Performing Department
Molecular Biology and Microbiology
Non Technical Summary
Most of the investigators utilize the tobacco chloroplast genome sequence published in 1986. Several recent publications question the validity of using heterologous sequences to investigate basic concepts in other plant species. Although only six crop chloroplast genomes were sequenced until 2004, some progress has been made recently. While several hundred non-crop chloroplast genomes have been sequenced for phylogenetic purposes supported by NSF, no federally funded project has been funded for sequencing crop chloroplast genomes. The Daniell laboratory has sequenced >20 crop chloroplast genomes in the past few years, including very important crops used in daily life: cotton, soybean, grape, orange, coffee, carrot, potato, tomato, cassava, cereals, etc. More than hundred foreign genes have been engineered via the tobacco chloroplast genome and demonstrated to be exceptionally functional, with highest levels of resistance to insects, diseases, drought or salt tolerance or phytoremediaion or production of human therapeutic proteins. However, useful agronomic traits have been introduced in only three crop species. In this proposal, we critically examine reasons for inadequate transfer of valuable concepts from tobacco to economically important crops and propose solutions. Inadequate understanding of basic concepts of foreign regulatory and recombination sequences has delayed transfer of research from model system to useful crops. Therefore, in this proposal, three economically important tree chloroplast genomes (chestnut, peach, cacao) will be sequenced and deposited into the public data base. The US Congress "Energy Independence and Security Act of 2007" set the goal for annual production of 16 billion gallons of cellulosic ethanol by 2022 and energy from renewable resources is one among the highest priorities of the Obama administration. European Union's requirement that 10% of all transport fuels come from renewable sources makes this a global challenge. About 145 billion gallons of gasoline are used in the US each year. All cars are E10 compatible. A blend of 10% ethanol equates to 14.5 billion gallons requirement per year. In the US, 25-30% of corn production is currently used for ethanol production, raising prices of corn and other food/feed sources. Therefore, renewable cellulosic biomass from agricultural waste is a better feedstock for biofuel production. There are two major hurdles in production of cellulosic ethanol: availability of adequate biomass and low cost enzymes to convert biomass into fermentable sugars. This proposal addresses both these concerns. Hormones play an important regulatory role in plant growth and development. Most of the steps in hormone biosynthesis and metabolism are irreversible except for the formation of inactive conjugates which can release active hormones by enzymatic hydrolysis. In this project, we propose to double the biomass by producing a biomass hydrolyzing enzyme in chloroplasts or vacuoles by releasing growth hormones stored within these cellular compartments. Results of these investigations will lead to enhanced biomass production and hydrolysis.
Animal Health Component
(N/A)
Research Effort Categories
Basic
65%
Applied
25%
Developmental
10%
Goals / Objectives
Chloroplast genetic engineering has conferred valuable agronomic traits and facilitated expression of human therapeutic proteins in tobacco but this concept has not yet been advanced to economically important crops. Lack of chloroplast genome sequence information for transgene integration/expression/recombination and inadequate tissue culture/selection procedures are among the two major limitations. While several hundred non-crop chloroplast genomes have been sequenced for phylogenetic purposes, no federally funded project has focused on sequencing crop chloroplast genomes. Therefore, in this proposal, three economically important tree chloroplast genomes (chestnut, peach, cacao) will be sequenced. Hormones play an important regulatory role in plant growth and development. Most of the steps in hormone biosynthesis and metabolism are irreversible except for the formation of inactive conjugates which can release active hormones by enzymatic hydrolysis. In this project, we propose to investigate the mechanism of hormone conjugation in chloroplasts or vacuoles. Results of these investigations will lead to enhanced biomass production and hydrolysis.
Project Methods
Chloroplast genomes of chestnut (Castanea mollissima), cacao (Theobroma cacao) and peach (Prunus persica), will be sequenced, annotated and deposited into GenBank and results will be published in peer reviewed journals. The tobacco or lettuce chloroplast genome will be transformed with Bgl-1 gene (from Trichoderma reesei). The nuclear genome will be transformed with Bgl-1 gene by targeting β-glucosidase to the vacuole, using three different vacuolar targeting sequences: CTPP of ConA, Chitinase A and NTPP of sporamin. Control and transplastomic or transgenic lines will be evaluated for biomass, height, leaf area, trichome density, release of plant hormones from their conjugates stored within chloroplasts or vacuole (GA53, GA44, GA19 and GA20, Zeatin, IAA) and culture protoplasts with or without exogenously supplied hormones or their conjugates. Investigate protection against insects conferred by released glucosides or sucrose esters.