From farm to fork and beyond! UV enhances Aryl hydrocarbon receptor-mediated activity of cruciferous vegetables in human intestinal cells upon colonic fermentation.

While the "farm to fork" strategy ticks many boxes in the sustainability agenda, it does not go far enough in addressing how we can improve crop nutraceutical quality. Here, we explored whether supplementary ultraviolet (UV) radiation exposure during growth of broccoli and Chinese cabbage can induce bioactive tryptophan- and glucosinolate-specific metabolite accumulation thereby enhancing Aryl hydrocarbon receptor (AhR) activation in human intestinal cells. By combining metabolomics analysis of both plant extracts and in vitro human colonic fermentation extracts with AhR reporter cell assay, we reveal that human colonic fermentation of UVB-exposed Chinese cabbage led to enhanced AhR activation in human intestinal cells by 23% compared to plants grown without supplementary UV. Thus, by exploring aspects beyond "from farm to fork", our study highlights a new strategy to enhance nutraceutical quality of Brassicaceae, while also providing new insights into the effects of cruciferous vegetables on human intestinal health.

Induction of early light-inducible protein gene expression in Pisum sativum after exposure to low levels of UV-B irradiation and other environmental stresses.

Plants are constantly subjected to environmental changes and have developed various defence mechanisms to facilitate their continued existence. Pisum sativum plants were exposed to low levels of UV-B radiation and ELIP (early light-inducible proteins) mRNA, with a probable protective function, was rapidly and strongly induced during this type of stress. To our knowledge, this is the only photosynthetic gene that is up-regulated following exposure to UV-B, and this result has to be compared with studies predominantly reporting down-regulation by UV-B of genes encoding proteins localised in the plastid. The expression pattern of ELIP mRNA in pea was also investigated during salt, wounding and ozone stress. The transcript levels of ELIP were induced after the salt and wounding treatments but not during ozone fumigation.

Ultraviolet-B radiation causes tendril coiling in Pisum sativum.

Low dose UV-B radiation (UV-B(BE,300) = 0.1 W m(-2)), but neither UV-A radiation, ozone and NaCl stress, nor wounding, caused tendril coiling in Pisum sativum. This coiling occurred with both attached and detached tendrils and can be used as a specific UV-B stress marker in pea.

Transcriptional activation of the parsley chalcone synthase promoter in heterologous pea and yeast systems.

Introduction by electroporation of different parsley (Petroselinum crispum) CHS-promoter/beta-glucuronidase(GUS)-reporter constructs into pea (Pisum sativum L.) protoplasts leads to a high constitutive GUS-expression and to the loss of the light-inducibility seen in the homologous parsley protoplast system. These results indicate that Unit 1 of the parsley CHS-promoter is only partly responsible for the GUS-expression detected. Instead, additional cis-elements, which are located downstream within 100 bp from the transcriptional start site, mediate the de-repression in pea protoplasts. In contrast, in yeast (Saccharomyces cerevisiae) cells, the GUS expression from the heterologous CHS/GUS construct is controlled by elements between Unit 1 and -100 bp. In both pea and yeast cells, transcription factors different from those regulating UV-responsiveness in parsley, are probably mediating the constitutive expression from the heterologous construct. The results with pea protoplasts imply that protoplastation of pea leaf cells itself induces de-repression as a result of stress to the protoplasts. This notion was strengthened by the finding that mRNA levels of the endogenous chalcone synthase were drastically increased as the result of the protoplastation procedure.

Molecular markers for UV-B stress in plants: alteration of the expression of four classes of genes in Pisum sativum and the formation of high molecular mass RNA adducts.

Sixteen ultraviolet-B radiation-regulated pea genes were identified. Functionally, the corresponding proteins were divided into four groups. (i) Chloroplast-localized proteins. Genes for these proteins were down-regulated, underlining the deleterious effects of UV-B on this organelle. A novel down-regulated photosystem I light-harvesting chlorophyll a/b-binding protein gene (PsLhcA4), was cloned and sequenced. (ii) Protein turnover enzymes. Levels of mature mRNAs for the PU1 and PsUBC4 genes, encoding proteins of the ubiquitin protein degradation pathway, were up- and down-regulated, respectively, implying alteration of plant cell protein content by changes in both gene expression and protein degradation. (iii) Proteins involved in intracellular signalling. Expression of genes for small GTPases, rab and rho homologues, were altered. (iv) Phenylpropanoid or flavonoid biosynthesis. Expression of three genes encoding enzymes in these pathways were up-regulated and one of them, the novel PsC450R1, was cloned and sequenced. Moreover, unexpected high molecular mass psbA RNA adducts were found to appear after UV-B exposure. In addition, a large increase in corresponding high molecular mass adducts were also found for PsLhcA4, and PsUBC4 mRNA and 23S rRNA. These RNA species do not contain protein and probably appear due to cross-linking of two or more RNA molecules, or are the result of UV-B-induced failure of transcription termination.

Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses.

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m(-2)) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.

The mRNA-binding ribosomal protein S26 as a molecular marker in plants: molecular cloning, sequencing and differential gene expression during environmental stress.

One condition for using a gene as a transcriptional marker for environmental stress is its specific and differential expression. In order to be used as such a marker, the ribosomal protein S26 cDNA from pea (Pisum sativum L.) was cloned and fully sequenced. The gene (PsRPS26) was shown to be differentially regulated by ozone and UV-B radiation in opposite ways. Ozone gave rise to increased mRNA levels, whereas UV-B led to a decrease in S26 transcript abundance. Thus, the expression of PsRPS26 can be used as a molecular marker to differentiate between these two environmental stresses.

Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon.

Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon [Tybulewicz, V. L. J., Falk, G. & Walker, J. E. (1984) J. Mol. Biol. 179, 185-214]. A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb. blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum. Although the protein is present during the entire life cycle of a Rb. blasticus culture, it is most abundant early during the stationary phase. Plasmid constructs of the URF6 gene for overexpression in E. coli were made. These constructs were designed to obtain proteins both with and without His-tagging. In both cases, a protein product was visible in induced cells. The His-tagged protein was purified to 85% on a Ni column and, further, to at least 95% by anion-exchange chromatography. By N-terminal sequencing of the His-tagged protein, its identity was confirmed.

Ultraviolet-B-radiation-induced changes in nicotinamide and glutathione metabolism and gene expression in plants.

Pea (Pisum sativum L. cv. Greenfeast) plants were exposed to supplementary ultraviolet-B (UV-B) radiation (biologically effective dose rates normalised to 300 nm, UV-B[BE,300]: 0.18, 0.32 or 1.4 W m[-2]). Leaf nicotinamide, trigonelline, GSHtot (total glutathione) and GSSG (oxidised glutathione) levels remained unchanged after exposure to the lowest dose rates. 1.4 W m(-2) UV-B(BE,300) gave rise to 60-fold and 4.5-fold increases in GSSG and GSHtot, respectively. 3.5-fold and 9.5-fold increases were found in nicotinamide and trigonelline, respectively. cab (Chlorophyll-a/b-binding protein) transcript levels decreased and CHS (chalcone synthase) and PAL (phenylalanine ammonia-lyase) mRNA increased after shorter UV-B exposures (hours) to the higher dose rate of UV-B, and after exposure to the intermediate dose rate. CHS and PAL mRNAs also increased after prolonged exposure to the lowest dose rate. cab transcripts completely disappeared, whereas CHS and PAL mRNA levels rose by 60-fold and 17-fold, respectively, after 12 h exposure at the highest dose rate and 12 h of development. Our results indicate that nicotinamide or trigonelline do not function as signalling compounds for CHS and PAL gene expression. Elevated nicotinamide and trigonelline levels occur in response to UV-B, but only at UV-B doses high enough to cause oxidative stress.

A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum.

A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55-60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.

UV-B- and oxidative stress-induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP-ribose)polymerase inhibitor in plant tissue.

Nicotinamide and trigonelline contents increased in Catharanthus roseus tissue culture after exposure to 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) or vanadylsulfate and in Pisum sativum leaves after exposure to UV-B radiation. Vanadylsulfate increased phenylalanine ammonia-lyase (PAL) activity and the content of reduced and oxidized gluthathione in C. roseus tissue culture. The increases in PAL activity caused by 2 mM AAPH or 0.2mM vanadylsulfate were prevented by 0.1 mM 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose)polymerase. Present results support the hypothesis [Berglund, T., FEBS Lett. (1994) 351, 145-149] that nicotinamide and/or its metabolites may function as signal transmittors in the response to oxidative stress in plants and that poly(ADP-ribose)polymerase has a function in the induction of defensive metabolism.

UV-B damage and protection at the molecular level in plants.

Influx of solar UV-B radiation (280-320 nm) will probably increase in the future due to depletion of stratospheric ozone. In plants, there are several targets for the deleterious UV-B radiation, especially the chloroplast. This review summarizes the early effects and responses of low doses of UV-B at the molecular level. The DNA molecules of the plant cells are damaged by UV due to the formation of different photoproducts, such as pyrimidine dimers, which in turn can be combatted by specialized photoreactivating enzyme systems. In the chloroplast, the integrity of the thylakoid membrane seems to be much more sensitive than the activities of the photosynthetic components bound within. However, the decrease of mRNA transcripts for the photosynthetic complexes and other chloroplast proteins are among very early events of UV-B damage, as well as protein synthesis. Other genes, encoding defence-related enzymes, e.g., of the flavonoid biosynthetic pathway, are rapidly up-regulated after commencement of UV-B exposure. Some of the cis-acting nucleotide elements and trans-acting protein factors needed to regulate the UV-induced expression of the parsley chalcone synthase gene are known.

Amino acid sequence similarities between the vacuolar proton-pumping inorganic pyrophosphatase and the c-subunit of F0F1-ATPases.

Comparison of the Arabidopsis thaliana vacuolar proton-pumping inorganic pyrophosphatase with three F0F1-ATPase c-subunits revealed a strong similarity between a stretch containing amino acids 227-245 of the H(+)-PPase and a transmembrane alpha-helix of the c-subunits which contains the glutamate which binds N,N'-dicyclohexylcarbodiimide.

Reduction in cab and psb A RNA transcripts in response to supplementary ultraviolet-B radiation.

The cab and psb A RNA transcript levels have been determined in Pisum sativum leaves exposed to supplementary ultraviolet-B radiation. The nuclear-encoded cab transcripts are reduced to low levels after only 4 h of UV-B treatment and are undetectable after 3 days exposure. In contrast, the chloroplast-encoded psb A transcript levels, although reduced, are present for at least 3 days. After short periods of UV-B exposure (4 h or 8 h), followed by recovery under control conditions, cab RNA transcript levels had not recovered after 1 day, but were re-established to ca. 60% of control levels after 2 more days. Increased irradiance during exposure to UV-B reduced the effect upon cab transcripts, although the decrease was still substantial. These results indicate rapid changes in the cellular regulation of gene expression in response to supplementary UV-B and suggest increased UV-B radiation may have profound consequences for future productivity of sensitive crop species.

Proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive inorganic pyrophosphate synthase from Rhodospirillum rubrum: purification, characterization, and reconstitution.

A new method has been developed for the isolation of the proton-pumping N,N'-dicyclohexylcarbodiimide-sensitive PPi synthase (H(+)-PPi synthase) from chromatophores of Rhodospirillum rubrum. The H(+)-PPi synthase was purified by extraction of chromatophores with a mixture of nonanoyl-N-methylglucamide and cholate, by fractionation with poly(ethylene glycol) 4000, hydroxyapatite chromatography, and affinity chromatography. The purified enzyme is homogeneous and has a specific activity of 20.4 mumol of PPi hydrolyzed min-1 mg-1 at pH 7.5 and 20 degrees C. The hydrolytic activity of the enzyme was stimulated by addition of phospholipids and Triton X-100. Of the lipids tested, cardiolipin proved to have the maximal activating effect. Reconstitution of the H(+)-PPi synthase by the freeze-thaw technique yielded an uncoupler-stimulated and N,N'-dicyclohexylcarbodiimide-sensitive PPi hydrolytic activity. The subunit composition of the purified H(+)-PPi synthase was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band was obtained after silver staining with an apparent molecular weight of 56,000. The oligomeric structure of the H(+)-PPi synthase is discussed.

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria.

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PPi) in three phototrophic bacteria. Formation of PPi was found in chromatophores from Rhodopseudomonas viridis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsulatus. The maximal rate of PPi synthesis in Rps. viridis was 0.15 µmol PPi formed/(min*µmol Bacteriochlorophyll) at 23°C. The synthesis of PPi was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PPi synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PPi after short light flashes was also studied. The yield of PPi after a single 1 ms flash was equivalent to approximately 1 µmol PPi/500 µmol Bacteriochlorophyll. In Rps. viridis chromatophores, PPi was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.

Amount and turnover rate of the F0F1-ATPase and the stoichiometry of its inhibition by oligomycin in Rhodospirillum rubrum chromatophores.

The amount of F1-ATPase in chromatophores from Rhodospirillum rubrum was determined by Western blotting using anti-RrF1 rabbit antibodies. 9.1 mmol F1 (mol bacteriochlorophyll)-1 was obtained or 14% of the total protein content of the chromatophores. The turnover rate of the F0F1-ATPase was 17 molecules ATP s-1 during synthesis, 2 molecules ATP s-1 during hydrolysis under coupled conditions with Mg2+ as the divalent cation, and 7 molecules ATP s-1 during hydrolysis in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Binding of 1 mol oligomycin/mol F0F1-ATPase was found to inhibit the activities of the enzyme completely. A single binding site was found with a Kd of approximately 2 microM.

Division of divalent cations into two groups in relation to their effect on the coupling of the F0F1-ATPase of Rhodospirillum rubrum to the protonmotive force.

Divalent cations are divided into two groups in relation to their ability to promote ATP synthase catalyzed reactions. In the presence of Mg2+, the following pattern rules: (i) uncoupler-stimulated ATP hydrolysis of Rhodospirillum rubrum chromatophores which shows an optimum concentration of the divalent cation; (ii) ATP-induced proton pumping in chromatophores; (iii) light-induced ATP synthesis in chromatophores; (iv) no or very low ATPase activity of purified F1-ATPase unmasked by diethylstilbestrol or n-octyl beta-D-glucopyranoside. In the presence of Ca2+, the following pattern occurs: (i) no stimulation of the ATP hydrolysis in chromatophores by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; (ii) no ATP-induced proton pumping; (iii) no light-induced ATP synthesis; (iv) a high ATPase activity of the purified F1-ATPase which is inhibited by diethylstilbestrol and n-octyl beta-D-glucopyranoside. Co2+, Mn2+, and Zn2+ are members of the "Mg2+-group", whereas Cd2+ is suggested to fall between the two groups. Intrinsic uncoupling of the membrane-bound ATP synthase has been suggested to account for the effect caused by Ca2+ in chloroplasts [Pick, U., & Weiss, M. (1988) Eur. J. Biochem. 173, 623-628]. Such an interpretation is consistent with our results on chromatophores. The uncoupling cannot occur at the level of the membrane since neither light-induced nor Mg-ATP-induced proton pumping is affected by Ca2+. A conformational change is suggested to be the reason for this intrinsic uncoupling, and it is proposed to be controlled by the diameters of the divalent cations (Ca2+ greater than Cd2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Mg2+).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of equisetin on energy-linked reactions in Rhodospirillum rubrum chromatophores.

Light-induced proton uptake, light-induced carotenoid absorbance shift, photophosphorylation, and hydrolysis of Mg-ATP, Ca-ATP, and PPi in Rhodospirillum rubrum chromatophores are shown to be inhibited by the antibiotic equisetin. The Mg- and Ca-ATPase activities of purified F0F1-ATPase are inhibited by equisetin. In contrast, only the Ca-ATPase activity of purified F1-ATPase is decreased by equisetin, whereas the Mg-ATPase is stimulated. Both equisetin and N,N'-dicyclohexylcarbodiimide (DCCD) inhibit the hydrolytic activity of the purified H+-PPase but not the hydrolytic activity of soluble PPase from R. rubrum and yeast. The I50 for the PPi hydrolysis is near 20 microM for both equisetin and DCCD. The action of equisetin on membranes is compared to the effect of Triton X-100 and carbonyl cyanide p-trifluoromethoxyhydrazone. On the basis of these new data, equisetin is proposed to act nonspecifically on membranes and hydrophobic domains of proteins.