Assessing the between-background stability of metabolic effects arising from lignin-related transgenic modifications, in two Populus hybrids using non-targeted metabolomics.

The advances in 'high-throughput' biology have significantly expanded our fundamental understanding of complex biological processes inherent to tree growth and development. Relative to the significant achievements attained with whole genome re-sequencing and transcriptomics efforts, the development and power of post-transcriptional tools such as proteomics and metabolomics continue to lag behind in tree biology. However, the inclusion of these powerful functional genomics platforms should substantially enable systems biology assessments of tree development, physiology and response(s) to biotic and abiotic stresses. Herein, we employ a non-targeted metabolomics platform to elucidate the metabolic plasticity of xylem lignification in distinct hybrid poplar genetic backgrounds, as well as in transgenic trees in these backgrounds expressing two common constructs: the first construct (C4H::F5H) augments monolignol content (syringyl:guaiacyl (S:G) ratio), while the second construct (C3'H-RNAi) reduces cell wall lignification. The results clearly show that genotype-specific metabolism exists, and provide an appropriate foundation for properly comparing the influence of background on the relationships between metabolic and specific phenotypic traits. Moreover, it was apparent that transgene-induced phenotypic gradients in cell wall chemical wood can be associated with global metabolism of secondary xylem biosynthesis, however in a genotype-specific manner. This result implies that the same may be true for phenotypic gradients arising through natural genetic variation, intensive breeding or environmental factors. It is also apparent that while distinct, at a global level the wood-forming metabolisms of different poplar hybrids can, to some extent, respond similarly to the influences of genetic manipulation of lignin-related genes. This further implies that with the correct approach, it may be possible to associate the emergence of specific wood traits from different genetic backgrounds-be they transgene-induced or otherwise-with stable metabolic signatures.

Predicting the regenerative capacity of conifer somatic embryogenic cultures by metabolomics.

Somatic embryogenesis in gymnosperms is an effective approach to clonally propagating germplasm. However, embryogenic cultures frequently lose regenerative capacity. The interactions between metabolic composition, physiological state, genotype and embryogenic capacity in Pinus taeda (loblolly pine) somatic embryogenic cultures were explored using metabolomics. A stepwise modelling procedure, using the Bayesian information criterion, generated a 47 metabolite predictive model that could explain culture productivity. The model performed extremely well in cross-validation, achieving a correlation coefficient of 0.98 between actual and predicted mature embryo production. The metabolic composition and structure of the model implied that variation in culture regenerative capacity was closely linked to the physiological transition of cultures from the proliferation phase to the maturation phase of development. The propensity of cultures to advance into this transition appears to relate to nutrient uptake and allocation in vivo, and to be associated with the tolerance and response of cultures to stress, during the proliferation phase.

Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling.

Determination of the physico-chemical attributes of plant cell walls, such as lignin content and composition, is of paramount importance in germplasm screening and for evaluating the results of plant breeding and genetic engineering. There are escalating needs for analyses to be robust, reproducible, accurate, and efficient. We have recently modified an established protocol for discrimination of lignin monomers, thioacidolysis, with the goal of increasing sample throughput while maintaining accuracy and reducing equipment load and consumption of reagents. Numerous methodological changes related to volume scaling, selection of the processing vessel, and sample handling were addressed. The revised protocol permitted rapid processing of some 50 or more samples per person per day. A direct comparison between methods using hybrid poplar (Populus alba x tremula) wood samples, resulted in quantities of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignin monomers that were equivalent to those derived from the original protocol. The revised methodology was then applied to quickly generate phenotypic trait data from 267 hybrid poplar trees (including wild type and eight C4H::F5H transgenic lines), for the development of a near-infrared-based model for predicting the proportion of lignin monomers across a broad phenotypic range of S:G. The resulting partial least squares regression model performed well under full cross-validation, giving strong, linear relationships between actual and predicted monomer proportions, and very high predictive accuracy for the predominant G and S monomers. This research brings considerable refinement to the thioacidolysis procedure, and establishes a method for rapidly and accurately quantifying cell-wall lignin composition that could effectively be employed in routine phenotypic screening platforms.

Metabolite profiling of Douglas-fir (Pseudotsuga menziesii) field trials reveals strong environmental and weak genetic variation.

The primary objective of this study was to assess metabolomics for its capacity to discern biological variation among 10 full-sib families of a Douglas-fir tree breeding population, replicated on two sites. The differential accumulation of small metabolites in developing xylem was examined through metabolite profiles (139 metabolites common to 181 individual trees) generated by gas chromatography mass spectrometry and a series of statistical analyses that incorporated family, site, and tree growth and quantitative phenotypic wood traits (wood density, microfibril angle, wood chemistry and fiber morphology). Multivariate discriminant, canonical discriminant and factor analyses and broad-sense heritabilities revealed that metabolic and phenotypic traits alike were strongly related to site, while similar associations relating to genetic (family) structure were weak in comparison. Canonical correlation analysis subsequently identified correlations between specific phenotypic traits (i.e. tree growth, fibre morphology and wood chemistry) and metabolic traits (i.e. carbohydrate and lignin biosynthetic metabolites), demonstrating a coherent relationship between genetics, metabolism, environmental and phenotypic expression in wood-forming tissue. The association between cambial metabolites and tree phenotype, as revealed by metabolite profiling, demonstrates the value of metabolomics for systems biology approaches to understanding tree growth and secondary cell wall biosynthesis in plants.

The potential of metabolite profiling as a selection tool for genotype discrimination in Populus.

Differences between wild-type Populus tremulaxalba and two transgenic lines with modified lignin monomer composition, were interrogated using metabolic profiling. Analysis of metabolite abundance data by GC-MS, coupled with principal components analysis (PCA), successfully differentiated between lines that had distinct phenotypes, whether samples were taken from the cambial zone or non-lignifying suspension tissue cultures. Interestingly, the GC-MS analysis detected relatively few phenolic metabolites in cambial extracts, although a single metabolite associated with the differentiation between lines was directly related to the phenylpropanoid pathway or other down-stream aspects of lignin biosynthesis. In fact, carbohydrates, which have only an indirect relationship with the modified lignin monomer composition, featured strongly in the line-differentiating aspects of the statistical analysis. Traditional HPLC analysis was employed to verify the GC-MS data. These findings demonstrate that metabolic traits can be dissected reliably and accurately by metabolomic analyses, enabling the discrimination of individual genotypes of the same tree species that exhibit marked differences in industrially relevant wood traits. Furthermore, this validates the potential of using metabolite profiling techniques for marker generation in the context of plant/tree breeding for industrial applications.

Direct-to-consumer pharmaceutical advertising: physician and public opinion and potential effects on the physician-patient relationship.

BACKGROUND: Previous studies have shown that direct-to-consumer (DTC) pharmaceutical advertising can influence consumer behavior and that many physicians have negative views of these advertisements. Physician and public opinions about these advertisements and how they may affect the physician-patient relationship are not well established. METHODS: Mail survey of 523 Colorado physicians and 261 national physicians and telephone survey of 500 Colorado households asking respondents to rate their agreement with statements about DTC advertising. RESULTS: Most physicians tended to view DTC advertisements negatively, indicating that such advertisements rarely provide enough information on cost (98.7%), alternative treatment options (94.9%), or adverse effects (54.8%). Most also believed that DTC advertisements affected interactions with patients by lengthening clinical encounters (55.9%), leading to patient requests for specific medications (80.7%), and changing patient expectations of physicians' prescribing practices (67.0%). Only 29.0% of public respondents agreed that DTC advertising is a positive trend in health care and 28.6% indicated that advertisements make them better informed about medical problems; fewer indicated that advertisements motivated them to seek care (10.5%) or led them to request specific medications from their physicians (13.3%). CONCLUSIONS: Most physicians have negative views of DTC pharmaceutical advertising and see several potential effects of these advertisements on the physician-patient relationship. Many public respondents have similarly negative views, and only a few agree that they change their expectations of or interactions with physicians. While these advertisements may be influencing only a few consumers, it seems that the impact on physicians and their interactions with patients may be significant.

Physician and public opinions on quality of health care and the problem of medical errors.

BACKGROUND: The 1999 Institute of Medicine report on medical errors proposed major changes to the health care system and gained widespread media attention, yet there is limited information on physician or public opinion regarding recommendations from that report. METHODS: Mail survey of 1000 Colorado physicians (n = 594) and 1000 national physicians (n = 304), and telephone survey of 500 Colorado households to assess agreement with several proposals and conclusions from the 1999 Institute of Medicine report. RESULTS: Most physicians believed that reduction of medical errors should be a national priority (69.7% of Colorado physicians). However, physicians were much less likely than the public to believe that quality of care is a problem (29.1% vs 67.6%; P<.001) or that a national agency is needed to address the problem of medical errors (24.1% vs 59.8%; P<.001). Uniformly, physicians believed that fear of medical malpractice is a barrier to reporting of errors and that greater legal safeguards are necessary for a mandatory reporting system to be successful. Nearly all physicians (92.9%) believed that more training in how to handle medical errors is needed, and 60.1% agreed that it is difficult to differentiate errors due to negligence from unintended errors. CONCLUSIONS: There appears to be widespread concern among physicians regarding medical errors, but only a minority in this survey believed that the problem is as significant as the Institute of Medicine and the public believe it to be. Our results suggest that physicians see several barriers to successful error reduction including difficulty defining errors, the need for more training in handling errors, and fear of malpractice litigation. Addressing these barriers will be a necessary step to increasing physician support for many of the changes proposed by the Institute of Medicine.

Association between use of complementary/alternative medicine and health-related behaviors among health fair participants.

BACKGROUND: The relationship between complementary and alternative medicine (CAM) and standard preventive care is not well defined. METHODS: We surveyed 1,593 health fair participants on their use of CAM and determined odds ratios for standard preventive care and healthy behaviors among users of provider-based CAM (e.g., chiropractic) and users of herbs or supplements. RESULTS: Users of provider-based CAM were no less likely than nonusers to receive standard preventive care, with the exception of lower likelihood of influenza vaccination among elderly CAM users (OR 0.2). Herbs or supplements were used by a majority of respondents (61%), and users were more likely to report engaging in healthy behaviors such as eating a low-fat diet (OR 1.5) and taking a daily multivitamin (OR 2.0). CONCLUSIONS: Those who use alternative therapies, including herbs or supplements, appear no less likely overall than nonusers to receive standard preventive care. In addition, users of herbs or supplements are more likely to engage in healthy behaviors and appear to be a more health-conscious group. Overall, our findings support the notion that unconventional therapies are more complementary than alternative to standard preventive care.