PsaI is the only subunit of PSI whose precise physiological function has not yet been elucidated in higher plants. While PsaI is involved in PSI trimerization in cyanobacteria, trimerization was lost during the evolution of the eukaryotic PSI, and the entire PsaI side of PSI underwent major structural remodelling to allow for binding of light harvesting complex II antenna proteins during state transitions. Here, we have generated a tobacco (Nicotiana tabacum) knockout mutant of the plastid-encodedpsaIgene. We show that PsaI is not required for the redox reactions of PSI. Neither plastocyanin oxidation nor the processes at the PSI acceptor side are impaired in the mutant, and both linear and cyclic electron flux rates are unaltered. The PSI antenna cross section is unaffected, state transitions function normally, and binding of other PSI subunits to the reaction centre is not compromised. Under a wide range of growth conditions, the mutants are phenotypically and physiologically indistinguishable from wild-type tobacco. However, in response to high-light and chilling stress, and especially during leaf senescence, PSI content is reduced in the mutants, indicating that the I-subunit plays a role in stabilizing PSI complexes.