Progress 10/01/07 to 09/30/10

Outputs
OUTPUTS: During the time period covered by this report, activities once again consist primarily of experiments conducted by research associate Dr. Huijun Yang whose salary was in part supported by this grant. The main goal of this project was to examine the production of foreign proteins in plants as an alternative to scale-up production in microbial fermentation systems. We are mostly using tobacco as a model plant as its chloroplast genome is easily transformed, but the research will be generally applicable to other plant species and algae. Indeed, Dr. Yang has generated the necessary tools to insert new gene sequences into two algae species and has begun to generate a collection of mutants for future work on algae projects. Growth and extraction of lipids from algae is viewed as a potentially important technology for biofuel production. Production of high value co-products in algae would improve the economics of biofuel production. Another output of our work during this period was the training of undergraduate students. Dr. Yang worked with three undergraduates Stefan Engst, Michael Melfi and Christine Curtis. Two of these students had no laboratory experience prior to working with Dr. Yang and both were working performing experiments fairly independently by the time they finished working on their projects. Stefan and Christine were trained to assay the activity of the enzymes being produced in the plant tissues and both were trained to perform immunoblots to measure the abundance of a specific protein. Michael has been working with Dr. Yang on the algae project. PARTICIPANTS: PI Ahner worked collaboratively with Research Associate Dr. Huijun Yang to perform experiments described in the summaries and to train undergraduate students to assist with experiments. TARGET AUDIENCES: The target audience for this research project is the scientific community interested in plant biotechnology and biotechnology companies that are seeking to improve enzyme production for biofuel production. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
All of the experiments were performed by Dr. Yang, who was partially funded by this Hatch grant, or were performed by my undergraduate students who were trained by Dr. Yang. Materials and supplies for performing the experiments were also purchased with funds from the grant. Experiments focused on the effects of nitrogen availability on the production of a foreign protein in two separate tobacco lines. Both of these new lines contain significantly more foreign protein than the GFP-containing tobacco (GFP stands for green fluorescent protein) that we originally proposed to use. In one tobacco line, we are able to measure high levels of the bacterial cellulase Cel6A, an enzyme which cleaves cellulose polymers into short glucose chains. In the other plant line we are able to measure high levels of the bacterial beta-glucosidase BglC, an enzyme which cleaves cellobiose into two glucose molecules. For both plant lines we are able to quantify the protein with two methods, including immunoblots and enzyme assays. The two measurement techniques yield statistically similar results suggesting that all or nearly all of the protein expressed by the transgenic plant is properly folded and active. As of the submission of this report, we have a paper that describes the BglC expressing plant lines "in press" at Plant Molecular Biology. Both of these enzymes are needed for the conversion of biomass (cellulose) into sugars (glucose), and then the sugars can transformed into biofuel. In order to test the effect of nitrogen availability on foreign protein expression, tobacco seeds from both plant lines (NPTII-BglC or TetC-Cel6A) were sewed into soil (Metromix 360). They were allowed to germinate and then were grown for two weeks in plant growth chamber. After two weeks, seedlings were rinsed gently with water to remove soil attached to the roots, and then were transferred to vermiculite watered with nutrient solution containing different nitrogen levels (a normal concentration typical of a model hydroponic solution, double normal concentration and with no added nitrogen). Seedlings were watered with different nutrient solutions and were grown for another two weeks. One leaf from each plant was collected and three or more leaves were pooled together as one sample. Our experiments revealed that while nitrogen availability had a significant effect on the growth of the seedlings and total protein in the seedling tissue, it did not alter the amount of foreign transgenic protein relative to the total extractable protein. This result is contrary to results published by others on transgenic tobacco seedlings in which the amount of transgenic protein remained high while total protein decreased. In these plants, which were expressing different proteins inserted at a different location in the chloroplast genome, the percent of foreign protein increased as the plants became more nitrogen limited. With our transgenic plants the percent of foreign protein remained constant with decreasing nitrogen. These results will be included in a publication that will be submitted to a peer-reviewed journal.

Publications

• No publications reported this period

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: During the time period covered by this report, activities once again consist primarily of experiments conducted by research associate Dr. Huijun Yang whose salary was in part supported by this grant and by graduate student Benjamin Gray. The main goal of this project is to examine the production of foreign proteins in plants as an alternative to scale-up production in microbial fermentation systems. We are using tobacco as a model plant as its chloroplast genome is easily transformed, but the research will be generally applicable to other plant species. We have continued our experimentation with tobacco chloroplast-transformants that contain the green fluorescent protein (GFP; a marker protein that is easily measured) that we obtained from Dr. Maureen Hanson's laboratory (line MR220 cv Petite Havana). We originally proposed to grow these GFP-expressing plants under constant light and temperature conditions and then to manipulate the composition of the hydroponic growth medium. In particular we proposed to alter the nitrogen content during a time course to see if we could stimulate the production of foreign protein. We also proposed to manipulate the levels of atmospheric carbon dioxide in the plant growth chamber with the aim of reducing the plant requirement for Rubisco, the most abundant protein in plant tissue, and therefore making additional nitrogen-containing protein building blocks available for foreign protein expression. These experiments were delayed during the time period of the proposal as we were waiting for Riley-Robb growth chambers to be available for experiments. During this time period instead we experimented with the cytokinin kinetin to see if this plant hormone could be used to maintain high levels of expressed foreign proteins in plant leaves of varying maturity since we and others have observed decreased protein stability in maturing leaves with some proteins. Both light and dark incubations were performed with leaf discs that had been suspended in buffered water containing 0, 1 and 10 micromolar kinetin for up to five days. Foreign protein levels were measured as a function of time. We used both GFP expressing line MR220 and 22XE2, a transformed tobacco line expressing the bacterial cellulase Cel6A at relatively low levels. We have also worked with new lines of plants as they have been developed in our collaborator's laboratory. These new lines contain significantly more foreign protein than the GFP-containing tobacco that we originally proposed to use. We have measured protein levels in plants that contain the bacterial cellulase Cel6A (several new lines in addition to 22XE2 mention above) and the bacterial beta-glucosidase BglC. We have developed experimental strategies for sampling and assaying the enzymes in these plants in preparation for when the growth chamber experiments are to be performed. PARTICIPANTS: Research associate Dr. Huijun Yang and fifth year graduate student Benjamin Gray worked on this project. Gray completed his PhD in June 2009. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The experiments funded by this grant during the time period covered and described in this report have resulted in a few interesting and publishable findings. In order for transgenic plants to be optimized for foreign protein expression we must understand how various external environmental variables and or chemical enhancers will influence protein accumulation. During the time period of this report we investigated the effects of one such chemical enhancer. In this set of experiments we used leaf tissues from two different transgenic lines expressing two entirely different proteins- GFP and Cel6A, the former being very stable in as leaves mature and the latter decreasing significantly as a percent of the total protein as the leaves mature. We found that low concentrations of the plant cytokinin kinetin can delay protein degradation in floating leaf discs for up to five hours whereas high concentrations accelerate the loss of protein compared to controls where no kinetin is added. These experiments will be repeated with new transgenic plant tissues and the effect of in vivo kinetin application will also be explored. Graduate student Benjamin Gray completed his thesis and received his Ph.D. in June 2009.

Publications

• No publications reported this period

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Activities consist primarily of experiments conducted by the research associate Dr. Huijun Yang and graduate student Benjamin Gray who have been supported by this grant with materials and supplies during the period of the grant. The main goal of our project is to examine the production of foreign proteins in plants as an alternative to production in microbial fermentation systems. We are using tobacco as a model plant as its chloroplast genome is easily transformed, but the research will be generally applicable to other plant species. We have done our initial experiments with tobacco chloroplast-transformants that contain the green fluorescent protein (GFP; a marker protein that is easily measured) that we obtained from Dr. Maureen Hanson's laboratory (MR220 cv Petite Havana). We proposed to grow these GFP-expressing plants under constant light and temperature conditions and then to manipulate the composition of the hydroponic growth medium. In particular we proposed to alter the nitrogen content during a time course to see if we could stimulate the production of foreign protein. We also proposed to manipulate the levels of atmospheric carbon dioxide in the plant growth chamber with the aim of reducing the plant requirement for Rubisco, the most abundant protein in plant tissue, and therefore making additional nitrogen-containing protein building blocks available for foreign protein expression. Initial experiments with nitrogen removal at various time intervals followed by various intervals of nitrogen re-supply have yielded results that are fairly ambiguous and we found a great deal of variability among replicates. Plants that experienced nitrogen removal have measurably less biomass than those receiving full nitrogen throughout the experiment, therefore small increases in the amount of foreign protein that may be affected by this particular nutrient manipulation may be outweighed by a decrease in total biomass. These experiments are being repeated to clarify our results. We are making significant progress toward the goals established in the original proposal. We are also beginning experiments with plants that contain the bacterial cellulase Cel6A since the levels of foreign protein produced in some of these newer plant lines exceeded those of the GFP-containing tobacco that we originally proposed to use. This plant material is also available from a collaboration with Dr. Hanson. The highest of these is a plant that expresses the cellulase with a particular short peptide on the beginning of the protein. We have determined that the activity of this enzyme is unaltered due to this fusion and that the level of protein appears to peak in mature leaves with only a slight decrease as the leaves become senescent. Experiments in greenhouses under various conditions of temperature and light with this plant line are underway. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have just completed the first year of this project therefore we have not yet entirely achieved our proposed outcomes. We are making significant progress toward our goals and have altered experimental design as described above as we analyze the data from initial experiments. We have established a collaboration with the NYS company CEA Systems together with other researchers here at Cornell and expect that our results will inform how their projects proceed.

Publications

• No publications reported this period