Z. N aturforsch. 4 7 c , 5 7 -6 2 (1992); received A ugust 6, 1990/July 9, 1991
Photoreduction o f N A D P +, Photosystem II, Pheophytin, M anganese, R eaction Center The effects o f reversible manganese extraction on N A D P + photoreduction were studied with higher plant subchloroplast preparations o f photosystem II (PS II). U nder anaerobic conditions, when the reaction centers (RCs) o f PS II are “closed” (i.e. in the state [P680 Pheo] QA), and in the presence o f fe r r e d o x in -fe r r e d o x in -N A D P + reductase, N A D P + reduction is observed at a rate o f 0 .8 -1 .1 nm ol/m g x chlorophyll x h. After com plete removal o f m an ganese from PS II, the rate o f N A D P + reduction is reduced 4 0 - 50-fold. U p on the addition o f M n at a concentration o f approx. 4 Mn atom s per reaction center, the N A D P + reduction is restored up to 8 5 -9 0 % o f the initial value. W hen half o f this am ount o f Mn is com bined with about 40 times o f the equivalent concentration o f other divalent ions (C a2+, Sr2+, M g2+ etc.) the reaction is also reactivated. D inoseb (10-6 m ) an inhibitor o f electron transfer in PS II pre vents N A D P + photoreduction. It is concluded that under conditions when the first quinone acceptor, Q A, is in its reduced state (QäX electrons are transferred from reduced pheophytin (P h eoT) to N A D P +, indicating that PS II can reduce N A D P + w ithout the participation o f PS I. On the basis o f these and literature data, an alternate pathway for electron phototransfer in PS II reaction centers o f higherplants is suggested. Som e problem s concerning the Z-scheme are discussed.
The location of ß-carotene-5,6-epoxide in the photosynthetic apparatus of higherplants is given in Tables II and III. The epoxide is mainly located in the PS I reaction centre complexes (CP 1 and CP 1 a) from oats, radish and barley thylakoid membrane. Isolated chloroplasts and thylakoids illuminated in the absence and presence of D C M U , show increased levels of the epoxide. It is only under such extreme conditions, where pigment destruction has taken place, that ß-carotene-5,6-epoxide is found in the PS II reaction centre (CPa). This is in contrast to the report of Ashikawa et al.  that ß-carotene mono epoxide is preferentially located in PS II particles. It is evident, however, that oxidation of ß-carotene to ß-carotene-5,6-epoxide takes place in the individual pigment-protein complexes of the thylakoid mem brane. It is reasonable to assume that differences between the results of our study and that of Ashika wa and co-workers  may be due, in part, to differ ences in the preparation of these complexes. Indeed Ashikawa et al.  suggest that the amount of the epoxide found in isolated thylakoid membranes may be dependent on conditions of harvesting and prepa ration.
Oxygenic photosystem II (PSII) is the only protein complex which can oxidize water and release molecular oxygen. Studies on PSII have been stimulated by its close homology to the purple bacteria reaction centre although this ancient form of the photosystem can not evolve oxygen. Although PSII is found in cyanobacteria (e.g. Synechocystis), green algae (e.g. Chlamydomonas reinhardtii) and higherplants, the overall structure is similar in all these organisms. PSII consists of a central reaction centre core surrounded by a light-harvesting antenna system. The first chemical reaction of PSII is a charge separation within the reaction centre and it is driven by absorbed light energy. The chemical nature of the primary donor, known as P680, is likely a dimer of chlorophyll a but its exact nature has still not been resolved. Subsequent electron transfer steps prevent the primary charge separation from recombining by transferring the electron to pheophytin and then to Q A which becomes Q A - . From Q A - the electron is transferred to another plastoquinone molecule, Q B , which after two photochemical turnovers becomes completely reduced (PQH 2 ). While the electron removed from P680 is rapidly sent away, an electron from a tyrosine residue (Y Z ) in the reaction centre protein D1 reduces P680 + . Electrons for the reduction of Y Z are extracted from water by the water-oxidizing complex. Since one absorbed photon drives the transfer of one electron, the overall photochemical reaction of PSII is
of the p y reth ro id cy p erm eth rin reduces oxygen evolution of the cell cultures to zero. This m eans th at the insecticide is capable of com pletely b lo ck ing the ph o to sy n th etic electro n tra n sp o rt chain in higherplants. Follow ing this o b servation, the q u es tion arises, w eth er the effect is based on a general and unspecific inhibition of the electro n tra n sp o rt as a whole. A n o th e r possibility w ould be th a t the chem ical exerts its effect in a specific region or even at a specific site which can be localized by analyzing specific and distinct parts of the electro n tra n sp o rt chain. In o rd e r to clarify this q uestion we m easured the oxygen gas exchange of tobacco chloroplasts in w ater —» m ethylviologen, w ater —> ferricyanide, w ater —* silicom olybdate and dichlor- op h en o lin d o p h en o l/asco rb ate —» m ethylviologen partial reactions of the electro n tran sp o rt. For this and for the follow ing ex p erim en ts we used two o th e r pyrethroids, nam ely fen v alerate and delta- m eth rin (Fig. 4). T hese substances have been c h o sen because of th e (possibly) relev an t stru ctu ral differences of the m olecules. In the case of delta- m eth rin the two chlorides on the dichlorovinyl-di- m ethylcyclopropyl side of th e m olecule are changed to two brom ides. F en v alerate shows a com pletely different stru ctu re in this p art of the m olecule. It contains an isobutyl co m p o n en t and a chlorophenyl ring instead. Fig. 5 clearly shows th at these py reth ro id s have the sam e inhibitory e f fect on the electron tra n sp o rt as p erm eth rin and cyperm ethrin have as far as the overall effect on the w hole chain is concerned. This effect m ust be
As shown in Table III deepoxidation of violaxan thin and the epoxidation of zeaxanthin could be achieved in isolated intact spinach chloroplasts at pH 7.6 w ithout adding any cosubstrates. These alterations in the xanthophyll composition of the thylakoid which occur in chloroplasts o f all higherplants were discovered in 1957 by Sapoznikov et al.  and elucidated in full detail by Hager and Stransky [3 -1 1 ] and Y am am oto et al. [12-17], From the observation that the deepoxidation of violaxanthin also occurred in red light and is in hibited in the presence of DCM U a strong connec tion between the xanthophyll cycle and photosyn thetic electron transport has been suggested [7, 19]. Moreover it was found that violaxanthin was deep- oxidized in uncoupled spinach thylakoids already in the dark if the pH of the incubation m edium was changed from 7 to 5 . This result as well as the pH-optim um (pH = 5.2) of the isolated violaxan- thin-deepoxidase  lead to the conclusion that the enzyme is located at the inner side of the thylakoid membrane.
In higherplants VLCFAs are structurally impor tant constituents of seed storage lipids, plant m em branes and epicuticular waxes, the latter including different long chain com ponents like acids, alde hydes, alcohols and esters, all biochemically deriv ing from VLCFA precursors (Cassagne et al., 1994; von Wettstein-Knowles, 1993). The chloroacet amide metolachlor has been reported to reduce the wax formation with its C28-C32 com ponents in Sorghum bicolor (Ebert and Ramsteiner, 1984). Möllers and Albrecht (1994) showed both the in hibition of 18:2 desaturation and 2 0 : 1 elongation
Z. Naturforsch. 44c, 504—508 (1989); received January 11, 1989 D edicated to P rofessor A chim Trebst on the occasion o f his 60th birthday Sulphate Assimilation, PAPS-Reductase, Thioredoxin, Sulphite Formation
Higher plant leaf protein was investigated for the enzyme activity catalyzing a thioredoxin- dependent reduction of 3'-phosphoadenylylsulphate (PAPS) to sulphite. The enzyme became detectable when heterologous thioredoxin from E scherichia coli was used substituting for the hitherto unidentified plant thioredoxin. The enzyme's cross-reactivity with heterologous thioredo xin enabled the partial purification and brief characterization. The molecular weight of the en zyme as estimated by HPLC size exclusion and gel filtration was 68—72 k. The protein reduced PAPS only when thioredoxin was present as cosubstrate. The function of this enzyme in the assimilation of inorganic sulphate by higherplants is discussed in comparison to the function of the respective enzymes from Escherichia coli and Saccharom yces cerevisiae.
It is possible that the gene duplication and fission events, establishing an alternative ICS copy and splitting apart this activity from the MenF module of PHYLLO product in the course of evolution of higherplants, represent a consequence of a need for at least one separate ICS gene outside of the PHYLLO context that is especially important under conditions of phytopathogenic attack to induce the metabolic flow from chorismate towards the synthesis of SA required for plant resistance (Wildermuth et al., 2001; Durrant and Dong, 2004; Brodersen et al., 2005). The SA acts as a central signal in the process of systemic and local acquired resistance and is associated with accumulation of pathogenesis-related proteins, which are though to contribute to plant defence. It has been demonstrated in Arabidopsis that the isochorismate pathway, initiated by the induction of the ICS1 gene, is the major source of SA during systemic acquired resistance (Wildermuth et al., 2001; Durrant and Dong, 2004). The present work provides the genetic evidence that ICS1 is also required for the synthesis of PhQ and exerts overlapping functions with ICS2. Therefore, duplication and inactivation of the menF module of PHYLLO by a gene split event generated a branching point between PhQ and SA biosynthesis (Fig. 25).
The presence of diverse RNA polymerases operating in plastids of higherplants is complemented by the existence of different enzyme-specific promoters and possibly also termination signals preceeding and following plastid transcription units, respectively. The PEP enzyme is known to initiate transcription from -10/-35 eubacterial-type promoters (Igloi and Kössel, 1992). Analyses of transcription in mutants with disrupted PEP genes or in plastids of non-photosynthetic tissue culture cells led to the identification of NEP-specific promoters that are reminiscent of promoters recognised by mitochondrial and T3/T7 phage RNA polymerases (Hajdukiewicz et al., 1997; Kapoor et al., 1997). In vitro studies identified a small three nucleotide motif (“CRT”) at position –6 to –8 as the NEP promoter core (Liere and Maliga, 1999). Based on analyses of wild-type and PEP-deficient plastids, transcription units have been operationally grouped into three principal classes. Some genes or operons have been suggested to be transcribed by either PEP or NEP, whereas others appeared to be transcribed by both enzymes (Hajdukiewicz et al., 1997). It was proposed that NEP preferentially drives transcription of genes for components of the plastid genetic system, whereas PEP transcribes genes for constituents of the photosynthetic machinery (Hajdukiewicz et al., 1997; Maliga, 1998). Consistently, the accumulation of transcripts of a selected set of photosynthesis-related genes was shown to be reduced dramatically in plants lacking PEP (Allison et al., 1996; Hajdukiewicz et al., 1997). Analysis of run-on transcription activities in PEP-deficient plastids, however, revealed that most segments of the plastid chromosome, independent of the encoded gene class, are transcribed even in the absence of PEP (Krause et al., 2000). Thus, the functional and phylogenetic implications of multiple transcription machineries in the plastids of higherplants is obviously much more complex than initially supposed.
Salad, soybeans, rye and tobacco plants, grown on field in the open air or in greenhouses con tain benzo (e) pyrene, benzo (a) pyrene, perylene, anthanthrene, benzo (ghi) perylene, dibenzo(a,h) anthracene, and coronene through air pollution. Plants of the same seeds, grown in a special room with air supply by filter-combination (cellulose filter + filtering charcoal + special filters) and with air lock for personnel (changed clothes) show non of the polycyclic hydrocarbons.
For ultrastructural analysis of actin distribution in healthy and stolbur-diseased S. lycopersicum plants, experiments using LRW embedded tissue and immunogold labeling of plant actin was performed as follows (modified after White et al. 1994). To block unspecific binding sites, grids were placed on droplets of blocking solution made up of normal goat serum (NGS) diluted 1:30 in 1% BSA in PBS, pH 7.6, for 2 h at RT. Then, grids were incubated overnight at 4° C with primary mouse monoclonal antibody against actin (MAB anti-actin, clone C11, Agrisera, Vännäs, Sweden), diluted 1:200 in blocking solution. Control grids were incubated in 1 % BSA/PBS without primary antibody. All grids were then rinsed with PBS, and treated for 1 h at RT with secondary goat antimouse antibody coated with colloidal 5 nm gold particles (GAM 5, Auro Probe EM GAM G5 Amersham, USA), diluted 1:40 in 1% BSA/PBS. Sections were stained in 3 % uranyl acetate and 0.1% lead citrate (Reynolds 1963) and observed under a PHILIPS CM 10 (FEI, Eindhoven, The Netherlands) Transmission Electron Microscope operating at 100 kV.
Three additional homologous genes of unknown function, APO2 to APO4, are present in the Arabidopsis genome. Interestingly, each of the four APO proteins possesses an orthologous counterpart in the rice genome (Figure 10B). Therefore, the APO gene family consists of four distinguishable groups that are present in both monocotyledonae and dicotyledonae. APO1 to APO4 show much fewer similarities in the N terminus than in the remaining part of the proteins, indicating different localizations and/or functions. APO2 is also predicted to be localized in the chloroplast. In rice (Oryza sativa), APO3 is computed to be present in the chloroplast as well, whereas the Arabidopsis protein is predicted to be localized in mitochondria. APO4 is computed to represent a mitochondrial protein in both organisms. All members of the previously unknown APO gene family contain a 100 amino acid residues - spanning region (APO motif 1) with conserved Cys, His, Gly, and acidic and basic amino acids (Figure 10). The highly conserved APO motif 1 is duplicated at the C terminus (designated APO motif 2). These two motifs are always separated from each other by a less- conserved spacer that is also variable in length but in groups 1 to 3 contains one conserved Met embedded within positively charged amino acid residues directly upstream of APO motif 2 (Figure 10). A similar sequence is also present downstream of APO motif 2 close to the C terminus in all groups. APO motif 1 is followed by a short stretch containing positive charges in groups 1 to 3. The highly conserved signature of both motifs in APO1, which fits to all members of the APO gene family in vascular plants, can be defined as C-x2-C-x3-(H,Q)-x4- GH-x4-C-x11-H-x-W-x6-D-x8- H-x(20-26)-PA-x2-E(L,I)C-x3-G. The conserved Cys in both motifs could provide ligands for tetranuclear Fe-S centers (Sticht and Rösch, 1998). Several conserved differences between the two motifs could indicate different functions (Figure 10B). For example, both motifs, APO 1 and 2, contain a conserved H in another position. Two conserved R residues are present in APO motif 1 and two K residues in motif 2. In addition, APO motif 2 contains conserved G, VW, YG, and A residues, which are not present in APO motif 1 in any of the groups (Figure 10).
mina, suggesting two different intermediates in the bio
synthesis o f these simple 2- or 5-hydroxy-l,4-naphtho- quinones.
The biosynthesis of certain higher plant naph thoquinones became clearer when it could be shown that all seven carbon atoms of shikimic acid were incorporated in toto into the naphthoqui nones juglone of Juglans regia and lawsone of
Bow ler C., Van Camp W., Van Montagu M. and Inze D. (1994), Superoxide dismutase in plants. C R C Critical Reviews in Plant Sciences 13, 1 9 9 -2 1 8 .
Cardinaels C., Put C., Van Assche F. and Clijsters H. (1984), The superoxide dismutase as a biochemical in dicator, discriminating between zinc and cadmium toxicity. A rch. Int. Physiol, et Biochem. 92 P F 2 7 - 2 8 . Chaudiere J. (1994), Some chemical and biochemical constraints of oxidative stress in living cells. In: Free Radical Damage and Its Control. New Comprehen sive Biochemistry, vol. 28, (Rice-Evans C. A. and Bur- don R. H ., eds.). Elsevier, Amsterdam, 2 5 - 6 6 . Ciscato M., Valcke R., Van Loven K., Clijsters H. and
The plant small heat shock protein genes have evolved from a single gene found in most animals and fungi into a large super gene family (Waters et al., 1996). The phylogenetic relationships of sHSP’s reveals that gene duplication, sequence divergence and gene conversation have all played a role in their evolution (Waters et al., 1996). In comparison to the large HSP genes, small heat-shock protein genes have evolved much more quickly. Plants have six sHSP’s gene families which are nuclear encoded. Evolutionary analysis shows that these classes arose prior to the divergence of the major groups of angiosperms sHSP’s are more related to proteins of the same class from divergent species than to other small heat shock proteins of the same species (Waters et al., 1996). The analysis of rate of evolution showed that sHSP gene families have evolved at unequal rates (Waters, 1995). In the early publications concerning the structure of HSP genes it was shown that some genes are free of introns (Yost and Lindquist, 1986). It was also demonstrated that abrupt heat stress interrupts intron processing of several gene transcrips in Drosophila (Yost and Lindquist, 1986) and S.cerevisiae (Yost and Lindquist, 1991). These facts led to the assumption that absence of introns in HSP genes is a mechanism employed to avoid heat- induced block in inhibition of splicing of HSP transcripts (Yost and Lindquist, 1986). However, several other heat-inducible HSP genes contain introns which are spliced efficiently under heat stress conditions (Russnak and Candido, 1985; Czarnecka et al., 1985; Bond, 1988; Minchiotti et. al., 1991; Takahashi et al., 1992). The first intron- containing small heat shock gene of plants with molecular weight 26 kDa was identified from soybean (Czarnecka et al., 1988). Later it was shown that chloroplast-localised sHSP’s from Arabidopsis thaliana, Nicotiana tabacum, N. sylvestris and N. tomentosiformis also possess a single intron (Osteryoung et. al., 1993; Lee et al., 1998a).
xanthin and zeaxanthin. T h e m ain q u in o n e con tained in chloroplasts and th e ir th y la k o id m e m branes was the electron ca rrie r and p ro to n tran s locator redox system p la sto q u in o n e -9 /p la sto h y d ro - quinone-9 . a-T o co p h ero l, suggested to serve as a structural com ponent and an tio x id a tiv e agent  was contained in leaves and chloroplasts in sim ilar am ounts as plastoquinone-9 (Tables II, III). a-Toco- quinone and phylloquinone were also obtained in thylakoids, although in m uch sm aller am ounts (Table II). As can be deduced from Tables I —III th e overall pigm ent and quin o n e com p o sitio n o f chloroplasts from different plan t species grow n in th e sam e natural environm ent does not change very m uch, but considerable changes can be ob serv ed betw een plants grown u n d er d iffe ren t in ten sities o f light ([24, 25], T ables II, III). An analysis concerning the d istrib u tio n o f chlorophylls, caro ten o id s and quinones in su b p la stid fractions revealed th a t virtually all pigm ents and quin o n es w ere contained exclusively in th e th y la k o id m e m b ra n e an d not in the envelope (T able IV). A lthough 5 m gs (spinach) and 4.1 mgs (ra d ish ) envelope p ro tein w ere assayed, the am o u n t o f chlorophylls, caro ten o id s and quinones contained in these fractions was in the range o f d etec tab ility and could only be estim ated q uantitatively by using high p e rfo rm an ce liq u id ch rom atography (Figs. 1, 2). As co m p ared to recent envelope p re p a ra tio n s no specific en rich m en t in violaxanthin was o b ta in e d b u t m ost surprisingly m ore po lar terp e n o id s like chlo ro p h y ll a and b, violaxanthin and n eo x a n th in w ere still detected. /7-carotene, lutein and an th e ra x a n th in w ere com pletely absent. T hese results m ost likely suggest that the very sm all am o u n ts o f pigm ents o b ta in ed in the isolated envelope fractions m ay d eriv e from a nonspecific b inding o f these m ore p o la r terp en o id s to the envelope m e m b ran e d u rin g c h lo ro p last isola tion.
According to the generally accepted endosymbiont theory mitochondria and chloroplasts are descendants of bacteria-like organisms. This theory was first developed for the origin of chloroplasts (Schimper, 1883), and subsequently for mitochondria (Wallin, 1927). By means of molecular biological analyses this theory has been essentially verified (Margulis, 1970; Gray, 1989; Gray, 1992). Based on this theory mitochondria have a common ancestor with aerobic α-proteobacteria (purple bacteria) that seem to have been embedded in a protoeukaryotic cell, a probably anaerobic single cell organism with high similarity to nowadays archaea. The symbiosis preadapted the cells to a change from a reducing to an oxidizing atmosphere about 1.5 billion years ago. The cells were now not only able to circumvent the toxicity of oxygen by metabolizing it into harmless by-products, but also to generate energy in a process called respiration (de Duve, 1996). The origin of plastids is based on a second symbiosis event, in which a progenitor of the present cyanobacteria was integrated into a eukaryotic cell. New studies provide evidence for a single origin of chloroplasts in red and green algae, from which the green plants evolved (Moreira et al., 2000; Palmer, 2000). An analogous singular origin of mitochondria is supported by several physiological and biochemical studies (Gray, 1999a; Gray, 1999b).
the expected size were considered to carry the transgene and were chosen for further analysis. Genomic DNA of these plants was used for Southern analysis to confirm the presence of the transgene and to determine as explained in the “Results” part whether the transgene was in a homozygous or heterozygous state. To get additional evidence for transgene homozygosity, genetic crosses between progeny plants of selfed primary transformants and SR1 wild-type plants were performed. Young buds were emasculated and pollen from wild-type tobacco was dusted onto the stigma. Seeds were collected and stored in Eppendorf tubes at 4°C. Two weeks after storage, about 100 seeds were sterilised and plated onto kanamycin-containing MS medium. Sterilisation was done by incubation of the seeds in 70% (v/v) ethanol for 2-3min followed by a 7% (v/v) sodium-hypocholride treatment (10min). Before plating the sterilised seeds onto the selection medium, they were washed with sterile water for at least 6 times. Subsequently, 10-20 seeds per plate were dispensed. The plates were incubated in a phytochamber under standard light conditions until seedlings were grown. By counting the number of germinating seeds versus non- germinating seeds, the germination percentage was calculated. The growth of germinating seeds that developed into seedlings was monitored. Due to the presence of the kanamycin, non-transgenic seedlings developed white cotyledons and died within few weeks. By contrast, seedlings from transgenic plants had green cotyledons and developed further. The ratio between developing and dying seedlings was counted. If all of the germinated seeds developed green cotyledons the parental T 1 transgenic plant was considered to carry the