Utilization of putrescine in tobacco cell lines resistant to inhibitors of polyamine synthesis

Three tobacco cell lines have been analyzed which are resistant to lethal inhibitors of either putrescine production or conversion of putrescine into polyamines. Free and conjugated putrescine pools, the enzymic activities (arginine, ornithine, and S-adenosylmethionine decarboxylases), and the growth characteristics during acidic stress were measured in suspension cultures of each cell line. One cell line, resistant to difluoromethylornithine (Dfr1) had a very low level of ornithine decarboxylase activity which was half insensitive to the inhibitor in vitro. Intracellular free putrescine in Dfr1 was elevated 10-fold which was apparently due to a 20-fold increase in the arginine decarboxylase activity. The increased free putrescine titer was not reflected in an increased level of spermidine, spermine, or putrescine conjugation. Dfr1 cultures survived acidic stress at molarities which were lethal to wild type cultures. Two other mutants, resistant to methylglyoxal bis(guanylhydrazone) (Mgr3, Mgr12), had near normal levels of the three decarboxylases and normal titers of free putrescine, spermidine, and spermine. Both mutants however had elevated levels of conjugated putrescine. Mgr12 had an increased sensitivity to acidic medium. These results suggest that increased levels of free putrescine production may enhance the ability of tobacco cells to survive acid stress. This was supported by the observation that cytotoxic effects of inhibiting arginine decarboxylase in wild type cell lines were dependent on the acidity of the medium.     

Ultraviolet mutagenesis and genetic analysis of resistance to methylglyoxal-bis (guanylhydrazone) in tobacco

Summary We have isolated cell lines of Nicotiana tabacum resistant to methylglyoxal-bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase. We obtained 31 resistant lines from ultraviolet light mutagenized cultures, representing at least 13 independent events. No resistant lines were obtained from non-mutagenized control cultures. The increase in mutation rate due to the ultraviolet light treatment was 22 to 62 fold increased over an estimate of the maximum possible spontaneous rate. In reconstruction experiments we reselected resistant cell lines from varying dilutions into a background of a constant concentration of wild type cells; at the minimum ratio of resistant cells to wild type cells, 1:125,000, we recovered resistant colonies at an estimated plating efficiency of 12.5%. A number of resistant lines have been regenerated into plants. All of the ones that flowered are male sterile, sometimes associated with morphological transformations. Some are female sterile as well. Meiotic genetic analysis of one resistant line, Mgr12, suggests that the MGBG resistance is segregating as a nuclear dominant trait. The male sterility and abnormal floral development of Mgr12 cosegregate with the MGBG resistance, suggesting the two phenotypes are coincident.     

Putrescine activated S-adenosylmethionine decarboxylase from Trypanosoma brucei brucei

Trypanosoma brucei brucei contained a S-adenosyl-L-methionine decarboxylase (AdoMetDC) strongly activated by putrescine. The enzyme was also activated to a lesser extent by cadaverine and 1,3-diaminopropane. Spermidine and spermine had no effect on basal activity of the enzyme. However, they interfered with putrescine activation of trypanosomal AdoMetDC. The trypanosomal enzyme could not be precipitated with antiserum against human AdoMetDC. The trypanosomal AdoMetDC enzyme subunit was labeled by reaction with ³⁵S-decarboxylated AdoMet in the presence of NaCNBH₄, and found to have a molecular weight of 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit was readily degraded on storage to a form with a molecular weight of 26 kDa. The specificity of labeling of AdoMetDC by this procedure was confirmed by the prevention of ³⁵S-decarboxylated S-adenosylmethionine (AdoMet) binding in the presence of specific AdoMetDC inhibitors [either methylglyoxal bis(guanylhydrazone (MGBG), a reversible inhibitor, or 5-deoxy-5-[(2-hydrazinoethyl)methylamino]adenosine (MHZEA), an irreversible inactivator]. As compared to human AdoMetDC, the trypanosomal enzyme showed weaker binding to a column of MGBG-Sepharose and also was significantly less sensitive to inhibition by MGBG and its congener ethylglyoxal bis(guanylhydrazone) (EGBG). Thus, the trypanosomal AdoMetDC differs significantly from its mammalian and bacterial counterparts and may therefore be exploited as a specific target for chemotherapy of trypanosomiasis.     

Chromosomal location of genes for resistance to powdery mildew in common wheat (Triticum aestivum L. em. Thell.) 4. Gene Pm 24 in Chinese landrace Chiyacao

 Chinese wheat landrace Chiyacao exhibited a response pattern different from that of the cultivars/lines possessing documented Pm genes after inoculation with 106 isolates of Erysiphe graminis f. sp. tritici. To characterize this resistance and to determine the chromosomal location of the gene or genes present, we crossed the landrace to susceptible cultivar ‘Chinese Spring’ and also to a set of 21 ‘Chinese Spring’ monosomic lines. Monosomic F₁ plants were allowed to self-pollinate and to produce F₂ seeds. Seedlings of F₂ plants and their parents were inoculated with isolates nos. 5 and 12 of Erysiphe graminis f. sp. tritici. The results revealed that one major dominant gene is located on chromosome 6D of Chinese common wheat landrace Chiyacao. The new gene is designated Pm 24. Received: 12 May 1997 / Accepted: 23 May 1997     

A 23-kDa, root exudate polypeptide co-segregates with aluminum resistance in Triticum aestivum

We previously reported that treatment with aluminum (Al) leads to the accumulation of several polypeptides (12-, 23-, and 43.5-kDa) in root exudates of an Al-resistant cultivar of Triticum aestivum. In this report, we examine the segregation of the 23-kDa, Al-induced polypeptide and the Al-resistant phenotype in single F₂ plants arising from a cross between Al-resistant and Al-sensitive doubled-haploid (DH) lines. Single plants and plant populations were screened for sensitivity/resistance to Al using synthesis of 1,3-β-glucans (callose) as a sensitive marker for Al injury. Callose production in the Al-sensitive cv. Katepwa was approximately 3-fold higher than observed in the Al-resistant cv. Maringa, or a near-isogenic line derived from Katepwa and Maringa (Alikat), over a broad range of Al concentrations (0–100 μM). Similar results were observed with DH lines developed from cv. Katepwa, which produced two–four times more callose than DH lines developed from cv. Alikat. When single plants from F₁ and F₂ populations derived from a cross between DH Katepwa and DH Alikat were scored for Al-induced callose production after 4 days exposure to 100 μM Al, all F₁ plants were Al-resistant and F₂ plants segregated approximately 3:1 for Al-resistance/sensitivity. A backcross population derived from crossing Al-resistant F₁ with Al-sensitive Katepwa, segregated 1:1 for Al-resistance/sensitivity. Thus, the Al-resistant phenotype is inherited in a monogenic, dominant fashion in our DH lines. Enhanced accumulation of the Al-induced, 23-kDa polypeptide in root exudates was a trait which co-segregated with the Al-resistant phenotype in F₂ populations. This polypeptide was strongly labeled with S-methionine after 3 days of Al exposure and 6 h labeling. When root exudate polypeptides were separated by immobilized metal ion affinity chromatography, the 23-kDa polypeptide demonstrated significant Al-binding capacity. This polypeptide has been purified to near-homogeneity, providing an opportunity to isolate the gene(s) encoding this polypeptide.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Targeted resequencing reveals geographical patterns of differentiation for loci implicated in parallel evolution

Parallel divergence and speciation provide evidence for the role of divergent selection in generating biological diversity. Recent studies indicate that parallel phenotypic divergence may not have the same genetic basis in different geographical locations – ‘outlier loci’ (loci potentially affected by divergent selection) are often not shared among parallel instances of phenotypic divergence. However, limited sharing may be due, in part, to technical issues if false‐positive outliers occur. Here, we test this idea in the marine snail Littorina saxatilis, which has evolved two partly isolated ecotypes (adapted to crab predation vs. wave action) in multiple locations independently. We argue that if the low extent of sharing observed in earlier studies in this system is due to sampling effects, we expect outliers not to show elevated F_ST when sequenced in new samples from the original locations and also not to follow predictable geographical patterns of elevated F_ST. Following a hierarchical sampling design (within vs. between country), we applied capture sequencing, targeting outliers from earlier studies and control loci. We found that outliers again showed elevated levels of F_ST in their original location, suggesting they were not generated by sampling effects. Outliers were also likely to show increased F_ST in geographically close locations, which may be explained by higher levels of gene flow or shared ancestral genetic variation compared with more distant locations. However, in contrast to earlier findings, we also found some outlier types to show elevated F_ST in geographically distant locations. We discuss possible explanations for this unexpected result.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

Ontogenetic change in the amount and position of slow-oxidative myotomal muscle in relationship to regional endothermy in juvenile yellowfin tuna Thunnus albacares

Myotomal slow-oxidative muscle (SM) powers continuous swimming and generates heat needed to maintain elevated locomotor muscle temperatures (regional endothermy) in tunas. This study describes how the amount and distribution of myotomal SM increases with fish size and age in juvenile yellowfin tuna Thunnus albacares in relationship to the development of regional endothermy. In T. albacares juveniles 40–74 mm fork length (L_F; n = 23) raised from fertilised eggs at the Inter-American Tropical Tuna Commission Achotines Laboratory in Panama and larger juveniles (118–344 mm L_F; n = 5) collected by hook and line off of Oahu, Hawaii, USA, SM was identified by histochemical staining for the mitochondrial enzyme succinic dehydrogenase or by colour (in the two largest individuals). The cross-sectional area of myotomal SM at 60% L_F, a position with maximal percentage of SM in larger T. albacares, increased exponentially with L_F. The percentage of total cross-sectional area composed of SM at 60% L_F increased significantly with both L_F and age, suggesting that SM growth occurs throughout the size range of T. albacares juveniles studied. In addition, the percentage of SM at 60% L_F that is medial increased asymptotically with L_F. The increases in amount of SM and medial SM, along with the development of the counter-current heat-exchanger blood vessels that retain heat, allow larger tuna juveniles to maintain elevated and relatively stable SM temperatures, facilitating range expansion into cooler waters.     

Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C₁₂H₂₅-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H₁₂-TAC) among many other detergents for extracting the yeast F₁F₀ ATP-synthase. H₁₂-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H₁₂-TAC or fluorinated surfactants such as C₂H₅-C₆F₁₂-C₂H₄-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H₂F₆-TAC) or C₆F₁₃-C₂H₄-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F₆-TAC), two surfactants exhibiting a comparable polar head to H₁₂-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H₁₂-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H₁₂-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H₂F₆-TAC and F₆-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F₆-TAC than with H₂F₆-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H₂F₆-TAC or F₆-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks.     

Drought-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through increased release of ethylene and changes in the ratio of polyamines in pakchoi

To study the mechanisms of drought inhibiting photosynthesis and the role of PAs and ethylene, the photosynthetic rate (P_n), the maximal photochemical efficiency of PSII (F_v/F_m), the intercellular CO₂ concentration (C_i), photorespiratory rate (P_r), the amount of chlorophyll (chl), antioxidant enzyme activity, ethylene levels, RuBPC (ribulose-1,5-bisphosphate carboxylase) activity and endogenous polyamine levels of pakchoi were examined, and an inhibitor of S-adenosylmethionine decarboxylase (SAMDC) and an inhibitor of ethylene synthesis and spermidine (Spd) were used to induce the change of endogenous polyamine levels. The results show that drought induced a decrease in P_n and RuBPC activity, an increase in the intercellular CO₂ concentration (C_i), but no change in the actual photochemical efficiency of PSII (Φ_PSII), and chlorophyll content. In addition, drought caused an increase in the free putrescine (fPut), the ethylene levels, a decrease in the Spd and spermine (Spm) levels, and the PAs/fPut ratio in the leaves. The exogenous application of Spd and amino oxiacetic acid (AOAA, an inhibitor of ethylene synthesis) markedly reversed these drought-induced effects on polyamine, ethylene, P_n, the PAs/fPut ratio and RuBPCase activity in leaves. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SAMDC resulting in the inability of activated cells to synthesize Spd and Spm, exacerbates the negative effects induced by drought. These results suggest that the decrease in P_n is at least partially attributed to the decrease of RuBPC activity under drought stress and that drought inhibits RuBPC activity by decreasing the ratio of PAs/fPut and increasing the release of ethylene.     

Inheritance studies with inbred lines of maize having different activity levels of the AP 1 controlled acid phosphatase isozymes

Summary The genetic control of acid phosphatase-1 (AP ₁) activity in pollen of maize was studied by crossing inbred lines having different AP ₁ isozymes and different activity levels of the A P ₁ enzyme. Usually, the intensities of the SS and FF isozyme bands were not equal in pollen of A P ₁ ^S /A P ₁ ^F heterozygous F ₁ hybrids, but the relative intensities of the two bands were not correlated to the activity levels of the parental lines. The A P ₁ ^S /A P ₁ ^S and A P ₁ ^F /A P ₁ ^F F ₂ populations differed in their mean level of activity. Both populations showed segregation in the activity levels indicating single gene control. The intensity ratios of the SS and FF bands in the different heterozygous A P ₁ ^S /A P ₁ ^F F ₂ plants did not segregate. The results support the competition model for gene regulation proposed by Schwartz (1971).     

Leaf gas exchange and grain yield of common bean exposed to spermidine under water stress

Three prevalent aliphatic polyamines (PAs) include putrescine, spermidine, and spermine; they are low-molecular-mass polycations involved in many physiological processes in plants, especially, under stressful conditions. In this experiment, three bean (Phaseolus vulgaris L.) genotypes were subjected to well-watered conditions and two moderate and severe water-stressed conditions with and without spermidine foliar application. Water stress reduced leaf relative water content (RWC), chlorophyll contents, stomatal conductance (gs), intercellular CO₂ concentration (C_i), transpiration rate, maximal quantum yield of PSII (F_v/F_m), net photosynthetic rate (P_N), and finally grain yield of bean plants. However, spermidine application elevated RWC, gs, C_i, F_v/F_m, and P_N, which caused an increase in the grain yield and harvest index of bean plants under water stress. Overall, exogenous spermidine could be utilized to alleviate water stress through protection of photosynthetic pigments, increase of proline and carotenoid contents, and reduction of malondialdehyde content.

     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Polyamines and Morphogenesis - Effects of Methylglyoxal-bis(guanylhydrazone)

The effects of methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis were studied on tuberization and cellular polyamine content using in vitro Solanum tuberosum (cv Binjte) plants. When MGBG was added to the culture medium, it produced a partial inhibition of the growth of stems and leaves; it totally blocked rhizogenesis and strongly stimulated tuber formation. Morphogenetic effects of MGBG were correlated to a 40 % decrease in free putrescine, spermidine, spermine content of the leaves and to a 28 % decrease in spermidine titer of the stems. In the tubers, this inhibitor did not change the free polyamine titer but increased by up to 85 % the titer of conjugated putrescine, spermidine, spermine. When the plants were grown in the dark, MGBG produced, like benzyladenine, a stimulation of the rate of tuberization and enhanced the content of conjugated polyamines in the tuber. These results support the hypothesis that polyamines play an important role in the morphogenesis of potato plants. The role of polyamine conjugation in tuber development is discussed.