Robert G. Rutledge

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

SummaryThe acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3′ end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.

Characterization and tissue‐specific expression of two lepidopteran farnesyl diphosphate synthase homologs: Implications for the biosynthesis of ethyl‐substituted juvenile hormones

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl‐substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl‐branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type‐I FPPSs display unique active site substitutions, including several in and near the first aspartate‐rich motif, whereas type‐II proteins have a more “conventional” catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type‐I enzymes are better suited than type‐II for generating ethyl‐substituted products, a quantitative real‐time PCR assessment of their relative abundance in insect tissues indicated that type‐I expression is ubiquitous whereas that of type‐II is essentially confined to the JH‐producing glands, where its transcripts are ∼20 times more abundant than those of type‐I. These results suggest that type‐II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity. Proteins 2006.

Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR

Background Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of ±25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4–8 determinations from each amplification reaction. Findings Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp) is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy. Conclusions Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the potential to fundamentally transform how real-time qPCR is conducted.

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

Background Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

Hygromycin resistance is an effective selectable marker for biolistic transformation of black spruce (Picea mariana)

Abstract Using particle bombardment of mature somatic embryos followed by the induction of secondary embryogenesis in the presence of hygromycin, we produced over 90 lines of transgenic embryonal masses expressing β-glucuronidase from two genotypes of black spruce. Transformation efficiencies of up to 7% (1 transgenic line per 14 embryos bombarded) were achieved by extending the period of selection from 8 to 12 weeks. Proliferation of transformed embryonal masses in the presence of hygromycin had no effect on either embryogenicity or embryo maturation. Southern blot hybridization and PCR amplification confirmed the presence of the hygromycin phosphotransferase gene in genomic DNA. The expression of the β-glucuronidase gene in the needles of regenerated seedlings support the potential for long-term transgene expression in spruce.

Characterization of a MYBR2R3 gene from black spruce (Picea mariana) that shares functional conservation with maize C1.

PCR amplification with degenerate primers targeted to highly conserved amino acid motifs within the MYB domain was used to demonstrate that black spruce (Picea mariana) possesses a diverse MYB gene family. Amino acid sequence comparisons revealed three broad MYB subfamilies, one of which shares extensive similarity with maize C1, a central regulator of anthocyanin biosynthesis. A cDNA clone encoding a MYBR2R3 protein from P. mariana with high levels of sequence homology to maize C1 was shown to transactivate the Bz2 promoter in combination with maize R in embryonal tissues of both black spruce and larch. Functional dependence on the maize R protein, and the presence of a conserved C-terminal GIDPxTH motif, support the conservation of MYBR2R3 function in conifers, and demonstrate that the basic components of MYBR2R3-dependent transcriptional regulation have been conserved between angiosperms and gymnosperms.

Hormonally regulated overexpression of Arabidopsis WUS and conifer LEC1 ( CHAP3A ) in transgenic white spruce: implications for somatic embryo development and somatic seedling growth

Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somatic embryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would induce somatic embryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-β-estradiol during stages of somatic embryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somatic embryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed.

Genetic Transformation of Conifers Utilizing Somatic Embryogenesis

Over the last 5 yr, the production of transgenic conifers has been greatly facilitated by the ability to transform somatic embryonal tissues (somatic embryos) via cocultivation with Agrobacterium tumefaciens. This has allowed us to develop protocols for the genetic transformation of several spruce species. Furthermore, these procedures can produce an average of 20 independent transgenic lines (translines) per gram fresh mass of embryonal tissue, providing for the first time the magnitude-of-scale required for implementing large-scale functional genomics studies in conifers. Combined with efficient regeneration of transgenic trees via somatic embryos, the potential for genetic engineering of conifers has been demonstrated by stable reporter gene expression (GUS or GFP) resulting from single insert T-DNA integration events.

Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca)

BackgroundAmong the many commercial opportunities afforded by somatic embryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees.ResultsMicroarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response.ConclusionsWhile additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction.

Gene Expression Profiling of Shoot-Derived Calli from Adult Radiata Pine and Zygotic Embryo-Derived Embryonal Masses

Background Although somatic embryogenesis has an unprecedented potential for large-scale clonal propagation of conifers, the ability to efficiently induce the embryonal cultures required for somatic embryo production has long been a challenge. Furthermore, because early stage zygotic embryos remain the only responsive explants for pines, it is not possible to clone individual trees from vegetative explants at a commercial scale. This is of particular interest for adult trees because many elite characteristics only become apparent following sexual maturation. Findings Shoot explants collected from adult radiata pine trees were cultured on four induction media differing in plant growth regulator composition, either directly after collection or from in vitro-generated axillary shoots. Six callus lines were selected for microscopic examination, which failed to reveal any embryonal masses (EM). qPCR expression profiling of five of these lines indicated that explant type influenced the absolute level of gene expression, but not the type of genes that were expressed. The analysis, which also included three EM lines induced from immature zygotic embryos, encompassed five categories of genes reflective of metabolic, mitotic and meristematic activity, along with putative markers of embryogenicity. Culture medium was found to have no significant impact on gene expression, although differences specific to the explant’s origin were apparent. Expression of transcriptional factors associated with vegetative meristems further suggested that all of the callus lines possessed a substantive vegetative character. Most notable, however, was that they all also expressed a putative embryogenic marker (LEC1). Conclusions While limited in scope, these results illustrate the utility of expression profiling for characterizing tissues in culture. For example, although the biological significance of LEC1 expression is unclear, it does present the possibility that these callus lines possess some level of embryogenic character. Additionally, expression of vegetative meristem markers is consistent with their vegetative origin, as are differences in expression patterns as compared with EM.