PI: Timothy Nelson, Yale University Co-PIs: Thomas Brutnell, Boyce Thompson Institute; Klaas van Wijk, Cornell University; Robert Turgeon, Cornell University; Qi Sun, Cornell University; Peng Liu, Iowa State University
C4-type plants such as maize, sorghum and several promising biofuel feedstocks possess a set of complex traits that greatly enhance their efficiency of carbon-fixation, water and nitrogen use, and performance in high temperatures and light intensities, in comparison to C3-type plants such as rice and many temperate grasses. The key C4 traits are (1) specialization and cooperation of two leaf photosynthetic cell types (mesophyll and bundle sheath) for carbon fixation and photosynthesis, (2) enhanced movement of metabolites between cooperating cells, and (3) very high density of leaf venation. These C4 traits appear to be regulatory enhancements of features already present in less-efficient C3 plants, but regulated in different patterns. Although C4 plants have evolved at least 50 times independently in various taxonomic groups, the molecular basis of key C4 traits is insufficiently understood to permit their introduction into important C3 plants to enhance their performance as agricultural or biofuel feedstocks.
This project will compare the leaves of rice (a C3 grass), maize (a moderate C4 grass) and sorghum (an extreme C4 grass). The abundance and spectrum of gene transcripts, proteins and metabolites will be compared along a developmental gradient from immature tissues at leaf base to mature tissues at the leaf tip. To align the gradients of the three species, markers for developmental time points in gene expression, protein accumulation, sink-source transition and cell wall specialization will be employed. Mesophyll and bundle sheath cells will be obtained from each leaf stage by laser microdissection, and their whole genome RNA transcripts, proteomes (including modifications), and selected metabolites (related to photosynthesis) will be profiled and compared. Two hypotheses will be tested by the comparative analysis of the corresponding C3 and C4 plant datasets: (1) To produce C4 traits, plants use networks of genes, proteins, and metabolites that are already present in C3 plants, and (2) C4 features are plastic and expressed in a degree that depends on environment and developmental stage. This analysis should identify the regulatory points that are potential targets for the production of C4 traits in C3 species.
Established and successful summer internship programs (http://bti.cornell.edu/pgrp/, www.yale.edu/stars/) will be used to involve undergraduates from under-represented groups in this research. PI/Co-PIs will also participate in curriculum development workshops for high school teachers. Teachers from New York and Connecticut will participate in a one-week program of lectures and discussions to introduce them to plant genomics tools and technologies. These teachers will work with plant science faculty on the Cornell campus to develop genomics-based modules for the classroom. Targeting high school teachers, rather than the students themselves, will greatly extend the reach of this program and will fulfill a primary mission of the NSF: to communicate the significance of the outcomes of plant genome research to society.
The project outcomes will be available through a project-specific public website (C3-C4DB, http://c3c4.tc.cornell.edu), and curated into the Gramene public database (www.gramene.org). Data will also be deposited at NCBI www.ncbi.nlm.nih.gov/ and EBI www.ebi.ac.uk/. Teaching modules developed at Cornell will be available through http://bti.cornell.edu/pgrp/pgrp.php?id=601.
Project Outcomes The introduction of C4 traits into conventional crops such as rice and wheat could have a positive impact on the efficiency of water, nitrogen and land use for agriculture, increasing yield of some crops by as much as 50%. The enhanced-efficiency C4 suite of traits includes (1) high efficiency C4-type photosynthesis, (2) increased number of leaf veins, (3) increased pores between cooperating photosynthetic cells, and (4) addition of a protective gas-impermeable cell wall layer for photosynthetic cells. The fact that this C4 suite has evolved independently in many plant groups suggests that it is the result of optimizing features of anatomy and biochemistry that preexist in all plants. To understand the mechanistic basis for C4 traits well enough to improve other plants, we designed a systems approach to evaluate the contributions of individual RNAs, proteins, metabolites and other molecules as the C4 traits are formed during leaf development. To compare progressive stages of development with and without the C4 scheme, we inventoried these molecules from the immature base to the mature tip of young leaves of maize (a C4 plant) and rice (non-C4). We also developed computational tools for integrating, viewing and analyzing the rich datasets of specific gene expression and protein accumulation that we obtained from each successive point in development. These new information resources for leaf development can now support gene discovery, regulation, and systems studies of any leaf biology feature. There are several key outcomes from this project: 1. Confirmed the hypothesis that C4 biology and anatomy is produced by redirecting and adapting resources from gene and protein networks also present in C3 species. This conclusion enables the general strategy of improving the efficiency of numerous C3 crops, such as rice, wheat and soybean, by re-regulating the existing genetic resources and pathways they already possess. 2. Obtained deep datasets of transcriptomes (genome-wide RNA inventories) and proteomes (genome-wide protein inventories) for progressive stages in the development of a C4 and a non-C4 leaf. 3. Developed computational and data-viewing resources that enable the rich datasets from developing leaves to enable a "systems" approach to any problem in leaf biology. 4. C4 biology: Molecular description of the sequence of events in leaf development, from building the infrastructure of cell types and anatomical features, through building of specialized chloroplasts and finally to the acquiring of functional C4 traits. See Figure 1 for depiction of these overlapping events. 5. C4 biology: tests of models. Descriptive molecular inventories from this project enable tests of models for pathways of gene regulation and metabolic regulation that explain the special efficiency of the C4 system. 6. Publications and other outputs. 14 publications, two of which present the core datasets and analysis. Additional manuscripts on rice developmental transcriptomes and proteomes, on maize-rice comparisons, on cell-specific cell transcriptomes and on developmental analysis of C4 metabolites are in preparation. In addition to sharing with community data portals, we share data and analytical tools on our C4 project website (C3-C4DB, http://c3c4.tc.cornell.edu). 7. Training: This project was the vehicle for research training of 3 undergraduates, 3 graduate students and 13 postdoctoral scientists. We also present a Bioinformatics workshop to teach the analysis of gene expression with NextGen methods, using our project data and computational resources. 8. Outreach. We developed several programs to communicate to a broader audience the importance of plant biology to economy, environment and health. Included were undergraduate research internships, a week-long summer workshop for high school teachers to introduce students to cutting-edge plant science research (www.bti.cornell.edu/educationTeacherPrograms.php), and the BrachyBio! distributed mutant-screening network, in which new mutations in the grass Brachypodium are supplied to high school students around the US for trait-screening, gene-mapping and eventual cloning.
