Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Vanillic acid, a metabolite of 4-hydroxy-3-methoxyphenylglycol and 4-hydroxy-3-methoxymandelic acid in man

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with ¹⁴C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 ± 1.1% (mean ± SEM) was excreted as ¹⁴C-labelled vantllic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium-labelled HMPG ([²H₃]HMPG) and HMMA ([²H₆]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [²H₃]VA from [²H₃]HMPG was 8.3 ± 2.1% and the recovery of [²H₆]VA from [²H₆]HMMA was 9.0 ± 2.1%. The ²H-labelled VAs were probably formed by a decar boxylation reaction, in the case of HMPG after previous oxidation to HMMA.     

Tracing the slow growth of anaerobic methane-oxidizing communities by 15N-labelling techniques

The anaerobic oxidation of methane (AOM) is an important methane sink in marine ecosystems mediated by still uncultured Archaea . We established an experimental system to grow AOM communities in different sediment samples. Approaches to show growth of the slow-growing anaerobic methanotrophs have been either via nucleic acids (quantitative PCR) or required long-term incubations. Previous long-term experiments with ¹³C-labelled methane led to an unspecific distribution of the ¹³C-label. Although quantitative PCR is a sensitive technique to detect small changes in community composition, it does not determine growth yield. Therefore, we tested an alternative method to detect a biomass increase of AOM microorganisms with ¹⁵N-labelled ammonium as N-source. After only 3 weeks, significant ¹⁵N-labelling became apparent in amino acids as major structural units of microbial proteins. This was especially evident in methane-containing incubations, showing the methane-dependent uptake of the ¹⁵N-labelled ammonium by microorganisms. Cell counts demonstrated a two- and fourfold increase at ambient or elevated methane concentrations. With denaturing gradient gel electrophoresis, over 6 months incubation no changes in community composition of sulphate-reducing bacteria and archaea were detected. These data indicate doubling times for AOM microorganisms between 2 and 3.4 months. In conclusion, the ¹⁵N-labelling approach proved to be a sensitive and fast way to show growth of extremely slow-growing microorganisms.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Chitinolytic bacteria and chitin mineralization in the marine waters and sediments along the Antartic Peninsula

Abstract Chitinolytic bacteria were enumerated and isolated from marine waters and sediments along the highly productive Antarctic Peninsula. Chitinolytic bacteria were found in low concentrations (approximately 1 cell per ml) in the water column and at much higher levels in marine surface sediments (10⁴–10⁵ per g). The predominant chitinolytic bacteria isolated from the water column were identified as psychrophilic Vibrio spp. Rates of chitin mineralization were measured by collection of ¹⁴CO₂ respired from ¹⁴C-labeled chitin synthesized from chitosan and [1-¹⁴C]acetic anhydride. Chitin mineralization rates were extremely low in the marine waters analyzed (0.00085–0.0019% of the added label respired in 48 h) and appreciably higher in the marine sediments (0.0039–0.01% per 48 h), suggesting that the sediments are much more important in chitin degradation. Such low mineralization rates suggest that chitin may be accumulating in Antarctic marine sediments, though animals may also play an important role in chitin degradation.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

Abstract: A ⁴⁵Ca²⁺ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N -methyl- d -aspartate (NMDA) receptors. Inhibition of glutamate-stimulated ⁴⁵Ca²⁺ influx by six glycine-site antagonists and inhibition of glycine-stimulated ⁴⁵Ca²⁺ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated ⁴⁵Ca²⁺ influx for recombinant NMDA receptors, inhibiting and stimulating ⁴⁵Ca²⁺ influx into cells expressing NR1a/NR2A receptors (IC₅₀ = 408 µ M ) and NR1a/NR2B receptors (EC₅₀ = 37.3 µ M ), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC₅₀ = 0.099 µ M ) compared with NR1a/NR2A receptors (IC₅₀ = 164 µ M ). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg²⁺ and Zn²⁺ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of ⁴⁵Ca²⁺ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

THE EFFECT OF DIABETES, INSULIN AND WALLERIAN DEGENERATION ON LEUCINE METABOLISM OF ISOLATED RAT SCIATIC NERVES

Abstract– ¹⁴CO₂ production and ¹⁴C incorporation into proteins was studied in isolated rat sciatic nerves during incubation with 0.1 mM-[1-¹⁴C]leucine. Rats were made diabetic with streptozotocin. Nerves from diabetic rats incubated with glucose oxidized more [¹⁴C]leucine than controls. This difference was abolished in the presence of insulin (1 mU/ml). The effects of diabetes and insulin on leucine oxidation could not be demonstrated in the absence of glucose. Insulin stimulated the incorporation of [¹⁴C] from leucine into proteins by nerves from controls and diabetic rats.
Nerves undergoing Wallerian degeneration showed a marked increase in DNA content and stimulated incorporation of [¹⁴C]leucine into proteins. ¹⁴CO₂ production from leucine proceeded at 75% of the rate observed in intact nerves. Neither insulin nor diabetes affected leucine metabolism in degenerating nerves.
Neither the extracellular space nor the concentration of free amino acids were significantly different in nerves obtained from control and diabetic rats, except for lower glutamine content in the latter.
In vitro leucine metabolism of nerves is affected by diabetes, insulin and the integrity of the axon. The Schwann cell is suggested as a possible site of the observed changes in leucine metabolism.     

Oligotrophic properties of heterotrophic bacteria and in situ heterotrophic activity in pelagic seawates

Abstract The distribution of heterotrophic bacteria in polluted coastal and unpolluted pelagic seawaters was studied using a ¹⁴C-MPN method with either five of seven kinds of ¹⁴C-organic compounds as substrates. The total number of heterotrophic bacteria in pelagic waters ranged from 9.2 × 10³ to 5.4. ¢ 10⁴ cell/ml and more than 85% of the heterotrophic bacteria were represented by obligate oligotrophs. In coastal waters, the number of heterotrophs was one order of magnitude higher (av. 3.5 ¢ 10⁵ cells/ml), and eutrophic and facultatively oligotrophic bacteria were predominant. Oligotrophs in pelagic waters had a high specificity for the utilization of amino acids, especially glycine, and acetate-utilizing bacteria were scarce. The in situ maximum uptake rates of glutamate and glycine were much higher than those of glycolate and acetate. Acetate uptake rates were extremely low or not detectable in pelagic waters. The specificity of uptake kinetics is assumed to depend on the existence of obligate oligotrophs as dominant bacteria in pelagic seawater.     

Heterotrophic prokaryotic production in ultraoligotrophic alpine karst aquifers and ecological implications

Spring waters from alpine karst aquifers are important drinking water resources. To investigate in situ heterotrophic prokaryotic production and its controlling factors, two different alpine karst springs were studied over two annual cycles. Heterotrophic production in spring water, as determined by [³H]leucine incorporation, was extremely low ranging from 0.06 to 6.83 pmol C L⁻¹ h⁻¹ (DKAS1, dolomitic-karst-spring) and from 0.50 to 75.6 pmol C L⁻¹ h⁻¹ (LKAS2, limestone-karst-spring). Microautoradiography combined with catalyzed reporter deposition-FISH showed that only about 7% of the picoplankton community took up [³H]leucine, resulting in generation times of 3–684 days. Principal component analysis, applying hydrological, chemical and biological parameters demonstrated that planktonic heterotrophic production in LKAS2 was governed by the respective hydrological conditions, whereas variations in DKAS1 changed seemingly independent from discharge. Measurements in sediments recovered from LKAS2, DKAS1 and similar alpine karst aquifers ( n =12) revealed a 10⁶-fold higher heterotrophic production (average 19 μmol C dm⁻³ h⁻¹) with significantly lower generation times as compared with the planktonic fraction, highlighting the potential of surface-associated communities to add to self-purification processes. Estimates of the microbially mediated CO₂ in this compartment indicated a possible contribution to karstification.     

Activation of rainbow trout macrophages

Rainbow trout peritoneal macrophages were stimulated in vitro using Concanavalin A (Con A) and in vivo using formalin-killed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA). Whether these cells had been activated was determined by the measurements of oxygen anions (NBT reduction), H₂O₂ production (oxidation of phenol red), RNA synthesis (³H-uridine incorporation), acid phosphatase activity and bactericidal activity.
In vitro -stimulated macrophages showed an increased NBT reduction and ³H-uridine incorporation over a range of Con A concentrations, compared with untreated control macrophages, but no detectable increases in H₂O₂ production or bactericidal activity were observed. On the other hand, in vivo -stimulated peritoneal cells showed increases in all the assays compared with FIA-elicited control cells, and were considered to have been activated.     

Size-dependent mortality of red sea bream, Pagrus major, juveniles released with fluorescent otolith-tags in News Bay, Japan

Totals of 2^.67 x 10⁵ and 7^.56 x 10⁵ juvenile red sea bream of three size groups (10, 20 and 40 mm t.l.) marked with a fluorescent substance in the otolith were released in News Bay, Oita Prefecture, Japan, in July 1987 and June 1988, respectively; the aim was to estimate growth and mortality of different developmental stages.
Of fish released in 1987 and 1988, 10 618 and 4413, respectively were recaptured during those two years. Released fish remained in the bay until the end of summer, and afterwards migrated out towards open waters. Fish of the 40-mm group released in 1987 grew to over 200 mm t.l. in one year. Mean growth rate for 19 days after release was higher in the 40-mm group (0^.87 mm day⁻¹) than in the 20-mm group (0^.74mm day⁻¹). Survival rates over 19 days were 59^.0 and 10^.1 % for 40-mm and 20-mm fish, respectively, in 1987, and those over 30 days were 69^.2, 3^.3 and 0^.0% for 40-mm, 20-mm and 10-mm fish, respectively, in 1988.
Cannibalism was indicated by the presence of marked otoliths for 20-mm fish in the stomachs of a few 40-mm individuals recaptured 2 days after release. Size-dependent growth and size-selective mortality were both noted in juvenile red sea bream, i.e. the relative size differential between larger and smaller individuals was maintained in the period between marking and recapture, and mortality was inversely proportional to size.     

APPARENT IN VIVO ACETYLCHOLINE TURNOVER RATE IN WHOLE MOUSE BRAIN: EVIDENCE FOR A TWO COMPARTMENT MODEL BY TWO INDEPENDENT KINETIC ANALYSES

Abstract— Acetylcholine turnover has been determined in whole mouse brain using a newly available high specific activity [³H]choline (70 Ci/mmol). Animals were killed at various time points (0.25–10 min) after pulse adminstration of [³H]choline (Ch) by microwave irradiation of the head. Steady-state levels of ACh were determined by radioenzymatic analysis as described by G oldberg & M c C aman (1973) as modified by M c C aman & S tetzer , 1977. Ch levels were determined by a modification of the method of M c C aman & S tetzer (1977). Radiolabelled metabolites of [³H]Ch were separated by selective extraction of [³H]Ch and [³H]ACh inio tetraphenylboron in 3-heptanone (C arroll et al. , 1977) coupled with an enzymatic separation of [³H]Ch from [³H]ACh. A precursor-product relationship was verified for Ch and ACh specific activities. Acetylcholine turnover rate was determined by the biosynthesis ratio method (S chuberth et al. , 1969, Method 1) and by the finite-differences method (N eff et al. , 1971, Method 2). Both methods of kinetic analysis revealed two distinct turnover rates for acetylcholine. In the first phase (0.25–1.5 min post-[³H]Ch), the ACh turnover rate averaged 22nmol/g/min (both methods). During the second phase, (2–10 min) acetylcholine turnover rates were significantly ( P < 0.05 and P < 0.01) lower; i.e. 7nmol/g/min (Method 1) and 5.9 nmol/g/min (Method 2). The data are consistent with a 2-compartment model for ACh turnover in whole mouse brain. Additionally, the method described for the separation of radiolabelled metabolites of [³H]Ch allows an accurate determination of ACh turnover in as little as 2 mg of tissue.     

Determination of xanthoxin by immunoassay

2- Cis (-)xanthoxin (XA) was linked to bovine serum albumin through a Schiff's base and the adduct stabilized by sodium borohydride reduction. The conjugate (molar coupling ratio: 3 mol XA per mol protein) was highly immunogenic in rabbits. Antisera contained antibodies binding XA with high affinity (K_a= 1.8 × 10⁸ M ⁻¹). [³H]-XA (2.2 × 10¹⁴ Bq mol⁻¹) was synthesized by oxidation of [³H]-XA alcohol with MnO₂ and used to set up a radioimmunoassay [RIA, detection limit, 1 pmol; measuring range, 1 to 200 pmol (0.3 to 60 ng) XA]. The sera were also suitable for enzyme-linked-immunosorbent assay (ELISA) using XA-alkaline phosphatase conjugates. The technique was more sensitive [detection limit, 0.1 pmol; measuring range, 0.1 to 50 pmol (0.05 to 15 ng) XA] than the radioimmunoassay, but less precise.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

Modulation of 1O2-mediated retrograde signaling by the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein, a central integrator of stress and energy signaling

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts, changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Extensive genetic screens aimed at identifying constituents involved in ¹O₂-mediated plastid-to-nucleus signaling have failed to identify extraplastidic signaling components. This finding suggests that ¹O₂-mediated signals are not translocated to the nucleus via a single linear pathway, but rather through a signaling network that is difficult to block by single mutations. The complexity of this signaling network has been tackled by mutagenizing a transgenic flu line expressing the luciferase reporter gene under the control of the promoter of a ¹O₂-responsive AAA-ATPase gene (At3g28580) and isolating second site mutants that constitutively express the reporter gene at a high level. One of the mutants was shown by map-based cloning and sequencing to contain a single amino acid change in the PLEIOTROPIC RESPONSE LOCUS 1 (PRL1) protein. PRL1 suppresses the expression of AAA-ATPase and other ¹O₂-responsive genes. PRL1 seems to play a major role in modulating responses of plants to environmental changes by interconnecting ¹O₂-mediated retrograde signaling with other signaling pathways.