The pyridazinone SAS 13-338 (SAN 9785) inhibits the desaturation sequence leading to polyunsaturated fatty acids, mainly of glycolipids. Parallel to the inhibition of fatty acid desaturation in the presence of the pyridazinone. changes in energy-distribution parameters have been observed. These data indicate that the amount of polyunsaturated fatty acids in glycolipids is strongly correlated with excitation, trapping, grouping and dissipation, but not with spillover. Functional changes in energy distribution induced by SAS 13-338 are interpreted as a consequence of structural changes in the lipid matrix, which may imply a structure-function relationship between pigment protein complexes and the surrounding lipid environment in thylakoids

Graf, Josef A.

Kull, Ulrich

Strasser, Reto J.

Online Publikationen der Universität Stuttgart

Structure-Fuuction-Relationship in Thylakoids InDuenced by the 
Pyridazinone BAS 13-338 (SAN 9785) 
Josef A. Grat. Reto 1. Strasser, and Ulrich Kull 
Biologischcs Institut. UniversiHiI Stuttgart. Ulmer Stral3c 227. 0·7000 Stuttgart 60. 
Bundcsrcpublik Deutschland 
Z. Naturforsch. 42c. 808-812 (1981); received CXtober 31 . 1986 
Pyridaz.inone. Fauy Acid Dcsaturation. Energy Distribution. Structure-Function Relationship 
The pyridazinonc SAS 13-338 (SAN 9785) inhibits the desaturation sequence leading to 
polyunsaturated fatty acids. mainly of glycolipids . Parallel to the inhibition of fatty acid desatura-
lion in the presence of the pyridazinone. changes in energy-distribution parameters have been 
observed. These data indicate that the amount of polyunsallu3tcd rany acids in glycolipids is 
strongly correlated with Cl(citation. trapping. grouping and dissipation . but not with spillover. 
Functional changes in energy distribution induced by SAS 13-338 are interpreted as a conse-
quence of structural changes in the lipid matrix . which may imply a structure-function relationship 
between pigment protein complexes and the surrounding lipid environment in thylakoids. 
Introduction 
First steps of light transforming reactions in photo-
synthesis take place within heterogeneously distrib-
uted pigment protein complexes in thylakoid mem-
branes (1) and include light absorption-. exciton 
transfer- and charge separation-processes. It has 
been proposed that the pattern of exciton transfer 
i.e. the energy distribution behaviour influences the 
photosynthetic efficiency (2) . In order to coordinate 
energy distribution. a high degree of membrane or-
ganization might be necessary. The membrane or-
ganization seems to be characterized not only by the 
arrangement of pigment protein complexes but also 
by the interactions with the lipid environment. 
Therefore , the thylakoid specific lipid complement 
which contains a high amount of polyunsaturated 
fatty acids may reflect the functional role of the lipid 
matrix in these membranes. Several attempts have 
been made to gain insight into the functional interac-
tions between acyl lipids and LHe. reaction centers 
Abbrev;ariolU: PS I. n. photosystem I. 11 : LHC. light har-
vesting complex: RC I. 11. reaction center r. 11; MGDG . 
monogalactosyldiacylglycerol; DGDG, digalactosyldiacyl-
glycerol; SODG. sulfoquinovosyldiacyJglycerol ; PG, phos-
p~a.tidylgly.cerol; PLo other phospholipids; F,!o). FI/~ " FU~II ' 
IRltlal . "anable and maximal fluorescence of PS I; F 110l' 
FZ(YI ' Ft/M)' initial . variable and maximal fluorescence of 
PS 11 : V. nonnalized variable fluorescence of PS 11 ; .f. 
integral of non-fluorescence: 18:3. linolenic acid; BAS 
13-338 (SAN 9785). 4-chloro-S-(dimethylamino)-2-
phen yl-3(2 H)pyridazinone . 
Reprint requests to Josef A. Grar. 
Vcrlag der Zeitschrih fUr Naturforschung, D-7400Tiibingcn 
0341-0382/87/0600-0808 S 01.30/0 
o r herbicide binding protein (3-51. The effect of 
high amounts of polyunsaturated fatty acids. espe-
cially 18:3 in acyl lipids on structure· function rela· 
tionship between lipid matrix and pigment protein 
complexes as well as on arrangement of these com-
plexes can be studied by inhibiting fatty acid desat-
uration sequence with 4.chloro-5-(dimethylamino)-
2·phenyl·3(2H)pyridazinone (BAS 13 -338. SAN 
9785). This compound has been shown to be a potent 
state change inhibitor {6} and a PS 11 electron trans-
pon inhibitor in vivo and in vitro (7) . However . it 
inhibits different desaturation steps in the synthesis 
of polyunsaturated fatty acids 18. 9] leading to an 
increase of appressedlnon-appressed regions in thyla-
koids [10[ . 
In this paper we show the effect of an altered lipid 
matrix induced by the pyridazinone SAS 13-338 
and the functional role of polyunsaturated fatly acids 
in acyl lipids on energy distribution in thylakoids. 
Materials and Methods 
4-6 week old Petunia hybrida plants (cyanidine 
type) were treated with 160 j.lM SAS 13-338 in glass 
chambers under 15 W /m2 light intensity. 20 ± 2 °c 
growth temperature and 80 ± 10% relative humidity 
for 7 d . Low temperature fluorescence kinetic meas-
urements of leaf discs were carried out using excita-
tion light of 633 nm (NeHe laser. 20 W/m1). a multi-
branched fiber optic and 2 photomultipliers. Fluo-
rescence signals measured at 695 nm (PS I1) and at 
735 nm (PS I) were digitized on line (IMSAI 8080 
microcomputer) and stored in core memory for data 
J. A. Gear ~t 01 . . Pyridazinone and Structuee-Function-Rclationship 809 
handling. Absolute 77 K fluorescence emission spec-
tra [11] measured from 600 to 800 nm were used to 
correct the F(o)- and F(M)-values obtained from the 
kinetics . The characteristics of energy distribution, 
i.e. trapping, grouping, spillover and dissipation 
were analyzed according to the energy flux theory 
112J. Lipids were extracted from purified thylakoids 
as described elsewhere (13) and isolated by TLC on 
silica gel 60 (Merck) using the acetone: benzene: 
water system (91 :30:8 v/v/v). Lipid bands were re-
vealed by spraying the plates with 0.003% (w/v) al-
kaline Rhodamin 6G solution. For quantitative fatty 
acid analysis. individual lipid bands were scraped 
from the plates and transmethylated according to 
(141 in the presence of methylpentadecanoate stand-
ard. After extraction. aliquots of acylmethylesters 
were separated by GC in to ' x 1/8H steel columns 
packed with 10% EGSS-X on Chromosorb W /AW-
DCMS (SO-lOO mesh) using a Varian Aerograph 
2700 fitted with CDS 111 integrator. Acyl lipid bands 
and fatty acid methyl esters were identified by com-
paring them with authentic standards. 
Results 
Fotly acid changes during pyridazinone incubation 
period 
Although the qualitative and quantitative acyl 
lipid complement as well as the chlorophyll content 
of thylakoids remain nearly constant (data not 
shown), changes in the fatty acid composition have 
been observed (Table I). The pyridazinone treat-
ment alters the unsaturated character of thylakoids 
by decreasing mainly linolenic acid in favour of satu-
rated. mono- and diunsaturated C16- and Cls-fatty 
acids. This leads to a drastic decrease of double bond 
indices. These effects are more pronounced in gly-
colipids (MGDG, DGDG , SQDG) than in phos-
pholipid fraclions (PG, PL). 
Low temperature fluorescence kinetics during 
pyridaz;none incubation period 
77 K fluorescence kinetics indicate a loss in the 
variable fluorescence F2(~) and FI (~). a decrease in the 
maximal fluorescence FUM) and an increase in the 
initial fluorescence F2(o) (Fig. 1), The ratio F2(v/F2(M) 
shown to be proportional to the trapping probability 
Plb in an ungrouped bipartite model [15]. decreases 
from 0.75 to 0.47. whereas aN which implies how 
much energy in PS I originates from its own light 
absorption remains nearly constant during a 7 d incu-
balion period wilh BAS 13-338 (SAN 9785). Based 
on a grouped bipartite model. however , a decrease in 
the ratio F2I,v/Fm.o suggests that PS II trapping andlor 
grouping between PS lI-LHC-units may be influ-
enced by the pyridazinone treatment. According to 
(121. the FI versus F2 plots yield straight lines with the 
same slopes, indicating that spillover rate constant is 
not affected by an altered lipid matrix in thyJakoids. 
The energy flux theory predicts that the nor-
malized variable fluorescence of PS 11 V versus the 
non-fluorescence s (area growth curve) is a horizon-
tal hyperboliC function of different curvatures at 
77 K in the case of grouping and non-grouping. On 
Table I. Effect of 160 j.lM BAS 13-338 (SAN 9785) on (he fatty acid composition of thylakoid Iipids from Petunia hybrida 
in percenlage or total ratty acid content. The values arc mean ± SD obtained Crom at least 4 experiments. 
1600 16:1 16:u 18:0 18: 1 1802 1803 18:4 
MGDO Control 4.1 ±O. I 1.1±O.1 LO±O. I 1.5±0.1 1.1 ±O.I 4.5±0.1 78.9 ± 1.0 7.2±O.8 SAS 13-338 1O.5±0.4 2.7±0.3 3.0±0.2 3.7±0.2 3.6±0.3 6.8±0.1 6O.5±0.7 6.2 ± 0.7 
DODO Control 1O.3±0.1 1.3 ± 0.1 1.5 ±O, ) 2.2±0.1 1.5±0.1 3.9±0.1 74.6± 1.7 3.8±0.2 
BAS 13-338 16.2 ± 1.0 3.1 ±O. I 3.7±O.1 5.1 ±0.2 4.3 ±O. 1 4.7±0.1 57.5± 1.3 3.0 ±O.2 
SODO Control 31.0±O.5 3.2 ±O.3 3.7±0.8 5. t ± O.3 3.6±0.2 6.4± 0.6 37.5± 1.9 5A±O.2 SAS 13-338 4O.0 ±0.7 5.3±0.5 6.4± 1.4 8.2±O.4 7.1 ±0.4 5.6±0.5 19.0±0.9 2.8±0.1 
PO Control 30.6 ± 2.6 17.0±0.7 2.9±0.1 4.8±0.4 4.2 j: 0.4 8.0±0.8 31.0±5.4 0.3±0.1 
BAS 13-338 31.1±2.6 16.9±0.7 4.6±0.1 8.2±0.6 7.5±0.6 7.2±O.7 16.3±2.9 1.7±0.1 
Pl Control 26.0± 1.8 4.0±0.3 4.4±0.7 5.6±0.6 4.8±0.3 IS.S± 1.0 32.5 ±2.5 l.l ±0.2 
BAS 13-338 28.7±2.0 4.5±0.3 4.8±0.B 1.6±0.8 6.5 ±0.5 11.3±0.7 25 .1 ±1.9 3,2±0.5 
810 J, A. Graf I!t al . . Pyridazinone and Structure-function-Relationship 
F, A ('iH~i 
B 1\-- Fl(0). ---. 8 = 
, , 
• 
" 
, , 
• 
" 
, , 
• :: , , • , 
• 
" ~ ~ ~ , 250 ms " ~ 
'" 
~ 
" § :;: " 
'" 
" ~ 
, 
: .!!:.! F2 
-'" ' ''-....:... "-A,\B 
~ 
, 
Fig. 1. Simultaneously measured nuoresccnce kinetics of 
PS I (F,) and PS II (F:) at n K. (A) Control : (B) 160..,M 
BAS 13-338 (SAN 9785) 7 d , 
this basis. the curvature of the experimental signals 
F:IIl contains the information for cooperativity of 
neighbouring PS II·LHC-units . Therefore. if K is 
known, the grouping probabilities Pn can be calcu-
lated from the intercept V, of the V versus VI5 plots as 
shown in Fig. 2. At any time when grouping is zero 
If------~+-4V 
Fig. 2. Calculations of the grouping probabilities p!! from 
the inu:rsept V, of the V versus VIs plots during a 7 d treat-
ment \Io'ith 160..,M SAS 13-338 (SAN 9785) . K=O. 14S . 
(k ~ 0, the slope of the plot is m, = K/(I-K) K, the 
temperature constant for charge transfer of a re-
duced reaction center to oxidized internal acceptors 
(pheophytin. QA) can be calculatcd. K has the value 
0. 145 [161 which is valid for most higher plants. The 
effect of an altered lipid matrix on energy distribu-
tion is shown in Fig. 3. As presented in Table n, 
nearly all energy distribution parameters are affected 
following treatment with BAS 13-338 (SAN 9785). 
As can be shown by apparent quantum yields rp" of 
an individual photosystem and by energy transfer 
prObabilities P'I' trapping as well as grouping are 
decreasing. whereas spillover and dissipation are 
increasing during the pyridazinone treatment. How-
ever. it has to be emphasized that grouping is almost 
zero whereas the energy transfe r probability P2b for 
PS 11 trapping seems to be unaffected by a n altered 
lipid matrix . Furthermore . decreases of the confor-
mation constants k'l for trapping, grouping and spill-
over as well as increases in the relative distances R'I 
between the different loci might reflect functional 
changes and/or a rearrangement of pigment protein 
complexes caused by structural changes in the lipid 
environment. 
',' 
~, ~, 
" 
om ~ " 0 ~: > ~ ~ " • 0, 
,,~ • ·n " 0, , ~ ~ , 
.. • ~ , o~ " ~. , 0. 
" 
R. 11 RIA J U All " " " " .. 08~ .. ~~ ., . 1.-0:0.-_, , , 
"11~S ' , . , '~'701IJ'S , 1 G 1 I I • S • 
Fig. 3. Energy distribution terms Ip,. and relative distances 
R", where (f" gl,·es the quantum yield of an energy nux E,. 
(E,/absorption o f PS n or PS I) . R,. represents the relaliv~ 
distances between the pigment protein complexes i and J 
calculated from the conformation comlno's using the 
FORSTER-rcsooancc-reJationship: k,~' = R, .~ . 0 Control : 
• 160 ~M BAS 13-338 (SAN 91R5) . 
• 
J. A. Gra! et al . . Pyridazinone and Stnlcture-Function-Relationship 811 
Table 11. Energy distribution terms following 7 d treatment with 160 ~ BAS 13-338 (SAN 9185) . Relative values are 
mean ± SO from at least 4 experiments. 
Energy distribution Term Control BAS 13-338 
Apparent quantum yields <p" of PS It or PS 1 
Excitation PS 11 
"" 
1.43±0.OS 1.00 ±O.03 
Trapping PS JJ 
""" 
0 .15±0.0I 0.47 ± 0.01 
Grouping PS II-LHC-units 'Pn O.43±D.Ol o.ooao.ool 
Dissipation PS 11 
""D O.09 ±O.OI 0.30 ±O.O2 
Spill over PS IJ - PS I "", O.16±0.O I 0.23 ±0.01 Dissipation PS I q>'D O.35±0.02 0.67 ±O.03 
Trapping PS I 
'1'" 0 .82±0.03 0.63 ±0.03 
Excitation PS I '1', 1.18 ± 0 .01 1.30 ±O.DI 
Energy transfer probabilities p" 
Trapping PS 11 P2b 0.52 ±O.OI 0.47 ±O.Dl 
Grouping PS lI·lHC·units P"12 O.30 ± O.01 0 .002 ± 0.001 
Dissipation PS 11 Pm 0.01±O.OI 0 .30 ±O.DI 
Spill over PS 11-PS I p" 0. 11 ±O.Dl 0.22 .:to.Ol 
DiiSipation PS 1 POD O.30±O.O2 0.52 ±O.D3 
Trapping PS I Ph O.70±0.~ 0.48 ±O.DJ 
Discussion 
It has been proposed that the shape of a given acyl 
lipid molecule is mainly determined by the ratio of 
polar head group and the type of acyl moieties (17). 
Therefore, lipid packing properties are expected to 
bring about asymmetrical distribution of lipid classes 
and lipid species in membranes. The pyridazinone 
SAS 13-338 (SAN 9785) induces alterations of the 
ultrastructure of thylakoid membranes by increasing 
the ratio of appressedlnonappressed regions in grana 
(10) . This effect may be attributed to changes in fatty 
acid composilion from a more unsaturated (high 
linolenic acid content) to a more saturated (Iow 
linolenic acid content) state of the thylakoid lipid 
matrix. Changes in the unsaturation of fally acids are 
expected to cause Changes in lipid shapes provided 
that the acyl lipid composition is constant. Such 
changes may be e.g. from cone-shaped 10 cylindrical-
shaped MGDG. Therefore . remarkable changes in 
the lipid shape are thought to induce alterations in 
the organization of the lipid matrix . Our calculations 
imply that the frequencies of 18:3/18:3 species in 
MGDG. DGDG and SODG are lowered by nearly 
50%. In addition, our results indicate that energy 
transfer between different pigment protein complex-
es depends on close distances between them and 
could be influenced by the state of the lipid matrix . 
These findings are in agreement with the proposed 
relationship between pyridazinone induced altera-
Energy distribution Tenn Control BAS 13-338 
Conformation constants k" 
Trapping PS 11 k~ 1.96±0.14 1.56 ±0.15 
Grouping PS II·LHCuniIS k21 4.54±0.32 0.008 ± 0.008 
Spillover PS 11_ PS I k" 1.10±O.lS o.n ±0.O1 
Trapping PS 1 kh 2.35 ±O.21 0 .94 ±O.OB 
Dissipation PS 11 km 1.00 1.00 
Dissipation PS t k'D 1.00 1.00 
Relative Distances R" 
PS II-RC II R", 0 .71 ± O.OI 0.93 ± 0.01 
PS II-LHC-PS II -LHC Ru O.18±0.Ol 2.25 ±0.02 
PS II-PS I R" 0.92±0.01 LOS ±0.01 
PS I_RC J R" 0.81±0.01 1.01 ±O.OI 
F2f. ';Fm.fl 0 .1S±0.D2 0.41 ±O.OI 
a, 0.S9± O.03 0.64 ± O.03 
tions in linolenic acid content and fluorescence in-
duction in vivo (18) . 
Calculations in [19] propose that in appressed re-
gions, there is a minimum set-up of acyllipids which 
favour energy transfer between pigment protein 
complexes. All our data suggest that the amount of 
linolenic acid in glycolipids is strongly correlated 
(significance 0.1-5% ) with excitation, trapping and 
dissipation of both PS 11 and PS I. with grouping of 
PS JI -LHC-units , but not with spillover. From these 
findings we conclude that a relationship exists be-
tween the structural organization of the lipid matrix 
and the functional state of energy distribution in 
thylakoids . 
Considering the possible mechanism of the 
BAS 13-338 (SAN 9785) effect on energy distribu-
tion. we assume that due to pyridazinone induced 
alterations in shape, glycolipids are insened into ap-
pressed rather than into non-appressed regions of 
thylakoids by lipid turnover metabolism. This might 
result in an altered lipid micro-environment as well 
as in a rearrangement of pigment protein complexes 
(increased distances between these complexes) in ap-
pressed regions. Structural changes in the organiza-
tion of the lipid matrix are expected to cause changes 
in the functional energy distribution behaviour by 
creating a new state of lipid-protein interaction. 
On the basis of our data , possible models for the 
effecI of the pyridazinone BAS 13-338 (SAN 9785) 
8J2 J. A . Graf et al . . Pyridazinone and Structure.function·Relationship 
LEECH et al. 
1985 
Thylakoids proposed: 
GRAF et al. 1986 
J PSU.LHC 
(( 
LEECH et al. 
J985 
) 
" 
" 
". 
Fig . .; . Grana models : according to Leech ~t 01. (1985) . Thylakoid models : the modds show a lh)'lakoid sac originating 
from dirrerent appressed regions of grana stacks. respc:cti\·el)' . Energy distribution models: data calculated from n K 
nuorescence kinetics are compiled in a s),ncrgetic way visualizing energy distribution con!.lellation in thylakoids afler 7 d 
treatment with 160 ~M BAS 13-338. J = absorption. T =. !rapping. G ..,. grouping. S = spillover. 0 :: dissipation. 
e' = electron lranspon chain . 
on thylakoids are proposed (Fig. 4). In these thy-
lakoid models. the structure of the lipid matrix is 
related to the functional slate of energy distribution 
in a grouped bipartite model. Both models are in 
agreement with the observed pyridazinone induced 
cbanges in grana size (101. 
(11 B. Andersson and J . M. Anderson. Biochim. Biophys. 
Acta 593. 427-440 (1980) . 
(21 R . J . Strasscr. Ber. Dcutsch. BOI . Gc!>. 98. 53 - 72 
(1985) . 
(3] W. P. WilIiams. K. Gounari!>. and P. J . Quinn . in : 
Advances in Photosynthesis Research III (C . S)'bes· 
ma , ed .), p . 123-130. NijhoWJunk Publishers. Thc 
Hague 19B.; . 
(41 A . Radunz. K. P. BOlder. and G . H . Schmid. Z . pnan· 
zenphysiol. 114. 227-231 (1984) . 
(510. J . Chapman. J . De·Fdice. and J . Barber. Planta 
166.280-285 (1985) . 
(6) J . A . Graf. R . J . Slr3sser. and U . KuU . in : Advances 
in Photosynthesis Research IV (c. Sybesma. ed . ). p . 
37-40. NijhoWJunk Publishers, The Hague 1984. 
(7) R . M. Mannan and S. Bese. Photoehem. Photobiol. 
41. 63-72 (1985) . 
(8) J . B. St. John and M. N. Christiansen, Plant Ph),siol. 
57.257-259 (1976). 
(9) N. W. Lem and J. P. Williams. Plant Physiol. 68. 
944- 949 (1981) . 
1101 R. M. Leech. C. A. Wallon. and N. R . Baker. Planta 
165.277-283 (1985) . 
Acknowledgem~nts 
We are grateful to BASF AG. Limburgerhof. and 
SANDOZ AG. Basel. for the supply of the pyridazi· 
none used in this study. 
Ill1 E. Weis. Biochim . Bioph)'S . Acta 807. 118-126 
(1985). 
(121 R. J . Stra!>!>er. in : Chloroplasl Development (G. 
Akoyunoglou and J. H . Argyroudi·Akoyunoglou . 
eds. ). p . 513-524. EIsevier . Amsterdam 1978. 
113) E . G . Bligh and W. J . Dyer. Can. J. Biochem. Ph)'Siol. 
37.911 - 917 (1959) . 
(14) U . Kull 3:nd K. Jeremias. Z . pnanzenphysiol. 68, 
55-62 (1972). 
(15) W. L. Butler. Ann . Rev. Plant Physiol. 29. 345-378 
(1978) . 
[16] J . A. Graf. R. J . Slr3:SScr. and U. Kull . in : Procecdin~s 
Vllth International Congress on PholOsynthesislll (J . 
Biggins . cd.). pp. 713-716. Nijhoff/Junk Publishers. 
The Hague 1986. 
11 710. J. MU'l'hy . FEBS Lott . 150. 19-26 (1983). 
(18) G . Laskay. E. Lehoczki. A. L. Dobi. and L. Szala),. 
Planta 169.123-129 (1986). 
(19) D. J . Murphy. Biochim. Biophys. Acta 864. 33-94 
(1986). 


Structure-function-relationship in thylakoids influenced by the pyridazinone BAS 13-338 (SAN 9785)

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Structure-function-relationship in thylakoids influenced by the pyridazinone BAS 13-338 (SAN 9785)

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