The Effect of Phytochrome and Proteinsynthesis-Inhibitors on The

The Effect of Phytochrome and Proteinsynthesis-Inhibitors on the Formation of Chlorophylls and Carotenoids in Radish Seedlings Treated with Photosystem II and Bleaching Herbicides K. H. Grumbach and M. Drollinger Botanisches Institut, Universität Karlsruhe, Kaiserstraße 12, D-7500 Karlsruhe

Z. Naturforsch. 35 c, 445-450 (1980); received February 8, 1980

Photosystem II Herbicides, Chlorotic Herbicides, Chlorophylls, Carotenoids, Phytochrome, Inhibitors Etioplasts of radish seedlings treated with photosystem II (DCMU, bentazon) and chlorotic herbicides (amitrole, SAN 6706) were tested on their ability to perform the phytochrome mediated chlorophyll and carotenoid biosynthesis. The cytoplasmic influence on the chloroplastic action of herbicides was also investigated by inhibition of protein synthesis either in the chloro plast with chloramphenicol or in the cytoplasm with actidion. In all herbicide treated radish seedlings a phytochrome mediated chlorophyll and carotenoid biosynthesis was obtained as found in control plants. In plants treated with DCMU the bio synthesis of carotenoids is enhanced compared to the control plants, while SAN 6706 significantly suppresses the carotenoid formation. It is concluded, that photosystem II and chlorotic herbicides do not interfere with the primary action of phytochrome but rather do develope their effects on the terpenoid metabolism through phytochrome. Chloramphenicol applied at the time of sowing very strongly suppresses the formation of chlorophylls and carotenoids in control plants. The sensitivity towards actidion is, however, very much lower. If chloramphenicol and actidion treated plants were also supplied with herbicides, the pigment pattern is completely different In SAN 6706-treated plants chloramphenicol acts synergistic, resulting in an even lower chlorophyll and carotenoid content than in plants supplied only with chloramphenicol. On the other hand SAN 6706 in combination with actidion enhances the formation of pigments, leading to a much higher chlorophyll and carotenoid content as in plants treated only with actidion. A stimulatory effect on the formation of chlorophylls and carotenoids was also found for DCMU-treated plants in combination with actidion. The observation, that in plants treated with SAN 6706 together with actidion, which inhibits protein synthesis in the cytoplasm, the herbicidal response in the chloroplast is completely abolished, gives evidence that the photooxidative action of SAN 6706 in the chloroplast is developed by the cytoplasm.

Introduction In this communication the influence of active phytochrome on the development of the herbicidal It is generally believed that the herbicidal re activity was investigated. It is known that chloro sponse is manifestated in the chloroplast. Photosys phyll and carotenoid biosynthesis are controlled by tem II herbicides like DCMU and bentazon develop phytochrome [7-9], but whether the action of her their toxicity via inhibition of the photosynthetic bicides interferes with phytochrome is unknown. By electron transport [1], while bleaching herbicides like using protein synthesis inhibitors it was also in amitrole and particularly SAN 6706 cause a photo- vestigated, whether the chloroplastic action of the in oxidative destruction of chlorophylls and carotenoids vestigated herbicides is developed or influenced by [2—5]. The primary action especially of bleaching the cytoplasm. herbicides is still unknown. It was assumed that chlorosis develops by the photooxidative destruction of chlorophylls induced by a carotenoid deficiency Materials and Methods [2]. But it was also proposed that in addition the in activation of peroxisomal enzymes [2] and changes Radish seedlings were grown for 6 d on 10_4m in the ratio of saturated to unsaturated fatty acids of DCMU and bentazon (Photosystem II herbicides) as the glycolipids [6] contribute to the photooxidative well as 10~* m amitrole and 10~5 m SAN 6706 (chlo damage of the chloroplast. rotic herbicides) in total darkness. The structure of the used herbicides is shown in Fig. 1. Reprint requests to Dr. K. H. Grumbach. Investigations concerning the influence of active 0341-0382/80/0500-0445 $01.00/0 phytochrome were performed by irradiating the her-

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 446 K. H. Grumbach and M. Drollinger • Herbicides and Terpenoid Metabolism

Fig. 1. Structure of herbicides assayed: DCMU, 3-(3,4-dichlorophenyl)-1,1 -dimethylurea; Bentazon, 3-isopropyl-l H-2,1,3- benzothiadiazin-4-(3 H-one-2,2-dioxide); Amitrole, 3-amino-1,2,4-triazole; SAN 6706, 4-chloro-5(dimethyl-amino)-2-a,a,ar- trifluoro-m-tolyl-3-pyridazinone.

bicide treated plants with 5 min red light, 5 min far Results red light and 5 min red followed by 5 min far red Etiolem ent light for 6 days every 24 h. For the investigation of the cytoplasmic influence Besides protochlorophyll(ide), all carotenoids con on the chloroplastic action of herbicides control and tained in etioplasts of control plants, were also found herbicide treated plants were also supplied with in plastids of etiolated radish seedlings supplied with lO^m chloramphenicol (for 6 days) and 10~4m DCMU, bentazon, amitrole and SAN 6706. While actidion (from the 3rd to the 6th day) in total DCMU-, bentazon- and amitrole-treated seedlings darkness and thereafter illuminated for 24 h with exhibit nearly the same carotenoid content as the fluorescent white light (0.8 W/cm2). control plants, SAN 6706 reduces the formation of Chlorophylls were extracted and determined ac xanthophylls (Fig. 4). cording to Ziegler and Egle [10]. Carotenoids were extracted into cold acetone, the crude extract sapon Phytochrome ified with KOH, chromatographied and quantita Chlorophyll and carotenoid biosynthesis are con tively determined according to Hager and Meyer- trolled by active phytochrome. It was, however, not Bertenrath [11] using an absorbance coefficient of yet investigated, whether herbicides paticularly £(1%/1 cm) = 2500. bleaching herbicides like amitrole and SAN 6706 in- K. H. Grumbach and M. Drollinger • Herbicides and Terpenoid Metabolism 447

CHLOROPHYLLS CAROTENES

CONTROL CONTROL HR+DR+HR HR+DR+HR } HR______HR | HR+DR HR+DR | 0 40 80 pg/IOOplants DR \ DCMU DP j HR+DR+HR 1 0 10 20 30 HR | pg / 100 plants HR+DR 0 40 00 pg/100 plants BENTAZON DCMU BENTAZON HR+DR+HR HR | HR+DR+HR | HR+DR+HR HR* DR HR HR -f- 0 40 80 pg/100 plants HR+DR HR+DR } AMITROLE DR DR f HR+DR+HR DD DD - f - HR 0 10 20 30 40 0 10 20 30 HR* DR pg /100 plants pg /100 plants 0 40 80 pg/100 plants SAN 6 7 0 6 HR+DR+HR AMITROLE SAN 6706 HR | HR+DR HR+DR+HR -(- HR+DR+HR } 0 40 60 p g /100 p la n ts HR ( HR Fig. 2. The effect of active phytochrome on the chlorophyll HR+DR -f- HR+DR-}- content of etiolated radish seedlings supplied with photo DR | DR system II and bleaching herbicides. DD | DD { 0 10 20 30 0 10 20 30 p g/1 00 pi ant s p g /1 0 0 plants Fig. 3. The effect of phytochrome on the carotene content terfere with the primary action of phytochrome. As of etiolated radish seedlings supplied with photosystem II shown in Figs 2 - 4 in all herbicide-treated plants and bleaching herbicides. chlorophyll and carotenoid formation is controlled by active phytochrome like in untreated radish seedlings, but the control is quantitative rather than and neoxanthin are enhanced in their biosynthesis qualitative. There are no differences in the accu by phytochrome, while the a-ionon xanthophyll mulation of chlorophyll between control and her lutein is formed to the same extent as in untreated bicide-treated plants except for DCMU which shows plants. an enhancement effect. The phytochrome mediated biosynthesis of chlo Inhibitors rophylls and carotenoids is enhanced in radish treat ed with DCMU, while in SAN 6706-treated plants It is known that actidion (cycloheximide) inhibits the accumulation of carotenoids is significantly sup protein synthesis in the cytoplasm, while chloram pressed (Figs 3, 4). Bentazon- and amitrole-treated phenicol inhibits protein synthesis in the chloroplasts plants exhibit nearly the same carotenoid content as and mitochondria. The application of inhibitors of control plants. It is of particular interest, that in translation should help to understand the action of radish seedlings supplied with the photosystem II herbicides on the chlorophyll and carotenoid accu herbicides DCMU and bentazon the /?-ionon xan- mulation particularly for the bleaching herbicide thophylls zeaxanthin, antheraxanthin, violaxanthin SAN 6706. 448 K. H. Grumbach and M. Drollinger • Herbicides and Terpenoid Metabolism

XANTHOPHYLLS Chloramphenicol applied at the time of sowing very strongly suppresses the formation of chloro CONTROL phylls and carotenoids in radish seedlings [12, 13]. The sensitivity towards actidion (applied after 3 d h r+d r+h r darkness) is, however, very much lower (Figs 5, 6). HR I f plants treated with chloramphenicol and actidion HR+DR DR were also supplied with a photosystem I I or bleach DD ing herbicide, the pigment pattern is completely dif 100 200 300 ferent from that of control plants. I f plants grown for p g / 100 p la n ts 6 days in total darkness were also supplied with 10-5 m SAN 6706 and thereafter irradiated for 24 h with fluorescent white light (0.8 W /m2) the chloro phyll and carotenoid content is lower than in radish DCMU BENTAZON supplied only with chloramphenicol (Figs 5, 6). This shows that chloramphenicol in SAN 6706-treated HR+DR+HR HR+DR+HR plants enhances the SAN-induced bleaching effect. HR HR On the other hand, if in SAN-treated radish seed HR+DR HR+DR lings protein synthesis in the cytoplasm is inhibited DR OR | after 3 days by 10-4 m actidion and plants are ir DD DD radiated after 6 days for 24 h with white light, then 100 200 300 0 100 2 00 300 pg/ 100 plants pg/ lOOplants the bleaching effect of SAN is completely abolished and the chlorophyll and carotenoid content is even higher in these plants than in radish treated only with actidion (Figs 5, 6). This result shows that protein synthesis in the AMITROLE SAN 6706 cytoplasm is required for the development of the HR+DR+HR HR+DR+HR herbicidal response. A stimulatory effect on the HR HR chlorophyll and carotenoid formation was also ob HR+DR HR+DR| tained for DCMU-treated plants in combination DR DR with actidion. DD DD 100 200 300 0 100 200 pg/ 100 plants pg /100 plants Discussion Fig. 4. The effect of phytochrome on the xanthophyll con tent of etiolated radish seedlings supplied with photosys Degradation of herbicide treated plants results tem II and bleaching herbicides. from changes in a large number of physiological

] water control

□ ♦icrfn A ctid io n

CHLOROPHYLLS J +10“^mChloramphenicol

! ! ■ 10" m SAN 6706

Fig. 5. The effect of chloramphenicol and actidion on the chlorophyll content of etiol ated radish seedlings supplied with the 1200 bleaching herbicide SAN 6706 after 24 h pg/100 plants illumination with fluorescent white light. K. H. Grumbach and M. Drollinger • Herbicides and Terpenoid Metabolism 449

Fig. 6. The effect of chloramphenicol and actidion on the carotenoid content of etiolated ] water control radish seedlings supplied with 10-5 m SAN 6706 after 24 h illumination with fluorescent □ ♦10"^m Actidion white light. ! J ♦10'^fn Chloramphenicol CAROTENES 10 m SAN 6706

60 p g/1 00 plants

LUTEI N + ZEAXANTHIN

100 120 pg/100 plants

ANTHER AX ANTHIN ♦ LUTEI N-5.6-EP0XI D

20 30 iO 50 p g /100 plants

VIOLA XANTHIN

H20 1 3 1 -} SAN

0 10 20 30 iO 50 60 pg/100 plants

NEOXANTHIN

0 10 20 jjg /100 p la n ts

reactions and mainly by blocking chloroplastic ones ceeds exclusively in the chloroplast. Therefore we in [14]. Ureas and benzothiadiazinones are known to in vestigated the action of phytochrome on the bio hibit the photosystem II dependent Hill reaction synthesis of chlorophylls and carotenoids in radish while triazoles and pyridazinones block the accu treated with photosystem II and bleaching herbi mulation of chlorophylls and carotenoids and dis- cides. The aim was, to obtain whether phytochrome turbe the assembly of the thylakoid membrane [2]. is involved in the development of the herbicidal Chlorophylls and carotenoids are essential con toxicity. stituents of the thylakoid membrane. Their biosyn As shown in Figs 2 - 4 in all herbicide treated thesis is controlled by active phytochrome and pro seedlings a phytochrome mediated chlorophyll and 450 K. H. Grumbach and M. Drollinger • Herbicides and Terpenoid Metabolism carotenoid biosynthesis takes place like in untreated of chlorophylls and carotenoids with an even higher control plants. Only SAN 6706 reduces the accumu concentration than in plants treated only with acti lation of carotenoids up to 50%. From this result we dion. From this it is concluded that the effect of conclude that photosystem II and bleaching herbi SAN 6706, which causes a photooxidative destruc cides do not interfere with the primary action of tion of pigments is enhanced by the simultaneous in phytochrome but do develop their toxicity under the hibition of protein synthesis in the chloroplast. The influence of phytochrome. This assumption is sup observation, that in plants treated with SAN 6706 in ported by the observation that the herbicidal re combination with actidion no photooxidation of sponse on the terpenoid metabolism of the chloro pigments occurs gives strong evidence that the pho- plast is established predominantly in continuous red tooxidative damage of the chloroplast under the in light [15], fluence of the bleaching herbicide SAN 6706 is Besides specific reactions in the chloroplast many developed by the cytoplasm. This means that SAN investigations reveal a herbicide effect in the cyto 6706 either is metabolized into a toxic form in the plasm and propose that the extrachloroplastic effects cytoplasm and thereafter transferred into the chloro contribute to the development of the herbicidal plast or that SAN 6706 can only develop its her toxicity in the chloroplast [2], To analyse this subject bicidal response in combination with a cytoplasmic further we inhibited protein synthesis of the herbi factor. Further investigations concerning this prob cide treated plants either in the chloroplast or in the lem are in progress. cytoplasm. As shown in Figs 5 and 6 inhibition of protein synthesis in the chloroplast enhances the Acknowledgements SAN 6706 induced bleaching effect. On the other hand application of actidion which inhibits protein Financial support by Deutsche Forschungsge synthesis in the cytoplasm completely abolishes the meinschaft to Prof. Dr. H. Lichtenthaler is gratefully bleaching effect of SAN leading to an accumulation acknowledged.

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