IV.1. 37 STATE-l STATE-2 TRANSITION INFLUENCED BY HERBICIDES WHICH MODIFY FATTY ACID COMPOSITION IN LEAVES . J.A. Graft R.J. Strasser. U. Kull Inst. of Biology,University of Stuttgart, Ulmerstr. 227, FRG INTRODUCTION Interactions of herbicides with photosyntheti c membranes are still not solved in many respects. Three different modes of action have been reported for pyridazinone herbicides : inhi- bition of (1) photosynthetic electron transport (2) carotenoid biosynthesis and (3) fatty acid desaturation in the galacto- lipid fraction of chloroplasts (ST. JOHN, HILTON 1976). The pyridazinone BASF 13-338 (=SAN 9785) used in our investigations has no effect on pigmentsynthesis and photosynthetic activity but affects fatty acid desaturation in leaves (TREBST, HARTH 1974). Cerulenin an antibiotic from the fungus Cephalosporium caerulens inhibits fatty acid synthesis generally. Both herbi- cides act indirectly on photosynthesis because they alter the mobility of photosynthetic units in the membrane. MATERIAL AND METHODS Plants of Petunia hybrida were treated with the herbiCides 4-chloro-5Idlmethylamino)-2-phenyl-3(2H)pyridazinone (BASF 13-338. SAN 9785) 40 or 160 ~M or Cerulenin 4.5 pM for la hours or up to 7 days. Kinetin (4.7, 47, 118 pM) was preincubated for 2 days before the herbicide was added at a final concentration of 40 pM. Fluorescence kinetic experiments at low as we ll as room temperature were car2ied out using exitation light of 633 nm (Ne He Laser, 5\'l/m ). The fluorescence signals which were measured at 694 nm (PSII) and 735 nm (PSI) were digitized and analyzed with an IMSAI B080 microcomputer. For state-l state-2 transition experiments leaves were kept dark for 15 minutes. The electron transports H20 to MV in red light and ASC to MV in red and far red ~705 nm) light in phosphorylated or nonphosphorylated stage were measured according to SOLIS, STRASSER 1983 in 10 mM MgCl . For fatty acid analysis lipids were extracted from dried ;jterial with chloroform/methanol 2:1 (v/v) esterfied in methanol with H2S04 conc. and analyzed by gas chromatography. RESULTS Figure 1 shows the influence of 4.5 pM Cerulenin and 160 pM BASF 13-338 on low temperature fluorescence kinetics. The ratio F2(V)/F2(M) decreases in both cases continuously while the con- trols remain constant for several days. The term~N which tells how much energy in PSI originates from own light absorption (1~ is the fraction due to spill over) is not change d signifi- cantly by the pyridazinone. Cerulenin however causes a decrease after 3 days treatment, that means, the spill over increases. State-1 state-2 transitions measured as room t e mperature fluores- Sybesma, C. (M.), Advances in Photosynthesis Research. Vol . IV. ISBN 90-147.194'.9. © 1984 MaNinus NUhoJ/lDr W. Junk Publishers. The Hague/ Boston/ uneaster. Printtd in TM Nttherlands. IV. I. 38 - -..-<: . , -- • ~ • - ..... , - ........... Figure 1 , - ...... In fl uence .. - .... 11·," of •• ./ Cerulenin ~ a nd .. BASF 13-338 o n low • •• temperature t- - fluores - .. cence • , kinetics •. depending .. • o n time of • " ~ -~-It . , - ,,_ .. '" • .. • • • " •• treatment. ~. • F, n F735 •• ~ - , ..... F, • F695 .. - - I}.~ • " cence kinetics are disturbed by both BASF 13- 338 and Cerulenin (Figure 2 A - 0). The division Fl / F 2 is besides a constant proportional to the spill over rate constant k2 1 (equation 1, see also LOMBARO, STRASSER 1983) . Fl/F2 is not increasing in plants (Fl / F2'experimental - k21 (equatio n 1) Figure 2 State -l state -2 transitions measured as room temperature fluorescence kinetics. • CONfAOl qO , COMTIIQL 10 , UfUTIH 'IUNCII8ATlOtI 20 • '''Sf U -"'8 I!!! - '1"2 • r 1U 2 - " • " -I " '1 Fl '2 u • - , " u so, "" "" • • (ERUlE'UII 110 , MAI(II P.(I~UI.nION 2D • .. us, 13-3 8 18" 0 '1 , 'I " • '2 2 - '1 , > • '1 '1"2 • r 1" 2 " - , 2 • SO, SO, IV.1. 39 treated with BASF 13-338 as compared to the controls. Leaves treated with the pyridazinone cannot hold new steady state (state-2) and Fl and F2 run through a minimum and increase again. t/2 the time necessary to reach half Fl/F2 ascent is reduced by both BASF 13-338 and Ceru l enin. Figure 3 shows the variation of state-l state-2 transitions measured as room temperature fluores- cence kinetics depending on the time of treatment with the pyrid- azinone BASF 13-338. The first effects are seen after a 1ag phase of 1 hour. After that time a minimum in the kinetics become evi- dent and the fluorescence signals increase again. Figure 2 E,F indicates that the phytohormone Kinetin influences the BASF 13-338 effect on state-1 state-2 transition. 2 days preincubation with the cytokinin using concentrations ranging from 4.7 to 118 ~M abolishes the herbicide effect clearly. L / 2 shows values comparable to those of untreated plants. \,lhen plants are simul- taneously treated with Kinetin and the pyridazinone for 18 hours the phytohormone has no influence on the herbicide effect (GRAF, unpublished) . The influence of BASF 13-338 on the phosphorylation of the light harvesting complex is shown in table 1. After light harvesting phosphorylation in the control chloroplasts the electron trans- port H 0 to MV (PSII + PSI) decreases in red light whereas the electr6n transport ASC to MV (PSI) in red and far red light i n- creases as compared to the nonphosphorylated state. In experi- ments with chloroplasts isolated from herbicide treated plants the electron transports remain unchanged in all cases. 40 »M BA SF 13-338 has no significant effect on the photosynthetic Hill activities measured as H20 to MV and H70 to FeCy in coupled as well as in uncoupled states (data nOt shown). After 18 hours incubation with BASF 13-338 the fatty acid composition of glyco- Figure 3 Variation of state-l Figure 4 Influence of BASF state-2 transitions depending 13-338 on fatty acid com- on time of treatment. position. ,. ~"" .. ..~ ~(~n ~u, ~ ,,';' ••. 0.5 (e., ...... . / u'" " . • " • iH " " " IH IH -f1 IrI k1'"CCIlI'IIII IH [)c0lf ..... r " ll-nt IH IH - IH 1100 IV.1. 40 lipids is changed but not of phospholipids (Figure 4). The ratios linolenic acid/linoleic acid (18:3/18:2) and hexadecatrienolc acidihexadecadienoic acid 116:3/16:2) decrease whereas the ratio palmitic acid/unsaturated C16 acids (16:0/16:u) increases, that means, desaturation is inhibited. Table 1 Effect of BASF 13-338 on electron transport in phos- phorylated and nonphosphorylated stages of the light harvesting complex (oMol 02 / mg Ch! min). PREPARATION ±ATP CONTROL +ATP CHLOROPL. 6' PREILL. -ATP BASF 13-338 +ATP CHLOROPL. 6 • PREILL. -ATP CONCLUSIONS 1 2 H O"MV ASC"MV 2RED RED 470 705 602 617 338 588 323 558 3 •• ASC"'MV 2 :1 FAR RED 2-: 1 558 2+:3+ 3+:1· 2-:3 3-:1 1.46 0.90 1.62 441 294 1.01 1.05 0.96 294 Based on the Energy Flux Theory for Blomembranes (STRASSER 1978, 1980) the low temperature fluorescence kinetics indicate that both BASF 13-338 and Cerulcnin force the energy flux constants k2b to decrease or/and k23 to increase. k21 is affected only after 3 days treatment 'with Cerulenin but not with BASF 13-338 because ~N remains constant. From state-1 state-2 transitions measured at room temperature as well as light harvesting phos- phorylation experiments wc conclude that the pyridazinone BASF 13-338 inhibits dynamic changes within and between photosynthetic units during state-l state-2 transition (STRASSER 1983) by altering the matrix structure of the membrane. The photosynthetic units are primarly not attacked because spill over remains constant although mobility in the membrane is changed. variations in the fatty acid composition alter the fluidity of the mem- branes. Kinetin which influences the fatty acid metabolism (GRAP et al. 1982) acts against the effect due to the pyridazinone herbicide by causing the synthesis of more unsaturated fatty acids thus maintaining mobility of photosynthetic units. REFERENCES GRAF JA, HQRSCH A, KULL U (1982), Plant Physiol. 69, Suppl. ST JOIIN JB, HILTON JL (19761 Weed ScL 24, 579 LOMBARD F, STRASSER RJ (19831. Proc. 6th Int. Conf. Photos. SQLIS B, STRASSER RJ (1983), Proc. 6th lnt. Conf. Photos. STRASSER RJ (1918), In Akoyunoglou C, cd., Chloroplast Development, 513, Elsevier, Holland STRASSER RJ (1980), In Akoyunoglou G, cd., Proe. 5th Int. Conf. Photos. 111, Balaban, USA STRASSER RJ (1983), Proe. 6th Int. Conf. Photos. TREBST A, HARTH E (19741, Z. Naturforsch. 29, 232