Polyamines and environmental challenges: recent development

Plant Science 140 (1999) 103–125

Review

Polyamines and environmental challenges: recent development

A. Bouchereau *, A. Aziz, F. Larher, J. Martin-Tanguy

Groupe de Physiologie et Biochimie Vegetales, UMR CNRS 6553, Uni6ersite de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France

Received 6 July 1998; received in revised form 9 November 1998; accepted 9 November 1998

Abstract

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis,catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe anddiscuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant responseto environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine,long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia,and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, mayconfer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyaminesand stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmentalpollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis ofputrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed inplants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (acompatible osmolyte) accumulation. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Arginine decarboxylase; Bound polyamines; Conjugated polyamines; Environmental challenges; Ornithine decarboxy-lase; Polyamine oxidation; Proline

1. Introduction

Polyamines (PAs), spermidine (Spd), spermine(Spm) and their diamine obligate precursor pu-trescine (Put), are small aliphatic amines that areubiquitous in all plant cells. Evidence gathered inrecent years in plant systems supports their role as

regulators of cell proliferation and differentiation,previously proposed for animal and bacterial cells[1,2]. PAs are basic molecules which are positivelycharged at physiological pH [3]. They have beenshown to bind strongly in vitro to negativelycharged nucleic acids [4], acidic phospholipids [5]and many types of proteins, including numerousenzymes whose activities are directly modulated bypolyamine binding [6]. These ionic interactions areimportant in regulating the structure and functionof biological macromolecules, as well as their syn-thesis in vivo [7]. The first step in PA biosynthesisin higher plants and bacteria is the decarboxyla-tion of either ornithine or arginine, in reactionscatalysed by the enzymes ornithine decarboxylase(ODC; EC 4.1.1.17) and arginine decarboxylase(ADC; EC 4.1.1.19) (see Fig. 1, reviewed in Ref.

Abbre6iations: ACC, 1-aminocyclopropane 1-carboxylic acid;ADC, arginine decarboxylase; Ag, aminoguanidine; Agm, agmatine;Cad, cadaverine; CHA, cyclohexylamine; DAO, diamine oxidase;Dap, diaminopropane; DFMA, difluoromethylarginine; DFMO, difl-uoromethylornithine; GABA, g-aminobutyric acid; LSD, lysine de-carboxylase; MGBG, methylglyoxal bis-(guanylhydrazone); MTA,5%-methylthioadenosine; PA, polyamine; PAO, polyamine oxidase;PDH, pyrroline dehydrogenase; Put, putrescine; SAM, S-adenosyl-methionine; SAM dec, S-adenosylmethionine decarboxylase; Spd,spermidine; Spm, spermine; Tyr, tyramine.

* Corresponding author.

0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0168 -9452 (98 )00218 -0

A. Bouchereau et al. / Plant Science 140 (1999) 103–125104

Fig. 1. Pathways of biosynthesis of the major plant polyamines (Putrescine, Spermidine and Spermine), relationships with ethylenebiosynthesis. 1, Arginine decarboxylase (ADC); 2, Ornithine decarboxylase (ODC); 3, Arginase; 4, Agmatine iminohydrolase; 5,N-carbamoyl putrescine amidohydrolase; 6, SAM decarboxylase (SAM DC); 7, Spermidine synthase; 8, Spermine synthase; 9,SAM synthase; 10, ACC synthase; 11, ACC oxydase. PA inhibitors are indicated in bubbles at their target sites (DFMA,difluoromethylarginine; DFMO, difluoromethylornithine; CHA, cyclohexylamine; MGBG, methyl-(glyoxal)-bis-guanylhydra-zone).

[8]). In mammals and fungi, only one pathway(ODC reaction) leads to Put formation (reviewedin Ref. [9]). Spd and Spm are formed by thesubsequent addition of an aminopropyl moietyonto Put and Spd, respectively, in reactionscatalysed by the enzymes Spd synthase (EC2.5.1.16) and Spm synthase (EC 2.5.1.22). Theaminopropyl moiety results from the decarboxyla-tion of S-adenosylmethionine (SAM) by the en-zyme S-adenosylmethionine decarboxylase (SAMdec; EC 4.1.1.50) (see Fig. 1). Polyamines andethylene synthesis are linked through this commonprecursor (SAM) (see Fig. 1). Considerable evi-dence exists that PAs and ethylene inhibit eachother’s biosynthesis and action, in part as a resultof sharing a common precursor (reviewed in Ref.[10]). SAM is also a donor for transmethylationreactions for a number of substrates (reviewed inRef. [10]). A variety of other related compounds

have been found in plants, including cadaverine(Cad). Caddiamine is synthesized predominantlyas the result of lysine decarboxylase (LDC; EC4.1.1.18) activity (reviewed in Ref. [8]). This di-amine is not as widely distributed as Put and ismainly found in Leguminoseae [11] and in theflowers of Arum lilies [12]. As in the case of anyplant growth regulator, the intracellular free PApool does not only depend on its synthesis, butalso on several other processes includingpolyamine degradation (oxidative deamination),polyamine conjugation and polyamine transport.PAs (diamines and polyamines) are oxidativelydeaminated by the action of amine oxidases.Amine oxidases include the copper diamine oxi-dases (DAO; EC 1.4.3.6), defined on the basis oftheir higher substrate specificity towards diaminesand the flavoprotein polyamine oxidases (PAO;EC 1.5.3.3.), which oxidise Spd and Spm at their

A. Bouchereau et al. / Plant Science 140 (1999) 103–125 105

Fig. 2. Schematic representation of polyamine degradation by amine oxidases (reviewed in Ref. [11]). DAO, diamine oxidase;GABA, 7-aminobutyric acid; PAO, polyamine oxidase; PDH, pyrroline dehydrogenase.

secondary amino groups (reviewed in Ref. [13]).These enzymes are associated with the cell walls oftissues, where lignification, suberization and wallstiffering occur [14]. According to the scheme inFig. 2, DAO reaction products from Put arepyrroline, hydrogen peroxide and ammonia, whilePAO yields pyrroline and 1,5-diabicyclononane,from Spd and Spm, respectively, along with di-aminopropane (Dap) and hydrogen peroxide. Dapcan be converted into b-alanine, whereas pyrrolinecan be further catabolized to g-aminobutyric acid(GABA) in a reaction catalysed by pyrroline dehy-drogenase (PDH) [15]. The g-aminobutyric acid issubsequently transaminated and oxidised to suc-cinic acid, which is incorporated into the Krebscycle. Thus, this pathway ensures the recycling ofcarbon and nitrogen from Put [16]. Far from beingonly a means of eliminating cellular PAs, theenzymes involved in PA catabolism and the prod-ucts deriving from their action, have been demon-strated to be involved in important physiologicalprocesses [17,18]. Rapid and large increases inGABA synthesis in response to diverse environ-

mental stimuli are well documented [19]. Recentdata have shown the existence in plants (as previ-ously demonstrated in animals) of oxidative inter-conversion pathways for Put from Spd (reviewedin Ref. [20]).

In nature, PAs often occur as free molecularbases, but they can also be associated with smallmolecules like phenolic acids (conjugated forms)and also to various macromolecules like proteins(bound forms) (reviewed in Ref. [18]). The mostcommon conjugated PAs are those which are co-valently linked to cinnamic acids. PAs are conju-gated by the formation of an amide linkage,utilizing esters of CoA for the provision of theactivated carboxyl groups [21,22]. Post-transla-tional covalent linkage of PAs to proteins is cata-lyzed by a class of enzymes known astransglutaminases (EC; 2.3.2.13), which have beenlocalized both intra- and extra-cellularly [23]. Inplants, the main characteristics of plant transglu-taminases have recently been reviewed [24].

Recent results clearly indicate that PA transportacross the plasmalema is energy-dependent and


that calcium is involved in the correspondingmechanism [25]. Chemical treatments which antag-onize calmodulin action or inhibit protein kinaseand phosphatase activities markedly reduced Ca2+

-activated Put uptake. All the data support thehypothesis that calcium influences the transportprocess through a cascade pathway involvingprotein kinase and phosphatase activities.

Plants are exposed to continually and rapidlychanging environmental factors (biotic and abioticstresses). Light, temperature, water and nutrientavailability fluctuate seasonally, even hourly andthese have a major impact on plant growth andproductivity.

In higher plants, PA metabolism is responsive toexternal conditions. PAs have been implicated inmolecular signalling events in plant pathogen in-teractions (reviewed in Ref. [26]). These com-pounds are also apparently involved in plantresponse to microbial symbionts important inplant nutrition [27]. On the other hand, the oldestknown and perhaps the most dramatic changes inPA content which characterize plant cells are thoseobtained with abiotic stresses (reviewed in Ref.[28]). It is clear that major shifts in nitrogen andPA metabolism can occur when plants are starvedfor nutrients, or exposed to osmotic shock oratmospheric pollutants (reviewed in Ref. [28]). At-tention has been focused mostly on the regulationof free PA levels and biosynthetic enzyme activi-ties during response to external stimuli. Most ofthe earlier studies omitted PA catabolism and theconjugated and bound forms of PAs. In fact,conjugated and bound forms of PAs and PAcatabolism appear important for plant develop-mental processes such as floral induction, repro-ductive processes and root formation (reviewed inRef. [18]). In addition, most studies relating tostress responses have been done as the externalsymptoms of abiotic challenges become acute.

In this review, we will try to summarize somerecent data concerning the changes in PAmetabolism (biosynthesis, catabolism and regula-tion) in higher plants subjected to a wide array ofenvironmental stress conditions and to describeand discuss some of the new advances concerningthe different proposed mechanisms of PA actionimplicated in plant response to environmentalchallenges. In recent years, the availability of spe-cific inhibitors of PA biosynthesis (see Fig. 1) hasallowed the probing of the mechanisms involved

and this may help uncover the physiological sig-nificance of PA responses to several environmentalchallenges. For example, a-difluoromethylor-nithine (DFMO) is a suicide inhibitor of ODC[29]. ADC can be inhibited by the suicide inhibitora-difluoromethylarginine (DFMA) [30]. DFMAand DFMO, combine irreversibly with the activesites of ADC and ODC, respectively, thus prevent-ing further enzyme activity. The antileukaemicagent methylglyoxal bis-(guanylhydrazone)(MGBG) is a powerful inhibitor of SAM-dec [31].Spd synthase is inhibited by cyclohexylamine,CHA [31]. Finally aminoguanidine (Ag) is astrongly specific inhibitor of DAO (reviewed inRef. [13]). Furthermore, few studies have exam-ined the specific relationships between PAs andethylene during an environmental injury. Thus, inthe current review we will describe the relation-ships between PAs and ethylene in response tosome external conditions.

2. Stress factors

2.1. Mineral nutrient deficiencies

Most studies relating PAs to mineral nutrientdeficiencies had been done in leaves and/or inseedlings, as the external symptoms of deficiencybecome acute. The accumulation of Put in leavesof K+deficient barley plants was first reported byRichards and Coleman [32] and subsequent studiesby others (reviewed in Ref. [28]) have establishedthat specific role of Put in maintaining a cation–anion balance in plant tissues. As a result ofK+starvation, this diamine accumulation (viaADC activation), is widespread among mono- anddicotyledonous species and may well be a univer-sal response. In all cases, maximum Put accumula-tion coincides with the appearance of severenutrient deficiency symptoms. In a recent report, itappears that in Arabidopsis the increase of ADCactivity due to potassium deficiency stress does notappear to involve changes in mRNA or proteinabundance [33].

The physiological rationale for Put increase isunclear. The induced high levels of Put might bethe cause of the stress injury. Put might also bebeneficial for the plants. Alternatively, high levelsof Put could be one of the many physiologicalchanges induced by mineral nutrient deficiencywithout any special significance.


Recently, Geny et al. [34] reported in severalorgans of fruiting cuttings of grapevine that PAcontent (especially conjugated and bound forms)was strongly influenced by K+deficiency beforevisual K+deficiency symptoms appeared in leaves(chlorosis developed in the leaf center at the stageof fruit ripening). These changes in PA composi-tion and content, induced by K+starvation, dif-fered according to tissue and stage of developmentand were associated with alterations in a numberof developmental processes. Thus, the time offlowering was delayed, fruit set process and fruitripening were retarded and the production ofberries per plant was reduced. In flowers at anthe-sis free PA (Put and Spd), levels remained rela-tively unchanged under K+deficiency. However,there was a close and inverse correlation betweenconjugated and bound Spd and potassium nutri-tion. In contrast, an opposite situation was ob-served with conjugated and bound Put. Theseresults reflect different rates of metabolism be-tween floral tissues of control and K+starvedplants and suggest that Put in these tissues wasmetabolized faster in K+deficient plants. This wasprobably due to increased conversion of Put inSpd. The high Spd content might have resultedfrom an enhancement of the activity of SAMdecarboxylase and Spd synthase. This increase ofSpd might rely on a metabolic adjustment causedby growth processes under K+deficiency. It hasoften been postulated that high Spd levels arecharacteristic of young meristematic and unelon-gated tissues, whereas Put levels increase duringcell elongation and ageing [35]. Possibly, duringthe period of the metabolic adaptation toK+deficiency, Spd plays a role in the stabilizationof membrane systems. In leaves at this stage ofdevelopment, free PAs (Put, Spd) remained unaf-fected under K+starvation. In green berries dur-ing fruit set, K+deficiency was associated with amassive production of conjugated and bound Dap.Free Dap increased drastically, whereas free Putand Spd remained unchanged. In leaves at thisstage of growth, K+deficiency led to a massiveproduction of conjugated and bound Dap. Thesecompounds do not normally occur in leaves ofcontrols. In these organs free PAs (Put, Spd, Dap)remained relatively unaffected. Thus, during fruitset the main changes concerned the levels of Dap.These data raised the question of the function(s)of Dap in the K+deficient tissues at this stage of

development. In red berries and leaves at fruitripening (when the visual K+deficiency symptomsappeared in leaves) there was an inverse correla-tion between free Put content and K+nutrition.Similarly, we observed in several organs of fruitingcuttings of grapevine that Mg2+[34] and nitrogendeficiencies [36] induced several changes in PAcontent (especially in conjugated and boundforms), before nutrient deficiency symptoms ap-peared in leaves (at the stage of fruit ripening).These changes differed according to tissue andstage of development and were associated withseveral alterations in a number of floral and repro-ductive processes (fruit set process and fruit ripen-ing were retarded, berry production was reduced).In several cases deprivation of Mg2+or nitrogencaused a PA pattern similar to that observedunder K+deficiency; however nitrogen and Mg2+

deficiencies had some opposite effects on PAmetabolism depending on tissue and stage of de-velopment. Thus, in flowers at anthesis nitrogenstarvation led to a drastic production of conju-gated Spm. In leaves, nitrogen starvation was notassociated with a significant effect on free PAcontent and composition at the appearance ofnutrient deficiency symptoms. However, at thisstage of development these organs massively accu-mulated bound Put. These compounds did notnormally occur in control leaves.

In all cases, these data raised the question of theaccumulation of bound and conjugated PAs inmineral nutrient deficiencies.

2.2. Osmotic stress, salinity and heat

2.2.1. Osmotic stressOsmotic treatments using sorbitol induce high

levels of Put and ADC in detached oat leaves [37].Spd and Spm show a dramatic decrease. Conven-tional water-stress imposed by withholding waterled to similar results [37]. The authors also reportthat osmotica with widely different assimilationroutes, such as sorbitol, mannitol, proline, betaineand sucrose, all induce a rise in Put. These changesare coincident with measurable signs of stress,such as wilting and protein loss. Pretreatment ofoat leaves with DFMA before an osmotic shockcauses a decrease of Put and an increase of Spmtiters [38]. This pretreatment also improved theprotoplast viability, supporting the view that a risein the Put/Spd ratios may contribute to difficulties


encountered in cereal protoplast cultures [38]. Pro-toplasts treated with DFMA show significantlyenhanced ability to synthesize protein and RNA,although they still fail to divide consistently [38].When peeled oat leaves are incubated on sorbitolin the dark, they lose chlorophyll and senescerapidly [38]. Senescence can be delayed by includ-ing Spm in the incubation medium. The senes-cence-retarding effect of Spm in oat protoplastsand in dark-induced excised leaves is correlatedwith increases in the incorporation of labeled pre-cursors into proteins, RNA and DNA. Such treat-ments also result in limited mitotic activity and theinhibition of senescence-induced rise in ribonucle-ase and protease activities. Osmotic shock in thedark induces an activation of the pathway cata-lyzed by ADC [38]. The magnitude of the osmoti-cally-induced increase in ADC activity is the samein the light or in the dark. However, Put accumu-lation in dark-stressed leaves is less dramatic thanunder light. The results suggest that the primaryevent in this stress-induced phenomenon occursvery rapidly and requires de novo proteinsynthesis.

A recent work provides evidence for regulationof ADC biosynthesis during an osmotic-stress:Borrell et al [39] have reported the regulation ofADC synthesis by Spm in osmotically-stressed oatleaves using a polyclonal antibody to oat ADCand a cDNA clone encoding oat ADC. Treatmentwith Spm in combination with osmotic-stress re-sulted in increased steady-state levels of ADCmRNA, yet the levels of ADC activity decreased.This absence of correlation is explained by the factthat Spm inhibits processing of the ADC proen-zyme, which results in increased levels of thisinactive ADC form and a subsequent decrease inthe ADC-processed form. In osmotically-stressedoat leaves, degradation of cytochrome thylakoidproteins and the enzyme Rubisco can be avoidedby addition of Spm to the incubation medium [39].Thus post-translational regulation of ADC synthe-sis by Spm may be important in explaining itsanti-senescence properties. Interestingly, overex-pression of oat ADC in tobacco resulted in similardetrimental effects to those observed by ADCactivation induced by osmotic-stress in the ho-mologous oat leaf stem (chlorosis and necrosis)[40]. Inducible overexpression of oat ADC intransgenic tobacco led to an accumulation ofADC mRNA, increased ADC activity and Put

level. In the context of the role of specific di andpolyamines in plant growth and development, theresults indicate that activation of the ADC path-way, leading to high levels of endogenous Put, istoxic for the vegetative growth of the plant [40].Transgenic lines, induced during vegetative stage,displayed different degrees of an altered pheno-types, the severity of which was correlated withPut content. It is tempting to consider that theeventual synthesis of PAs from Put is the keyprotective factor for the stressed cell. Further-more, when cell cultures of drought-tolerant al-falfa lines were exposed to water-deficit caused byPEG, there was a trend toward increased accumu-lation of Spd and Spm and a loss of Put [41].Under stress in these cultures, a whole new seriesof PAs appear mostly symmetrical analogs of Spdand Spm. The most common of these long-chainedPAs are norspermidine (caldine) and norspermine(thermine) [41]. The corresponding structures areshown in Fig. 3. These uncommon long-chainedPAs were previously reported in thermophilic bac-teria when challenged with heat-stress [42,43].They were also found in halophilic bacteria grownin extreme environments [44]. In these bacteria,long-chained PAs are required for activation ofthe ribosomal ternary complex during protein syn-thesis and they influence the rate of chain elonga-tion [44]. The biosynthesis pathway leading to theuncommon PAs, caldine and thermine, requiresDap as a metabolic precursor [41] (see Fig. 3).PAO is a pivotal enzyme that generates Dap is thispathway [41]. A general picture has emerged fromthese studies: susceptible organisms tend to accu-mulate Put and/or show an inability to maintaintotal PA (Spd and/or Spm) pools upon osmotic-stress and to shift these pools to higher molecularweight PAs sometimes at the expense of Put.

In rape leaf discs the response to osmotic-stresshas been found to be associated with increases inPut and Dap and decreases in Spd levels [45]. Incontrast, Spm levels showed small changes whileCad accumulated massively. Similar results wereobserved in whole rape seedlings subjected todrought conditions. This is the first report of Cadaccumulation in osmotic-stressed tissues of higherplants. The observation that in control rape leafexplants DFMA, but not DFMO, provoked adecrease in the amount of PAs strongly suggeststhat the ADC pathway is constitutively involvedin Put and Spd biosynthesis. When the osmotically


Fig. 3. Pathway of the major long-chained PAs found in higher plants. DFMA, difluoromethylarginine; DFMO, difluoromethy-lornithine; CHA, cyclohexylamine; MGBG, methyl-(glyoxal)-bis-guanylhydrazone.

shocked discs were supplied with DFMA orDFMO, their PA contents decreased. This couldindicate that in stressed leaf explants both ADCand ODC are required for the synthesis of Put.This is in contrast with results of the studiesconcerning osmotic-stress-induced PA responses,which have been assumed to rely mainly on ADCstimulation. This is indeed the first report suggest-ing that ODC activation is also involved in theobserved changes in the PA levels during osmotictreatment. The biosynthesis of Spd from Put andSAM in rape leaf explants submitted to high os-motic treatment was found to continue. This is invariance with most studies suggesting that Putaccumulation under high osmotic shock resultsnot only from ADC activation, but also from adecrease in Spd synthase activity (reviewed in Ref.[28]). Furthermore, under osmotic-stress rape leafcells were induced to synthesize Dap through acti-vation of PAO. The results raise the question ofthe function(s) of Dap in the osmotic-stressedtissues. A typical scenario of the predicted conse-quences of metabolic adaptation that occur at thelevel of the diamines and their related metabolitesin rape leaf discs subjected in vitro to high os-motic-stress is shown in Fig. 4.

In stressed but not in unstressed rape leaf tis-sues, the PA contents (except for Agm and Dap)were depressed by treatment with ethephon, a

compound that releases ethylene [45]. The datasupport the view that in unstressed tissues ethep-hon treatment provoked an increase in ADCwhereas in stressed tissues it caused a decrease ofODC. In control tissues, the inhibitors of ethylenebiosynthesis induced an increase in Spd and adecrease in Put. This adjustment is consistent witha shift in the utilization of SAM in favour of PAbiosynthesis. In contrast, in stressed tissues theblock of ethylene synthesis provoked not only adecrease in Put level, but also a decline in that ofPAs. These observations suggest that the fluxes ofSAM towards either ethylene or PAS are ex-tremely responsive to environmental challenges.

2.2.2. SalinityThe effects of saline conditions on PA

metabolism is not always clear cut and the mecha-nism involved is generally less understood than thechange caused by osmotic-stress.

Differences in PA (Put, Spd, Spm) responseunder salt-stress have been reported among andwithin species. For example, according to Prakashet al. [46], endogenous levels of PAs (Put, Spd andSpm) decreased in rice seedlings under NaCl-stress, whereas Basu et al. [47] reported that salin-ity results in accumulation of these compounds inthe same material. Krishnamurthy and Bhagwat[48] reported that salt-tolerant rice cultivars drasti-


Fig. 4. Changes in the pathways of polyamine metabolism in rape leaf discs (control plants) as related to an upshockosmotic-stress (stressed plants). The width of the arrows represents the importance of the metabolic fluxes through the varioussteps of the pathways. DFMA, difluoromethylarginine; DFMO, difluoromethylornithine; CHA, cyclohexylamine; MGBG,methyl-(glyoxal)-bis-guanylhydrazone); DAO, diamine oxidase; GABA, aminobutyric acid; PAO, polyamine oxidase; PDH,pyrroline dehydrogenase; LDC, lysine decarboxylase.

cally accumulated high levels of Spd and Spmresulting in enhanced level of total PA, with arelative decrease in Put content. The most signifi-cant aspect of salt-sensitivity in rice cultivars was

their excessive accumulation of Put and their in-ability to maintain high levels of Spd and Spm intheir root systems when exposed to saline environ-ment. In sorghum, Spd and Spm accumulation


occurred during salt-stress and was considered asan adaptive response [49]. Santa-Cruz [50] re-ported that the (Spd+Spm)/Put ratios increasedwith salinity in the salt-tolerant tomato species(Lycopersicon pennellii, Carrel D’Arcy) but not inthe salt-sensitive tomato species (L. esculentum). Inboth species, stress treatments decreased the levelsof Put and Spd. The Spm levels did not decreasewith salinity in L. pennellii (Carrel D’Arcy) overthe salinization period, whereas they greatly de-creased in L. esculentum. These results support theprevious findings in plant leaf sections and proto-plast cultures, where sensitivity to osmotic-stresswas associated with a rise in Put and an apparentimpairment in the capacity to synthesize Spd fromPut. Consequently, the Put accumulation observedin stress-sensitive plants in response to stress mighthave resulted from an enhancement of Put synthe-sis or, alternatively, from an inhibition of theactivity of the enzyme SAM decarboxylase, thusallowing Put to accumulate. The accumulation ofPut could be favoured by the absence of Putdegradation (via DAO).

The effects of different NaCl concentrations onmaize embryogenic cells derived from immatureembryo cultures of a salt-sensitive inbred line (cv,w64) and a resistant hybrid (cv Arizona) have alsobeen reported [51]. A significant decrease in thegrowth of calluses in relation to increased saltconcentrations was observed. Embryogenic Ari-zona calluses showed the lowest growth inhibitionand calluses w64 the highest inhibition after sev-eral days of culture in saline medium. At high saltconcentrations (1.2–2%), calluses showed a signifi-cant increase in total PA (Put, Spd) content, espe-cially caused by a rise in Put. This increase wasproportionally higher for Arizona than for w64calluses. Whereas the Spd content of Arizona cal-luses was greatly augmented with salt, a dramaticSpd decrease was observed for w64 from 0.8%NaCl.

In most of the studies concerning salt-stress, theinduced PA response has been assumed to relymainly on ADC activation (reviewed in Ref. [28]).In a recent work, the effect of salinity-stress on theactivity of ADC, as well as its transcript level, wascompared in salt-sensitive and salt-tolerant ricecultivars [52]. Treatment of 72 h grown seedlings,either with increasing concentrations of NaCl orwith 150 mM NaCl for different period of time,showed a gradual increase of ADC activity in the

salt-resistant cultivar. In the salt-sensitive cultivaran intermediate increase was followed by sharpdecrease beyond 6 h or above 150 mM NaCl. Inseedlings of the salt-resistant cultivar ADC activityincreases and its transcript also accumulates dur-ing the prolonged salinity-stress. This mechanismis absent in the salt-sensitive rice cultivar, where aprolonged period of salinity-stress down-regulatesboth ADC activity and its transcript level. Usinginhibitors of Put synthesis, we have reported thatin tomato leaf explants ADC pathway is operatingunder both control and stress conditions, whereasODC is promoted only in response to salt-stress[53]. DFMA induces a marked decrease in the Putlevel in both control and stressed tissues, whileDFMO causes a strong decrease in Spd, Spm andDap only in salinized discs. In these tissues, theresponse to salt-stress has been found to be associ-ated with decreases in Put and Spd, accumulationof Cad and strong increases in Agm, Dap, Spmand Tyramine (an aromatic amine derived fromtyrosine). Using inhibitors of SAM decarboxylaseand Spd synthase activities, the results support theview that salt-stress in tomato did not block nei-ther SAM decarboxylase nor Spd synthase activi-ties, respectively. However, the increased levels ofDap reflected a salt-stress promoted PA oxidation.This is consistent with the findings of Smith [54],reporting a stimulation on DAO and PAO activi-ties in salt-stressed oat seedlings.

On the other hand, tomato leaf explants submit-ted to salt-stress accumulate very high amounts ofproline [53]. This amino acid is perhaps the mostwidely distributed compatible osmolyte (reviewedin Ref. [55]). In organisms from bacteria to plants,there is a strong correlation between increasedcellular proline levels and the capacity to surviveboth water deficit and the effects of high environ-mental salinity (reviewed in Ref. [55]). In salinizedexplants the decrease in Put and Spd contents istemporally correlated with the start of prolineaccumulation [53]. It is found that DFMA, whichblocks Put synthesis under both control and stressconditions, behaves also as an inhibitor of prolineaccumulation. In contrast, DFMO, which stronglyreduces the levels of Spd, Spm and Dap in salt-stressed leaf explants, does not exert significanteffect on proline content. In accordance with thesefindings, Put exogenously supplied at low concen-trations stimulates the accumulation of proline.Inhibition of Spd biosynthesis does not affect pro-


line accumulation under saline conditions. Exoge-nous application of Spd has no effect on prolineaccumulation. Treatment with aminoguanidine, aspecific inhibitor of diamine oxidases, strongly in-hibits proline accumulation. These results suggestthat in stressed tissues, Put and proline are con-nected by a precursor–product relationship via theactivity of DAO and GABA metabolism. In con-trast, proline accumulation is partially suppressedby exogenously supplied Spm. In all cases nomajor changes are observed in the proline contentof non-stressed leaf explants upon amine treat-ment [53].

2.2.3. HeatPlant cells have the ability to synthesize uncom-

mon long-chained PAs (caldine, thermine) previ-ously reported only in thermophilic bacteria, whenchallenged with heat-stress. In bacteria, these un-common long-chained PAs are apparently essen-tial for continued protein synthesis at hightemperatures (both in vitro and in vivo). Thermusthermophilus produces a new PA composed of fiveaminopropyl residues, which has been named cal-dopentamine (Fig. 3) [43]. Pollen and cell culturesof heat-tolerant cotton genotypes accumulatecaldine and thermine when exposed to high tem-peratures [56]. A compound, identified as cal-dopentamine, also appears in these conditions [57].Caldopentamine, like caldine and thermine, re-quires Dap as a metabolic precursor [57] (see Fig.3). Under heat-stress tolerant plants tend to main-tain or to increase total PA (Spd and Spm) pools,at the expense of Put [57].

Roy and Ghosh [58] have reported that heat-stress in rice callus was associated with the pro-duction of long-chained PAs and several changesin PA metabolism. The levels of free and boundPAs, as well as ADC and PAO activities, werehigher in tolerant than in sensitive callus undernon-stressed conditions. Heat-stress caused greateraccumulation of free and bound PAs in callus ofthe heat-tolerant cultivar than in that of the heat-sensitive cultivar. The uncommon PAs caldine andthermine were detected only in the heat-tolerantcultivar. ADC and PAO activities increased to alarger extent in the heat-tolerant cultivar than inthe heat-sensitive cultivar during stress, activitiesthat were well correlated with the increased levelsof common (Spd and Spm) and uncommon PAs.Increased levels of transglutaminase activity indi-

cated the high content of bound PAs. All thesedata show a correlation between heat-stress toler-ance, ADC, PAO and transglutaminase activities.Thus, the data raised the question of the functionsof long-chained and bound PAs under heat-stress.

2.3. Chilling stress

Chilling injury is thought to involve alterationof membrane structure. Raison and Lyons [59],proposed that the primary event causing chillinginjury is a phase transition in the molecular order-ing of membrane lipids. The membrane phasetransition would have many deleterious effects onthe tissues, including increases in membrane per-meability and alteration of the activity of mem-brane proteins. Exposure to low temperature hasbeen reported to induce Put accumulation in sev-eral species (reviewed in Ref. [26]). In wheat therewas an increase in Put induction parallel to anincrease in frost resistance [60]. Some data clearlydemonstrate that several plants responded to lowtemperature acclimation with a uniform and sub-stantial increases in Spd (reviewed in Ref. [28]). Incherimoya fruit (Annona cherimola Mill.) stored at6°C (a chilling, injury-inducing temperature) therewas a dramatic decrease in conjugated Spm andbound Spd [61]. The chilling injury of Zucchinisquash (Cucurbito pepo) can be reduced by precon-ditioning the fruit for 2 days at 10°C [62]. Precon-ditioning led to a significant increase in Spd andSpm, but not in Put levels. The increases in Spdand Spm were correlated with elevated SAM de-carboxylase activity [63]. In the controls, chillinginjury was correlated with increases in Put butdecreases in Spd and Spm. Therefore, in this caseit is the PA (Spd, Spm) proper and not the di-amine that appear associated with a protectivemechanism underlying preconditioning. Based onthe results, the authors suggest that Spd and Spmmay prevent chilling injury in squash by a mecha-nism involving protection of membrane lipids. Insupport of these correlations, pressure infiltrationwith Spd or Spm resulted in a significant increasein fruit firmness. This treatment also inhibited thedevelopment of chilling injury (brown core) [63].

All the results support the view that Spd andSpm may inhibit chilling injury by retarding lipidperoxidation. On the other hand, the reduction ofchilling injury by these compounds could be aresult of their antioxidant properties. In variance,


recently Lee et al. [64] reported that in riceseedlings of a chilling-tolerant cultivar, levels ofPut and activity of ADC in both shoots and rootsand levels of Spd and Spm and activity of SAMdecarboxylase in shoots increased after exposureto chilling. In a chilling-sensitive cultivar level ofPut and activity of ADC in shoots increasedslightly after exposure to chilling while those ofroots decreased drastically. Activity of ODC inboth cultivars remained unchanged after exposureto chilling. DFMA, but not DFMO inhibited freePut accumulation in chilled seedlings of the chill-ing-tolerant cultivar and resulted in a decrease inchilling tolerance, a decrease in survival and anincrease in electrolyte leakage. These effects werereversed by the addition of Put. Interestingly, theauthors showed that in the chilling-tolerant-culti-var, chilling induced an increase of free ABAlevels first, then ADC activity and finally free Putlevels. Fluridone, an inhibitor of ABA synthesis,inhibited the increase of free ABA levels, ADCactivity and free Put levels in chilled seedlings ofthe chilling-tolerant cultivar. These effects resultedin a lower tolerance to chilling and could bereversed by the pre-chilling treatment with ABA.Application of ABA for 48 h before chilling notonly rendered the chilling-sensitive cultivar toler-ant to chilling but also induced a change of PA(Put, Spd, Spm) pattern to be similar to chilledseedlings of the chilling-tolerant cultivar.

It seems that, for the materials used in theabove study, one of the strategies of rice seedlingsto resist chilling-stress is to raise ABA levels and,in turn, one of ABA’s functions is to enhance theADC-mediated Put synthesis. The data raise thefunction (s) of Put in chilling tolerance of wheatand rice.

An interesting question relates the positive rela-tion between ABA and ADC. In animal systems,not only PA biosynthesis (Put, Spd and Spm)considerably increased in response to hormonesbut the PAs produced as a result are necessary forthe hormone action (reviewed in Ref. [1]). Inplants, many data suggest that endogenous titersof PA change in response to exogenous hormones(namely auxins, cytokinins and gibberellins) (re-viewed in Ref. [65]). This had led some researchersto suggest that PAs may mediate the action ofplant hormones or are part of their signalresponse.

2.4. Hypoxia

Several data obtained both with plant shoots inhypoxic conditions and with seedlings of differentGramineae species subjected to lack of oxygen,provide evidence of an association between toler-ance and the capacity to accumulate Put [66]. Yet,an association with the differential tolerance toanoxia of various species was also observed withthe ADC activity of the tissues. Species such asrice and barnyard grass, which are adapted togerminate in an oxygen deprived environment,showed a greater capacity of Put accumulationthan the anoxia-intolerant species [67]. This con-sideration supports the hypothesis for a role of Putas a protective compound against hypoxia [68].The elongation of rice coleoptiles under such con-dition is closely related to the Put titer, but it isrelatively unresponsive to auxin [69]. If ADC isblocked by DFMA then Put titer and coleoptileelongation are diminished; if Put is then added,both inhibitions are reversed [69]. It appears, fromthese experiments, that Put is required for theanaerobic elongation of rice coleoptiles. Interest-ingly, Put has no effect on aerobic elongation ofrice coleoptiles where auxin is active [69]. Thisaccumulation of Put during anoxia, besides beingthe result of accelerated synthesis and inhibition ofSAM decarboxylase, could be favoured by theabsence of Put degradation (via DAO). Thebiosynthesis and accumulation of Put would serveas a homeostatic buffering mechanism to stabilizethe intracellular pH [68,69]. A stabilized pH dur-ing the anoxia-stress might permit the shoot toelongate.

In rice and wheat shoots the induction of Putaccumulation by decreasing oxygen concentrationcoincided with the beginning of elongation reduc-tion [67]. In both species, with a decrease in oxy-gen concentration, the conjugated Put becamepredominant in comparison with the free forms(80% at 0.3% oxygen) [67]. There was a negativecorrelation between Put accumulation (speciallyunder conjugated forms) and shoot elongation.

Recently [70], the role of PAs in the elongationof shoots of Scirpus under hypoxia was investi-gated. Under submergence, the levels of free Putand the activities of ADC and ODC increased, butthe levels of free Spd and Spm and the activity ofSAM decarboxylase decreased. The increases infree Put and shoot elongation in submerged shoots


diminished from the base to the apex. The increasein free Put in submerged shoots was coincidentwith the increase in shoot length. The hypoxia-in-duced increases in free Put and shoot elongationwere inhibited by both DFMA and DFMO andthe inhibitory effects were reversed by Put. Theseoverall results indicate that ADC- and ODC-medi-ated Put synthesis are essential for the elongationof Scirpus shoots grown under submergence.

3. Environmental pollutants

Ozone (O3) is a major component of air pollu-tion and is considered to have serious effects onthe vegetation in Europe and North America.Even when applied at low levels, O3-stress can leadto a significant decline in net photosynthesis, causeleaf injury and accelerate senescence [71]. Subacutelevels of O3 can induce a number of biochemicalchanges, some of which may form part of theplant’s defense mechanism(s). Reactions to thisoxidative-stress include increases in ascorbic acid,peroxidases, phenolic compounds, ethylene andpolyamines [71]. When exogenous PAs were fed totomato and tobacco plants, there was a significantsuppression of O3-induced leaf injury [72]. In O3-treated barley leaves, ADC activity increased be-fore the injury was apparent [73]. Spd contentincreased significantly during O3 treatment, bothin young and old leaves. When DFMA was ap-plied to the leaves, the rise in ADC activity wasprevented and injury to ozone exposure was con-siderably enhanced; little visual injury was ob-served in the leaves of O3 free controls treatedwith DFMA. These results suggest that PAs mayhave a protective role against O3 damage. PAs(Spd, Spm) have also been observed to accumulatein O3-treated Norway spruce and wheat [74,75].The mechanism involved is not clear, but becauseO3 effects may be largely due to oxidative damage,the possibility that PAs may be involved in freeradical scavenging has been investigated. The lev-els of superoxide radical formed enzymaticallywith xanthine oxidase or chemically from ri-boflavin or pyrogallol were inhibited in vitro byPut, Spd or Spm at 10–50 mM [75]. Superoxideradical protection and superoxide-dependent con-version of ACC were also inhibited by PAs addedto microsomal membrane preparations [75]. Theinhibition was dose-dependent and related to the

number of amino-group on the scavengers (asdescribed above). These findings, together with invivo studies showing that PAs can retard senes-cence and inhibit lipid peroxidation (reviewed inRefs. [8] and [28]), were attributed to the radical-scavenging ability of these compounds. A laterstudy however, has disputed these conclusions[76]. Leaf injury, caused by O3 in the tobaccocultivar Bel W3, could be prevented by feedingPut, Spd or Spm through the root. These exoge-nous treatments were correlated with a 2- to 3-foldincrease in soluble conjugated Put and Spd (mono-caffeoyl forms). Conjugated Put and Spd associ-ated with cell wall and membrane fractions wereincreased 4- to 6-fold. When free PAs were as-sayed in vitro for their radical-scavenging proper-ties, very low rate constants were found. On theother hand, PA conjugates had relatively high rateconstants. It was thus concluded that free PAscould not account for the protection against O3

damage, assuming a role as free radical scav-engers. It was suggested that the protective effectof exogenous free PAs was mediated by their priorconversion to conjugated forms. Consistent withthis hypothesis, it was found that monocaffeoylPut, an effective scavenger of oxyradicals, waspresent in the apoplastic fluid of tobacco leavesexposed to O3 [76]. This extracellular locationcould allow free radical scavenging at the sitewhere they are regenerated. The conjugated PAshave scavenging properties comparable to those ofknown radical efficient scavengers, e.g. ascorbate[76]. Acclimation to O3 may also involve down-regulation of ethylene biosynthesis by diversion ofSAM to PAs (Spd, Spm) [77]. Ethylene was postu-lated to be mediator of ozone toxicity [77]. On theother hand, PAs (Spd and Spm) may stabilize theplasma membrane. In a comparison of an O3-sen-sitive tobacco variety with an O3-tolerant one (re-viewed in Ref. [78]), in vivo Spd and Spm levelsrose in response to O3 in the tolerant variety,whereas stress ethylene was induced in the sensi-tive variety. A relationship between ethylene emis-sion and PA biosynthesis was found in O3-treatedpotato and tobacco plants, the leaves of whichshow early senescence in response to the pollutant[78]. In the presence of O3, all compounds ofethylene biosynthetic pathway in tobacco leaveswere up-regulated. Put and Spd levels also in-creased, as did ODC activity.


High extracellular levels of oxyradicals and per-oxides are not only found upon ozone exposure,but have also been detected as a rapid responseupon microbial infection to plants [79]. Ozone andpathogens appear to induce similar biochemicalreactions that are at least partially localized in theextracellular space, e.g. newly synthesized stressproteins such as b-glucanase, phenolic compoundsand lignin [79]. It is speculated that under bothtypes of stress, high concentrations of radicals andperoxides can initiate plant defense reactions andinclude PAs as an important component.

In addition, oxidative-stress resistance inConyza bonariensis has been correlated with highlevels of antioxidant enzyme activities (superoxidedismutase, catalase, peroxydase, etc.) [80]. Theantioxidant enzyme activities are constitutivelyhigher in the resistant biotypes than in the sensi-tive biotypes [80]. Higher levels of PAs correlatedwith paraquat oxidant resistance in Conyzacanadensis [81]. Ye et al. [82] extended their resultsby finding that constitutively elevated PA levels(under free and conjugated forms) were correlatedwith elevated levels of Put-generating enzymes(ADC and ODC) in an oxidant-stress-resistant C.canadensis. Their activities were specially elevatedin the resistant biotypes. The variations in levels ofPAs and ADC and ODC activities paralleled theconstitutive variation of antioxidant enzyme activ-ities, as well as oxidant resistance. The level ofparaquat induced oxidant resistance can be furtherenhanced by exogenous application of polyamines.The results suggest that ADC and ODC are in-volved in synthesizing the PAs necessary for oxi-dative-stress, albeit at different stages ofdevelopment. The results complement the reportsof Minton et al. [83], that paraquat toxicity isincreased when Put and Spd synthesis of Es-cherichia coli is defective. On the other hand, PAsmay be involved in inhibiting paraquat uptake orthe transport system, as found by Hart et al. [84]in an isolated root system. Furthermore, Zhelevaet al. [85] have demonstrated the alleviation ofinhibitory effects of herbicide (atrazine) on chloro-phyll content and protein synthesis by PAs.

Furthermore, pretreatment of Arabidopsis withDFMA prevented the rise of Spd induced byultraviolet C (UV-C) and increased the sensitivityof the plant to UV-C irradiation [86]. Applicationof Spd to plants partially prevented the damagingeffects induced by UV-C. On the other hand, UV

light has been shown to stimulate Put accumula-tion in cucumber [87].

4. Physiological impact and significance

4.1. Spermidine and spermine and stress tolerance

All the data support the view that Put andderived PAs (Spd, Spm, long-chained PAs) mayhave several functions during environmental chal-lenges. In several systems (except during hypoxiaand chilling tolerance of wheat and rice) an induc-tion of PAs (Spd, Spm) not Put accumulation,may confer a stress tolerance. Several findingssuggest that Put may have different effects thanthose reported for Spd and/or Spm. Put has beenreported to cause depolarization of membranesand increase potassium leakage [88]. In someplants (barley, horse bean, cotton and pea), feed-ing Put in excess of 3 mM leads to loss of turgorand causes formation of necrotic spots, whichmimic the symptoms of extreme salinization [28].Spd and Spm were shown to reduce ethylenesynthesis by inhibition of ACC synthase and con-version of ACC to ethylene (reviewed in Ref. [20]).Addition of Ca2+could reduce these effects, indi-cating that the sites of action of PAs (Spd, Spm)on the membrane are the same (reviewed in Ref.[9]). Water-stress in drought-sensitive plants pro-moted ethylene production [89]. Furthermore, in-creasing salt concentrations in salt-sensitive plantsgradually raised ethylene production rates [90].Ethylene appeared to be a factor in promoting leafsenescence (reviewed in Ref. [10]). Spd and Spmwere also effective in reducing chlorophyll break-down in the dark in many cereal and dicot leaves,the order of activity being Spm\Spd (reviewed inRef. [10]).

Spd and Spm might interact with membraneseither by inhibiting transbilayer movement ofphospholipids [91], or by stabilizing molecularcomplexes of thylakoid membranes [92,93]. Bothlipoxygenase (EC; 1.13.11.12) activity and enzyme-protein levels were reduced by incubation withSpm [88]. PAs could directly or indirectly functionas free radical scavengers [94]. Spm, which hasfour amino groups, was a more effective scavengerof superoxide than the triamine Spd, suggestingthe involvement of amino groups. In addition, theproduction of superoxide-dependent radicals by


senescing microsomal membranes was inhibited byPAs (especially by Spm), as was the superoxide-de-pendent conversion of ACC to ethylene (reviewedin Ref. [10]). On the other hand, Spd and Spminactivate the fructose-1,6 bis-phosphate phos-phatase of chloroplasts in vitro [6] and enhancethe Mehler reaction of the ferredoxin reductase inchloroplasts [95].

Increased PAs (Spd, Spm) biosynthesis duringthe transition from the G1 to the S phase of thecell cycle preceding the onset of the DNA synthe-sis in dividing cells, appears to be a universalphenomenon in animals and plants [96]. The ratioof Spd+Spm/Put is positively related to the activ-ity of macromolecular biosynthesis [97]. Spm ac-celerates mitotic activity in isolated embryonicaxes of chick-pea seeds [98]. The fact that inhibi-tion of PA (Spd and Spm) biosynthesis blocks thecells in the G1 stage of the cell cycle, supports theview that PA biosynthesis is required for nucleicacid synthesis and for the completion of the mi-totic cycle [99]. Cells progress to the S phase whenPAs are added [100]. Inhibition of mitotic initia-tion has been detected in pea roots subjected toosmotic-stress [101]. A control, acting to preventprogress from G1 phase under water-stress, wasdetected in cells of wheat leaves at the distalmargin of the meristem, where G1 phase cellsincrease from 60 to 95% in the 3- to 6-mm regionafter 48 h of stress [102]. The transition of cellsfrom G1 to mitosis is known to depend on theactivity of a multi-protein complex comprised ofprotein kinase 34cdc2 and cyclin [103,104]. Cdc(cell-division-cycle) 2 kinase activity is depressedby water-stress [102]. The response begin early andis completed rapidly. Thus, deactivation to 50%occurs within 3 h of stress imposition in cells atthe base of the meristematic zone. Furthermore, aportion of a mitotic cyclin, sequence termed BvcycII, has been isolated from Beta 6ulgaris [100]. Theexpression of Bvcyc II in quiescent sugar beet cellsin suspension culture is induced by PAs (Spd,Spm) [100]. Induction of Bvcyc II occurs within 6h of the addition of PAs. This effect is not duesimply to replenishment for a nitrogen source tothe nutrient starved cells [100]. This can indicatethat PAs have a role in controlling gene expressionand more specifically in controlling cell division.Spm has been reported in the nuclei and to playan important role in the configuration of DNA[105]. Spm and Spd, but not Put, readily change

the B-DNA to Z-DNA equilibrium [106] and alsocause DNA bending [107]. Spd is less effectivethan Spm. Different PAs (Spd, Spm) can exhibitdifferent physiological effects. It was shown [108]that some characteristics of chromatin, isolatedfrom PA-depleted human cervical carcinoma cells‘HeLa cells’ (slower sedimentation, higher DNasesensitivity and Mg solubility), could be restored bythe addition of Spm but not of Spd and Put. Lowlevels of Spm (500 mM) specifically release a set ofDNA-binding proteins (SRPs, spermine-releasedproteins) from chromatin in rice seedlings [109]. Itwas shown that several of these proteins bindDNA. The DNA-binding activity of SRPs wasdecreased upon phosphorylation. The effect ofSpm was very specific. Spd was far less effectiveand Put was essentially ineffective in releasingthese proteins. The most abundant Spm-releasedprotein was shown to be homologous to the maizeHMG1 protein. The authors propose that Spmcauses the release of SRPs by changing torsional-stress via a B- to Z-DNA transition.

Furthermore, during an osmotic-stress a post-translational regulation of ADC synthesis by Spmwas described (see above).

In animal systems, several data suggest a func-tional relationship between PAs (Spd and Spm)and cytoskeletal structures, e.g. PA starvation inPA auxotrophic CHO (Chinese hamster ovary)cells inhibited the organization of actin filamentsand of microtubules [110]. Moreover, the inhibi-tion of PA biosynthesis (Spd, Spm) also blockedcytokinesis [111].

Spm has been found to inhibit the in vitroactivity of polygalacturonase [6] and to stimulatethe activity of d-glucose-6-phosphate cyclohydro-lase, an enzyme which is involved in the synthesisof pectic substances and hemicelluloses [112].Polygalacturonase is the major enzyme responsiblefor pectin disassembly in ripening fruit [113]. Ex-pression of the polygalacturonase gene is ethyleneregulated [114]. This hormone may exert overex-pression of polygalacturonase mRNA [115].

Spd and especially Spm interact with the nega-tive charges of pectic substrates, which can condi-tion the binding of the pectin methyl esterase[116]. Messian et al. [117] propose that some of thenumerous physiological effects of PAs could bemediated by their differential ionic interactionwith pectic fragments that are known to act onmorphogenesis and pathogenesis in plants. The


ionic selectivity sequence was Spm4+\Spd3+\Ca2+\Put2+. This selectivity sequence is par-tially explained by the cationic charge and thegeometry of the PAs and the inability of diaminesto adsorb on all of the uronates of the wall andpartly by the heterogeneity of the exchange sitesthat distribute between high- and low-affinity sites.

Put is required for the anaerobic elongation ofrice coleoptile, elongation of shoots of Scirpusunder hypoxia and chilling tolerance in riceseedlings (see above). These date raise the questionof function(s) of Put during these processes. Dur-ing chilling tolerance, Put could bind to antioxy-dant enzymes, such as superoxide dismutase, or beconjugated to small antioxydant molecules andallow them to permeate to the sites of oxidative-stress within the cells. A covalent Put-superoxidedismutase complex was 20-fold more membranepermeable than superoxide dismutase alone, facili-tating oxidant protection in a mammalian system[118]. Endogenous or constitutive Put levels mightaffect the redox state of plant cells. Nagele et al.[119] reported that the superoxide dismutase-mimetic copper complex of copper-Put-pyridinehas many functions in mammalian cells. The com-plex can dismutate superoxide with high efficiencyand enhance oxidation of glutathione. It can pro-tect against peroxide-induced cell injury.

4.2. Bound polyamines and stress tolerance

An interesting question relates to the origin andfunction of bound PAs under several environmen-tal stresses and mineral nutrient deficiencies. Inmung beans, the ratio of the free PAs to boundPAs was high during the growth stages and de-creased with the aging of the tissues [120]. Somereports [121,122] demonstrated high level of en-dogenous binding of Put and Spd to proteins inactively dividing young tissues and the lower bind-ing in mature non-dividing tissues. Protein-conju-gated PAs have been found to be involved inphotosynthetic functions. Thus, the enzyme Ru-bisco has been identified as a substrate for transg-lutaminase [123]. Its activity is Ca2+andlight-stimulated and is responsible for the incorpo-ration of PAs into thylakoid and stromal proteins[123]. The protein substrates were also some apo-proteins of the chlorophyll a/b antenna complex[124]. Purified native light-harvesting complex(LHC) II was incubated with purified transglutam-

inase in the light and in the dark. The apoproteinwas covalently linked mainly in the light withdecreasing efficiency to Spm, Spd and Put, butonly if the LHC II pigments were linked to theapoprotein. Thus, the conformation of the an-tenna proteins could affect the PA linkage, or viceversa, it could be PA-regulated, with a possibleeffect on light harvesting efficiency [124].The in-duction of transglutaminase activity at woundedsites of excised or bruised leaves suggest a poten-tial role in wound repair [125]. Alternatively, thediscovery of PAs linked to cell wall polysaccha-rides or membranous fractions indicate prospectsfor a function in formation and anchoring of theplant cell wall polysaccharide net to the plasmamembrane, or in coupling membrane proteins tocytoplasmic structural protein [126]. A number ofbiological functions involving protein cross-linkinghave been proposed for extracellular transglutami-nases in animal systems including formation of thefibrin clot as a terminal step in the bloodclottingcascade [127]. On the other hand, in mammaliansystems proposed role for transglutaminase in-clude an involvement in stabilization of the extra-cellular matrix [128] in exocytosis [129] and intransmembrane signal mediation, acting as a G-protein [130].

4.3. Conjugated polyamines and stress tolerance

In several cases the response to abiotic injuryand mineral nutrient deficiency is associated withthe production of conjugated PAs. Are they stor-age forms for the PAs or biologically active sub-stances? Slocum and Galston [131] suggested thatthere is limited exchange between the free and theconjugated PAs. Likewise, Flores and Filner [16]claimed that conjugation to cinnamic acids mightbe a way of regulating the free PA pool in theplant cell. It has also been proposed that theconjugates may act as means for PA translocation[132,133] and that they could be preferred sub-strates for amine oxidases (reviewed in Ref. [18]).In addition, these compounds are good substratesfor peroxidases in cells of tobacco leaves [134].Peroxidases which utilize PA conjugates may re-move H2O2 in the apoplast. Conjugation of PAsmay regulate their interactions with inorganiccations such as Ca2+, which might have an impli-cation for the proposed role of PA on membranestabilization. Conjugation reactions could regulate


PA functions by, for example, affecting their bind-ing and interaction with proteins, nucleic acids orphospholipids. If the latter interaction stabilizesmembranes, then amine conjugation may lead tomembrane destabilization and thereby contributeto senescence. Then PA conjugation may have acausal effect in development. Previous resultsdemonstrated that high levels of PAs and espe-cially Put conjugates, inhibit cell multiplicationand suppress differentiation in a number of species(reviewed in Ref. [18]). Furthermore, elevated lev-els of these compounds can contribute to abnor-mal floral development (reviewed in Ref. [18]). Thepossibility also exists that PA conjugation to acinnamoyl moiety would be important in thedetoxicating phenolic compounds known to in-hibit growth (reviewed in Ref. [18]). In addition,hydroxycinnamic acids are antioxidants andphenylpropanoid accumulation could be involvedin preventing Al toxicity [135]. It was proposedthat the protective effect of exogenous PAs againstthe damage of the superoxides was dependent ontheir prior conversion to conjugated forms (asdescribed above).

4.4. ADC/ODC and stress tolerance

In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants)recent results indicate that both ADC and ODCare required for the synthesis of Put and PAs (Spdand Spm). This is in contrast with results of moststudies concerning stress-induced polyamine re-sponses, which have been assumed to rely on ADCstimulation (see above). To what extent do alter-nate pathways, leading to Put or PA synthesis,play a role in biochemical adaptation? What deter-mines the flux rates through the ADC versus ODCpathways?

Increases in ODC in response to stress may bephysiologically important, considering the com-partmentation of both ADC and ODC. ODC isknown to be located in the nucleus [131], whileADC could be rather associated with either thecytosol or the chloroplasts [136]. With a localiza-tion of ADC in chloroplasts, Borrell et al. [136]concluded that the role of the PA synthesized maybe to maintain photosynthetic activity, preventingosmotic-stress induced senescence. Several reports(reviewed in Ref. [18]) suggest that ODC could beinvolved in the regulation of cell division in active

growing tissues, while that of ADC could bemainly concerned with cell extension. In S. cere-6isiae it has been shown that when the ODC geneis inactivated, cell division stops but the cell ex-pansion continues [137]. The relative contributionof ADC and ODC to the biosynthesis of Put andconsequently of PAs, might also be dependent oncell water content at the cell level.

The pathway via ODC is a major one for pro-duction of conjugated and bound PAs and isrequired for floral development, root formationand formation of undifferentiated callus (reviewedin Ref. [18]). On the other hand, several free PAsderived from ADC may have an application invegetative development (reviewed in Ref. [18]).

Another interesting question relates the produc-tion of Cad in a response to an abiotic injury. Therole of Cad in plant tissue is not known but it issuggested that when Put synthesis is blocked,lysine is channelled into Cad formation to com-pensate for the lack of PA formation, as wasshown for other organisms [138].

4.5. Polyamine oxidation and stress tolerance

In several cases, the response to abiotic injuryand mineral nutrient deficiency is found to beassociated with a stimulation of PA oxidation(leading to an increase in Dap content and/or to aproduction of uncommon long-chained PAs). Dapis found to be especially active in the reduction ofsenescence [139]. Dap indeed inhibits the rise inprotease activity and the loss of chlorophyll asso-ciated with senescence [139,140]. Dap was effectivein retarding the loss of cytochrome f from thethylakoid membranes as well as Chl from the leaftissue (reviewed in Ref. [141]). Dap may bind topolyribosomes and effect protein synthesis, whichmay account for the decrease in protease activity(reviewed in Ref. [141]). Retardation of senescenceby Dap, which could imply membrane binding(reviewed in Ref. [141]), might also be related toinhibition of both ACC synthesis and the conver-sion of ACC to ethylene (reviewed in Ref. [141]).Exogenous Dap was able to reduce the level ofmalondialdehyde in dark-incubated A6ena sati6aL. cv Victory leaves [142]. On the other hand PAOactivity produces H2O2 as a reaction product andit has been suggested that this activity might influ-ence growth, via coupled H2O2-dependent peroxi-dase-mediated cross-linking of cell wall polymers


such as lignin [143]. In microbial systems, long-chained PAs show specific protection against os-motic lysis [44] and seem to protect enzymesagainst heat denaturation [144]. They promotepolymerization of cytoskeletal components andare essential for in vitro protein synthesis at ele-vated temperature (as described above). Owing tothe marked stabilizing properties of the highermolecular weight tetraamines and pentaamines onmembranes [145], nucleic acids [46] and theprotein synthesizing apparatus [146], there is someevidence to support the hypothesis that the long-chained uncommon PAs may serve as drought-stress and/or heat-stress protectants from theseconditions in plants.

A new and interesting study demonstrates thatunder salt-stress Put catabolism (via DAO) cancontribute to proline accumulation (see above). Itwill be interesting to study in several environmen-tal challenges with different plants, the relation-ship between Put catabolism and prolineaccumulation. The major pathway for proline syn-thesis in plants is from glutamate (not from Put),through g-glutamyl phosphate and glutamyl-g-semialdehyde (a two-step reaction that is catalyzedin plants and animals by a single enzyme, D1-pyrroline-5-carboxylate synthetase: P5CS). Inplants, the role of proline may not be restricted tothat of a compatible osmolyte. For example, pro-line synthesized during water deficit and salt-stressmay serve as an organic nitrogen reserve that canbe utilized during recovery [147]. There is alsoevidence that the degradation of proline is directlycoupled to the respiratory electron transport sys-tem and ATP production [148]. Proline degrada-tion could therefore improve the energy status ofcells recovering from the water deficit (reviewed inRef. [149]).

In addition, the results from several studiesappear to confirm the importance of interactionsbetween PAs and ethylene in response to someexternal conditions.

5. Concluding remarks and directions for futureresearch

PAs appear to be involved in a myriad of pro-cesses in plants, including the regulation of DNAreplication, transcription of genes, cell division,organ development, fruit ripening, leaf senescenceand environmental challenges.

With the availability of some of the key genesinvolved in the biosynthetic pathways andcatabolic processes, it has become possible to ma-nipulate PA metabolism using antisense and sensetransgenic approaches [150–154]. This will permitdirect investigation of the molecular mechanismsby which the various environmental challenges acton PA metabolism. There are several advantageswith these techniques over the inhibitor-based ex-periments. First, unlike inhibitor experiments, an-tisense and sense methods are highly specific to thetarget genes. Second, the overexpression of specificenzymes provides a means of changing the biosyn-thetic flux, which is not generally possible with theapplication of inhibitors.

Furthermore, it is essential to establish severalkey points:� How the biosynthetic and catabolic pathways

are regulated at the transcriptional, transla-tional and post-transcriptional levels.

� How they are transported from the site ofsynthesis to other cells and organs.

� Where they localize within cells.� How they regulate specific metabolic processes.

Cloning of genes coding for transglutaminasesin plants will in the future allow the study of themechanisms controlling PA conjugation toproteins as well as to establish the molecularmechanisms by which these bound PAs play a rolein the photosynthetic process and/or structuralfunctions.

Furthermore, current uncertainties concerningthe proposed relationships between PAs andethylene could be resolved by the development anduse of appropriate mutants and transgenic plants.Mutants might be selected that show delayedsenescence or unusually high or low levels of PAs.In addition, transformants could be developedthat contain antisense genes for ACC synthase,Spd synthase or SAM decarboxylase linked topromoters that are temporally expressed and/orreadily inducible by environmental cues. Suchstudies may help develop crops which are betterable to withstand environmental insult. For all theexcitement that awaits us, it would be wise toconstantly remind ourselves of the need to inte-grate PA physiology within the context of theirmetabolic and functional interactions with plantgrowth regulators.

Recent studies have shown the existence of linksbetween the PA content in foods and tumor


growth [155]. Therefore, the development of trans-genic food plants with high or low PA levels mayincrease their nutritional and health values. Withthis in mind, manipulation of the PA biosyntheticpathway may deserve closer attention in the futurefrom a biotechnological point of view.

Acknowledgements

We thank P. Lemesle for typing the manuscript.

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Polyamines and environmental challenges: recent development

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