The major research goals for year one of this project can be divided in two sections, experimental and bioinformatics. The Noble Foundation Staff is responsible for the experimental portion of the project.

Experimental - (Noble Foundation)

• to define optimal conditions for elicitation of Medicago truncatula cell suspension cultures for production of phenylpropanoid and triterpenoid natural products
• to generate the large amounts of elicited cell culture material for parallel analysis of the transcriptome, proteome and metabolome
• to establish preliminary microarray capabilities to facilitate initial analyses of the M. truncatula transcriptome prior to the availability of the community unigene set
• to purchase and set up a Q-TOF mass spectrometry capability
• to optimize protocols for 2-D gel electrophoresis and peptide mass mapping of proteins from M. truncatula cell suspension cultures
• to optimize LC/MS and GC/MS- based metabolite profiling approaches for M. truncatula cell suspension cultures

These goals have been attained, in spite of a delay in hiring some of staff. Other personnel fully funded by The Noble Foundation helped in these activities.

Optimization of cell culture conditions (Dixon)

To optimize conditions for cell culture elicitation, we first analyzed a number of potential elicitor molecules with different M. truncatula cell culture lines. We found that root-derived cell suspension cultures of M. truncatula responded to both yeast elicitor (for induction of phenylpropanoid natural products) and to methyl jasmonate (MeJA) (for production of triterpene saponins). Concentrations of these elicitors were optimized for maximum response.

Cell culture elicitations (Dixon, May, Sumner)

Large-scale elicitations to provide the cell culture material for the data generation phase of the project have now been carried out and the material is stored in replicate batches in a number of -80oC freezers. For each elicitation condition (yeast elicitor, MeJA and high UV light) we have collected 30-40 g of elicited material (from three replicate treatments per time point) at a range of times post-elicitation as outlined in the original proposal (in fact adding two extra time points). An alteration from the proposal is that each elicitation was accompanied by a control cell culture, instead of a single control for all three elicitations. This results in a total of 6 time courses.

Preliminary microarray conditions (Dixon, May)

In order to be able to initate microarray analyses of the elicited material prior to the availability of the Medicago community unigene set, we have made DNA chips using selected 70 mer oligonucleotides corresponding to all known genes of the phenylpropanoid, flavonoid, isoflavonoid and lignin pathways in the Medicago EST collections, in addition to the entry point enzymes for triterpene synthesis and a range of other metabolic processes and controls. The Noble Foundation covered the cost of the 288 oligonucleotides made for us by Operon Technologies. Using the oligo arrays, we have generated preliminary data on the induction of known natural product pathway genes in response to a range of elicitors in M. truncatula cell suspension cultures.

Isolation of time-course RNA (May, Dixon)

The May and Dixon groups are isolating total RNA from the MeJa-, UV- and yeast- elicited time courses.  Details including absorbance, 260/280 ratios, concentrations and images of samples separated by agarose gel electrophoreses have been recorded and will be made readily accessible to the research community. 

A modification to the proposed research is the inclusion of a reference RNA source in the experimental design. Twenty milligrams of reference RNA was isolated from M. truncatula cell cultures. This amount of reference RNA is sufficient to twice replicate all time course experiments. Pair-wise comparisons of RNA from control and elicited time course samples will be made against the reference RNA. It is anticipated that this approach will better enable inter-time course comparisons (see Figure N)

Figure N. Schematic detailing elicited time course experimental parameters and expression profiling against a reference RNA for enabling inter-time course comparisons.

Generation of an M. truncatula 70-mer oligonucleotide microarrays (May)

M. truncatula genome-wide microarrays are being generated using the Medicago Array-Ready Oligonucleotide Set (GS-1700-02) Version 1.0 (Operon). A 384 M. truncatula oligonucleotide evaluation plate has also been made available. The development of these oligonucleotide sets was a collaborative effort between, Gregory May, Noble Foundation, Kate Vandenbosch, University of Minnesota, Chris Town, TIGR, scientists at Qiagen Operon, and members of the international Medicago research community.  

Approximately 16,000, amino-linked, 70-mer oligonucleotides are being printed onto aminosilane-coated ?Superamine? slides (Telechem), using Telechem type SMP3 printing pins in Dr. David Galbraith?s laboratory at the University of Arizona. Operon has agreed to update the Medicago oligonucleotide genome set as additional M. truncatula EST and genome sequence information becomes available. Supplemental oligonucleotide sets will be made available to researchers that purchase the Medicago Array-Ready Oligonucleotide Set Version 1.0. M. truncatula 16K oligonucleotide arrays have been made available to the research community on a collaborative basis through the Noble Foundation

Purchase and configuration of microarray software (May)

Hybridized microarrays are being scanned on a GSI Lumonics ScanArray 4000 two-color microarray analysis system.  Data analysis is being performed using Axon?s GenePix (4.1) and Iobion?s GeneTraffic Duo (Version 2.5) software. 

Optimization of microarray protocols (May)

Expression analysis protocols have been optimized. Briefly, 20 µg of total RNA are labeled using the Amino Allyl cDNA labeling kit (Ambion Inc, USA) according to the supplied protocol. The final labeled product is resuspended in 50 µL of SlideHyb #1 hybridization buffer (Ambion Inc, USA) and denatured at 95-100°C for two minutes. The denatured samples are pipetted onto the oligonucleotide microarrays and a coverslip (Corning, USA) is applied before sealing in a hybridization chamber (Corning, USA). Sealed chambers are wrapped in aluminum foil and incubated in a 42°C water bath for 16-24 hours. Arrays are subsequently washed with 1X SSC, 0.1% SDS, followed by a wash in 0.5X SSC, 0.01% (wt/vol) SDS, and a third wash with 0.05X SSC, at room temperature for five minutes each. Slides are dried by centrifugation and scanned.

Purchase of a QTOF mass spectrometer (Sumner)

An Q-Star Pulsar Mass Spectrometer with both ESI and MALDI ion sources was ordered from ABI and is currently being installed. This instrument is equipped with an LC-Packings HPLC and includes a remote processing computer for high throughput protein identifications. Installation is expected to be complete by June 31, 2002. This is an alteration from the original proposal, where it was stated that an equivalent instrument from Micromass would be purchased. Several criteria led to the choice of the ABI instrument:

i) the ABI instrument is capable of the same performance than the Micromass instrument as specified originally

ii) ABI added a number of features additional to the Micromass specification without increase of price

iii) Micromass recently lost a lawsuit to ABI which resulted in its QTOF instrument to have considerably reduced performance, compared with the ABI. Overall we purchased an instrument capable of all the features specified in the original proposal with a few extra capabilities (notably a MALDI source, allowing one to carry out MALDI-Q-TOF mass spectrometry of protein fragments).

Optimization of protocols for proteomics (Sumner)

A survey of the proteome of Medicago truncatula tissues by two-dimensional polyacrylamide gel electrophoresis (2-DE) was carried out. This survey included 2-DE protein profiling of specific tissues such as leaf, stem, root, flower, seed pod, and cell suspension cultures. As part of this survey, methods have been established for protein extraction, 2-DE, spot excision, in-gel digestion, and protein identification using peptide mass mapping. Preliminary experiments have also been conducted to quantify the level analytical and biological variance associated with 2-DE of Medicago truncatula to provide a statistical basic for the prediction of protein expression differences. Final experiments are now being performed to evaluate a large format (24cm x 24cm) 2-DE that should provide an enhanced separation and visualization of the proteins.

Optimization of protocols for metabolite profiling (Sumner, Smith)

The approach being utilized for metabolomics at The Noble Foundation includes multiple profiling technologies for a more complete visualization of the metabolome. The profiling technologies are GC/MS, LC/MS, CE/UV, and CE/MS. Currently GC/MS is being used for both polar extracts following derivatization and hydrophobic compounds following a single biphasic extraction. Capillary electrophoresis (CE) and CE/MS methods are also being developed by the Smith Group at Southeastern Oklahoma State University. Methods are being developed for carbohydrate analysis and also expanded to include anionic/cationic species and amino acids. These expanded regions will hopefully provide greater visualization of the metabolome, not originally included the proposed project. LC/MS is being utilized primarily to profile phenolic conjugates including flavonoids and saponins. Profiling of these conjugates is not possible by GC/MS. We have recently documented the use of LC/MS for saponin profiling and identification as standards for this class of compounds are not available commercially.

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