Supporting Dataset for "A synthetic C4 shuttle via the β-hydroxyaspartate cycle in C3 plants"

<p>Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO₂&nbsp;fixation. However, especially in C3 plants, photosynthetic yield is reduced by the formation of 2-phosphoglycolate, a toxic oxygenation product of Rubisco, which needs to be recycled in a high-flux&ndash;demanding metabolic process called photorespiration. Canonical photorespiration dissipates energy and causes carbon and nitrogen losses. Reducing photorespiration through carbon-concentrating mechanisms, such as C4 photosynthesis, or bypassing photorespiration through metabolic engineering is expected to improve plant growth and yield. The &beta;-hydroxyaspartate cycle (BHAC) is a recently described microbial pathway that converts glyoxylate, a metabolite of plant photorespiration, into oxaloacetate in a highly efficient carbon-, nitrogen-, and energy-conserving manner. Here, we engineered a functional BHAC in plant peroxisomes to create a photorespiratory bypass that is independent of 3-phosphoglycerate regeneration or decarboxylation of photorespiratory precursors. While efficient oxaloacetate conversion in&nbsp;<em>Arabidopsis thaliana</em>&nbsp;still masks the full potential of the BHAC, nitrogen conservation and accumulation of signature C4 metabolites demonstrate the proof of principle, opening the door to engineering a photorespiration-dependent synthetic carbon&ndash;concentrating mechanism in C3 plants.</p> <p>Data analysis was performed in R. For analysis of gas exchange measurements, the &ldquo;plantecophys&rdquo; package was used (<a href="https://www.pnas.org/doi/full/10.1073/pnas.2022307118#core-r55">55</a>). The data are summarized in&nbsp;<a href="http://www.pnas.org/lookup/doi/10.1073/pnas.2022307118#supplementary-materials">Datasets S1&ndash;S10</a>. All other study data are included in the article and/or supporting information, available at&nbsp;<a href="https://doi.org/10.1073/pnas.2022307118">https://doi.org/10.1073/pnas.2022307118</a></p> <p>Dataset S1: Enzymatic activity of BHAC enzymes in Arabidopsis rosette leaves. For ISR the rate of percentual 15N label enrichment in aspartate was quantified. Shown mean and standard deviation (SD).</p> <p>Dataset S2: Metabolome of BHAC plants. Shown is mean and standard deviation (SD) of the calculated relative amount per mg fresh weight of four biological replicates per genotype for each condition.</p> <p>Dataset S3: Ammonium quantification in BHAC plants. Shown is mean and standard deviation (SD) for four biological replicates per genotype per condition measured in technical triplicates.</p> <p>Dataset S4: Phenotyping of BHAC plants. Shown is mean and standard deviation (SD) of five biological replicates per genotype per condition.</p> <p>Dataset S5: A/Ci curve measurements of BHAC plants. Shown is mean of four biological replicates per genotype.</p> <p>Dataset S6: Light response measurements of BHAC plants. Shown is mean of four biological replicates per genotype.</p> <p>&nbsp;Dataset S7: Metabolite levels of phosphorylated intermediates and glyoxylate in air-grown plants. Shown is mean and standard deviation of &ge; 3 replicates.</p> <p>Dataset S8: Metabolome of ggt1-1 complementation lines with AGAT. Shown is mean and standard deviation (SD) of four biological replicates.</p> <p>Dataset S9: Enzymatic activity of AGAT and GGT&nbsp; in Arabidopsis rosette leaves of the ggt1-1 complemention lines. Shown mean and standard deviation (SD) of three biological replicates measured in technical triplicates.</p> <p>Dataset S10: O2-Dependency of CCP was measured at 4% O2. Shown is the mean &plusmn;SD of n &ge; 3.</p>