Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments.

Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the 'mustard bomb' reaction in reducing pungency upon the mastication of fresh tissue-a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.

Influence of Alcohol ß-Fluorination on Hydrogen-Bond Acidity of Conformationally Flexible Substrates.

Rational modulations of molecular interactions are of significant importance in compound properties optimization. We have previously shown that fluorination of conformationally rigid cyclohexanols leads to attenuation of their hydrogen-bond (H-bond) donating capacity (designated by pKAHY ) when OH⋅⋅⋅F intramolecular hydrogen-bond (IMHB) interactions occur, as opposed to an increase in pKAHY due to the fluorine electronegativity. This work has now been extended to a wider range of aliphatic ß-fluorohydrins with increasing degrees of conformational flexibility. We show that the observed differences in pKAHY between closely related diastereomers can be fully rationalized by subtle variations in populations of conformers able to engage in OH⋅⋅⋅F IMHB, as well as by the strength of these IMHBs. We also show that the Kenny theoretical Vα (r) descriptor of H-bond acidity accurately reflects the observed variations and a calibration equation extended to fluorohydrins is proposed. This work clearly underlines the importance of the weak OH⋅⋅⋅F IMHB in the modulation of alcohol H-bond donating capacity.

SHAPE-directed discovery of potent shRNA inhibitors of HIV-1.

The RNA interference (RNAi) pathway can be exploited using short hairpin RNAs (shRNAs) to durably inactivate pathogenic genes. Prediction of optimal target sites is notoriously inaccurate and current approaches applied to HIV-1 show weak correlations with virus inhibition. In contrast, using a high-content model for disrupting pre-existing intramolecular structure in the HIV-1 RNA, as achievable using high-resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing information, we discovered strong correlations between inhibition of HIV-1 production in a quantitative cell-based assay and very simple thermodynamic features in the target RNA. Strongest inhibition occurs at RNA target sites that both have an accessible "seed region" and, unexpectedly, are structurally accessible in a newly identified downstream flanking sequence. We then used these simple rules to create a new set of shRNAs and achieved inhibition of HIV-1 production of 90% or greater for up to 82% of designed shRNAs. These shRNAs inhibit HIV-1 replication in therapy-relevant T cells and show no or low cytotoxicity. The remarkable success of this straightforward SHAPE-based approach emphasizes that RNAi is governed, in significant part, by very simple, predictable rules reflecting the underlying RNA structure and illustrates principles likely to prove broadly useful in understanding transcriptome-scale biological recognition and therapeutics involving RNA.

Architecture and secondary structure of an entire HIV-1 RNA genome.

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS and other serious health threats. Viral replication is regulated at many levels, including the use of conserved genomic RNA structures. Most potential regulatory elements in viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests that RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions, including splice site acceptors and hypervariable regions. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by previously unrecognized regulatory motifs and that extensive RNA structure constitutes an important component of the genetic code.

Status of the Riparian ecosystem in the upper San Pedro River, Arizona: application of an assessment model.

A portion of Arizona's San Pedro River is managed as a National Riparian Conservation Area but is potentially affected by ground-water withdrawals beyond the conservation area borders. We applied an assessment model to the Conservation Area as a basis for monitoring long-term changes in riparian ecosystem condition resulting from changes in river water availability, and collected multi-year data on a subset of the most sensitive bioindicators. The assessment model is based on nine vegetation bioindicators that are sensitive to changes in surface water or ground water. Site index scores allow for placement into one of three condition classes, each reflecting particular ranges for site hydrology and vegetation structure. We collected the bioindicator data at 26 sites distributed among 14 reaches that had similar stream flow hydrology (spatial flow intermittency) and geomorphology (channel sinuosity, flood-plain width). Overall, 39% of the riparian corridor fell within condition class 3 (the wettest condition), 55% in condition class 2, and 6% in the driest condition class. Condition class 3 reaches have high cover of herbaceous wetland plants (e.g., Juncus and Schoenoplectus spp.) along the perennial stream channel and dense, multi-aged Populus-Salix woodlands in the flood plain, sustained by shallow ground water in the stream alluvium. In condition class 2, intermittent stream flows result in low cover of streamside wetland herbs, but Populus-Salix remain abundant in the flood plain. Perennial wetland plants are absent from condition class 1, reflecting highly intermittent stream flows; the flood plain is vegetated by Tamarixa small tree that tolerates the deep and fluctuating ground water levels that typify this reach type. Abundance of herbaceous wetland plants and growth rate of Salix gooddingii varied between years with different stream flow rates, indicating utility of these measures for tracking short-term responses to hydrologic change. Repeat measurement of all bioindicators will indicate long-term trends in hydro-vegetational condition.

Ligand-mediated anticodon conformational changes occur during tRNA methylation by a TrmD methyltransferase.

Orthologs of TrmD, G37 tRNA methyltransferases, have been analyzed with regard to post-tRNA binding events required to move the residue G37 in proximity to bound AdoMet for catalysis. This was approached initially by probing tRNA with T2 nuclease or Pb acetate in the presence, then absence, of Escherichia coli TrmD protein. Cleavage patterns clearly show that portions of the anticodon loop phosphodiester backbone are protected from cleavage only in the presence of sinefungin, a potent AdoMet analogue. This demonstrates that there must be considerable movement of the loop region and/or protein as the AdoMet site is occupied. Florescence energy transfer experiments were employed to better assess the movement of the G37 and G36 base residues in response to occupancy of the AdoMet site. When the Streptococcus pneumoniae TrmD protein was bound to synthetic tRNA(1)(Leu) substituted with 2-aminopurine at positions 36 and 37, fluorescence energy transfer analysis showed that a decrease in 2-aminopurine fluorescence occurs only when AdoMet is present. Taken together, these results suggest that the base to be methylated by the TrmD protein is mobilized into the active center after tRNA binding only when the AdoMet site is occupied.

Characterization of Streptococcus pneumoniae TrmD, a tRNA methyltransferase essential for growth.

Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, tRNA(Leu)(CAG). Other heterologous nonsubstrate tRNA species, like, tRNA (Thr)(GGT), tRNA(Phe), and tRNA (Ala)(TGC), bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).

Insights into catalysis by a knotted TrmD tRNA methyltransferase.

The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution. TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer. Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain. The two bound AdoHcy moieties are buried at the bottom of deep clefts. The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur. In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis. The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring. Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.

Cluster analysis of structural stage classes to map wildland fuels in a Madrean ecosystem.

Geospatial information technology is changing the nature of fire mapping science and management. Geographic information systems (GIS) and global positioning system technology coupled with remotely sensed data provide powerful tools for mapping, assessing, and understanding the complex spatial phenomena of wildland fuels and fire hazard. The effectiveness of these technologies for fire management still depends on good baseline fuels data since techniques have yet to be developed to directly interrogate understory fuels with remotely sensed data. We couple field data collections with GIS, remote sensing, and hierarchical clustering to characterize and map the variability of wildland fuels within and across vegetation types. One hundred fifty six fuel plots were sampled in eight vegetation types ranging in elevation from 1150 to 2600 m surrounding a Madrean 'sky island' mountain range in the southwestern US. Fuel plots within individual vegetation types were divided into classes representing various stages of structural development with unique fuel load characteristics using a hierarchical clustering method. Two Landsat satellite images were then classified into vegetation/fuel classes using a hybrid unsupervised/supervised approach. A back-classification accuracy assessment, which uses the same pixels to test as used to train the classifier, produced an overall Kappa of 50% for the vegetation/fuels map. The map with fuel classes within vegetation type collapsed into single classes was verified with an independent dataset, yielding an overall Kappa of 80%.