Location: Global Change and Photosynthesis Research
Project Number: 5012-21000-030-040-C
Project Type: Cooperative Research and Development Agreement
Start Date: Sep 1, 2022
End Date: Mar 31, 2023
Objective:
1. Reduce the cost of photorespiration.
a. Optimize bypass pathways.
b. Design novel pathways.
c. Down-regulate native pathway.
d. Improve the native photorespiratory pathway.
e. Field test most promising transgenic lines.
f. Incorporate successful synthetic bypass pathways into rice, cowpea and cassava.
2. Improve crop canopy light distribution.
a. Achieve greater reductions in leaf chlorophyll.
b. Characterize the resulting plants from canopy alteration to improve canopy models for more precise predictions of the most effective combination of traits.
3. Evaluate carbon metabolic pathways associated with tobacco and/or other plants engineered for increased photosynthetic efficiency.
Approach:
Previous results identified two novel metabolic pathways to convert glycolate to 3- phosphoglycerate within chloroplasts. Level 0 DNA constructs have been generated which will be used to build novel versions of photorespiratory bypass with T-DNA constructs for plant transformation.
Once a fully operational bypass is installed in which glycolate flux to the native pathway is prevented by inactivating both the PLGG1 and BASS6 transporters, RNAi will be used to target steps in the native photorespiratory pathway downstream of glycine decarboxylase. A photorespiratory flux model will guide this work.
Improving the native photorespiratory pathway. Using a metabolic model of photorespiration< under development for catalase kinetics and temperature responses, the correlation between carbon dioxide assimilation and yield and catalase activity will be evaluated. This model will be parameterized with kinetics determined in vitro from species of catalase expressed in Escherichia coli. Catalase isoforms will be transformed into tobacco and screened via gas exchange at 25°C and 35°C for reductions in the CO2 photocompensation point. The model will be further validated by comparison to steady-state metabolic profiles determined in a custom-built gas exchange system that allows rapid and multiple samplings of illuminated leaf material.
Strategies will be focused on those that further suppress leaf chlorophyll content without impacting biochemical capacity for CO2 assimilation. Tobacco will be transformed with new constructs that simultaneously target multiple steps in the biosynthetic pathway to prevent large amounts of any individual intermediate from accumulating. We will also investigate the use of inducible promoters to initiate chlorophyll reduction later in the plant’s growth stage to avoid the highly sensitive seedling stage. Once optimal chlorophyll reduction is achieved without loss of leaf photosynthetic capacity, these constructs will be combined with those for up-regulating other photosynthetic genes to make use of the nitrogen released from lower chlorophyll.
Refine canopy light distribution models. For all approaches to canopy alteration, characterization of the resultant plants will be used to inform and improve our canopy models to allow more precise prediction of the most effective combinations of traits to achieve improved crop photosynthetic efficiency of light, water, and nitrogen use.
The carbon metabolic pathways associated with tobacco and/or other plants engineered for increased photosynthetic efficiency under this Agreement will be evaluated. Studies will include isotopic labeling and metabolic flux analysis of C3 plants with 13CO2 and potentially other isotopes to quantify carbon assimilation and the operation of photorespiratory pathways that have been rewired for the benefit of crop productivity.
Isotopic labeling will be measured in central intermediates using mass spectrometry. Computational models will be developed to assess flux.