In vitro reconstitution of light-harvesting POR-protochlorophyllide complex with protochlorophyllides a and b

NADPH:protochlorophyllide oxidoreductase (POR; EC1.1.33.1) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999)Nature 397, 80–84). Here we report the in vitro reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a andb and galacto- and sulfolipids. Low temperature (77 K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-ms flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a had been photoactive and was converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Nondenaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complexin vivo, suggesting that both complexes may be identical.

Independent evolution of the prochlorophyte and green plant chlorophyll a/b light-harvesting proteins

The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins. We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II. The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins. Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Induction of Ig light chain gene rearrangement in heavy chain-deficient B cells by activated Ras

During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by μ HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of κ LC gene rearrangement and a preference for κ over λ LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement.

Induction of pre-B cell proliferation after de novo synthesis of the pre-B cell receptor

The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig μ heavy chain (μH-chain), the surrogate light (SL) chain, and the Igα/β dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which μH-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of μH-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of μH-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.

Induction of Acclimative Proteolysis of the Light-Harvesting Chlorophyll a/b Protein of Photosystem II in Response to Elevated Light Intensities1

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

Differential Control of Xanthophylls and Light-Induced Stress Proteins, as Opposed to Light-Harvesting Chlorophyll a/b Proteins, during Photosynthetic Acclimation of Barley Leaves to Light Irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 μmol m−2 s−1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.

Ultraviolet-B photodestruction of a light-harvesting complex.

Cyanobacteria are important contributors to global photosynthesis in both marine and terrestrial environments. Quantitative data are presented on UV-B-induced damage to the major cyanobacterial photosynthetic light harvesting complex, the phycobilisome, and to each of its constituent phycobiliproteins. The photodestruction quantum yield, phi295 nm, for the phycobiliproteins is high (approximately 10(-3), as compared with approximately 10(-7) for visible light). Energy transfer on a picosecond time scale does not compete with photodestruction. Photodamage to phycobilisomes in vitro and in living cells is amplified by causing dissociation and loss of function of the complex. In photosynthetic organisms, UV-B damage to light-harvesting complexes may significantly exceed that to DNA.

Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability.

In early seedling development, far-red-light-induced deetiolation is mediated primarily by phytochrome A (phyA), whereas red-light-induced deetiolation is mediated primarily by phytochrome B (phyB). To map the molecular determinants responsible for this photosensory specificity, we tested the activities of two reciprocal phyA/phyB chimeras in diagnostic light regimes using overexpression in transgenic Arabidopsis. Although previous data have shown that the NH2-terminal halves of phyA and phyB each separately lack normal activity, fusion of the NH2-terminal half of phyA to the COOH-terminal half of phyB (phyAB) and the reciprocal fusion (phyBA) resulted in biologically active phytochromes. The behavior of these two chimeras in red and far-red light indicates: (i) that the NH2-terminal halves of phyA and phyB determine their respective photosensory specificities; (ii) that the COOH-terminal halves of the two photoreceptors are necessary for regulatory activity but are reciprocally inter-changeable and thus carry functionally equivalent determinants; and (iii) that the NH2-terminal halves of phyA and phyB carry determinants that direct the differential light lability of the two molecules. The present findings suggest that the contrasting photosensory information gathered by phyA and phyB through their NH2-terminal halves may be transduced to downstream signaling components through a common biochemical mechanism involving the regulatory activity of the COOH-terminal domains of the photoreceptors.

Excavations in the upper Little Colorado drainage, eastern Arizona / [by] Paul S. Martin [and] John B. Rinaldo.

v.51:no.1(1960)