Fredy Alpeter, coordinator of the plant graduate student competition, announced this year’s winners. Cash awards of $250, $150, and $50 were provided for the first, second, and third place winners respectively. The awards were made possible by ad hoc donations from Kitchen Culture Kits, Inc. (Carol Stiff), Caisson Laboratories LLC (Mike Hanson and Gordon Reese), as well as the general PBS membership. Fredy thanked this year’s judges (Neal Stewart, Vincent Wingate, Tom Clemente) for their time and encouragement of the student participants.
Mrinalini Muralidharan
First place – Characterizing Cholinesterase like Enzyme in A. thaliana
Acetylcholinesterase (AChE) is an enzyme that hydrolyses the neurotransmitter acetylcholine in the neural synapse and neuromuscular junctions in mammals. There is evidence of non-neuronal functions of AChE including roles in development and stress physiology. The presence of AChE in other species such as plants, fungi etc has been determined indirectly by looking for AChE activity. Although a maize gene purported to be encoding for acetylcholinesterase was recently cloned (Sagane et al, 2005, Plant Physiol 138: 1359-1371)., little is known about the function of AChE in plants. At3g26430 is the Arabidopsis thaliana ortholog of the maize gene and it is currently annotated as lipase in the TIGR and TAIR databases. We expressed the At3g26430 gene in a variety of systems including bacterial, yeast, plant and mammalian expression systems. To establish its biochemical identity as a cholinesterase, we analyzed the ability of the enzyme to hydrolyze various esterase and lipase substrates and looked at its inhibition profile. Existing microarray data were mined to determine the expression patterns of this protein and suggest that the protein could be part of a plant defense mechanism. Its subcellular localization using GFP fusions will be presented. We have created A.thaliana plants that are over-expressers and obtained knockouts for the gene to help us elucidate its function
Mrinalini Muralidharan,School of Life Sciences and The Biodesign Institute, Arizona State University PO Box 4601, Tempe, AZ 85287. In Vitro Cellular and Developmental Biology, 44:S66, 2008
Second place – Risk Assessment of Transgenic, Apomictic Forage and Turf Grass (Paspalum notatum Flugge)
Sukhpreet Sandhu
The commercially important bahiagrass cultivar “Argentine” is an obligate apomict. Its asexual reproduction resulting in uniform seed progeny could reduce the risk of unintended gene dispersal by pollen. The primary objective of the present study was to investigate pollen-mediated gene transfer from apomictic tetraploid bahiagrass to sexual diploid bahiagrass using glufosinate resistance as a marker. Glufosinate-resistant, fertile transgenic bahiagrass was generated by biolistic gene transfer and used as pollen donor in a field trial (USDA-APHIS permit no. 05-365-01r) in Marianna, FL, with two replications. Primary transgenics and the apomictic seed progeny was characterized by Southern blot analysis, immunochromatographic assay, and glufosinate application. Open-pollinated seeds were harvested from wild-type, sexual-diploid bahiagrass surrounding the transgenic glufosinate-resistant bahiagrass at 1 m distance. Gene transfer frequency was determined by application of 0.14% glufosinate to germinated seedlings using the wild type as the negative control and the transgenic parent as the positive control. Very low hybridization frequency of less than 0.1%(26 hybrids out of 28,000 seedlings analyzed) was observed between the apomictic transgenic and wild-type bahiagrass. Bar gene integration and expression in the hybrids were confirmed by Southern blot and immunochromatographic analysis and herbicide application, respectively. All six of the so far analyzed hybrids have been confirmed as triploids by both flow cytometry and root-tip chromosome counting. Embryo sac analysis detected both apomictic and sexual embryo sacs, suggesting facultative apomixis. Fertility of hybrids will be evaluated. All triploid hybrids showed reduced vigor compared to diploid or tetraploid bahiagrass. The results suggests that using the apomictic bahiagrass cultivar Argentine as a target for bahiagrass transformation provides a high level of transgene containment compared to open-pollinating sexual-diploid turf and forage grasses.
Sukhpreet Sandhu, Agronomy Department, PMCB, Genetics Institute, University of Florida-IFAS, Gainesville, FL 32611. In Vitro Cellular and Developmental Biology, 44:S62-63, 2008
Nancy J. Engelmann
Third place – Tailoring Herbicide Treatments of Tomato Suspension Cultures to Produce 14C-Carotenoids for Animal Metabolic Tracing
Consumption of tomato food products is associated with a decreased risk of prostate cancer, and carotenoids are believed to play an integral role in this diet-disease relationship. Lycopene (LYC) is often credited as the main bioactive carotenoid in tomatoes, but two other prominent colorless carotenoids, phytoene (PE) and phytofluene (PF) may play an active role as well. Utilization of 14C carotenoid tracers will facilitate building knowledge on LYC, PE, and PF metabolism and bioavailability, the basis for understanding disease prevention mechanisms. Norflurazon (NORF), and 2-(4-chlorophenyl-thio) triethylamine (CPTA) are bleaching herbicides, which lead to marked increases in PE and PF or LYC, respectively, by interrupting carotenogenesis. Cherry ‘VFNT’ tomato cell cultures grown 14 d in Murashige & Skoog liquid media with growth regulators IAA (5 mg/L) and all-trans zeatin (2 mg/L) were treated with a combination (COMBO) of NORF (day 7, 0.75 mg/L) and CPTA (day 1, 0.075 g/L) or each herbicide alone. Analysis of concentrations of PE, PF, and LYC revealed that CPTA alone produced the highest combined total of the carotenoids (0.20, 0.19, and 1.62 mg/L), NORF alone produced the highest levels of PE (0.90, 0.05, and 0), and the COMBO resulted in the most uniform production across the three targeted carotenoids (0.40, 0.15, and 0.50 mg/L). The COMBO treatment was used to radiolabel these carotenoids by the addition of 363 uCi U-14C-sucrose to 80 ml media inoculated with 4 ml of tomato cells with 8 ml spent media. Twelve labeled cultures were grown in an enclosed chamber system designed to aerate cultures 6x/d and trap 14CO2 in NaOH for the 14 d growth cycle. Of the initial 14C-sucrose dose provided, 20% accumulated in the cells, 52% remained in the media, and 16% was lost to respiration. Using the methods described here, herbicide treatments can be tailored for the targeted production of radiolabeled carotenoids. Labeled cultures will be extracted for 14C-carotenoids for use in carotenoid metabolism investigations. (Supported by NIH/NCI CA 112649-01A1)
Nancy J. Engelmann, Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801. In Vitro Cellular and Developmental Biology, 44:S75, 2008
