Towards spruce-type photosystem II: consequences of the loss of light-harvesting proteins LHCB3 and LHCB6 in Arabidopsis

The largest stable photosystem II (PSII) supercomplex in land plants (C₂S₂M₂) consists of a core complex dimer (C₂), two strongly (S₂) and two moderately (M₂) bound light-harvesting protein (LHCB) trimers attached to C₂ via monomeric antenna proteins LHCB4–6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C₂S₂M₂ supercomplex. Here, we performed structure–function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the “spruce-type” PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The “spruce-type” PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C₂S₂ semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C₂S₂ arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.

Photosystem II supercomplexes in Arabidopsis lacking antenna proteins LHCB3 and LHCB6 differ from their spruce counterparts and form potentially photoprotective semi-crystalline arrays in thylakoids.     

De-epoxidation of violaxanthin in the minor antenna proteins of photosystem II, LHCB4, LHCB5, and LHCB6

The conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4-6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein.     

Light‐dependent N‐terminal phosphorylation of LHCSR3 and LHCB4 are interlinked in Chlamydomonas reinhardtii

Phosphorylation dynamics of LHCSR3 were investigated in Chlamydomonas reinhardtii by quantitative proteomics and genetic engineering. LHCSR3 protein expression and phosphorylation were induced in high light. Our data revealed synergistic and dynamic N‐terminal LHCSR3 phosphorylation. Phosphorylated and nonphosphorylated LHCSR3 associated with PSII‐LHCII supercomplexes. The phosphorylation status of LHCB4 was closely linked to the phosphorylation of multiple sites at the N‐terminus of LHCSR3, indicating that LHCSR3 phosphorylation may operate as a molecular switch modulating LHCB4 phosphorylation, which in turn is important for PSII‐LHCII disassembly. Notably, LHCSR3 phosphorylation diminished under prolonged high light, which coincided with onset of CEF. Hierarchical clustering of significantly altered proteins revealed similar expression profiles of LHCSR3, CRX, and FNR. This finding indicated the existence of a functional link between LHCSR3 protein abundance and phosphorylation, photosynthetic electron flow, and the oxidative stress response.     

The SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LHCB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis in Arabidopsis seedlings

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons. SCO2 encodes a chloroplast-localized protein disulphide isomerase, hypothesized to be involved in protein folding. Analysis of co-expressed genes with SCO2 revealed that genes with similar expression patterns encode chloroplast proteins involved in protein translation and in chlorophyll biosynthesis. Indeed, sco2-1 accumulates increased levels of the chlorophyll precursor, protochlorophyllide, in both dark grown cotyledons and leaves. Yeast two-hybrid analyses demonstrated that SCO2 directly interacts with the chlorophyll-binding LHCB1 proteins, being confirmed in planta using bimolecular fluorescence complementation (BIFC). Furthermore, ultrastructural analysis of sco2-1 chloroplasts revealed that formation and movement of transport vesicles from the inner envelope to the thylakoids is perturbed. SCO2 does not interact with the signal recognition particle proteins SRP54 and FtsY, which were shown to be involved in targeting of LHCB1 to the thylakoids. We hypothesize that SCO2 provides an alternative targeting pathway for light-harvesting chlorophyll binding (LHCB) proteins to the thylakoids via transport vesicles predominantly in cotyledons, with the signal recognition particle (SRP) pathway predominant in rosette leaves. Therefore, we propose that SCO2 is involved in the integration of LHCB1 proteins into the thylakoids that feeds back on the regulation of the tetrapyrrole biosynthetic pathway and nuclear gene expression.     

Oka: Behind the Barricades: Kanehsatake: 270 Years of Resistance: Spudwrench: Kahnawake Man: My Name is Kahentiiosta: Acts of Defiance

Oka: Behind the Barricades. 1998. 312 minutes. For more information contact the National Film Board of Canada, 3155 Côte de Lisse Rd., St. Laurent, Quebec H4N 2N4. Series of four videos:
Kanehsatake: 270 Years of Resistance. 1993. 120 minutes. Directed by Alanis Obomsawin .
Spudwrench: Kahnawake Man. 1997. 58 minutes. Directed by Alanis Obomsawin .
My Name is Kahentiiosta. 1995. 30 minutes. Directed by Alanis Obomsawin .
Acts of Defiance. 1992. 104 minutes. Directed by Alec MacLeod .     

Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A,G:G:A:A,and A:A:A:A tetrads

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG₃(TTAG₃)₃ did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three‐tetrad scaffold, as proved by CD and PAGE in both Na⁺ and K⁺ solutions. Thermodynamic data showed that, in Na⁺ solution, the dG₃(TTAG₃)₃ quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced syn‐guanosines. In physiological K⁺ solution, the highest destabilization was caused by the 4A tetrad. In K⁺, only the unmodified dG₃(TTAG₃)₃ quadruplex rearranged into a K⁺‐dependent quadruplex form, none of the multiple adenine‐modified structures did so. This may imply biological consequences for nonrepaired A‐for‐G mutations. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 880–886, 2010.     

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.     

Ultrastructure of the spermatozoon of Megaselia scalaris Loew (Diptera:Brachycera:Cyclorrhapha:Phoridea:Phoridae)

When viewed by scanning electron microscopy (SEM), the spermatozoon of the phorid dipteran Megaselia scalaris appears threadlike, lacking distinct head and tail areas. These areas can be observed, however, in appropriately stained material. Measurements of Feulgen-stained material reveal average lengths of the head, tail, and total cell of 18.7, 128.7, and 147.4 μm, respectively. When tested for sulfhydryl and disulfide groups, the head displays only disulfide groups. Transmission electron microscopy (TEM) reveals 12 different regions: three (1–3) in the head, four (9–12) in the tail, and five (4–8) in a short zone of overlap between the head and tail. Most of the cell lies in regions 9 and 11 of the tail and 3 of the head, accounting for, respectively, 37.3%, 45.7%, and 11.2% of the total length. A tubelike acrosome indents the anterior end of the nucleus. The tail originates asymmetrically in relation to the long axis of the cell as a peglike structure associated with the dorsolateral region of the nucleus. No centriole is visible, and the nucleus has a notched appearance in longitudinal sections. Two mitochondrial derivatives and an axoneme displaying a 9+9+2 microtubule configuration and ATPase activity extend throughout most of the tail. In regions 9 and 10, an asymmetrically arranged accessory body is also present. Features having possible taxonomic utility include the asymmetrically arranged accessory body, the size and shape of the acrosome, and the notched appearance of the nucleus. The present report is apparently the first to describe the spermatozoon of a cyclorrhaphous dipteran which is not a member of the Schizophora.