Dynamic QTL-based ecophysiological models to predict phenotype from genotype and environment data.

BACKGROUND: Predicting the phenotype from the genotype is one of the major contemporary challenges in biology. This challenge is greater in plants because their development occurs mostly post-embryonically under diurnal and seasonal environmental fluctuations. Most current crop simulation models are physiology-based models capable of capturing environmental fluctuations but cannot adequately capture genotypic effects because they were not constructed within a genetics framework. RESULTS: We describe the construction of a mixed-effects dynamic model to predict time-to-flowering in the common bean (Phaseolus vulgaris L.). This prediction model applies the developmental approach used by traditional crop simulation models, uses direct observational data, and captures the Genotype, Environment, and Genotype-by-Environment effects to predict progress towards time-to-flowering in real time. Comparisons to a traditional crop simulation model and to a previously developed static model shows the advantages of the new dynamic model. CONCLUSIONS: The dynamic model can be applied to other species and to different plant processes. These types of models can, in modular form, gradually replace plant processes in existing crop models as has been implemented in BeanGro, a crop simulation model within the DSSAT Cropping Systems Model. Gene-based dynamic models can accelerate precision breeding of diverse crop species, particularly with the prospects of climate change. Finally, a gene-based simulation model can assist policy decision makers in matters pertaining to prediction of food supplies.

Cost-effective detection of genome-wide signatures for 2,4-D herbicide resistance adaptation in red clover.

Herbicide resistance is a recurrent evolutionary event that has been reported across many species and for all major herbicide modes of action. The synthetic auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely used since the 1940s, however the genetic variation underlying naturally evolving resistance remains largely unknown. In this study, we used populations of the forage legume crop red clover (Trifolium pratense L.) that were recurrently selected for 2,4-D resistance to detect genome-wide signatures of adaptation. Four susceptible and six derived resistant populations were sequenced using a less costly approach by combining targeted sequencing (Capture-Seq) with pooled individuals (Pool-Seq). Genomic signatures of selection were identified using: (i) pairwise allele frequency differences; (ii) genome scan for overly differentiated loci; and (iii) genome-wide association. Fifty significant SNPs were consistently detected, most located in a single chromosome, which can be useful for marker assisted selection. Additionally, we searched for candidate genes at these genomic regions to gain insights into potential molecular mechanisms underlying 2,4-D resistance. Among the predicted functions of candidate genes, we found some related to the auxin metabolism, response to oxidative stress, and detoxification, which are also promising for further functional validation studies.

Understanding the Complexity of Cold Tolerance in White Clover using Temperature Gradient Locations and a GWAS Approach.

White clover ( L.) is the most important grazing perennial forage legume in temperate climates. However, its limited capacity to survive and restore growth after low temperatures during winter constrains the productivity and wide adoption of the crop. Despite the importance of cold tolerance for white clover cultivar development, the genetic basis of this trait remains largely unknown. Hence, in this study, we performed the first genome-wide association study (GWAS) analyses in white clover to identify quantitative trait loci (QTL) for cold-tolerance-related traits. Seeds from 192 divergent genotypes from six populations in the Patagonia region of South America were collected and seed-derived plants were further clonally propagated. Clonal trials were established in three locations representing temperature gradient associated with elevation. Given the allotetraploid nature of the white clover genome, distinct genetic models (diploid and tetraploid) were tested. Only the tetraploid parameterization was able to detect the 53 loci associated with cold-tolerance traits. Out of the 53 single nucleotide polymorphism (SNP) trait associations, 17 controlled more than one trait or were stable across multiple sites. This work represents the first report of QTL for cold-tolerance-related traits, providing insights into its genetic basis and candidate genomic regions for further functional validation studies.

A Predictive Model for Time-to-Flowering in the Common Bean Based on QTL and Environmental Variables.

The common bean is a tropical facultative short-day legume that is now grown in tropical and temperate zones. This observation underscores how domestication and modern breeding can change the adaptive phenology of a species. A key adaptive trait is the optimal timing of the transition from the vegetative to the reproductive stage. This trait is responsive to genetically controlled signal transduction pathways and local climatic cues. A comprehensive characterization of this trait can be started by assessing the quantitative contribution of the genetic and environmental factors, and their interactions. This study aimed to locate significant QTL (G) and environmental (E) factors controlling time-to-flower in the common bean, and to identify and measure G × E interactions. Phenotypic data were collected from a biparental [Andean × Mesoamerican] recombinant inbred population (F11:14, 188 genotypes) grown at five environmentally distinct sites. QTL analysis using a dense linkage map revealed 12 QTL, five of which showed significant interactions with the environment. Dissection of G × E interactions using a linear mixed-effect model revealed that temperature, solar radiation, and photoperiod play major roles in controlling common bean flowering time directly, and indirectly by modifying the effect of certain QTL. The model predicts flowering time across five sites with an adjusted r-square of 0.89 and root-mean square error of 2.52 d. The model provides the means to disentangle the environmental dependencies of complex traits, and presents an opportunity to identify in silico QTL allele combinations that could yield desired phenotypes under different climatic conditions.

Development of a QTL-environment-based predictive model for node addition rate in common bean.

KEY MESSAGE: This work reports the effects of the genetic makeup, the environment and the genotype by environment interactions for node addition rate in an RIL population of common bean. This information was used to build a predictive model for node addition rate. To select a plant genotype that will thrive in targeted environments it is critical to understand the genotype by environment interaction (GEI). In this study, multi-environment QTL analysis was used to characterize node addition rate (NAR, node day- 1) on the main stem of the common bean (Phaseolus vulgaris L). This analysis was carried out with field data of 171 recombinant inbred lines that were grown at five sites (Florida, Puerto Rico, 2 sites in Colombia, and North Dakota). Four QTLs (Nar1, Nar2, Nar3 and Nar4) were identified, one of which had significant QTL by environment interactions (QEI), that is, Nar2 with temperature. Temperature was identified as the main environmental factor affecting NAR while day length and solar radiation played a minor role. Integration of sites as covariates into a QTL mixed site-effect model, and further replacing the site component with explanatory environmental covariates (i.e., temperature, day length and solar radiation) yielded a model that explained 73% of the phenotypic variation for NAR with root mean square error of 16.25% of the mean. The QTL consistency and stability was examined through a tenfold cross validation with different sets of genotypes and these four QTLs were always detected with 50-90% probability. The final model was evaluated using leave-one-site-out method to assess the influence of site on node addition rate. These analyses provided a quantitative measure of the effects on NAR of common beans exerted by the genetic makeup, the environment and their interactions.

Plastic expression of heterochrony quantitative trait loci (hQTLs) for leaf growth in the common bean (Phaseolus vulgaris).

Heterochrony, that is, evolutionary changes in the relative timing of developmental events and processes, has emerged as a key concept that links evolution and development. Genes associated with heterochrony encode molecular components of developmental timing mechanisms. However, our understanding of how heterochrony genes alter the expression of heterochrony in response to environmental changes remains very limited. We applied functional mapping to find quantitative trait loci (QTLs) responsible for growth trajectories of leaf area and leaf mass in the common bean (Phaseolus vulgaris) grown in two contrasting environments. We identified three major QTLs pleiotropically expressed under the two environments. Further characterization of the temporal pattern of these QTLs indicates that they are heterochrony QTLs (hQTLs) in terms of their role in influencing four heterochronic parameters: the timing of the inflection point, the timing of maximum acceleration and deceleration, and the duration of linear growth. The pattern of gene action by the hQTLs on each parameter was unique, being environmentally dependent and varying between two allometrically related leaf growth traits. These results provide new insights into the complexity of genetic mechanisms that control trait formation in plants and provide novel findings that will be of use in studying the evolutionary trends.

Punctuated distribution of recombination hotspots and demarcation of pericentromeric regions in Phaseolus vulgaris L.

High density genetic maps are a reliable tool for genetic dissection of complex plant traits. Mapping resolution is often hampered by the variable crossover and non-crossover events occurring across the genome, with pericentromeric regions (pCENR) showing highly suppressed recombination rates. The efficiency of linkage mapping can further be improved by characterizing and understanding the distribution of recombinational activity along individual chromosomes. In order to evaluate the genome wide recombination rate in common beans (Phaseolus vulgaris L.) we developed a SNP-based linkage map using the genotype-by-sequencing approach with a 188 recombinant inbred line family generated from an inter gene pool cross (Andean x Mesoamerican). We identified 1,112 SNPs that were subsequently used to construct a robust linkage map with 11 groups, comprising 513 recombinationally unique marker loci spanning 943 cM (LOD 3.0). Comparative analysis showed that the linkage map spanned >95% of the physical map, indicating that the map is almost saturated. Evaluation of genome-wide recombination rate indicated that at least 45% of the genome is highly recombinationally suppressed, and allowed us to estimate locations of pCENRs. We observed an average recombination rate of 0.25 cM/Mb in pCENRs as compared to the rest of genome that showed 3.72 cM/Mb. However, several hot spots of recombination were also detected with recombination rates reaching as high as 34 cM/Mb. Hotspots were mostly found towards the end of chromosomes, which also happened to be gene-rich regions. Analyzing relationships between linkage and physical map indicated a punctuated distribution of recombinational hot spots across the genome.

From flower to seed: identifying phenological markers and reliable growth functions to model reproductive development in the common bean (Phaseolus vulgaris L.).

The lack of dependable morphological indicators for the onset and end of seed growth has hindered modeling work in the common bean (Phaseolus vulgaris L.). We have addressed this problem through the use of mathematical growth functions to analyse and identify critical developmental stages, which can be linked to existing developmental indices. We performed this study under greenhouse conditions with an Andean and a Mesoamerican genotype of contrasting pod and seed phenotypes, and three selected recombinant inbred lines. Pods from tagged flowers were harvested at regular time intervals for various measurements. Differences in flower production and seed and pod growth trajectories among genotypes were detected via comparisons of parameters of fitted growth functions. Regardless of the genotype, the end of pod elongation marked the beginning of seed growth, which lasted until pods displayed a sharp decline in color, or pod hue angle. These results suggest that the end of pod elongation and the onset of color change are reliable indicators of important developmental transitions in the seed, even for widely differing pod phenotypes. We also provide a set of equations that can be used to model different aspects of reproductive growth and development in the common bean.

Induced fit on heme binding to the Pseudomonas aeruginosa cytoplasmic protein (PhuS) drives interaction with heme oxygenase (HemO).

Iron, an essential nutrient with limited bioavailability, requires specialized cellular mechanisms for uptake. Although iron uptake into the cytoplasm in the form of heme has been well characterized in many bacteria, the subsequent trafficking is poorly understood. The cytoplasmic heme-binding proteins belong to a structurally related family thought to have evolved as "induced fit" ligand-binding macromolecules. One member, Pseudomonas aeruginosa cytoplasmic protein (PhuS), has previously been shown to be important for delivering heme to the iron regulated heme oxygenase (HemO). Spectroscopic investigations of the holo-PhuS complex revealed a dynamic heme environment with overlapping but distinct heme-binding sites with alternative coordinating heme ligands, His-209 or His-212. In the present work we establish a mechanism for how heme is transferred from PhuS to its partner, HemO. Using surface plasmon resonance and isothermal titration calorimetry, we have discovered that holo-PhuS, but not apo-PhuS, forms a 1:1 complex with HemO. Sedimentation velocity and limited proteolysis experiments suggest that heme binding to PhuS induces a conformational rearrangement that drives the protein interaction with HemO. Hydrodynamic analysis reveals that the holo-PhuS displays a more expanded hydrodynamic envelope compared with apo-PhuS, and we propose that this conformational change drives the interaction with HemO. We further demonstrate that replacement of His-212 by Ala disrupts the interaction of holo-PhuS with HemO; in contrast, the His-209-Ala variant can still complex with HemO, albeit more weakly. Together, the present studies reveal a mechanism that couples a heme-dependent conformational switch in PhuS to protein-protein interaction, the subsequent free energy of which drives heme release to HemO.

Catalytic turnover dependent modification of the Pseudomonas aeruginosa heme oxygenase (pa-HO) by 5,6-O-isopropyledine-2-O-allyl-ascorbic acid.

Heme oxygenase (HO) catalyzes the NADPH dependent conversion of heme to biliverdin with the release of iron and CO via three successive oxygenation steps. The oxidation of heme in the presence of alternate reductants, such as ascorbic acid, has been used extensively to characterize the mechanism of oxygen activation in HO without altering the chemistry of the reaction. NADPH-dependent cytochrome P450 reductase (CPR) and ascorbic acid mediated reactions are mechanistically very similar, in that both use molecular oxygen to initiate the reaction. In the present manuscript, we report on an ascorbic acid derivative, 5,6-O-isopropyledine-2-O-allyl-ascorbic acid, that during catalysis partitions the reaction between the conversion of heme to biliverdin, and an alternate pathway that traps the verdoheme intermediate as a result of protein modification. We propose that following activation of 5,6-O-isopropyledine-2-O-allyl-ascorbic acid to the cation radical, protein modification results via alkylation of an active site nucleophile (Asp or Glu), trapping the Fe(III)-verdoheme intermediate. The potential site of the modification and the relevance to the mechanism of Fe(III)-verdoheme conversion to biliverdin is discussed.

Identification of two heme-binding sites in the cytoplasmic heme-trafficking protein PhuS from Pseudomonas aeruginosa and their relevance to function.

PhuS is a cytoplasmic, 39 kDa heme-binding protein from Pseudomonas aeruginosa. It has previously been shown to transfer heme to its cognate heme oxygenase. It is expressed from the phu operon, which encodes a group of proteins known to actively internalize and transport heme from host organisms. This study combines the spectral resolution of resonance Raman spectroscopy with site-directed mutagenesis to identify and characterize the heme-bound states of holo-PhuS. This combined approach has identified a site in monomeric PhuS having alternate His ligands at positions 209 and 212. A second distinct binding site is present in dimeric PhuS. This site supports six-coordinate, low-spin heme, even when both His209 and His212 are mutated to Ala. The presence of conserved His and Tyr residues in all of the homologs characterized to date suggest that the dimer could be of the domain-swapped type in which two protein molecules are cross-linked by bound heme. The multiple heme-bound states and their sensitivity to pH suggest the possibility that these cytoplasmic heme-binding proteins have multiple functions that are toggled by variations in intracellular conditions.

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.

P(450)/NADPH/O(2)- and P(450)/PhIO-catalyzed N-dealkylations are mechanistically distinct.

A high-valent iron-oxo species analogous to the compound I of peroxidases has been thought to be the activated oxygen species in P450-catalyzed reactions. Spectroscopic characterization of the catalytically competent iron-oxo species in iodosobenzene (PhIO)-supported model reactions and parallels between these model reactions and PhIO- and NADPH/O2-supported P450 reactions have been taken as strong evidence for this proposal. To support this proposal, subtle differences observed in regio- and chemoselectivities, isotope effects, and source of oxygen, etc., between NADPH/O2- and PhIO-supported P450 reactions have been generally attributed to reasons other than the mechanistic differences between the two systems. In the present study, we have used a series of sensitive mechanistic probes, 4-chloro-N-cyclopropyl-N-alkylanilines, to compare and contrast the chemistries of the NADPH/O2- and PhIO-supported purified CYP2B1 N-dealkylation reactions. Herein we present the first experimental evidence to demonstrate that the NADPH/O2- and PhIO-supported P450 N-dealkylations are mechanistically distinct and, thus, the P450/PhIO system may not be a good mechanistic model for P450/NADPH/O2-catalyzed N-dealkylations.

Evidence for a hydrogen abstraction mechanism in P450-catalyzed N-dealkylations.

The experimental evidence presented in this manuscript suggest against the widely accepted single electron/proton transfer mechanism for P450 catalyzed N-dealkylations and provides strong support for a hydrogen atom abstraction mechanism.

Functional deactivations: change with age and dementia of the Alzheimer type.

Young adults typically deactivate specific brain regions during active task performance. Deactivated regions overlap with those that show reduced resting metabolic activity in aging and dementia, raising the possibility of a relation. Here, the magnitude and dynamic temporal properties of these typically deactivated regions were explored in aging by using functional MRI in 82 participants. Young adults (n = 32), older adults without dementia (n = 27), and older adults with early-stage dementia of the Alzheimer type (DAT) (n = 23) were imaged while alternating between blocks of an active semantic classification task and a passive fixation baseline. Deactivation in lateral parietal regions was equivalent across groups; in medial frontal regions, it was reduced by aging but was not reduced further by DAT. Of greatest interest, a medial parietal/ posterior cingulate region showed differences between young adults and older adults without dementia and an even more marked difference with DAT. The temporal profile of the medial parietal/posterior cingulate response suggested that it was initially activated by all three groups, but the response in young adults quickly reversed sign, whereas DAT individuals maintained activation throughout the task block. Exploratory whole-brain analyses confirmed the importance of medial parietal/posterior cingulate cortex differences in aging and DAT. These results introduce important opportunities to explore the functional properties of regions showing deactivations, how their dynamic functional properties relate to their baseline metabolic rates, and how they change with age and dementia.

Microsomal P450-catalyzed N-dealkylation of N,N-dialkylanilines: evidence for a C(alpha)-H abstraction mechanism.

The early proposal that P450-catalyzed N-dealkylation of N,N-dialkylamines proceeds through a single-electron-transfer (SET) mechanism was later challenged in favor of the C(alpha)-H abstraction mechanism. In the present study, a series of N-alkyl-N-cyclopropyl-p-chloroaniline probes have been used to examine whether the P450-catalyzed N-dealkylations proceed through a C(alpha)-H abstraction and/or a SET mechanism, using phenobarbital-induced rat liver microsomal P450 enzymes as a model system. While the findings are highly consistent with a C(alpha)-H abstraction mechanism, further experimental evidence may be necessary to completely rule out the SET mechanism.