The Photoprotective Role of Spermidine in Tomato Seedlings under Salinity-Alkalinity Stress

Polyamines are small, ubiquitous, nitrogenous compounds that scavenge reactive oxygen species and stabilize the structure and function of the photosynthetic apparatus in response to abiotic stresses. Molecular details underlying polyamine-mediated photoprotective mechanisms are not completely resolved. This study investigated the role of spermidine (Spd) in the structure and function of the photosynthetic apparatus. Tomato seedlings were subjected to salinity-alkalinity stress with and without foliar application of Spd, and photosynthetic and morphological parameters were analyzed. Leaf dry weight and net photosynthetic rate were reduced by salinity-alkalinity stress. Salinity-alkalinity stress reduced photochemical quenching parameters, including maximum photochemistry efficiency of photosystem II, quantum yield of linear electron flux, and coefficient of photochemical quenching (qP). Salinity-alkalinity stress elevated nonphotochemical quenching parameters, including the de-epoxidation state of the xanthophyll cycle and nonphotochemical quenching (NPQ). Microscopic analysis revealed that salinity-alkalinity stress disrupted the internal lamellar system of granal and stromal thylakoids. Exogenous Spd alleviated the stress-induced reduction of leaf dry weight, net photosynthetic rate, and qP parameters. The NPQ parameters increased by salinity-alkalinity stress were also alleviated by Spd. Seedlings treated with exogenous Spd had higher zeaxanthin (Z) contents than those without Spd under salinity-alkalinity stress. The chloroplast ultrastructure had a more ordered arrangement in seedlings treated with exogenous Spd than in those without Spd under salinity-alkalinity stress. These results indicate that exogenous Spd can alleviate the growth inhibition and thylakoid membrane photodamage caused by salinity-alkalinity stress. The Spd-induced accumulation of Z also may have an important role in stabilizing the photosynthetic apparatus.


Introduction
Salinity-alkalinity stress is becoming one of the most serious abiotic stresses affecting crop growth and yield [1], and causes considerable economic losses. Our previous studies clearly show that plants are more sensitive to salinity-alkalinity stress than to neutral salt stress [1,2]. Previous reports show that salinityalkalinity stress strongly inhibits assimilation of photosynthetic carbon [3][4][5], which leads to an unfavorable dissipation of excess light energy. When photosystem II (PSII) is exposed to excess light, reactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ), hydroxyl ions (OHN), and H 2 O 2 are formed by the interaction of molecular oxygen and triplet chlorophyll [6,7], which causes photoinhibition and photodamage [8]. ROS directly affects chloroplasts and inhibits the repair of photodamaged PSII [7]. Salinity stress reduces photosynthesis and electron transport activity, thereby exacerbating problems arising from photoinhibition and photodamage of the photosynthetic apparatus [9].
Polyamines (PAs) are organic, low molecular weight, nitrogencontaining compounds; they include spermidine (Spd), spermine (Spm), and their diamine obligate precursor putrescine (Put) [10]. PAs function as plant growth regulators or intracellular messengers that regulate plant growth, development, and responses to abiotic stresses [11]. They also function as antioxidant, scavengers of ROS and free radicals [12], inhibit lipid peroxidation reactions [13] due to a combination of their anion-and cation-binding properties [14], and respond to a wide range of abiotic stresses. PAs also stabilize the structure and function of the photosynthetic apparatus in response to unfavorable environment factors [15][16][17]. PAs protect the structure of chloroplasts by keeping the thylakoid membranes in an orderly arrangement and maintaining high photosynthetic efficiency [18]. Exogenous Spd may regulate the synthesis of xanthophyll components and promote the conversion of violaxanthin (V) to zeaxanthin (Z), which can protect LHCII reaction centers against salinity-induced oxidative damage and injury from excess light energy [19].
The xanthophyll cycle pigment Z is formed from V via the intermediate anthoxanthin (A) during abiotic stress. Z is thought to be involved in photoprotective thermal dissipation and nonphotochemical quenching (NPQ) [20,21]. The thermal dissipation process protects PSII against photodamage by decreasing excess redox potential of PSII and the electron transfer chain [22]. Z has an important antioxidant function in the lipid phase of the membrane and protects chloroplasts from photo-oxidative stress [23]. Z reduces the formation of ROS, or detoxifies alreadyformed ROS, to minimize photo-oxidative damage of the photosynthetic apparatus [21].
Previous studies showed that exogenous application of PAs partially alleviated the salinity-induced decline in photosynthetic efficiency [24]. However, the molecular mechanism of Spdmediated photoprotection of the photosynthetic apparatus and the relationship between Spd and the xanthophyll cycle in response to salinity-alkalinity stress were not completely resolved. Therefore, we sought to determine whether exogenous Spd could protect tomato seedlings from photoinhibition and photodamage and interact with the endogenous xanthophyll cycle under salinityalkalinity conditions.

Plant Materials and Growth Conditions
Tomato (Solanum lycopersicum cv. Jinpengchaoguan) seeds were germinated at 27uC in Petri plates lined with two layers of filter paper moistened with sterile distilled water. The germinated seeds were then sown in commix medium (Xintiandi Co., Yangling, Shanxi, China). Seedlings were grown in a controlled environmental greenhouse at Northwest A & F University, where the air temperature was maintained at 2561uC/1561uC (day/ night). A 12-h light/12-h dark photoperiod was imposed with a daylight intensity of 800 mmol?m 22 s 21 . Seedlings were transferred into 40-L containers of one-half-strength Hoagland solution (pH 6.560.1; electrical conductivity, 1.421.8 ds?m 21 ; dissolved oxygen, 6.060.2 mg?L 21 ) when the third true leaves were fully expanded.

Salinity-alkalinity Conditions and Spermidine Treatment
When seedlings had five true leaves, they were treated with 75 mM complex neutral and alkali salts [NaCl:Na 2 SO 4 :NaH-CO 3 :Na 2 CO 3 = 1:9:9:1] and the foliage was sprayed with 0.25 mM Spd. The experiments included the following four treatments: 0 mM salinity-alkalinity plus 0 mM Spd (CK); 0 mM salinity-alkalinity plus 0.25 mM Spd (CS); 75 mM salinityalkalinity plus 0 mM Spd (S); and 75 mM salinity-alkalinity plus 0.25 mM Spd (SS). Each treatment group included 48 plants and four replicates. Plant containers were arranged in a randomized block design. The solutions were renewed every 3 days. All experiments were performed at least three times with similar results.

Plant Growth Measurements
Four days after the salinity-alkalinity treatment, plants were washed with sterile distilled water and dissected into leaves, stems, and roots. The fresh weights of dissected tissues were determined. The leaves, stems, and roots were dried at 105uC for 15 min and then at 75uC for 72 h to constant mass, and their dry masses were recorded. The shoot weight was equal to the sum of the leaf weight and the stem weight. The root/shoot ratio (R/S) was calculated as (R/S) = root weight/shoot weight. Four plants were measured per treatment.

Gas-Exchange Measurements
Gas-exchange parameters were measured using a portable photosynthesis system (LI-6400, LI-COR Inc, USA). The photosynthetic rate was measured at 380610 mmol?mol 21 CO 2 , 25uC, 70% relative humidity, and 800 mmol?m 22 s 21 light intensity. Stomatal limitation (L s ) was calculated as L s = 12C i /C a (C i and C a represent the intercellular and ambient CO 2 concentration, respectively) according to Farquhar et al. [25].

Pigments Analysis
Pigment composition was determined according to the procedure of Chen et al. with some minor modifications [27]. Xanthophyll pigments were extracted with 100% acetone under weak light. The extract was centrifuged at 12,0006g for 10 min. The supernatant was passed through a 0.2-mm syringe filter into an amber HPLC vial for immediate analysis. Twenty-five microliters of the filtered supernatant was analyzed with a Shimadzu LC-20A HPLC system (Kyoto, Japan). The Waters Table 1. Effects of exogenous spermidine on tomato seedling fresh weight under salinity-alkalinity stress.

Treatment
Shoot fresh weight (g) Root fresh weight (g) Stem fresh weight (g)

Transmission Electron Microscopy of Chloroplasts
The second fully expanded leaves from the top of the plants were randomly selected for microscopic examination. The leaf samples were cut into pieces of approximately 1 mm 2 and fixed overnight with 4% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4). The fixed samples were washed three times with the same solution for 10 min each. The samples were then post-fixed in 1% osmium tetroxide in cacodylate buffer for 2 h, and then washed three times in 0.1 M PBS (pH 7.4). The samples were dehydrated in a graded ethanol series (50%, 70%, 90%, and 100%), and then in absolute acetone for 15 min. The samples were embedded in Spurr's resin, and ultra-thin sections were cut and stained with uranium acetate and lead citrate, in series. The ultra-thin sections were mounted on copper grids and examined with a HITACHI HT7700 transmission electron microscope.

Statistical Analysis
All treatments and measurements were conducted at least in triplicate. All data were statistically analyzed with statistical software SAS (version 8.0, SAS Institute, Cary, NC) using Duncan's multiple range test at the P less than 0.05 level of significance.

Plant Growth
The fresh and dry weights of tomato seedlings were significantly reduced by salinity-alkalinity stress compared with controls after 4 days of treatment (Tables 1 and 2). Reductions in dry leaf and shoot weights were alleviated by addition of exogenous Spd ( Table 2). The S treatment had higher R/S than any other treatment ( Table 2). There were no significant differences between S and SS treatments with respect to dry root weight and dry stem weight ( Table 2). These results indicated that tomato seedling leaf growth was inhibited by salinity-alkalinity stress, and this inhibition was alleviated by exogenous Spd. However, exogenous Spd did not significantly affect the fresh weight measurements of tomato seedlings with or without salinity-alkalinity stress (Table 1).

Gas-Exchange Parameters
Salinity-alkalinity stress significantly reduced the net photosynthetic rate (P n ), stomatal conductance (G s ), and C i , and increased L s , compared with those of controls (Fig. 1). Exogenous Spd alleviated reductions in P n and G s , and increase in L s . Exogenous Spd had no significant effect on C i under salinity-alkalinity stress (SS treatment) compared with that of S treatment. No significant differences in P n , G s , C i , or L s were detected in CS and CK treatments.

Chlorophyll Fluorescence
F v 9/F m 9, WPSII, and qP were substantially reduced under salinity-alkalinity conditions compared with controls (Fig. 2). The addition of exogenous Spd alleviated these effects (Fig. 2). The maximum quantum yield of PSII (F v /F m ) was reduced in plants subjected to salinity-alkalinity stress. The addition of exogenous Spd did not protect the quantum yield of PSII. Plants subjected to salinity-alkalinity stress had significantly elevated NPQ, and this increase was alleviated by exogenous Spd. Exogenous Spd did not affect F v /F m , F v 9/F m 9, WPSII, qP, or NPQ in the absence of salinity-alkalinity stress.

Xanthophyll Cycle Components
The Z content and the total V+A+Z pool size increased in response to salinity-alkalinity stress, and exogenous Spd induced an even higher increase in the contents of these metabolites. The V content markedly decreased in response to salinity-alkalinity stress, and this decrease was greater in leaves without exogenous Spd than in those with Spd. The (A+Z)/(V+Z+A) ratio increased in response to salinity-alkalinity stress in plants treated with exogenous Spd, but it was lower than in those without Spd. These results indicate that Spd increased the total V+A+Z pool size and the constitutive accumulation of Z in response to salinityalkalinity stress.

Ultrastructural Changes in Chloroplasts
A typical elongated chloroplast was observed in controls (Fig. 3B), which had intact double membranes and a regular arrangement of granal and stromal thylakoids. Under salinityalkalinity stress (Fig. 3G-I), chloroplasts were swollen and contained more plastoglobuli than those in controls (Fig. 3A-C). The chloroplast membrane structures became dilatant, undefined, and in some cases appeared disintegrated (Fig. 3H). The disoriented lamellar system of the granal and stromal thylakoids became swollen and unclear (Fig. 3I). The numbers of grana were considerably reduced. Exogenous Spd alleviated the salinityalkalinity-mediated damage of the chloroplast photosynthetic apparatus, and more normal chloroplast ultrastructure was observed (Fig. 3J-L). No significant differences in chloroplast ultrastructure were observed in controls with or without the application of Spd (Fig. 3A-F).

Discussion
PAs are low molecular weight, ubiquitous, nitrogenous compounds that function in many cellular processes in numerous organisms [28]. Due to their polycationic nature and positive charge at physiological pH, PAs bind to macromolecules such as proteins and nucleic acids [14,29]. In this way, PAs are involved in regulating the physical and chemical properties of membranes, stabilizing nucleic acid structure and function, and modulating enzyme activities [30]. Chloroplasts are the primary sites of photosynthetic reactions in higher plants. Some types of stress reduce photochemical efficiency and electron transport activity, which might reflect changes in the structure of the photosynthetic apparatus [31]. Previous studies report that PAs stabilize the photosynthetic apparatus in response to stress [15].
In the present study, the deleterious effects of salinity-alkalinity stress on leaf and shoot dry weights in tomato seedlings were partly counteracted by the addition of exogenous Spd ( Table 1). The improved dry matter accumulation in shoots and leaves might be associated with Spd-mediated improvement in P n in tomato seedlings under salinity-alkalinity conditions (Fig. 1). A similar result was reported by Zhang et al. in salt-stressed cucumber plants sprayed with Put, which enhanced leaf area, plant height, and fresh and dry weight accumulation [24]. These authors demonstrated that Put likely enhanced photosynthetic production of saltstressed plants, and this increased production appeared to effectively support the enhance growth. In tomato plants subjected to salinity-alkalinity stress and treated with exogenous Spd, our R/ S results showed that most photosynthetic products were utilized to produce shoots rather than roots (Table 2).
Salinity-alkalinity stress reduced P n and G s , and increased L s (Fig. 1). Previous studies evaluated the relationship between L s and nonstomatal factors. If both C i and G s decreased, P n was limited primarily by stomatal conductance. If G s decreased but C i did not change or increased, the P n decrease could be ascribed to nonstomatal factors [19,24,32,33]. In the present study, salinityalkalinity-induced reductions in P n and G s and increase in L s were alleviated by exogenous Spd. However, exogenous Spd had no significant effect on C i with or without stress (Fig. 1). These observed changes in G s , C i , and L s imply that exogenous Spd alleviates both L s and nonstomatal limitations under salinityalkalinity conditions.
In general, F v /F m , F v 9/F m 9, WPSII, and qP are parameters that reflect photochemical quenching, whereas NPQ reflects nonphotochemical quenching [26]. In this study, the values of F v /F m , F v 9/ F m 9, WPSII, and qP were reduced by salinity-alkalinity treatment, and these decreases were alleviated by exogenous Spd. A previous report suggested that salt-stress-induced reductions in F v 9/F m 9 and WPSII might reflect damage to PSII electron transport [19]. These authors demonstrated that inhibition of electron transport may occur during transfer from the primary acceptor plastoquinone (QA) to the secondary acceptor plastoquinone (QB) at the acceptor side of PSII. Although exogenous Spd had no significant effect on F v /F m of tomato seedlings with or without salinity-alkalinity stress, it alleviated the stress-induced reductions in F v 9/F m 9 and WPSII. This result indicates that exogenous Spd alleviates the stressinduced inhibition of photosynthetic electron transport.
Exogenous Spd alleviated the salinity-alkalinity-induced decrease in qP and increase in NPQ. This suggests that exogenous Spd may increase qP at the expense of NPQ under stress conditions, thereby mitigating the dissipation of excitation energy in the PSII antennae [34]. This is supported by the observation that exogenous Spd improved P n and decreased the de-epoxidation status of the xanthophyll cycle in tomato seedlings under salinity-alkalinity stress (Fig. 2). The xanthophyll cycle serves as an essential photoprotective process in the dissipation of excess energy [35,36]. This thermal dissipation is an important function of NPQ to alleviate excess excitation energy on PSII reaction centers by converting excess excitation energy into heat [37]. Previous work showed that, to a certain extent, severe stress might result in higher levels of NPQ and (A+Z)/(V+Z+A) [38,39]. Consequently, under salinity-alkalinity conditions, leaves treated with Spd suffered less photoinhibition than those without Spd. Exogenous Spd may protect the photosynthetic apparatus against over-excitation, perhaps by preventing a loss of thylakoid membrane integrity [40]. The observed chloroplast ultrastructures support the proposal that exogenous Spd alleviates injury to the photosynthetic membrane caused by salinity-alkalinity stress (Fig. 4J-L). This result may be attributed to increases in the total V+A+Z pool size and the Spd-induced increases in Z content.
Z serves another important function as an antioxidant in the lipid phase of the thylakoid membrane to block photo-oxidative stress [21]. Photoinhibition occurs even under moderate light conditions if excess light energy cannot be appropriately dissipated [41]. 1 O 2 is one of the most important ROS involved in photoinhibition and photo-oxidative damage [42]. Nonproteinbound Z enhances photoprotection by blocking the formation of 1 O 2 and 1 O 2 -related oxidation products [43,44], which reduces lipid peroxidation. ROS scavenging protects chloroplasts from direct injury and diminishes photon (electron)-related stress [7]. In our study, exogenous Spd increased the amount of Z under salinity-alkalinity stress, which stabilized thylakoid membrane structure and improved photosynthetic function.
Chloroplasts are crucial plant organelles because they are the site of photosynthesis. Chloroplast ultrastructure can be substantially disrupted by salinity stress [45]. In the present study, increased numbers of plastoglobuli were observed in mesophyll cells subjected to salinity-alkalinity stress, and some of these were larger than those in controls (Fig. 4G-I). Increases in plastoglobuli numbers may be caused by thylakoid membrane degradation [46], and individual plastoglobuli may be larger when chloroplasts are under oxidative stress [47]. Chloroplasts are a major source of ROS, including 1 O 2 , OHN, and H 2 O 2 [7], which cause membrane lipid peroxidation and cellular abnormalities when plants are exposed to environmental stresses [48]. Tian et al. showed that exogenous Spd enhanced the activities of ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD), and catalase when cucumber seedlings were subjected to high temperatures [49]. Exogenous Spm alleviated salt-induced chloroplast membrane injury by increasing the levels of antioxidant metabolites (including dehydro-L-ascorbic acid, ascorbic acid, glutathione-S-S-glutathione, and glutathione-stimulating hormone) and enzyme activities (including SOD, POD, and APX) in chloroplasts [16]. PAs also bind membrane proteins, including polypeptides of the light-harvesting complex (LHC) [50]. Exogenous PAs with a high net positive charge can stabilize PSII proteins such as D1 and D2 under photoinhibition conditions [51]. PA binding to membrane proteins may provide stability for protein structure during stress, and consequently preserve photosynthetic activity. Our results indicate that Spd may serve a protective role in chloroplasts by enhancing ROS scavenging and stabilizing photosynthetic membrane proteins.
In conclusion, our results show that salinity-alkalinity stress induces photoinhibition and photodamage in tomato seedlings. Exogenous Spd alleviates the stress-induced inhibition of plant shoot growth and P n by reducing ROS and stabilizing thylakoid membrane structure. Exogenous Spd indirectly improves total V+ A+Z pool size, especially the amount of Z, which acts as an antioxidant in the lipid phase of the thylakoid membrane to block photo-oxidative stress. However, under salinity-alkalinity stress, Spd-induced increases in Z content did not increase NPQ compared with that in seedlings without Spd. This may be because Spd protects leaves from light-induced damage due to qP, which differs from heat dissipation.