Paul Loida

Discovery of imidazole glycerol phosphate dehydratase inhibitors through 3-D database searching

Imidazole glycerol phosphate dehydratase (IGPD) has become an attractive target for herbicide discovery since it is present in plants and not in mammals. Currently no knowledge is available on the 3-D structure of the IGPD active site. Therefore, we used a pharmacophore model based on known inhibitors and 3-D database searches to identify new active compounds. In vitro testing of compounds from the database searches led to the identification of a class of pyrrole aldehydes as novel inhibitors of IGPD.

Design and synthesis of β-carboxamido phosphonates as potent inhibitors of imidazole glycerol phosphate dehydratase

We describe the synthesis and enzymatic activity of a library of beta-carboxamido phosphonates as inhibitors of imidazole glycerol phosphate dehydratase (IGPD). Biological results suggest the presence of an enzymatic interaction site not previously observed for other inhibitors of IGPD.

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Involvement of the N-terminal B-box Domain of Arabidopsis BBX32 Protein in Interaction with Soybean BBX62 Protein

Background: AtBBX32 is a member of the B-box protein family from A. thaliana. Its molecular mechanism is poorly understood. Results: We demonstrate functional interactions of AtBBX32 with soybean BBX62 (GmBBX62). Conclusion: The N-terminal B-box domain plays a key role in mediating the interaction between AtBBX32 and GmBBX62. Significance: Our data offer novel insight into the role of B-box domains in mediating protein-protein interactions between different plant B-box proteins. Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn2+-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.

Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity

Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.

Breeding and Biotech Approaches Towards Improving Yield in Soybean

Soybean (Glycine max (L.) Merrill) is the most widely cultivated oilseed crop accounting for more than 50 % of the world’s oilseed production. Yield gain in soybean estimated to be 0.5–0.7 % per year in North America has been driven by the adoption of agronomic or management practices and genetic improvement. While genetic improvement through breeding will continue to play a significant role in enhancing yield by the development of cultivars adapted to a wide range of latitudes, biotech traits such as enhanced insect protection and weed control contribute indirectly to yield improvement. An understanding of physiological traits associated with genetic gain in yield offers vast opportunities for further advances in yield improvement. Potential targets for genetic improvement include source capacity (leaf area index, leaf area duration, carbon and nitrogen assimilation, and dry matter partitioning), sink strength (number of primary and secondary yield components, seed-filling rate and duration), and tolerance to suboptimal conditions (water limitation and high/low temperature). Manipulating single or multiple traits using breeding and biotechnology approaches will help to improve intrinsic yield potential and yield stability traits in soybean. Application of multiple technologies to improve yield gain is vital, with the changing climatic conditions and increasing global demand for food and feed.