lable at ScienceDirect

Plant Physiology and Biochemistry 73 (2013) 7e15

Contents lists avai

Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Effect of drought and combined drought and heat stress on polyaminemetabolism in proline-over-producing tobacco plants

Milena Cvikrová*, Lenka Gemperlová, Olga Martincová, Radomira VankováInstitute of Experimental Botany AS CR, Rozvojová 263, 165 02 Prague 6, Czech Republic

a r t i c l e i n f o

Article history:Received 16 July 2013Accepted 12 August 2013Available online 29 August 2013

Keywords:DroughtHeat stressPolyaminesProlineTobacco plants

Abbreviations: ADC, arginine decarboxylase; DAOmass; FM, fresh mass; MDA, malondialdehyde; N-norspermine; ODC, ornithine decarboxylase; PAO, poamines; Put, putrescine; ROS, reactive oxygen species;SAMDC, S-adenosylmethionine decarboxylase; SM,spermidine; Spm, spermine.* Corresponding author.

E-mail address: cvikrova@ueb.cas.cz (M. Cvikrová)

0981-9428/$ e see front matter � 2013 Published byhttp://dx.doi.org/10.1016/j.plaphy.2013.08.005

a b s t r a c t

The roles of proline and polyamines (PAs) in the drought stress responses of tobacco plants wereinvestigated by comparing the responses to drought alone and drought in combination with heat in theupper and lower leaves and roots of wild-type tobacco plants and transformants that constitutively over-express a modified gene for the proline biosynthetic enzyme D1-pyrroline-5-carboxylate synthetase(P5CSF129A; EC 2.7.2.11/1.2.1.41). In both genotypes, drought stress coincided with a decrease in relativewater content (RWC) that was much less severe in the upper leaves than elsewhere in the plant. Thedrought also increased proline levels in both genotypes. A brief period of heat stress (2 h at 40 �C) at theend of the drought period did not significantly influence the proline levels in the upper leaves and rootsbut caused a further increase in the lower leaves of both genotypes. The rate at which these elevatedproline levels returned to normal during the post-stress recovery period was slower in the transformantsand plants that had been subjected to the combined stress. In both genotypes, drought stress signifi-cantly reduced the levels of spermidine (Spd) and putrescine (Put) in the leaves and roots relative tothose for controls, and increased the levels of spermine (Spm) and diaminopropane (Dap, formed by theoxidative deamination of Spd and Spm). Spd levels may have declined due to its consumption in Spmbiosynthesis and/or oxidation by polyamine oxidase (PAO; EC 1.5.3.11) to form Dap, which became moreabundant during drought stress. During the rewatering period, the plants’ Put and Spd levels recoveredquickly and the activity of the PA biosynthesis enzymes in their leaves and roots increased substantially;this increase was more pronounced in transformants than WT plants. The high levels of Spm observed indrought stressed plants persisted even after the 24 h recovery and rewatering phase. The malondial-dehyde (MDA) contents of the lower leaves of WTs increased substantially during the drought stressperiod; a less pronounced increase occurred in the transformants and after the application of thecombined stress. After the post-stress recovery period, the MDA contents in the leaves of both genotypeswere higher than those in the corresponding controls. The MDA contents of the upper leaves in plants ofboth genotypes remained relatively constant throughout, indicating that these leaves are preferentiallyprotected against the adverse effects of oxidative stress and demonstrating the efficiency of the plants’induced antioxidative defense mechanisms.

� 2013 Published by Elsevier Masson SAS.

1. Introduction

Drought stress affects water retention in plants at the cellular,tissue and organ levels, causing both specific and unspecific

, diamine oxidase; DM, drySpd, norspermidine; N-Spm,lyamine oxidase; PAs, poly-RWC, relative water content;water-saturated mass; Spd,

.

Elsevier Masson SAS.

reactions as well as potentially damaging tissues and inducingadaptive responses [1]. Plants cope with droughts by activatingdefense mechanisms against water deficits [2]. These include sto-matal closure, which reduces the rate of transpiration and photo-synthesis [3]; the synthesis of new proteins; and the accumulationof osmolytes [4]. Plants’ interactions with the environment aremediated, at least in part, by phytohormones. The most importantphytohormone in dehydration responses is abscisic acid (ABA [5]).ABA mediates both rapid responses based on modulating the ac-tivity of ion channels to induce stomatal closure and slowermetabolic changes that coincide with the activation of defensepathways. Recently, an increasing amount of evidence has emergedto indicate that cytokinins and auxins, plant hormones that are

Table 1Relative water content (RWC) in tobacco wild type (WT) and M51-1 transformantsdetermined in upper (U) and lower (L) leaves at control conditions, after 5-d, 10-ddrought stress (D), combined drought and heat stress (D þ HS) and after rewater-ing (Rew).

RWC (%)

Control 5-d D 10-d D 10-d D þ HS Rew D Rew D þ HS

WT U 84.35 73.06 56.29 48.68 79.61 74.18L 85.13 70.09 42.68 40.34 76.52 73.11

M51-1 U 83.96 81.92 64.73 51.96 84.91 76.91L 84.91 77.91 48.92 40.85 82.35 78.24

Table 2Alterations in shoot fresh weights of tobacco wild type (WT) and M51-1 trans-formants after 5-d, 10-d drought stress (D), combined drought and heat stress(D þ HS) and after rewatering (Rew). Data represent the percentages of values oftreated plants as compared to control plants which were taken as 100%.

Relative shoot fresh weight (%)

Control 5-d D 10-d D 10-d D þ HS Rew D Rew D þ HS

WT 100 78.9 50.9 46.2 85.3 74.2M51-1 100 93.0 61.4 54.3 87.6 75.7

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e158

primarily associated with the stimulation of cell division and thecontrol of plant growth and development, play important roles instress responses [6].

The exposure of plants to environmental stresses such as heatstress and drought causes the generation of reactive oxygen species(ROS [7]). High levels of ROS seem to function as a signal thattriggers protective responses; however, they are also potentiallyharmful to all cellular components and negatively influence cellularmetabolic processes [8]. In order to maintain redox homeostasis,plant cells respond to elevated ROS levels by increasing theexpression and activity of ROS-scavenging enzymes [9] and byincreasing their production of lipophilic and hydrophilic scav-enging molecules [10].

Osmotic adjustment is a mechanism for the maintenance ofwater potential in plant cells during periods of water deficiency. Itinvolves the accumulation of a range of osmotically active mole-cules/ions such as soluble sugars, sugar alcohols, proline, organicacids, calcium, potassium, and chloride ions within the cell. Prolineaccumulation has been demonstrated to correlate with tolerance ofdrought and salt stress in plants [11], and occurs due primarily viade novo synthesis [12] together with the suppression of prolineoxidation [13]. A common method for obtaining plants withelevated proline levels involves overexpression of the D1-pyrroline-5-carboxylate synthetase(s) (P5CS) gene. However, the native P5CSprotein is subject to feedback inhibition by its product (proline).This feedback loop can be circumvented by site-directed muta-genesis [14]. In P5CS from Vigna aconitifolia, this was achieved bysubstituting the phenylalanine residue at position 129with alanine.Using this construct, tobacco plants with significantly elevatedproline levels were prepared [15].

Another group of osmotically active substances that is impor-tant in drought tolerance is the polyamines (PAs). Their roles inosmotic adjustment, the maintenance of membrane stability andfree radical scavenging have been emphasized [16]. The expressionof many genes that encode enzymes involved in polyamine meta-bolism has been analyzed under several stress conditions [17].Biosynthesis of the three most common PAs e putrescine (Put),spermidine (Spd) and spermine (Spm) e is initiated either by thedirect decarboxylation of ornithine by the enzyme ornithinedecarboxylase (ODC) or by the decarboxylation of arginine byarginine decarboxylase (ADC) via the intermediacy of agmatine andN-carbomoylputrescine. Another essential enzyme in PA synthesisis S-adenosylmethionine decarboxylase (SAMDC), which isrequired for the production of the aminopropyl group found in Spdand Spm. Diamine oxidase (DAO) and polyamine oxidase (PAO) arethought to play major roles in the production of H2O2 via PAcatabolism in plant tissues (for a review see Ref. [18]). Plant cellsunder osmotic stress typically contain both free PAs and PA con-jugates with hydroxycinnamic acids, which are referred to ashydroxycinnamic acid amides. Stress-tolerant plants generally havea greater capacity to synthesize polyamines in response to stressthan do susceptible plants, and can increase their endogenouspolyamine levels by a factor of two or three in response to stress[19]. It has been demonstrated that treatment with exogenous Spmconfers enhanced tolerance of drought and salt stress in Arabidopsis[20]. Moreover, it has been suggested that transgenic plants withelevated levels of proline and PAs exhibit enhanced stress tolerance[21]. However, the levels of PAs and proline may be interrelatedbecause their biosynthetic and catabolic pathways have some in-termediates in common [22].

The study presented herein was undertaken to investigate theroles of proline and PAs in the drought responses of tobacco plantsand to determine how (if at all) elevated proline levels affectdrought tolerance and PA metabolism in tobacco plants. The stressresponses of plants from a tobacco line that over-expresses a

modified P5CS gene (P5CSF129A) and therefore has elevated prolinelevels were compared to those for the corresponding wild type(WT), enabling us to estimate the impact of increased proline levelson the dynamics of PA levels under stress conditions.

2. Results

2.1. Relative water content

The relative water content values (RWC) did not differ signifi-cantly between the two genotypes under control conditions(Table 1). A minor decrease (3e8%) in RWC was observed intransformant plants subjected to a 5-d drought period; under thesame conditions, the RWC of the WT plants decreased by 14e18%.After a prolonged drought stress of 10-d, much more significantreductions in RWC were observed in both genotypes. In WT plants,the RWC fell by 33% and 50% in the upper and lower leaves,respectively; the corresponding percentages for the transformantswere 23 and 42%. The water deficit was thus much more pro-nounced in lower leaves. The application of a combined droughtand heat stress (10-d drought þ 2 h at 40 �C) caused a furtherdecrease in RWC, with only minor differences between the testedgenotypes. During the 24-h recovery period, the RWC values forboth genotypes rose more rapidly after a 10-d drought than theydid after the combined drought and heat stress. Notably, the RWC ofthe transformants returned to approximately the level observed incontrol leaves within 24 h of the termination of the drought stress(Table 1).

2.2. Plant growth under stress conditions

Changes in the fresh weights of the shoots of WT and trans-formant plants were monitored during the 10-d drought stressperiod, at the end of the combined stress period, and after rewa-tering (Table 2). The reductions in shoot freshweight observed afterdrought periods of 5 and 10 days and after the combined stresstreatment were in good agreement with the corresponding RWCvalues. The inhibitory effects of both stress treatments on shootfresh weight were more pronounced in WT plants.

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e15 9

2.3. Proline content

Under control conditions, the transformants accumulated 40and 25 times more proline in their upper and lower leaves,respectively, than did the WTs, and 10 times more in their roots(Fig. 1). In both genotypes, water deficits caused considerable in-creases in proline content (P < 0.05 relative to the values observedin the controls). After 5 days of water stress, the amount of prolinein the upper and lower leaves and roots of the WTs had increasedsubstantially relative to those in the controls (by factors of 25, 6 and10, respectively). The proline levels in the leaves and roots of thetransformants also increased relative to the corresponding controls,albeit less dramatically: they rose by factors of 3, 9 and 7 in theupper and lower leaves and roots, respectively. Extending theduration of the water deficit to ten days caused further increases inthe proline contents of the leaves and roots in both the wild typeplants and the transformants. The combined stress treatment, inwhich plants were subjected to a ten day drought and then to a 2 hperiod of heat stress, did not significantly affect the proline levels inthe upper leaves and roots but did cause further increases in thelower leaves in both genotypes. During the 24 h post-stress re-covery period (rehydration at 25 �C), the amount of prolinedecreased significantly in the leaves of theWT plants. However, theproline levels in the lower leaves of the transformants were higherthan those immediately after the end of the combined stresstreatment. The proline levels in the leaves and roots of both culti-vars remained higher than those in the control plants throughoutthe post-stress recovery period. Proline levels fell more slowly inthe transformants and in plants that had been subjected to thecombined stress.

2.4. Activities of ODC, ADC and SAMDC

The activities of ODC and ADC in the upper leaves of the WTcontrol plants were almost identical to those in the upper leaves ofthe transformant controls. However, the enzyme activities for thetwo genotypes differed slightly in the lower leaves and significantlyin the roots (Fig. 2). The variation in ODC activity over the course ofthe stress period in theWT plants was very similar to that observedin the transformants: ODC activity in the leaves decreased slightlyduring the first five days of drought stress but then increasedslightly over the full ten day period. Subsequent exposure to hightemperatures during the combined stress treatment then reducedODC activity. In contrast, drought and the combined stress treat-ment caused a pronounced increase in ODC activity in the roots ofboth the WT plants and the transformants. During the post-stress

Fig. 1. Proline levels in the upper (U) and lower leaves (L) and roots (R) of wild type (WT) toperiods of drought stress (D-5d, D-10d), after a combined stress treatment (D-10d followerecovery phase after combined stress (RewD þ HS). Data indicate the means � SE (n ¼ 3)stressed plants and those at the end of the recovery phase, and between treated and contr

recovery period, the ODC activities in the leaves were lower thanthose in the untreated controls for both genotypes. There were nosignificant changes in the ADC activity in the upper leaves of eitherthe WT plants or the transformants during the stress period.However, in the lower leaves of both genotypes, ADC activityincreased over the ten day drought period and then decreased atthe end of the combined stress period. In the roots of both geno-types, ADC activity rose significantly during the first five days of thedrought period and peaked on the tenth day (Fig. 2). The increasewas more pronounced in the WTs than in the transformants. Incontrast to the ODC activity, the ADC activity in the roots of bothcultivars remained high until the end of the post-stress recoveryperiod. Furthermore, the ADC activity in the lower leaves of thetransformants increased rapidly and substantially after rewatering;by the end of the recovery period, it was almost 25% higher thanthat in the corresponding untreated controls. Drought and thecombined stress treatment also caused a significant increase inSAMDC activity in the roots of both cultivars. This increase in ac-tivity persisted through the post-stress recovery period. In lowerleaves of both genotypes, SAMDC activity decreased slightly duringthe stress period but then rose during the recovery period.

2.5. Activities of DAO and PAO

The highest DAO and PAO activities in the WT and transformantcontrol plants were observed in the roots (Fig. 3). In both geno-types, these enzymes were more active in the lower leaves than theupper ones. Five days of drought stress caused decreases in DAOactivity in the roots of WT plants and the lower leaves of both ge-notypes, but the DAO activity in the roots of both genotypesremained significantly higher than in the leaves (Fig. 3). Longerperiods of drought caused significant (P < 0.05) and pronouncedreductions in DAO activity in the leaves of both genotypes relativeto the levels observed in control plants. In both genotypes, thechanges in PAO activity over time were similar to those observedfor DAO during the stress period, i.e. its activity declined markedlyin the roots and lower leaves and slightly in the upper leaves.During the post-stress recovery period, DAO activity increasednoticeably (relative to their levels at the end of the drought period)in the lower leaves of both genotypes; this increase was morepronounced in WT plants than in transformants. However, this wasaccompanied by a reduction in DAO activity in the roots. Rewater-ing also caused a significant increase in PAO activity (relative to thatobserved in stressed plants) in the lower leaves of the trans-formants. The activities of both amine oxidases in the leaves androots of both genotypes at the end of the 24 h rehydration period

bacco plants and M51-1 transformants under control conditions, during 5- and 10-dayd by a heat shock at 40 �C for 2 h; D þ HS), after rewatering (RewD), and during the. There were significant differences (P < 0.05) between the proline levels observed inol plants throughout the experiment.

U L R U L R U L R U L R U L R U L R

To

tal O

DC

activity [p

kat.m

g-1 p

ro

tein

]

0

20

40

60

80

100

U L R U L R U L R U L R U L R U L R

0

20

40

60

80

100

U L R U L R U L R U L R U L R U L R

To

tal A

DC

activity [p

kat.m

g-1 p

ro

tein

]

0

10

20

30

U L R U L R U L R U L R U L R U L R

0

10

20

30

U L R U L R U L R U L R U L R U L RTo

tal S

AM

DC

activity [p

kat.m

g-1

p

ro

tein

]

0

2

4

6

8

10

12

U L R U L R U L R U L R U L R U L R

0

2

4

6

8

10

12

Control D-5dD-10d D+HS RewDRewD+HS ControlD-5d D-10dD+HS RewD RewD+HS

WT M51-1

WT

WT

M51-1

M51-1

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

**

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

**

*

**

*

*

*

*

Fig. 2. Activities of the biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase (SAMDC) in the upper (U)and lower leaves (L) and roots (R) of wild type (WT) tobacco plants andM51-1 transformants under control conditions, during 5- and 10-day periods of drought stress (D-5d, D-10d),after a combined stress treatment (D-10d followed by a heat shock at 40 �C for 2 h; D þ HS), after rewatering (RewD), and during the recovery phase after combined stress(RewD þ HS). Data indicate the means � SE (n ¼ 3). Asterisks above the bars indicate significant differences (P < 0.05) between the enzyme activities observed in stressed plantsrelative to those for the corresponding controls.

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e1510

were low. The DAO activity in the upper and lower leaves and theroots of the WT plants decreased by about 70%, 30% and 65%,respectively, relative to those observed in control plants; the cor-responding reductions in the transformants were 60%, 50%, 25%,respectively. The PAO activity in the leaves and roots of the WTplants decreased relative to those in the controls, by 85%, 75% and70% in the upper, lower leaves and roots, respectively, while thosein the transformants fell by 60%, 75%, 65% relative to the controls(Fig. 3).

2.6. Polyamine content

Under non-stressed conditions, the transformants had higherPA contents than the WT plants. After 5 d of drought stress, thelevels of Put in the WT plants did not change significantly butthose of Spd decreased (P < 0.05) relative to those observed in thecontrols. The levels of Spm in the lower leaves and roots increasedsignificantly (P < 0.05) relative to the controls; those in the upperleaves also increased but not significantly. There were furtherstatistically significant (P < 0.05) changes in the polyamine con-tent of the WTs (relative to those for the control plants) at the end

of the ten day drought and combined stress periods: Spd and Putbecame less abundant while Spm and 1,3-diaminopropane (Dap)became more abundant in the leaves and roots (Fig. 4). In thetransformants, the only change in polyamine levels observed after5 days of drought was a reduction in the abundance of Put in theroots (relative to the level observed in control plants). At the end ofthe combined stress period, the transformants had significantly(P < 0.05) lower levels of Spd and Put relative to control plants andhigher levels of Spm and Dap their leaves and roots (Fig. 4). Afterrewatering, the Put and Spd contents of both genotypes increasedsubstantially relative to the levels seen in the stressed plants. ThePut and Spd contents of the upper leaves of both genotypesincreased to approximately the levels seen in the controls. How-ever, the lower leaves of both genotypes and the roots of the WTplants had significantly (P < 0.05) higher Put concentrations thanwere found in non-stressed plants. The levels of Spm and Dap inthe leaves and roots of both genotypes remained higher than inthe controls. In both genotypes, the increase in PA levels duringthe post-stress recovery period was faster in plants that had onlyexperienced drought stress than in those that had been subjectedto the combined stress.

U L R U L R U L R U L R U L R U L R

DA

O activity [p

kat . m

g-1 p

ro

tein

]

0

20

40

60

80

100

120

140

160

U L R U L R U L R U L R U L R U L R0

20

40

60

80

100

120

140

160

U L R U L R U L R U L R U L R U L R

PA

O activity [p

kat . m

g-1 p

ro

tein

]

0

10

20

30

U L R U L R U L R U L R U L R U L R0

10

20

30

WT

WT

M51-1

M51-1

ControlD-5d D-10d D+HS RewD RewD+HSControl D-5d D-10d D+HS RewDRewD+HS

*

*

**

*

* **

*

*

*

*

**

*

*

*

*

*

* * **

*

*

*

*

*

**

*

* *

*

**

*

*

***

*

*

*

*

*

*

*

**

Fig. 3. Activities of the catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) in the upper (U) and lower leaves (L) and roots (R) of wild type (WT) tobacco plantsandM51-1 transformants under control conditions, during 5- and 10-day periods of drought stress (D-5d, D-10d), after a combined stress treatment (D-10d followed by a heat shockat 40 �C for 2 h; D þ HS), after rewatering (RewD), and during the recovery phase after combined stress (RewD þ HS). Data indicate the means � SE (n ¼ 3). Asterisks above the barsindicate significant differences (P < 0.05) between the enzyme activities observed in stressed plants relative to those for the corresponding controls.

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e15 11

2.7. Malondialdehyde content

Under control conditions, the reactive oxygen species contents(calculated based on their MDA contents) of the upper leaves androots of the WT plants were almost identical to those for thetransformants, and the two genotypes differed only slightly withrespect to the ROS contents of their lower leaves (Fig. 5). Five daysof drought stress caused slight reductions in the MDA contents ofthe upper leaves of the transformants and WT plants relative tocontrols; the change was significant (P < 0.05) for the WT plants.This was accompanied by a slight increase in the MDA contents ofthe lower leaves in both genotypes. The MDA contents of the lowerleaves of the WT plants increased substantially after ten days ofdrought stress and after the combined stress period; similar butless pronounced increases occurred in the transformants. After thepost-stress recovery period, the MDA levels in the leaves of bothgenotypes remained higher than those observed in the corre-sponding controls.

3. Discussion

Proline plays an important role in plants’ defenses againstabiotic stresses, and plants with elevated proline levels are knownto be particularly tolerant to such stresses [31,32]. Similarly toprevious reports (e.g. Ref. [33]), the tobacco plants examined in thiswork responded to water stress by accumulating large quantities ofPro, particularly in their upper leaves. The Pro level was notablyhigher in the stressed 35S:P5CSF129A transformants than in theWTplants. However, due to the higher basal proline content of thetransformants, the relative increase in Pro levels in response todrought stress was more pronounced in the WT plants (Fig. 1). Theapplication of a combined drought and heat stress, in which plantswere first subjected to ten days of drought and then exposed to

high temperatures for 2 h caused further increases in the prolinecontents of the lower leaves. In both genotypes, drought stresscaused a reduction in RWC, although this reduction was less pro-nounced in the upper leaves than in the other parts of the plants.Notably, in the transformant line, the RWC fell at a later stageduring the drought period than it did in the WT plants, andreturned to its starting levels more rapidly during the recoveryperiod. By the end of the recovery period, the RWC values for thetransformants’ leaves were approximately the same as those forcontrol plants (Table 1). This strongly suggests that the trans-formant is more stress-tolerant than the WT. Moreover, the freshweight of the transformants’ shoots did not decrease as much or asrapidly as was observed for the WT plants during the droughtperiod; this also indicates their greater stress tolerance (Table 2).Recently, several studies have demonstrated that PA metabolism isimportant in plants’ responses to abiotic stresses, especially saltand drought stress [16,34]. As was the case in our studies on to-bacco BY-2 cells [35], the results obtained in this work stronglysuggest that ODC is the most important enzyme in Put biosynthesisin tobacco (Fig. 3). Prolonged drought stress caused increases in theactivity of both ODC and ADC in the lower leaves and roots of WTplants; however, in the transformants, the activities of these en-zymes only increased significantly in the roots (Fig. 2).

A moderate water deficit caused by five days of drought stressreduced the Spd content of theWT plants (relative to controls) whileincreasing their Spm contents. No significant changes in the PAcontents of the transformants occurred under these conditions, withthe exception of a slight reduction in the Put contents of their roots(Fig. 4). After ten days of drought, the levels of Spd and Put in both theWT plants and the transformants decreased significantly relative tothose seen in control plants. Thiswas accompanied by correspondingincreases in the levels of Spm and Dap, which are formed by theoxidative deamination of Spd and Spm. The combined stress

0

1000

2000

3000

4000

0

1000

2000

3000

4000

Put

Spd

Spm

Dap

0

500

1000

1500

2000

0

500

1000

1500

2000

2500

3000

0

500

1000

1500

2000Put

Spd

Spm

Dap

M51-1

RWT

M51-1

M51-1

U

L

R

Co

nten

t o

f p

olyam

in

es [n

mo

l . g

-1 D

W]

0

500

1000

1500

2000

2500

3000

WT

WT

L

U

D-5dD-5dControl

Control

Control

Control

Control

Control

D-5d D-5d

D-5dD-5d

D-10d D-10d

D-10d D-10d

D-10dD-10d

D+HS D+HS

D+HS D+HS

D+HSD+HS

RewD RewD

RewDRewD

RewD RewD

RewD+HS

RewD+HS

RewD+HS

RewD+HS RewD+HS

RewD+HS

Put

Spd

Dap

**

*

** *

*

**

**

**

*

*

*

* *

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

**

*

*

*

**

*

*

*

*

*

*

*

***

*

*

*

* *

*

**

*

*

**

*

**

*

*

*

*

** **

*

*

*

*

*

**

*

*

*

***

*

**

Spm

Fig. 4. Levels of free polyamines in the upper (U) and lower leaves (L) and roots (R) of wild type (WT) tobacco plants and M51-1 transformants under control conditions, during 5-and 10-day periods of drought stress (D-5d, D-10d), after a combined stress treatment (D-10d followed by a heat shock at 40 �C for 2 h; D þ HS), after rewatering (RewD), and duringthe recovery phase after combined stress (RewD þ HS). Data indicate the means � SE (n ¼ 3). Asterisks above the bars indicate significant differences (P < 0.05) between thecontents observed in stressed plants relative to those for the corresponding controls.

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e1512

treatment caused a further reduction in the levels of Put and Spd inthe lower leaves and roots of the transformants; again, this wasaccompanied by increases in the abundance of Spm and Dap, whichis indicative of enhanced oxidative stress. The decline in Spd levelsmay have been due to its use in Spmbiosynthesis or it may have been

U L R U L R U L R U L R U L R U L R

Co

nten

t o

f M

DA

[n

mo

l . g-1 D

W]

0

50

100

150

200

250

300

Control D-5d D-10d D+HS RewD RewD+HS

WT

*

*

*

*

*

*

*

*

Fig. 5. Content of malondialdehyde in the upper (U) and lower leaves (L) and roots (R) of wi5- and 10-day periods of drought stress (D-5d, D-10d), after a combined stress treatment (Dduring the recovery phase after combined stress (RewD þ HS). Data indicate the means � Svalues observed in stressed plants relative to those for the corresponding controls.

oxidized by PAO to formDap, the levels of which increased during thedrought stress period. These results suggest that the oxidation ofSpm by PAO was partially responsible for the fall in its levels afterrewatering (Figs. 3 and 4). This is consistent with the results of aprevious study on bean seedlings under salt stress [36]. Although the

U L R U L R U L R U L R U L R U L R

0

50

100

150

200

250

300

Control D-5d D-10d D+HS

M51-1

*

*

*

*

**

RewD RewD+HS

ld type (WT) tobacco plants and M51-1 transformants under control conditions, during-10d followed by a heat shock at 40 �C for 2 h; D þ HS), after rewatering (RewD), andE (n ¼ 3). Asterisks above the bars indicate significant differences (P < 0.05) between

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e15 13

precise role of Put in plants under adverse stress conditions is stillcontroversial, many reports have shown that Spm helps to protectplants against drought stress [37,20]. It has been suggested that theaccumulation of free Spm is characteristic of plants’ responses tolong-term salt stress [38]. The high levels of Spm observed in droughtstressed leaves persisted even after the 24 h recovery phase, duringwhich the RWC of the plants was returning to the levels seen in thecontrols (Fig. 3); this may reflect the role of Spm in modulating theactivity of ion channels [39]. In this context, it is noteworthy that PAssuch as Spm inhibit stomatal opening and induce their closure byregulating Kþ channel activity, as was observed in Vicia faba guardcells [40]. Marked increases in the levels of Put and Spd coincidedwith increases in the activity of PA biosynthetic enzymes in theleaves and roots after the rewatering of the plants. These increaseswere more pronounced in the transformants than in the WT plants(Fig. 2) and were probably due to general increases in metabolicactivity. The early stages of recovery are associated with elevatedmetabolic activity due to a sharp increase in the demand for re-sources throughout the plant. Proline degradation may serve as animportant source of reducing agents in this process, which wouldsupport oxidative phosphorylation in the mitochondria and thus theformation of the ATP that is required by the processes involved inactive stress recovery [41]. Some of the accumulated prolinemight bedegraded by proline dehydrogenase, which catalyzes the conversionof proline to pyrroline-5-carboxylate and whose expression is stim-ulated during stress recovery [42]. In light of the well-known cor-relation between the proline and PA biosynthetic pathways, it ispossible that glutamate might serve as a substrate in PA biosynthesisand could also be involved in the pronounced increase in Put and Spdlevels during the recovery period (Figs. 1 and 4, [43]).

Drought stress is one of the most important types of abioticstress and induces the production of various ROS. The nature of therelationship between PAs and ROS is rather controversial. On onehand, it has been suggested that PAs may protect cells against ROS;on the other, that their catabolism generates ROS [44]. It has alsobeen proposed that the protective effect of exogenous PAs againstoxidative damage caused by superoxides is dependent on theirprior conversion to conjugates [45]. We believe that the decreasesin Put and Spd levels that occurred during the drought stress pe-riods in our experiments were primarily due to their consumptionin PA conjugation reactions. This hypothesis is supported by thesubstantial decreases in DAO activity that occurred in the upper andlower leaves of both genotypes during prolonged droughts and thecombined stress period, i.e. under conditions of high oxidativestress (Fig. 3). The results presented by [46] suggest that Putoxidation by DAO occurs only during the early stages of droughttreatment in Arabidopsis. The importance of PA conjugation in thecontrol of free PA levels in tobacco BY-2 cells under oxidative stresshas been further discussed by [47]. In addition, we have previouslyconfirmed the ability of PA conjugates to modulate free PA levels inalfalfa cell suspension cultures and in oak embryogenic cultures[48,49]. However, further studies will be required to properlyinvestigate the potential roles of hydroxycinnamic acid amides inthe control of PA homeostasis in response to stress.

In general, ROS are produced on a continuous basis in plants(even under physiological steady state conditions) in cell organellesinvolved in active electron transport. These ROS must be efficientlyscavenged and maintained at non-damaging levels in order topermit cell survival. However, they are also useful signaling mole-cules that are important in stress signal transduction and theactivation of acclimation and defense mechanisms [50]. Measuredrates of lipid peroxidation (which are determined by monitoringchanges in the levels of MDA) can be related to the antioxidantactivity balance within a given cell or tissue. The MDA levels in theleaves of the transformed and the WT plants did not vary greatly

under control conditions. However, the MDA contents of the lowerleaves of the transformants increased substantially over the courseof the ten day drought period, and a similar but much less pro-nounced increase was observed in the transformants. The imposi-tion of a 2 h period of heat stress at the end of the ten day droughtperiod caused a significant increase in oxidative stress, as demon-strated by pronounced increases in the MDA contents of the upperand lower leaves of the WT plants and the lower leaves of thetransformants. After the post-stress recovery period, the MDAlevels in the leaves of both genotypes decreased but neverthelessremained higher than those in the corresponding controls (Fig. 5).The higher levels of MDA observed after rewatering may indicatepersistent stress-induced damage. No significant increases in MDAlevels were observed in the upper leaves of the transformantsduring the stress period, which may indicate that the plants’ de-fense mechanisms are particularly active in these tissues.

In summary, the results of this study indicate that the elevatedproline and PA levels in the transformed tobacco plants had mild butdistinct positive effects on their abiotic stress tolerance. The upperleaves seem to be preferentially protected, as demonstrated by theirnear-constant levels of MDA during the drought periods. This alsodemonstrates the efficiency of the plants’ native defensemechanismsagainst oxidative damage and is consistent with the suggestion thatthe young and reproductive tissues are particularly well protected.

4. Materials and methods

4.1. Plant material and stress application

Wild-type (Nicotiana tabacum L. cv. M51) and transgenic35S:P5CSF129A tobacco plants (for detailed information see Ref. [15]were grown in soil in a growth chamber (SANYO MLR 350H, Osaka,Japan) for 6 weeks with a 16-h photoperiod at 130 mmol m�2, day/night temperatures of 25/23 �C, and a relative humidity (RH) of ca.80%. Drought stress was imposed by the cessation of watering for 10days at reduced RH (35%). A combined stress was imposed by trans-ferring the plants to high temperature conditions (40 �C, 2 h) at thevery end of drought period. The recovery of the stressed plants wasdetermined 24 h after re-watering at 25 �C. Samples of the upperleaves (specifically, the two youngest unfolded leaves) and lowerleaves were collected, cut into pieces, and immediately frozen inliquid nitrogen after removal of the main vein. Root samples wereshaken to remove residual soil, washed briefly with cold tap water(cca. 1 min), dried with filter paper and frozen in liquid nitrogen. Foreach treatment, a total of four (control) or five (stress) plants wereharvested. Three independent experiments were performed.

4.2. Determination of relative water content

The relative water content (RWC) of the sampled leaves wasmeasured as follows: individual cut leaves were weighed to deter-mine their fresh mass (FM), saturated with water in beakers for 12 h,re-weighed to determine their water-saturated mass (SM), dried at80 �C, andfinallyweighedagain to give thedriedmass (DM). TheRWCwas then calculated as: RWC (%) ¼ [(FM � DM)/(SM � DM)] � 100.

4.3. Determination of free proline levels

The levels of free proline in the samples were determined ac-cording to the method of [23].

4.4. Ornithine decarboxylase, arginine decarboxylase and S-adenosylmethionine decarboxylase assays

The activities of ornithine decarboxylase (ODC; EC 4.1.1.17), argi-nine decarboxylase (ADC; EC 4.1.1.19) and S-adenosylmethionine

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e1514

decarboxylase (SAMDC; EC 4.1.1.50) activitieswere determined by theradiochemicalmethod developed by [24], modified according to [25].

The protein contents of the samples were measured withBradford’s method using bovine serum albumin as a standard [26].

4.5. Diamine oxidase assay

Diamine oxidase (DAO, EC 1.4.3.6) activity was assayed by aspectrophotometric method based its ability to catalyze the conver-sion of cis-1,4-diamino-2-butene into pyrrole via the correspondingamino aldehyde [25]. Samples were homogenized in 0.1 M TriseHClbuffer, pH 8.5, containing 2 mM mercaptoethanol and 1 mM EDTA,and centrifuged at 20,000� g for 15min at 4 �C. The reactionmixturecontained 0.1 M TriseHCl buffer, pH 8.5, catalase (25 mg) and 0.01 Mcis-1,4-diamino-2-butene. The reaction was started by adding 0.2 mlof supernatant, incubated for 1 h at 37 �C and stopped by adding 1mlof Ehrlich’s reagent. The reaction mixture was then incubated at50 �C for 5 min after which it was chilled on an ice bath beforereading the absorbance of the newly-formed pyrrole at 563 nm.Enzymatic activity is expressed in pkat mg�1 prot.

4.6. Polyamine oxidase assays

The activity of polyamine oxidase (PAO; EC 1.5.3.11) was esti-mated using a modification of the radiometric method of [27], with[1,4-14C] spermidine (4.37 GBq mmol�1, Amersham PharmaciaBiotech, UK) as the substrate. Samples for the measurement of PAOactivity were extracted in 3 volumes of ice-cold 0.1 M TriseHClbuffer (pH 8.5) containing 2 mM b-mercaptoethanol and centri-fuged at 20,000� g for 30min at 4 �C. Both the supernatant (solublefraction) and the resuspended pellet (particulate fraction) wereused to determine the sample’s PAO activity. The assay mixturescontained 0.3 ml of extract, 1 mM unlabeled Spd, and 7.4 kBq of[1,4-14C]Spd. After incubation at 37 �C for 30 min, the reaction wasterminated by adding 60 ml of saturated sodium carbonate. The D1

[14C] pyrroline in the samples was immediately extracted using4 ml toluene. The radioactivity of the extracted toluene solutionwas counted using a Tri-Carb 2900 TR liquid scintillation analyzer(Packard). Enzymatic activity is expressed in pkat mg�1 prot.

4.7. Polyamine analysis

Extraction and HPLC analysis of benzoylated polyamines wasperformed according to [28].

4.8. Malondialdehyde assay

The malondialdehyde (MDA) content of the samples wasdetermined using the NWLSS-Malondialdehyde Assay kit (cat. no.NWK-MDA01, Northwest Life Science Specialties, LLC, Vancouver,Canada) as described in detail by [29]. The assay is based on thereaction of MDA with thiobarbituric acid (TBA), which forms anMDAeTBA2 adduct that absorbs strongly at 532 nm. The absor-bance of the supernatant was measured at 532 nm and correctedfor non-specific turbidity by subtracting the sample’s absorbance at600 nm [30]. The quantity of MDA in the samplewas determined byreference to a five-point standard curve.

4.9. Statistical analyses

Three independent experiments were carried out, in whichsimilar results were obtained. The mean values (�the standarderror, SE) obtained in one experiment with three replicates areshown in the figures. Data were analyzed using Student’s t-test.

Acknowledgments

We thank Sees-editing Ltd. for linguistic editing. We are verygrateful to Dr. Jozef Gubis (Research Institute of Plant Production,Piestany, Slovakia) for kindly donating seeds of the 35S:P5CSF129Atransformed tobacco line. This work was supported by the Ministryof Education, Youth and Sports CR (Project No. OC08013) and by theCzech Science Foundation (Project No. 206/09/2062).

References

[1] E.H. Beck, S. Fettig, C. Knake, K. Hartig, T. Bhattarai, Specific and unspecificresponses of plants to cold and drought stress, J. Biosci. 32 (2007) 501e510.

[2] H.A. Pinheiro, F.M. DaMatta, A.R.M. Chaves, M.E. Loureiro, C. Ducatti,Drought tolerance in relation to protection against oxidative stress inclones of Coffea canephora subjected to long-term drought, Plant Sci. 167(2004) 1307e1314.

[3] T.C. Hsiao, E. Acevedo, Plant responses to water deficits, water-use efficiency,and drought resistance, Agric. Meteorol. 14 (1974) 59e84.

[4] E.A. Bray, Drought-stress-induced polypeptide accumulation in tomato leaves,Plant Cell Environ. 13 (1990) 531e538.

[5] J.A.D. Zeevaart, R.A. Creelman, Metabolism and physiology of abscisic acid,Annu. Rev. Plant Physiol. Plant Mol. Biol. 39 (1988) 439e473.

[6] J. Dobra, V. Motyka, P. Dobrev, J. Malbeck, I.T. Prasil, D. Haisel, A. Gaudinova,M. Havlova, J. Gubis, R. Vankova, Comparison of hormonal responses to heatdrought and combined stress in tobacco plants with elevated proline kontent,J. Plant Physiol. 167 (2010) 1360e1370.

[7] S. Munne-Bosch, J. Penuelas, Photo- and antioxidative protection, and a rolefor salicylic acid during drought and recovery in field-grown Phillyreaangustifolia plants, Planta 217 (2003) 758e766.

[8] F.V.E. Breusegem, J.F. Vranova, D.I. Dat, The role of active oxygen species inplant signal transduction, Plant Sci. 161 (2001) 405e414.

[9] M. Farooq, T. Aziz, M. Hussain, H. Rehman, K. Jabran, M.B. Khan, Glycinebetaineimproveschillingtolerance inhybridmaize, J. Agric.CropSci. 194 (2008)152e160.

[10] P.M. Hasegawa, R.A. Bressan, J.K. Zhu, H.J. Bohnert, Plant cellular and molec-ular responses to high salinity, Annu. Rev. Plant Physiol. Plant Mol. Biol.(2000) 463e499.

[11] A.J. Delauney, D.P.S. Verma, Proline biosynthesis and osmoregulation inplants, Plant J. 4 (1993) 215e223.

[12] G.S. Voetberg, R.E. Sharp, Growth of the maize primary root at low waterpotentials. 3. role of increased proline deposition in osmotic adjustment, PlantPhysiol. 96 (1991) 1125e1130.

[13] Z. Peng, Q. Lu, D.P.S. Verma, Reciprocal regulation of delta(1)-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls prolinelevels during and after osmotic stress in plants, Mol. Gen. Genet. 253 (1996)334e341.

[14] C.S. Zhang, Q. Lu, D.P.S. Verma, Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthase, a bifunctional enzyme catalyzing the first2 steps of proline biosynthesis in plants, J. Biol. Chem. 270 (1995) 20491e20496.

[15] J. Gubis, R. Vanková, V. Cervená, M. Dragunová, M. Hudcovicová,H. Lichtnerovia, T. Dokupil, Z. Jureková, Transformed tobacco plants withincreased tolerance to drought, S. Afr. J. Bot. 73 (2007) 505e551.

[16] A. Bouchereau, A. Aziz, F. Lahrer, J. Martin-Tanguy, Polyamines and environ-mental challenges: recent development, Plant Sci. 140 (1999) 103e125.

[17] X.P. Wen, X.M. Pang, N. Matsuda, M. Kita, H. Inoue, Y.J. Hao, C. Honda,T. Moriguchi, Over-expression of the apple spermidine synthase gene in pearconfers multiple abiotic stress tolerance by altering polyamine titers, Trans-genic Res. 17 (2008) 251e263.

[18] H.M. Wallace, A.V. Fraser, A. Hughes, A perspective of polyamine metabolism,Biochem. J. 376 (2003) 1e14.

[19] Y. Kasukabe, L.X. He, K. Nada, S. Misawa, I. Ihara, S. Tachibana, Overexpressionof spermidine synthase enhances tolerance to multiple environmentalstresses and up-regulates the expression of various stress regulated genes intransgenic Arabidopsis thaliana, Plant Cell Physiol. 45 (2004) 712e722.

[20] K. Yamaguchi, Y. Takahashi, T. Berberich, A. Imai, T. Takahashi,A.J. Michael, T. Kusano, A protective role for the polyamine apermineagainst drought stress in Arabidopsis, Biochem. Biophys. Res. Commun.352 (2007) 489e490.

[21] L. Simon-Sarkadi, G. Kocsy, A. Varhegyi, G. Galiba, J.A. de Ronde, Effect ofdrought stress at supraoptimal temperature on polyamine concentrations intransgenic soybean with increased proline levels, Z. Naturforsch: C-A Biosci.61 (2006) 833e839.

[22] A. Aziz, J. Martin-Tanguy, F. Larher, Stress-induced changes in polyamine andtyramine levels can regulate proline accumulation in tomato leaf discs treatedwith sodium chloride, Physiol. Plant 104 (1998) 195e202.

[23] L.S. Bates, R.P. Waldren, I.D. Teare, Rapid determination of free proline forwater-stress studies, Plant Soil 39 (1973) 205e207.

[24] A. Tassoni, M. van Buuren, M. Franceschetti, S. Fornale, N. Bagni, Polyaminecontent and metabolism in Arabidopsis thaliana and effect of spermidine onplant development, Plant Physiol. Biochem. 38 (2000) 383e393.

M. Cvikrová et al. / Plant Physiology and Biochemistry 73 (2013) 7e15 15

[25] L. Gemperlová, J. Eder, M. Cvikrová, Polyamine metabolism during the growthcycle of tobacco BY-2 cells, Plant Physiol. Biochem. 43 (2005) 375e381.

[26] M.M. Bradford, A rapid and sensitive method for quantification of micro-quantities of protein utilizing the principle of protein dye binding, Anal.Biochem. 72 (1976) 248e254.

[27] K.A. Paschalidis, K.A. Roubelakis-Angelakis, Sites and regulation of poly-amine catabolism in the tobacco plant. Correlations with cell division/expansion, cell cycle progression, and vascular development, Plant Physiol.138 (2005) 2174e2184.

[28] R.D. Slocum, H.E. Flores, A.W. Galston, L.H. Weinstein, Improved method forHPLC analysis of polyamines, agmatine and aromatic monoamines in planttissue, Plant Physiol. 89 (1989) 512e517.

[29] M. Cvikrová, L. Gemperlová, O. Martincová, I.T. Prásil, J. Gubis, R. Vanková,Effect of heat stress on polyamine metabolism in proline-over-producingtobacco plants, Plant Sci. 182 (2012) 49e58.

[30] E. Esteban, E. Moreno, J. Penalosa, J.I. Cabrero, R. Millan, P. Zornoza, Short andlong-term uptake of Hg in white lupin plants: kinetics and stress indicators,Environ. Exp. Bot. 62 (2008) 316e322.

[31] H.B.C. Molinari, C.J. Marur, J.C. Bespalhok Filho, A.K. Kobayashi, M. Pileggi,R.P. Leite Junior, L.F.P. Pereira, L.G.E. Vieira, Osmotic adjustment in transgeniccitrus rootstock Carrizo citrange (Citrus sinensis Osb. � Poncirus trifoliata L.Raf.) overproducing proline, Plant Sci. 167 (2004) 1375e1381.

[32] P.B. Kishor, S. Sangam, R.N. Amrutha, P. Sri Laxmi, K.R. Naidu, K.R.S.S. Rao, S. Rao,K.J. Reddy, P. Theriappan, N. Sreenivasulu, Regulation of proline biosynthesis,degradation, uptake and transport in higher plants: its implication in plantgrowth and abiotic stress tolerance, Curr. Sci. 88 (2005) 424e438.

[33] L. Szabados, A. Savoure, Proline: a multifunctional amino acid, Trends PlantSci. 15 (2010) 89e97.

[34] R. Alcázar, T. Altabella, F. Marco, C. Bortolotti, M. Reymond, C. Koncz,P. Carrasco, A.F. Tiburcio, Polyamines: molecules with regulatory functions inplant abiotic stress tolerance, Planta 231 (2010) 1237e1249.

[35] M. Cvikrová, L. Gemperlová, J. Eder, E. Zazimalová, Excretion of polyamines inalfalfa and tobacco suspension-cultured cells and its possible role in mainte-nanceof intracellular polyamine contents, Plant Cell Rep. 27 (2008) 1147e1156.

[36] N.I. Shevyakova, L.I. Musatenko, L.A. Stetsenko, N.P. Vedenicheva,L.P. Voitenko, K.M. Sytnik, V.V. Kuznetsov, Effects of abscisic acid on thecontents of polyamines and proline in common bean plants under salt stress,Russ. J. Plant Physiol. 60 (2013) 200e211.

[37] H.P. Liu, B.H. Dong, Y.Y. Zhang, Z.P. Liu, Y.L. Liu, Relationship between osmoticstress and the levels of free, conjugated and bound polyamines in leaves ofwheat seedlings, Plant Sci. 166 (2004) 1261e1267.

[38] K. Urano, Y. Yoshiba, T. Nanjo, Y. Igarashi, M. Seki, F. Sekiguchi, K. Yamaguchi-Shinozaki, K. Shinozaki, Characterization of Arabidopsis genes involved inbiosynthesis of polyamines in abiotic stress responses and developmentalstages, Plant Cell Environ. 26 (2003) 1917e1926.

[39] T. Kusano, T. Berberich, C. Tateda, Y. Takahashi, Polyamines: essential factorsfor growth and survival, Planta 228 (2008) 367e381.

[40] K. Liu, H. Fu, Q. Bei, S. Luan, Inward potassium channel in guard cells as atarget for polyamine regulation of stomatal movements, Plant Physiol. 124(2000) 1315e1326.

[41] P.D. Hare, W.A. Cress, Metabolic implications of stress-induced proline accu-mulation in plants, Plant Growth Regul. 21 (1997) 79e102.

[42] J. Dobrá, R. Vanková, M. Havlová, A.J. Burman, J. Libus, H. �Storchová, Tobaccoleaves and roots differ in the expression of proline metabolism-related genesin the course of drought stress and subsequent recovery, J. Plant Physiol. 168(2011) 1588e1597.

[43] T. Nanjo, M. Fujita, M. Seki, T. Kato, S. Tabata, K. Shinazoki, Toxicity of freeproline revealed in an Arabidopsis T-DNA-tagged mutant deficient in prolinedehydrogenase, Plant Cell Physiol. 44 (2003) 541e548.

[44] S. Mohapatra, R. Minocha, S. Long, S.C. Minocha, Putrescine overproductionnegatively impacts the oxidative state of poplar cells in culture, Plant Physiol.Biochem. 47 (2009) 262e271.

[45] D.R. Walters, Polyamines in plant-microbe interactions, Physiol. Mol. PlantPathol. 57 (2000) 137e146.

[46] R. Alcázar, M. Bitrián, D. Bartels, C. Koncz, T. Altabella, A.F. Tiburcio, Polyaminemetabolic canalization in response to drought stress in Arabidopsis and theresurrection plant Craterostigma plantagineu, Plant Signaling Behav. 6 (2011)243e250.

[47] A. Kuthanová, L. Gemperlová, S. Zelenková, J. Eder, I. Machá�cková, Z. Opatrný,M. Cvikrová, Cytological changes and alterations in polyamine contentsinduced by cadmium in tobacco BY-2 cells, Plant Physiol. Biochem. 42 (2004)149e156.

[48] M. Cvikrová, P. Binarová, J. Eder, M. Vágner, M. Hrubcová, J. Zo�n,I. Machá�cková, Effect of inhibition of phenylalanine ammonia-lyase activity ongrowth of alfalfa cell suspension culture: alterations in mitotic index, ethyleneproduction, and contents of phenolics, cytokinins, and polyamines, Physiol.Plant 107 (1999) 329e337.

[49] M. Cvikrová, J. Malá, M. Hrubcová, J. Eder, J. Zo�n, I. Machá�cková, Effect of in-hibition of biosynthesis of phenylpropanoids on sessile oak somaticembryogenesis, Plant Physiol. Biochem. 41 (2003) 251e259.

[50] J.M. McCord, The evolution of free radicals and oxidative stress, Am. J. Med.108 (2000) 652e659.