Polyamine oxidase activity and polyamine content in maize during seed germination

Polyamine oxidase (PAO, EC 1.5.3.3) activity and polyamine content in the cell wall and soluble fractions obtained from embryos, endosperms and shoots and roots of etiolated or green seedlings of maize ( Zea mays L. cv. WF9) during the first 7 days of germination were investigated. Polyamine content was also determined in the trichloroacetic acid-soluble (free polyamines) and trichloroacetic acid insoluble (bound polyamines) fraction obtained from the same tissues. PAO activity, determined by the radiometric method based on the recovery of the labelled reaction product 1-pyrroline, was mostly localized in the cell wall fraction. The activity was very low in embryos and endosperms and present in traces in roots. In etiolated shoots PAO activity increased sharply, while in green shoots it was low and increased slowly. No polyamines were found in the cell wall fraction and only putrescine was detected in the soluble fraction, with the exception of the embryo, where spermidine and spermine were also present. In the TCA-soluble fraction of embryos, putrescine increased during imbibition, while spermidine and spermine decreased; in the endosperm no relevant changes in polyamines occurred. In the same fraction of green and etiolated seedlings, putrescine increased, giving a peak at days 3–5, while spermidine decreased to very low levels. The amount of bound polyamines was 1–4% of the free ones. The pattern of PAO activity seems to be unrelated to endogenous free polyamine content, which is the same in shoots and roots of etiolated and green seedlings. Enzyme activity, very low in ungerminated seeds, increased continuously during the progression of germination, especially in etiolated shoots, indicating a possible involvement in cell wall formation.     

Distribution of polyamines and their related catabolic enzyme in etiolated and light-grown leguminosae seedlings

Diamine-oxidase (DAO; EC 1.4.3.6) activity and di-and polyamine levels were estimated along the epicotyl and root of light-grown and etiolated lentil (Lens culinaris Medicus) and pea (Pisum sativum L.) seedlings. The activity of DAO was higher in etiolated epicotyls than in lightgrown ones. In both species there was a positive correlation between DAO activity and the diamine (putrescine and cadaverine) levels along the whole epicotyl and root. Polyamine (spermine and spermidine) distribution seemed to be associated with the meristematic and elongating zone of the epicotyl and root. The physiological function of DAO is discussed in relation to its possible role in providing hydrogen peroxide to peroxidase-dependent reactions occurring in the cell wall.Abbreviations CAD cadaverine - DA diamine - DAO diamine oxidase - PA polyamine - PUT putrescine - SPD spermidine - SPM spermine     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

Polyamine-phospholipid interaction probed by the accessibility of the phospholipidsn-2 ester bond to the action of phospholipase A2

Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV _max while the apparentK _m was slightly increased. In the presence of 4mm CaCl₂ inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl₂ polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl₂ reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries.     

Changes in polyamines and related enzymes with loss of viability in rice seeds

Putrescine, spermidine and spermine of high vigour, low vigour and non-viable (classes 1, 2 and 3 respectively) seeds of Oryza sativa increased with loss of viability. The largest concentration of spermine was found in non-viable embryos. Spermine was absent in the husks of all the three categories of seeds. Arginine decarboxylase was greatest in high vigoured seeds and its activity gradually declined with loss of viability. However, diamine oxidase and polyamine oxidase activities gradually increased with the loss of viability of the seeds while DNA, RNA and protein contents decreased. The total content of polyamines increased on kinetin treatment but declined on ABA treatment. DNA, RNA and protein followed the same trend as polyamines. The polyamine contents increased by ca 3- and 4-fold, respectively, in high vigoured and low vigoured seeds on 10^?4 M kinetin treatment. The activity of ADC followed the same change as that of the polyamines in both cases, but the reverse was observed for the activities of diamine and polyamine oxidases.     

Polyamines, both common and uncommon, under heat stress in rice (Oryza sativa) callus

Enhanced production and accumulation of free and conjugated polyamines as well as increased activities of their biosynthetic enzymes in plants have been associated with heat stress. Perchloric acid-soluble free, as well as conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated polyamines, as well as arginine decarboxylase (EC 4.1.1.19) and polyamine oxidase (EC 1.4.34) activities were higher in tolerant than in sensitive callus under non-stressed conditions. Heat stress caused greater accumulation of free and conjugated polyamines in callus of the heat-tolerant cultivar N22 than in that of the heat-sensitive cultivar IR8. In particular, the uncommon polyamines norspermidine and norspermine were detected in cv. N22, which increased appreciably during stress, but they were not detected in callus of cv. IR8. Arginine decarboxylase and polyamine oxidase activities increased to a larger extent in N22 than in IR8 callus during stress, activities that were well correlated with the increased levels of common and uncommon polyamines. Increased levels of transglutaminase activity indicated the high titre of conjugated polyamines.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Inhibition of polyamine biosynthesis and growth in plant pathogenic fungi in vitro

Polyamine (PA) biosynthesis inhibitors, difluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal bis-(guanylhydrazone) (MGBG) and bis-(cyclohexylammonium) sulphate (BCHA) have been tested for their effects on colony diameters at different intervals after inoculation of four plant pathogenic fungi (Helminthosporium oryzae, Curvularia lunata, Pythium aphanidermatum and Colletotrichum capsici). All these inhibitors, except DFMA had strongly retarded the growth of four fungi in a dose- and species-dependent fashion, and H. oryzae and C. lunata were found to be most sensitive to the effects of PA inhibitors. P. aphanidermatum and C. capsici were relatively insensitive and required rather high concentrations of inhibitors to get greater inhibition of mycelial growth, except DFMA which had stimulatory effect on the growth of these two fungi. However DFMA had greatly suppressed the growth of H. oryzae and C. lunata. The effect was generally more pronounced with MGBG than with DFMO and BCHA, and 1 mM Put completely prevented the inhibitory effects of 1 and 5 mM DFMO. Analysis of free and conjugated PAs in two sensitive fungi (H. oryzae and C. lunata) revealed that Put was present in highest concentrations followed by Spd and Spm and their levels were greatly reduced by DFMO application, and such inhibitions were totally reversed by exogenously supplied Put; in fact, PA titers were considerably increased by 1 mM Put alone and in combination with 1 mM DFMO. These results suggest that PA inhibitors, particularly DFMO and MGBG may be useful as target-specific fungicides in plants.     

Involvement of putrescine in the inductive rooting phase of poplar shoots raised in vitro

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence.
The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ¹-pyrroline pathway, in the inductive phase of rooting in poplar shoots.