Somatic mutation-mediated evolution of herbicide resistance in the nonindigenous invasive plant hydrilla (Hydrilla verticillata)

Hydrilla (Hydrilla verticillata L.f. Royle) was introduced to the surface water of Florida in the 1950s and is today one of the most serious aquatic weed problems in the USA. As a result of concerns associated with the applications of pesticides to aquatic systems, fluridone is the only USEPA-approved chemical that provides systemic control of hydrilla. After a decrease in fluridone's efficacy at controlling hydrilla, 200 Florida water bodies were sampled to determine the extent of the problem and the biological basis for the reduced efficacy. Our studies revealed that hydrilla phenotypes with two- to six-fold higher fluridone resistance were present in 20 water bodies. Since fluridone is an inhibitor of the enzyme phytoene desaturase (PDS), the gene for PDS (pds) was cloned from herbicide-susceptible and -resistant hydrilla plants. We report for the first time in higher plants three independent herbicide-resistant hydrilla biotypes arising from the selection of somatic mutations at the arginine 304 codon of pds. The three PDS variants had specific activities similar to the wild-type enzyme but were two to five times less sensitive to fluridone. In vitro activity levels of the enzymes correlated with in vivo resistance of the corresponding biotypes. As hydrilla spread rapidly to lakes across the southern United States in the past, the expansion of resistant biotypes is likely to pose significant environmental challenges in the future.     

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.     

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method

Photosynthesis Research - A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic...     

Intragenic Enhancers and Suppressors of Phytoene Desaturase Mutations in Chlamydomonas reinhardtii

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis-the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.     

ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

Phytoene desaturase (PDS) is a rate‐limiting enzyme in carotenoid biosynthesis. Algal PDS is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. Specific point mutations in PDS confer resistance to the herbicide norflurazon, suggesting that mutated PDS could be used as a dominant selectable marker for genetic engineering of algae, for which very few selective markers are available. In this study, we report the isolation and characterization of the PDS gene from the astaxanthin‐producing green alga Chlorella zofingiensis Dönz. The open reading frame (ORF) of this PDS gene, interrupted by six introns, encoded a polypeptide of 558 amino acid residues. The deduced protein sequence showed significant homology to phytoene desaturases of algae, cyanobacteria, and higher plants. Expression of the PDS gene in Escherichia coli demonstrated that the enzyme was able to convert phytoene to ζ‐carotene. The PDS gene in Chlorella was shown to be up‐regulated by high light and glucose treatment. With a single amino acid change (L516R), the mutated PDS‐L516R was still active and exhibited ～36‐fold greater resistance to the bleaching herbicide norflurazon than the unaltered enzyme. Thus, the modified PDS gene could be a useful tool for genetic engineering of carotenoid biosynthesis in C. zofingiensis and perhaps also in other algae.     

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.     

Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation.

Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.     

The molecular basis of resistance to the herbicide norflurazon

We have cloned and sequenced a gene, pds, from the cyanobacterium Synechococcus PCC7942 that is responsible for resistance to the bleaching herbicide norflurazon. A point mutation in that gene, leading to an amino acid substitution from valine to glycine in its polypeptide product, was found to confer this resistance. Previous studies with herbicide-resistant mutants have indicated that this gene encodes phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids. A short amino acid sequence that is homologous to conserved motifs in the binding sites for NAD(H) and NADP(H) was identified in PDS, suggesting the involvement of these dinucleotides as cofactors in phytoene desaturation.     

Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of β-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon

Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of β-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial‐scale algal production facilities against contamination by organisms sensitive to these broad‐spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×‐fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO‐inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene‐based gene sequences found in various norflurazon‐resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large‐scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm , respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large‐scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.     

Barley stripe mosaic virus-induced gene silencing in a monocot plant

RNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modified BSMV to express untranslatable foreign inserts downstream of the gammab gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA amplified from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, norflurazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green fluorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the first demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.     

Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to zeta-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids

A phytoene desaturase (PDS) gene was cloned and characterized from the unicellular green microalga Chlamydomonas reinhardtii. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery displayed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.     

Regulation of carotenoid biosynthesis during tomato development.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.     

Herbicide resistance of transgenic rice plants expressing human CYP1A1

Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.     

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.