Inhibition by Sethoxydim of Chloroplast Biogenesis,
Development and Replication in Barley Seedlings
H artm u t K. L ichtenthaler and D ieter M eier
Botanisches Institut der Universität, Kaiserstraße 12, D-7500 Karlsruhe 1, Bundesrepublik
Deutschland
Z. Naturforsch. 39 c, 115 —122 (1984); received N ovem ber 17, 1983
Chloroplast Biogenesis, Inhibition of Chloroplast Replication, Sethoxydim.
Sun-Type Chloroplast
Sethoxydim not only blocks leaf growth and developm ent of barley seedlings but also inhibits
chloroplast biogenesis at all stages of development from proplastids to prochloroplasts, young
and mature chloroplasts. Not only thylakoid synthesis, thylakoid multiplication and grana for­
mation are affected, but also chloroplast replication. The chloroplasts o f secondary leaves
which before the sethoxydim application are in the stage o f young, developing chloroplasts,
remain in this differentiation stage when treated with sethoxydim. With their "ultrastructural
characteristics (e.g. lower stacking degree, higher proportion o f exposed mem branes, a lower
thylakoid frequency etc.) they resemble sun-type chloroplasts. In the shoot meristem
sethoxydim-treated plants contain only proplastids, whereas the plastids in the shoot meristem o f
control plants are already in the developmental stage between prochloroplasts to young
chloroplasts. Mesophyll cells o f sethoxydim-treated plants contain only one third of the
chloroplasts found in the controls.
Introduction
Sethoxydim ,
a
cyclohexendione
derivative
(N P 5 5 ; BAS-9052 O H ), (Fig. 1) is a very efficient
herbicide for postem ergence control o f an n u al and
perenniel grass w eeds (Avena fatua, Agropvron
repens, Alopecurus myosuroides, Echinochloa crusgalli, Cynpodon dactylon, Sorghum halepense etc.) in
a w ide range o f dicotyledonous crop plants (soy­
beans, flax, sugar beet, pea nuts, tom atos, cotton)
[1], The structurally related substance alloxydim
Setho xydim ;
*fH3
h 2c ^
s^
0H
*fH3
N - 0 - C H 2- C H 3
A - c - c h 2- c h 2- c h 3
c h ^ ch
A llo x y d im :
(Enol-Form )
0H
(N P 4 8 ) exhibits very sim ilar effects an d is also
applied in b ro ad leaf crops [2]. A bsorbed by the
leaves the system ic h erbicide sethoxydim is rapidly
translocated (phloem ) to the m eristem atic zones o f
leaves, roots an d the shoot apex w here it blocks
grow th an d yields necrotic tissue.
The reason for its selectivity tow ards grass w eeds
an d also tow ards gram in eo u s crop plants, seem s to
be a differential u p tak e an d a rap id break d o w n o f
sethoxydim in b ro a d le a f crops. The exact m ode o f
action o f sethoxydim or alloxydim are at present,
how ever, not know n [2 -4 ],
Plastid (chloroplast) d ifferen tiatio n an d m u ltip li­
cation are basic p aram eters o f le a f grow th and
developm ent. In o rd er to o b ta in m ore in form ation
on its possible m ode o f actio n we stu d ied the effect
o f sethoxydim on ch lo ro p last biogenesis an d d evel­
o p m en t in barley seedlings.
N - 0 ~ C H 2- C H = C H 2
C H 2 —CH 2— CH 3
CH3 / U * 0
ch3 1
C0 0 - C H 3
Fig. 1. Chemical structures of the two related herbicides
sethoxydim and alloxydim.
Reprint requests to Prof. Dr. H. K. Lichtenthaler
0341-0382/84/0100-0115 S 01.30/0
M aterials and M ethods
Barley seedlings ( Hordeum vulgare L. cv. Breuns
Villa) were g erm in ated for 3 days in the dark then
placed in a d ay -n ig h t rhythm (14 h H -10 h) and
grown (22 °C, 65% relative h um idity) at a m edium
light intensity o f 20 W - m ' 2 ( ~ 6 klux). O n the 8 th
day the plants w ere sprayed w ith sethoxydim in a
dose o f 300 g • h a ' 1, w hich co rresponds to spraying
Unauthenticated
Download Date | 6/17/17 4:37 AM
116
H. K. Lichtenthaler and D. Meier • Sethoxydim Inhibition of Chloroplast Biogenesis
alm ost fully been developed. The tertiary le a f being
an area o f 500 cm 2 w ith 10 ml o f a ca. 4.4 X 10- 4 m
form ed in the control plants from day 8 to day 1 1
sethoxydim solution.
after germ ination does not a p p e a r in the h erbicideOn day 11 le a f segm ents w ere infiltrated and
treated plants. This indicates that sethoxydim
fixed in a buffered 5% g lu tard iald eh y d e (pH 7.4) at
prim arily affects the growing parts o f leaves.
4 °C , postfixed in 2% 0 s 0 4 and further p rep a re d for
L eaf d evelopm ent o f barley proceeds like in
the electron-m icroscope investigation as described
o th er gram ineous plants, via an intercalaric m eri[5]. Poststaining w ith 10% uranyl acetate and lead
stem on the leaf basis. At the tim e o f h erbicide
citrate was applied [6 ], The biom etrical analyses
treatm en t the top o f the secondary le a f blade
(Table 1) were p erform ed on 30 m edian lo n g itu ­
dinal chloroplast sections. The stacking degree o f contains the old er m ature le af cells w ith fully
developed chloroplasts. In the m iddle le a f region o f
thylakoids was d eterm in ed by m easuring the total
the control plants there occurs from d ay 8 to 1 1
length o f thylakoids and the length o f stacked
the final cell elongation an d the plastids go th ro u g h
thylakoid regions. From the length and the w idth o f
the final steps o f chloroplast d ev elo p m en t (fu rth er
m edian chloroplast sections we calculated the
thylakoid m ultiplication an d grana form ation). In
chloroplast volum e by assum ing an ellipsoidal
rotation body, revolving on the axis a. V = 4 /3 j i ■ the low er and younger p arts o f the b lad e o f the
secondary leaf the cells an d chloroplasts are in a
a • b2 (a = h a lf w idth an d b = h a lf length).
m uch earlier stage o f developm ent.
Since ad u lt green leaf cells contain a m uch
Results and D iscussion
higher n u m b er o f chloroplasts per cell th an the
The developm ent o f the le a f b lade o f the second ­ younger, m ore m eristem atic cells [7], o n e can
ary leaf o f 8 day old barley seedlings is blocked by
expect in this youngest p art o f the leaf b lad e ( 2 nd
spraying o f the plants w ith sethoxydim . This refers
leaf) not only d evelopm ental ch loroplast stages b e­
to growth in length an d in w idth o f the le a f blade
tw een proplastids to young chloroplasts, b u t also a
(Fig. 2). There is little effect on the prim ary leaf,
significantly low er n u m b e r o f plastids (ch lo ro ­
w hich at the tim e o f herbicide trea tm e n t had
plasts) per cell. In ord er to study th e effect o f
sethoxydim on chloroplast d evelopm ent we a n a l­
ysed the leaf m esophyll cells in the top, m id d le an d
low er region o f the second le af blade.
In the control plants the chloroplasts from the
upper, m iddle and low er p art o f the second le af
blade on 1 1 d ay old barley seedlings show ed a
sim ilar ultrastructure w ith well developed strom a
and grana thylakoids o f m atu re chloroplasts (Fig. 3).
The leaf tip (2nd leaf) o f the seth o x y d im -treated
plants did not show any difference to those o f
control plants. The m iddle and low er p arts o f the
leaf blade ( 2 nd leaf), how ever, co n tain ed ch lo ro ­
plasts which w ere not elongated as in controls b u t
rou n d ed off (Fig. 3 b). They also show ed a low er
thylakoid frequency (|im thylakoids p er 1 0 ^ m 2
chloroplast section), a low er stacking degree o f
thylakoids, as well as a low er w idth an d height o f
grana stacks. This was as expected m ore p ro ­
nounced in the younger, low er p art o f the leaf
blade than in the m iddle region (Table 1).
These results clearly show th at the final stages o f
chloroplast differentiation are blocked by sethoxy­
Fig. 2. Appearance o f 11 day old barley seedlings without
dim . Sim ilar differences in chloroplast u ltrastru c­
(control) and with sethoxydim -treatm ent (300 g - h a -1) on
ture betw een control and seth o x y d im -treated plants
the 8 th day after germ ination.
Unauthenticated
Download Date | 6/17/17 4:37 AM
Fig. 3. Chloroplasts of the secondary leaf (lower region) from 11 day old barley seedlings a) controls and b) three days
after treatm ent with sethoxydim. P = plastoglobuli; st = starch; bar = 0.5 (.im.
Unauthenticated
Download Date | 6/17/17 4:37 AM
H. K. Lichtenthaler and D. Meier • Sethoxydim Inhibition of Chloroplast Biogenesis
V
118
Fig. 4. Section through mesophyll cells o f the secondary leaf (lower region) o f 11 day old barley seedlings (control plants)
with many chloroplasts. M = mitochondria; P = chloroplasts; V = vacuole; bar = 5 ^im.
Unauthenticated
Download Date | 6/17/17 4:37 AM
H. K. Lichtenthaler and D. Meier • Sethoxydim Inhibition o f Chloroplast Biogenesis
Fig. 5. Section through mesophyll cells of the secondary leaves of 11 day old barley seedlings, three days after treatm ent
with sethoxydim (only few chloroplasts) N = nucleus; P = choroplasts; V = vacuole; bar = 5 ^m.
are found betw een shade (low -light) chloroplasts
an d sun (high-light) chloroplasts o f plants or leaves
grown at different light quan ta fluence rates [6 , 8 , 9].
The sethoxydim -induced block o f the final thylakoid
m u ltiplication an d g rana form ation thus results in
the ap p e aran c e o f sun-type-like chloroplasts in
younger parts o f the secondary le a f w hich upon
h erbicide ap p licatio n are n ot fu rth er developed.
Sethoxydim also affects ch lo ro p last replication.
The m esophyll cells o f the le af b lad e (2nd leaf) o f
control plants (Fig. 4) also contain m ore chloro­
plasts th an those o f the h erb icid e-treated plants
Unauthenticated
Download Date | 6/17/17 4:37 AM
120
H.K. Lichtenthaler and D. Meier ■ Sethoxydim Inhibition of Chloroplast Biogenesis
(Fig. 5). As co m pared to control plants in the
average only one third o f the chloroplast num bers
per sectioned le a f cell are detected. In control
plants sim ilary sm all n um bers o f chloroplasts are
found only in the still grow ing zones o f the devel­
oping tertiary leaf. These results thus dem o n strate
that sethoxydim not only blocks chloroplast dif­
ferentiation, but also chloroplast division. This also
explains the larger average volum e o f the ch lo ro ­
plasts from the second le af o f sethoxydim -treated
plants (Table I). In contrast, the chloroplasts o f
control plants, w hich undergo chloroplast division,
possess a sm aller volum e.
Since sethoxydim absorbed by the leaves is also
rapidly translocated to the shoot apex, we studied
its effect on plastid developm ent in the m eristem atic
regions behind the apex o f barley seedlings. In
control plants one finds there transitional stages
betw een proplastids an d y oung chloroplasts w hich
we have term ed pro-chloroplasts (Fig. 6 ). They are
characterized by vesicles and p ro tu b u la r structures
Table I. Differences in ultrastructural param eters o f chlo­
roplasts from 11 day old barley seedlings, which were treat­
ed on the 8 th day after germination with sethoxydim.
Secondary leaf blade
a) middle leaf region:
thylakoid frequency++
stacking degree of
thylaKoids in %
ratio of stacked to
exposed mem brandes
average width of grana (|im)
volume o f chloroplasts (|j,m3)
b) younger, lower leaf region:
thylakoid frequency
stacking degree of
thylakoias in %
ratio of stacked to
exposed membranes
average width o f grana (^im)
volume o f chloroplasts (^im3)
Controls
-1- Sethoxydim
194
62
124
49
1.6
1.0
0.61
51
0.46
74
170
62
95
54
1.6
1.1
0.58
27
0.52
70
++ |am thylakoid length per 10 |im 2 median chloroplast sec­
tion.
Unauthenticated
Download Date | 6/17/17 4:37 AM
H. K. Lichtenthaler and D. Meier • Sethoxydim Inhibition o f Chloroplast Biogenesis
and also contain som e starch, a few prothylakoids
and plastoglobuli; such plastid stages are regular
com ponents o f chlo roplast d evelopm ent in m any
plants [ 1 0 - 1 2 ].
In the sethoxydim -treated plants the m eristem atic
regions next to the shoot apex do not contain any o f
these transitional stages in chloroplast developm ent.
O nly proplastid stages are found (Fig. 7). This d e m ­
121
onstrates th at sethoxydim not only blocks ch lo ro ­
plast replication and the final stages o f ch loroplast
differentiation, b u t also the early steps o f plastid
and chloroplast d evelopm ent at the prochloroplast.
C oncerning the u ltrastru ctu re o f m ito ch o n d ria
there ap p e ar to be no differences betw een control
and herb icid e-treated plants. The process o f p h o to ­
synthesis is also not affected by sethoxydim as has
Fig. 7. Proplastids from the shoot meristem of 11 day old barley seedlings three days after sethoxydim treatm ent. M = mi­
tochondrion; bar = 0.5 i^m.
Fig. 6 . Developing plastids in the shoot meristem of 1 day old barley seedlings (controls): prochloroplasts with protubular
structures, starch and plastoglobuli. G = Golgi apparatus; M = mitochondrion; P = plastoglobuli; PTB = protubular body;
bar = 0.5 |j.m.
Unauthenticated
Download Date | 6/17/17 4:37 AM
122
H. K. Lichtenthaler and D. Meier • Sethoxydim Inhibition o f Chloroplast Biogenesis
been show n by m easuring the chlorophyll fluores­
cence induction kinetics (K autsky effect) o f
w hole leaves [12]. O u r results indicate that chloro­
plast biogenesis, d evelopm ent and replication are a
m ajor target for the herbicide action. Cell m ulti­
plication (the tertiary le af is not form ed) and
chloroplast m ultiplication (-(-developm ent) are in ­
hibited sim ultaneously by sethoxydim . Since leaf
grow th and the develo p m en t and differentiation o f
cells and chloroplasts are p rotein-dependent, this
points to a possible interaction o f sethoxydim in the
sensitive gram ineous plants with the process o f
protein form ation. W hether the prim ary m ode o f
action o f sethoxydim is in the plastid a n d /o r cyto­
plasm can only be decided in fu rth er investigations.
[1] Poast, Technical Data Sheet, pp. 1-23, BASF-AG,
Agricultural Research Station, D-6703 Lim burgerhof
1982.
[2] P. Veerasekaran and A. H. Catchpole, Pestic. Sei.
13, 452-462(1982).
[3] K. K. Hatzios, Plant Cell Reports 1, 8 7 -9 0 (1982).
[4] N. K. Asare-Boamah and R. A. Fletcher, Weed
Science 31, 4 9 -5 5 (1983).
[5] D. Meier and H. K. Lichtenthaler, Protoplasma 107,
195-207 (1981).
[6 ] E. S. Reynolds, J. Cell Biol. 17, 208-212 (1963).
[7] T. Butterfaß, Patterns of Chloroplast Reproduction,
Springer Verlag. Wien 1979.
[8 ] H. K. Lichtenthaler, C. Buschmann, M. Döll, H. J.
Fietz, T. Bach, U. Kozel, D. Meier, and U. Rahmsdorf, Photosynth. Res. 2, 115-141 (1981).
[9] H. K. Lichtenthaler, G. Kuhn, U. Prenzel, C. Busch­
mann, and D. Meier, Z. Naturforsch. 37c, 464-475
(1982).
[10] J. M. Whatley. New Phytologist 78, 4 07-420 (1977).
[11] A. R. Wellburn, Intern. Rev. Cytology 80, 133-191
(1982).
[12] H. K. Lichtenthaler, Z. Naturforsch. 39 c, in press
(1984).
A cknowledgements
This w ork was sponsored in p art by a g ran t from
the D eutsche F orschungsgem einschaft. We wish to
thank Drs. D. M angold and G. Retzlaff. L im burgerh o f for a gift o f sethoxydim an d Mrs. G. Ihrig for
assistance.
Unauthenticated
Download Date | 6/17/17 4:37 AM