polyamine accumulation in transgenic tomato enhances the tolerance to high temperature stress

Journal of Integrative Plant Biology 2009, 51 (5): 489–499

Polyamine Accumulation in Transgenic TomatoEnhances the Tolerance to High Temperature Stress

Lin Cheng, Yijing Zou, Shuli Ding, Jiajing Zhang, Xiaolin Yu, Jiashu Cao and Gang Lu∗

(Key Laboratory of Horticultural Plant Growth, Development and Biotechnology, Ministry of Agriculture, Department of Horticulture,

Zhejiang University, Hangzhou 310029, China)

Abstract

Polyamines play an important role in plant response to abiotic stress. S-adenosyl-l-methionine decarboxylase (SAMDC)is one of the key regulatory enzymes in the biosynthesis of polyamines. In order to better understand the effect ofregulation of polyamine biosynthesis on the tolerance of high-temperature stress in tomato, SAMDC cDNA isolated fromSaccharomyces cerevisiae was introduced into tomato genome by means of Agrobacterium tumefaciens through leafdisc transformation. Transgene and expression was confirmed by Southern and Northern blot analyses, respectively.Transgenic plants expressing yeast SAMDC produced 1.7- to 2.4-fold higher levels of spermidine and spermine than wild-type plants under high temperature stress, and enhanced antioxidant enzyme activity and the protection of membranelipid peroxidation was also observed. This subsequently improved the efficiency of CO2 assimilation and protected theplants from high temperature stress, which indicated that the transgenic tomato presented an enhanced tolerance to hightemperature stress (38 ◦C) compared with wild-type plants. Our results demonstrated clearly that increasing polyaminebiosynthesis in plants may be a means of creating high temperature-tolerant germplasm.

Key words: heat stress; Lycopersicon esculentum; polyamines; Saccharomyces cerevisiae; S-adenosylmethionine decarboxylase; transformation.

Cheng L, Zou Y, Ding S, Zhang J, Yu X, Cao J, Lu G (2009). Polyamine accumulation in transgenic tomato enhances the tolerance to hightemperature stress. J. Integr. Plant Biol. 51(5), 489–499.

Available online at www.jipb.net

Tomato (Lycopersicon esculentum Mill.) is one of the mostpopular and widely consumed vegetables grown worldwide.High temperature is a major factor limiting productivity and itadversely affects the vegetative and reproductive phases oftomato, ultimately reducing yield and fruit quality (Dinar andRudich 1985; Sato et al. 2001; Pressman et al. 2002).

Polyamines (PAs) are small ubiquitous compounds that havebeen implicated in the regulation of many physiological pro-cesses and a variety of stress responses in plants (Bouchereau

Accepted 21 Jun. 2008 Received 28 Dec. 2008

Supported by the State Key Basic Research and Development Plan of

China (2009CB119000), the National Natural Science Foundation of China

(30571268), and the Hi-Tech Research and Development Plan of China

(G2006AA100108).∗Author for correspondence.

Tel: +86 571 8697 1349;

Fax: +86 571 8697 1188;

E-mail: <[email protected]>.

C© 2009 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1744-7909.2009.00816.x

et al. 1999; Yang et al. 2007). PAs, spermidine (Spd), spermine(Spm) and putrescine (Put) accumulate under abiotic stressconditions. The enhanced level of polyamines plays an impor-tant role in the protective response of plants to various abioticstresses (Kumar et al. 2006). However, the mechanism of ac-tion of PAs in abiotic stress response is not clearly understood.It has been suggested that PA involvement in abiotic stressadaptation could be due to their roles in osmotic adjustment,membrane stability, free-radical scavenging and regulation ofstomatal movements (Liu et al. 2007).

The genetic manipulation of polyamine metabolism is one ofthe approaches to elucidate the functional role that polyaminesplay under stress. Unfortunately, there are only few reportson responses of plants expressing PA metabolic genes toenvironmental stress condition (Rajam et al. 1998; Kumar et al.2006), although it has been shown that an increased toler-ance to environmental stress was observed overexpressing PAbiosynthetic genes, such as arginine decarboxylase (Masgrauet al. 1997; Roy and Wu 2001; Capell et al. 2004), ornithine de-carboxylase (Kumria and Rajam 2002), S-adenosylmethioninedecarboxylase (Torrigiani et al. 2005) and spermidine synthase(Franceschetti et al. 2004; Kasukabe et al. 2004, 2006) in rice,tobacco, Arabidopsis and sweet potato plants.

490 Journal of Integrative Plant Biology Vol. 51 No. 5 2009

S-Adenosyl-l-methionine decarboxylase (SAMDC, EC4.1.1.50) is one of the key regulatory enzymes inthe biosynthesis of polyamines. SAMDC catalyzes S-adenosylmethionine (SAM) to form decarboxylated SAM,which provides the aminopropyl groups for subsequent Spdand Spm. It is believed that the synthesis of Spd and Spm ismainly regulated by the level of SAMDC. Overexpression ofheterologous SAMDC in plants generally results in improvingthe tolerance to abiotic stress, including salt (Roy and Wu2002), drought (Waie and Rajam 2003), acidic and oxidantstress (Wi et al. 2006). However, there are few reports on itsrole in heat-stress protection in higher plants. The SAMDCprimary sequences in higher plants are similar. Therefore,the SAMDC gene isolated from Saccharomyces cerevisiaewas selected and introduced to tomato plants to avoid thehomologous depression in the present work. The response oftransgenic plants to high temperature stress was investigatedin order to obtain some fundamental information on the roleof PAs during heat stress. Indeed, overexpression of yeastSAMDC gene (ySAMDC) in transgenic tomato plants led to anincrease in PAs content and enhanced the tolerance to hightemperature stress. Our results provide helpful information

Figure 1. The yeast S-adenosyl-l-methionine decarboxylase (ySAMDC) transgenic plantlets of tomato were obtained by Agrobacterium tumefaciens

mediated method.

(A) HygR buds were induced.

(B) Screen HygR shoots.

(C) HygR shoots were formed from buds.

(D) Rooting of HygR shoots.

(E) Regenerated plants transferred to the pot.

(F) Fruit of HygR plant.

in order to better understand the physiological function ofpolyamines under abiotic stress in tomato plant.

Results

Transformation and regeneration

The visible shoots emerged in the co-cultivated explants after7–10 d of culture on selection medium. The growth of shootsderived from leaf explants was slow and the shoots rootedin rooting medium after more than 80 d of culture. However,the plantlets grew well when transferred to pots for 20–30 d,and all of the transgenic plants showed normal flower and fruitformation (Figure 1).

Transgene integration and expression

The presence of ySAMDC in putative transgenic plants wasconfirmed by polymerase chain reaction (PCR) analysis. Theexpected amplified product of 1.2 kb, specific to the ySAMDCgene was obtained (Figure 2A). The PCR positive plants were

Polyamine Accumulation and Heat Stress 491

analyzed by Southern hybridization to identify the integrationand copy number of the transgene. The transgenics showedone or two copy insertions of the transgene in tested plants(Figure 2B).

The transgenic lines showed high transgene expression atthe transcript level by reverse transcription (RT)-PCR usingySAMDC gene-specific primers, while not in wild type plants(Figure 2C). The inserted ySAMDC was further proved byNorthern blot analysis, transgenic plants showed high transgeneexpression at the transcript level (Figure 2D). The expressionlevels were, however, variable among the transgenic lines. Thehighest transcription level was observed in A7 and Q5, whilerelatively low in B2.

Electrolyte leakage

Leaf electrolyte leakage (EL) was measured to evaluate cellmembrane stability, so EL is suggested to be closely related toheat tolerance in higher plant. The amount of leakage increasedwith time of exposure to heat stress condition in all lines;however, EL in transgenic plants was significantly (P < 0.05)less than in wild-type plants after 4 h treatment stress. After 12 htreatment, the EL in wild-type plants was 87.7%, whereas thatin transgenic tomato lines B2 and Q5 was 64.3% and 43.1%,respectively (Figure 3), which suggested that overexpression ofSAMDC in tomato plants resulted in enhanced protection of cellmembrane permeability.

Polyamine metabolism

The transgenic lines showed significant increase in Spd andSpm levels when compared with wild-type plants and pCAM-BIA1301 plants (Table 1). In wild-type plants, the Spd andSpm contents were 35.2% and 40%, respectively, higher underhigh temperature stress than under non-stressed conditions.However, in the transgenic lines, Spd contents were increasedby 104% and 145% for B2 and Q5, respectively, while Spmcontents increased by 93.9% and 51.7%, when compared withnon-stressed conditions. The levels of accumulated polyamineswere much higher in all transgenic tomato plants than in wild-type ones. On average, Spd and Spm in transgenic lines wereincreased by 134% and 66.1%, respectively, under high tem-perature conditions compared with wild-type plants. Differenttransgenic lines showed variations in the increased levels ofPAs when exposed to high temperature. Q5 showed higherincrease of PAs than B2, which may correspond to the highlevel of SAMDC RNA shown in Figure 2.

Transgenic lines showed significant difference in Put levelgrown under control conditions. Put content increased 25.3%in Q5 but decreased 19.4% in B2 compared with the wild-type plants (Table 1). Under high temperature treatment,Put increased 72.3% and 23%, respectively, in B2 and Q5

Figure 2. Molecular characterization of transgenic tomato plants.

(A) Polymerase chain reaction (PCR) analysis of putative transgenic

plants using primers specific to the yeast S-adenosyl-l-methionine decar-

boxylase (ySAMDC) gene. 100 bp DNA ladder (M), DNA from wild-type

(WT) plant, transformed plantlet with the empty plasmic pCAMBIA1301,

different putative transgenic plants and positive p35S-SAMDC vector.

(B) Southern analysis of EcoRI-digested genomic DNA for checking

transgene copy number using the hpt gene as a probe. DNA from wild-

type plants and different transgenic lines (A7, B2, B6, Q5, T9).

(C) Reverse transcription (RT)-PCR analysis for transgene expression

at transcript level using primers specific to yeast SAMDC gene (upper)

and actin primer. The RNA from wild-type plants and different transgenic

plants (A7, B2, Q5 and T9).

(D) Northern blot with the probe specific to ySAMDC gene. RNA from

wild-type plants and different transgenic plants (A7, B2, Q5 and T9).


0

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1 2 4 6 8 10 12

Duration (h)

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B2

Q5

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Figure 3. Electrolyte leakage of leaf discs of wild-type (WT), and trans-

genic yeast S-adenosyl-l-methionine decarboxylase (ySAMDC) (Lines

B2 and Q5) tomato plants following heat stress (42 ◦C) of varying

durations (1, 2, 4, 6, 8, 10 and 12 h).

Values are means of four repeats ± standard error (SE). Different letters

(in column) indicate significant differences (P < 0.05) between means

within each sampling time with Duncan’s multiple range test.

compared with the control conditions, and there was no signifi-cant difference between two transgenic lines. On the contrary,Put level in the non-transformed plants and pCAMBIA1301plants remained unchanged.

CO2 assimilation and chlorophyll fluorescence parameters

Photosynthetic gas exchange parameters were examined intransgenic plants, which were kept at 28 ◦C for 3 d after havingbeen exposed to high temperature of 38 ◦C for 4 d (Figure 4).Net photosynthetic rate (Pn) was not significantly different intransgenic lines compared with wild-type plants grown under

Table 1. Polyamine level in leaves from in vivo-grown tomato plants of non-transgenic plants and transgenic lines B2 and Q5 with or without high

temperature treatment

Spermidine Spermine PutrescinePlants

Control Stress Control Stress Control Stress

Non-transgenic 128 ± 32a,b 173 ± 25a 40 ± 9.4a 56 ± 7.2a 170 ± 26a,b 203 ± 39a,b

Transformed plant with pCAMBIA1301 113 ± 21a 165 ± 37a 32 ± 5.9a 47 ± 12a 201 ± 34a,b 196 ± 17a

Transgenic B2 184 ± 43b 376 ± 55b 49 ± 11a,b 95 ± 16b 137 ± 43a 236 ± 33a,b

Transgenic Q5 177 ± 35b 434 ± 64b 60 ± 14b 91 ± 11b 213 ± 45b 262 ± 42a

Values (nmol/g fresh weight) represent average of data from three independent experiments and are shown as means ± standard error (SE) Different

letters in columns indicate significant differences (P < 0.05) between means of different transgenic lines and wild-type with Duncan’s multiple range test.

control conditions. Although Pn was 20.9% lower in B2 than Q5,the difference was not significant (P = 0.064). Pn significantlydecreased in all plant types when exposed to 38 ◦C for 4 d. How-ever, the decrease in Pn was much greater in wild-type plantsthan in transgenic plants. Pn in transgenic plants was signifi-cantly higher than wild-type plants after high temperature treat-ment. Moreover, Pn in transgenic lines recovered near to normallevels when the plants were brought to optimal growth conditionsfor 3 d, while not in wild-type plants. Similar results were alsoobserved in the stomatal conductance (Gs) and internal CO2

concentration (Ci). Gs decreased 31.3% and 20% in B2 andQ5, respectively, compared with 56.3% in wild-type plants underhigh temperature. Ci decreased by 23.6% and 13% in line B2and Q5, respectively (Figure 4), while it reached 37.9% in wild-type plants. Gs and Ci in transgenic plants were significantlyhigher than in wild-type plants after high temperature treatment.These results indicated that the tolerance of CO2 assimilationrate to high temperature was greatly increased in transgenicplants.

The transpiration rate (Tr) was not significantly affected intransgenic plants under control growth conditions. Increased Trwas observed in wild-type and transgenic tomato plants whenthey grew under high temperature of 38 ◦C for 4 d. However,Tr increase in transgenic lines was significantly lower thanthat in wild-type plants. After 3-d recovery, Tr decreased inall examined plants to near the normal level and showed nosignificant difference between transgenic and non-transgenicplants.

The ratio of variable fluorescence (Fv) to maximum fluo-rescence (Fm) was measured to estimate leaf photochemicalefficiency. The maximum quantum efficiency of photosystemII (PSII), as given by Fv/Fm, was not influenced by transgeneof ySAMDC under normal growth conditions. Fv/Fm decreasedslightly after high-temperature treatment for 4 d. Such declinewas not observed in transgenic line Q5 (Figure 5). However,the difference between transgenic plants and non-transgenicplants was not significant under high temperature conditionsand during the period of recovery.


Figure 4. Net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and internal CO2 concentration (Ci) in tomato leaves from

the non-transgenic plants (WT), transgenic line B2, and Q5 treated with high temperature conditions (38/30 ◦C) for 4 d (4 d), and then transferred to

normal conditions (28/22 ◦C) for recovery for 3 d (7 d).

The control plants were grown in normal conditions (28/22 ◦C) at all times. Data are the means of three independent repeats ± standard error (SE)

shown by vertical error bars. Different letters indicate significant differences (P < 0.05) between means within each sample day with Duncan’s multiple

range test.

Activities of antioxidant enzymes andmalondialdehyde content

Antioxidant enzymes including superoxide dismutase (SOD),catalases (CATs), peroxidase (POD) and ascorbate peroxidase(APX) scavenge active oxygen species to protect plant cellsthrough a complex antioxidant system (Scandalios 1993). Theactivities of SOD, APX, guaiacol peroxidase (G-POD) andCAT significantly increased in all lines after exposure to hightemperature (Figure 6). GPOD and CAT were even increasedby more than twofold. In general, the activities of SOD, APX,GPOD and CAT increased in transgenic lines; however, theextent of increase of SOD and CAT in two transgenic lines wassignificantly different. SOD activity was significantly higher inQ5 than that in B2 and wild-type plants, while CAT activity washigher in B2 than in Q5. Activities of SOD, APX, GPOD and CAT

decreased after recovery in normal temperature for 3 d. Therewas no significant difference between transgenic and wild-typeplants after recovery.

Malondialdehyde (MDA) is the product of membrane lipidperoxidation. Increasing MDA content in plant cells indicatesdamage of cell membranes (Scandalios 1993), which canlead to inhibition of photosynthesis and respiration processes,and thus reduced plant growth. MDA content was significantlyincreased and almost doubled in wild-type plants after hightemperature treatment for 4 d (Figure 6). MDA also increasedin transgenic lines after heat-stress, but the increase wassignificantly lower than wild-type plants. After recovering innormal temperature for 3 d, MDA content in all tested plantsdecreased approximately to the normal level, and there wasno significant difference between transgenic and wild-typeplants.


Figure 5. Effects of high temperature treatment and recovery on Fv/Fm

in tomato leaves from the non-transgenic plants (WT), transgenic lines

B2 and Q5.

Data are the mean of three independent repeats ± standard error (SE)

shown by vertical error bars.

Discussion

It is known that the stress tolerance is associated with mainte-nance of high concentrations of polyamines. Increased levelsof Spd and Spm under stress conditions have been implicatedin radical scavenging mechanisms (Lester 2000). Transgenicplants overexpressing PAs gene with increased PA levelsshowed tolerance to salinity, drought, cold or acidic stress (Royand Wu 2002; Waie and Rajam 2003; Wi et al. 2006). Recently,Prabhavathi and Rajam (2007) reported that transgenic egg-plants with oat arginine decarboxylase (ADC) gene exhibitedincreased polyamine content and enhanced tolerance levels tomultiple abiotic stresses.

In the present study, transformation with ySAMDC in tomatoplant significantly increased PAs accumulation, especially Spmand Spd under high temperature conditions. Spd and Spmincreased 2.4-fold and 1.7-fold, respectively, on average intransgenic lines compared with wild-type plants (Table 1).Increased Spd and Spm levels are usually associated withenhanced plant tolerance to unfavorable conditions (Jimenez-Bremont et al. 2007). Spm accumulation is associated with thestabilization of the membranes and cell constituents throughbinding with negatively charged groups (Ha et al. 1998).On the other hand, we found that increase in Put was notsignificantly different between transgenic plants and wild-typeplants, although Put also increased when exposed to hightemperature. These results suggested that plants overexpress-ing SAMDC genes with high endogenous PA levels wouldbe very important for abiotic stress tolerance, which may be

implemented by PA involvement of signal transduction path-ways associated with this process (Sairam and Tyagi 2004;Kasukabe et al. 2004; Vinocur and Altman 2005; Alcazar et al.2006).

Although the genetic engineering of the synthesis ofpolyamine to tolerate abiotic stress appears promising (Alcazaret al. 2006; Vinocur and Altman 2006), there are still noreports on the enhanced tolerance of photosynthesis to hightemperature stress. Photosynthesis is, among the plant func-tions, the most sensitive to high temperature damage (Havaux1993; Camejo et al. 2005). Heat-stress caused a significantreduction in Pn. However, this was only partly a result ofstomatal closure, since carboxylation was also limited (Figure 4).However, the decline in assimilation rates in transgenic plantswas smaller compared with wild-types, which suggested thatplants in these treatments were able to better use internalcarbon dioxide. Such a protective influence of these compoundsmay explain the higher stomatal conductance, carboxylation ef-ficiency (Figure 4). On the other hand, the antitranspirant actionobserved in this study confirmed previous reports for polyamine.A lower Tr rate helped to conserve water in heat-stressedplants (Rajasekaran and Blake 1999). The results showed thatPn responses to high temperature were partially reversed byenhanced polyamine level in SAMDC tomato plants; however,the promotive effects of polyamine were not the result of amore favorable PSII. High temperatures induced no significantdamage to PSII, as given by Fv/Fm. SAMDC transgene had nosignificant effect on the chlorophyll fluorescence parameter. Ourresults are in agreement with previous studies that PSII is notaffected at moderately high temperature (35 ◦C–45 ◦C) althoughPSII has long been considered the most temperature-sensitivecomponent of photosynthesis (Havaux 1993; Haldimann andFeller 2005). Hence, the promoting action of polyamine on Pnmay have been triggered by other processes. There are manydata that strongly support the hypothesis that polyamines play amore complex role in the regulation of structure and function ofthe photosynthetic apparatus. Thus, there was strong indicationthat polyamines hold a pivotal role in photosynthesis, since theyhave been reported to be capable of simulating a photosyntheticapparatus adapted to high temperature conditions throughthe enhanced thermostability of thylakoid membranes (Kusanoet al. 2007). Another possible explanation is that the apparentincreased protection of photosynthesis from SAMDC couldresult from an effect on carotene accumulation, which directlyaffected the xanthophyll cycles. Enhanced carotene content inSAMDC transgenic plants provides the clues that polyaminecould apparently affect carotene biosynthesis (Mehta et al.2002).

As antioxidants, polyamines may protect against oxidativedegradation and membrane damage, resulting in maintenanceof homeostasis in plant cells (Rodrıguez-Kessler et al. 2006).Enhancing the PA accumulation was found to be associ-ated with increased antioxidant enzyme activities under stress


Figure 6. Effects of high temperature treatment on the activity of superoxide dismutase (SOD), guaiacol peroxidase (G-POD), ascorbate peroxidase

(APX), catalase (CAT) and malondialdehyde (MDA) content in tomato leaves from the non-transgenic plants (WT), transgenic lines B2 and Q5.

Data are the mean of three independent repeats ± standard error (SE) shown by vertical error bars.

conditions. Wi et al. (2006) proved that overexpression ofSAMDC in tobacco could induce high mRNA levels of severalantioxidant enzymes, such as ascorbate peroxidase, super-oxide dismutase and glutathione S-transferase in transgenicplants. PAs were shown to function mainly as a scavengerof free superoxide radicals under conditions of weak stress,whereas under conditions of strong stress they mainly acted aspositive modulators of antioxidant genes (Tkachenko and Nes-terova 2004; Liu et al. 2007). In the present study, an increase in

Spd, Spm and the total free polyamine level were found underhigh temperature treatment, which was accompanied by themarkedly increased antioxidant enzyme activity and decreasedMDA content in transgenic plants. The stress increased lipidperoxidation and this was significantly alleviated by overexpres-sion of the ySAMDC gene. There was less lipid peroxidationrelative to wild-type plants. These results suggested that apossible mechanism of heat tolerance was due to an increasein polyamines with marked increases of antioxidant enzyme


activities and alleviation of the membrane damage caused byreactive oxygen species (ROS) during heat stress. Liu et al.(2007) have demonstrated that Spd and Spm levels in tomatoleaves could have a protective role against heat stress-inducedROS.

In summary, in the present paper, an ySAMDC cDNA fromyeast was expressed in tomato, the transgenic progeny (T2), be-longing to two different lines, were compared with wild-type andthe empty vector-transformed (pCAMBIA1301) controls in termsof PA metabolism, photosynthetic parameters and response toheat stress. The introduction of the ySAMDC gene into tomatoincreased the PAs accumulation and antioxidant enzyme activityand also enhanced CO2 assimilation, and therefore enhancedthe tolerance to heat stress.

Materials and Methods

Plant material and plasmid

The seeds of tomato (Lycopersicon esculentum Mill.) variety“zhongshu No.6” were obtained from the Institute of Veg-etables and Flowers, Chinese Academy of Agricultural Sci-ences, Beijing, China. The Agrobacterium tumefaciens strainLBA4404 containing binary plasmid pCAMBIA1301 with theSAMDC gene from Saccharomyces cerevisiae under the controlof cauliflower mosaic virus CaMV35S promoter and nopalinesynthase terminator, and hygromycin phosphotransferase (hpt)as plant selection marker was used for tomato transformation(Ding et al. 2006). An empty pCAMBIA1301 vector was alsotransferred into the A. tumefaciens strain LBA4404 as a positivecontrol.

Tomato transformation and regeneration

Cotyledon explants, collected from about 6–7 d seedlings,were used for transformation. The Agrobacterium culture grownto an optical density (O.D.) (A600) of 0.1–0.2 was used forinfection (10 min) on 2-d-old precultured explants, grown onshoot regeneration medium (SRM), Murashige and Skoog (MS)medium supplemented with 1 mg/L benzylaminopurine (BAP)and 0.15 mg/L indole-3-acetic acid (IAA) for 2 d. After in-fection, the explants were co-cultured on SRM for 2 d, andthen transferred to selection medium (i.e. SRM containing7.0 mg/L hygromycin[Hyg] + 300 mg/L Cefotaime [Cef]) andcultured for 35 d with one sub-culture. The small shootsobtained on selection medium were subjected to proliferationon MS medium fortified with 0.5 mg/L BAP and 0.1 mg/LIAA. The well-grown shoots were excised and transferredto the rooting medium (half-strength MS with 0.05 mg/L 1-naphthaleneacetic acid (NAA) + 7.0 mg/L Hyg + 300 mg/LCef). The rooted plants were transferred to pots containing peat: perlite in a 2:1 ratio and covered with polythene bags for 1

week to maintain high humidity for hardening in the growthchamber and then transferred to a greenhouse (Ding et al.2006).

Polymerase chain reaction

The putative transgenic plants were analyzed by PCR for theintegration of the transgene. DNA was isolated from the leafexplants by the Cetyl Trimethyl Ammonium Bromide (CTAB)method. About 100 ng of DNA from untransformed plants, aswell as putative transgenic lines, was taken and mixed with100 mM of primer pair, 7.5 μL PCR buffer, 100 mmol/L dNTP mixand 0.5 U of Taq polymerase (MBI Fermentas, Burlington, On-tario, Canada).The PCR program included denaturation at 94 ◦Cfor 5 min followed by 30 cycles of denaturation at 94 ◦C for 30 s,annealing at 56 ◦C for 45 s and synthesis at 72 ◦C for 45 s and fi-nally one cycle of 7 min at 72 ◦C. The primer pairs specific for theamplification of a 1.2-kb fragment of the ySAMDC gene were 5′-CGGAGCTCACATGGCTGTCACCATAAAAGAATTGA-3′ and5′-GCCGCGGATCCTTTTCATATTTTCTTCTGCAATTTC-3′.

Southern blot hybridization

Tomato genomic DNA (10 μg) was restricted with EcoRI todetect the copy number of the transgene. Southern blotswere prepared by the standard procedure (Sambrook et al.1989) using Hybond-N Nylon membrane (Amersham Pharma-cia Biotech, Piscataway, NJ, USA). The hpt gene probes wereprepared using 32P-labeled dCTP by nick translation as themanufactures guidelines (Gibco-BRL, Gaithersburg, MD, USA).Hybridization was carried out for 18–22 h at 65 ◦C. Signals weredetected by exposure of storage phosphor screens, which werescanned in a Typhoon 9400 (GE Healthcare, Piscataway, NJ,USA).

RNA extraction, RT-PCR and Northern blot

Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA,USA) as a template and the cDNA was made using SMART PCRcDNA Synthesis Kit according to the manufacturer’s instructions(Clontech, Mountain View, CA, USA). The 20 μL of reactionmixture included 1× PCR reaction buffer, 400 mmol/L of dNTPs,1.2 mmol/L of each primer, 2 U Taq Polymerase and 1.2 μLcDNA. The reaction mixture was heated to 95 ◦C for 10 min,followed by 30 cycles of 30 s denaturation at 94 ◦C, annealingat 53 ◦C for 30 s, extension at 72 ◦C for 1 min and finalextension for 10 min. The PCR products were analyzed on 1%agarose gel.

Twenty micrograms of total RNA were electrophoresedthrough a 1.2% (w/v) denaturing formaldehyde/agarose gel,blotted to a Nitran filter by the capillary blot method.Prehybridization and hybridization were carried out in 50%


formamide buffer using an [32P]-labeled SAMDC cDNA probe at42 ◦C. Yeast specific SAMDC cDNA fragment was labeled usingRandom Priming Labeling Kit (Promega, Shanghai, China) withα-[32P] dCTP. Washing of the filter was carried out first with2× standard saline citrate (SSC) , 0.5% sodium dodecyl sulfate(SDS) for 7 min and then 1× SSC, 0.5% SDS at 55 ◦C for 4 min.

Electrolyte leakage

The T1 seeds obtained from primary transgenic plants werescreened with 7 mg/L hygromycin in seedlings for PCR analysis.Two independent lines (B2 and Q5) of the hygromycin-resistantprogenies (T2) from self-fertilized T1 transgenic tomato plantswere selected for the following study. The leaf discs (0.80 cm indiameter) were punched out with a cork borer from the youngestfully expanded leaves of two T2 transgenic lines and the wild-type plants of 40-d-old plantlets grown under normal conditions(28 ◦C/22 ◦C). The leaf discs were immersed in a test tubecontaining 10 mL of deionized, distilled water. The base of thetube was submerged in a water bath at 42 ◦C and removed after1, 2, 4, 6, 8, 10 and 12 h for testing. Following heat treatment,electrolyte leakage was measured using a conductivity meter(Greisinger Electronic, Regenstauf, Germany). Determinationof percent electrolyte leakage was calculated according to themethod of Ahn et al. (1999). Means for all values are an averageof three subsamples in each plant with four replications.

High temperature treatment

The seeds of T2 transgenic lines, empty vector-transformedline and wild-type plants were surface-sterilized and sowed invermiculite-filled plastic egg trays in the greenhouse of ZhejiangUniversity, China. After 3 weeks, the seedlings were transferredto 10 cm individual plastic pots with the media of peat, perlite andvermiculite mixture (7:2:1 in volume). The pots were incubatedin a greenhouse maintained at 28 ◦C/22 ◦C (day/night) withphotosynthetic photon flux density (PPFD) of 1 000 μmol/m2

per s, a relative humidity of 75% to 80% and a light : darkcycle of 14:10 h. Nutrient solution were supplied once every 3–4 d, and the solution included Ca(NO3)2 5.0 mmol/L, KNO3

4.0 mmol/L, KH2PO4 1.0 mmol/L, MgSO4 2.0 mmol/L, Fe-ethylenediaminetetraacetic acid (EDTA)70.0 μmol/L, MnSO4

10.0 μmol/L, H3BO3 50.0 μmol/L, ZnSO4 0.7 μmol/L, CuSO4

0.2 μmol/L, (NH4)6Mo7O24 0.01 μmol/L.To avoid complications resulting from differences in plant size

and reproduction stage, all stress assays were carried out with35-d-old seedlings. At this stage no apparent differences inplant size and growth were observed between the wild-typeand transgenic plants (data not shown).

The treatments were conducted in a climate chamber with38 ◦C/30 ◦C (day/night), 80% relative humidity, 16 h light/day(photonflux of 350 μmol/m2 per s) After 4 d of high-temperature

treatment, the seedlings were recovered for 3 d at the temper-ature 28 ◦C/22 ◦C. Samples on the 4th (stressed) and 7th days(recovered) after high-temperature treatment were used for theevaluation of photosynthesis characteristics and heat tolerance.The control plants were maintained under a constant temper-ature of 28 ◦C/22 ◦C. All of the measurements on physiologicaland biochemical parameters were carried out on the youngestfully expanded leaves.

Analysis of polyamines

Polyamines were estimated in seedlings of the wild-type andtransgenic lines with and without high temperature treatmentaccording to the protocol of Minocha et al. (1990). Tomatoleaves (0.4 g) from five seedlings were pooled and poweredwith nitrogen, then extracted in 10 volumes of 4% perchloric acid(PCA) and centrifuged at 20 000g for 30 min at 4 ◦C. Aliquots(0.2 mL) of the supernatant containing free polyamines weredansylated, extracted in tobuene and analyzed by high perfor-mance liquid chromatography (HPLC) (Shimadzu CS-9301, LC-7A, Japan) on a reverse phase C18 column (Spherisorb ODS2,5-μm particle size, 4.6 × 250 mm, Waters, Wexford, Ireland)using a programmed acetonitrile : water step gradient, respec-tively, with a 1 mL/min flow rate. Eluted peaks were detected witha fluorescence spectrophotometer at excitation and emissionwavelengths of 360 and 506 nm and their areas were recordedand integrated relative to those of standard PAs (Sigma, St.Louis, MO, USA). Three extractions of polyamines were madefrom each sample and each extraction was quantified by HPLCin duplicate.

Photosynthetic and chlorophyll fluorescence parameter

Gas exchange parameters were determined on a fully ex-panded attached leaf of tomato seedlings by using an in-fra red gas analyzer (CIRAS-1-PP systems, Hitchin, Herts,UK). For the measurement of net photosynthetic rate (Pn),intracellular CO2 content (Ci) and stomatal conductance (Gs),air temperature, relative humidity, CO2 concentration andPPFD were maintained at 25 ◦C, 80–90%, 400 μL/L and1 000 μmol/m2 per s, respectively. The measurements onthese photosynthetic parameters lasted approximately 10 min,during which no significant recovery was observed on theseparameters.

Chlorophyll fluorescence was measured using a portablepulse modulated fluorimeter (Model FMS-2, Hansatech, Norfolk,UK). After a dark adaptation period of 20 min, minimum fluores-cence (Fo) was determined under a weak pulse of modulatinglight over 0.8 s and maximal fluorescence (Fm) was inducedby a saturating pulse of light (8 000 μmol/m2 per s) appliedover 0.8 s. The measurements were carried out on the attachedleaves of tomato seedlings. The maximal efficiency of PSII


photochemistry was determined as the ratio of variable tomaximal chlorophyll fluorescence (Fv/Fm) (Krause and Weis1991).

Activities of antioxidant enzymes and MDA content

For the enzymatic activity analysis, 0.5 g fresh leaf sam-ples were homogenized in an ice bath in 5 mL 25 mmol/LHEPES buffer (contained 0.2 mmol/L EDTA, pH 7.8) and2% polyvinylpyrrolidone. The homogenate was centrifuged at13 000 g for 20 min at 4 ◦C. Supernatant obtained was used forenzyme analysis. All assays were carried out in a UV/visible lightspectrophotometer (Shimadzu UV-2410 PC). Protein contentwas determined according to the method of Bradford (1976),which uses bovine serum albumin as the standard. Four sepa-rate extractions were carried out for each treatment for assayof the activities of all enzymes. The APX, CAT and SOD weredetermined according to our previous report (Lu et al. 2008).The method according to Cakmak and Marschner (1992) wasused to determine the activity of G-POD with some modifica-tions. The contents of the reaction mixture were: 25 mmol/Lphosphate buffer (pH 7.0), 0.05% guaiacol, 10 mmol/L H2O2

and enzyme extract. Increase in absorbance at 470 nm causedby guaiacol oxidation was used to measure activity.

Thiobarbituric acid (TBA) test was used to measure MDA.Leaves were homogenized, centrifuged in a potassium phos-phate buffer (pH 7.8) for 20 min at 12 000g, and 1 mL of thesupernatant was incubated in boiling water for 30 min. The tubeswere placed in an ice bath to stop the reaction after whichthe samples were centrifuged at 1 500g for 10 min and theabsorption read at 532 nm. The value for non specific absorptionat 600 nm was subtracted.

Data analysis

The data reported in tables and figures are means of thevalues with standard error (SE) and examined statistically byANOVA using SAS software (SAS, 1996, SAS Institute, Cary,NC, USA). Means were compared for significant differencesbetween treatments according to Duncan’s multiple range testat P < 0.05.

Acknowledgements

The authors are thankful to Dr Riaz Ur Reman (Quaid-i-AzamUniversity) for his kind proofreading of this article.

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polyamine accumulation in transgenic tomato enhances the tolerance to high temperature stress

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