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In plants, abiotic stressors are frequently encountered during growth and development. To counteract these challenges, zinc finger proteins play a critical role as transcriptional regulators. The EgrZFP6 gene, which codes for a zinc finger protein of the C2H2 type, was shown to be considerably elevated in the leaves of Eucalyptus grandis seedlings in the current study when they were subjected to a variety of abiotic stimuli, including heat, salinity, cold, and drought. Analysis conducted later showed that in EgrZFP6 transgenic Arabidopsis thaliana, EgrZFP6 was essential for causing hyponastic leaves and controlling the stress response. Furthermore, the transgenic plants showed elevated levels of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H2O2). Additionally, in EgrZFP6-overexpressing plants, transcriptome sequencing analysis demonstrated a considerable downregulation of many genes involved in photosynthesis, decreasing electron transport efficiency and perhaps promoting the buildup of ROS. Auxin levels were higher and auxin signal transduction was compromised in the transgenic plants. Stress-related genes were also upregulated in Arabidopsis as a result of EgrZFP6 overexpression. It is hypothesized that EgrZFP6 can downregulate photosynthesis, which would cause the production of ROS in chloroplasts. As a result, this protein may alter plant stress responses and leaf morphology via a retrograde mechanism driven by ROS. These results highlight the significance of zinc finger proteins in this sophisticated process and advance our understanding of the complex link between gene regulation, ROS signaling, and plant stress responses.

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As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 to C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis to C4 photosynthesis remains elusive. Here, we provide evidence suggesting a role of higher α-ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increased in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increases the concentrations of C4-related metabolites in C3-C4 and C4 species but not the activity of C4-related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitated the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanism. All these results provide insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

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C4 photosynthesis evolved from ancestral C3 photosynthesis by recruiting pre-existing genes to fulfill new functions. The enzymes and transporters required for the C4 metabolic pathway have been intensively studied and well documented; however, the transcription factors (TFs) that regulate these C4 metabolic genes are not yet well understood. In particular, how the TF regulatory network of C4 metabolic genes was rewired during the evolutionary process is unclear. Here, we constructed gene regulatory networks (GRNs) for four closely evolutionarily related species from the genus Flaveria, which represent four different evolutionary stages of C4 photosynthesis: C3 (F. robusta), type I C3-C4 (F. sonorensis), type II C3-C4 (F. ramosissima), and C4 (F. trinervia). Our results show that more than half of the co-regulatory relationships between TFs and core C4 metabolic genes are species specific. The counterparts of the C4 genes in C3 species were already co-regulated with photosynthesis-related genes, whereas the required TFs for C4 photosynthesis were recruited later. The TFs involved in C4 photosynthesis were widely recruited in the type I C3-C4 species; nevertheless, type II C3-C4 species showed a divergent GRN from C4 species. In line with these findings, a 13CO2 pulse-labeling experiment showed that the CO2 initially fixed into C4 acid was not directly released to the Calvin-Benson-Bassham cycle in the type II C3-C4 species. Therefore, our study uncovered dynamic changes in C4 genes and TF co-regulation during the evolutionary process; furthermore, we showed that the metabolic pathway of the type II C3-C4 species F. ramosissima represents an alternative evolutionary solution to the ammonia imbalance in C3-C4 intermediate species.

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BACKGROUND: Photosynthesis close interacts with respiration and nitrogen assimilation, which determine the photosynthetic efficiency of a leaf. Accurately quantifying the metabolic fluxes in photosynthesis, respiration and nitrogen assimilation benefit the design of photosynthetic efficiency improvement. To accurately estimate metabolic fluxes, time-series data including leaf metabolism and isotopic abundance changes should be collected under precisely controlled environments. But for isotopic labelled leaves under defined environments the, time cost of manually sampling usually longer than the turnover time of several intermediates in photosynthetic metabolism. In this case, the metabolic or physiological status of leaf sample would change during the sampling, and the accuracy of metabolomics data could be compromised. RESULTS: Here we developed an integrated isotopic labeling and freeze sampling apparatus (ILSA), which could finish freeze sampling automatically in 0.05 s. ILSA can not only be used for sampling of photosynthetic metabolism measurement, but also suit for leaf isotopic labeling experiments under controlled environments ([CO2] and light). Combined with HPLC-MS/MS as the metabolic measurement method, we demonstrated: (1) how pool-size of photosynthetic metabolites change in dark-accumulated rice leaf, and (2) variation in photosynthetic metabolic flux between rice and Arabidopsis thaliana. CONCLUSIONS: The development of ILSA supports the photosynthetic research on metabolism and metabolic flux analysis and provides a new tool for the study of leaf physiology.