In photosynthetic organisms, photons are captured by light-harvesting antenna complexes, and energy is transferred to reaction centers where photochemical reactions take place. We describe here the isolation and characterization of a fully functional megacomplex composed of a phycobilisome antenna complex and photosystems I and II from the cyanobacterium Synechocystis PCC 6803. A combination of in vivo protein cross-linking, mass spectrometry, and time-resolved spectroscopy indicates that the megacomplex is organized to facilitate energy transfer but not intercomplex electron transfer, which requires diffusible intermediates and the cytochrome b6f complex. The organization provides a basis for understanding how phycobilisomes transfer excitation energy to reaction centers and how the energy balance of two photosystems is achieved, allowing the organism to adapt to varying ecophysiological conditions.

Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.

The Orange Carotenoid Protein (OCP) plays a similar photoprotective role in cyanobacterial photosynthesis to that of non-photochemical quenching in higher plants. Under high-light conditions, OCP binds to the phycobilisome (PBS) and reduces energy transfer to the photosystems. The protective cycle starts from a light-induced activation of OCP. Detailed information on the molecular mechanism of this process as well as the subsequent recruitment of active OCP to the phycobilisome is not known. We report here our investigation on OCP photoactivation from the cyanobacterium Synechocystis sp. PCC 6803 by using a combination of native mass spectrometry (MS) and protein cross-linking. We demonstrate that Native MS is able to capture OCP with its intact pigment and further reveal that OCP undergoes a dimer-tomonomer transition upon light illumination. The reversion of activated form of OCP to inactive, dark form was also observed by using native MS. Furthermore, in vitro reconstitution of OCP and PBS allowed to perform protein chemical cross-linking experiments. LC-MS/MS analysis identified cross-linking species between OCP and the PBS core components. Our result indicates that the N-terminal domain of OCP is closely involved in the association with a site formed by two allophycocyanin trimers in the basal cylinders of the phycobilisome core. This report helps to understand the activation mechanism of OCP and the structural binding site of OCP during the cyanobacterial non-photochemical quenching process.

Interfering RNA was used to suppress the expression of the genes At1g06680 and At2g30790 in Arabidopsis thaliana, which encode the PsbP-1 and PsbP-2 proteins, respectively, of photosystem II (PS II In higher plants, algae, and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by PS II 2 (1-3). These are CP47, CP43, the D1 and D2 proteins, and the ␣ and ␤ subunits of cytochrome b 559 . Insertional inactivation or deletion of the genes for these components results in the disassembly of the PS II complex and the complete loss of oxygen evolution activity (for review, see Ref. 4). Additionally, a number of low molecular mass, intrinsic membrane protein components are associated with PS II (5-7), although the functions of many of these proteins remain obscure. Although PS II complexes containing only these intrinsic components can evolve oxygen in vitro, they do so at low rates (ϳ25-40% of control), are extremely susceptible to photoinactivation, and require high, nonphysiological levels of calcium and chloride for maximal activity (1, 3).)In higher plants and green algae, three extrinsic proteins, with apparent molecular masses of 33, 24, and 17 kDa, are required for high rates of oxygen evolution at physiological inorganic cofactor concentrations. The 33-kDa component, the PsbO protein, has been termed the manganese-stabilizing protein due to its stabilization of the manganese cluster during exposure to low chloride concentrations or to exogenous reductants. In vitro, the 24-and 17-kDa proteins (termed the PsbP and PsbQ proteins, respectively) appear to modulate the calcium and chloride requirements for efficient oxygen evolution. The precise roles of these proteins in oxygen evolution and PS II assembly/stability in vivo, however, remain unclear. These three extrinsic components interact with intrinsic membrane proteins and possibly with each other to yield fully functional oxygen-evolving complexes.The mature PsbP protein is highly conserved (8) in higher plants. In Arabidopsis, there are two putative genes, At1g06680 and At2g30790, which encode PsbP-1 and PsbP-2, respectively. It should be noted that initially only PsbP-1 was observed in Arabidopsis (9, 10) using two-dimensional IEF-SDS-PAGE. Recently, however, PsbP-2 has been detected during two-dimensional difference gel electrophoresis (11). In the cyanobacterium Synechocystis 6803, mutants in which the homologue of the psbP gene had been deleted exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl 2 -limiting conditions (7, 12), whereas in Chlamydomonas, a mutant which did not accumulate PsbP was deficient in photoactivation (13).RNAi is a post-transcriptional gene-silencing process in which double-stranded RNA induces the degradation of homologous mRNA sequences (14). RNAi has been successfully applied as a powerful gene-silencing tool in a variety of organisms, including Caenorhabditis elegans and Drosophila melanogaster, and in mouse oocytes. It has also become a pop-* T...

Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn 4 Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27ΔctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3ΔctpAΔpsb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27ΔctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as