The experimental herbicide CGA 325'615 inhibits synthesis of crystalline cellulose and causes accumulation of non-crystalline beta-1,4-glucan associated with CesA protein.

Developing cotton (Gossypium hirsutum) fibers, cultured in vitro with their associated ovules, were used to compare the effects of two herbicides that inhibit cellulose synthesis: 2,6-dichlorobenzonitrile (DCB) and an experimental thiatriazine-based herbicide, CGA 325'615. CGA 325'615 in nanomolar concentrations or DCB in micromolar concentrations causes inhibition of synthesis of crystalline cellulose. Unlike DCB, CGA 325'615 also causes concomitant accumulation of non-crystalline beta-1,4-glucan that can be at least partially solubilized from fiber walls with ammonium oxalate. The unusual solubility of this accumulated glucan may be explained by its strong association with protein. Treatment of the glucan fraction with protease changes its size distribution and leads to precipitation of the glucan. Treatment of the glucan fraction with cellulase digests the glucan and also releases protein that has been characterized as GhCesA-1 and GhCesA-2--proteins that are believed to represent the catalytic subunit of cellulose synthase. The fact that cellulase treatment is required to release this protein indicates an extremely tight association of the glucan with the CesA proteins. In addition, CGA 325'615, but not DCB, also causes accumulation of CesA protein and a membrane-associated cellulase in the membrane fraction of fibers. In addition to the effects of CGA 325'615 on levels of both of these proteins, the level of both also shows coordinate regulation during fiber development, further suggesting they are both important for cellulose synthesis. The accumulation of non-crystalline glucan caused by CGA 325'615 mimics the phenotype of the cellulose-deficient rsw1 mutant of Arabidopsis that also accumulates an apparently similar glucan (T. Arioli, L. Peng, A.S. Betzner, J. Burn, W. Wittke, W. Herth, C. Camilleri, H. Hofte, J. Plazinski, R. Birch et al. [1998] Science 279: 717).

Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside.

Glycosylation of endogenous secondary plant products and abiotic substances such as herbicides increases their water solubility and enables vacuolar deposition of these potentially toxic substances. We characterized and compared the transport mechanisms of two glucosides, isovitexin, a native barley flavonoid C-glucoside and hydroxyprimisulfuron-glucoside, a herbicide glucoside, into barley vacuoles. Uptake of isovitexin is saturable (Km = 82 microM) and stimulated by MgATP 1.3-1.5-fold. ATP-dependent uptake was inhibited by bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, but not by vanadate. Transport of isovitexin is strongly inhibited after dissipation of the DeltapH or the DeltaPsi across the vacuolar membrane. Uptake experiments with the heterologue flavonoid orientin and competition experiments with other phenolic compounds suggest that transport of flavonoid glucosides into barley vacuoles is specific for apigenin derivatives. In contrast, transport of hydroxyprimisulfuron-glucoside is strongly stimulated by MgATP (2.5-3 fold), not sensitive toward bafilomycin, and much less sensitive to dissipation of the DeltapH, but strongly inhibited by vanadate. Uptake of hydroxyprimisulfuron-glucoside is also stimulated by MgGTP or MgUTP by about 2-fold. Transport of both substrates is not stimulated by ATP or Mg2+ alone, ADP, or the nonhydrolyzable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Our results suggest that different uptake mechanisms exist in the vacuolar membrane, a DeltapH-dependent uptake mechanism for specific endogenous flavonoid-glucosides, and a directly energized mechanism for abiotic glucosides, which appears to be the main transport system for these substrates. The herbicide glucoside may therefore be transported by an additional member of the ABC transporters.

An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast.

Glutathione S-transferases (GSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max L.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic GST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic GST but did not affect the intracellular isoforms. The discovery of apoplastic inducible GST will be discussed in light of the putative function of these enzymes in plants.

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

Induction of cytochrome P450 genes by ethanol in maize.

Ethanol treatment of etiolated maize seedlings led to a marked enhancement in metabolism of the herbicides metolachlor and prosulfuron. cDNA clones representing eight genes that encode putative cytochrome P450 enzymes were isolated from maize. They fall into three families and are designated CYP71C5, CYP73A6, A7, and A8, and CYP81A1, A2, A3, and A4. Ethanol treatment induced the CYP81A subfamily at the mRNA level in both roots and shoots of etiolated seedlings.

A herbicide antidote (safener) induces the activity of both the herbicide detoxifying enzyme and of a vacuolar transporter for the detoxified herbicide.

In plants potentially toxic compounds are ultimately deposited in the large central vacuole. In this report we show that isolated barley mesophyll vacuoles take up the glucoside conjugate of the herbicide derivate [5-hydroxyphenyl]primisulfuron. Transport is stimulated by Mg-ATP and is distinct from that previously described for glutathione conjugates. Treatment of barley with different herbicide antidotes (safeners) revealed that the safener cloquintocet-mexyl doubles the vacuolar transport activities for both the glutathione and glucoside conjugates. Stimulation of the uptake of the metolachlor-glutathione conjugate was the result of an increased uptake velocity whereas the Km remained unaltered, suggesting that the higher activity was due to a higher expression of the transporter. These results indicate that modulation of vacuolar transport activities are an integral part of the detoxification mechanism of plants.

Light-dependent, but phytochrome-independent, translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgare L.).

This work reports on the regulation of synthesis of the P700 chlorophyll-a apoprotein of photosystem I in barley. The mRNA for the P700 apoprotein is almost exclusively confined to the plastid membrane-bound polysomes. However, the mRNA for the 32-kDa herbicide-binding protein of photosystem II is found in both the soluble and membrane-bound polysomes.The mRNA for the P700 apoprotein is found in similar amounts in dark-grown and light-grown wild-type as well as mutant xantha-l(81) barley. The latter mutant is deficient in chlorophyll biosynthesis. However, while wild-type leaves accumulate the P700 chlorophyll-a protein only in the light, mutant leaves never accumulate the P700 apoprotein.A more sensitive approach was taken using isolated plastids to study P700 apoprotein synthesis. Etioplasts did not synthesize detectable P700 apoprotein even when the etioplasts were exposed to light. However, only a 1-min exposure of leaves to light was necessary to induce P700 apoprotein synthesis by isolated plastids.Phytochrome involvement in controlling P700 apoprotein synthesis was tested by using red/farred light treatment of leaves. These treatments showed no far-red reversibility of red-induced P700-apoprotein synthesis in isolated plastids even after 3 h of darkness after the light treatments. From these data we conclude that the accumulation of P700 apopootein is not under the control of phytochrome and that the light induction of P700 apoprotein is most likely mediated through the protochlorophyllide/chlorophyllide system. This control, however, may also involve cytoplasmic signals as the synthesis of the P700 apoprotein is not turned on in illuminated etioplasts.

The light-dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center in barley. Evidence for translational control.

The light-dependent accumulation of the P700 chlorophyll a protein of the photosystem I reaction center has been studied in greening barley (Hordeum vulgare L.) seedlings. Immunoblot analysis of total cellular protein fractions and immunogold labelling of the P700 chlorophyll a protein in ultrathin sections of Lowicryl-embedded leaf tissue revealed that the concentration of this chlorophyll-binding protein in plastids of dark-grown barley seedlings is below the limit of detection. Upon illumination with white light, a rapid accumulation of this protein is induced. This light effect seems not to be regulated at the level of transcription. The gene for the P700 chlorophyll a protein has been mapped within the large single-copy region of the plastid DNA of barley. High levels of transcripts of this gene are present already in dark-grown seedlings and remain fairly constant throughout an extended illumination period. Polysomes were isolated from etioplasts and chloroplasts. The same high relative concentration of mRNA encoding the P700 chlorophyll a protein was present in both polysome fractions. This result suggests that the light-dependent accumulation of the P700 chlorophyll a protein during chloroplast formation in barley seedlings is regulated at the translational, or posttranslational, level.

The implication of a plastid-derived factor in the transcriptional control of nuclear genes encoding the light-harvesting chlorophyll a/b protein.

In carotenoid-deficient albina mutants of barley and in barley plants treated with the herbicide Norflurazon the light-dependent accumulation of the mRNA for the light-harvesting chlorophyll a/b protein (LHCP) is blocked. Thus, the elimination of a functional chloroplast, either as a result of mutation or as a result of herbicide treatment, can lead to the specific suppression of the expression of a nuclear gene encoding a plastid-localized protein. These results confirm and extend earlier observations on maize [Mayfield and Taylor (1984) Eur. J. Biochem. 144, 79-84]. The inhibition of mRNA accumulation appears to be specific for the LHCP; the mRNAs encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the NADPH: protochlorophyllide oxidoreductase are relatively unaffected. The failure of the albina mutants and of Norflurazon-treated plants to accumulate the LHCP mRNA is not exclusively caused by an instability of the transcript but rather by the inability of the plants to enhance the rate of transcription of the LHCP genes during illumination. Several chlorophyll-deficient xantha mutants of barley, which are blocked after protoporphyrin IX or Mg-protoporphyrin, and the chlorophyll-b-less mutant chlorina f2 accumulate the LHCP mRNA to almost normal levels during illumination. Thus, if any of the reactions leading to chlorophyll formation is involved in the control of LHCP mRNA accumulation it should be one between the formation of protochlorophyllide and the esterification of chlorophyllide a. While the nature of the regulatory factor(s) has not been identified our results suggest that, in addition to phytochrome (Pfr), plastid-dependent factors are required for a continuous light-dependent transcription of nuclear genes encoding the LHCP.

Synthesis of prenyl lipids in cells of spinach leaf. Compartmentation of enzymes for formation of isopentenyl diphosphate.

Purified spinach chloroplasts incorporate [1-14C]isopentenyl diphosphate into prenyl lipids in high yields. The immediate biosynthetic precursors of isopentenyl diphosphate (hydroxymethylglutaryl-CoA, mevalonate, mevalonate-5-phosphate, mevalonate-5-diphosphate), on the other hand, are not accepted as substrates and the corresponding enzymes hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase are not present in the organelles. These enzymes can only be detected in a membrane-bound form at the endoplasmic reticulum (hydroxymethylglutaryl-CoA reductase) and as soluble activities in the cytoplasm. The concept is developed that isopentenyl diphosphate is formed in the cytoplasm as a 'central intermediate' and is distributed then to other cellular compartments (endoplasmic reticulum, plastids, mitochondria) for further biosynthetic utilization.

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts.

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-(14)C]isopentenyl pyrophosphate) or [(14)C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-(14)C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.

The site of carotenogenic enzymes in chromoplasts from Narcissus pseudonarcissus L.

The membranes from the chromoplasts of Narcissus pseudonarcissus L. which are derived from the inner envelope membrane are the site of ß-carotene synthesis from [1-(14)C]isopentenyl diphosphate. The enzymes involved are partly peripheral membrane proteins (prenyltransferase, phytoene synthase) and partly integral membrane proteins (cis-trans isomerase, dehydrogenase(s), cyclase(s)). Metabolic channeling is suggested.

Chlorophyll synthetase in chlorophyll-free chromoplasts.

A considerable incorporation of [1-(14)C]isopentenyl diphosphate into chlorophyll in chromoplast preparations from daffodil flowers (Narcissus pseudonarcissus L.) was observed when exogenous chlorophyllide a was added. The enzyme chlorophyll synthetase showed properties of a peripheral membrane protein.

On the compartmentation of isopentenyl diphosphate synthesis and utilization in plant cells.

Purified spinach chloroplast and daffodil chromoplast preparations do not use mevalonate, phosphomevalonate, and diphosphomevalonate for the synthesis of isopentenyl diphosphate. Isopentenyl diphosphate, on the other hand, is incorporated into plastidal polyprenoids in large amounts. In the presence of a cytoplasmic supernatant, however, mevalonate and the phosphomevalonates were incorporated into the plastidal polyprenoids in equally large amounts, which demonstrates that the enzymes mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and diphosphomevalonate decarboxylase (EC 4.1.1.33) are soluble cytoplasmic enzymes and that they apparently do not occur as isoenzymes within the plastids. The concept is developed that isopentenyl diphosphate is a central intermediate in plant polyprenoid formation which is channeled into several compartment for different biosynthetic pathways.

ß-Carotene synthesis in isolated chromoplasts from Narcissus pseudonarcissus.

A system has been established from isolated intact chromoplasts of Narcissus pseudonarcissus flowers that synthesizes geranylgeraniol, an unknown polyprenoid alcohol, phytoene, and ß-carotene from [1-(14)C]isopentenyl pyrophosphate in a good yeild. Long chain pyrophosphates are not accumulated. San 6706 inhibits the dehydrogenation of phytoene, whereas nicotine does not lead to an accumulation of lycopene. Separation and identification of polyprenoid lipids was performed by HPLC. The properties and advantages of the chromoplast system are discussed.