The thylakoid proteome of chloroplasts contains multiple proteins involved in antioxidative defense, protein folding, and repair. To understand this functional protein network, we analyzed the quantitative response of the thylakoid-associated proteome of Arabidopsis (Arabidopsis thaliana) wild type and the ascorbate-deficient mutant vtc2-2 after transition to high light (HL; 1,000 micromol photons m(-2) s(-1)). The soluble thylakoid proteomes of wild type and vtc2-2 were compared after 0, 1, 3, and 5 d of HL using two-dimensional gels with three independent experiments, followed by a multivariant statistical analysis and tandem mass spectrometry. After 5 d of HL, both wild-type and vtc2-2 plants accumulated anthocyanins, increased their total ascorbate content, and lost 10% of photosystem II efficiency, but showed no bleaching. Anthocyanin and total ascorbate concentrations in vtc2-2 were respectively 34% and 20% of wild type, potentially leading to enhanced oxidative stress in vtc2-2. Forty-five protein spots significantly changed as a consequence of genotype, light treatment, or both. Independent confirmation was obtained from western blots. The most significant response was the up-regulation of thylakoid YCF37 likely involved in photosystem I assembly, and specific fibrillins, a flavin reductase-like protein, and an aldolase, each located in thylakoid-associated plastoglobules. Fe-superoxide dismutase was down-regulated in vtc2-2, while Cu,Zn-superoxide dismutase was up-regulated. vtc2-2 also showed a systematic up-regulation of a steroid dehydrogenase-like protein. A number of other stress-related proteins, several thylakoid proteases, and lumenal isomerases did not change, while PsbS increased in wild type upon light stress. These findings are discussed in terms of plastid metabolism and oxidative stress defense, and emphasize that understanding of the chloroplast stress-response network must include the enzymatic role of plastoglobules.