REMARKABLE CONSERVATION OF INTERNALLY TRANSCRIBED SPACER SEQUENCES OF ARTHROSPIRA (“SPIRULINA” ) (CYANOPHYCEAE,CYANOBACTERIA) STRAINS FROM FOUR CONTINENTS AND OF RECENT AND 30‐YEAR‐OLD DRIED SAMPLES FROM AFRICA1

The internally transcribed spacer (ITS) sequences of 21 Arthrospira clonal strains from four continents and assigned to four different species (A. platensis, A. maxima, A. fusiformis, A. indica) in the culture collections were determined. Two main clusters, I and II, were differentiated by 49 positions out of 475 nt or 477 nt, respectively. Each cluster was further subdivided into two subclusters. Subclusters I.A and I.B were separated by two substitutions, whereas subclusters II.A and II.B were distinguished by four substitutions. After direct sequencing of the PCR products, three dried samples from Chad aged between 3 months and 35 years yielded a sequence belonging to subcluster I.A, as did a recent commercial product. The strains grown in production plants belonged to the same (sub)clusters as strains from culture collections, mainly I.A and II. PCR primers specific for each cluster and subcluster were designed and tested with crude cell lysates of Arthrospira strains. One dried sample (“dihé” 1) and a herbarium sample from Lake Sonachi (Kenya) only contained I.A sequences, whereas the commercial product was a mixture of the four genotypes and the other two dried samples contained minor polymorphisms characteristic of different clusters. Five clonal Arthrospira strains, thought to be duplicates, showed the simultaneous presence of the two forms of the four diagnostic positions that distinguish subclusters genotype II.A and genotype II.B. This is likely to be caused by multiple copies of the rDNA operon, in a intermediate stage of homogenization between subcluster II.A and subcluster II.B. The high conservation of ITS sequences is in contrast with the assignment to four different species, the great morphological variability of the strains, and their wide geographic distribution.     

Effects of short-term ozone fumigation on tobacco plants: response of the scavenging system and expression of the glutathione reductase

The role that the constituents of the ascorbate–glutathione cycle play in the mechanism of contrasting ozone sensitivities was examined in mature and old tobacco leaves after acute ozone-fumigation (150 p.p.b., 5 h). Levels of the enzyme activities associated with the detoxifying system were lower in ozone-sensitive Bel W3 control plants than in unfumigated ozone-tolerant Bel B plants. In particular, the endogenous activities of ascorbate peroxidase (APX) and glutathione reductase (GR), and the metabolites ascorbic acid (AA) and reduced glutathione (GSH) were more abundant in Bel B than Bel W3 control plants. These results suggest that the higher tolerance of Bel B to O₃ is associated with a greater initial content of the antioxidant enzymes or metabolites. Only in the mature leaves of the ozone-tolerant Bel B cv. did fumigation trigger activation of APX and, weakly, of dehydroascorbate reductase (DHAR). The activity of these enzymes was significantly lower after ozone treatment in both mature and old leaves of Bel W3 than in control plants. Fumigation had little effect on the ascorbate content. Its main effects on the glutathione pool were that it boosted the oxidized form and lowered the reduced form, particularly in mature Bel W3 leaves. Extractable GR activity remained unchanged in both Bel B and Bel W3 immediately after fumigation, but increased slightly 24 h later, particularly in mature leaves of Bel W3. Exposure to O₃ caused a sharp decline in chloroplastic GR mRNA levels in both cultivars. However, as Western blot analysis failed to detect any major changes in GR protein content at this time, the protein must be highly stable. There is therefore a good correlation between tolerance to O₃ and high endogenous levels of antioxidant metabolites such as AA and GSH in tobacco. In addition, the degree of inducibility of the system discriminates the two cultivars investigated.     

The effects of endogenous cytokinin content on benzyladenineenhanced nitrate reductase induction

Foliar application of benzyladenine (BA) has been shown to enhance nitrate-dependent induction of nitrate reductase (NR; EC 1.6.6.1) in etiolated wheat ( Triticum aestivum L.) seedlings. Whether similar enhancement occurs in light-grown plants, or whether endogenous cytokinin content affects this enhancement is unknown. Since the cytokinin content of etiolated plants probably differs from that of light-grown seedlings, the NR response of each to exogenous root- or shoot-applied BA in wheat (cv. Red Bob) was examined. Endogenous cytokinins present in untreated control tissues prior to BA application and changes that occurred after a 22 h (12 h dark followed by 10 h of light) period were determined using a combined HPLC-immunoassay method. Shoot application of BA enhanced the induction of NR in etiolated seedlings in a concentration-dependent manner but failed to enhance NR induction in light-grown plants. Root-applied BA enhanced NR induction in both etiolated and light-grown seedlings. Endogenous root cytokinin levels were similar in both etiolated and light-grown plants. In contrast, shoots of 6 day-old light-grown seedlings contained at least 20 times the amount of total cytokinins measured in shoots from etiolated plants of the same age. Total cytokinin content of the light-grown plants diminished after the 22-h period while that measured in etiolated seedlings increased. The responsiveness of seedlings to BA was correlated with endogenous cytokinin levels in that enhancement of NR induction by exogenous BA was low in tissues which contained high concentrations of cytokinin at the time of BA application. These results may prove useful in interpretation of gene responses to exogenous plant growth regulators.     

Effects of NADH and thiol compounds on wheat leaf nitrate reductase

The initial activity of wheat leaf nitrate reductase was depressed on inclusion of the following thiol compounds; dithiothreitol, dithioerythreitol or mercaptoethanol, but not cysteine and glutathione. This thiol effect simply resulted from an interference with the chemical determination of nitrite. Preincubation of the enzyme with NAD⁺ and these thiols enhanced the inhibition of nitrate reductase activity. This effect was mediated by NADH production by the thiol reduction of NAD⁺. The inactivation by NAD⁺ in the presence of thiol compounds which was enhanced by cyanide ions could be reversed by ferricyanide, as has been observed previously for NADH-mediated inactivation of nitrate reductase.     

31P-NMR STUDIES ON INORGANIC POLYPHOSPHATES IN MICROALGAE1

Using “P nuclear magnetic resonance analysis, total inorganic polyphosphate in algae could be quantitatively estimated, For this purpose the algal suspension, which had been kept in cold trichloroacetic acid, was further treated with 6 mM EDTA, or the cells were kept in 2 N KOH containing 100 mM EDTA for 18 h at 37°C. These simple methods avoid hydrolysis of cellular inorganic polyphosphate and, therefore, are useful for the study of phosphorus metabolism in algae. The effects of these treatments on visualization of the signal for inorganic polyphosphate in nuclear magnetic resonance spectra were discussed in comparison with in vivo, ‘P nuclear magnetic resonance spectra of algae.     

Relations between uptake and utilization of NO−3 in Pisum growing exponentially under nitrogen limitation

The utilization and translocation of nitrogen was investigated in exponentially growing, nitrogen-limited Pisum sativum L. cv. Marma. The plants were given N daily at exponentially increasing, although suboptimal, relative nitrogen addition rates (R_N) calculated to yield a relative increment in N of 0.06 day^?1 and 0.12 day^?1. After 10 days of NO^?₃ additions (26 days after sowing), the relative growth rate more or less equaled R_N. Uptake of NO^?₃ was several-fold higher than the N requirement for the growth rate set by R_N. The daily addition of NO^?₃ was taken up after 7 to 8 h, resulting in a cyclic behaviour in the NO^?₃ utilization. During the phase of net NO^?₃ influx, the filling phase (0 to 8 h), in vitro nitrate reductase activity (NR activity) and intracellular levels of soluble N in the root increased. In the phase of no net influx of NO^?₃ the depletion phase (8 to 24 h), the plants were entirely dependent on stored N. During this phase both in vitro NR activity and intracellular levels of soluble N decreased. Also the calculated actual rate of NO^?₃ reduction was high in the filling phase, while it was close to zero in the depletion phase. The pattern of these fluctuations indicates that the regulation of NO^?₃ utilization involves an interplay between transmembrane fluxes of NO^?₃, the cytosolic NO^?₃ concentration and NR activity. Cyclic fluctuations in N-containing compounds were also found in the xylem. Nitrogen was mainly transported as amino acids. The pattern of NO^?₃ transport in the xylem and the fluctuations in the shoot of in vitro NR activity indicate that a reasoning similar to that for the regulation of NO^?₃ assimilation in the root also applies for the shoot. The results also indicate a substantial supply of amino acids to the xylem through recirculation from the shoot.     

Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.     

Variation in the activity of some enzymes of photorespiratory metabolism in C4 grasses

BACKGROUND AND AIMS: Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. METHODS: The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. KEY RESULTS: The NADP-malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD-malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP-malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. CONCLUSIONS: There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses.     

The role of molybdenum in agricultural plant production

BACKGROUND: The importance of molybdenum for plant growth is disproportionate with respect to the absolute amounts required by most plants. Apart from Cu, Mo is the least abundant essential micronutrient found in most plant tissues and is often set as the base from which all other nutrients are compared and measured. Molybdenum is utilized by selected enzymes to carry out redox reactions. Enzymes that require molybdenum for activity include nitrate reductase, xanthine dehydrogenase, aldehyde oxidase and sulfite oxidase. SCOPE: Loss of Mo-dependent enzyme activity (directly or indirectly through low internal molybdenum levels) impacts upon plant development, in particular, those processes involving nitrogen metabolism and the synthesis of the phytohormones abscisic acid and indole-3 butyric acid. Currently, there is little information on how plants access molybdate from the soil solution and redistribute it within the plant. In this review, the role of molybdenum in plants is discussed, focusing on its current constraints in some agricultural situations and where increased molybdenum nutrition may aid in agricultural plant development and yields. CONCLUSIONS: Molybdenum deficiencies are considered rare in most agricultural cropping areas; however, the phenotype is often misdiagnosed and attributed to other downstream effects associated with its role in various enzymatic redox reactions. Molybdenum fertilization through foliar sprays can effectively supplement internal molybdenum deficiencies and rescue the activity of molybdoenzymes. The current understanding on how plants access molybdate from the soil solution or later redistribute it once in the plant is still unclear; however, plants have similar physiological molybdenum transport phenotypes to those found in prokaryotic systems. Thus, careful analysis of existing prokaryotic molybdate transport mechanisms, as well as a re-examination of know anion transport mechanisms present in plants, will help to resolve how this important trace element is accumulated.     

Influence of near null magnetic field on in vitro growth of potato and wild Solanum species

The influence of near null magnetic field on in vitro growth of different cultures of potato and related Solanum species was investigated for various exposure times and dates. Potato (Solanum tuberosum L. cv. Désirée) in vitro cultures of shoot tips or nodal segments were used. Three different exposure periods revealed either stimulation or inhibition of root, stem, or leaf in vitro growth after 14 or 28 days of exposure. In one experiment the significant stimulation of leaf growth was also demonstrated at biochemical level, the quantity of chlorophyll a and b and carotenoids increasing more than two-fold. For the wild species Solanum chacoense, S. microdontum, and S. verrucosum, standardized in vitro cultures of nodal stem segments were used. Root and stem growth was either stimulated or slightly inhibited after 9 days exposure to near null magnetic field. Callus cultures obtained from potato dihaploid line 120/19 were maintained in near null magnetic field in 2 different months. For these experiments as well as for Solanum verrucosum, callus cultures recorded either slight inhibition or no effect on fresh weight. For all experiments significant growth variation was brought about only when geomagnetic activity (AP index) showed variations at the beginning of in vitro growth and when the explant had at least one meristematic tissue. Moreover longer maintenance in near null magnetic field, 28 days as compared to 14 days or the controls, can also make a difference in plant growth in response to geomagnetic field variations when static component was reduced to zero value. These results of in vitro plant growth stimulation by variable component of geomagnetic field also sustain the so-called seasonal "window" effect.