Methanococcus jannaschii uses a pyruvoyl-dependent arginine decarboxylase in polyamine biosynthesis

The genome sequence of the hyperthermophilic methanogen Methanococcus jannaschii contains homologs of most genes required for spermidine polyamine biosynthesis. Yet genomes from neither this organism nor any other euryarchaeon have orthologs of the pyridoxal 5'-phosphate-dependent ornithine or arginine decarboxylase genes, required to produce putrescine. Instead, as shown here, these organisms have a new class of arginine decarboxylase (PvlArgDC) formed by the self-cleavage of a proenzyme into a 5-kDa subunit and a 12-kDa subunit that contains a reactive pyruvoyl group. Although this extremely thermostable enzyme has no significant sequence similarity to previously characterized proteins, conserved active site residues are similar to those of the pyruvoyl-dependent histidine decarboxylase enzyme, and its subunits form a similar (alphabeta)(3) complex. Homologs of PvlArgDC are found in several bacterial genomes, including those of Chlamydia spp., which have no agmatine ureohydrolase enzyme to convert agmatine (decarboxylated arginine) into putrescine. In these intracellular pathogens, PvlArgDC may function analogously to pyruvoyl-dependent histidine decarboxylase; the cells are proposed to import arginine and export agmatine, increasing the pH and affecting the host cell's metabolism. Phylogenetic analysis of Pvl- ArgDC proteins suggests that this gene has been recruited from the euryarchaeal polyamine biosynthetic pathway to function as a degradative enzyme in bacteria.     

Arabidopsis polyamine biosynthesis: absence of ornithine decarboxylase and the mechanism of arginine decarboxylase activity

Unlike other eukaryotes, which can synthesize polyamines only from ornithine, plants possess an additional pathway from arginine. Occasionally non-enzymatic decarboxylation of ornithine could be detected in Arabidopsis extracts; however, we could not detect ornithine decarboxylase (ODC; EC 4. 1.1.17) enzymatic activity or any activity inhibitory to the ODC assay. There are no intact or degraded ODC sequences in the Arabidopsis genome and no ODC expressed sequence tags. Arabidopsis is therefore the only plant and one of only two eukaryotic organisms (the other being the protozoan Trypanosoma cruzi) that have been demonstrated to lack ODC activity. As ODC is a key enzyme in polyamine biosynthesis, Arabidopsis is reliant on the additional arginine decarboxylase (ADC; EC 4.1.1.9) pathway, found only in plants and some bacteria, to synthesize putrescine. By using site-directed mutants of the Arabidopsis ADC1 and heterologous expression in yeast, we show that ADC, like ODC, is a head-to-tail homodimer with two active sites acting in trans across the interface of the dimer. Amino acids K136 and C524 of Arabidopsis ADC1 are essential for activity and participate in separate active sites. Maximal activity of Arabidopsis ADC1 in yeast requires the presence of general protease genes, and it is likely that dimer formation precedes proteolytic processing of the ADC pre-protein monomer.     

Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death

Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.     

SLAMseq resolves the kinetics of maternal and zygotic gene expression during early zebrafish embryogenesis

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Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana

Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 m M NaCl before transfer to 125 m M NaCl, but not in plants that were directly transferred to 125 m M NaCl without prior treatment with 100 m M NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.     

Somatic embryogenesis of Scots pine: cold treatment and characteristics of explants affecting induction

This is the first report on Scots pine (Pinus sylvestris L.) somatic embryo plants regenerated and growing in a greenhouse. The present work focused on improving somatic embryogenesis of the species by studying the factors affecting culture induction. Developmental stage of explants that were immature female gametophytes, including the zygotic embryos with suspensor tissues, was investigated in detail. The genetic background of the material, cold treatments (14 d, 1 or 2 months at +5C) of cones including explants, as well as the plant growth regulator composition of the initiation medium, were also examined. When initiation of somatic embryogenesis was successful, the zygotic embryos in the explants were either proembryos or early embryos. Cold treatment of the cones had no significant effect on induction, nor were there any differences among the treatments with different duration, thus improving the practical applicability of the culture technique. The explants in cold-stored cones probably retained their initiation capacity due to the conversion of starch to sugars. This was observed as decreased number and size of starch grains in the megagametophytes compared with the controls. The seed family and the medium significantly affected induction success, the medium with auxin (9.1 or 13.6 M 2,4-dichlorophenoxyacetic acid) and cytokinin (2.2 M 6-benzylaminopurine) being better than the medium with cytokinin (5 M 6-benzylaminopurine) alone. The significance of the genetic background of the explants and the initiation medium indicate that it might be possible to improve the initiation rates by using explants from controlled crossings between competent genotypes, and by developing more specific media for important seed families.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

Interactions during in vitro germination of Scots pine pollen

Pre-zygotic pollen competition is believed to play an important role in nonrandom mating, i.e., the unequal success of different pollen donors. We studied pollen–pollen interactions of Scots pine in vitro using multiwell plates with freely permeable inserts. Six genotypes were included in our experiments: three from northern Finland and three from southern Finland. We conducted control experiments by placing pollen of each genotype in both the well and its insert. In competition experiments each southern genotype was incubated in insert with each northern genotype in well, and vice-versa. Samples for the germinability observations were taken from inserts. The mean germination percentage of northern genotypes was lower when incubating with southern genotypes in competition experiments than in control experiments. In one case, a northern genotype showed lower mean germination percentage in competition experiment than in control experiment and in another case a southern genotype showed a higher value. Our results suggest a chemically mediated interaction between pollen from different genotypes and one which can vary among genotypes.     

Transgene expression in regenerating cotyledons and embryogenic cultures of Scots pine

Regenerating cotyledons and embryogenic cultures of Scots pine (Pinus sylvestris L.) were used as targets for gene delivery via particle bombardment. Both target tissues differed in their response to selective agents, regulative sequences of transferred genes, and conditions optimal for particle bombardment. Of the gene constructs tested, the pB1410 including CaMV 35S promoter, AMV-translation enhancer and gusA:npt reporter and selectable marker gene fusion was the most suitable for cotyledon transformation, resulting on average in 3.7 ( 0.1 SE) -glucuronidase (GUS) expression units per cotyledon. In embryogenic cell masses the pB1221.1 gene construct including the gusA reporter gene driven by the 35S promoter gave the highest transient expression, 63 (plusmn; 15 SE) GUS expression units of g^-1 fresh weight of embryogenic cell mass. Kanamycin and geneticin concentrations suitable for selection of cotyledons were 10-15 mg dm^-3 and 0.5-1.0 mg dm^-3, respectively. Kanamycin (10 mg dm^-3) and phosphinotricin (1 mg dm^-3) as selectors caused a significant decline in growth, but geneticin did not significantly affect the growth of the embryogenic cultures during the 8 week cultivation period. The production of transgenic plantlets seems to be more dependent on the regeneration and multiplication efficiency of organogenesis and somatic embryogenesis of Scots pine than on gene delivery into regenerating tissues.     

Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis.

The Arabidopsis thaliana LEC1 gene regulates embryo morphology and seed maturation. For a better understanding of its function, we isolated a carrot (Daucus carota L. cv. US-Harumakigosun) counterpart of this gene, C-LEC1, from a cDNA library of carrot somatic embryos, since carrot is a better model plant for preparing large quantities of somatic embryos at the same developmental stage. The predicted amino acid sequence of C-LEC1 is similar to that of LEC1 and contains regions that are conserved in the heme-activated protein 3 (HAP3) subunit of plants, animals and microorganisms. C-LEC1 expression was detected in embryogenic cells, somatic embryos, and developing seeds. In situ hybridization analysis revealed C-LEC1 expression in the peripheral region of the embryos but not in the endosperm. Expression of C-LEC1 driven by Arabidopsis LEC1 promoter was able to complement the defects of the Arabidopsis lec1-1 mutant. These results suggest that C-LEC1 is a functional homolog of Arabidopsis LEC1, an important regulator of zygotic and somatic embryo development.     

Diurnal changes in polyamine content, arginine and ornithine decarboxylase, and diamine oxidase in tobacco leaves

Changes in the contents of polyamines (PAs) in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under 16 h photoperiod were correlated with arginine and ornithine decarboxylase (EC 4.1.1.19 and EC 4.1.1.17) and diamine oxidase (EC 1.4.3.6) activities. The maximum of free and soluble conjugated forms of PAs occurred 1-2 h after the middle of the light period and was followed by two distinct peaks at the end of the light and at the beginning of the dark phase. Putrescine was the most abundant and cadaverine the least abundant PA in both free and PCA-soluble forms. However, cadaverine was predominant in PCA-insoluble conjugates, followed by putrescine, spermidine, and spermine. Both arginine and ornithine decarboxylases are involved in putrescine biosynthesis in tobacco leaves. Light dramatically stimulated the activity of ornithine decarboxylase, while no photoinduction of arginine decarboxylase activity was observed. Ornithine decarboxylase was found mainly in the particulate fraction. Only one peak, just after light induction, occurred in the cytosolic fraction, with 35% of the total ornithine decarboxylase activity. By contrast, the total arginine decarboxylase activity was equally divided between the soluble and pellet fractions. A sharp increase in diamine oxidase activity occurred 1 h after exposure to light, concomitant with the light-induced increase in ornithine decarboxylase activity. After a decline, diamine oxidase activity increased again, together with the rise in the amount of free Put. The roles of both conjugation of PAs with hydroxycinnamic acids and oxidative degradation of putrescine in maintaining free PA levels during the 24 h light/dark cycle are discussed. The presented results have shown that the parameters studied here followed rhythmical changes and were not only affected by light.     

Characteristics of gene expression in morphologically abnormal needles of Scots pine

Expression of 373 genes in Scots pine morphologically abnormal needles has been analyzed with microarray analysis. It was found microarray technique allows detecting the minor changes in gene expression. The results obtained showed that changes in expression of 42 genes occur during the process of abnormal morphogenesis in the Pinus sylvestris needles. The increasing of expression level of genes encoding ubiquitin-conjugating enzyme and L-ascorbate peroxidase was revealed for all samples analyzed.