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

Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO2 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–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 β-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 Arabidopsis thaliana 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–concentrating mechanism in C3 plants.

Data analysis was performed in R. For analysis of gas exchange measurements, the “plantecophys” package was used (55). The data are summarized in Datasets S1–S10. All other study data are included in the article and/or supporting information, available at https://doi.org/10.1073/pnas.2022307118

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).

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.

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.

Dataset S4: Phenotyping of BHAC plants. Shown is mean and standard deviation (SD) of five biological replicates per genotype per condition.

Dataset S5: A/Ci curve measurements of BHAC plants. Shown is mean of four biological replicates per genotype.

Dataset S6: Light response measurements of BHAC plants. Shown is mean of four biological replicates per genotype.

 Dataset S7: Metabolite levels of phosphorylated intermediates and glyoxylate in air-grown plants. Shown is mean and standard deviation of ≥ 3 replicates.

Dataset S8: Metabolome of ggt1-1 complementation lines with AGAT. Shown is mean and standard deviation (SD) of four biological replicates.

Dataset S9: Enzymatic activity of AGAT and GGT  in Arabidopsis rosette leaves of the ggt1-1 complemention lines. Shown mean and standard deviation (SD) of three biological replicates measured in technical triplicates.

Dataset S10: O2-Dependency of CCP was measured at 4% O2. Shown is the mean ±SD of n ≥ 3.

植物依赖核酮糖-1,5-二磷酸羧化酶/加氧酶（ribulose-1,5-bisphosphate carboxylase/oxygenase，Rubisco）进行二氧化碳固定。然而，尤其在C3植物中，Rubisco的氧合反应会生成有毒产物2-磷酸乙醇酸，该物质需通过一种高耗能的代谢过程——光呼吸——进行循环回收，进而降低光合产量。典型光呼吸会消耗能量，并造成碳、氮元素的流失。通过碳浓缩机制（如C4光合作用）降低光呼吸，或通过代谢工程绕过光呼吸，有望提升植物生长量与产量。β-羟基天冬氨酸循环（β-hydroxyaspartate cycle，BHAC）是近年发现的微生物代谢途径，可高效将植物光呼吸的代谢产物乙醛酸转化为草酰乙酸，同时实现碳、氮与能量的节约利用。本研究在植物过氧化物酶体中构建了具有功能的BHAC，以此构建了不依赖3-磷酸甘油酸再生或光呼吸前体脱羧的光呼吸旁路途径。尽管在拟南芥（Arabidopsis thaliana）中草酰乙酸的高效转化尚未完全发挥BHAC的全部潜力，但氮元素保留与特征性C4代谢物的积累已验证了该途径的原理可行性，为在C3植物中构建依赖光呼吸的人工碳浓缩机制奠定了基础。

数据分析基于R语言完成。气体交换测量数据的分析采用了"plantecophys"软件包（参考文献55）。所有数据集汇总于数据集S1至S10。其余研究数据已收录于本文及/或补充材料，可通过https://doi.org/10.1073/pnas.2022307118获取。

数据集S1：拟南芥莲座叶中BHAC酶的酶活性测定。本研究通过同位素示踪法（ISR）量化了天冬氨酸中15N标记富集的百分比水平，结果以平均值及标准差（SD）呈现。
数据集S2：BHAC转基因植物的代谢组分析。结果以各处理条件下每个基因型的4次生物学重复样本的单位鲜重相对含量平均值及标准差（SD）呈现。
数据集S3：BHAC转基因植物的铵盐含量定量分析。各处理条件下每个基因型的4次生物学重复样本均进行3次技术重复测定，结果以平均值及标准差（SD）呈现。
数据集S4：BHAC转基因植物的表型分析。各处理条件下每个基因型的5次生物学重复样本的结果以平均值及标准差（SD）呈现。
数据集S5：BHAC转基因植物的A/Ci曲线测定。结果以每个基因型的4次生物学重复样本的平均值呈现。
数据集S6：BHAC转基因植物的光响应曲线测定。结果以每个基因型的4次生物学重复样本的平均值呈现。
数据集S7：空气培养条件下植物的磷酸化代谢中间产物与乙醛酸含量分析。结果以≥3次重复样本的平均值及标准差呈现。
数据集S8：携带AGAT的ggt1-1互补株系的代谢组分析。结果以4次生物学重复样本的平均值及标准差（SD）呈现。
数据集S9：ggt1-1互补株系的拟南芥莲座叶中AGAT与GGT的酶活性测定。3次生物学重复样本均进行3次技术重复测定，结果以平均值及标准差（SD）呈现。
数据集S10：在4%氧气浓度下测定了CCP的氧依赖性。结果以n≥3次重复的平均值±标准差呈现。