Transcriptome analysis of embryo maturation in maize.

BACKGROUND: Maize is one of the most important crops in the world. With the exponentially increasing population and the need for ever increased food and feed production, an increased yield of maize grain (as well as rice, wheat and other grains) will be critical. Maize grain development is understood from the perspective of morphology, hormone responses, and storage reserve accumulation. This includes various studies on gene expression during embryo development and maturation but a global study of gene expression of the embryo has not been possible until recently. Transcriptome analysis is a powerful new tool that can be used to understand the genetic basis of embryo maturation. RESULTS: We undertook a transcriptomic analysis of normal maturing embryos at 15, 21 and 27 days after pollination (DAP), of one elite maize germplasm line that was utilized in crosses to transgenic plants. More than 19,000 genes were analyzed by this method and the challenge was to select subsets of genes that are vitally important to embryo development and maturation for the initial analysis. We describe the changes in expression for genes relating to primary metabolic pathways, DNA synthesis, late embryogenesis proteins and embryo storage proteins, shown through transcriptome analysis and confirmed levels of transcription for some genes in the transcriptome using qRT-PCR. CONCLUSIONS: Numerous genes involved in embryo maturation have been identified, many of which show changes in expression level during the progression from 15 to 27 DAP. An expected array of genes involved in primary metabolism was identified. Moreover, more than 30% of transcripts represented un-annotated genes, leaving many functions to be discovered. Of particular interest are the storage protein genes, globulin-1, globulin-2 and an unidentified cupin family gene. When expressing foreign proteins in maize, the globulin-1 promoter is most often used, but this cupin family gene has much higher expression and may be a better candidate for foreign gene expression in maize embryos. Results such as these allow identification of candidate genes and promoters that may not otherwise be available for use. mRNA seq data archived in NCBI SRA; Accession number: ACC=SRA060791 subid=108584.

Heterologous expression of cellobiohydrolase II (Cel6A) in maize endosperm.

The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

Manipulating corn germplasm to increase recombinant protein accumulation.

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.