Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii.

The response of the antioxidative systems of leaf cell mitochondria and peroxisomes of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species Lycopersicon pennellii (Lpa) to NaCl 100 mM stress was investigated. Salt-dependent oxidative stress was evident in Lem mitochondria as indicated by their raised levels of lipid peroxidation and H2O2 content whereas their reduced ascorbate and reduced glutathione contents decreased. Concomitantly, SOD activity decreased whereas APX and GPX activities remained at control level. In contrast, the mitochondria of salt-treated Lpa did not exhibit salt-induced oxidative stress. In their case salinity induced an increase in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione-dependent peroxidase (GPX). Lpa peroxisomes exhibited increased SOD, APX, MDHAR and catalase activity and their lipid peroxidation and H2O2 levels were not affected by the salt treatment. The activities of all these enzymes remained at control level in peroxisomes of salt-treated Lem plants. The salt-induced increase in the antioxidant enzyme activities in the Lpa plants conferred cross-tolerance towards enhanced mitochondrial and peroxisomal reactive oxygen species production imposed by salicylhydroxamic acid (SHAM) and 3-amino-1,2,4-triazole (3-AT), respectively.

The high oxygen atmosphere toward the end-Cretaceous; a possible contributing factor to the K/T boundary extinctions and to the emergence of C(4) species.

Angiosperm plants were grown under either the present day 21 kPa O(2) atmosphere or 28 kPa, as estimated for the end-Cretaceous (100-65 MyBP). CO(2) was held at different levels, within the 24-60 Pa range, as also estimated for the same period. In C(3) Xanthium strumarium and Atriplex prostrata, leaf area and net photosynthesis per unit leaf area, were reduced by the high O(2), while the whole-plant respiration/photosynthesis ratio increased. The high O(2) effects were strongest under 24 Pa, but still significant under 60 Pa CO(2). Growth was reduced by high O(2) in these C(3) species, but not in Flaveria sp., whether C(3), C(4), or intermediary grown under light intensities <350 micromol m(-2) s(-1) PPF. Photosynthesis of C(3) Flaveria sp. was reduced by high O(2), but only at light intensities >350 micromol m(-2) s(-1) PPF. It is concluded that the high O(2) atmosphere at the end-Cretaceous would have reduced growth of at least some of the vegetation, thus adversely affecting dependent fauna. The weakened biota would have been predisposed to the consequences of volcanism and the K/T boundary bolide impact. Conversely, photosynthesis and growth of C(4) Zea mays and Atriplex halimus were little affected by high, 28 kPa, O(2). This suggests an environmental driver for the evolution of C(4) physiology.

Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings.

The roles of light and of the putative plastid signal in glycolate oxidase (GLO) gene expression were investigated in tobacco (Nicotiana tabacum cv. Samsun NN) seedlings during their shift from skotomorphogenic to photomorphogenic development. GLO transcript and enzyme activities were detected in etiolated seedlings. Their respective levels increased three- and six-fold during 96 h of exposure to light. The GLO transcript was almost undetectable in seedlings in which chloroplast development was impaired by photooxidation with the herbicide norflurazon. In transgenic tobacco seedlings, photooxidation inhibited the light-dependent increase in GUS activity when it was placed under the regulation of the GLO promoter P(GLO). However, even under these photooxidative conditions, a continuous increase in GUS activity was observed as compared to etiolated seedlings. When GUS expression was driven by the CaMV 35S promoter (P35S), no apparent difference was observed between etiolated, deetiolated and photooxidized seedlings. These observations indicate that the effects of the putative plastid development signal and light on GUS expression can be separated. Translational yield analysis indicated that the translation of the GUS transcript in P(GLO)::GUS seedlings was enhanced 30-fold over that of the GUS transcript in P35S::GUS seedlings. The overall picture emerging from these results is that in etiolated seedlings GLO transcript, though present at a substantial level, is translated at a low rate. Increased GLO transcription is enhanced, however, in response to signals originating from the developing plastids. GLO gene expression is further enhanced at the translational level by a yet undefined light-dependent mechanism.

News & notes: detection of microorganisms with overall cellulolytic activity.

A modification is described of the plate method for the detection of microorganisms with overall cellulolytic activity, including those like Cytophaga, in which the activity is cell bound. Within a few days of incubation colonies of cellulose-degrading bacteria formed holes in discs of lens paper placed on freshly inoculated agar plates.

Promoter activity of the 5' flanking region of a tobacco glycolate oxidase gene in transgenic tobacco plants.

A clone harboring the 5' flanking region of a tobacco glycolate oxidase (GLO) gene was isolated from a lambda EMBL3-tobacco genomic DNA library. Primer extension analysis indicated two major transcripts with 76 and 81 bp 5' UTRs. An RT-PCR assay mapped the major mRNA transcription initiation site to thymine at position 81 upstream of the translation initiation codon. A putative TATA box spanning positions -56 to -50 upstream of the transcription initiation site was found. Promoter activity of the 5' flanking region (-3.0 kb to +82 bp) was demonstrated in tobacco plants transformed with a GLO-beta-glucuronidase (GUS) chimeric gene. Furthermore, in these transgenic plants, GUS expression patterns mimicked the expression patterns of the endogenous GLO.

Growth hormone receptors in avian epiphyseal growth-plate chondrocytes.

Growth hormone receptor (GH-R) gene expression was evaluated in avian growth-plate cartilage by Northern blot and hybridization using the avian GH-R probe. A single transcript of approximately 5.2 kb was demonstrated in cultured growth-plate chondrocytes as well as in growth-plate extracts. GH receptor gene expression was inhibited by chicken GH (cGH) in a dose- and time-dependent manner. Chicken GH was more potent in down-regulating the GH-R gene expression than hGH, but on the other hand cGH exhibited a lower affinity to avian chondrocytes receptor than did the human hormone. Addition of ascorbic acid to the culture media caused cell differentiation: induction of alkaline phosphatase activity and attenuation of collagen type II gene expression. No differences in the GH-R gene expression were observed in the nondifferentiated cells compared with the differentiated cells. Chicken GH did not form any complex with the purified hGH binding protein (hGHBP), did not bind to human lymphocytes GH receptor, and did not affect Nb2 cell proliferation. These systems represent somatogenic and lactogenic types of GH receptors, respectively. In summary, avian growth-plate chondrocytes in situ and in culture exhibit GH-R and these receptors are capable of binding GH. Thus, the failure of GH to affect avian chondrocytes' proliferation was not due to either the absence of receptors on the cell membrane or to a lack in its binding activity, but rather may be due to events farther downstream.

Wheat (Triticum aestivum L.) [gamma]-Gliadin Accumulates in Dense Protein Bodies within the Endoplasmic Reticulum of Yeast.

Following their sequestration into the endoplasmic reticulum (ER), wheat storage proteins may either be retained and packaged into protein bodies within this organelle or transported via the Golgi to vacuoles. We attempted to study the processes of transport and packaging of wheat storage proteins using the heterologous expression system of yeast. A wild-type wheat [gamma]-gliadin, expressed in the yeast cells, accumulated mostly within the ER and was deposited in protein bodies with similar density to natural protein bodies from wheat endosperm. This suggested that wheat storage proteins contain sufficient information to initiate the formation of protein bodies in the ER of a heterologous system. Only a small amount of the [gamma]-gliadin was transported to the yeast vacuoles. When a deletion mutant of the [gamma]-gliadin, lacking the entire N-terminal repetitive region, was expressed in the yeast cells, the mutant was unable to initiate the formation of protein bodies within the ER and was completely transported to the yeast vacuole. This strongly indicated that the information for packaging into dense protein bodies within the ER resides in the N-terminal repetitive region of the [gamma]-gliadin. The advantage of using yeast to identify the signals and mechanisms controlling the transport of wheat storage proteins and their deposition in protein bodies is discussed.

The carboxy-terminal end of glycolate oxidase directs a foreign protein into tobacco leaf peroxisomes.

The carboxy-terminal residues of several peroxisomal proteins were shown to act as a peroxisomal targetting signal. This study was conducted to test whether the C-terminus of glycolate oxidase, a key enzyme in the glycolate metabolism pathway, is functioning as a targetting signal that directs proteins into plant leaf peroxisomes. A chimeric gene coding for a fusion protein composed of the C-terminal-truncated beta-glucuronidase, a synthetic linker of four amino acids and the last six C-terminal amino acids of glycolate oxidase, was constructed. Transformation of tobacco plants with the chimeric gene resulted in expression of beta-glucuronidase enzymic activity. About 50% of the transgenic beta-glucuronidase activity was localized to the peroxisomes. The results indicate that the six C-terminal amino acid residues of glycolate oxidase act as a targetting signal that is recognized by leaf peroxisomes.

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.

Spinach carbonic anhydrase primary structure deduced from the sequence of a cDNA clone.

A cDNA clone 1,156 base pairs in length was selected by screening a lambda gt11 library with antibodies directed against spinach chloroplast carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1). Sequence analysis revealed an open reading frame of 957 base pairs encoding a polypeptide containing 319 amino acids with a molecular weight of 34,569. This polypeptide is of sufficient size to represent the precursor of spinach chloroplast carbonic anhydrase. The polypeptide contains a sequence of 19 amino acids identical to the sequence of a cyanogen bromide fragment from spinach carbonic anhydrase. In addition, Escherichia coli was transformed with a plasmid that expresses spinach carbonic anhydrase. Lysates prepared from transformed E. coli contain acetazolamide-inhibitable carbonic anhydrase activity. The amino acid sequence of spinach carbonic anhydrase is distinct from those reported for the mammalian isozymes.

The primary structure of spinach glycolate oxidase deduced from the DNA sequence of a cDNA clone.

A cDNA clone encoding the peroxisomal enzyme glycolate oxidase (EC 1.1.3.15) was identified by probing a cDNA library of spinach with synthetic oligonucleotides based on the partial amino acid sequence of the enzyme. Determination of the DNA sequence of the 1526-nucleotide cDNA indicated a 1107-nucleotide open reading frame which encodes a polypeptide of 40,282 daltons. The polypeptide produced by in vitro transcription and translation of the cDNA insert had the same apparent subunit molecular mass as the enzyme purified from leaves, indicating that the cDNA encodes a full-length polypeptide and that no cleavage of the polypeptide is required for uptake of the polypeptide by peroxisomes. Comparison of the deduced amino acid sequence with those of two other plant peroxisomal proteins revealed a region of homology which may be involved in directing proteins to the peroxisome.

Plant regeneration from leaf protoplasts of Solanum torvum.

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10(4) per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1-2 weeks and 4 weeks later p-calli were 1-3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.

Photosynthesis and Inorganic Carbon Accumulation in the Acidophilic Alga Cyanidioschyzon merolae.

The intracellular pH and membrane potential were determined in the acidophilic algae Cyanidoschyzon merolae as a function of extracellular pH. The alga appear to be capable of maintaining the intracellular pH at the range of 6.35 to 7.1 over the extracellular pH range of 1.5 to 7.5. The membrane potential increase from -12 millivolts (negative inside) to -71 millivolts and thus DeltamuH(+) decreased from -300 to -47 millivolts over the same range of extracellular pH. It is suggested that the DeltamuH(+) may set the upper and lower limits of pH for growth. Photosynthetic performance was also determined as a function of pH. The cells appeared to utilize CO(2) from the medium as the apparent K(m(co(2))) was 2 to 3 micromolar CO(2) over the pH range of 1.5 to 7.5 C. merolae appear to possess a ;CO(2) concentrating' mechanism.

Is HCO(3) Transport in Anabaena a Na Symport?

Na(+) strongly promoted HCO(3) (-) transport in Anabaena variabilis. The effect was highly specific to this cation. Kinetic analysis indicated a progressive decrease in the K(m) (HCO(3) (-)) of the transport system with increasing Na(+) concentration. V(max) was also affected. We raise the possibility that the transport is a Na(+)-HCO(3) (-) symport; alternatively, that a Na(+)-H(+) antiport (or Na(+)-OH(+) symport) system mediates the efflux of the OH(-) ions derived from the entering HCO(3) (-) ions, and that this antiport can rate-limit HCO(3) (-) influx.

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis.

The nature of the inorganic carbon (C(i)) species actively taken up by cyanobacteria CO(2) or HCO(3) (-) has been investigated. The kinetics of CO(2) uptake, as well as that of HCO(3) (-) uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO(2) was higher than that for HCO(3) (-). Though the calculated V(max) was the same in both cases, the maximum rate of uptake actually observed was higher when HCO(3) (-) was supplied. C(i) uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO(2) was the species supplied. Observations of photosynthetic rate as a function of intracellular C(i) level (following supply of CO(2) or HCO(3) (-) for 5 seconds) led to the inference that HCO(3) (-) is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C(i) transport apparatus binds CO(2) and releases HCO(3) (-) at or near a membrane porter. The latter transports HCO(3) (-) ions to the cell interior.

Is modulation of the rate of proton pumping a key event in osmoregulation?

The net uptake of 3-O-methylglucose into leaf segments obtained from Senecio mikanioides Otto, and net proton efflux from the segments, were both promoted when the osmotic potential of the medium was decreased by addition of mannitol, sorbitol, or polyethylene glycol (optimal osmolarity, 0.3 Osmolar for mannitol and sorbitol). The effect was not due to promotion of ;aging', since the antibiotic cerulenin suppressed aging without reducing the size of the mannitol stimulation; further, mannitol did not accelerate aging. Neither was the effect ascribable to diminished efflux (i.e. reduced ;leak' because: first, visualization of the unidirectional sugar fluxes by double labeling indicated that the effect of added osmoticum was to promote influx rather than to reduce efflux; second, compartment analysis did not suggest any effect of mannitol on the rate constants for efflux from either the slowly equilibrating or more rapidly equilibrating compartment. The effect was not specific to poly-ols since it was also obtained with betaine and choline chloride. Since methyl glucose is not taken up into the phloem it could not be ascribed to a turgor effect on phloem loading. We conclude that the effect may reflect osmoregulation. As the sugar flux is probably driven by protonmotive force, it is likely that the effects on proton flux and on sugar flux are related. We suggest that the plasmalemma-sited proton pump is sensitive to the hydrostatic pressure gradient across the plasmalemma-cell wall complex, and functions both as detector and as effector in osmoregulation.

Nature of the rate-limiting step in the supply of inorganic carbon for photosynthesis in isolated asparagus mesophyll cells.

Accumulation of acid-stable and acid-labile C has been studied in cells isolated from cladophylls of Asparagus sprengeri regel, as a function of the concentrations of the various inorganic C (C(i)) species in the external medium.The rate of CO(2) fixation was higher when C(i) was supplied as CO(2) as opposed to HCO(3) (-), at the same concentration. Participation of HCO(3) (-) was indicated when the external CO(2) and HCO(3) (-) concentrations were chosen such that, owing to interconversion between C(i) species, the same CO(2) concentrations would be reached at a certain point in time, regardless of which species was supplied initially; at this point, a higher fixation rate was observed in the case of HCO(3) (-) supply.In the presence of carbonic anhydrase, the apparent affinity for C(i) was raised. This enzyme raised fixation rate even under steady-state conditions, but only at limiting external C(i) concentrations. The decrease in external CO(2) concentration was correlated with a decreasing internal C(i) level when CO(2) was the species supplied.When 10 mum CO(2) was supplied initially fixation rate was almost independent of pH. However, when HCO(3) (-) was supplied at concentrations calculated to yield 10 mum CO(2) at equilibrium, fixation rate rose with pH. Carbonic anhydrase raised the fixation rate over the entire pH range when HCO(3) (-) was the species supplied.It is concluded that CO(2) was the major C(i) species permeating from the medium to the carboxylating site. Further, our results bring clear evidence that formation of CO(2) from HCO(3) (-) in the unstirred layer, and the diffusion of both species in this layer, rate limit CO(2) fixation by these isolated cells.

Evidence for Mediated HCO(3) Transport in Isolated Pea Mesophyll Protoplasts.

The kinetics of (14)C fixation, and inorganic C (C(inorg)) accumulation, have been followed in isolated pea mesophyll protoplasts. NaH(14)CO(3) was supplied to the protoplasts in media the pH of which was varied between 7 and 8.When (14)CO(2) fixation was plotted against the calculated concentration of free CO(2) in the media, the apparent K(m) for CO(2) was observed to rise as external pH increased. The V(max) did not alter significantly. Similarly, when C(inorg) uptake, either in the light or in the dark, was plotted against external CO(2) concentration the slope of the curves was steeper at higher external pH.Investigation of the time course of uptake showed that internal C(inorg) concentration rose throughout the experimental period, and that in the light it surpassed the external C(inorg) concentration after about 3 minutes. Irradiation of protoplasts previously taking up (14)C(inorg) in the dark brought about a sharp increase in the rate of (14)C(inorg) accumulation which was sustained for at least 20 minutes.Estimates of internal pH based on the distribution of labeled 5,5-dimethyloxazoladine-2,4-dione (DMO) between protoplast and medium suggested that internal pH altered relatively little with change in external pH. The values for internal pH as calculated from C(inorg) distribution were always higher than those calculated from DMO distribution, i.e. the internal C(inorg) concentration was higher than would be predicted on the assumption of passive distribution in accordance with pH.Addition of carbonic anhydrase to the external solution was without effect either on rate of (14)CO(2) fixation or C(inorg) accumulation.Various possible interpretations of the results are considered. It is concluded that the most reasonable explanation, consistent with all the data, is that HCO(3) (-) ions can cross the protoplast membranes, and that their passage is mediated by a transfer mechanism.