Inder S. Sheoran

Proteome profile and functional classification of proteins in Arabidopsis thaliana (Landsberg erecta) mature pollen

Proteome analysis of mature Arabidopsis thaliana (Landsberg erecta ecotype) pollen was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 960 spots were resolved on pH 4–7 IPG strips and 110 distinct proteins were identified from 150 spots analyzed. The identified proteins were categorized based on their functional role in the pollen, which included proteins involved in energy regulation, defense-related mechanisms, calcium-binding and signaling, cytoskeletal formation, pollen allergens, glycine-rich proteins (GRPs), and late embryogenesis abundant (LEA) proteins. These proteins potentially play important roles in pollen function at maturity and during subsequent germination and tube growth. Some of the proteins identified were related to known pollen-specific transcripts, while some were similar to proteins found in the seed. In this study, 66 new proteins were identified which were not reported in two other recent studies on Arabidopsis pollen, 17 proteins were common in all three studies, and 35 or 26 proteins reported here had an overlap with one or the other two studies. These differences may be attributed to the methods of protein extraction, spot selection for analysis, and the ecotype used. Together, the three studies provide a broad spectrum of the Arabidopsis pollen proteome.

Differential expression of proteins in the wild type and 7B-1 male-sterile mutant anthers of tomato (Solanum lycopersicum): A proteomic analysis

In the 7B-1 male-sterile mutant of tomato, pollen development breaks down prior to meiosis in microspore mother cells (MMCs). We have used the proteomic approach to identify differentially expressed proteins in the wild type (WT) and mutant anthers with the objective of analyzing their roles in normal pollen development and in male sterility. By using 2-DE and DIGE technologies, over 1800 spots were detected and of these 215 spots showed 1.5-fold or higher volume ratio in either WT or 7B-1 anthers. Seventy spots, either up-regulated in WT, or in 7B-1, were subjected to mass spectrometry and 59 spots representing 48 distinct proteins were identified. The proteins up-regulated in WT anthers included proteases, e.g., subtilase, proteasome subunits, and 5B-protein with potential roles in tapetum degeneration, FtsZ protein, leucine-rich repeat proteins, translational and transcription factors. In 7B-1 anthers, aspartic protease, superoxide dismutase, ACP reductase, ribonucleoprotein and diphosphate kinase were up-regulated. Also, cystatin inhibitory activity was high in the mutant and correlated with the expression of male sterility. Other proteins including calreticulin, Heat shock protein 70, glucoside hydrolase, and ATPase, were present in both genotypes. The function of identified proteins in tapetum and normal pollen development, and in male sterility is discussed.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture, hyphal morphogenesis, and asexual development.

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeA Delta strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmA Delta strain. As for the ugmA Delta strain, ugeA Delta colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeA Delta strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeA Delta, and ugmA Delta hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmA Delta strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.

Dynamics of protein expression during pollen germination in canola (Brassica napus).

The proteome of mature (MP) and in vitro germinating pollen (GP) of canola (Brassica napus) were analyzed using the DIGE technology with the objective of identifying proteins and their function in pollen germination. Of the 2,238 protein spots detected in gel images, 344 were differentially expressed in MP and GP samples of which 165 were subjected to MALDI-TOF/TOF and 130 were successfully identified using the NCBInr and Brassica EST databases. The major proteins up-regulated in GP, relative to MP, have roles in carbohydrate metabolism, protein metabolism, and cell wall remodeling. Others with roles in cytoskeleton dynamics, nucleotide and amino acid metabolism, signal transduction, and stress response also showed higher expression in GP. Proteins concerned with transcriptional regulation and ion transport were similar in MP and GP, and some catalases and LEA proteins were down-regulated in GP. A number of proteins including, oleosin, cruciferin, and enolase, were released into the pollen germination medium indicating their potential role in pollen–stigma interaction. Glycosylated proteins were also identified in MP and GP, but their protein profiles were not different. This study has documented the dynamics of protein expression during pollen germination and early tube growth in B. napus and provides insights into the fundamental mechanisms involved in these processes, and in cell growth, cell–cell communication, and cell signaling.

Elucidation of Substrate Specificity in Aspergillus nidulans UDP-Galactose-4-Epimerase.

The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD+ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.

Preliminary X-ray crystallographic studies of UDP-­glucose-4-epimerase from Aspergillus nidulans

UDP-glucose-4-epimerase (GALE) from Aspergillus nidulans was overexpressed in Escherichia coli, purified via His-tag affinity chromatography and cocrystallized with UDP-galactose using the microbatch method. The crystals diffracted to 2.4 Å resolution using synchrotron radiation on the Canadian Light Source 08ID-1 beamline. Examination of the data with d*TREK revealed nonmerohedral twinning, from which a single lattice was ultimately extracted for processing. The final space group was found to be C2, with unit-cell parameters a = 66.13, b = 119.15, c = 161.42 Å, β = 98.48°. An initial structure solution has been obtained via molecular replacement employing human GALE (PDB entry 1hzj) as a template model.