RESEARCH PLAN

Agave americana Mesembryanthemum crystallinum Opuntia ficus-indica

First, necessary omics data (e.g., RNA-Seq, DNA-Seq, ChIP-Seq and metabolite) will be generated in Aim 1 to support CAM gene module discovery in Aim 2 by constructing co-expression networks and gene regulatory models further refined by protein structure and molecular dynamic simulations. The gene network modeling will be evaluated with RNAi knockdown loss-of-function lines and transient expression analysis of protein-protein and protein-DNA interactions. The verified CAM gene modules will then be used in Aim 3 to engineer Arabidsopsis and Populus plants for drought-inducible CAM expression. Finally, the transgenic plants will be characterized in Aim 4 for aspects of gene expression, CAM biochemistry/physiology, photosynthesis, WUE, and biomass productivity.

Aim 1. Integrated omics data generation and primary analysis

The goal of Aim 1 is to generate and perform primary analysis of the necessary data to facilitate predictions on the enzymatic and regulatory requirements for CAM performance. The successful engineering CAM into a C3 species will likely require the design and transfer of fully functional modules that permit the temporal separation of carboxylation and decarboxylation processes, along with a day/night pattern of stomatal closure/opening. Therefore, experimentation and data collection will be conducted in a temporal and tissue-specific manner (e.g., epidermal peels, mesophyll) to generate the regulatory networks and tissue comparisons necessary for accurate module design predictions. Analysis of both mesophyll and guard cell types will be essential to identify regulatory controls responsible for the inverse pattern of stomatal regulation in CAM species as there is currently uncertainty as to the requirements for proper stomatal regulation.

Aim 2. Integrative characterization of CAM modules

The goal of this aim is to use the data from Aim 1 for bioinformatic workflows and modeling endeavors to aid in the design and preliminary corroboration of carboxylation, decarboxylation, and stomatal modules. Successful module design will take into account the enzymatic and regulatory requirements for CAM operation. Multiple modeling services and analyses of comparative genomics, co- expression networks, comparative networks, gene regulatory networks, protein structure predictions, and molecular dynamic simulations, together with the expertise of CAM physiologists (Borland Lab), biochemists (Cushman Lab) and molecular biologists (Hartwell Lab), will be integrated during the module design process. The temporal and tissue-specific conditions in which Aim 1 data is collected will allow us to model the unique behavior of CAM physiology and thereby aid in module design.

Aim 3. Engineering of CAM Modules into Arabidsopsis and Populus

Current genetic engineering approaches can either transfer a limited number of genes (fewer than 10) at a time or stack genes one-by-one via time-consuming sequential transformation. The goal of this aim is to develop new technologies for CAM pathway engineering by integration of multigene transformation vectors and site-specific stacking of the gene modules. Our approach is to assemble CAM gene modules using an iterative cloning system and to stack the three CAM modules into a predefined single locus of the target plant genome including Arabidsopsis as a simple, readily transformable model, and Populus as a dedicated bioenergy crop.

Aim 4. Phenotypic Characterization of Transgenic Modules

The primary goal is to identify transformants with a CAM-like phenotype and the secondary goal is to establish whether introducing a CAM-like phenotype into Populus maintains productivity while improving WUE. The rationale here is to characterize the physiology of C3 plants engineered with specific CAM carboxylation and decarboxylation modules and that possess a CAM-like phenotype as evidenced by nocturnal acid accumulation, nocturnal net CO2 uptake, and daytime stomatal closure.