Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium

Environmental and Experimental Botany 72 (2011) 157–166

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Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium Xiao-Peng Wen a,b,1 , Yusuke Ban c,1 , Hiromichi Inoue a , Narumi Matsuda d , Masayuki Kita e , Takaya Moriguchi a,∗ a

National Institute of Fruit Tree Science, Tsukuba, Ibaraki 305-8605, Japan Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, Guizhou, PR China c National Institute of Fruit Tree Science, Higashihiroshima, Hiroshima 739-2494, Japan d Yamagata General Agricultural Research Center, Horticultural Experimental Station, Sagae, Yamagata 991-0043, Japan e National Institute of Fruit Tree Science, Okitsu, Shizuoka 424-0292, Japan b

a r t i c l e

i n f o

Article history: Received 24 August 2010 Received in revised form 24 February 2011 Accepted 2 March 2011 Keywords: Abiotic stress Antioxidant European pear (Pyrus communis L. ‘Ballad’) Polyamines Spermidine synthase (SPDS) Transgene

a b s t r a c t Three transgenic European pear (Pyrus communis L.) lines with reduced spermidine synthase (SPDS) expression and spermidine (Spd) titers were developed using a construct containing an apple SPDS gene (MdSPDS1) in antisense orientation. After exposure to either salt or cadmium stress, growth inhibition was more severe in the antisense lines than in the wild-type (WT). The antioxidant system, as shown by glutathione (GSH) content, activity of glutathione reductase (GR) and superoxide dismutase (SOD), and proline accumulation, was not effectively induced under stress in the antisense lines as compared with the WT. The reduction in antioxidant system function in the antisense lines was accompanied by a greater accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation. Growth inhibition, Spd level, and parameters indicative of the antioxidant system were significantly ameliorated by exogenous Spd application. Under either salt or cadmium stress, GSH content, GR and SOD activity, and proline accumulation were positively correlated with Spd, putrescine (Put), and total polyamine titers. Conversely, MDA level showed a significantly negative correlation with these polyamines under both stress conditions. Thus, the responses to stress treatments were first identified in the SPDS antisense European pears, and the results provide further evidence for the important role of polyamines in both salt and cadmium stress tolerance, in which the polyamines act, at least in part, by influencing the antioxidant system. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) are low-molecular-weight organic cations that are found in a wide range of organisms from bacteria to plants and animals. In plants, polyamines are involved in various physiologi-

Abbreviations: ADC, arginine decarboxylase; CaMV, cauliflower mosaic virus; DIG, digoxigenin-dUTP; FWI, fresh weight increment (%); GR, glutathione reductase; GSH, glutathione; MDA, malondialdehyde; Put, putrescine; ROS, reactive oxidative species; SAMDC, S-adenosylmethionine decarboxylase; SHI, shoot height increment (%); SOD, superoxide dismutase; SPDS, spermidine synthase; Spd, spermidine; Spm, spermine; TBA, thiobarbituric acid. ∗ Corresponding author at: National Institute of Fruit Tree Science, Research Team for Effects of Global Warming on Fruit Trees, 2-1 Fujimoto, Tsukuba, Ibaraki 3058605, Japan. Tel.: +81 29 838 6500; fax: +81 29 838 6437. E-mail address: [email protected] (T. Moriguchi). 1 These authors contributed equally to this work. 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.03.001

cal events such as development, senescence, and stress responses; in particular, the involvement of polyamines in environmental stress alleviation has been examined in detail (Bouchereau et al., 1999; Pang et al., 2007; Alcázar et al., 2010). During the past few years, various approaches had been employed to unravel key functions of different polyamines in the regulation of abiotic stress tolerance. Exogenous applications of polyamines and polyamine biosynthetic inhibitors have been tested in a variety of plant species (e.g., Songstad et al., 1990; Shen et al., 2000; Tang et al., 2004; Zhao and Qin, 2004; Verma and Mishra, 2005; Liu et al., 2006). These methods, however, suffer from some drawbacks: (i) absorption and catabolic flow of exogenously applied compounds cannot be precisely regulated, and (ii) application of inhibitors causes non-specific and pleiotropic effects on plant metabolism (Noh and Minocha, 1994; Bhatnagar et al., 2001; Duan et al., 2007). Alternatively, the use of transgenic plants is a powerful means to investigate the correlation between modulation of a single gene and its specific role. The response to environmental stress has

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been examined using transgenic plants overexpressing various polyamine biosynthesis genes. For example, it has been reported that plants show high stress tolerance with overexpression of arginine decarboxylase (ADC) (Capell et al., 2004; Prabhavathi and Rajam, 2007), spermidine synthase (SPDS) (Kasukabe et al., 2004), or S-adenosylmethionine decarboxylase (SAMDC) (Wi et al., 2006). Nevertheless, the precise molecular mechanism by which polyamines control plant responses to stress stimuli, are largely unknown. Previously, we have generated transgenic European pear lines overexpressing apple SPDS (MdSPDS1), which has enhanced tolerance to multiple environmental stresses such as salt, hyperosmosis, and heavy metals, by inducing an antioxidant effect (He et al., 2008; Wen et al., 2008, 2010), which suggests that polyamines likely participate in the amelioration of oxidative status under abiotic stress. The combination of gain- and loss-of-function analyses is a comprehensive and circumspect strategy to reveal the function of a target gene. Thus, in addition to overexpression of a particular polyamine biosynthetic gene, introduction of the corresponding antisense transgene provides an excellent way to address the contribution of the polyamine to abiotic stress alleviation and to confirm the amelioration of oxidative status by comparison with the results from the sense transgene. Toward this end, we further developed transgenic European pear with reduced SPDS expression and Spd titer via antisense technology, and subjected these transgenic antisense lines and the wild-type (WT) to environmental stresses. To enable us to build on the results previously obtained in the MdSPDS1-overexpressing European pear (Wen et al., 2008, 2010), we followed methods described in those previous reports by exposing the antisense lines to either salt or cadmium stress and by measuring the same parameters as those in the previous study (Wen et al., 2008, 2010), with the addition of proline content. We also investigated the effects of exogenous application of Spd on the restoration of the antioxidant parameters in the antisense lines which were subjected to either salt or cadmium stress. To our best knowledge, this is a first report for the investigation in SPDS antisense plants. The results presented herein further demonstrate a role for polyamines in stress tolerance in European pear, by increasing expression and function of the antioxidant system.

2. Materials and methods 2.1. Generation and confirmation of the antisense transgenes MdSPDS1 (AB072915) was excised with BamHI/KpnI from pBluescript-MdSPDS1 (Zhang et al., 2003) and then ligated into binary vector pBI121 in the antisense direction under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Agrobacterium tumefaciens strain LBA4404 was transformed with the binary vector construct and then used to transform European pear (Pyrus communis L. ‘Ballad’). The preparation of explant material of in vitro pear shoots, Agrobacterium inoculation, and selection procedures followed the method described by Matsuda et al. (2005). RNA gel blotting was used to detect mRNA abundance in the antisense lines, with the WT included as a control. The sense- and antisense-strand RNA probes were labeled using the full-length sequence of MdSPDS1 as a template and according to procedures supplied by the manufacturer of the digoxigenin-dUTP (DIG) RNA labeling kit (Roche Diagnostics, Mannheim, Germany). Other procedures for RNA gel blotting were performed according to the method of Ban et al. (2007). Based on the RNA gel blotting result, lines representing different reduced levels of MdSPDS1 expression were selected and analyzed with PCR and Southern blotting, so as to further confirm integration of the MdSPDS1 transgene. Total genomic DNA

was isolated from three selected antisense lines (AS21, AS22, and AS33) and the WT according to the method described by Porebski et al. (1997). PCR was conducted using a CaMV 35S-F (5 -TGT GAT AAC ATG GTG GAG CA-3 ) and MdSPDS1 C-F (5 -TGA TTG ACG CAA AAG CAA AG-3 ) primer set, which produces an expected band of 824 bp. The thermal conditions were 35 cycles of 30 s at 94 ◦ C, 30 s at 58 ◦ C, and 90 s at 72 ◦ C. For Southern blotting, 10 ␮g of genomic DNA was digested with DraI, which has no restriction sites within MdSPDS1. After digestion, the DNA was separated on 1% agarose gels overnight, then neutralized and denatured, followed by transfer to nylon Hybond N membrane (Amersham Biosciences, Piscataway, NJ, USA) via capillary transfer. After the membrane was UV-crosslinked with a crosslinker (UV StrataLinker 2400, Stratagene Japan, Tokyo, Japan), it was subjected to prehybridization at 42 ◦ C for 3 h in high-SDS hybridization buffer. The probe was the full-length sequence of MdSPDS1 labeled with DIG (Roche Diagnostics) by PCR. Hybridization and subsequent procedures were performed according to the method of Ban et al. (2007). Membranes were used to expose X-ray film (Fuji Photo Film, Tokyo, Japan) to detect hybridization. 2.2. Stress treatment In vitro cultured shoots of the selected antisense lines (AS21, AS22, and AS33) and the WT were maintained under 16 h/8 h light (photon flux density 55.6 ␮mol s−1 m−2 )/dark conditions at 25 ◦ C in MS medium (Murashige and Skoog, 1962) containing B5 organic components (Gamborg et al., 1968), 3% sucrose, 1.0 ␮M indole butyric acid, 4.0 ␮M zeatin, 4.0 ␮M N6 -benzylaminopurine, and 0.8% agar. The cultures were transferred to new medium at 4-week intervals. Age-matched shoots 5–6 cm in height from antisense lines and the WT were planted in bottles and cultured with or without salt (150 mM NaCl) or cadmium (150 ␮M CdCl2 ) according to the results of previous reports (Wen et al., 2008, 2010). For confirmation of exogenous Spd effects, antisense lines and the WT were also subjected to salt + Spd (150 mM NaCl + 1.0 mM Spd) or cadmium + Spd (150 ␮M CdCl2 + 0.5 mM Spd). 2.3. Growth of the antisense lines To elucidate the response of the transgenic lines to long-term stress, growth parameters were investigated after 10 days for salt (salt + Spd) stress or 21 days for cadmium (cadmium + Spd) stress because plants are often subjected to such stresses for a long time. The shoot height and fresh weight were measured at the start and end of the experiment. The shoot height increment (%) (SHI) during the treatment period was calculated using the following equation: SHI = ([SHat the end − SHat the start ]/SHat the start ) × 100. The fresh weight increment (%) (FWI) was calculated by substituting FW for SH in the equation. After measurement of the net increment, shoots from the same bottle were sampled, the bottom ends of the shoots in contact with the medium were removed, and the shoots were either used for malondialdehyde (MDA) quantification or immediately frozen in liquid nitrogen and stored at −80 ◦ C for further analysis. Meanwhile, another set of shoots was subjected to 30-day stress treatments for morphological observations. 2.4. Measurement of polyamines by HPLC Free polyamines were quantified according to the method described by Song et al. (2002). Briefly, polyamines were extracted by homogenizing the shoots with 5% (w/v) perchloric acid. After centrifugation, the supernatant was preserved and the pellet was resuspended in 5% perchloric acid after several washes with the same solution. After dansylation, polyamines in the supernatant were quantified via HPLC. 1,6-Hexanediamine was used as an inter-

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nal standard. Free polyamine content was expressed as nmol g−1 FW. 2.5. Measurement of antioxidant status and activity To further evaluate the oxidative status and responses to the tested stresses, we focused on alterations in glutathione (GSH) content, as well as the activities of superoxide dismutase (SOD) and glutathione reductase (GR), because SOD catalyzes dismutation of the superoxide anion into H2 O2 . H2 O2 can be reduced to H2 O via the ascorbate–glutathione cycle, in which GR plays an important role in the production of GSH (del Río et al., 1998). These parameters were quantified according to the protocols described in our previous study (Wen et al., 2010). 2.6. Measurement of MDA and proline Lipid peroxidation in shoots after the two stresses was determined as the amount of MDA as measured by the thiobarbituric acid (TBA) reaction, according to the method documented by Liu et al. (2006). Proline was measured according to a method modified from Wren and Wiggall (1965) and Chandler and Thorpe (1987). About 0.5 g of shoot tissue was homogenized in 4 ml of methanol:chloroform:water (12:5:1, v/v/v). After addition of 1.5 ml water and 1 ml chloroform, the solution was mixed and centrifuged at 10,000 × g for 5 min at 4 ◦ C. An aliquot (0.2 ml) of the upper phase was diluted with 0.8 ml water, 2.5 ml of a 3:2 (v/v) mixture of 4 ␮mol−1 ml glycine in acetic acid and 6 M phosphoric acid, and 2.5 ml 40 mg−1 ml ninhydrin. The solution (6.0 ml) was boiled at 95 ◦ C in a water bath for 40 min. After cooling at room temperature, 5 ml of toluene were added, the solution was mixed and centrifuged at 10,000 × g for 5 min at 4 ◦ C, and the absorbance at 520 nm was measured. The proline content was calculated using a standard curve. 2.7. Statistical analysis For SHI and FWI investigations, one treatment consisted of one shoot and measurements were repeated nine times, while for other investigations of antioxidant parameters, one experiment consisted of three shoots and measurements were repeated three times. Data are the mean values of nine replicates (SHI and FWI) or of three replicates (antioxidant parameters), and are expressed as mean ± SE. Statistical analysis was performed using one-way ANOVA (for all parameters excluding the correlation coefficient), or Fisher’s exact test of significance (correlation coefficient). Relationships between polyamine content and antioxidant parameters were plotted, and coefficients were computed based on the corresponding data pairs. 3. Results 3.1. Generation of antisense transgenic pear lines and determination of polyamine titers An antisense construct based on apple SPDS1 was introduced into European pear to reduce SPDS expression and thus, reduce Spd titer. A total of seven independent kanamycin-resistant pear lines were obtained from transformation with antisense MdSPDS1. RNA gel-blotting analysis with sense- and antisense-strand probes revealed that the expression levels of SPDS varied among the antisense lines (Fig. 1a). As expected, the sense probe of MdSPDS1 (apple) did not hybridize to RNA from the WT, indicating that no antisense strand was being produced. The sense probe detected

Fig. 1. (a) RNA gel blot analysis of antisense and sense MdSPDS1 expression in the pear transformants. Bottom panel shows rRNA stained with ethidium bromide. (b) PCR confirmation of the presence of the MdSPDS1 antisense construct in pear transformants. (c) DNA gel blot analysis of MdSPDS1 in the pear transformants using a cDNA probe generated based on conserved regions of plant SPDS.

antisense RNA in some of the transformed lines, although in others (e.g., AS19 and AS30), no signal was detected. Conversely, the antisense probe hybridized with endogenous SPDS mRNA in European pear (as found in the WT), possibly due to the high homology between apple and European pear sequences for SPDS. These results indicated that antisense MdSPDS1 could repress the expression of endogenous SPDS, and its repression could be monitored by RNA gel blotting. Among the seven kanamycin-resistant lines, three (AS21, AS22, and AS33) represented different levels of reduced expression of SPDS caused by the introduction of antisense MdSPDS1 (Fig. 1a). To further verify the presence of the antisense transgene, the three candidate antisense lines and the WT were used for PCR and Southern blotting analysis (Fig. 1b, c). A band with the expected size (824 bp) was successfully amplified in the three antisense lines but not in the WT (Fig. 1b). Southern blotting showed integration of MdSPDS1 into the three antisense lines: a single copy of MdSPDS1 was found in AS21, but several copies were found in AS22 and AS33 (Fig. 1c). This result was further confirmed with HindIII digestion (data not shown). To confirm reduction of the Spd titer by introduction of antisense MdSPDS1, polyamine titers were analyzed by HPLC. HPLC

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Table 1 Polyamine titers (nmol g−1 FW) in the antisense lines and the WT under salt stress for 10 days. Values within a column followed by the same letter are not significantly different at P < 0.05.

WT

AS21

AS22

AS33

Treatment

Put

Control Salt Salt + Spd Control Salt Salt + Spd Control Salt Salt + Spd Control Salt Salt + Spd

126.8 144.5 180.2 110.5 40.4 118.4 75.9 28.5 100.8 161.6 71.5 114.0

Spd ± ± ± ± ± ± ± ± ± ± ± ±

18.5 bc 30.3 ab 26.1 a 22.8 cd 7.7 e 12.5 c 16.3 de 3.1 f 20.3 cd 25.7 ab 17.5 de 11.6 c

analysis demonstrated that free Spd titer was reduced by antisense MdSPDS1 compared with the WT under the nonstressed (control) conditions (Tables 1 and 2). The ratios of Spd titers of AS21, AS22, and AS33 to those of the WT were 66.4–69.3%, 42.6–44.2%, and 54.8–54.7% under the control conditions (Tables 1 and 2). Thus, we successfully reduced Spd titers in the transgenic pear lines using antisense technology. Interestingly, the Spm titer was also reduced by the introduction of MdSPDS1 under the control conditions, but no particular trend with respect to the Put titer was observed, except for AS22, which showed a reduction of the Put titer compared to the WT in both experiments (Tables 1 and 2). 3.2. Alteration of polyamine titers under stress conditions Alterations of the polyamine titers were triggered by salt or cadmium stress. Salt stress significantly reduced the Spd titer in the WT. Salt stress had no significant effect on Spd in the antisense lines, although Spd in the salt-stressed antisense lines was significantly lower than that in the salt-stressed WT (Table 1). Salt stress significantly inhibited Put accumulation in the antisense lines; however, no significant alteration was observed in the WT. A significant increase in the Spm titer under salt stress was observed in the AS22 antisense line, but the Spm titer in the WT was significantly decreased upon salt stress. The only significant change in total polyamine titer (Spd + Spm + Put) was in the WT, in which the titer decreased after salt stress. In addition, the total polyamine titer in the antisense lines was significantly lower than in the WT under both salt-stressed and control conditions. After application of Spd along with salt stress (salt + Spd), the titers of Put, Spd, and total polyamines of all three antisense lines were significantly enhanced compared with the plants given only salt stress without Spd. Less of an increase was seen for Spm, and the increase was statistically significant in only one of the three antisense lines (AS22). The effects of exogenous application of Spd in the WT were similar to those in

192.4 158.5 322.3 127.8 116.8 207.6 82.0 76.9 238.3 105.5 93.6 234.2

Spm ± ± ± ± ± ± ± ± ± ± ± ±

4.4 c 9.9 d 35.2 a 4.8 ef 20.3 fg 22.5 bc 4.4 h 6.8 h 48.2 b 5.4 fg 18.5 gh 24.1 b

136.8 65.9 173.0 76.2 97.3 125.8 41.9 97.5 157.0 75.8 123.6 130.1

Total ± ± ± ± ± ± ± ± ± ± ± ±

39.8 bc 11.4 e 17.2 a 9.4 de 16.4 cd 7.4 c 6.3 f 16.2 cd 17.8 ab 13.8 de 30.2 cd 23.1 bc

456.0 368.8 675.5 314.4 254.4 451.8 199.8 202.9 496.1 342.8 288.7 478.2

± ± ± ± ± ± ± ± ± ± ± ±

18.9 b 43.4 c 78.5 a 33.2 cd 44.0 de 42.3 b 24.9 e 23.7 e 73.9 b 23.8 cd 64.7 de 44.3 b

the antisense lines, with significantly increased titers of Spd, Spm, and total polyamines compared to salt stress alone. Cadmium stress also caused diverse changes in polyamine titers among the antisense lines; the only consistent change was a decrease in Put, which was significant in two of the three lines (Table 2). In the WT, both Put and Spd titers were significantly enhanced by cadmium. When the total polyamine titer was examined after cadmium stress, it was not significantly changed in any of the lines except for AS21 and the WT, in which the former showed a significant reduction and the latter showed a significant induction. After application of Spd (cadmium + Spd), all polyamine titers (Put, Spd, Spm, and total) were significantly increased in both the antisense lines and the WT compared with the plants in the treatment with cadmium alone. The exogenous application of Spd enhanced the total polyamine titer in the antisense and WT lines, to a similar level, except that the final level of total polyamines under cadmium + Spd treatment in AS21 (542.5 nmol g−1 FW) was considerably lower than that in the other lines. The ratio of (Spd + Spm) to Put in the antisense lines significantly increased under both stresses, whereas that in the WT decreased (Table 3). After exogenous application of Spd, the ratio in saltstressed WT plants increased to the control level, but the ratio in cadmium-stressed WT plants decreased further. In antisense lines AS21 and AS22, Spd application under both stress conditions reduced the ratios to approximately half the values seen after stress treatment alone. AS33 showed a smaller but still significant reduction for cadmium stress + Spd, and no significant change for salt stress + Spd, compared with the ratios seen for the stress treatments alone. 3.3. Growth of the antisense lines The selected antisense transgenic lines displayed phenotypic changes (supplementary data). At day 30, the antisense lines

Table 2 Polyamine titers (nmol g−1 FW) in the antisense lines and the WT under cadmium stress for 21 days. Values within a column followed by the same letter are not significantly different at P < 0.05.

WT

AS21

AS22

AS33

Treatment

Put

Control Cadmium Cadmium + Spd Control Cadmium Cadmium + Spd Control Cadmium Cadmium + Spd Control Cadmium Cadmium + Spd

132.3 170.6 337.6 111.7 54.2 206.7 76.5 46.2 289.2 158.1 113.6 233.9

Spd ± ± ± ± ± ± ± ± ± ± ± ±

28.6 f 4.9 de 36.8 a 19.7 fg 11.6 gh 37.3 d 17.6 g 3.0 h 19.4 ab 35.4 de 15.9 fg 37.5 bc

186.9 221.4 252.4 129.7 110.2 225.2 82.6 127.7 259.9 102.3 135.2 231.7

Spm ± ± ± ± ± ± ± ± ± ± ± ±

9.6 c 10.4 b 18.1 a 3.2 de 19.0 def 28.9 ab 5.7 g 11.2 def 19.8 a 7.8 f 13.6 d 19.1 ab

139.7 89.8 101.2 77.3 55.2 110.7 42.2 76.5 100.3 74.0 96.1 157.4

Total ± ± ± ± ± ± ± ± ± ± ± ±

29.7 ab 6.3 cde 8.0 bc 9.2 de 12.6 e 12.7bc 7.1 e 17.4 d 10.9 bc 17.6 de 13.0 cd 14.4 a

458.9 481.9 691.2 318.7 219.6 542.5 201.3 250.4 649.4 334.5 344.9 623.0

± ± ± ± ± ± ± ± ± ± ± ±

6.0 d 12.0 c 59.7 a 25.9 e 42.8 f 78.3 b 28.6 f 31.0 f 45.0 a 50.3 e 39.1 e 70.1 ab

X.-P. Wen et al. / Environmental and Experimental Botany 72 (2011) 157–166 Table 3 The ratio of (Spd + Spm) to Put in the WT and the antisense lines under salt or cadmium stress. Values within a row followed by the same letter are not significantly different at P < 0.05. (Spd + Spm)/Put (%) Control Salt WT AS21 AS22 AS33 Cadmium WT AS21 AS22 AS33

Stress

Stress + Spd

258.7 189.1 166.6 114.4

± ± ± ±

47.6a 18.8c 13.8c 13.3b

158.9 532.7 616.2 305.3

± ± ± ±

16.0b 16.6a 49.2a 16.5a

275.8 282.0 400.0 318.9

± ± ± ±

6.0a 4.5b 52.9b 8.3a

266.6 196.5 171.3 117.2

± ± ± ±

42.1a 22.2b 9.7b 15.1c

182.6 306.5 441.0 204.6

± ± ± ±

4.9b 8.1a 23.0a 10.2a

105.1 163.6 124.7 167.7

± ± ± ±

3.7c 5.3b 4.8c 7.5b

showed somewhat weaker growth under the control conditions than did WT, indicating the importance of polyamines for normal growth. When subjected to salt stress, some shoots of the antisense lines, especially AS22 and AS33, deteriorated, whereas AS21 and the WT showed milder symptoms (supplementary data). The same trends were observed after exposure to cadmium. The phenotypic damage in antisense lines exposed to either salt or cadmium was considerably mitigated by Spd application, especially in AS22 and AS33 (supplementary data). To quantitatively evaluate biomass under stress conditions, SHI and FWI of the antisense lines and the WT were investigated after stress treatments. Compared with the control conditions, both stress treatments caused significant inhibition of SHI in the antisense lines (Fig. 2a, b). WT treatment with salt also caused a significant inhibition of SHI; the value for the WT under cadmium stress was lower than that of the control, but the decrease was not significant (Fig. 2a, b). SHI of the antisense lines under salt stress was only about 30% of the value under the control conditions (Fig. 2a), and SHI under cadmium stress was about 50% of that under the control conditions (Fig. 2b). SHI significantly increased in both antisense lines and the WT under salt stress after Spd application, though they did not reach the levels seen under the control conditions (Fig. 2a). A similar trend was seen for cadmium stress, but only the increases in AS21 and AS33 were significant (Fig. 2b). Similar patterns of reduction of FWI after stress alone and recovery in the presence of stress + Spd were also observed in the antisense lines, irrespective of stress type (Fig. 2c, d). 3.4. Antioxidant level and activity To elucidate the antioxidant response of the antisense lines to salt or cadmium stress, GSH levels and the activities of GR and SOD were investigated. Compared with the control conditions, salt treatment caused a significant increase in GSH content in AS33 and the WT, with a slight, non-significant increase in AS21 and AS22 (Fig. 3a). GSH levels of all three antisense lines under salt stress were significantly enhanced after application of exogenous Spd (up to 80% elevation), with a non-significant increase measured in the WT. In contrast to salt stress, cadmium stress caused a significant reduction in GSH levels of the antisense lines (Fig. 3b). GSH status in the cadmium-stressed antisense lines was significantly enhanced after Spd application. In WT plants treated with cadmium or cadmium + Spd, the same trends were observed but the changes were not statistically significant. Salt treatment caused no significant alteration in GR activity in the antisense lines, but did significantly increase GR activity in the WT plants. Conversely, cadmium caused a large inhibition of GR activity in both antisense and WT plants compared with that under the control conditions (Fig. 3d). When exogenous Spd was applied

161

Table 4 Correlations between the polyamine titers and the antioxidant parameters under salt or cadmium stress. One or two asterisks stand for a significant correlation at P < 0.05 or 0.01, respectively. Stress

Parameter

Put

Spd

Spm

Total

Salt

GR SOD GSH MDA Proline GR SOD GSH MDA Proline

0.73* 0.89** 0.81** −0.78** 0.66* 0.87** 0.91** 0.91** −0.79** 0.76*

0.90** 0.93** 0.82** −0.80** 0.89** 0.86** 0.91** 0.85** −0.90** 0.89**

0.65* 0.68* 0.64 −0.50 0.66* 0.73* 0.62 0.39 −0.30 0.50

0.89** 0.96** 0.86** −0.84** 0.89** 0.92** 0.94** 0.88** −0.80** 0.83**

Cadmium

along with either salt stress (Fig. 3c) or cadmium stress (Fig. 3d), GR activities of the antisense lines and the WT were elevated compared with those of the stress treatments alone; all increases except for that in the WT under salt stress + Spd were statistically significant. Furthermore, no significant differences in GR activities were observed among the antisense lines and the WT after Spd application in the presence of either stress (Fig. 3c, d). SOD activities of the antisense lines were less than those in the WT under the control conditions (Fig. 3e, f). When either WT or antisense lines were exposed to salt or cadmium stress, changes in SOD activity were relatively small compared with those seen for the other oxidation parameters measured. The only significant changes were an increase in salt-stressed AS33 (Fig. 3e), and a decrease in cadmium-stressed AS21 and AS22 (Fig. 3f). In addition, antisense lines showed significantly lower SOD activities in comparison with that in the WT regardless of salt or cadmium status (Fig. 3e, f). After exogenous Spd application, SOD activities of both the antisense lines and the WT were significantly enhanced, irrespective of stress, although in most cases, the SOD activities in the antisense lines were lower than those in the WT (Fig. 3e, f). 3.5. MDA and proline content MDA is generally considered an indicator of lipid peroxidation under stress. In most cases, no significant differences were found between the antisense lines and the WT under the control conditions (Fig. 4a, b). Significant MDA accumulation was observed when both the antisense lines and the WT were exposed to either salt (Fig. 4a) or cadmium (Fig. 4b) stress, with the greatest accumulation seen in the antisense lines. With Spd application, MDA accumulation in the antisense lines was significantly reduced under both salt and cadmium stresses (Fig. 4a, b). Significant reduction of MDA was also observed in the WT in the cadmium + Spd treatment, with no significant change seen for the salt + Spd treatment (Fig. 4a, b). Proline levels were investigated in the antisense lines and in the WT (Fig. 4c, d). Under the control conditions, the only significant differences measured in proline were between the WT and AS33. Proline levels were significantly elevated in the antisense lines and the WT subjected to salt or cadmium stress, although the increase after salt stress was significantly lower in the antisense lines than in the WT (Fig. 4c). After Spd application, the proline levels of the antisense lines and the WT were significantly enhanced under both stress conditions (Fig. 4c, d). 3.6. Relationship between polyamine and antioxidant parameters To further elucidate the mode of polyamine action in alleviation of oxidative stress after salt or cadmium stress, correlations between polyamine titers and antioxidant enzyme activity, GSH, proline, and MDA content were calculated for all the lines (Table 4). Under salt and cadmium, Spd had a significant positive correlation

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Fig. 2. Net increase in the shoot height increment (a, b) and in the fresh weight increment (c, d) of the antisense lines and the WT subjected to salt (10 days after stress) or cadmium (21 days after stress) stress. Data are means ± SE (n = 9). Values within the same panel followed by the same letter are not significantly different at P < 0.05.

with GR, SOD, GSH, and proline, and a significant negative correlation with MDA. Put and total polyamines also showed a positive relationship with these antioxidant parameters. Conversely, correlation coefficients between Spm and the antioxidant parameters were comparatively low and many were non-significant. 4. Discussion It has been recognized for some time that polyamines play pivotal roles in plant stress tolerance. Recent evidence have shown that many plants accumulate polyamines under abiotic stresses and that stress-tolerant genotypes possess enhanced ability to synthesize polyamines in response to abiotic stress compared with stresssensitive genotypes (Alcázar et al., 2006; Groppa and Benavides, 2008). However, the role of each polyamine type and the precise mechanisms underlying stress tolerance remain largely a matter of speculation. In previous studies, we demonstrated the alleviation of salt or cadmium stress in MdSPDS1-overexpressing European pear shoots (Wen et al., 2008, 2010). Therefore, in this study, we intended to expand on our previous results by studying antisense MdSPDS1 transgenic European pear lines, which was the first trial in this field. 4.1. Generation of the antisense lines and measurement of polyamine titers In this study, the expression levels of endogenous SPDS in European pear were reduced to various extents in the transgenic lines (Fig. 1a). The successful repression of accumulation of SPDS transcripts indirectly indicates that the coding sequences of SPDS in European pear and apple could be very similar. Spd levels in the three lines (AS21, AS22, and AS33) were reduced through antisense effects, but the reduction in the Spd titer was not as great as the reduction in RNA levels (Tables 1 and 2). Reduced Spd titer in TDNA insertion mutants of Arabidopsis SPDS1 and SPDS2 was shown to hinder normal embryo development (Imai et al., 2004). There-

fore, we assumed that an extremely low Spd titer could be lethal to plants. Conversely, when MdSPDS1 in the sense orientation was introduced into plants, including European pear, transgenic plants showed Spd titers only 1.5–3 times the values in the WT, despite the utilization of the constitutive CaMV 35S promoter (Kasukabe et al., 2004; Wen et al., 2008). It has been suggested that the toxicity of Spd places a cap on the maximum levels that can be achieved in transformed cells (Noh and Minocha, 1994; Schipper et al., 2000). Based on these findings, we speculate that adventitious shoots with either excessively high Spd (in a sense experiment) or extremely low Spd (in an antisense experiment) would not survive. This homeostatic situation also suggests that exogenous application of polyamines will not always lead to stress alleviation. When the exogenously applied polyamines are in excess, the polyamines might be catabolized by polyamine oxidase (PAO) bound to cell walls (Angelini et al., 1995; Tavladoraki et al., 1998) to maintain in vivo polyamine concentrations at a desirable level (Moschou et al., 2008a). In the current study, exogenous Spd was effective for stress alleviation in both the antisense lines and the WT, which may suggest insufficient Spd concentrations in these plants. Indeed, additional Spd treatment of a MdSPDS1-overexpressing line (#32; Wen et al., 2008) caused deterioration in growth and stress tolerance (unpubl. data). Moreover, in our preliminary experiment, all shoot tips except in AS22 died after exposure to cadmium + 1 mM Spd (data not shown), while with exogenous application of 0.5 mM Spd, tolerance to cadmium was significantly elevated in this study. Interestingly, in the antisense lines treated with either salt or cadmium stress, Put levels decreased and Spm levels increased, although these changes were not statistically significant in all cases (Tables 1 and 2). Theoretically, the Put titer should be retained at a high level because Put is not efficiently converted to Spd, and the Spm titer should be reduced because of the reduced expression of the SPDS gene in the antisense lines. It is difficult to give a reasonable explanation for this observation in the current study, except by assuming that in vivo polyamine concentrations are under subtle homeostatic regulation, as suggested by Bhatnagar et al. (2002).

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Fig. 3. GSH content (a, b) and enzyme activities of GR (c, d) and SOD (e, f) in the antisense lines and the WT subjected to salt (10 days after stress) or cadmium (21 days after stress) stress. Data are means ± SE (n = 3). Values within the same panel followed by the same letter are not significantly different at P < 0.05.

Therefore, the measurement of all types of polyamines, such as conjugated polyamines, may be necessary to elucidate this issue. Here, it is noteworthy that both the Put and Spm titers were increased after exogenous Spd application. In the case of Put accumulation, back-conversion of Spd to Put by PAO (Moschou et al., 2008b) could be involved, whereas in the case of Spm accumulation, exogenously applied Spd is expected to be utilized by spermine synthase for the biosynthesis of Spm. Obtaining direct evidence regarding this issue is an important objective for future work. 4.2. Relationship of the Spd titer with growth and antioxidant parameters Under salt stress, the antisense lines showed significantly lower FWI and SHI than in the WT plants (Fig. 2a, c). FWI and SHI in the antisense lines were also significantly lower than in the WT plants under cadmium stress (Fig. 2b, d), except for AS21, in which a non-significant increase in FWI was measured (Fig. 2d). In most

cases, the SHI and FWI values, which were reduced by salt or cadmium stress, were restored to levels similar to those in the WT by exogenous Spd application, which could suggest alleviation of stress effects through increased levels of Spd. Therefore, the growth characteristics of the antisense lines reflected their susceptibility to salt or cadmium stress and were corroborated by the reduced Spd titer compared to WT plants. Moreover, the Spd titer (Tables 1 and 2), antioxidant parameters such as GSH, GR, and SOD (Fig. 3), and proline (Fig. 4) in the antisense lines concomitantly showed less effective response to the stresses compared with the WT. It is possible that a decrease in GR activity in the antisense lines reduces the NADP+ /NADPH ratio, thereby restraining the availability of NADP+ to accept electrons and resulting in more electron flow to O2 for the generation of reactive oxygen species (ROS). This decreased GR activity may also affect the ratio of GSH to glutathione disulfide, which is required for the regulation of ascorbate threshold levels and detoxification of heavy metals (Foyer and Halliwell, 1976; Gasic and Korban,

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Fig. 4. MDA (a, b) and proline (c, d) contents in the antisense lines and the WT subjected to salt (10 days after stress) or cadmium (21 days after stress) stress. Data are means ± SE (n = 3). Values within the same panel followed by the same letter are not significantly different at P < 0.05.

2007). Conversely, MDA levels in the antisense lines were significantly higher than those in the WT (Fig. 4), reflecting more severe lipid peroxidation in the antisense lines under salt or cadmium stress. Consequently, the antisense lines demonstrated reduced tolerance to the tested stresses. It has been reported that exogenous Spd treatment caused a substantial reduction in ROS and thereby mitigated oxidative stress in barley leaves under water deficits (Kubi´s, 2005). Furthermore, Kubi´s (2008) reported that Spd treatment enhanced the activities of the scavenging system enzymes in water-stressed cucumber leaves. In the current investigation, antioxidant enzyme activities in antisense lines significantly improved with Spd application under either salt or cadmium stress (Fig. 3). Together, these results indicated the broad-spectrum involvement of Spd in salt or cadmium stress alleviation, which was ascribed to the oxidative alleviation, thereby resulted in the positive correlations between the Spd titer and the antioxidant parameters (Table 4). Thus, improved Spd status may greatly contribute to mitigating stress damage: therefore, to attenuate stresses, exogenous application of Spd may be successfully employed in crop cultivation as long as Spd does not reach a toxic level. 4.3. Differences in polyamine titers in the sense and antisense studies The ratio of (Spd + Spm) to Put in our previous study using sense (overexpressing) transgenic line #32 increased after stress treatments. Under salt stress, the ratio increased from 99.7% to 162.7%; in the presence of mannitol, the ratio increased from 99.7% to 151.4%; under copper stress, the ratio increased from 115.0% to 216.7% (Wen et al., 2008). Wang et al. (2007) demonstrated that endogenous Spd and Spm can promote copper tolerance in Nymphoides peltatum because of the alleviation effects of exogenous application of Spd and Spm, and suggested that the elevation of the (Spd + Spm)/Put ratio is critical in improving copper tolerance

of this plant. In the present study, this ratio significantly increased in the antisense lines after both salt and cadmium stress (Table 3); this increase can be largely ascribed to the significant decrease in the Put titer (Tables 1 and 2). Nevertheless, these stress treatments caused negative effects such as inhibition of shoot growth and less induction of the antioxidant system in the antisense lines. These results seemed to contradict our previous report (Wen et al., 2008) and the results by Wang et al. (2007). Furthermore, exogenous application of Spd alleviated the negative effects of both salt and cadmium despite reducing the (Spd + Spm)/Put ratio, compared with that of the stress condition alone. Our hypothesis is that in addition to the optimal absolute contents of the polyamines, there exists a desirable ratio of (Spd + Spm) to Put, which may vary depending on the plant species and kind of stress. When this ratio becomes out of the desirable range, as seen in the antisense lines after stress treatment, it may negatively affect growth and function of the antioxidant system. Therefore, in this study, the reduction of the ratio by exogenous Spd (Table 3) could return the ratio to the desirable range and alleviate the stress damage through a mechanism by which the Put titer is increased (Tables 1 and 2). These results may therefore indicate that not only individual polyamine concentrations, but also the balance between the different types of polyamine molecules, could be crucial for stress alleviation. Positive relationships between the Spd and Spm titers and the antioxidant parameters were observed in the sense transgenic study (Wen et al., 2010), whereas such relationships were observed for Put but not for Spm in the current antisense investigation. The fact that the effect of a type of polyamine species differed between the sense and antisense studies might also support the importance of the polyamine concentrations and their balance. In the green alga, protection of photosynthetic apparatus from stress has been achieved through the adjustment of polyamine balance (Sfichi et al., 2004; Demetriou et al., 2007). To test this assumption, exogenous application of Put and Spm to the sense and antisense lines and measurement of PAO activity could

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be helpful to uncover this dilemma, and we are currently planning this study. In conclusion, the antisense transgenic lines showed the opposite responses to salt or cadmium stress in comparison with the sense line (Wen et al., 2008, 2010), as expected. Exogenous Spd application alleviated the negative impact in the antisense lines to some extent, possibly by adjusting the balance between the concentrations of different polyamines as well as enhancing internal Spd titer. All of these results provide evidence for the important role of polyamines in alleviation of both salt and cadmium stress in European pear. Although the mechanism underlying this stress alleviation by polyamines has not yet been fully elucidated, it has been reported that there is a different mode of action between Put and higher polymines, Spd and Spm: Put enhances the photochemical quenching of the absorbed light energy mainly through the luminal buffering and the strong enhancement of chemiosotic ATP synthesis (Ioannidis et al., 2006), while Spd and Spm regulate positively the non-photochemical quenching (Ioannidis and Kotzabasis, 2007). These are also likely one of the contributing factors for the minimization of the free radicals in addition to the increase in the antioxidant system. Acknowledgments This work was undertaken when X.-P. Wen was a foreign researcher at the National Institute of Fruit Tree Science (Japan). This work was supported by Japan Society for the Promotion of Science (No. 21380028, No. 18·06621). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.envexpbot.2011.03.001. References Alcázar, R., Altabella, T., Marco, F., Bortolotti, C., Reymond, M., Koncz, C., Carrasco, P., Tiburcio, A.F., 2010. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 231, 1237–1249. Alcázar, R., Marco, F., Cuevas, J.C., Patron, M., Ferrando, A., Carrasco, P., Tiburcio, A.F., Altabella, T., 2006. Involvement of polyamines in plant response to abiotic stress. Biotechnol. Lett. 28, 1867–1876. Angelini, R., Federico, R., Bonfante, P., 1995. Maize polyamine oxidase: antibody production and ultra-structural localization. J. Plant Physiol. 145, 686–692. Ban, Y., Honda, C., Hatsuyama, Y., Igarashi, M., Bessho, H., Moriguchi, T., 2007. Isolation and functional analysis of a myb transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol. 48, 958–970. Bhatnagar, P., Glasheen, B.M., Bains, S.K., Long, S.L., Minocha, R., Walter, C., Minocha, S.C., 2001. Transgenic manipulation of the metabolism of polyamines in poplar cells. Plant Physiol. 125, 2139–2153. Bhatnagar, P., Minocha, R., Minocha, S.C., 2002. Genetic manipulation of the metabolism of polyamines in poplar cells. Plant Physiol. 128, 1455–1469. Bouchereau, A., Aziz, A., Larher, F., Martin-Tanguy, J., 1999. Polyamines and environmental challenges: recent developments. Plant Sci. 140, 103–125. Capell, T., Bassie, L., Christou, P., 2004. Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress. Proc. Natl. Acad. Sci. U.S.A. 101, 9909–9914. Chandler, S.F., Thorpe, T.A., 1987. Characterization of growth, water relations, and proline accumulation in sodium sulfate tolerant callus of Brassica napus L. cv Westar (canola). Plant Physiol. 84, 106–111. del Río, L.A., Pastori, G.M., Palma, J.M., Sandalio, L.M., Sevilla, F., Corpas, F.J., Jiménez, A., López-Huertas, E., Hernández, A., 1998. The activated oxygen role of peroxisomes in senescence. Plant Physiol. 116, 1195–1200. Demetriou, G., Neonaki, C., Navakoudis, E., Kotzabasis, K., 2007. Salt stress impact on the molecular structure and function of the photosynthetic apparatus – the protective role of polyamines. Biochim. Biophys. Acta Bioenerg. 1767, 272–280. Duan, J., Li, J., Guo, S., Kang, Y., 2007. Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances short-term salinity tolerance. J. Plant Physiol. 165, 1620–1635. Foyer, C.H., Halliwell, B., 1976. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25. Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158.

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