Almost all plants fix CO2 into carbohydrate using an ancient photosynthetic process termed C3 photosynthesis (i.e., C3 plants). A subset of plants utilize a more efficient photosynthetic process termed C4 photosynthesis (i.e., C4 plants). In open sunny environments the rate of CO2 fixation, biomass accumulation, and grain yield in C4 plants is nearly twice that of C3 plants. The increased photosynthetic performance of C4 versus C3 plants is due in part to the evolution of an additional set of photosynthetic CO2 fixation enzymes referred to as the C4 pathway. Despite the inherently higher productivity of C4 versus C3 plants, there are only four agronomically important C4 crop species: maize, Sorghum, sugar cane, and switch grass. All other crop species (e.g., rice, soybean, wheat) are C3 plants. Thus an ongoing research goal of plant scientists is to breed or engineer C4 photosynthesis into C3 crop plants for improving yields. Efforts to date have fallen short in part because of the lack of understanding on how the C4 pathway is controlled in leaves for optimizing use of available sunlight. The ultimate aim of this project is to elucidate how such regulation works. A first step in this endeavor, and the goal of this one-year project, is to develop maize plants that are deficient in two interrelated light-responsive C4 pathway enzymes, pyruvate phosphate dikinase (PPDK) and the PPDK regulatory protein (RP). It is hypothesized that these enzymes control the rate of the CO2 fixation by the C4 pathway in response to sunlight. Using RNA interference, a genetic engineering method, stable lines of maize plants will be isolated with reduced levels of PPDK or RP enzyme. These plants will be subsequently assessed for photosynthetic performance. From these analyses, an accurate picture of how PPDK and RP confer sunlight-responsive C4 pathway regulation is expected to emerge. This knowledge will be critical for projects now underway that seek to introduce a functional C4 pathway into leaves of C3 crop species. A major education and training component of the project is the participation of undergraduate Biochemistry/Biotechnology degree students in fulfillment of their senior thesis research requirement.
C4 photosynthesis (C4 PS) is a recently evolved form of photosynthesis (~30 million BCE) found in a minority of terrestrial plant species. Plants that fix CO2 using C4 photosynthesis (C4 plants) are also inherently more productive in terms of biomass accumulation and grain yield. However, only three crop species, maize, Sorghum, and sugarcane, are C4 plants. All other crop species including rice, wheat, and soybeans, use the less efficient C3 photosynthesis (C3 plants). Because of the inherently higher productivity of crop plants that use C4 PS, government and private initiatives are underway to bioengineer this process into rice, a C3 crop species. For such applications to be realized, a few remaining details underpinning the C4 PS process must first be elucidated. This project was initiated to elucidate one of these details, namely how variation in light intensity incident on C4 plant leaves controls the rate of CO2 fixation into the C4 fixation pathway. One of the suspected components of this light-control mechanism, pyruvate phosphate dikinase (PPDK), an enzyme in the C4 fixation pathway, was the focus this project. The rate of PPDK is known to be proportionally increased or decreased in response to light intensity. Another enzyme, the PPDK regulatory protein (PDRP), changes the rate of PPDK in response to light variation by reversibly modifying the enzyme's structure thus serving as the light-intensity sensor for PPDK. While the relationship of PPDK and PDRP has been deduced in vitro, just how vital these two enzymes are to the overall C4 PS light-control mechanism in the intact leaf is unknown. This project sought to resolve this question by developing a set of transgenic maize lines deficient in PDRP and PPDK. In turn, the seed lines from these maize plants will serve as the basis for a subsequent proposal that will deduce the relevance of PPDK and PDRP to the C4 PS light-control mechanism in intact leaves using advanced physiological methods. To this end, 33 transgenic maize lines deficient in PDRP were isolated during the project's time. Thirteen of these were shown to have a greater than 50% reduction in leaf PDRP enzyme. Another 19 transgenic PPDK mutant maize lines were isolated but these have yet to be assessed for reduction of PPDK enzyme. Preliminary observations of phenotype of PDRP-deficient and candidate PPDK-deficient maize plants were also made. The most common phenotype exhibited by these plants was a progressive loss of chlorophyll in leaves and overall loss of vigor. These preliminary observations are consistent with the hypothesis that PDRP regulation of PPDK in leaves is required for robust CO2 fixation by the C4 PS process. Another significant outcome of the project is in the education and training of undergraduate students. During the life of the grant, ten undergraduate students participated in the project's research. Eight of the students used their research to fulfill their senior-thesis research requirement for the B.S. degree in Biochemistry and Biotechnology. Of those students who have graduated, three have entered Ph.D. programs in biochemistry or molecular biology, one is in an M.D.-Ph.D. program, with the remainder employed in the health or biotechnology industry. *"This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the
