Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

Alfalfa (Medicago sativa L.) clones tolerant to salt stress: in vitro selection

In order to quickly and efficiently evaluate the salt tolerance of alfalfa, salinity tests were conducted on Medicago sativa L. var. australis, var. icon, var. loi, and var. gea, under in vitro conditions. Pregerminated seeds of four varieties were subjected to five different NaCl concentrations (0, 50, 100, 150, 200 mM). The influence of saline stress was estimated on the basis of survival percentage, growth parameters, and electrolyte leakage. The seedlings surviving on the medium enriched with salt at the highest concentration were presumed to be tolerant and represented the mother plants for the production of in vitro clones. In the following step, the clones were evaluated in vitro to confirm the salt tolerance. The influence of mild salt stress (75 mM NaCl) on the growth parameters of selected clones was examined. At the end of this trial, the proline accumulation and sodium content in alfalfa shoots were also quantified. The results suggest an increased level of proline promotes salt tolerance. Medicago sativa L. var. icon is highly tolerant in comparison with the other varieties tested. In vitro selection of M. sativa L. varieties on salt-containing media allowed us to obtain clones with increased salinity tolerance.     

The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants.2. An Arrhenius plot of the photoreduction of NADP⁺ from water by chloroplasts isolated from tomato (Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole^?1. Between 11 and 3 °C the activation energy increases to 22 kcal·mole^?1. Photoreduction of NADP⁺ by chloroplasts from another chilling-sensitive plant, bean (Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole^?1 below about 12 °C.3. The photoreduction of NADP⁺ by chloroplasts isolated from two chilling-resistant plants, lettuce (Lactuca sativa var. winter lake) and pea (Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole^?1, respectively, over the temperature range 3–25 °C.4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP⁺ from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP⁺ reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C.5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible.6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP⁺, would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.     

11,13-Dehydrodesacetylmatricarin and other sesquiterpene lactones from Artemisia ludoviciana var. Ludoviciana and the identity of artecanin and chyrsartemin B

11,13-Dehydrodesacetylmatricarin, achillin, parishin-C, vulgarin and artecanin were isolated from A. ludoviciana var. ludoviciana. The identity of artecanin and chrysartemin-B was confirmed and ¹³ C NMR and ¹H NMR data are described.     

Variation in morphological and physiological parameters in herbaceous perennial legumes in response to phosphorus supply

Change in morphological and physiological parameters in response to phosphorus (P) supply was studied in 11 perennial herbaceous legume species, six Australian native (Lotus australis, Cullen australasicum, Kennedia prorepens, K. prostrata, Glycine canescens, C. tenax) and five exotic species (Medicago sativa, Lotononis bainesii, Bituminaria bituminosa var albomarginata, Lotus corniculatus, Macroptilium bracteatum). We aimed to identify mechanisms for P acquisition from soil. Plants were grown in sterilised washed river sand; eight levels of P as KH₂PO₄ ranging from 0 to 384 μg P g^?1 soil were applied. Plant growth under low-P conditions strongly correlated with physiological P-use efficiency and/or P-uptake efficiency. Taking all species together, at 6 μg P g^?1 soil there was a good correlation between P uptake and both root surface area and total root length. All species had higher amounts of carboxylates in the rhizosphere under a low level of P application. Six of the 11 species increased the fraction of rhizosphere citrate in response to low P, which was accompanied by a reduction in malonate, except L. corniculatus. In addition, species showed different plasticity in response to P-application levels and different strategies in response to P deficiency. Our results show that many of the 11 species have prospects for low-input agroecosystems based on their high P-uptake and P-use efficiency.     

Ammonia regulation of carbon metabolism in photosynthesizing leaf discs

Alfalfa (Medicago sativa L., var. El Unico) leaf discs, floating on buffer containing NH₄Cl and photosynthesizing with ¹⁴CO₂, produced more labeled amino acid and less sucrose than did control discs (no added NH₄Cl). The level of pyruvate increased and that of phosphoenolpyruvate decreased. These and other changes in levels of labeled compounds led us to conclude that pyruvate kinase was activated by ammonia, resulting in increased transfer of photosynthetically incorporated carbon to synthesis of amino acid skeletons at the expense of sucrose synthesis. Carbon flow through enzymes catalyzing the anaplerotic reactions was apparently stimulated.     

(+)-Tsugacetal,a lignan acetal from Taiwan hemlock

By the evidence of ¹H and ¹³C NMR spectra, and single crystal structure determination, a novel lignan acetal, (+)-tsugacetal, isolated from Tsuga chinensis var. formosana, was found to have an α-conidendrin-related structure with an acetal methoxy group at the β-position.     

Propionate precursors in the biosynthesis of daunomycin and adriamycin: A 13C nuclear magnetic resonance study

A ¹³C-NMR study of the biosynthesis of daunomycin adriamycin from propionate[1-¹³C] has been carried out in cultures of Streptomyces peucetius var. caesius. Results give direct support for the postulate that a propionate ‘starter’ is involved in the biosynthesis of both metabolites.     

Combined Effects of Ultrahigh Vacuum and Temperature on the Viability of Some Spores and Soil Organisms

Considerably fewer spores of Bacillus stearothermophilus, B. megaterium, and Clostridium sporogenes were recovered than were spores of B. subtilis var. niger and Aspergillus niger after 4 to 5 days at 53 and 60 C in ultrahigh vacuum. There were no significant differences in the recoveries of these five organisms at 25 C and atmospheric pressure, and after exposure to 25 and -190 C in vacuum. At 60 C, a far greater decrease in viability was demonstrated for B. stearothermophilus, B. megaterium, and C. sporogenes in ultrahigh vacuum than at atmospheric pressure. Viable B. subtilis var. niger spores were not detected in an initial 10⁷ spores after retention at 90 C and ultrahigh vacuum, and 10⁴ spores were viable after 5 days at 90 C and atmospheric pressure from an initial 10⁶ spores. Molds and actinomycetes in soil were particularly resistant up to 69 C in vacuum. Actinomycetes were the only soil organisms recovered so far at 120 C.     

Comparative Genomic Analyses of the Vibrio Pathogenicity Island and Cholera Toxin Prophage Regions in Nonepidemic Serogroup Strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTX), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPI. In order to determine the prevalence of horizontal transfer of VPI and CTX among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI⁺ strains have also acquired the CTX. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTX prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTX prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTX independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

N-methyltyramine: Formation in Opuntia clavata and metabolism in Coryphantha macromeris var. Runyonii

-Administration of tyramine-[1-¹⁴C] to Opuntia clavata resulted in the formation of labeled N-methyltyramine. This procedure established the biosynthetic origin of the major alkaloid in this cactus as well as providing a radiolabeled chemical that was not commercially available. The N-methyltyramine-[1-¹⁴C] was in turn administered to Coryphantha macromeris var. runyonii to determine its metabolic role in the biosynthesis of the psychoactive cactus alkaloid normacromerine (N-methyl-3,4-dimethoxy-β-hydroxyphenethylamine). This feeding experiment established N-methyltyramine as a precursor to normacromerine.     

2-β-d-Glucopyranosyloxy-2-methylpropanol in Acacia sieberana var. woodii

A new diol glucoside, 2-β-d-glucopyranosyloxy-2-methylpropanol, the first reported naturally occurring monoglucoside of an aliphatic dihydric alcohol, was isolated from pods of Acacia sieberana var. woodii. Structure elucidation was based on ¹ H and ¹³C NMR spectroscopy, and enzymatic analyses. The compound was hydrolysed very slowly by almond β-glucosidase, but cleaved by a β-glucuronidase enzyme complex from Helix pomatia.     

Biosynthesis of α-spinasterol from (2- 14C (4R)-4-3H1) mevalonic acid by Spinacea oleracea and Medicago sativa

Leaves of Spinacea oleracea and Medicago sativa were incubated with (2-¹⁴C, (4R)-4³H₁ mevalonic acid and the sterols isolated. Cycloartenol had a ³H: ¹⁴C atomic ratio of 6:6 whilst oxidation to cycloartenone resulted in a ratio of 5:6 showing that tritium was present in the 3α-position and that the cycloartenol was symmetrically labelled. Separation of the 4-demethyl sterols gave α-spinasterol and a mixture of stigmast-7-enol and 24-methylcholest-7-enol, which had ³H: ¹⁴C atomic ratios of 3:5. Ozonolysis of α-spinastery] acetate gave the terminal side chain fragment as 2-ethyl-3-methyl butanoic acid. The acid contained ¹⁴C but no tritium thus showing that the C-24 hydrogen of cycloartenol is lost during the alkylation reactions leading to the C-24 ethyl group of α-spinasterol.     

Phage Resistance in a Phage-Insensitive Strain of Streptococcus lactis: Temperature-Dependent Phage Development and Host-Controlled Phage Replication

Streptococcus lactis ME2 is a dairy starter strain that is insensitive to a variety of phage, including 18. The efficiency of plating of 18 on ME2 and N1 could be increased from <1 × 10⁻⁹ to 5.0 × 10⁻² and from 7.6 × 10⁻⁷ to 2.1 × 10⁻², respectively, when the host strains were subcultured at 40°C before plating the phage and the phage assay plates were incubated at 40°C. Host-dependent replication was demonstrated in N1 at 30°C and in N1 and ME2 at 40°C, suggesting the operation of a temperature-sensitive restriction and modification system in ME2 and N1. The increased sensitivity of ME2 and N1 to 18 at 40°C was also demonstrated by lysis of broth cultures and increased plaque size. ME2 grown at 40°C showed an increased ability to adsorb 18, indicating a second target for temperature-dependent phage sensitivity in ME2. Challenge of N1 with a 18 preparation that had been previously modified for growth on N1 indicated that at 40°C phage development was characterized by a shorter latent period and larger burst size than at 30°C. The evidence presented suggests that the high degree of phage insensitivity expressed by ME2 consists of a variety of temperature-sensitive mechanisms, including (i) the prevention of phage adsorption, (ii) host-controlled restriction of phage, and (iii) suppression of phage development. At 30°C these factors appear to act cooperatively to prevent the successful emergence of lytic phage active against S. lactis ME2.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage φadh

The temperate bacteriophage adh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10^-8 to 10^-10 transductants per PFU. BglII-generated DNA fragments from phage adh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage adh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10²- to 10⁵-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of adh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the adh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::adh molecule. In addition to strain ADH, pTRK170 could be transduced via adh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Alkaline phytase activity in nonionic detergent extracts of legume seeds

Alkaline phytase activity, with a pH optimum of 8, was recovered from detergent extracts of dormant seeds of nine varieties of Phaseolus vulgaris L., Pisum sativum L. var. Early Alaska, and Medicago sativa L. This alkaline phytase of legume seeds was activated by calcium and differed from most seed phytases in its relative insensitivity to inhibition by fluoride.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

LED irradiance level affects growth and nutritional quality of Brassica microgreens

This study examines the effect of irradiance level produced by solid-state light-emitting diodes (LEDs) on the growth, nutritional quality and antioxidant properties of Brassicaceae family microgreens. Kohlrabi (Brassica oleracea var. gongylodes, ‘Delicacy Purple’) mustard (Brassica juncea L., ‘Red Lion’), red pak choi (Brassica rapa var. chinensis, ‘Rubi F₁’) and tatsoi (Brassica rapa var. rosularis) were grown using peat substrate in controlled-environment chambers until harvest time (10 days, 21/17°C, 16 h). A system of five lighting modules with 455, 638, 665 and 731 nm LEDs at a total photosynthetic photon flux densities (PPFD) of 545, 440, 330, 220 and 110 µmol m^?2s^?1 respectively were used. Insufficient levels of photosynthetically active photon flux (110 µmol m^?2 s^?1) suppressed normal growth and diminished the nutritional value of the Brassica microgreens studied. In general, the most suitable conditions for growth and nutritional quality of the microgreens was 330–440 µmol m^?2 s^?1 irradiation, which resulted in a larger leaf surface area, lower content of nitrates and higher total anthocyanins, total phenols and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging capacity. High light levels (545 µmol m^?2 s^?1), which was expected to induce mild photostress, had no significant positive impact for most of investigated parameters.     

Effects of irradiance and salinity on the growth of carpospore-derived tetrasporophytes of Gracilaria edulis and Gracilaria tenuistipitata var liui (Rhodophyta)

Gracilaria edulis and Gracilaria tenuistipitata var liui are agarophytes with high commercial value which are currently cultivated in countries like India and Thailand. They have great potential for mariculture in Malaysia. Experiments were carried out to study carpospore germination and determine the effects of irradiance and salinity on the growth of these two species. Both species showed the Dumontia type of carpospore development. Both species showed increased daily growth rate (% day^?1) with increasing irradiance and tolerance for a wide range of salinity with a preference for low salinity. G. edulis grew best at 100 μmol photons m^?2 s^?1 and 15 psu while G. tenuistipitata var liui grew best at 60–130 μmol photons m^?2 s^?1 and 15 psu. The highest growth rate obtained for G. edulis and G. tenuistipitata var liui was 13.57 and 19.7 % day^?1 respectively. tenuistipitata var liui. ANOVA showed that both irradiance and salinity have significant effect on the growth of both species (P?<?0.05). The results showed that G. tenuistipitata var liui is a good candidate for mass cultivation in Malaysian brackish waters. Besides, this study also showed the feasibility of using spore culture to provide stocks for sustainable farming of Gracilaria.     

Reactive oxygen species derived from impaired quality control of photosystem II are irrelevant to plasma-membrane NADPH oxidases

Protein quality control plays an important role in the photosynthetic apparatus because its components receive excess light energy and are susceptible to photooxidative damage. In chloroplasts, photodamage is targeted to the D1 protein of Photosystem II (PSII). The coordinated PSII repair cycle (PSII disassembly, D1 degradation and synthesis, and PSII reassembly) is necessary to mitigate photoinhibition. A thylakoid protease FtsH, which is formed predominantly as a heteromeric complex with two isoforms of FtsH2 and FtsH5 in Arabidopsis, is the major protease involved in PSII repair. A mutant lacking FtsH2 (termed var2) shows compromised D1 degradation. Furthermore, var2 accumulates high levels of chloroplastic reactive oxygen species (cpROS), reflecting photooxidative stress without functional PSII repair. To examine if the cpROS produced in var2 are connected to a ROS signaling pathway mediated by plasma membrane NADPH oxidase (encoded by AtRbohD or AtRbohF), we generated mutants in which either Rboh gene was inactivated under var2 background. Lack of NADPH oxidases had little or no impact on cpROS accumulation. It seems unlikely that cpROS in var2 activate plasma membrane NADPH oxidases to enhance ROS production and the signaling pathway. Mutants that are defective in PSII repair might be valuable for investigating cpROS and their physiological roles.Key words: reactive oxygen species (ROS), photosystem II repair cycle, chloroplast, FtsH, NADPH oxidase, D1 protein, protein turnoverPhotosynthetic apparatus components receive excess light energy that can ultimately engender photoinhibition.^1,2 Chloroplasts are therefore equipped with molecular systems to minimize accumulation of photodamaged proteins.³ Photosystem II, a large pigment-protein complex located in the thylakoid membrane, transfers electrons to plastoquinone and drives oxidation of water molecules using light energy.^4,5 Because PSII is an initial and rate-limiting step of electron flow, photosynthetic organisms have evolved a unique mechanism of protein quality control (PSII repair cycle), in which the damage is centralized into the reaction center D1 protein, and in which PSII is recycled efficiently.^6,7 Several lines of evidence from genetic and biochemical studies indicate that a prokaryotic ATP-dependent metalloprotease FtsH plays a critical role in D1 turnover of the PSII repair.^8–12In chloroplasts, FtsH forms a heteromeric complex with two major isoforms.^13,14 Mutants lacking either major isoform (var2 lacking FtsH2 or var1 lacking FtsH5) show leaf variegation forming white sectors that contain cells with aberrant plastids.^8,9,11,15 The variegated phenotype implies that FtsH is involved not only in D1 degradation but also in thylakoid development.^16,17 We conducted in vivo D1 degradation assays using “non-variegated” var1 and var2 mutants (owing to a trans-acting suppressor mutation fug1).^18,19 Results showed that both D1 degradation and PSII electron transport rates were impaired in these non-variegated lines.¹⁹ Collectively, our results corroborate the important role of chloroplast FtsH in the PSII repair cycle. We also infer that the variegation phenotype in var mutants is separable from the defect in the PSII repair.Two important observations concomitant with impaired D1 degradation were made in our recent study.¹⁹ One is the accumulation of PSII partial complexes in var2. Two-dimensional blue-native SDS-PAGE analysis demonstrated that thylakoid-membrane fractions from var2 chloroplasts contained fewer PSII supercomplexes (representing functional PSII) and more partial PSII complexes (representing disassembled intermediates in the PSII repair cycle). These results indicate, although indirectly, that the impaired D1 degradation affects the disassembly/ reassembly step of the PSII repair cycle. The other important observation is the accumulation of reactive oxygen species (ROS), such as superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in var2. Results of NBT staining indicated that O₂⁻ is specially localized in chloroplasts of var2 green sectors in a light-dependent manner. No NBT staining was detected in wild type under identical conditions. Similarly, DAB staining indicated that H₂O₂ is detectable in var2 green sectors. High ROS in var2 therefore demonstrates that chloroplasts suffer from photooxidative stress without a functional PSII repair system. Where these ROS are generated within chloroplasts remains unclear. We raise one possibility: that PSII partial complexes in var2 contribute to ROS production because they potentially accumulate excitation energy that might not be used for water oxidation.A considerable amount of chloroplastic O₂⁻ in var2 might be converted rapidly to H₂O₂, which can then be exported to cytosol or to other organelles for detoxification. Simultaneously, H₂O₂ in cytosol might act as a signaling molecule and consequently affect responses to environmental stress.²⁰ We raised one possibility: cpROS in var2 are influenced by an apoplastic oxidative burst that is mediated by plasma membrane-bound NADPH oxidases and which further activates downstream signaling cascades. For example, cpROS produced in guard cells of ozonetreated Arabidopsis were shown to activate certain NAPDH oxidases through the action of heterotrimeric G protein signaling. ²¹ Although Gα subunit activated by cpROS is primarily involved in oxidative bursts, Gβγ complexes appear to act on further production of cpROS.²¹ These observations led us to examine whether high cpROS in var2 are regulated by NADPH oxidases.Ten genes for NADPH oxidases (AtRbohA to AtRbohJ) are reported in Arabidopsis.²² Among these, AtRbohD and AtRbohF are expressed in mesophylls and are involved in cpROS signaling in guard cells.^22–24 To investigate the effect of these NADPH oxidases on cpROS accumulation in var2, we generated double mutants (var2/atrbohD and var2/atrbohF). The degrees of leaf variegation were similar in var2 and the double mutants. Single atrbohD and atrbohF mutants did not accumulate detectable ROS (not shown). We observed strong signals in var2/ atrbohD and var2/atrbohF double mutants both in NBT and DAB stains (Fig. 1). Overall, results showed no significant difference in the accumulation of cpROS between var2 and the double mutants. Furthermore, results of our microarray analyses demonstrated that expression levels of AtRbohD and AtrbohF are similar between var2 green sectors and wild type (unpublished data). Taken together, these results suggest that no apparent NADPH oxidase activities in plasma membranes contribute to cpROS detected in var2.Open in a separate windowFigure 1ROS accumulation in var2 and var2/atrboh mutants. (A) In situ detection of superoxide by staining with NBT (blue, bottom panels) in four-week-old wild type (Columbia), var2, var2/atrbohD, var2/atrbohF leaves. Bar = 1 mm. (B) In situ detection of hydrogen peroxide by DA B staining (dark brown, bottom panels) in four-week-old wild type (Columbia), var2, var2/rbohD, var2/rbohF leaves. Bar = 1 mm.Actually, ROS transiently generated by apoplastic NADPH oxidases are known to regulate a cell-death signaling pathway such as a hypersensitive response against pathogen infection.²⁵ Based on results of our current genetic and microarray analyses, we reason that, in var2, constitutive cpROS do not activate the ROS-mediated signaling pathway. Nevertheless, involvement of cpROS in signaling cascades has been suggested in other experimental systems. The mutants described in this report might be valuable for use in future studies.