Abstract
Global demand for plant biomass for food, feed, and energy is rising, requiring increased yields and sustainability. Yet, yields of major crops are stagnating under climate change. Enhancing photosynthetic efficiency is a key strategy to boost yields and ensure sustainable food security. Photosynthesis involves light reactions and the Calvin cycle (CBC), both limited by environmental and intrinsic factors, including redox regulation. While thiol-based mechanisms regulating carbon assimilation during dark-to-light transitions are known, the impact of light dynamics on redox control and carbon fixation under natural conditions remains unclear. This project aims to clarify how photosynthetically derived redox signals affect carbon fixation and productivity under varying light conditions. The project will analyze electron transport and carbon fixation in the model bryophyte Physcomitrium patens, focusing on: (i) linking altered electron flow to redox signaling and CBC efficiency; (ii) investigating redox control of FBPase and SBPase; (iii) expressing modified enzyme isoforms to enhance productivity. Any significant results can be translated to crops and microalgae with industrial applications with higher productivity and adaptability to modern agriculture.
Results achieved
During the PRIN 2022 PNRR – IPERAFIX project, an integrated experimental platform was established to investigate the redox regulatory mechanisms of the Calvin-Benson-Bassham (CBB) cycle and their implications for photosynthetic metabolism in the moss Physcomitrium patens. Through the close collaboration between the University of Bologna (UNIBO) and the University of Padova (UNIPD), complementary biochemical, structural, and physiological approaches were successfully integrated, providing new insights into the function of key CBB cycle enzymes and their interplay with photosynthetic electron transport. One of the major achievements of the project was the optimization of the heterologous expression and purification of the photosynthetic fructose-1,6-bisphosphatase from P. patens (PpFBPase). The high yield and purity of the recombinant protein enabled an extensive biochemical characterization, including the determination of its main kinetic parameters and the investigation of the redox dependence of enzyme activity. Size-exclusion chromatography analyses unexpectedly revealed the coexistence of dimeric and tetrameric forms of PpFBPase. Subsequent functional studies demonstrated that the tetrameric form represents the catalytically active conformation, suggesting a previously unrecognized regulatory mechanism based on modulation of the enzyme oligomeric state. To elucidate the molecular determinants underlying this regulatory mechanism, several site-directed cysteine variants were generated, purified, and biochemically characterized. Comparative analyses of the recombinant proteins identified the contribution of individual cysteine residues to both structural stability and redox regulation of enzymatic activity. In parallel, experimental protocols based on alkylation assays were optimized to monitor the redox state of the protein, while structural investigations were initiated through computational analyses and crystallization trials. Although the crystals obtained have not yet yielded diffraction-quality data, the optimized experimental conditions provide a solid foundation for future high-resolution structural studies. On the physiological side, advanced methodologies were established to characterize photosynthetic performance in P. patens. Gas-exchange measurements were optimized using the LI-COR 6800 system, while chlorophyll fluorescence analyses were performed using the Dual-PAM-100 instrument, enabling the coordinated assessment of Photosystem I and Photosystem II activities, non-photochemical quenching, and carbon assimilation. These methodologies were subsequently applied to both wild-type plants and transgenic lines with altered FBPase expression, allowing correlations to be established between changes in carbon metabolism and photosynthetic performance. Another important outcome of the project was the optimization of methodologies for the detection of reactive oxygen species (ROS). Following the evaluation of different analytical approaches, increasingly sensitive methods were implemented, culminating in the application of Electron Paramagnetic Resonance (EPR) spectroscopy, which enabled a more accurate characterization of the redox dynamics associated with alterations in photosynthetic electron transport. From a genetic perspective, several transgenic P. patens lines overexpressing PpFBPase, together with lines expressing enzyme variants lacking regulatory cysteine residues, were successfully generated. Their phenotypic and physiological characterization provided a valuable experimental platform for investigating the role of FBPase in regulating photosynthetic efficiency and metabolic homeostasis. Finally, the project broadened its scope by initiating the biochemical and structural characterization of sedoheptulose-1,7-bisphosphatase (PpSBPase), another key enzyme of the CBB cycle. Expression and purification protocols for the recombinant protein were successfully established, laying the groundwork for future comparative studies aimed at elucidating the regulatory mechanisms governing enzymes involved in photosynthetic carbon fixation. Overall, the project established a comprehensive experimental framework for investigating the regulation of CBB cycle enzymes in Physcomitrium patens. The results obtained have advanced our understanding of the complex interplay between photosynthetic electron transport, redox metabolism, and carbon fixation, providing novel insights into the mechanisms that regulate photosynthetic efficiency in plants and establishing a strong foundation for future research aimed at improving photosynthetic performance.Project details
Unibo Team Leader: Mirko Zaffagnini
Unibo involved Department/s:
Dipartimento di Farmacia e Biotecnologie
Coordinator:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Total Eu Contribution: Euro (EUR) 247.760,00
Total Unibo Contribution: Euro (EUR) 127.522,00
Project Duration in months: 24
Start Date:
30/11/2023
End Date:
28/02/2026
