Photorespiratory Engineering from an Evolutionary Perspective: A Constraint-Aligned Evaluation and Validation Framework

More than two billion years of photorespiratory evolution have shaped three sets of constraints: catalytic constraints associated with Rubisco oxygenase activity; compartmental topology constraints arising from compartmental organization, transport processes, and cofactor coupling; and physiological and ecological constraints arising from integration with metabolic and signaling networks. Photorespiratory engineering has emerged as a key strategy for improving photosynthetic efficiency in C3 crops, yet existing studies have not systematically assessed the practical implications of these constraints. Evaluation metrics remain inconsistent, the scope and depth of stability and risk assessments vary, and methodological heterogeneity hinders comparisons across studies. To address these issues, we synthesize the evolutionary history of photorespiration and findings from previous research to develop an evaluation and validation framework aligned with these constraints. This framework comprises eight assessment dimensions defining what to report, a context matrix specifying the conditions under which assessments should be conducted, and a five-tier validation pipeline defining the sequence of validation steps and the criteria for classifying outcomes. Retrospective application to five published case studies demonstrates that the framework enables consistent classification of outcomes and identifies distinct priorities for further improvement across studies involving different crop species, engineering strategies, and evaluation endpoints. By providing a practical evidence structure that can be updated as new findings emerge, this framework supports the transition from empirical exploration to systematic photorespiratory engineering and facilitates cross-study comparisons and the cumulative synthesis of evidence across assessment dimensions, experimental contexts, and outcomes.