Presence Of Norflurazon Research Articles

Pigment Production of Chlamydomonas Strains in Response to Norflurazon and ZnO Nanoparticles

Numerous species of microalgae have been utilized for pigment production. More and more species are gaining popularity due to their ability to accumulate pigments with varying chemical compositions and the fact that some have distinctive byproducts that can be co-produced. Despite the fact that many of the species have unique by-products and traits, they are not being used economically due to high production costs. Utilizing agricultural and industrial wastewater for algae cultivation is one way to lower manufacturing costs. Herbicide-contaminated wastewater can result from agricultural contamination. Norflurazon is a popular pesticide frequently used for weed control. The presence of norflurazon in water renders that water unusable and requires proper treatment. Nanoparticles of ZnO (ZnO NPs), on the other hand, are utilized in a variety of industrial productions of numerous household goods. Water contaminated with ZnO NPs can present potential risks to human health and the environment. In this study, two field isolates of the green microalga Chlamydomonas reinhardtii, a widely used model organism, were examined for their reaction to these two compounds in order to assess the responses of different natural strains to environmental stresses. Norflurazon at 10 µM had a higher inhibitory effect on growth and pigment production than ZnO NPs at 200 mg L−1. Although both norflurazon and ZnO NPs inhibit cell growth and pigmentation, they do so through distinct processes. Norflurazon induces oxidative stress in cells, resulting in photosystem damage. ZnO nanoparticles, on the other hand, did not cause photosystem damage but rather mechanical cell damage and disintegration. In addition, the physiological responses of the two Chlamydomonas strains were distinct, supporting the utilization of natural algal strains for specific types of environmental pollutants.

The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae.

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

1O2-Mediated and EXECUTER-Dependent Retrograde Plastid-to-Nucleus Signaling in Norflurazon-Treated Seedlings of Arabidopsis thaliana

Chloroplast development depends on the synthesis and import of a large number of nuclear-encoded proteins. The synthesis of some of these proteins is affected by the functional state of the plastid via a process known as retrograde signaling. Retrograde plastid-to-nucleus signaling has been often characterized in seedlings of Arabidopsis thaliana exposed to norflurazon (NF), an inhibitor of carotenoid biosynthesis. Results of this work suggested that, throughout seedling development, a factor is released from the plastid to the cytoplasm that indicates a perturbation of plastid homeostasis and represses nuclear genes required for normal chloroplast development. The identity of this factor is still under debate. Reactive oxygen species (ROS) were among the candidates discussed as possible retrograde signals in NF-treated plants. In the present work, this proposed role of ROS has been analyzed. In seedlings grown from the very beginning in the presence of NF, ROS-dependent signaling was not detectable, whereas, in seedlings first exposed to NF after light-dependent chloroplast formation had been completed, enhanced ROS production occurred and, among others, (1)O2-mediated and EXECUTER-dependent retrograde signaling was induced. Hence, depending on the developmental stage at which plants are exposed to NF, different retrograde signaling pathways may be activated, some of which are also active in non-treated plants under light stress.

‘happy on norflurazon’ (hon) mutations implicate perturbance of plastid homeostasis with activating stress acclimatization and changing nuclear gene expression in norflurazon‐treated seedlings

Various mutant screens have been undertaken to identify constituents involved in the transmission of signals from the plastid to the nucleus. Many of these screens have been performed using carotenoid-deficient plants grown in the presence of norflurazon (NF), an inhibitor of phytoene desaturase. NF-treated plants are bleached and suppress the expression of nuclear genes encoding chloroplast proteins. Several genomes uncoupled (gun) mutants have been isolated that de-repress the expression of these nuclear genes. In the present study, a genetic screen has been established that circumvents severe photo-oxidative stress in NF-treated plants. Under these modified screening conditions, happy on norflurazon (hon) mutants have been identified that, like gun mutants, de-repress expression of the Lhcb gene, encoding a light-harvesting chlorophyll protein, but, in contrast to wild-type and gun mutants, are green in the presence of NF. hon mutations disturb plastid protein homeostasis, thereby activating plastid signaling and inducing stress acclimatization. Rather than defining constituents of a retrograde signaling pathway specifically associated with the NF-induced suppression of nuclear gene expression, as proposed for gun, hon mutations affect Lhcb expression more indirectly prior to initiation of plastid signaling in NF-treated seedlings. They pre-condition seedlings by inducing stress acclimatization, thereby attenuating the impact of a subsequent NF treatment.

In‐depth analysis of the distinctive effects of norflurazon implies that tetrapyrrole biosynthesis, organellar gene expression and ABA cooperate in the GUN‐type of plastid signalling

Application of norflurazon (NF) damages plastids, induces photobleaching and represses expression of the nuclear LHCB1.2 gene encoding a light-harvesting protein. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of NF. The mutants gun2, gun4 and gun5 exhibit perturbations in tetrapyrrole biosynthesis, but gun1 is defective in organellar gene expression (OGE). How gun mutations affect nuclear gene expression (NGE) and why the signals elicited by the two types evoke the same response remains unknown. Here we show that the carotenoid biosynthesis inhibitors amitrole and flurochloridone can replace NF in gun assays, whereas novel tetrapyrrole pathway mutations do not provoke a gun phenotype. Changes in haem levels also do not account for LHCB1.2 derepression in NF-treated gun mutants. Pigment measurements indicated that gun mutants are not resistant to NF, but gun2, gun4 and gun5 retain low levels of lutein, as well as of neoxanthin and violaxanthin, the precursors of abscisic acid (ABA). This might explain the enhanced ABA sensitivity of gun4 and gun5 plants found in germination assays. Metabolite profiling and analyses of reactive oxygen species and cellular redox state failed to suggest a link between gun mutations and altered LHCB1.2 expression. However, in contrast to NF-treated wild-type plants, gun mutants retain to a marked extent the capability to express the plastome-encoded proteins AtpB and RbcL. This, together with the finding that application of ABA can partially restore LHCB1.2 expression in NF-treated wild-type plants, supports the view that tetrapyrrole, OGE and ABA signalling are interconnected.

β-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis

The cauliflower ( Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of β-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced β-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.

Coordination of plastid and nuclear gene expression.

The coordinated expression of genes distributed between the nuclear and plastid genomes is essential for the assembly of functional chloroplasts. Although the nucleus has a pre-eminent role in controlling chloroplast biogenesis, there is considerable evidence that the expression of nuclear genes encoding photosynthesis-related proteins is regulated by signals from plastids. Perturbation of several plastid-located processes, by inhibitors or in mutants, leads to decreased transcription of a set of nuclear photosynthesis-related genes. Characterization of arabidopsis gun (genomes uncoupled) mutants, which express nuclear genes in the presence of norflurazon or lincomycin, has provided evidence for two separate signalling pathways, one involving tetrapyrrole biosynthesis intermediates and the other requiring plastid protein synthesis. In addition, perturbation of photosynthetic electron transfer produces at least two different redox signals, as part of the acclimation to altered light conditions. The recognition of multiple plastid signals requires a reconsideration of the mechanisms of regulation of transcription of nuclear genes encoding photosynthesis-related proteins.

Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides.

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in zeta-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against zeta-carotene desaturase inhibitors.

Characterization of a norflurazon-resistant mutant of Chlamydomonas reinhardtii

A norflurazon-resistant mutant has been isolated from Chlamydomonas reinhardtii that showed a three-fold factor of resistance over wild type cultures. In comparison to wild type cultures, the mutant showed better retention of chlorophylls and carotenoids when grown in light in the presence of norflurazon. When grown in the dark, chlorophyll losses were similar, while carotenoid losses were lower than in the wild type cultures. Higher levels of phytoene accumulated in the wild type cultures in the presence of norflurazon than in the resistant cultures. The resistant cultures also showed cross tolerance to EMD-IT 5914, a herbicide with a similar mode of action. Norflurazon resistance in this alga appears to arise from alterations in the target enzyme phytoene desaturase.

Role of carotene in the rapid turnover and assembly of photosystem II in Chlamydomonas reinhardtii

Inhibitors of the phytoene desaturase in carotene biosynthesis were tested in the enhanced rapid turnover of the D1 protein of photosystem II in high light exposure of Chlamydomonas reinhardtii cells. After 1 h high light on heterotrophically grown cells in the presence of norflurazon or fluridone, photosynthesis activity in vivo and PS II activity in vitro is lost. The D1 protein has disappeared. PS I activity is not affected, nor is the D2 protein. It is concluded that β-carotene is essential for the assembly of the D1 protein into functional photosystem II. It is suspected that bleaching of β-carotene in the reaction center of PS II by high light destabilizes the structure and triggers the degradation of the D1 protein.

Transcription initiation site of a NADP-specific glutamate dehydrogenase gene and potential use of its promoter region to express foreign genes in ammonium-culturedChlorella sorokiniana cells

Chlorella sorokiniana may prove to be a useful organism in which to express genes/cDNAs from certain higher plants encoding enzymes catalyzing the synthesis of compounds of medical or industrial importance. This organism has a NADP-specific glutamate dehydrogenase (NADP-GDH) nuclear-gene encoding a precursor mRNA that appears to be alternatively spliced to yield mRNAs differing in size and encoding enzyme subunits with differing affinities for ammonium. The promoter of this gene may be useful to drive the expression of heterologous genes. The transcription initiation site of the NADP-GDH gene was identified. This site delineates the 3′-border of the promoter region, and 1285 bp upstream from this site have been sequenced. To identify the promoter 5′-border by deletion analysis, this region (coupled to a reporter gene in a plasmid vector) was to be introduced by electroporation intoC. sorokiniana protoplasts. Cells were treated with a mixture of hydrolytic enzymes; however, non-hydrolyzable component(s) in the cell wall inhibited protoplast formation. The cell wall was subsequently shown to contain acetolysis-resistant component(s) identified as sporopollenin-like in other algae. Since sporopollenin was reported earlier to be a polymer of carotenoids/carotenoid-esters, the cells were cultured in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Whereas synthesis of total carotenoids was inhibited by 75%, synthesis of the acetolysis-resistant component(s) in the cell wall was not inhibited. In addition to the above studies, a revised model for the regulation of expression of theC. sorokiniana NADP-GDH gene is presented.

Deletion analysis of a nitrite-reductase promoter from spinach in transgenic tobacco

Deletion analysis of the nitrite-reductase (NiR) promoter from spinach (Spinacia oleracea L.) fused to the β-glucuronidase (GUS) reporter gene and introduced into tobacco (Nicotiana tabacum L., cv. Coker 176) indicates that basic elements required for light- and nitrate-dependent expression of the reporter are located within the promoter sequence -200/+131 relative to the transcription-initiation site. Detailed analysis indicates that positive regulatory elements exist between -200 and-330 as well as between -1450 and -1730, stimulating the level of GUS gene expression under all experimental conditions. Induction/reversion light-pulse experiments show that the promoter sequence -200/+131 suffices for phytochrome-mediated expression of the reporter gene. The observation that the NiR promoter from spinach exhibits full reversibility in transgenic tobacco confirms the previous conclusion that the NiR promoter from spinach fused to a GUS reporter gene and introduced into tobacco responds to nitrate and phytochrome as would be expected for tobacco (host) and not as would be expected for spinach (donor). When the plastids were damaged by photooxidation in the presence of Norflurazon, GUS activity levels were reduced to the same extent for all NiR-promoter/GUS fusions tested, indicating that the promoter region involved in the action of the ‘plastidic factor’ is between -200 and +131. The GUS gene expression under the control of the CaMV-35S promoter is not affected by light, nitrate or the ‘plastidic factor’.

Mutagenesis of an Amino Acid Responsible in Phytoene Desaturase from Synechocystis for Binding of the Bleaching Herbicide Norflurazon

Synechocystis mutants were selected resistant against the bleaching herbicide norflurazon. In vitro analysis of phytoene desaturation demonstrated that they all possess a resistant enzyme. Sequencing of the corresponding pds gene revealed that three different point mutations were obtained. In all cases, the same amino acid, Arg195, was modified either into Cys, Pro, or Ser. The degree of resistance determined via carotenoid formation in cells as well as cell-free phytoene desaturation in the presence of norflurazon was highest when Arg was changed to Ser. A modification to Pro or to Cys resulted in a gradually less pronounced degree of resistance. Cross-resistance toward other bleaching herbicides like fluridone, flurtamone, or fluorochloridone was also studied. However, there was only a slight resistance against fluridone in one and against flurtamone in two of the mutants. In contrast to other cyanobacterial mutants previously described, impairment of catalytic activities of the mutated enzymes was negligible.

The promoter of the Arabidopsis thaliana plastocyanin gene contains a far upstream enhancer-like element involved in chloroplast-dependent expression.

Plastocyanin is part of the photosynthetic electron transport chain in the chloroplast and is encoded in the nucleus. Expression of the Arabidopsis thaliana plastocyanin gene is organ specific: high mRNA levels are observed in young green parts of the plant. Furthermore, expression is dependent on the presence of light and functional chloroplasts. When grown in the presence of norflurazon under white light conditions, resulting in the photo-oxidative destruction of the chloroplast, plastocyanin mRNA levels are strongly reduced. A -1579 to -9 promoter fragment confers light-regulated and chloroplast-dependent expression to the beta-glucuronidase reporter gene in transgenic tobacco plants. This suggests that regulation takes place at the level of transcription. A plastocyanin promoter deletion series ranging from -1579 to -121 which was also tested in tobacco, revealed the presence of a strong positive regulating element (PRE) in the -1579 to -705 region. Deletion of this part of the promoter resulted in a approximately 100-fold reduction of GUS expression as measured in mature leaves. Surprisingly, this enhancer-like element was capable of stimulating transcription from a position downstream of its reporter. Moreover, it could also activate a truncated CaMV 35S promoter. Deletion of this element coincides with the loss of chloroplast-dependency of reporter gene expression, as judged by norflurazon treatment of transgenic seedlings. So, the activity of the PRE itself might depend on the presence of functional chloroplasts.

Carotenoids in photosynthesis: Protection of D1 degradation in the light

Photosynthesis has been determined with mutants of Anacystis which form different amounts of carotenoids. With these cultures a highly significant correlation between photosynthetic oxygen evolution and the amounts of synthesized carotenoids was observed. In addition, the influence of carotenoids on light-dependent degradation of thylakoid proteins was investigated with Scenedesmus cultures grown in darkness in the presence of norflurazon, an inhibitor of carotenoid biosynthesis. Pre-illumination of cells resulted in decrease of photosynthetic activity accompanied by loss of the D1 protein. This effect is dependent on the length of illumination, and the light intensity, and increased when carotenoid content was lowered during previous growth of the norflurazon-treated cultures.

Regulation of Plastid Gene Expression during Photooxidative Stress

We have used the carotenoid biosynthesis inhibitor norflurazon to study the relationship between chloroplast and nuclear gene expression and the mechanisms by which plastid mRNA accumulation is regulated in response to photooxidative stress. By treating 4-week-old hydroponic spinach plants (Spinacea oleracea), we were able to determine the response at two distinct stages of chloroplast development. For all parameters studied, differences were found between the norflurazon-treated young and mature leaves. Young leaves lost essentially all pigment content in the presence of norflurazon, whereas mature leaves retained more than 60% of their chlorophyll and carotenoids. The accumulation of plastid mRNA was determined for several genes, and we found a decrease in mRNA levels for all genes except psbA in herbicide-treated young leaves. For genes such as atpB, psbB, and psaA, there was a corresponding change in the relative level of transcription, but for psbA and rbcL, transcription and mRNA accumulation were uncoupled. In norflurazon-treated mature leaves, all plastid mRNAs except psaA accumulated to normal levels, and transcription levels were either normal or higher than corresponding controls. This led to the conclusion that plastid mRNA accumulation is regulated both transcriptionally and posttranscriptionally in response to photooxidative stress. Although direct photooxidative damage is confined to the plastid and peroxisome, there is a feedback of information controlling the transcription of nuclear-encoded plastid proteins. Considerable evidence has accumulated implicating a "plastid factor" in this control. Therefore, the expression of several nuclear-encoded plastid proteins and the corresponding mRNAs were determined. Although the levels of both the small subunit of ribulose-1,5-bisphosphate carboxylase and the light harvesting chlorophyll a/b-binding protein and corresponding mRNAs were reduced, a 28-kilodalton chloroplast RNA-binding protein and corresponding mRNA were at normal levels in norflurazon-treated plants. Changes in mRNA and protein levels were not the result of a general loss due to photooxidation but rather the result of selective stabilization of certain components. The response of both genomes to photooxidative stress is discussed in terms of the postulated plastid factor.

Photooxidative Destruction of Chloroplasts Leads to Reduced Expression of Peroxisomal NADH-Dependent Hydroxypyruvate Reductase in Developing Cucumber Cotyledons

Photooxidative destruction of chloroplasts by exposure of norflurazon-treated cucumber (Cucumis sativus L.) seedlings to white light leads to reduced levels of the nuclear-encoded, peroxisomal enzyme hydroxypyruvate reductase. The partial reduction in hydroxypyruvate reductase activity under photooxidative conditions is accompanied by reductions in levels of hydroxypyruvate reductase protein and transcript. The low level of hydroxypyruvate reductase gene expression in the dark is not affected by norflurazon, and nonphotooxidizing far-red light is able to induce significant increases in hydroxypyruvate reductase expression even in the presence of norflurazon. We conclude that intact plastids are required for maximal expression of hydroxypyruvate reductase in the light and that the plastids affect hydroxypyruvate reductase gene expression at a pretranslational level.

Appearance of nitrite-reductase mRNA in mustard seedling cotyledons is regulated by phytochrome

Nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) are the key enzymes of nitrate reduction. It is well established that the appearance of these enzymes is "induced" by nitrate, and it is generally believed that NR is cytosolic while NIR is plastidic. In mustard (Sinapis alba L.) cotyledons we observed two isoforms of NIR (NIR1 and NIR2) using a chromato-focusing technique. Only one of them (NIR2) disappeared when the plastids were damaged by photooxidation in the presence of Norflurazon. It is concluded that NIR2 is plastidic while NIR1 is extraplastidic and not affected by photooxidation of the plastids. Both isoforms appear to have the same molecular weight (60 kilodaltons, kDa). Two distinct translation products which could be immunoprecipitated with NIR antiserum produced against total NIR from mustard were observed which differed slightly in molecular weight (60 versus 63 kDa). The 63-kDa polypeptide was considered to be the precursor of NIR2. While synthesis of NIR protein depended largely on nitrate, the levels of in-vitro-translatable NIR mRNAs were found to be either independent of nitrate and light (NIR1) or controlled by phytochrome only (NIR2). It appears that phytochrome strongly stimulates the level of mRNA while significant enzyme synthesis (NIR2) takes place only in the presence of relatively large amounts of nitrate. Since an increased enzyme level was strictly correlated with an increase of immunoresponsive NIR protein it is improbable that activation of a precursor plays a role. Rather, it is concluded that, in situ, nitrate controls translation.

Stereoisomers of β-Carotene and Phytoene in the Alga Dunaliella bardawil

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of beta-carotene when grown outdoors under defined conditions. The beta-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans beta-carotene and 40% 9-cis beta-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of beta-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of beta-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.

THE EFFECT OF NORFLURAZON ON PHOTOTROPIC REACTION IN Phaseolus aureus Roxb. SEEDLINGS

Abstract. The phototropic response of norflurazon‐treated mung bean seedlings has been studied to evaluate the possible role of carotenoids, carotenoid‐derived growth substances, or other factors in the perception/reaction system. Phototropism was slowed significantly in plants grown in white and blue light in the presence of norflurazon. This effect was evident in norflurazon‐bleached seedlings, as well as in those whose pigment system was not affected, due to a shorter period of herbicide action. The possible modes of norflurazon action are discussed.