Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0 ×10⁴ clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indicajaponica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Molecular cloning and characterization of 5-enolpyruvylshikimate-3-phosphate synthase gene from Convolvulus arvensis L.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS), the target enzyme for glyphosate inhibition, catalyzes an essential step in the shikimate pathway for aromatic amino acid biosynthesis. The full-length cDNA of 1,751 nucleotides (CaEPSPS, Genbank accession number: EU698030) from Convolvulus arvensis was cloned and characterized. The CaEPSPS encodes a polypeptide of 520 amino acids with a calculated molecular weight of 55.5 kDa and an isoelectric point of 7.05. The results of homology analysis revealed that CaEPSPS showed highly homologous with EPSPS proteins from other plant species. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in stems, leaves and roots, with lower expression in roots. CaEPSPS expression level could increase significantly with glyphosate treatment, and reached its maximum at 24 h after glyphosate application. We fused CaEPSPS to the CaMV 35S promoter and introduced the chimeric gene into Arabidopsis. The resultant expression of CaEPSPS in transgenic Arabidopsis plants exhibited enhanced tolerance to glyphosate in comparison with control.     

Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells

Two distinct cDNAs for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) were obtained from a glyphosate-tolerant tobacco cell line. The cDNAs were 89% identical and the predicted sequences of the mature proteins were greater than 83% identical with EPSPS proteins from other plants. Tobacco EPSPS proteins were more similar to those from tomato and petunia than Arabidopsis. One cDNA clone, EPSPS-1, represented a gene that was amplified in glyphosate-tolerant cells, while the gene for EPSPS-2 was unaltered in these cells. Consequently, EPSPS-1 mRNA was more abundant in tolerant than unselected cells, whereas EPSPS-2 mRNA was at relatively constant levels in these cell lines. Exposure of unselected cells and tobacco leaves to glyphosate produced a transient increase in EPSPS mRNA. However, glyphosate-tolerant cells containing amplified copies of EPSPS genes did not show a similar response following exposure to glyphosate. A significant proportion of the EPSPS gene amplification was maintained when tolerant cells were grown in the absence of glyphosate for eight months. Plants regenerated from these cells also contained amplified EPSPS genes.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

Molecular characterization of the rbcS multi-gene family of Petunia (Mitchell)

Summary The small subunit (RbcS) of ribulose bisphosphate carboxylase (RuBPCase) is encoded by eight genes in Petunia (Mitchell). These genes can be divided into three subfamilies (51, 117 and 71) based upon hybridization to three petunia rbcS cDNA clones. The nucleotide sequence of six of the eight petunia rbcS genes is presented here and the structure of the genes is discussed with respect to their genomic linkage and their expression levels in petunia leaf tissue. The rbcS genes belonging to the same subfamily encode an identical mature RbcS polypeptide, however the different subfamilies encode distinguishable polypeptides. All the genes, except one, contian two introns within the mature subunit coding region; one gene contains one extra intron within the coding region. There are large regions of nucleotide sequence homology within the introns of genes within a subfamily, but significantly less homology between the introns of genes of different subfamilies. A complex pattern of homology within the multiple genes of the 51 subfamily is observed. There are regions within these genes which share high levels of sequence homology; this homology does not extend throughout the whole gene and the regions of homology do not always occur in adjacent genes. Two 3 rbcS gene fragments which we isolated from the petunia genome show high levels of homology to two of the intact rbcS genes.     

Co-suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem

The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.     

Bacterial glyphosate resistance conferred by overexpression of an <Emphasis Type="Italic">E. coli</Emphasis> membrane efflux transporter

Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.     

Aldo‐keto reductase enzymes detoxify glyphosate and improve herbicide resistance in plants

In recent years, concerns about the use of glyphosate‐resistant crops have increased because of glyphosate residual levels in plants and development of herbicide‐resistant weeds. In spite of identifying glyphosate‐detoxifying genes from microorganisms, the plant mechanism to detoxify glyphosate has not been studied. We characterized an aldo‐keto reductase gene from Pseudomonas (PsAKR1) and rice (OsAKR1) and showed, by docking studies, both PsAKR1 and OsAKR1 can efficiently bind to glyphosate. Silencing AKR1 homologues in rice and Nicotiana benthamiana or mutation of AKR1 in yeast and Arabidopsis showed increased sensitivity to glyphosate. External application of AKR proteins rescued glyphosate‐mediated cucumber seedling growth inhibition. Regeneration of tobacco transgenic lines expressing PsAKR1 or OsAKRI on glyphosate suggests that AKR can be used as selectable marker to develop transgenic crops. PsAKR1‐ or OsAKRI‐expressing tobacco and rice transgenic plants showed improved tolerance to glyphosate with reduced accumulation of shikimic acid without affecting the normal photosynthetic rates. These results suggested that AKR1 when overexpressed detoxifies glyphosate in planta.     

Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimic acid-3-phosphate synthase gene

Summary A Daucus carota cell line selected as resistant to N-(phosphonomethyl)-glycine (glyphosate) was found to have increased levels of 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) activity of 5.5 times over wild-type carrot and an EPSPS protein level increase of 8.7 times as confirmed by Western hybridization analysis. Southern blot hybridization using a petunia EPSPS probe showed increases in the number of copies of EPSPS genes in the glyphosate-resistant line which correlated with the higher levels of the EPSPS enzyme. The mechanism of resistance to glyphosate is therefore due to amplification of the EPSPS gene. To examine the stability of the amplified genes, cloned lines selected as doubly resistant to Dl-5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) were fused with the amplified EPSPS glyphosate-resistant cell line. Somatic hybrids expressed resistances to 5MT in a semidominant fashion while A2C and glyphosate resistance was expressed as dominant, or semi-dominant traits, in a line-specific manner. The hybrid lines possessed additive chromosome numbers of the parental lines used and no double minute chromosomes were observed. The glyphosate-resistant parental line and most somatic hybrids retained the amplified levels of EPSPS in the absence of selection pressure over a 3-year period.     

Engineering carpel‐specific cold stress tolerance: a case study in Arabidopsis

Climate change predictions forecast an increase in early spring frosts that could result in severe damage to perennial crops. For example, the Easter freeze of April 2007 left several states in the United States reporting a complete loss of that year's peach crop. The most susceptible organ to early frost damage in fruit trees is the carpel, particularly during bloom opening. In this study, we explored the use of a carpel‐specific promoter (ZPT2‐10) from petunia (Petunia hybrida var. Mitchell) to drive expression of the peach dehydrin PpDhn1. In peach, this gene is exceptionally responsive to low temperature but has not been observed to be expressed in carpels. This study examined carpel‐specific properties of a petunia promoter driving the expression of the GUS gene (uidA) in transgenic Arabidopsis flowers and developed a carpel‐specific ion leakage test to assess freezing tolerance. A homozygous Arabidopsis line (line 1‐20) carrying the petunia ZPT2‐10 promoter::PpDhn1 construct was obtained and freezing tolerance in the transgenic line was compared with an untransformed control. Overexpression of PpDhn1 in line 1‐20 provided as much as a 1.9°C increase in carpel freezing tolerance as measured by electrolyte leakage.     

Identification of the first glyphosate transporter by genomic adaptation

The soil bacterium Bacillus subtilis can get into contact with growth-inhibiting substances, which may be of anthropogenic origin. Glyphosate is such a substance serving as a nonselective herbicide. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, which generates an essential precursor for de novo synthesis of aromatic amino acids in plants, fungi, bacteria and archaea. Inhibition of the EPSP synthase by glyphosate results in depletion of the cellular levels of aromatic amino acids unless the environment provides them. Here, we have assessed the potential of B. subtilis to adapt to glyphosate at the genome level. In contrast to Escherichia coli, which evolves glyphosate resistance by elevating the production and decreasing the glyphosate sensitivity of the EPSP synthase, B. subtilis primarily inactivates the gltT gene encoding the high-affinity glutamate/aspartate symporter GltT. Further adaptation of the gltT mutants to glyphosate led to the inactivation of the gltP gene encoding the glutamate transporter GltP. Metabolome analyses confirmed that GltT is the major entryway of glyphosate into B. subtilis. GltP, the GltT homologue of E. coli also transports glyphosate into B. subtilis. Finally, we found that GltT is involved in uptake of the herbicide glufosinate, which inhibits the glutamine synthetase.     

Functional characterization of Class II 5-enopyruvylshikimate-3-phosphate synthase from <Emphasis Type="Italic">Halothermothrix orenii</Emphasis> H168 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Although a large number of AroA enzymes (5-enopyruvylshikimate-3-phosphate synthase [EPSPS]) have been identified, cloned and tested for glyphosate resistance, only AroA variants derived from Agrobacterium tumefaciens strain CP4 have been successfully used commercially. We have now used a polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method to synthesize an aroA gene (aroA _{H. orenii} ) from Halothermothrix orenii H168 encoding a new EPSPS similar to AroA _{A. tumefaciens CP4.} AroA _{H. orenii} was then expressed in Escherichia coli and key kinetic values of the purified enzyme were determined. Kinetic analysis of AroA _{H. orenii} indicated that the full-length enzyme exhibited increased tolerance to glyphosate compared with E. coli AroA _{E. coli} while retaining a high affinity for the substrate phosphoenolpyruvate. Transgenic Arabidopsis plants containing aroA _{H. orenii} were resistant to 15 mM glyphosate. Site-directed mutagenesis showed that residues Thr355Ser affected the affinity of AroA _{H. orenii} for glyphosate, providing further evidence that specific amino acid residues are responsible for differences in enzymatic behavior among different AroA enzymes.     

Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways to valine, isoleucine, and leucine. It is the target of two structurally unrelated classes of herbicides, the sulfonylureas and the imidazolinones. Genomic clones encoding ALS have been isolated from the higher plants Arabidopsis thaliana and Nicotiana tabacum, using a yeast ALS gene as a heterologous hybridization probe. Clones were positively identified by the homology of their deduced amino acid sequences with those of yeast and bacterial ALS isozymes. The tobacco and Arabidopsis ALS genes have approximately 70% nucleotide homology, and encode mature proteins which are approximately 85% homologous. Little homology is seen between the amino acid sequences of the presumptive N-terminal chloroplast transit peptides. Both plant genes lack introns. The tobacco ALS gene was isolated from a line of tobacco which is resistant to the sulfonylurea herbicides due to an alteration in ALS. The tobacco gene which was isolated codes for an ALS that is sensitive to the herbicides, as assayed by transformation of the gene into sensitive tobacco cells.     

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.     

Characterization of centrin genes from Ochromonas danica (Chrysophyceae) and Scytosiphon lomentaria (Phaeophyceae)

Centrin, the EF‐hand Ca²⁺‐binding protein is localized at the basal apparatus of flagella and in centrioles in many eukaryotic cells. In the present study, centrin genes of the heterokont algae have been clarified for the first time. We isolated and analyzed cDNA and genomic DNA of centrin genes from the crysophycean alga Ochromonas danica Prings (UTEX LB1298) and the brown alga Scytosiphon lomentaria (Lyngbye) Link. The centrin gene of Ochromonas contained an open reading frame of 163 amino acids. The deduced protein, named Odcen, exhibited 85%, 78% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. The centrin genes of Scytosiphon contained an open reading frame of 164 amino acids. The deduced protein, named Slcen, exhibited 84%, 77% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. Both Odcen and Slcen possessed N‐terminal extensions before the conserved amino acid among various centrins, four EF‐hand domains and an aromatic amino acid at the C‐terminus. Southern blot hybridization suggested that the centrin gene occurs as a single copy gene in both Ochromonas and Scytosiphon genomes. Comparison of the sequence of the cDNA and the genomic DNA revealed that the Odcen gene was split into three fragments by introns and Slcen gene consisted of five fragments. The junctions of all introns of both genes conformed to the GT–AG rule. The introns of Slcen gene were considerably long and, as a result, the Slcen gene was approximately seven times longer than Odcen gene.     

Structure and molecular evolutionary analysis of a plant cytochrome c gene: Surprising implications forArabidopsis thaliana

Summary We have isolated a cytochrome c gene fromArabidopsis thaliana (cv. Columbia), which is the first cytochrome c gene to be cloned from a higher plant. Genomic DNA blot analysis indicates that there is only one copy of cytochrome c inArabidopsis. The gene consists of three exons separated by two introns. Gene features such as regulatory regions, codon usage, and conserved splicing-specific sequences are all present and typical of dicotyledonous plant nuclear genes. We have constructed phenograms and cladograms for cytochrome c amino acid sequences and histone H3, alcohol dehydrogenase, and actin DNA sequences. For both cytochrome c and histone H3,Arabidopsis clusters poorly with other higher plants. Instead, it clusters withNeurospora and/or the yeasts. We suggest that perhaps this observation should be considered when usingArabidopsis as a model system for higher plants.