Protein Allostery at Atomic Resolution.

Protein allostery is a phenomenon involving the long range coupling between two distal sites in a protein. In order to elucidate allostery at atomic resoluion on the ligand-binding WW domain of the enzyme Pin1, multistate structures were calculated from exact nuclear Overhauser effect (eNOE). In its free form, the protein undergoes a microsecond exchange between two states, one of which is predisposed to interact with its parent catalytic domain. In presence of the positive allosteric ligand, the equilibrium between the two states is shifted towards domain-domain interaction, suggesting a population shift model. In contrast, the allostery-suppressing ligand decouples the side-chain arrangement at the inter-domain interface thereby reducing the inter-domain interaction. As such, this mechanism is an example of dynamic allostery. The presented distinct modes of action highlight the power of the interplay between dynamics and function in the biological activity of proteins.

Amyloid Aggregates Arise from Amino Acid Condensations under Prebiotic Conditions.

Current theories on the origin of life reveal significant gaps in our understanding of the mechanisms that allowed simple chemical precursors to coalesce into the complex polymers that are needed to sustain life. The volcanic gas carbonyl sulfide (COS) is known to catalyze the condensation of amino acids under aqueous conditions, but the reported di-, tri-, and tetra-peptides are too short to support a regular tertiary structure. Here, we demonstrate that alanine and valine, two of the proteinogenic amino acids believed to have been among the most abundant on a prebiotic earth, can polymerize into peptides and subsequently assemble into ordered amyloid fibers comprising a cross-ß-sheet quaternary structure following COS-activated continuous polymerization of as little as 1 mm amino acid. Furthermore, this spontaneous assembly is not limited to pure amino acids, since mixtures of glycine, alanine, aspartate, and valine yield similar structures.

Extensive functional pleiotropy of REVOLUTA substantiated through forward genetics.

In plants, genes may sustain extensive pleiotropic functional properties by individually affecting multiple, distinct traits. We discuss results from three genome-wide association studies of approximately 400 natural poplar (Populus trichocarpa) accessions phenotyped for 60 ecological/biomass, wood quality, and rust fungus resistance traits. Single-nucleotide polymorphisms (SNPs) in the poplar ortholog of the class III homeodomain-leucine zipper transcription factor gene REVOLUTA (PtREV) were significantly associated with three specific traits. Based on SNP associations with fungal resistance, leaf drop, and cellulose content, the PtREV gene contains three potential regulatory sites within noncoding regions at the gene's 3' end, where alternative splicing and messenger RNA processing actively occur. The polymorphisms in this region associated with leaf abscission and cellulose content are suggested to represent more recent variants, whereas the SNP associated with leaf rust resistance may be more ancient, consistent with REV's primary role in auxin signaling and its functional evolution in supporting fundamental processes of vascular plant development.

Genome-wide association implicates numerous genes underlying ecological trait variation in natural populations of Populus trichocarpa.

In order to uncover the genetic basis of phenotypic trait variation, we used 448 unrelated wild accessions of black cottonwood (Populus trichocarpa) from much of its range in western North America. Extensive data from large-scale trait phenotyping (with spatial and temporal replications within a common garden) and genotyping (with a 34 K Populus single nucleotide polymorphism (SNP) array) of all accessions were used for gene discovery in a genome-wide association study (GWAS). We performed GWAS with 40 biomass, ecophysiology and phenology traits and 29,355 filtered SNPs representing 3518 genes. The association analyses were carried out using a Unified Mixed Model accounting for population structure effects among accessions. We uncovered 410 significant SNPs using a Bonferroni-corrected threshold (P<1.7×10(-6)). Markers were found across 19 chromosomes, explained 1-13% of trait variation, and implicated 275 unique genes in trait associations. Phenology had the largest number of associated genes (240 genes), followed by biomass (53 genes) and ecophysiology traits (25 genes). The GWAS results propose numerous loci for further investigation. Many traits had significant associations with multiple genes, underscoring their genetic complexity. Genes were also identified with multiple trait associations within and/or across trait categories. In some cases, traits were genetically correlated while in others they were not.

Geographical and environmental gradients shape phenotypic trait variation and genetic structure in Populus trichocarpa.

• Populus trichocarpa is widespread across western North America spanning extensive variation in photoperiod, growing season and climate. We investigated trait variation in P. trichocarpa using over 2000 trees from a common garden at Vancouver, Canada, representing replicate plantings of 461 genotypes originating from 136 provenance localities. • We measured 40 traits encompassing phenological events, biomass accumulation, growth rates, and leaf, isotope and gas exchange-based ecophysiology traits. With replicated plantings and 29,354 single nucleotide polymorphisms (SNPs) from 3518 genes, we estimated both broad-sense trait heritability (H(2)) and overall population genetic structure from principal component analysis. • Populus trichocarpa had high phenotypic variation and moderate/high H(2) for many traits. H(2) ranged from 0.3 to 0.9 in phenology, 0.3 to 0.8 in biomass and 0.1 to 0.8 in ecophysiology traits. Most traits correlated strongly with latitude, maximum daylength and temperature of tree origin, but not necessarily with elevation, precipitation or heat : moisture indices. Trait H(2) values reflected trait correlation strength with geoclimate variables. The population genetic structure had one significant principal component (PC1) which correlated with daylength and showed enrichment for genes relating to circadian rhythm and photoperiod. • Robust relationships between traits, population structure and geoclimate in P. trichocarpa reflect patterns which suggest that range-wide geographical and environment gradients have shaped its genotypic and phenotypic variability.

Network analysis reveals the relationship among wood properties, gene expression levels and genotypes of natural Populus trichocarpa accessions.

High-throughput approaches have been widely applied to elucidate the genetic underpinnings of industrially important wood properties. Wood traits are polygenic in nature, but gene hierarchies can be assessed to identify the most important gene variants controlling specific traits within complex networks defining the overall wood phenotype. We tested a large set of genetic, genomic, and phenotypic information in an integrative approach to predict wood properties in Populus trichocarpa. Nine-yr-old natural P. trichocarpa trees including accessions with high contrasts in six traits related to wood chemistry and ultrastructure were profiled for gene expression on 49k Nimblegen (Roche NimbleGen Inc., Madison, WI, USA) array elements and for 28,831 polymorphic single nucleotide polymorphisms (SNPs). Pre-selected transcripts and SNPs with high statistical dependence on phenotypic traits were used in Bayesian network learning procedures with a stepwise K2 algorithm to infer phenotype-centric networks. Transcripts were pre-selected at a much lower logarithm of Bayes factor (logBF) threshold than SNPs and were not accommodated in the networks. Using persistent variables, we constructed cross-validated networks for variability in wood attributes, which contained four to six variables with 94-100% predictive accuracy. Accommodated gene variants revealed the hierarchy in the genetic architecture that underpins substantial phenotypic variability, and represent new tools to support the maximization of response to selection.

Genome-wide association mapping for wood characteristics in Populus identifies an array of candidate single nucleotide polymorphisms.

Establishing links between phenotypes and molecular variants is of central importance to accelerate genetic improvement of economically important plant species. Our work represents the first genome-wide association study to the inherently complex and currently poorly understood genetic architecture of industrially relevant wood traits. Here, we employed an Illumina Infinium 34K single nucleotide polymorphism (SNP) genotyping array that generated 29,233 high-quality SNPs in c. 3500 broad-based candidate genes within a population of 334 unrelated Populus trichocarpa individuals to establish genome-wide associations. The analysis revealed 141 significant SNPs (α ≤ 0.05) associated with 16 wood chemistry/ultrastructure traits, individually explaining 3-7% of the phenotypic variance. A large set of associations (41% of all hits) occurred in candidate genes preselected for their suggested a priori involvement with secondary growth. For example, an allelic variant in the FRA8 ortholog explained 21% of the total genetic variance in fiber length, when the trait's heritability estimate was considered. The remaining associations identified SNPs in genes not previously implicated in wood or secondary wall formation. Our findings provide unique insights into wood trait architecture and support efforts for population improvement based on desirable allelic variants.

The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus.

• The formation of secondary cell walls in cell types such as tracheary elements and fibers is a defining characteristic of vascular plants. The Arabidopsis transcription factor KNAT7 is a component of a transcription network that regulates secondary cell wall biosynthesis, but its function has remained unclear. • We conducted anatomical, biochemical and molecular phenotypic analyses of Arabidopsis knat7 loss-of-function alleles, KNAT7 over-expression lines and knat7 lines expressing poplar KNAT7. • KNAT7 was strongly expressed in concert with secondary wall formation in Arabidopsis and poplar. Arabidopsis knat7 loss-of-function alleles exhibited irregular xylem phenotypes, but also showed increased secondary cell wall thickness in fibers. Increased commitment to secondary cell wall biosynthesis was accompanied by increased lignin content and elevated expression of secondary cell wall biosynthetic genes. KNAT7 over-expression resulted in thinner interfascicular fiber cell walls. • Taken together with data demonstrating that KNAT7 is a transcriptional repressor, we hypothesize that KNAT7 is a negative regulator of secondary wall biosynthesis, and functions in a negative feedback loop that represses metabolically inappropriate commitment to secondary wall formation, thereby maintaining metabolic homeostasis. The conservation of the KNAT7 regulatory module in poplar suggests new ways to manipulate secondary cell wall deposition for improvement of bioenergy traits in this tree.

Atomic resolution protein allostery from the multi-state structure of a PDZ domain.

Recent methodological advances in solution NMR allow the determination of multi-state protein structures and provide insights into structurally and dynamically correlated protein sites at atomic resolution. This is demonstrated in the present work for the well-studied PDZ2 domain of protein human tyrosine phosphatase 1E for which protein allostery had been predicted. Two-state protein structures were calculated for both the free form and in complex with the RA-GEF2 peptide using the exact nuclear Overhauser effect (eNOE) method. In the apo protein, an allosteric conformational selection step comprising almost 60% of the domain was detected with an "open" ligand welcoming state and a "closed" state that obstructs the binding site by changing the distance between the ß-sheet 2, α-helix 2, and sidechains of residues Lys38 and Lys72. The observed induced fit-type apo-holo structural rearrangements are in line with the previously published evolution-based analysis covering ~25% of the domain with only a partial overlap with the protein allostery of the open form. These presented structural studies highlight the presence of a dedicated highly optimized and complex dynamic interplay of the PDZ2 domain owed by the structure-dynamics landscape.

ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation in Arabidopsis.

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana). abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.

Yield quantitative trait loci from wild tomato are predominately expressed by the shoot.

Plant yield is the integrated outcome of processes taking place above and below ground. To explore genetic, environmental and developmental aspects of fruit yield in tomato, we phenotyped an introgression line (IL) population derived from a cross between the cultivated tomato (Solanum lycopersicum) and a wild species (Solanum pennellii). Both homozygous and heterozygous ILs were grown in irrigated and non-irrigated fields and evaluated for six yield components. Thirteen lines displayed transgressive segregation that increased agronomic yield consistently over 2 years and defined at least 11 independent yield-improving QTL. To determine if these QTL were expressed in the shoots or the roots of the plants, we conducted field trials of reciprocally grafted ILs; out of 13 lines with an effect on yield, 10 QTL were active in the shoot and only IL8-3 showed a consistent root effect. To further examine this unusual case, we evaluated the metabolic profiles of fruits from both the homo- and heterozygous lines for IL8-3 and compared these to those obtained from the fruit of their equivalent genotypes in the root effect population. We observed that several of these metabolic QTL, like the yield QTL, were root determined; however, further studies will be required to delineate the exact mechanism mediating this effect in this specific line. The results presented here suggest that genetic variation for root traits, in comparison to that present in the shoot, represents only a minor component in the determination of tomato fruit yield.

A review of varietal change in roots, tubers and bananas: consumer preferences and other drivers of adoption and implications for breeding.

This review of the literature on varietal change in sub-Saharan Africa looks in detail at adoption of new varieties of bananas in Uganda, cassava in Nigeria, potato in Kenya, sweetpotato in Uganda and yams in Côte d'Ivoire. The review explored three hypotheses about drivers of varietal change. There was a strong confirmation for the hypothesis that insufficient priority given to consumer-preferred traits by breeding programmes contributes to the limited uptake of modern varieties (MVs) and low varietal turnover. Lack of evidence meant the second hypothesis of insufficient attention to understanding and responding to gender differences in consumer preferences for quality and post-harvest traits was unresolved. The evidence on the third hypothesis about the informal seed system contributing to slow uptake of MVs was mixed. In some cases, the informal system has contributed to rapid uptake of MVs, but often it appears to be a barrier with inconsistent varietal naming a major challenge.

A prebiotic template-directed peptide synthesis based on amyloids.

The prebiotic replication of information-coding molecules is a central problem concerning life's origins. Here, we report that amyloids composed of short peptides can direct the sequence-selective, regioselective and stereoselective condensation of amino acids. The addition of activated DL-arginine and DL-phenylalanine to the peptide RFRFR-NH2 in the presence of the complementary template peptide Ac-FEFEFEFE-NH2 yields the isotactic product FRFRFRFR-NH2, 1 of 64 possible triple addition products, under conditions in which the absence of template yields only single and double additions of mixed stereochemistry. The templating mechanism appears to be general in that a different amyloid formed by (Orn)V(Orn)V(Orn)V(Orn)V-NH2 and Ac-VDVDVDVDV-NH2 is regioselective and stereoselective for N-terminal, L-amino-acid addition while the ornithine-valine peptide alone yields predominantly sidechain condensation products with little stereoselectivity. Furthermore, the templating reaction is stable over a wide range of pH (5.6-8.6), salt concentration (0-4 M NaCl), and temperature (25-90 °C), making the amyloid an attractive model for a prebiotic peptide replicating system.

Towards Prebiotic Catalytic Amyloids Using High Throughput Screening.

Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-ß-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.

Comparison of Resistance Level to Tomato Yellow Leaf Curl Virus Among Commercial Cultivars and Breeding Lines.

The effect of tomato yellow leaf curl virus (TYLCV) on total yield and yield components of various resistant F1 tomato cultivars and new breeding lines was evaluated in the field. Seeds of the F1 hybrids 8484, 3761, Fiona, and Tyking and the new breeding lines TY172 and TY197 were sown in an insect-proof greenhouse. When the seedlings reached the first-leaf stage, they were inoculated with TYLCV by means of the whitefly vector Bemisia tabaci. Noninoculated plants of the same cultivar or line which were exposed to nonviruliferous whiteflies served as controls. After a short recovery period, the plants were transplanted to the field. The inoculated plants of each cultivar or line were compared with their respective control, noninoculated plants, in terms of total yield, average fruit weight and number, and plant fresh weight. Disease symptom development and virus accumulation in the inoculated plants were monitored. There were substantial differences among the different entries tested in the extent of yield loss relative to the corresponding noninoculated control plants as well as viral DNA accumulation levels. Plants of TY172 and TY197 suffered the least relative yield loss and contained the lowest level of viral DNA. Therefore, these two lines exhibited the highest level of resistance. These results clearly demonstrate the ability of the various resistant tomato cultivars and lines to inhibit the effects of the virus, even following inoculation at a very early stage of plant development.

SNP discovery in black cottonwood (Populus trichocarpa) by population transcriptome resequencing.

The western black cottonwood (Populus trichocarpa) was the first tree to have its genome fully sequenced and has emerged as the model species for the study of secondary growth and wood formation. It is also a good candidate species for the production of lignocellulosic biofuels. Here, we present and make available to the research community the results of the sequencing of the transcriptome of developing xylem in 20 accessions with high-throughput next generation sequencing technology. We found over 0.5 million putative single nucleotide polymorphisms (SNPs) in 26,595 genes that are expressed in developing secondary xylem. More than two-thirds of all SNPs were found in annotated exons, with 18% and 14% in regions of the genome annotated as introns and intergenic, respectively, where only 3% and 4% of sequence reads mapped. This suggests that the current annotation of the poplar genome is remarkably incomplete and that there are many transcripts and novel genes waiting to be annotated. We hope that this resource will stimulate further research in expression profiling, detection of alternative splicing and adaptive evolution in poplar.

Quantitative determination of NOE rates in perdeuterated and protonated proteins: practical and theoretical aspects.

Precision and accuracy are the limiting factors in extracting structural and dynamic information from experimental NOEs. In this study, error sources at all stages of such an analysis are identified and errors are estimated. The data set of H(N)-H(N) cross-relaxation rates obtained from triple-labeled ubiquitin presented in [B. Vögeli, T.F. Segawa, D. Leitz, A. Sobol, A. Choutko, D. Trzesniak, W. van Gunsteren, R. Riek, J. Am. Chem. Soc. 131 (47) (2009) 17215-17225] is extended to rates obtained from a double-labeled sample. Analog data sets are presented for GB3. It is shown that quantitative NOE rates can be determined with high accuracy from both triple-labeled as well as double-labeled samples. The quality of experimental cross-relaxation rates obtained from 3D HXQC-NOESY and NOESY-HXQC experiments is discussed. It is shown that NOESY-HXQC experiments provide rates of the same quality as HXQC-NOESY if both diagonal and cross peaks for a spin pair can be resolved. Expressions for cross-relaxation rates for anisotropically tumbling molecules exhibiting fast and slow motion are derived. The impact of anisotropy on the prediction of cross-relaxation rates and on the conversion of experimental rates into effective distances is discussed. For molecules with anisotropy D(II)/D( perpendicular) up to five the distance error is smaller than 2%. Finally, "averaged order parameters" are calculated for specific secondary-structural elements showing similar trends for ubiquitin and GB3.

Microarray gene expression profiling of developmental transitions in Sitka spruce (Picea sitchensis) apical shoots.

The apical shoot drives the yearly new stem growth of conifer trees, is the primary site for the establishment of chemical and physical defences, and is important in establishing subsequent perennial growth. This organ presents an interesting developmental system, with growth and development progressing from a meristematic tip through development of a primary vascular system, to a base with fully differentiated and lignified secondary xylem on the inside and bark tissue with constitutive defence structures such as resin, polyphenolic phloem parenchyma cells, and sclereids on the outside. A spruce (Picea spp.) microarray containing approximately 16.7K unique cDNAs was used to study transcript profiles that characterize the developmental transition in apical shoots of Sitka spruce (Picea sitchensis) from their vegetative tips to their woody bases. Along with genes involved in cell-wall modification and lignin biosynthesis, a number of differentially regulated genes encoding protein kinases and transcription factors with base-preferred expression patterns were identified, which could play roles in the formation of woody tissues inside the apical shoot, as well as in regulating other developmental transitions associated with organ maturation. Preferential expression of known conifer defence genes, genes encoding defence-related proteins, and genes encoding regulatory proteins was observed at the apical shoot tip and in the green bark tissues at the apical shoot base, suggesting a commitment to constitutive defence in the apical shoot that is co-ordinated with rapid development of secondary xylem.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome.

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.