Engineering CAM Photosynthetic Machinery into Bioenergy Crops for Biofuels Production in Marginal Environments
CAM provides an excellent opportunity to engineer enhanced photosynthetic performance and water-use efficiency (WUE) into bioenergy crops. Our vision was to understand the enzymatic and regulatory pathways required for CAM in order to engineer CAM photosynthetic machinery into Arabidopsis and Populus. Major advances were made in understanding the molecular “parts list” necessary for engineering CAM through the sequencing the genomes and transcriptomes of several model and agronomically important CAM species including Agave, Ananas, Clusia, Kalanchoe, Mesembryanthemum, and Opuntia. Comparison of diel co-expression network analyses in Agave, Ananas, and Kalanchoe with expression patterns in C3 photosynthesis species revealed the rescheduling of gene expression associated with stomatal opening and closing and led to the identification of many candidate CAM-related genes for further functional testing. Both RNAi-mediated gene silencing and CRISPR/Cas genome editing for gene knockout were used to investigate over 70 CAM-related enzymes, regulatory kinases/phosphatases, and transporters and 25 transcription factors implicated in playing key regulatory roles in CAM. Knockdown of key C4 cycle enzymes within the carboxylation and decarboxylation modules of CAM not only perturbed CAM itself, but also altered the expression of core circadian clock components demonstrating that loss of CAM function also perturbs the central circadian clock and alters the regulatory patterns of key guard cell signaling genes that are linked with the characteristic inverse pattern of stomatal opening and closing during CAM. An advanced system for joining D.AN fragments from a universal library was developed that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, regions of sequence homology, amplification (or mutation for domestication) of fragments to function systems for the construction of gene circuits. Alternatively, Gibson assembly was used to create gene circuits consisting of up to 15 unique gene expression cassettes expressing either the core C4 cycle of CAM comprising carboxylation or decarboxylation modules or both modules together. The expression of each gene cassette was driven by a drought-stress inducible 5’ regulatory region or promoter (and cognate 3’ terminator region) from A. thaliana so that CAM gene expression would be enhanced under conditions of water-deficit stress. These gene circuits were introduced into the C3 photosynthesis model species A. thaliana. Expression of the nocturnal carboxylation module resulted in increased biomass and seed yield arising from net CO2 fixation at night and malate accumulation typical of a CAM plant along with daytime CO2 fixation typical of a C3 photosynthesis plant. To create a leaf anatomy suited for the optimal performance of CAM in a C3 photosynthesis plant, increased tissue succulence was engineered in A. thaliana (and Nicotiana sylvestris), which also improved biomass and seed yield and drought attenuation and improved salinity tolerance. The basic CAM Biodesign principles described in this report can be extended to increase the productivity and WUE of food and bioenergy crops and are expected to have broad applicability and ensure sustainable biofuel feedstock production in the face of the global climate crisis.
- Research Organization:
- Univ. of Nevada, Reno, NV (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Contributing Organization:
- University of Newcastle; University of Tennessee, Knoxville
- DOE Contract Number:
- SC0008834; FOA-0000640; 11099732
- OSTI ID:
- 1576592
- Report Number(s):
- DOE-UNR-ORNL-SC0008834
- Country of Publication:
- United States
- Language:
- English
Similar Records
Transcript, protein and metabolite temporal dynamics in the CAM plant Agave
The pineapple genome and the evolution of CAM photosynthesis