Abstract
Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.
Highlights
Photosystem II (PSII),5 a supramolecular pigment-protein complex of photosynthetic organisms, utilizes absorbed light energy to oxidize water, releasing dioxygen and electrons that serve as the major source of reducing power in photosynthetic activity
The fact that most low molecular weight subunits (LMWs) of PSII have been highly conserved throughout the evolution implies that they perform essential functions, as this has been established for PsbI, PsbT, the ␣ and  subunits of the two-chain cytochrome b559, PsbE and PsbF, respectively, as well as for PsbL and PsbJ, which fulfill crucial structural and functional roles (20 –28)
Lower NPQ values were detected in the mutant at both light intensities indicating that mutant PSII is more susceptible to thermal dissipation (Table 1). These results indicated that PSII levels are comparable in mutant and wild type (WT), the quantum yield of PSII is significantly lower in ⌬psbM
Summary
Clone Construction to Inactivate the psbM Gene in Tobacco Chloroplasts—The recombinant plasmid B20 (tobacco plastome clone bank) [34] containing a 17,235-bp insertion (nucleotide positions 26,191– 43,426 in the plastid chromosome, accession number Z00044) in the vector pBR322 was digested with BamHI, and the resulting 2,096-bp fragment containing psbM was subcloned into the singular BamHI restriction site of pBluescript II KSϪ (Stratagene Inc., La Jolla, CA). For measurements of the intensity of the B band emission oscillations (QBϪ/ S2,S3 charge recombination) as a function of the numbers of single turnover excitations, a train of flashes (1– 6 flashes, about 300-ms interval between flashes) was given at 0 °C followed by rapid freezing. The kinetics of the QBϪ decay in darkness was measured following excitation of the dark-adapted sample by a single flash at 20 °C, followed by further incubation in darkness for the indicated periods, and followed by rapid freezing to Ϫ20 °C and starting the heating of the sample and photon-counting process. Measurements of Photoinhibition and Recovery Process—The sensitivity of PSII to oxidative stress has been determined with leaf disks (10 mm diameter, 5 disks per sample) of WT and ⌬psbM plants exposed to 500 E mϪ2 sϪ1 heterochromatic light. The recovery was followed in low light (3 E mϪ2 sϪ1) for up to 6 h measuring the Fv/Fm level every 1 h
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
