A stable LHCII-PSI aggregate and suppression of photosynthetic state transitions in the psae1-1 mutant of Arabidopsis thaliana

Abstract

During photosynthetic state transitions, a fraction of the major light-harvesting complex (LHCII) shuttles between photosystems II (PSII) and I (PSI), depending on whether or not it is phosphorylated. Its phosphorylation state in turn depends on the relative activity of the two photosystems, which is a function of redox state and illumination parameters. In the psae1-1 mutant of Arabidopsis thaliana (L.) Heynh., amounts of the PSI subunits E, C, D, H and L are decreased. A fraction of LHCII is stably associated with PSI when plants are exposed to low light conditions, giving rise to a high-molecular-mass protein-pigment complex detectable in native protein gels. The formation of this abnormal LHCII-PSI complex is associated with an almost complete suppression of state transitions, a drastic increase in the levels of phosphorylated LHCII under all light regimes tested, and a permanent reduction in PSII antenna size. All these observations suggest that the altered polypeptide composition of PSI perturbs the docking of phosphorylated LHCII, making psae1-1 a unique mutant for the study of PSI-LHCII interactions and additional effects of the mutation, such as a decrease in grana stacking and increased adenylate kinase activity.

Photosystem II core phosphorylation and photosynthetic acclimation require two different protein kinases

“…Mutant lines from the Salk collection 9 were identified by searching the SiGNAL database (http://signal.salk.edu/ tabout.html). Methods for plant propagation have been described elsewhere [25][26][27] . Chlorophyll fluorescence and pigment analysis.…”

“…Chlorophyll fluorescence and pigment analysis. Photosynthetic electron transport, state transitions and leaf pigment composition were measured as described [25][26][27] (see Supplementary Information for details). Analysis of LHCII phosphorylation in vivo and in vitro.…”

“…To determine the degree of LHCII phosphorylation in vivo, dark-adapted leaves from 4-week-old plants were incubated in the presence of [ 33 P]orthophosphate for 1 h and subsequently exposed for 2 h to levels of illumination favouring phosphorylation (low light, 80 mmol photons m 22 s 21 ) or dephosphorylation (high light, 800 mmol photons m 22 s 21 ). Identical amounts of thylakoid proteins (equivalent to 200 mg of fresh leaf) were prepared in the presence of 10 mM NaF 25 and fractionated by SDS-PAGE (14% polyacrylamide); labelled proteins were detected by phosphorimaging (Typhoon; Amersham Biosciences). For the assay in vitro, thylakoids isolated from dark-adapted leaves (30 mg of fresh leaf) were incubated with [g-33 P]ATP under reducing conditions in the dark 25 .…”

“…Identical amounts of thylakoid proteins (equivalent to 200 mg of fresh leaf) were prepared in the presence of 10 mM NaF 25 and fractionated by SDS-PAGE (14% polyacrylamide); labelled proteins were detected by phosphorimaging (Typhoon; Amersham Biosciences). For the assay in vitro, thylakoids isolated from dark-adapted leaves (30 mg of fresh leaf) were incubated with [g-33 P]ATP under reducing conditions in the dark 25 . Separation and detection of thylakoid proteins were performed as described for the in vivo assay.…”

“…Leaves from 4-week-old plants were harvested after overnight dark adaptation or light exposure as described above. Thylakoids were prepared in the presence of 10 mM NaF, fractionated on an SDS-polyacrylamide-gradient gel (8-25% polyacrylamide) and transferred to poly(vinylidene difluoride) membranes 25 . Filters were then probed with antibodies specific for phosphothreonine (Biolabs) and signals were detected by enhanced chemiluminescence (Amersham Biosciences).…”