of ageing is rendered more difficult by the lictpro- J
geneity of higher plant organs such as the corolla of fpomoru. After IIaxson and l\HN'lfI
Q
(J975) detected lli.it isolated segments For ribs (the particular tissue which isrcspo.NSiW'
Cn
C
o
30'
for the curling up of the corolla) behave in vitro as they do in vivo, work with a coin*
paralively simple system became possible. In the present paper we report on result
26i
from experiments concerning the mechanism of curling up of rib segments.
22i
Material and Methods
18'
Plant iua to rial,
Ipomom tricolor (I'av.) was grown in the greenhouse nn garden soil. "
su111 it11t. i hi' plants were, kept under natural photoperiodic conditions, whereas in winter, siipi'"'
meiitary illuniinatimi was provided in ;i pimtoperiod nf i I |j.
Isolated segments n[ ribs.
Klowcrs were collected after .m thesis and segments of''" ;
nhs, to mm in lt*n*;tIt, were exeised accordiiii: to llvxsnx .-mil Kknoi; |l!>7.">). A snip of intereosl
h,.:
tissue, 1 nun in width, remained attached to each side of the rib.
Si ts nf exeised segment's of ribs wen' floated on ."> ml of solution in l'.'i ml Ivrlenmevri flask" >'"!"
Wg.
pared with rubber eaps. Appropriate volumes of ithylene wen1 injeeted into the gas phase via,I'1'
curli.
rubber eaps. The incubations uric perforincil id l\v V in the dark.
:
:
i
Measurement of the anjrles cf culling.
The degree of curling was determined ',:
measuring the angle as indicated in h'ig. 2(11 vxsnx and Ki-:x m. I{>".">). The straight ribs of the nppflw
rcaci
corolla correspond In an angle \
this
exhibit angles up to 100
ISO . After completion of the curling in vitro Hu isolated fi"5 J
lion
I 10 .
'I
Of c
Up i
'I
Iran
JS6"
norn
*
ot mm
# ii
180°
270°
rig. L\ Ihtcrviiuiilinn ufniiijles of vurlhuj in isolated tri/meitls <,[ ribs.
390°
lo (J,
Mieiii
ami
On the Fading of an Wphcmeral Flower
:i()i
wcUl
Results and Discussion
TILE
n be
Isolated segments of ribs begin to curl up at about the same lime of day as the in-
ases
(acl corolla begins to fade. In addition, they can be induced to curl up prematurely
l/tmited with 10 ppm ethylene. It appears from Fig. .*'> that after the tissue is exposed
pi ethylene about !)() min elapse before the angle of curling starts to increase rapidly.
the
975,
i-'ii-
This corresponds exactly to the lading reaction indii/ed by ethylene in the intact corolla
,Ki:xi)K and BauMGARTNER 1974).
f to
1)74)
Cjfy at: 0830
£20
i'Ue-
eoug
ICIIC.
c?380
•lesis
!
loro:ndi:
sible
300
•oin-
suits
260
220
•II.
C^ a: ony
Ill
time
;pplr-
-i
0900
1
1100
i_
-j
1300
•
•
1500
time of day
10U0
>.:.,p.
U00
1600
lime of day
I till'
costal
1200
fig. 3. Induction of curlimj by ethylene mid it? inhibition by cyclohexiniuie in snjmmts of ribs.
fig. 4. Determination ofthe optimal concentration of cycloheximide for inliibilimj tin rthylene-iudaced
(iirlimj of segments of ribs.
-a ill,.
.! ley
peiU'il
•I rills;
In order to investigate a possible involvement of protein synthesis in the purlin"
reaction triggered by ethylene the effect of cycloheximide was tested. Kitrprisinglv,
,1,1s inhibitor of protein synthesis at the level of translation causes a complete iuhibilion of ihe curling process if supplied to segments al aconcentration of l()/*g ml^g.:'.).
The same effect is obtained with a ten limes lower concentration, whilst at 0.M//g/ml
0fcycloheximide the response of the ribs to exogeneons ethylene as well as ihe curling
Up of the control segments are only partially inhibited (Kig, 4).
Tin- inhibitory effect of cycloheximide is perfectly reversible. If ihe segments are
transferred to distilled water alter various periods of treatment with cycloheximide a
normal ethylene induced curling reaction can be observed. The proecs? of curling seem-;
iudepend upon a continuous synthesis ..f protein. This can be concluded from experi
!!|(.nis in which segments wen- transferred from water lo ryclnhexiinidi' at ihe tinsel
jnd iii ihe curse nf ihe curling process, respectively (Kig. :*)). The rale nf eurliiu' is re
-
.
....
l
,
,-•..
..
•..:.•
,.
. . .
PwnwMBPWMBWBBp
H. llArSIIIAKTXKJI, .1. Ill 1:11 i; .,,1.1 I'. .\l Mill
,,uml ," m" "•'»'
I.v BO mill if cycloheximide is given :W mill after the nii«trf 1
curl.,,g. Ii the beginning of treatment coincides with the unset of curling the inhibit „!"'
is complete.
The nature "f tin-proteins \vln\-h havV aruiuMioi. in the pn)cess.»r curling is uukii.»«i
"I IIn- enzymes known In be synthesized de novo in the course of ratling of ih- c,,r..ll;i. |
tile
«t !• •
•Hi.' glycosidases(\Vii;itKi:x and Wikmkkx l»7fj) which are possiblv involved in rrl ; (i'0I
wall lytic processes deserve particular attention. Observation* reported bv IhxsoS ,
;iml Kk.\iik(MI7i ithe effecMoF/W Inpon the curling oF segments snggesl that, i.ulee.1. ' ,„,'.
,l"';,,',""! "r ,m,ml wixvmni c-cmlcl be an importaul Factor in the cHiug reaction. Like- ! ,.,','
wise, il,.- dependence of ihe ran- „l ,„,ling „„ temperature points |p Hip iuvoh .-mclil
oii/.Vines.
Km-t-lic »r/#ll nutl c-lliyh- MllieeurlingoFsrg„„Milsaresl.Mwniii Fig..; I: ;ii.,.e:.rHial Hie exposure -if the ribs ... cilrale-phosphat,. buFFer. /.II I.;,, has alum., ,!„' «i
c,rr,,cl ;,s ,l;is ""' "•'•''"""'"' with ethylene. A. ,,|| .,.:, ,|„ M „r ,,„.,;„„ ,, |luirkwl„
'''T^'11 VV,lh r,,"'',nl '" ""• i'lliylt-iM-iptNilftl control. b„i still premature as eou.n.ii
«•'"' •"'" m.trealed control. Aeouspieuous ell,,, ,,f ,,|| «,„ ,|„. ,,,,,. „,- ,„,.,;„„. mP
;lls" rn"" lllr (i;il;l n »''wl '»' I'fe. «'»• Since Ihe tissue is severelv damaged al /ill valrn*
Iwlow 4.5. Hi,, analysis of //Il effects is restricted to (he n.uge above ,,|| | ;, |, |„•v,(,,di'(l ",»I»I«' ' Vidence that (he elTrel of buffer solutions depends ,,„ ihe buff.-r enif
1300
1500
time of doy
!'*-"• I:i1"' "' '•-'/'/"/'"-''''"'''
0900
1100
1300
\itt
Ida:
<an
cap;
Lie;
r
,
03c
time ol
V t* ol rib, /„,/,„,,/ „•;„, ,.„, hll, hi
t
""?* NW""• ,M,:, ,M* "i,ll,,,N-"-'H-!l"-M.,n,,::l,llllf!ll,,sp,1,1,,I1/:ill,, Ir, .....
solutions confanun- IW:IM| „f ryeloheximidc a, vari.ms stages of enrliu,
Kfe.li. (Untinyofsnjuiclsof ribs i„ //„ ,„,. , ,,,„.,„,,
., ,
.
! i•_•.
.rn/»«
T|,,
i »n IIn K.-i.lii,;.' nf .in Kpl
ic unset uf
• inhibition
Ili.M'iWll,
;/n' curol/a,
.•il in cell
• hansox
.1. indeed,
:•" . Like1 elUi'Ul of
•'• appears
- iln- sa'nte
• markedly
compared
it ajipoara
i .<ll values
••••">• 11 has
bllUel" CO],.
nil |>'|i
:;o:;
tcntratiOJI in addition to pi J. |„ ihe presence of 0.01 Mbulkr Hie curling proceeds to
lilt' normal final angle of 400 440° only at the optimal ,,ll ..I I,/,. .\t /v|| li.;, ;,„d 7.:»
lk curling is incomplete in the presence, and does not even begin in ihe absence ,.i
fihyleiic. IF- however, the buffer concentration is \viU\i-n\ to i ,,iM i-oniplc-fc curlings
(All be observed over the entire range of ,,|| values tested (Kig. 7). I'ud.-r these eomlili.ais the/ill determines only ihe ousel audi//I I;..;, and (i.o) the initial rale nf eurlin".
Hanson and Kkxiie (1D75) investigated the ethylene-induced efflux of mier.uuolefulcs and ions from isolated segments of ribs. Shut acid buffer solutions have >miilar
Pffects on ihe curling process as has ihe treatment will, ethylene, il mav be concluded
lluil this eihylene-indnced efflux res. ills incham.es of ,,11 i„ ,|„. ,,,„ ,,,| space which, in turn,
fjaises the change of activities of mural enzymes. In the presence of buffer solul ions Hie
rhanges of ihe actual mural r\\ and the curling reaction should depend on ihe b.ilTer
rapacity. Our results obtained with iw«, buffer concentrations are compatible with this
idea. Moreover, recent results obtained by LOsciikh (unpublished) deinonslrah that'
ihescgments are capable or adjtisting the/ill of the solution on which iIioa ure float ii .
The ethylene-inducible i'1'll.ix of niicrouioleeules was interpreted bvll.i.xxox and
KkM'K (ISITfi) in terms of turgor changes which are ultimately responsible |o,- ||,e
curving and curling of ihe corolla ribs, h, fad. turgor changes' are involved in this
control
....
and
/
1mM A (0.0) M !
/a/
o.380
•
/' /n
.
',. 340
Na(
r-*
/
•
/'
260
0.1 M
. .
lan -
'.500
-
•
.-900
1100
l
1300
1500
♦
09U0
t'rne ol doy
• ''"il n,
RV-'- /'''"'"/
/ '*' ""'"'" "!' "I - !
.„ irbicb Uf 'i an floalril.
.0M
d
lien
1300
lin
' »/ »7» riu //„ ,,// .., //„ / . , ,o.Ol|| !/
|j2.s. /•//"•' "/' -V'"'i ">i tin rurliwj ofs,,j„„„h ••/ ribs.
ri,eMilurioos for floatini! the lissu. contained citrale-ph.Kpl,,.!. i,„ii. , ,0.001 M. ,,|l 1.., ;„„! . ,,,,.
litrurimis uf Na("l .is indie; i .'.
• «••;
HH5 ••"'.: ' .1 ...X.... MMMMhMWM
i •••<!•
, : 1
m
;;oi
II. llAt'MOAUTXKII, J. IllMtTKIl and I'. Matilk
ecl dogi
turgor |
process. Astrong inhibition and delay of curling occurs if I m.M buffer /)H 4.;") issiip*
plcmented will. 0.1 m NaCI. A tenfold lower concentration of NaCI has onlv asinal
effect, whereas in ihe presence of I MNaCI Ihe curling is completely blocked (Fig• •
Experiments in which the involvement of turgor changes and cell wall modification*
I''
Mont • •
to dot.-
curling
in the curling reaction were further investigated were performed with osmotica such*
glucose and sorbitol. If curled up segments (c\ .".00°| are exposed to I Mglucose !''•'
resulting reduction of turgor causes the reversion of curling (Fig. 9). However, theft"
H£_
lOppm Cft-
O -
H2°
^) 1Mglucose
f'hai
dcducft
'I'-pend
conditio
Periods
M glucose
'lemon-:
'iicnts n
POllcilt
lias bee
v-sQS Msorbitol
H,0
10ppmC2H4
H^
1.5 hr
H20
2.0 M sorbitol
lOppm C2HL o
^S l hf
15 hr
— 0
fit:, f). Effect of ijlucose on curled up aeymciils of ribs.
Effect of ;, temporary exposure to sorbitol of curled up segments of ribs showing (he complete
vnsiliility ,,f tIn- osmotic effect on curling.
I
version is incomplete; il proceeds only to a;i angle of about 250° and then conies'
a stop, Even in the presence of 2M sorbitol the probably complete compensation ol
turgor does not result in a complete reversion of curving of the segments. This find
demonstrates that the curling is associated with changes in the physical properties ol
the cell wall skeleton of the tissue. These changes are responsible for the shape of tfl«
tissue in the absence of turgor. Another means of demonstrating the occurrence of cell
wall changes is the killing of the cells. This was don.- by extracting segments at varioui
'I'llC lis-.;
stages of curling with hot 80% elhanol. The result is presented in h'ig. |0. || appear*
that extraction has a similar effect as has the exposure to osmotically active solution*
In all cases a reversion of curling for about 20 00 was observed. IIbviously. this limit-
'" ill! OKI
U'eci
HO
30min
90min
i
lOppm £>H.
' '30mirT O gOmin ^
J'
i
i
1
with wi
show a •
J
bo the ci
the ri,i:
turgor i
30 soc 80% ethonol
[
—
<Mh
;*
—
^
o
o
Kir. I", t'hnnyes ,,/ //„ deyree of curvature upon Ho ivlnitlhm uf sujmruls of ribs with hoi SO" vlhnd
IIn- extraction was performed at. various stages nf curliu» as indicated.
!
tissue 'i
of the ri
side of rl
Plasticity
Justin, :
'-'" liiochci
On the fading of an Ephemeral I'll
er /'I I 4.5 is suphas only a small
blocked (Fig. 8).
all modifications
;smotiea such as
I M glucose the
Iiowever. the re-
.'505
cd degree of reversion represents the elastic component of ihe cell wall structure which
turgor pressure transposes into curving. Another means of demonstrating the involve
ment of turgor is the cancelling of membrane scmipormenbility by exposing Ihoscmcnts
to detergents. In the presence of Triton-x-100 or sodium dodoeylsnliatc (0.1%) the
curling is completely inhibited.
Changes in the physical properties of ceil walls in the course of curling can also be
deduced from the Following experiments. The successful osmotic reversion of curling
depends on the period during which the segments are allowed to slav in the rolled up
condition. Fully curled up segments (a =400-440°) were incubated in water For
periods up to 2hand then transferred to I Mglucose. The results depicted in [<V. .11
demonstrate that the degree, of osmotic reversion is markedly reduced when tluTsegmentS remain in the Fully curled state for I h or longer. In other words, the elastic com
ponent of cell wall structure disappears gradually when the movement of the tissue
has been completed.
' ~^\ 1M glucose
120 rnin
-O
OOmin
••• i li«'
iplctc ri-
H20 3.5 hr
lOppm C2H,
D^
^GOmin
^5min
.d Ihen comes to
compensation (,f
ieal properties rjj
the shape of the
if. 10. it appears
active solutions.
•oiisly. this limit.
Cy
" Oi „.„, ~C/3o~in- O
ois. This finding
iceui'renee of ce||
ooiii- at various
W 601
DIM glueur.e —>.
S^ir,
^--/ 120 min
'(J
foil. Stabilization ofthe final */*«/« ofcurled op scymeuts uf ribs.
mtissue was allowed In stay in the curled up stale fo. vtiri.nu penuds „| |„M, ,,,,1 .!„•„ suhjected
10 al! osmotic treat incut.
Recent work done by llmi.NKU (unpublished) suggests thai onlv a fraction of the
,isstic u<vd in the above experiments is responsible For Ihe curling.'lOxcised segments
rfthc ndgCS to half the depth of the leaf exhibit the same curling reaction as observed
,iih whole ribs. The histological examination of longitudinal sections of these ridges
••!",u':l C0,»P»U*1 l^sue c posed of long, spindle shaped cells. Curling must therefore
»the consequence of differential growth of these cells on the inner and outer side 0f
frridges. We postulate that the initial curving of Ihe (issue is caused bv differential
„igpr changes which result in the elastic extension of cells on (ho outer side (ventral
' """•• flhn,,i,l
s*of the leaf) of the ridges. Kurlher extension requires Ihe enhancement of cell wall
,|,sticilv which possibly involves the action of cell wall hlie en/.vmes. (Jonlinuous ad-
^tinenl ol Ihe It.rgor and of cell wall modifications sec, |„ be necessary For the com, niofll'iil. I'liyxiul. I'lliiiui'ii. Ilil. O.-.
•
.
.
:
306
It. ItAOMOAUTNi it, .1. liuiiTKR and P. Matii.i:. On the Fading of an Ephemeral Flower
plctc curling. At the end of this process the elasticity of the cell walls is reduced?*1
'"'"•lien. p|n
that the final shape of Ihe tissue is stabilized.
Ethylene appears to induce a complex of metabolic processes associated with fcUli":
of the Ipomoeu corolla. Apart from turgor changes which are possiblvassociated^
I'll! [I
changes of permeability properties of the tonoplasts (Haxsox and Kkxim:. 1*975) th1
synthesis of enzymes is controlled by this hormone. It is likely thai Ihe postulated'H
wall modifications involved in the curling of ribs comprehend both degradation «"'
synthesis of cell wall constituents. In any case the fading of the ephemeral IpuW*
appears to be Far from being the uncontrolled decay of an organ. It appears as the fin*1
phase of development which is as highly organized and strictly controlled as is tlici'
velopnienl of Ihe flower primordia leading to Ihe beautiful flower.
K">- Terni
I
Acknowledgements
'
The present work was supported by the Swiss National Science Foundation, hv the Ken ten
funds I'jTJIZ unit by .1. I*". Hurras ,v Oie., Manufacture de Cigarettes, Roncourt, Switzerland. TI
authors arc iiuleptod to Miss Sonja Turler and Mrs. Dcirti Furrer for their help with the hi.ohimi'F
ami to Mr. I'aul Frick who skillfully cultured the plants.
The foiutli
120
.,.,
...
W
r,,o pathwavs
,||vPrge when .
tw°siib-t..rniii
i
l|,;if is presi nl
References
t'MCAKTNKit, I!.. Kkmii . II., and Matii.k. I'ii.. RXnsp in ageing Japanese moruiii!: «;lorv. I'' 1'
'
•
r'siol. 55, 734
" that stage.
'" hring „I, „,
••illrl
787 (I'.175).
., A. D., and Kk.vuk, II., Fthylenc-cnhanced inn and sucrose efflux in mornim; pUw\ fl"*1'
''Uriels ti
"vl'i'i itne :•; || ,
•. Plant Physiol. 53, (i(j3—609 (lf)7f>).
II., and l!.\iM<i aii im at, I!., Regulation of ageing in flower.-, of fjiomoea hicolor hv i thvli'"'
Hkntytd, '-'7'.) 28!1 (l!»7-l).
I'll., ami WiMvi.Mt \i ii, I'., Fuiictinn id lysnsoincs and lysosomal in the seiicsciug n>t""J
e morning glory i Ipomoi <i purpart a). .1. cxp. lint. 22, i.">ii
711 (l!)7l).
'.K-firinuwa, V. Wikmkkn, A., mid Matii.k. I'ii., Mobilisation von Zcllwands'ofiin in"'
wcJJcmulpn lllute von Ipouioen tricolor (Cav.). IManta 115. "Jin 3t)7 (107-t).
\Vjj<jikkx, V., mid Wii:mm:\. A., Diclitcmarkiciuui: von //-(Jlycosidasen in wclkcndi u I'.lutcn v'Ipomoea tricolor (Guv.). 'A. I'Nair/enphysiol. 75, IN) IDO {I!l7y).
'"Pinnate ,-,.,.
"laity speeies
"f bipinnate
Received February 10, I'.'T.'i.
Aversion fr<
Autlior's address: Dr. II. I>.\i• mc;.\kr.xi:o, University of California, San Diego, Dcpaitmcnl
Biology, I'lii; ion, La Julia, California 1)2037 (USA), I'm!. Dr. I'll. Mutile, Department of Gcn«
Jlot.-inv, Swiss Federal Institute of Technology, Zurich and Dr. .1. Iluitrini Section of RiocUentisW?
"f the envin,
Swiss Federal Research St aIion For Arboriculture, Viticulture ami llortii illtu re, Witdenswil (SwiW'
land).
N
Striking f
l(,ast in ti,,. ,_.
naturally in,~.
;i niimlicr of
fecting leafs'
H,li'H.). |„ s.
C'nulga'-j I1
f«c first four
(c. 20T) ih.
•'lIKKC,.; 1070
"'pinnate (Fie
I :."
effect of lemp
20'