NDH-Mediated Cyclic Electron Flow Around Photosystem I is Crucial for C4 Photosynthesis.

C4 photosynthesis exhibits efficient CO2 assimilation in ambient air by concentrating CO2 around ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) through a metabolic pathway called the C4 cycle. It has been suggested that cyclic electron flow (CEF) around PSI mediated by chloroplast NADH dehydrogenase-like complex (NDH), an alternative pathway of photosynthetic electron transport (PET), plays a crucial role in C4 photosynthesis, although the contribution of NDH-mediated CEF is small in C3 photosynthesis. Here, we generated NDH-suppressed transformants of a C4 plant, Flaveria bidentis, and showed that the NDH-suppressed plants grow poorly, especially under low-light conditions. CO2 assimilation rates were consistently decreased in the NDH-suppressed plants under low and medium light intensities. Measurements of non-photochemical quenching (NPQ) of Chl fluorescence, the oxidation state of the reaction center of PSI (P700) and the electrochromic shift (ECS) of pigment absorbance indicated that proton translocation across the thylakoid membrane is impaired in the NDH-suppressed plants. Since proton translocation across the thylakoid membrane induces ATP production, these results suggest that NDH-mediated CEF plays a role in the supply of ATP which is required for C4 photosynthesis. Such a role is more crucial when the light that is available for photosynthesis is limited and the energy production by PET becomes rate-determining for C4 photosynthesis. Our results demonstrate that the physiological contribution of NDH-mediated CEF is greater in C4 photosynthesis than in C3 photosynthesis, suggesting that the mechanism of PET in C4 photosynthesis has changed from that in C3 photosynthesis accompanying the changes in the mechanism of CO2 assimilation.

Enhanced Ascorbate Regeneration Via Dehydroascorbate Reductase Confers Tolerance to Photo-Oxidative Stress in Chlamydomonas reinhardtii.

The role of ascorbate (AsA) recycling via dehydroascorbate reductase (DHAR) in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress was examined. The activity of DHAR and the abundance of the CrDHAR1 (Cre10.g456750) transcript increased after moderate light (ML; 750 µmol m-2 s-1) or high light (HL; 1,800 µmol m-2 s-1) illumination, accompanied by dehydroascorbate (DHA) accumulation, decreased AsA redox state, photo-inhibition, lipid peroxidation, H2O2 overaccumulation, growth inhibition and cell death. It suggests that DHAR and AsA recycling is limiting under high-intensity light stress. The CrDHAR1 gene was cloned and its recombinant CrDHAR1 protein was a monomer (25 kDa) detected by Western blot that exhibits an enzymatic activity of 965 µmol min-1 mg-1 protein. CrDHAR1 was overexpressed driven by a HSP70A:RBCS2 fusion promoter or down-regulated by artificial microRNA (amiRNA) to examine whether DHAR-mediated AsA recycling is critical for the tolerance of C. reinahartii cells to photo-oxidative stress. The overexpression of CrDHAR1 increased DHAR protein abundance and enzyme activity, AsA pool size, AsA:DHA ratio and the tolerance to ML-, HL-, methyl viologen- or H2O2-induced oxidative stress. The CrDHAR1-knockdown amiRNA lines that have lower DHAR expression and AsA recycling ability were sensitive to high-intensity illumination and oxidative stress. The glutathione pool size, glutathione:oxidized glutathione ratio and glutathione reductase and ascorbate peroxidase activities were increased in CrDHAR1-overexpressing cells and showed a further increase after high-intensity illumination but decreased in wild-type cells after light stress. The present results suggest that increasing AsA regeneration via enhanced DHAR activity modulates the ascorbate-glutathione cycle activity in C. reinhardtii against photo-oxidative stress.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons.

A novel light-dependent selection marker system in plants.

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H2O2 during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.

Molecular analysis of a homogentisate phytyltransferase gene from Lactuca sativa L.

Tocochromanols, usually known as vitamin E, play a crucial role in human and animal nutrition. The enzyme homogentisate phytyltransferase (HPT) performs the first committed step of the vitamin E biosynthetic pathway. The full-length cDNA encoding HPT was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPT, was 1,670 bp long containing an open reading frame (ORF) of 1,185 bp which encoded a protein of 395 amino acids. Sequence analysis indicated that the deduced protein, named as LsHPT, shared high identity with other dicotyledonous HPTs. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPT was preferentially expressed in mature leaves compared with other tissues. When lettuce plants were subjected to drought and high-light stress treatments, LsHPT expression was markedly increased. Expression of LsHPT in Arabidopsis showed that LsHPT could enhance the α-tocopherol biosynthesis in Arabidopsis. Transient expression of LsHPT via agroinfiltration resulted in 9-fold increase in LsHPT mRNA level and nearly 18-fold enhancement in α-tocopherol content compared with the negative controls.

Specific domain structures control abscisic acid-, salicylic acid-, and stress-mediated SIZ1 phenotypes.

SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

Cell variants showing differential susceptibility to ultraviolet light--induced transformation.

Six variant clones isolated from a subclone of BALB/3T3-A31 clone were classified into three groups according to their different susceptibilities to cell transformation by ultraviolet light irradiation: highly susceptible, intermediately susceptible, and resistant. All variant clones showed similar susceptibility to cytotoxic effects induced by ultraviolet light.

Observation of Phototropic Responses in the Liverwort Marchantia polymorpha.

The liverwort species, Marchantia polymorpha, shows environment-dependent morphological plasticity throughout its life cycle. Thalli, representing the predominant body form throughout most of this bryophyte's life cycle, grow with repeated dichotomous branching at the apex and develop horizontally under sufficient light intensity. Spores, after germination, produce a mass of cells, called sporelings, which then grow into thalli. Both thalli and sporelings, if grown under weak light conditions, form narrow shapes, and their apices grow toward the light source. These phototropic responses are specific to blue light and dependent on the blue-light receptor phototropin. This chapter provides several basic procedures, along with some tips, for designing and performing experiments with M. polymorpha to observe their phototropic responses, as well as methods for observing the localization of the phototropin "Mpphot" with a fluorescent protein tag.

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.

Enhanced transformation of human cells by UV-irradiated pSV2 plasmids.

Irradiating the plasmid pSV2-gpt with UV (254 nm) doses up to 200 J m-2 caused a dose-dependent increase in the yield of Gpt+ transformants when the plasmid was introduced into human cells by calcium phosphate coprecipitation. UV doses greater than 1 kJ m-2 were required to reduce the efficiency of transformation below that obtained with unirradiated DNA.

Impact of gamma irradiation on the transformation efficiency for extracellular plasmid DNA.

Extracellular DNA is omnipresent in aquatic environments and is thought to be a genetic material for horizontal gene transformation between microorganisms. We studied the impact of gamma irradiation on the transformation efficiency (transformants number per ng of DNA per ml) of extracellular DNA. Plasmid pEGFP as a model extracellular DNA was irradiated by gamma rays. The transformation efficiency decreased with the increase in radiation dose. A total dose of 10Gy is normally not lethal for microorganisms but certainly affects the transformation efficiency of extracellular DNA. The decrease in the efficiency would be induced by strand breaks of extracellular DNA because the yield of both single-strand breaks (SSBs) and double-strand breaks (DSBs) increased with the increase in radiation dose. The relative transformation efficiency of SSBs and DSBs to that of covalently closed circles (CCCs) was 30.3% and 0.2%, respectively. This impact on natural transformation suggests an inability of microorganisms to acquire new characteristics which should be normally acquired.

Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation.

Irradiation at 334 and 365 nm of a highly purified preparation of thymine-labeled transforming DNA from Haemophilus influenzae produced a photo product containing label from thymine but different from the cyclobutane dimer. The photoproduct is soluble in water and in ethanol and Rf values in a number of solvents are presented. The photoproduct has properties similar in a number of respects to those of the spore photoproduct, 5-thyminyl-5,6-dihydrothymine. The near ultraviolet photoproduct is more likely to affect the oxygen independent inactivation of transforming DNA rather than its mutagenesis, as judged by the quantitative relationship between amount of photboproduct and inactivation and mutagenesis.

Studies on DNA repair in Bacillus subtilis. I. A cellular factor acting on gamma-irradiated DNA and promoting its priming activity for DNA polymerase I.

Initiation of new DNA synthesis was observed in B. subtilis cells upon gamma-ray irradiation followed by toluene treatment and incubation in the presence of the four deoxynucleotide triphosphates and Mg2+. This DNA synthesis took place in the absence of ATP and was refractory to 6-(p-hydroxyphenylazo)-uracil which is a specific inhibitor for the type III polymerase of Bacillus subtilis. This repair-type DNA synthesis was greatly reduced in mutant cells deficient in DNA polymerase I. Restoration of transforming activity of cellular DNA was found to occur in parellel with the above repair type DNA synthesis. A protein factor which enhances the priming activity of gamma-irradiated DNA for DNA polymerase I was detected in DNA-free extracts prepared from B. subtilis cells by means of lysis with a buffer containing lysozyme, Brij-58 and EDTA.

Dictyostelium discoideum transformation by oscillating electric field electroporation.

Dictyostelium discoideum has been used as a genetically tractable model organism to study many biological phenomena. High-efficiency transformation is a prerequisite for successful genetic screens such as mutant complementation, identification of suppressor genes, or insertional mutagenesis. Although exponential decay electroporation is the standard transformation technique for D. discoideum, its efficiency is relatively low and its reproducibility is weak. Here we optimized the oscillating electroporation technique for D. discoideum transformation and compared it to the exponential decay electroporation. A 20-fold increase in the efficiency was resproducibly achieved. This alternative electroporation technique should facilitate future genetic approaches in D. discoideum.

Quantitative and molecular analyses of radiation-induced mutation in AS52 cells.

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.

DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. I. Kinetics of enhancement.

The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA.

Photolesions in DNA upon 193 nm excitation.

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single-and double-strand break formation, interstrand cross-links, locally denatured sites, (6-4)photoproducts and biological inactivation (phi ssb, phi dsb, phi icl, phi lds, phi 6-4 and phi ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are phi ssb approximately 1.5 x 10(-3), phi dsb approximately 0.06 x 10(-3) (dose: 10-200 J m-2), phi icl approximately phi lds approximately 0.1 x 10(-3) and phi 6-4 = 0.5 x 10(-3). Both phi ssb and phi dsb decrease strongly with increasing concentrations of TE buffer (0.01-10 mM). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and AB2480 uvr rec; the phi ina values are 1.4 x 10(-3), 2.1 x 10(-3) and 3 x 10(-3), respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.

alpha-Terthienyl photosensitizes damage to pBR322 DNA.

alpha-Terthienyl photosensitizes single strand breaks in pBR322 DNA. Almost identical results were observed under oxygen and under argon. In the presence of oxygen, this DNA nicking was enhanced by histidine and was not affected by superoxide dismutase, catalase, or the antioxidant BHT. Although chemical damage to DNA treated with alpha-terthienyl plus near-UV was clearly demonstrated in vitro, transformation in E. coli with this damaged pBR322 DNA still took place. Likewise, Haemophilus influenzae DNA transforming activity was not significantly decreased by photosensitization with alpha-terthienyl.

Cu(II) sensitizes pBR322 plasmid DNA to inactivation by UV-B (280-315 nm).

Copper(II), in the presence of UV-B radiation (280-315 nm), can generate single-strand breaks in the sugar-phosphate backbone of pBR322 plasmid DNA. A low level of single-strand backbone breaks occurs in the presence of Cu(II) alone, but UV-B irradiation increases the rate by the more than 100-fold. Concomitant with the damage to the DNA backbone is a loss of transforming activity. Oxygen is required for generation of the single-strand breaks but not for the loss of transforming activity. A DNA glycosylase (Fpg), which participates in the repair of certain DNA nitrogenous base damage, does not repair plasmid DNA damaged by Cu(II). The hydroxyl radical scavenging compound DMSO is only somewhat effective at protecting the physical and biological properties of the DNA. These results with Cu(II) are compared to those obtained previously with pBR322 plasmid DNA in the presence of Fe(III) and UV-A.

Preliminary investigation of resistance of plasmid DNA to neon ion beams using transformation into recipient Escherichia coli cells.

The sensitivity of the vector plasmid pKmK8 DNA to neon ion beams was studied under dry conditions. This plasmid contains the cloning vector pJKKmf(-) and a 3.6 kbp segment encoding the Escherichia coli crp gene that can be used in mutagenesis analysis. The survival curve of the plasmid indicated an exponential profile and a D10 value of about 16 kGy in the E. coli wild-type strain for DNA repair capability. This was similar to the D10 value for the shuttle vector plasmid pZ189 DNA. Therefore, it was assumed that the excision repair system in E. coli was not effective for repairing DNA lesions induced by irradiation with heavy ion beams.