2020 Spring.Includes bibliographical references.The growing world population has been putting considerable strain on energy and food demands. To address the ever-growing need to meet these demands and service the global populous, emphasis has been placed on developing new methods to generate additional fuel and food. Plants play a unique role in this challenge, as they offer a means to create sustainable sources of energy as well as provide a source of food. It is important to investigate ways to increase plant productivity so that society can dually and effectively address these needs. Plant material can be converted into numerous combustible fuel sources. High lignin content in plant secondary cell walls is desirable for thermochemical conversion, while low lignin content is more advantageous for enzymatic approaches targeting cellulose embedded in the cell wall matrix - both are substrates for converting biomass to energy. As essential building blocks of plant cell walls, increasing plant biomass fundamentally increases the energy stored in plant tissues. Our original approach to increase biomass focused on manipulation of the source-to-sink transport of carbon. More specifically, we aimed to increase plant biomass by engineering a transfer DNA (T-DNA) expression cassette that drives the overexpression of sucrose transport in rice phloem. The hypothesis simply suggested transporting more carbon (sucrose) from leaf tissue to heterotrophic tissues in the plant would increase the biomass of individual plants. Rice was used in these experiments because it is a model grass and staple crop, and is also a useful source for translational biology because it is closely related to bioenergy feedstocks such as sorghum, switchgrass, and Miscanthus. Through the screening of a large population of transgenic rice plants, we discovered a single plant that was significantly larger than the wild-type control. Further investigation revealed that this transgenic line not only showed an increase in biomass but also exhibited an increase in seed yield as well. Additionally, the extent of growth enhancement varied in the presence of stress, where this plant yielded higher biomass than wild-type plants under various stressors. This rice was more robust during optimal conditions, and even more so during stressed conditions compared to wild-type plants. Sequencing the DNA region around and including the T-DNA insertion event in this plant revealed that only a portion of the expression cassette was successfully inserted. Remarkably, the insertion did not contain the sucrose transporter gene that had been engineered into the cassette. Thus, the phenotype of the transgenic plant is not the result of the expressed transgene. Because integration of T-DNA into a chromosome can be a mutagenic event, we hypothesized that the insertion might have altered the expression of a nearby gene(s) that is responsible for the increased biomass, seed yield, and stress tolerance phenotype. In support of this hypothesis, we showed that the expression cassette that inserted in the genome (monitored through molecular analysis of the insertion site) segregated with the phenotype across multiple generations. Due to the increase in biomass we refer to this mutant as mpg1 (makes plants gigantic-1). Examining the expression of neighboring genes via semi-quantitative RT-PCR we discovered that one gene, a transcription factor from the APETALA 2-Ethylene-Responsive Element-Binding Protein (AP2/EREBP) transcription factor superfamily, has markedly increased expression in mpg1 compared to wild-type plants. This transcription factor belongs specifically to the AP2/ERF subfamily, which members have been shown to play a role in growth, development, and stress response. The mpg1 plants exhibit a pleiotropic phenotype consisting of greater plant height, larger stems, larger leaves, increased seed yield, delay in flowering, enhanced ratooned growth, and degrees of stress tolerance compared to wild-type plants. Transcriptomic analysis of homozygous mpg1 and wild-type null segregants taken during the vegetative growth period prior to and during our ability to measure the biomass increases revealed a large-scale difference in gene expression from numerous genes that play roles in transcription factor activity, flower development, response to stress, DNA metabolism, cell cycle, defoliation response, cell wall metabolism, and hormone regulation. Identification of the mechanism(s) responsible for the increased biomass, seed yield, and degrees of stress tolerance may lead to strategies that could be applied to other plants to aid in both energy and food security alike
