Plant Biology ISSN 1435-8603

RESEARCH PAPER

Genotype differences in the metabolism of proline and polyamines under moderate drought in tomato plants nchez-Rodrıguez D. Montesinos-Pereira, Y. Barrameda-Medina, L. Romero, J.M. Ruiz & E. Sa Department of Plant Physiology, Faculty of Science, University of Granada, Granada, Spain

Keywords polyamines; proline; stress tolerance; tomato; water stress. Correspondence D. Montesinos-Pereira, Department of Plant Physiology, Faculty of Science, University of Granada, 18071 Granada, Spain. E-mail: [email protected] Editor J. Sparks Received: 21 January 2014; Accepted: 7 February 2014 doi:10.1111/plb.12178

ABSTRACT Water stress is one of the most important factors limiting the growth and productivity of crops. The implication of compatible osmolytes such as proline and polyamines in osmotic adjustment has been widely described in numerous plants species under stress conditions. In the present study, we investigated the response of five cherry tomato cultivars (Solanum lycopersicum L.) subjected to moderate water stress in order to shed light on the involvement of proline and polyamine metabolism in the mechanisms of tolerance to moderate water stress. Our results indicate that the most water stress-resistant cultivar (Zarina) had increased degradation of proline associated with increased polyamine synthesis, with a higher concentration of spermidine and spermine under stress conditions. In contrast, Josefina, the cultivar most sensitive to water stress, showed a proline accumulation associated with increased synthesis after being subjected to stress. In turn, in this cultivar, no rise in polyamine synthesis was detected. Therefore, all the data appear to indicate that polyamine metabolism is more involved in the tolerance response to moderate water stress.

INTRODUCTION Approximately a third of the world’s arable land undergoes chronic periods of drought stress (Massacci et al. 2008). A major unfavourable abiotic factor that affects plants during growth and development is drought, which is also a major cause of osmotic stress. In turn, water stress can affect all metabolic processes and often results in a severe reduction in crop productivity, especially in arid and semiarid regions such as the Mediterranean area (Giorgi & Lionello 2008). The adaptation of plants to drought involves a series of complex physiological processes and changes that can occur over the short term, such as stomatal closure, thereby limiting carbon assimilation and long-term alterations more related to acclimation processes (Chaves et al. 2002). The response of plants to drought consists of the coordination of processes to alleviate both cell hyperosmolarity as well as osmotic imbalance. Thus, under water stress conditions, the plant accumulates compatible osmoprotective solutes to prevent water loss and to re-establish cell turgour (Kumar et al. 2008). The solutes that accumulate during osmotic adjustment include sugars, alcohols, amino acids (e.g., proline) and quaternary amines (e.g., glycine betaine and polyamines; Parida et al. 2008). These compounds can act as antioxidants, eliminating reactive oxygen species (ROS) formed in the cell, and finally help the plant to avoid or tolerate water deficit stress (Seki et al. 2007). Proline is perhaps the compatible osmolite that is most accumulated in plants in response to salinity, cold and water stress. Several authors have shown a strong correlation between the rise in proline levels and capacity to survive water deficit and salinity conditions. In plants, the function of proline is not restricted only to that of a compatible osmolite, but rather also 1050

protects cytoplasmic enzymes, e.g., those used for nitrogen and carbon uptake in growth after a stress period; acts as a stabiliser of cell membranes and of the machinery for protein synthesis; and serves as an energy sink to regulate redox potential (Szabados & Savoure 2010). Moreover, proline controls gene expression of proteins involved in the stress response (Kuznetsov & Shevyakova 1999). Although proline is considered a metabolite with protective functions, there is some controversy as to whether its accumulation can be considered an adaptive response or rather a stress symptom. Thus, it has been confirmed that, under certain conditions, the exogenous application of proline can prove toxic to plants, inhibiting growth and cell division (Maggio et al. 2002). In potato the increase in proline concentration was more pronounced in sensitive cultivars submitted to drought conditions (Schafleitner et al. 2007). In contrast, other authors found that proline accumulation has a protective role. In this sense, Saeedipour (2013) found that the tolerant wheat cultivar Zagros, which gave higher yields under water stress conditions, contained a higher concentration of proline than did the drought-sensitive cultivar Marvdasht. Another osmoprotective compound that plants can accumulate under stress conditions is polyamines. These are low-molecular weight nitrogenous compounds, and the main polyamines in plants are putrescine, spermidine and spermine. In turn, these can be found in a free form, conjugated with low-molecular weight compounds such as phenols, or bound to macromolecules such as proteins (Groppa & Benavides 2008). In plants, polyamines are involved in many physiological processes, such as organogenesis, embryogenesis, development, cell division, differentiation, proliferation, cell death, DNA and protein synthesis and gene expression, among others (Seiler & Raul 2005; Kusano et al. 2008). Many studies show a relationship

Plant Biology 16 (2014) 1050–1057 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Montesinos-Pereira, Barrameda-Medina, Romero, Ruiz & S anchez-Rodrıguez

between polyamine metabolism and plant response to different stress conditions (Radyukina et al. 2009; Zhang et al. 2009). In turn, polyamines act in osmotic adjustment, which is considered an important component of tolerance mechanisms to water stress in plants (Zhang et al. 1999). Another important effect is that of ROS detoxification. Work with tobacco and tomato plants that overexpress the synthesis enzyme arginine decarboxylase has shown that transgenic plants have higher drought stress tolerance and that ROS levels are drastically reduced (Wang et al. 2011). It has also been confirmed that low levels of polyamines in soy plants, especially of putrescine and spermidine, are related to increased damage from stress and decreased water content (Nayyar & Chander 2004). Glutamate is a common precursor in the biosynthesis of proline and polyamines and is converted into proline in two reactions catalysed by glutamate dehydrogenase and D1-pyrroline-5-carboxylate synthetase (P5CS). Another precursor of proline synthesis is ornithine, which is transaminated to pyrroline-5-carboxylate by ornithine-d-aminotransferase (OAT; Verbruggen & Hermans 2008). On the other hand, the metabolism and accumulation of proline depends also on its degradation, which is catalysed by the enzyme proline dehydrogenase (Alcazar et al. 2010). In the case of polyamines, glutamate is a more distant precursor, converted first into ornithine or arginine, which in turn can serve as substrates for two enzymes, ornithine decarboxylase and arginine decarboxylase, respectively. The following addition of aminopropyl groups to putrescine in two reactions catalysed by spermidine synthase and spermine synthase leads to the formation of spermidine and spermine, respectively (Groppa & Benavides 2008). Free polyamine levels in plant cells depend not only on their synthesis but also on their transport, degradation and conjugation. Putrescine degradation is catalysed by diamine oxidase, whereas spermidine and spermine are oxidised by a polyamine oxidase (Groppa & Benavides 2008). Therefore, high constitutive or stress-induced levels of polyamines in plants would prevent active proline accumulation and vice versa. Larher et al. (1998) showed that in response to osmotic stress, rapeseed leaves accumulated proline, and treatment with spermine suppressed this accumulation. However, opposite results have also € urk & Demir 2003), where plant treatment been published (Ozt€ with polyamines stimulated proline accumulation in spinach leaves. Thus, it is not known to what extent changes in pathways linked to polyamine metabolism are involved in control of metabolic responses related to osmotic adjustment in higher plants, such as that responsible for proline accumulation under water stress conditions. Given that tomato cultivation is concentrated in semiarid zones, where plants frequently undergo water stress, it is important to elucidate the physiological responses of this species under stress conditions. Our group has designed an experimental model with five commercial cherry tomato cultivars that show different degrees of tolerance to moderate water stress (Sanchez-Rodrıguez et al. 2010). Thus, the aim of the present work was to examine the effect of water stress on the metabolism of proline and polyamines and their possible role in water stress tolerance. To further clarify the kind of relationship between the biosynthetic routes of these two molecules (proline and polyamines) and determine whether it is a competitive or a synergistic interaction.

Genotype differences in droughted tomato

MATERIAL AND METHODS Plant material and growth conditions Five cherry tomato cultivars were used: Kosaco, Josefina, Katalina, Salome and Zarina (Solanum lycopersicum L., formerly Lycopersicon esculentum Mill.). The seeds of these cultivars were germinated and grown for 30 days in a tray with wells (each well 3 cm 9 3 cm 9 10 cm) in a nursery Saliplant S.L. (Carchuna, Granada). Afterwards, the seedlings were transferred to a cultivation chamber at the Plant Physiology Department, University of Granada, under controlled conditions with relative humidity of 50  10%, at 25 °C/15 °C (day/night), a 16-h/8-h photoperiod, with a PPFD (photosynthetic photon flux density) of 350 lmolm2s1 (measured with an SB quantum 190 sensor, Li-Cor Inc., Lincoln, NE, USA). Under these conditions, the plants grew in individual 8-l pots (25-cm upper diameter, 1-cm lower diameter, 25-cm high) filled with a 1:1 perlite:vermiculite mixture. Throughout the experiment, the plants were grown in a complete nutrient solution containing: 4 mM KNO3, 3 mM Ca (NO3)2, 2 mM MgSO4, 6 mM KH2PO4, 1 mM NaH2PO4, 2 lM MnCl2, 1 lM ZnSO4, 0.25 lM CuSO4, 0.1 lM Na2MoO4, 5 lM Fe-EDDHA, 50 lM H3BO3. The nutrient solution (pH 5.8) was renewed every 3 days and the substrate was partially rinsed with distilled water to avoid nutrient accumulation. The water stress treatments began 45 days after germination and were maintained for 22 days. The control treatment received 100% field capacity irrigation (500 ml), whereas moderate water stress corresponded to 50% of field capacity (250 ml). The experimental design consisted in a randomised complete block with two treatments, six plants per treatment arranged in individual pots with three replications. Furthermore, the experiment was repeated three times under the same conditions (n = 9). Plant sampling and determination of relative growth rate (RGR) and leaf relative water content (LRWC) All plants were at the late vegetative stage when harvested. Shoots were harvested, frozen immediately in liquid N2, and kept at 80 °C until used. To determine RGR, shoots from three plants per cultivar were sampled on day 45 after germination, immediately before starting the water stress treatment (Ti). Shoots were dried in a forced-air oven at 70 °C for 24 h, and the dry weight (DW) recorded as grams per plant. The remaining plants were sampled 67 days after germination (22 days of treatment, Tf). The relative growth rate was calculated from the increase in leaf DW at the beginning and at the end of the water stress treatment, using the equation RGR ¼ ðln DWf  ln DWiÞ=ðTf  TiÞ where T is time and the subscripts denote the final and initial sampling (i.e., days 0 and 22, respectively, after the water stress treatment; Bellaloui & Brown 1998). LRWC was measured following the method of Barrs & Weatherley (1962). Enzyme extraction and assay for proline metabolism Extraction of D1-pyrroline-5-carboxylate synthetase (P5CS) was carried out according to Sumithra et al. (2006). Leaves

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Genotype differences in droughted tomato

Montesinos-Pereira, Barrameda-Medina, Romero, Ruiz & Sanchez-Rodrıguez

were homogenised with extraction buffer containing 100 mM Tris-HCl (pH 7.5), 10 mM b-mercaptoethanol, 10 mM MgCl2 and 1 mM phenylmethylsulphonyl fluoride, and then centrifuged at 10,000 g for 15 min. The supernatant was used for enzyme assays. P5CS activity was measured as describe in Charest & Ton Phan (1990). The reaction mixture contained: 100 mM Tris-HCl (pH 7.2), 25 mM MgCl2, 0.4 mM NADPH, 5 mM ATP and the enzyme extract. The reaction was initiated by addition of 75 mM sodium glutamate. The activity was measured as the rate of consumption of NADPH, monitored as a decreased in absorbance at 340 nm. For ornithine-d-aminotransferase (OAT) and proline dehydrogenase extraction, leaves were homogenised in 100 mM potassium phosphate buffer (pH 7.8). The homogenate was filtered and centrifuged at 12,000 g for 20 min (4 °C). OAT was assayed according to Charest & Ton Phan (1990) in 0.2 M Tris-KOH buffer (pH 8.0) containing 5 mM ornithine, 10 mM a-ketoglutarate and 0.25 mM NADH. The decrease in absorbance of NADH was monitored at 340 nm for 1 min after initiating the reaction with addition of enzyme extract. Proline dehydrogenase activity was assayed as a reduction of NAD+ at 340 nm (Charest & Ton Phan 1990). The reaction mixture contained 0.15 M Na2CO3-HCl buffer (pH 10.3) containing 2.67 mM L-proline and 10 mM NAD+. Glutamate dehydrogenase was assayed by measuring the decrease in absorbance due to consumption of NADH at 340 nm (Kanamori et al. 1972). The reaction mixture contained 0.2 M Tris-HCl buffer (pH 8.0), 1.5 M NH4Cl, 0.5 M a-ketoglutaric acid, 3 mM NADH and 0.2 ml of enzyme extract. The protein concentration of the extracts was determined according to the method of Bradford (1976), using bovine serum albumin as standard.

dialysed extract was used for enzyme assay. Arginine decarboxylase reaction mixtures contained 100 mM Tris-HCl buffer (pH 7.5), 40 lM pyridoxal phosphate, 5 mM DTT, 5 mM EDTA, 40 mM L-arginine (or ornithine for ornithine decarboxylase determination) and the dialysed enzyme extract for arginine decarboxylase determination. The reaction mixture was incubated at 37 °C for 60 min and centrifuged at 3000 g for 10 min; 0.5 ml of the supernatant was mixed with 1 ml 2 mM NaOH, then benzoylated in accordance with the method of Aziz & Larher (1995). HPLC conditions were the same as during the measurement of polyamines. The assay mixture for spermidine synthase contained 0.1 M Tris-HCl buffer, pH 8.0, 30 lM putrescine, 25 lM dcSAM, 20 lM adenine and the enzyme solution in a total volume of 0.2 ml. After incubation at 37 °C for 30 min, the reaction was stopped by adding 20 ll 1.2 M perchloric acid then centrifuged (Yonn et al. 2000). For detection of spermidine after enzymatic reaction, HPLC conditions were the same as for the measurement of polyamines. Diamine oxidase and polyamine oxidase activities were determined by measuring the generation of H2O2, a product of the oxidation of polyamines, as described in Su et al. (2005), with some modifications. Plant material (0.7 g) was homogenised in 100 mM potassium phosphate buffer (pH 6.5). The homogenate was centrifuged at 10,000 g for 20 min at 4 °C. The supernatant was used for enzyme assay. The reaction mixture contained 2.5 ml potassium phosphate buffer (100 mM, pH 6.5), 0.2 ml 4-aminoantipyrine/N,N-dimethylaniline reaction solution, 0.1 ml horseradish peroxidase (250 Uml1) and 0.2 ml of the enzyme extract. The reaction was initiated by addition of 0.15 ml 20 mM putrescine for diamine oxidase determination and 20 mM spermidine + spermine for polyamine oxidase determination. A change in OD of 0.001 absorbance units at 555 nm per min was considered one unit of enzyme activity.

Proline content For determination of the free proline concentration, leaves were homogenised in 5 ml 96% ethanol. The insoluble fraction of the extract was washed with 5 ml 70% ethanol. The extract was centrifuged at 3500 g for 10 min and the supernatant preserved at 4 °C for proline determination (Irigoyen et al. 1992). An aliquot of this supernatant was taken and, after adding reactive ninhydrin acid reagent (ninhydrin, 6 M phosphoric acid, 60% glacial acetic acid) and 99% glacial acetic acid, was placed in a bath at 100 °C. After 45 min, the tubes were cooled, 5 ml benzene added and absorbance of the organic phase measured. The resultant proline concentration was expressed as lgg1 dry weight (DW). Enzyme extraction and assay for polyamine metabolism Diamine oxidase and arginine decarboxylase activities were determined according to Birecka et al. (1985), with some modifications. Plant material (0.7 g) was homogenised in 50 mM potassium phosphate buffer (pH 6.3) containing 5 mM EDTA, 0.1 mM PMSF, 40 lM pyridoxal phosphate, 5 mM DTT, 20 mM ascorbic acid and 0.1% polyvinylpyrrolidone. The homogenate was centrifuged at 12,000 g for 40 min at 4 °C and the supernatant was dialysed at 4 °C against 1 ml 10 mM potassium phosphate buffer (pH 6.3) containing 0.05 mM pyridoxal phosphate, 1 mM DTT, 0.1 mM EDTA for 24 h in darkness. The

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Content of polyamine forms Plant material (0.7 g) was homogenised in 1 ml 6% (v/v) cold perchloric acid, kept on ice for 1 h, and then centrifuged at 21,000 9 g for 30 min. The pellet was extracted twice with 1 ml 5% perchloric acid and centrifuged again. The three supernatants were pooled and used to determine levels of free polyamines. The supernatant were benzoylated in accordance with the method of Aziz & Larher (1995). The benzoyl derivatives were separated and analysed using HPLC (Agilent HPLC 1100 series, Agilent Technologies, Waldbronn, Germany). A sample of 10 ll acetonitrile solution of benzoyl polyamines was injected into a 5-lm particle size, C18 reverse-phase column. Column temperature was maintained at 30 °C. Samples were eluted from the column with 40% acetonitrile at a flow rate of 1 mlmin1. Polyamine peaks were detected with a UV detector at 254 nm; 1,6-hexanediamine was used as an internal standard. Statistical analysis For statistical analysis, data compiled were submitted to ANOVA, and differences between the means were compared with Fisher’s least significant difference (LSD; P > 0.05).

Plant Biology 16 (2014) 1050–1057 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Montesinos-Pereira, Barrameda-Medina, Romero, Ruiz & S anchez-Rodrıguez

RESULTS Biomass, RGR and LRWC All the cultivars studied underwent a decline, both in total biomass and in total and foliar RGR (Table 1) when submitted to water stress conditions. However, cv. Zarina underwent a lower reduction of these parameters than cv. Josefina, which had a more severe decrease in biomass and RGR (Table 1). Tomato cv. Zarina reached a higher LRWC than did cv. Josefina under water stress conditions, while the other cultivars showed intermediate values (Fig. 1). Proline metabolism For an understanding of the involvement of proline in the response to water stress, we studied its metabolism. With respect to glutamate dehydrogenase, activity increased only under water stress conditions in cv. Salome (Table 2). Tomato cv. Josefina showed a rise of 66% and 75% in activity of the enzymes P5CS and OAT, respectively, while in cv. Zarina OAT activity fell 44% (Table 2). In cv. Kosaco, there was a significant increase in activity of the enzyme P5CS, although only 14% (Table 2). With respect to proline degradation, there was a fall in proline dehydrogenase activity for cv. Josefina but a rise for cv. Zarina. There were no significant differences detected in the other cultivars (Table 2). The proline concentrations in the studied cultivars were analysed, and there was a significant increase in this amino acid in all cultivars under water stress, except cv. Zarina, in which there were significant differences (Fig. 2). Polyamine metabolism In terms of polyamine metabolism, ornithine decarboxylase activity increased in cv. Kosaco under stress conditions, while

Genotype differences in droughted tomato

in the other cultivars there were no significant differences (Fig. 3). However, a significant rise in arginine decarboxylase activity was found in cv. Zarina (Fig. 3). With regard to enzymes that degrade polyamines, our results show a general decline in diamine oxidase activity in all cultivars studied, except cv. Zarina, in which there was an 88% increase in activity under water stress conditions (Fig. 4). Meanwhile, polyamine oxidase activity significantly fell in cv. Katalina but rose significantly in cv. Zarina (Fig. 4). There were no changes in putrescine concentration in any of the cultivars subjected to stress conditions (Table 3). With respect to spermidine and spermine concentrations, there was an increase in cv. Kosaco, as well as an increase in spermine concentration in cv. Katalina, whereas in cv. Josefina both spermidine and spermine decreased (Table 3). On the other hand, cv. Zarina presented higher quantities of spermidine and spermine under water stress. Finally, in terms of total free polyamines, there was an increase in cv. Zarina but a decrease in cv. Josefina under stress conditions (Table 3).

DISCUSSION Biomass, RGR and LRWC A growth reduction under water stress has been widely reported in many plant species, e.g., potato (Yooyongwech et al. 2013) and rapeseed (Shahsavari & Rad 2013). Thus, Abdoli & Saeidi (2012), after analysing several wheat cultivars submitted to water stress, concluded that cv. Chamran was more tolerant to drought than cv. Parsi, based on growth parameters such as biomass. In our study, tomato cv. Zarina was the cultivar with the smallest loss in biomass under water stress (Table 1). Therefore, in agreement with Abdoli & Saeidi (2012), this cultivar could be defined as more tolerant to water deficit, as it had less biomass loss and a higher RGR than the

Table 1. Dry weight and RGR in plants of five cultivars of cherry tomato either well-watered or subjected to moderate water stress. cultivar/treatment Kosaco well watered water stress LSD0.05 Josefina well watered water stress LSD0.05 Katalina well watered water stress LSD0.05 Salom e well watered water stress LSD0.05 Zarina well watered water stress LSD0.05

total biomass (g)

total RGR (gg1day1)

shoot biomass (g)

shoot RGR (gg1day1)

12.83  1.15 8.40  0.57* 3.58

0.087  0.0012 0.068  0.0023* 0.0080

11.76  0.72 7.54  0.91* 3.25

0.087  0.0001 0.064  0.0006* 0.001

12.76  1.46 7.63  0.71* 4.51

0.084  0.0050 0.061  0.0003* 0.0161

11.66  0.57 6.77  1.15* 3.59

0.082  0.0032 0.054  0.0002* 0.008

13.29  1.09 8.51  0.57* 3.44

0.083  0.0005 0.063  0.0010* 0.0051

11.85  0.50 7.65  0.43* 1.84

0.087  0.0006 0.064  0.0017* 0.0051

12.17  0.64 7.51  0.57* 2.39

0.100  0.0007 0.078  0.0005* 0.0025

11.22  0.87 6.77  0.45 2.74

0.100  0.0007 0.076  0.0009* 0.003

11.38  0.62 8.83  0.68 2.58

0.093  0.0081 0.079  0.0092 0.0140

10.87  0.57 7.95  0.63* 2.38

0.093  0.0023 0.080  0.0003* 0.006

Values are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant difference from controls.

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Genotype differences in droughted tomato

Fig. 1. Leaf relative water content (LRWC) in cherry tomato plants under water stress. Columns are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant differences from controls.

Fig. 2. Proline content in cherry tomato plants subjected to water stress. Columns are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant differences from controls.

Table 2. Activity of enzymes in the metabolism of proline in five cherry tomato cultivars either well watered or subjected to moderate water stress. cultivar/ treatment

GDH

P5CS

OAT

PDH

(DAbs min1mg1 prot)

Kosaco well watered

0.68  0.17

7.14  0.34

0.05  0.01

0.06  0.02

water stress

0.54  0.07

8.50  0.20*

0.08  0.01

0.02  0.00

0.4

0.9

0.03

0.04

well watered

0.88  0.09

1.42  0.11

0.08  0.002

0.09  0.01

water stress

0.56  0.12

2.36  0.12*

0.14  0.003*

0.04  0.01*

LSD0.05

0.32

0.35

0.009

0.04

1.55  0.23

1.78  0.49

0.09  0.02

0.08  0.01

1.29  0.21

2.45  0.39

0.12  0.02

0.07  0.00

0.66

1.34

0.07

0.02

well

0.23  0.02

0.88  0.06

0.11  0.02

0.06  0.00

watered water

0.49  0.04*

0.78  0.25

0.09  0.03

0.08  0.01

stress LSD0.05

0.11

0.56

0.09

0.03

LSD0.05 Josefina

Katalina well watered water stress LSD0.05 Salome

Zarina well

0.37  0.17

1.08  0.22

0.18  0.02

0.06  0.01

watered water

0.64  0.22

1.17  0.44

0.08  0.01*

0.15  0.02*

stress LSD0.05

0.61

1.05

0.06

0.06

GDH, glutamate dehydrogenase; P5CS, D1-pyrroline-5-carboxylate synthetase; OAT, ornithine-d-aminotransferase; PDH, proline dehydrogenase. Values are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant differences from control.

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Fig. 3. Activity of the enzymes of polyamine synthesis (ornithine decarboxylase and arginine decarboxylase) in cherry tomato plants subjected to water stress. Columns are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant differences from controls.

Plant Biology 16 (2014) 1050–1057 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Montesinos-Pereira, Barrameda-Medina, Romero, Ruiz & S anchez-Rodrıguez

Genotype differences in droughted tomato

Table 3. Content of the different free polyamines [putrescine (Put), spermidine (Spd) and spermine (Spm)] in five cherry tomato cultivars either wellwatered or subjected to moderate water stress. free polyamines (mgg1 DW) cultivar/treatment Kosaco well watered water stress LSD0.05 Josefina well watered water stress LSD0.05 Katalina well watered water stress LSD0.05 Salome well watered water stress LSD0.05 Zarina well watered water stress LSD0.05

Put

Spd

Spm

0.15  0.04 0.24  0.09 0.18

0.34  0.01 0.44  0.02* 0.09

0.057  0.00 0.145  0.02* 0.05

0.25  0.05 0.12  0.05 0.22

0.35  0.01 0.29  0.01* 0.03

0.083  0.01 0.019  0.00* 0.04

0.14  0.01 0.13  0.30 0.11

0.33  0.01 0.30  0.02 0.06

0.020  0.00 0.065  0.01* 0.03

0.23  0.00 0.21  0.00 0.10

0.42  0.00 0.35  0.40 0.12

0.00  0.00 0.00  0.00 0

0.096  0.01 0.215  0.05 0.14

0.28  0.00 0.34  0.01* 0.05

0.00  0.00 0.07  0.02* 0.05

Values are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant differences from control.

Fig. 4. Activity degradation enzymes of polyamines in cherry tomato plants subjected to water stress. Columns are means (n = 9) and differences between means were compared with Fisher’s LSD (P = 0.05). Asterisk (*) indicates significant differences from controls.

other cultivars studied (Table 1). LRWC is considered a good indicator of the capacity of a plant to return to a favourable state after water deficit (Deivanai et al. 2010). In fact, cv. Zarina had the highest LRWC value (Fig. 1), confirming the tolerance of this cultivar under the growth conditions of the present experiment. By contrast, cv. Josefina had the lowest LRWC value (Fig. 1) and therefore can be defined as the most sensitive cultivar to water stress. Proline metabolism With respect to proline biosynthesis, many studies indicate that P5CS and OAT are the critical enzymes in proline synthesis (Yamada et al. 2005). The data compiled for enzymatic activities in proline synthesis could explain its accumulation, since in cv. Josefina activity of P5CS and OAT increased (Table 2). These data coincide with findings in cotton plants reported by Parida et al. (2008), showing that increased P5CS and OAT activity is related to higher proline accumulation under salinity or drought stress. In contrast, cv. Zarina showed no differences in P5CS activity, while OAT activity even fell (Table 2). In terms of the enzyme proline dehydrogenase, which is in involved in degradation, cv. Zarina had the highest activity and cv. Josefina the lowest (Table 2). These data agree with those of

Ruiz et al. (2002), who suggest that the activity of proline dehydrogenase can determine proline concentration in green bean. In the present work, there was a rise in the proline concentration in cv. Josefina, the most sensitive cultivar, while a fall was found in cv. Zarina, the most tolerant cultivar (Fig. 2). These data disagree with Hmida-Sayari et al. (2005) who suggested that proline accumulation is involved in water stress tolerance, but agree with other researchers (Juan et al. 2005; Szabados & Savoure 2010) who consider proline is produced in response to several abiotic stresses, including water stress. On the other hand, cv. Zarina had a higher LRWC (Fig. 1) and a lower proline concentration (Fig. 2) than the more sensitive cultivars, as in Rampino et al. (2006), who found that increased drought tolerance in wheat was related to higher LRWC and lower proline content. Finally, although there were no significant differences in activity of enzymes involved in synthesis of proline in cv. Katalina and cv. Salome (Table 2), we observed an increase in the concentration of proline in these cultivars under moderate water stress (Fig. 2). This increase could be due to transport of proline from the roots to the leaves under the imposed stress conditions, since in cv. Katalina and cv. Salome, although we observed an increase in proline in roots via P5CS, proline concentration in roots did not reach that in control conditions (data not shown). Polyamine metabolism It has been firmly demonstrated that polyamine metabolism is involved in plant response to abiotic stress, mainly salinity and

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Montesinos-Pereira, Barrameda-Medina, Romero, Ruiz & Sanchez-Rodrıguez

drought (Groppa & Benavides 2008). Plants have two polyamine synthesis pathways (both result in decarboxylation), the first reaction of which is catalysed by the enzyme ornithine decarboxylase, which forms putrescine from ornithine, and the other is catalysed by the enzyme arginine decarboxylase, which forms putrescine from arginine (Groppa & Benavides 2008). Recent studies state that the arginine decarboxylase pathway predominates under stress conditions (Bassard et al. 2010). There was an increase in arginine decarboxylase activity in cv. Zarina under stress conditions (Fig. 3). These data agree with other authors who demonstrated that the increased activity of this enzyme correlates with drought tolerance in rice plants and in Arabidopsis (Alcazar et al. 2010). The content of free polyamines depended not only on their biosynthesis but also on their transport, conjugation and degradation. Polyamines are degraded by two enzymes: diamine oxidase, which catalyses the conversion of putrescine to 1-pyrroline, and polyamine oxidase, which transforms spermidine and spermine into 1,3 diaminepropane or into 1-pyrroline, and both pathways collaterally produce hydrogen peroxide (Cona et al. 2006). Both of these enzymes showed significant activity in cv. Zarina (Fig. 4). The higher activity of degradation enzymes in cv. Zarina resulted in the production of hydrogen peroxide, which could act as a signal to trigger mechanisms involved in the antioxidant response in plants, as demonstrated in Wimalasekera et al. (2011). Indeed, previous studies from our research group confirm that cv. Zarina has the strongest antioxidant response under stress conditions (Sanchez-Rodrıguez et al. 2010). In many cases, stress is accompanied by a rise in the free polyamine content, indicating that its metabolism is a major component of stress tolerance mechanisms (Bouchereau et al. 1999). Regarding the content of different types of free polyamine (Table 3), no significant differences were found in the quantity of putrescine in any of the cultivars studied. However, concentrations of spermidine and spermine fell in the

most sensitive cultivar (Josefina) under stress conditions, as opposed to the most tolerant cultivar (Zarina), in which concentrations rose (Table 3). Multu & Bozcuk (2005) reported similar results, with an increase in spermine in salinity-tolerant sunflower plants, while putrescine remained constant or even fell under salinity conditions. On the other hand, Krishnamurthy & Bhagwat (1989) observed that salinity-tolerant rice cultivars maintained high levels of spermidine and spermine, while putrescine accumulated in sensitive cultivars. This could indicate that spermidine and spermine play a major role in water stress tolerance (Yamada et al. 2007; Kubis 2008). Finally, there was a 59% fall in total free polyamines in cv. Josefina as opposed to cv. Zarina, which recorded a 63% rise under stress conditions (Table 3), reflecting the importance of accumulation of these polyamines for resistance to the moderate water stress applied in the present experiment. In conclusion, this work demonstrates that under moderate water stress conditions, the metabolic pathways of proline and polyamines are competitive. Thus, the most water stress-resistant cultivar (Zarina) had increased degradation of proline associated with increased polyamine synthesis, with a higher concentration in spermidine and spermine under stress conditions. In contrast, cv. Josefina, the most water stress sensitive cultivar, showed a proline accumulation associated with increased synthesis when subjected to stress; in turn, no rise in polyamine synthesis was detected in this cultivar. Therefore, all the data appear to indicate that polyamine metabolism is more involved in the tolerance response to moderate water stress in cherry tomato.

REFERENCES Abdoli M., Saeidi M. (2012) Using different ındices for selection of resistant wheat cultivars to post anthesis water deficit in the west of Iran. Annals of Biological Research, 3, 1322–1333. Alcazar 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. Aziz A., Larher F. (1995) Changes in polyamine titers associated with the proline response and osmotic adjustment of rape leaf discs submitted to osmotic stress. Plant Science, 112, 175–186. Barrs H.D., Weatherley P.E. (1962) A re-examination of the relative turgidity technique for estimating water deficits in leaves. Australian Journal of Biological Sciences, 15, 413–428. Bassard J.E., Ullmann P., Bernier F., Werck-Reichhart D. (2010) Phenolamides: bridging polyamines to the phenolic metabolism. Phytochemistry, 71, 1808–1824. Bellaloui N., Brown P.H. (1998) Cultivar differences in boron uptake and distribution in celery (Apium graveolens), tomato (Lycopersicon esculentum) and wheat (Triticum aestivum). Plant and Soil, 198, 153–158. Birecka H., Bitonti A.J., McCann P.P. (1985) Assaying ornithine and arginine decarboxylases in some plant species. Plant Physiology, 79, 509–514.

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ACKNOWLEDGEMENTS This work was financed by the PAI programme (Plan Andaluz de Investigaci on, Grupo de Investigaci on AGR161).

Bouchereau A., Aziz A., Larher F., Martin-Tanguy J. (1999) Polyamines and environmental challenges: recent development. Plant Science, 140, 103–125. Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72, 248–254. Charest C., Ton Phan C. (1990) Cold acclimation of wheat (Triticum aestivum): properties of enzymes involved in proline metabolism. Physiologia Plantarum, 80, 159–168. Chaves M.M., Pereira J.S., Maroco J., Rodrigues M.L., Ricardo C.P.P., Os orio M.L., Carvalho I., Faria T., Pinheiro C. (2002) How plants cope with water stress in the field: photosynthesis and growth. Annals of. Botany, 89, 907–916. Cona A., Rea G., Angelini R., Federico R., Tavladoraki P. (2006) Functions of amine oxidases in plant development and defence. Trends in Plant Science, 11, 80–88. Deivanai S., Sheela Devi S., Sharrmila Rengeswari P. (2010) Physiochemical traits as potential indicators for determining drought tolerance during active tillering stage in rice (Oryza sativa L.). Pertanika Journal of Tropical Agricultural Science, 33, 61–70. Giorgi F., Lionello P. (2008) Climate change projections for the Mediterranean region. Global Planetary Change, 63, 90–104.

Groppa M.D., Benavides M.P. (2008) Polyamines and abiotic stress: recent advances. Amino Acids, 34, 35– 45. Hmida-Sayari A., Gargouri-Bouzid R., Bidani A., Jaoua L., Savoure A., Jaoua S. (2005) Overexpression of D1-pyrroline- 5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants. Plant Science, 169, 746– 752. Irigoyen J.J., Emerich D.W., Sanchez-Diaz M. (1992) Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants. Physiologia Plantarum, 84, 55–60. Juan M., Rivero R.M., Romero L., Ruiz J.M. (2005) Evaluation of some nutritional and biochemical indicators in selecting salt-resistant tomato cultivars. Environmental and Experimental Botany, 54, 193– 201. Kanamori T., Konishi S., Takahashi E. (1972) Inducible formation of glutamate dehydrogenase in rice plant roots by the addition of ammonia to the media. Physiologia Plantarum, 26, 1–6. Krishnamurthy R., Bhagwat K.A. (1989) Polyamines as modulators of salt tolerance in rice cultivars. Plant Physiology, 91, 500–504. Kubis J. (2008) Exogenous spermidine differentially alters activities of some scavenging system enzymes,

Plant Biology 16 (2014) 1050–1057 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Montesinos-Pereira, Barrameda-Medina, Romero, Ruiz & S anchez-Rodrıguez

H2O2 and superoxide radical levels in water-stressed cucumber leaves. Journal of Plant Physiology, 165, 397–406. Kumar A., Bernier J., Verulkar S., Lafitte H.R., Atlin G.N. (2008) Breeding for drought tolerance: direct selection for yield, response to selection and use of drought-tolerant donors in upland and lowland-adapted populations. Field Crops Research, 107, 221–231. Kusano T., Berberich T., Tateda C., Takahashi Y. (2008) Polyamines: essential factors for growth and survival. Planta, 228, 36–381. Kuznetson V.V., Shevyakova N.I. (1999) Proline under stress: biological role, metabolism and regulation. Russian Journal of Plant Physiology, 46, 274–289. Larher F., Aziz A., Deleu C., Lemesle P., Ghaffar A., Bouchard F., Plasman M. (1998) Suppression of the osmoinduced proline response of rapeseed leaf discs by polyamines. Physiologia Plantarum, 102, 139–147. Maggio A., Miyazaki S., Veronese P., Fujita T., Ibeas J.I., Damsz B., Narasimhan M.L., Hasegawa P.M., Joly R.J., Bressan R.A. (2002) Does proline accumulation play an active role in stress-induced growth reduction? The Plant Journal, 31, 699–712. Massacci A., Nabiev S.M., Pietrosanti L., Nematov S.K., Chernikova T.N., Thor K., Leipner J. (2008) Response of the photosynthetic apparatus of cotton (Gossypium hirsutum) to the onset of drought stress under field conditions studied by gas-exchange analysis and chlorophyll fluorescence imaging. Plant Physiology and Biochemistry, 46, 189–195. Mutlu F., Bozcuk S. (2005) Effects of salinity on the content of polyamines and some other compounds in sunflower plants differing in salt tolerance. Russian Journal of Plant Physiology, 52, 29–34. Nayyar H., Chander S. (2004) Protective effects of polyamines against oxidative stress induced by water and cold stress in chickpea. Journal of Agronomy and Crop Science, 190, 355–365. € urk L., Demir Y. (2003) Effects of putrescine and Ozt€ ethephon on some oxidative stress enzyme activities and proline content in salt stressed spinach leaves. Plant Growth Regulation, 40, 89–95. Parida A.K., Dagaonkar V.S., Phalak M.S., Aurangabadkar L.P. (2008) Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery. Acta Physiologiae Plantarum, 30, 619–627.

Radyukina N.L., Mapelli S., Ivanov Y.V., Kartashov A.V., Brambilla I., Kuznetsov V.V. (2009) Homeostasis of polyamines and antioxidant systems in roots and leaves of Plantago major under salt stress. Russian Journal of Plant Physiology, 56, 323–331. Rampino P., Pataleo S., Gerardi C., Mita G., Perrota C. (2006) Drought stress response in wheat: physiological and molecular analysis of resistant and sensitive genotypes. Plant, Cell and Environment, 29, 2143– 2152. Ruiz J.M., Sanchez E., Garcıa P.C., L opez-Lefebre L.R., Rivero R.M., Romero L. (2002) Proline metabolism and NAD kinase activity in greenbean plants subjected to cold-shock. Phytochemistry, 59, 473– 478. Saeedipour S. (2013) Relationship of grain yield, ABA and proline accumulation in tolerant and sensitive wheat cultivars as affected by water stress. Proceedings of the National Academy of Sciences, India, Section B: Biological Sciences, 83, 311–315. Sanchez-Rodrıguez E., del Mar Rubio-Wilhelmi M., Cervilla L.M., Blasco B., Rios J.J., Leyva R., Romero L., Ruiz J.M. (2010) Study of the ionome and uptake fluxes in cherry tomato plants under moderate water stress conditions. Plant and Soil, 335, 339–347. Schafleitner R., Gaudin A., Rosales R.O.G., Aliaga C.A.A., Bonierbale M. (2007) Proline accumulation and real time PCR expression analysis of genes encoding enzymes of proline metabolism in relation to drought tolerance in Andean potato. Acta Physiologiae Plantarum, 29, 19–26. Seiler N., Raul F. (2005) Polyamines and apoptosis. Journal of Cellular and Molecular Medicine, 9, 623–642. Seki M., Umezawa T., Urano K., Shinozaki K. (2007) Regulatory metabolic networks in drought stress responses. Current Opinion in Plant Biology, 10, 296–302. Shahsavari N., Rad A.H.S. (2013) Assessment of drought tolerance in new rapeseed (Brassica napus L.) cultivars. Advanced Science Letters, 19, 3675– 3678. Su G.X., An Z.F., Zhang W.H., Liu Y.L. (2005) Light promotes the synthesis of lignin through the production of H2O2 mediated by diamine oxidases in soy bean hypocotyls. Journal of Plant Physiology, 162, 1297–1303. Sumithra K., Jutur P.P., Carmel B.D., Reddy A.R. (2006) Salinity-induced changes in two cultivars of Vigna radiate: responses of antioxidative and

Plant Biology 16 (2014) 1050–1057 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Genotype differences in droughted tomato

proline metabolism. Plant Growth Regulation, 50, 11–22. Szabados L., Savoure A. (2010) Proline: a multifunctional amino acid. Trends in Plant Science, 15, 89– 97. Verbruggen N., Hermans C. (2008) Proline accumulation in plants: a review. Amino Acids, 35, 753–759. Wang B.-Q., Zhang Q.-F., Liu J.-H., Li G.-H. (2011) Overexpression of PtADC confers enhanced dehydration and drought tolerance in transgenic tobacco and tomato: effect on ROS elimination. Biochemical and Biophysical Research Communications, 413, 10– 16. Wimalasekera R., Tebartz F., Scherer G.F.E. (2011) Polyamines, polyamine oxidases, and nitric oxide in development, abiotic and biotic stresses. Plant Science, 181, 593–603. Yamada M., Morishita H., Urano K., Shiozaki N., Yamaguchi-Shinozaki K., Shinozaki K., Yoshiba Y. (2005) Effects of free proline accumulation in petunias under drought stress. Journal of Experimental Botany, 56, 1975–1981. Yamada M., Morishita H., Urano K., Shiozaki N., Yamaguchi-Shinozaki K., Shinozaki K., Yamaguchi K., Takahashi Y., Berberich T., Imai A., Takahashi T., Michael A.J., Kusano T. (2007) A protective role for the polyamine spermine against drought stress in Arabidopsis. Biochemical and Biophysical Research Communications, 352, 486–490. Yonn S.O., Lee Y.S., Lee S.H., Cho Y.D. (2000) Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes. Biochimica et Biophysica Acta, 1475, 17– 26. Yooyongwech S., Theerawitaya C., Samphumphuang T., Cha-um S. (2013) Water-deficit tolerance identification in sweet potato genotypes (Ipomoea batatas (L.) Lam.) in vegetative developmental stage using multivariate physiological indices. Scientia Horticultura, 162, 242–251. Zhang J., Nguyen H.T., Blum A. (1999) Genetic analysis of osmotic adjustment in crop plants. Journal of Experimental Botany, 50, 291–302. Zhang W., Jiang B., Li W., Song H., Yu Y., Chen J. (2009) Polyamines enhance chilling tolerance of cucumber (Cucumis sativus L.) through modulating antioxidative system. Scientia Horticultura, 122, 200–208.

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Genotype differences in the metabolism of proline and polyamines under moderate drought in tomato plants.

Water stress is one of the most important factors limiting the growth and productivity of crops. The implication of compatible osmolytes such as proli...
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