Regular paper LHC II protein phosphorylation in leaves of Arabidopsis thaliana mutants deficient in non-photochemical quenching Hanna-Leena Breitholtz, Renu Srivastava, Esa Tyystja¨rvi & Eevi Rintama¨ki* Department of Biology, University of Turku, 20014 Turku, Finland; *Author for correspondence (e-mail: evirin@utu.fi; fax: +358-2-3335549) Received 29 September 2004; accepted in revised form 20 January 2005 Key words: LHC II kinase, npq1-2 and npq4-1 mutants, redox regulation, ROSs, thioredoxin Abstract Phosphorylation of the light-harvesting chlorophyll a/b complex II (LHC II) proteins is induced in light via activation of the LHC II kinase by reduction of cytochrome b 6 f complex in thylakoid membranes. We have recently shown that, besides this activation, the LHC II kinase can be regulated in vitro by a thioredoxin- like component, and H 2 O 2 that inserts an inhibitory loop in the regulation of LHC II protein phosphor- ylation in the chloroplast. In order to disclose the complex network for LHC II protein phosphorylation in vivo, we studied phosphorylation of LHC II proteins in the leaves of npq1-2 and npq4-1 mutants of Arabidopis thaliana. In comparison to wild-type, these mutants showed reduced non-photochemical quenching and increased excitation pressure of Photosystem II (PS II) under physiological light intensities. Peculiar regulation of LHC II protein phosphorylation was observed in mutant leaves under illumination. The npq4-1 mutant was able to maintain a high amount of phosphorylated LHC II proteins in thylakoid membranes at light intensities that induced inhibition of phosphorylation in wild-type leaves. Light intensity-dependent changes in the level of LHC II protein phosphorylation were smaller in the npq1-2 mutant compared to the wild-type. No significant differences in leaf thickness, dry weight, chlorophyll content, or the amount of LHC II proteins were observed between the two mutant and wild-type lines. We propose that the reduced capacity of the mutant lines to dissipate excess excitation energy induces changes in the production of reactive oxygen species in chloroplasts, which consequently affects the regulation of LHC II protein phosphorylation. Abbreviations: DTT – dithiothreitol; LHC II – light-harvesting chlorophyll a/b complex II; NEM – N-ethyl- maleimide; NPQ – non-photochemical quenching; PFD – photon flux density; PG – propyl gallate; PS – Photosystem; ROS – reactive oxygen species; WT – wild-type Introduction Light reactions generate reactive intermediates and oxygen species that potentially damage photosyn- thetic machinery, especially in excess light. To prevent the detrimental effects of damaging sub- stances, plants have evolved a number of protec- tion mechanisms – including antioxidant systems and processes for thermal dissipation of light energy. An increase in the proton gradient across the thylakoid membrane triggers feedback non- photochemical quenching (NPQ), a specific type of thermal dissipation of excess energy in PS II that can be measured from chlorophyll fluorescence (reviewed in Demmig-Adams and Adams 1996; Horton et al. 1996). Characterization of the npq Photosynthesis Research (2005) 84: 217–223 Ó Springer 2005