HPPD Gene Research Articles

Biosynthesis of Hesperetin, Homoeriodictyol, and Homohesperetin in a Transcriptomics-Driven Engineered Strain of Streptomyces albidoflavus.

Flavonoids exhibit various bioactivities including anti-oxidant, anti-tumor, anti-inflammatory, and anti-viral properties. Methylated flavonoids are particularly significant due to their enhanced oral bioavailability, improved intestinal absorption, and greater stability. The heterologous production of plant flavonoids in bacterial factories involves the need for enough biosynthetic precursors to allow for high production levels. These biosynthetic precursors are malonyl-CoA and l-tyrosine. In this work, to enhance flavonoid biosynthesis in Streptomyces albidoflavus, we conducted a transcriptomics study for the identification of candidate genes involved in l-tyrosine catabolism. The hypothesis was that the bacterial metabolic machinery would detect an excess of this amino acid if supplemented with the conventional culture medium and would activate the genes involved in its catabolism towards energy production. Then, by inactivating those overexpressed genes (under an excess of l-tyrosine), it would be possible to increase the intracellular pools of this precursor amino acid and eventually the final flavonoid titers in this bacterial factory. The RNAseq data analysis in the S. albidoflavus wild-type strain highlighted the hppD gene encoding 4-hydroxyphenylpyruvate dioxygenase as a promising target for knock-out, exhibiting a 23.2-fold change (FC) in expression upon l-tyrosine supplementation in comparison to control cultivation conditions. The subsequent knock-out of the hppD gene in S. albidoflavus resulted in a 1.66-fold increase in the naringenin titer, indicating enhanced flavonoid biosynthesis. Leveraging the improved strain of S. albidoflavus, we successfully synthesized the methylated flavanones hesperetin, homoeriodictyol, and homohesperetin, achieving titers of 2.52 mg/L, 1.34 mg/L, and 0.43 mg/L, respectively. In addition, the dimethoxy flavanone homohesperetin was produced as a byproduct of the endogenous metabolism of S. albidoflavus. To our knowledge, this is the first time that hppD deletion was utilized as a strategy to augment the biosynthesis of flavonoids. Furthermore, this is the first report where hesperetin and homoeriodictyol have been synthesized from l-tyrosine as a precursor. Therefore, transcriptomics is, in this case, a successful approach for the identification of catabolism reactions affecting key precursors during flavonoid biosynthesis, allowing the generation of enhanced production strains.

Optimization of annealing temperature for amplification of the exon one region of the HPPD gene in HA1 accession sunflowers

The HPPD gene is one of the genes that is actively involved in the biosynthesis of tocopherol, which is the main component of Vitamin E. Sunflower is raw materials for natural medicine since their oil contains vitamin E. To obtain complete information regarding the whole sequences of the 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) gene in sunflower accession HA1, the exon one region needs to be identified using a PCR-based method. One factor affecting the success rate of PCR is the annealing temperature. The optimum annealing temperature will produce specific target gene products. This study aimed to determine the optimal temperature for amplifying the exon 1 region of the HPPD gene using the PCR method. This research began with primer design activities, followed by in vitro amplification (PCR), then optimization of annealing temperature using gradient PCR. The results showed that the optimum temperature for amplifying fragment one was an annealing temperature of 53 °C, fragment two was at an annealing temperature of 45 °C, and fragment three had an annealing temperature of 57 °C with the expected size of PCR products are 306 bp, 506 bp, and 534 bp respectively.

Rapid metabolism and increased expression of CYP81E8 gene confer high level of resistance to tembotrione in a multiple-resistant Palmer amaranth (Amaranthus palmeri S. Watson)

Herbicides, such as tembotrione, that inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme are used to control broad spectrum of weeds, primarily in corn, as this crop can metabolize these herbicides via cytochrome P450 activity. In 2018, a Palmer amaranth (Amaranthus palmeri) population, KCTR was found to be resistant to multiple herbicides including, tembotrione in Kansas (KS), USA. However, the mechanism of tembotrione resistance in KCTR is not known. The objective of this study was to characterize the level of tembotrione resistance and investigate the mechanism of resistance to this herbicide in KCTR using a known susceptible Palmer amaranth population (KSS). Tembotrione dose response experiments revealed that KCTR Palmer amaranth is 23 times more resistant to this herbicide, than KSS. No difference in absorption or translocation of [14C] tembotrione between KSS and KCTR was found. However, the time required to metabolize 50% of tembotrione was shorter in KCTR than in KSS. More than 95% of tembotrione was metabolized at 6 hours after treatment (HAT) in the KCTR, compared to only 50% in KSS plants. Application of cytochrome P450-inhibitors (e.g., malathion or piperonyl butoxide), along with tembotrione reversed the resistance in KCTR. Furthermore, the KCTR plants showed 35-fold increase in constitutive expression of CYP81E8 gene compared to KSS. Nonetheless, the HPPD gene expression was not altered in KCTR Palmer amaranth. Our results suggest that enhanced metabolism of tembotrione possibly due to increased expression of CYP81E8 gene contribute to tembotrione resistance in KCTR. Metabolic resistance to herbicides is a challenge for weed management as such resistance predisposes weeds to evolve resistance to unknown herbicides even without selection.

Recurrent Selection with Sub-Lethal Doses of Mesotrione Reduces Sensitivity in Amaranthus palmeri.

Amaranthus palmeri, ranked as the most prolific and troublesome weed in North America, has evolved resistance to several herbicide sites of action. Repeated use of any one herbicide, especially at lower than recommended doses, can lead to evolution of weed resistance, and, therefore, a better understanding of the process of resistance evolution is essential for the management of A. palmeri and other difficult-to-control weed species. Amaranthus palmeri rapidly developed resistance to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors such as mesotrione. The objective of this study was to test the potential for low-dose applications of mesotrione to select for reduced susceptibility over multiple generations in an A. palmeri population collected from an agricultural field in 2001. F0 plants from the population were initially treated with sub-lethal mesotrione rates and evaluated for survival three weeks after treatment. All F0 plants were controlled at the 1× rate (x = 105 g ai ha−1). However, 2.5% of the F0 plants survived the 0.5× treatment. The recurrent selection process using plants surviving various mesotrione rates was continued until the F4 generation was reached. Based on the GR50 values, the sensitivity index was determined to be 1.7 for the F4 generation. Compared to F0, HPPD gene expression level in the F3 population increased. Results indicate that after several rounds of recurrent selection, the successive generations of A. palmeri became less responsive to mesotrione, which may explain the reduced sensitivity of this weed to HPPD-inhibiting herbicides. The results have significance in light of the recently released soybean and soon to be released cotton varieties with resistance to HPPD inhibitors.

Assessing the Effects of β-Triketone Herbicides on the Soil Bacterial and hppd Communities: A Lab-to-Field Experiment.

Maize cultivators often use β-triketone herbicides to prevent the growth of weeds in their fields. These herbicides target the 4-HPPD enzyme of dicotyledons. This enzyme, encoded by the hppd gene, is widespread among all living organisms including soil bacteria, which are considered as “non-target organisms” by the legislation. Within the framework of the pesticide registration process, the ecotoxicological impact of herbicides on soil microorganisms is solely based on carbon and nitrogen mineralization tests. In this study, we used more extensive approaches to assess with a lab-to-field experiment the risk of β-triketone on the abundance and the diversity of both total and hppd soil bacterial communities. Soil microcosms were exposed, under lab conditions, to 1× or 10× the recommended dose of sulcotrione or its commercial product, Decano®. Whatever the treatment applied, sulcotrione was fully dissipated from soil after 42 days post-treatment. The abundance and the diversity of both the total and the hppd bacterial communities were not affected by the herbicide treatments all along the experiment. Same measurements were led in real agronomical conditions, on three different fields located in the same area cropped with maize: one not exposed to any plant protection products, another one exposed to a series of plant protection products (PPPs) comprising mesotrione, and a last one exposed to different PPPs including mesotrione and tembotrione, two β-triketones. In this latter, the abundance of the hppd community varied over time. The diversity of the total and the hppd communities evolved over time independently from the treatment received. Only slight but significant transient effects on the abundance of the hppd community in one of the tested soil were observed. Our results showed that tested β-triketones have no visible impact toward both total and hppd soil bacteria communities.

Cloning of a novel topramezone-resistant 4-hydroxyphenylpyruvate dioxygenase gene and improvement of its resistance through pressure acclimation

Topramezone is a new 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide that is widely used on corn to control annual grass weeds and broadleaf weeds. Due to its broad-spectrum weed control capacity, improved activity, excellent crop selectivity, low mammalian toxicity and high environmental safety, topramezone is considered an ideal target herbicide for transgenic engineering of herbicide tolerance. In this study, a topramezone-resistant strain, Burkholderia sp. BW-1, was screened from soil, and a novel topramezone-resistant HPPD gene (Bkhppd) was cloned from this strain. Purified BkHPPD displayed relatively high HPPD activity and topramezone resistance with a half-maximal inhibitory concentration (IC50) of 572.0 nM. Two BkHPPD mutants designated as BkHPPDt31 and BkHPPDt76 were screened through pressure acclimation. BkHPPDt31 contained three amino acid substitutions (H65D, N160 T and N258S), whereas BkHPPDt76 contained four amino acid substitutions (H65D, N160 T, N258S and N343 T). The topramezone IC50 values of BkHPPDt31 and BkHPPDt76 were 1.1- and 2.3-fold higher, respectively, than that of wild-type BkHPPD. In addition, site-directed mutagenesis indicated that the increased resistance conferred by BkHPPDt31 resulted from the synergistic effects of the three site mutations rather than a single site mutation, and that substitution of asparagine 343 with threonine significantly decreased catalytic efficiency and affinity but increased topramezone resistance. In summary, this study provides a novel topramezone-resistant HPPD gene for the engineering of genetically modified herbicide-resistant crops and facilitates further elucidation of the resistance mechanism of BkHPPD and improvement of resistance through directed evolution.

Heterologous production of pyomelanin biopolymer using 4-hydroxyphenylpyruvate dioxygenase isolated from Ralstonia pickettii in Escherichia coli

Here, we report a novel melanin-type pyomelanin pigment derived from Ralstonia pickettii. Melanin-pigment producing R. pickettii was screened from soil sources; its specific growth rates were observed to be 0.143 h−1 and 0.091 h−1 in LB and NB media, respectively. The highest pigment production was 90.9 ± 5.3 mg/L (1.5 mg/L/h) in M9 minimal medium supplemented with l-tyrosine and copper ions. Purified melanin pigments exhibited a distinct spectral feature of λmax at 290 nm and a 108.9 μg/mL IC50 value for ABTS radical scavenging capacity. To identify the genes involved in pigment production, the gene sequences were annotated using sequence analysis. We identified four putative 4-hydroxyphenyl pyruvate dioxygenase (HPPD)-encoding genes responsible for pyomelanin synthesis from l-tyrosine. One hppD gene was amplified and expressed in E. coil BL21(DE3) KSYH. The HPPD protein was expressed in the soluble fraction, and the recombinant E. coli strain could produce pyomelanin at levels up to 315.5 ± 12.9 mg/L (13.1 mg/L/h) in 24 h. Pyomelanin production was highly dependent on metal ions. Additionally, the gene annotation results revealed that R. pickettii could produce eumelanin through a tyrosinase dependent pathway, and the l-DOPA (L-dihydroxyphenylalanine) intermediate was observed during LC/MS analysis, suggesting that the isolated strain produced eumelanin and pyomelanin simultaneously. These findings provide valuable information regarding melanin synthesis and applications for industrial use.

Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19.

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The Km and kcat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 μM and 15.0 s-1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and Ki values of 5.2 and 2.5 μM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 μM; Ki: 6.0 μM) and mesotrione (IC50: 4.2 μM; Ki: 1.3 μM) and moderate resistance to tembotrione (IC50: 2.5 μM; Ki: 1.0 μM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.

Mechanism of atrazine resistance in atrazine- and HPPD inhibitor-resistant Palmer amaranth (Amaranthus palmeri S. Wats.) from Nebraska

Palmer amaranth (Amaranthus palmeri S. Wats.) is one of the most problematic weed species in agronomic crops in the United States. A Palmer amaranth biotype multiple-resistant to atrazine and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors was reported in a seed corn production field in Nebraska. Rapid detoxification mediated by cytochrome P450 monooxygenases and increased HPPD gene expression were reported as the mechanisms of mesotrione resistance in atrazine- and HPPD inhibitor-resistant Palmer amaranth biotype from Nebraska; however, the mechanism of atrazine resistance is unknown. The objectives of this study were to investigate target site or non-target site based mechanisms conferring atrazine resistance in Palmer amaranth from Nebraska. 14C-atrazine absorption and translocation studies revealed that reduced atrazine absorption or translocation were not involved as one of the mechanisms of atrazine resistance. Instead, greater 14C-atrazine absorption and recovery in treated leaves were observed in resistant compared with susceptible Palmer amaranth. No known mutations including Ser264Gly substitution in the psbA gene causing target site based atrazine resistance were observed. However, the parent 14C-atrazine was metabolized rapidly <4 h after treatment in resistant plants, conferring enhanced atrazine metabolism as the mechanism of resistance.

In-Silico analysis of missense SNPs in Human HPPD gene associated with Tyrosinemia type III and Hawkinsinuria

HPPD gene codes a dioxygenase enzyme involved in catalysis of different molecules such as tyrosine and phenylalanine by oxidizing them to produce energy. A single change in protein can trigger serious genetic disorders like Tyrosinemia type III and Hawkinsinuria. This study aims to identify the functional missense SNPs of the HPPD gene by using multiple computational tools. All deleterious missense SNPs retrieved from Ensembl and OMIM database were evaluated through six different software. Ultimately, out of 148 missense SNPs, only 27 were confirmed as diseasecausing SNPs by developing a consensus approach. These damaging SNPs were further examined to evaluate their impact on protein stability and energy including their evolutionary conservation. Native and mutated proteins structures were also designed and superimposed by I-TASSER and PyMol respectively. This work results in narrowing down missense SNPs which are still not confirmed experimentally and demands the confirmation by GWAS data. Thus, these missense SNPs could directly or indirectly destabilize the amino acid interactions causing functional deviations of protein.

Isolation of pyomelanin from bacteria and evidences showing its synthesis by 4-hydroxyphenylpyruvate dioxygenase enzyme encoded by hppD gene

Various types of pigments are produced by different bacterial species and play multifunctional role including their role as a protective agent against environmental stresses. The present study deals with the isolation and characterization of melanin pigment from bacteria. Three isolates showed pigment production and identified as Klebsiella pneumoniae S-1, Alcaligenes faecalis PBI and Enterobacter sp. ABI on the basis of 16S rRNA gene sequencing. The maximum production of brown pigment (λmax 405 nm) was attained after 6 days of growth in liquid medium containing 3 g L−1 of l-tyrosine. Solubility test, FTIR and ESI-MS analysis suggested the nature of pigment as melanin. Identity of pigment type from all the three bacteria was made by HPLC analysis, results showed the presence of homogentisic acid (HGA), a precursor of pyomelanin. All the bacteria showed high activity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) suggesting its role in HGA synthesis. PCR amplification of gene(s) also showed the presence of 4-hydroxyphenylpyruvate dioxygenase (hppD) gene coding for HPPD enzyme suggesting its role in pyomelanin synthesis. This study deals with only three bacterial isolates, isolation of such pigment from a large number of bacteria growing in harsh habitats may prove useful in finding novel type of melanin.

Effect of allelochemical tricin and its related benzothiazine derivative on photosynthetic performance of herbicide-resistant barnyardgrass

Despite increasing knowledge of allelochemicals as leads for new herbicides, relatively little is known about the mode of action of allelochemical-based herbicides on herbicide-resistant weeds. In this study, herbicidal activities of a series of allelochemical tricin-derived compounds were evaluated. Subsequently, a benzothiazine derivative 3-(2-chloro-4-methanesulfonyl)-benzoyl-hydroxy-2-methyl-2H-1,2-benzothiazine-1,1-dioxide with 4-hydroxyphenyl-pyruvate dioxygenase (HPPD) inhibiting activity was identified as a target compound on photosynthetic performance of penoxsulam-resistant versus -susceptible barnyardgrass (Echinochloa crus-galli). Regardless of barnyardgrass biotype, the benzothiazine derivative greatly affected chlorophyll fluorescence parameters (Fv/Fm, ETR1min and NPQ1min), reduced the chloroplast fluorescence levels and expression of HPPD gene. In particular, the benzothiazine derivative interfered with photosynthetic performance of resistant barnyardgrass more effectively than the allelochemical tricin itself. These results showed that the benzothiazine derivative effectively inhibited the growth of resistant barnyardgrass and its mode of action on photosynthesis system was similar to HPPD-inhibiting sulcotrione, making it an ideal lead compound for further development of allelochemical-based herbicide discovery.

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling.

A novel amperometric biosensor for ß-triketone herbicides based on hydroxyphenylpyruvate dioxygenase inhibition: A case study for sulcotrione

An amperometric biosensor was designed for the determination of sulcotrione, a β-triketone herbicide, based on inhibition of hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme allowing the oxidation of hydroxyphenylpyruvate (HPP) in homogentisic acid (HGA). HPPD was produced by cloning the hppd gene from Arabidopsis thaliana in E. coli, followed by overexpression and purification by nickel-histidine affinity. The electrochemical detection of HPPD activity was based on the electrochemical oxidation of HGA at +0.1V vs. Ag/AgCl, using a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate-modified screen-printed electrode. Assays were performed at 25°C in 0.1M phosphate buffer pH 8 containing 0.1M KCl. The purified HPPD was shown to display a maximum velocity of 0.51µMHGAmin−1, and an apparent KM of 22.6µM for HPP. HPPD inhibition assays in presence of sulcotrione confirmed a competitive inhibition of HPPD, the calculated inhibition constant KI was 1.11.10−8M. The dynamic range for sulcotrione extended from 5.10−10M to 5.10−6M and the limit of detection (LOD), estimated as the concentration inducing 20% of inhibition, was 1.4.10−10M.

Isolation and identification of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856

We describe the isolation and identification of a gene encoding 4-hydroxyphenylpyruvate dioxygenase (HPPD (EC 1.13.11.27)) from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856. HPPD directs the synthesis of 2,5-dihydroxyphenylacetic acid (homogentisic acid (HGA)). The sequence of the deduced peptide showed homology to HPPDs from other strains of Alteromonas; the greatest similarity was to the hypothetical HPPD from Alteromonas sp. SN2. As observed for HPPDs from other sources, expression of the A. stellipolaris HPPD gene in Escherichia coli cells could be detected by the gradual development of a brown pigment in cultures as a result of the spontaneous oxidation and polymerization of HGA.

Vliv exprese genu pro HPPD na obsah vitaminu E v ječmeni jarním

The enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) has a very important role in the biosynthetic pathway of vitamin E. Its activity influences the final level of tocols in plant tissues. Seven barley cultivars with different vitamin E level were grown under control conditions and activity of HPPD gene was measured four, eight and twelve days after pollination of ear tissues. It was found that activity of HPPD gene corresponded with vitamin E content detected in grains (r = 0.77*). The relationship between the gene activity for HPPD eight and twelve days after pollination and vitamin E content was also confirmed for analyzed cultivars grown in the field conditions (r = 0.85*).

Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.

With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.

Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria–Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific β-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.

Generation and characterization of soybean and marker‐free tobacco plastid transformants over‐expressing a bacterial 4‐hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.

Overexpression of the enzyme p-hydroxyphenolpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis

The enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisic acid (HGA), the aromatic precursor for the biosynthesis of vitamin E (α-tocopherol) and plastoquinone. In order to determine if increased HPPD activity could positively impact tocopherol yields, transgenic plants were generated that overexpressed the gene encoding Arabidopsis HPPD. Transgenic plants exhibiting high levels of HPPD expression were identified by increased tolerance to a competitive inhibitor of HPPD, the herbicide sulcotrione. HPPD gene expression in these transgenic lines, as determined at the RNA, protein and activity levels, was at least 10-fold higher than that of wild-type plants. Subsequent tocopherol analysis of leaf and seed material revealed that the increased HPPD expression resulted in up to a 37% increase in leaf tocopherol levels and a 28% increase in seed tocopherol levels relative to control plants. These results demonstrate that HPPD activity, and likely HGA levels, are at least one factor limiting the production of tocopherols in photosynthetic and non-photosynthetic plant tissues.