Photosynthetic traits of plants and the biochemical profile of tomato

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Agricultural and Environmental Chemistry

Photosynthetic traits of plants and the biochemical profile of tomato fruits are influenced by grafting, salinity stress and growing season Nina KACJAN MARSIC, Dominik Vodnik, Maja Mikulic-Petkovsek, Robert Veberic, and Helena Sircelj J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00169 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Journal of Agricultural and Food Chemistry

Photosynthetic traits of plants and the biochemical profile of tomato fruits are influenced by grafting, salinity stress and growing season Nina Kacjan Marsic*1, Dominik Vodnik2, Maja Mikulic-Petkovsek1, Robert Veberic1 and Helena Sircelj2 1

Biotechnical Faculty, Department of Agronomy, Chair for Fruit, Wine and Vegetable Growing and

2

Biotechnical Faculty, Department of Agronomy, Chair for Applied Botany, Ecology, Plant Physiology and

Informatics; University of Ljubljana, Jamnikarjeva 101, SI-1000 Vegetable Growing, Jamnikarjeva 101, SI1000 Ljubljana, Slovenia

1

Corresponding author (e-mail: [email protected], Fax: +386 1 423 10 88, Tel: +386 1 320 31 13)

ACS Paragon Plus Environment


ABSTRACT Changes in the photosynthetic traits of plants and metabolic composition of fruits of two tomato cultivars, grafted onto two rootstocks, grown in 3 salinity levels were studied in two growing periods during the season. Increased salinity stress conditions lowered water potential, stomatal conductance and transpiration rate of grafted tomato plants, in both growing period. Water deficit induced stomatal closure, which resulted in stomatal limitation of photosynthesis. The proline content in tomato leaves increased and was closely correlated with salinity. Some of the quality parameters of tomato fruits were affected by rootostock. The sugar/acid ratio was the highest in fruits of ‘Belle’/‘Maxifort’ grafts. With increasing salt stress conditions from 40 to 60 mM NaCl, the lycopene content increased and ascorbic acid content decreased in fruits of ‘Gardel’/’Maxifort’ grafts, indicating the ability of this scion/ rootstock combination to mitigate the toxicity effect of salinity stress. A higher phenolics concentration in fruits from the first growing period may be an additional indicator of stress, caused by higher temperatures and solar radiation, compared to the later period. Keywords: grafting, metabolites, NaCl, photosynthetic traits, proline, Solanum lycopersicum,


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INTRODUCTION Tomato (Solanum lycopersicum L.) is sensitive to moderate levels of salt in the soil.1 In areas with an optimal climate for tomato, salinity is a serious constraint, because inadequate irrigation management leads to salinization of water resources and soil.2 Salt in soil water inhibits plant growth for two reasons. Firstly, it reduces the plant’s ability to take up water, which is known as the osmotic or water-deficit effect.3 Water-deficit induces stomatal closure, which lowers the transpiration rate and results in stomatal limitation of photosynthesis.4 Secondly, salt can enter the transpiration stream and affect metabolic processes such as photosynthesis, protein synthesis and energy and lipid metabolism.5 It can eventually injure cells in the transpiring leaves, further reducing growth.3 In order to cope with salinity, tomato plants are able to produce osmotically active organic substances, mainly amino acids and sugars, which help to alleviate the salinity-mediated osmotic stress.1,

2

Many osmolytes and stress proteins with unknown functions probably

detoxify plants by scavenging reactive oxygen species (ROS) or prevent them from damaging cellular structures. Proline is a compatible solute known to accumulate in plants subjected to unfavourable environmental conditions.1 The concentration of this amino acid has been used in experiments as a measure of the stress imposed on tomato plants grown at different nutrient concentrations.6 One way of avoiding or reducing losses in production caused by environmental stresses in vegetables could be to graft them onto rootstocks capable of reducing the effect of external stresses on the shoot.7,

8

Grafted plants appear to have developed various mechanisms for

avoiding the physiological damage that can be caused by excessive accumulation of salt ions in the leaves.7, 9 The ability of rootstock to minimise perturbations in scion water status seems to be the most important factor.10 The breeding of vigorous rootstocks with broad abiotic stress resistance is hampered by the lack of practical selection tools, such as genetic markers,



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because knowledge about the physiology behind a successful rootstock (root–shoot interaction) is still very limited.8 On the other hand, rootstock selection and breeding have responded to the demand or extensive forms of stress resistance and for improved plant productivity, arguably at the expense of fruit quality.8,

11, 12

Most tomato quality

characteristics are strongly affected by the interaction of the rootstock-scion combination, and other factors such as climate, culture practice, water and nutrient availability, and duration and intensity of stress.13 In a temperate climate, tomato production takes place mainly in greenhouses, while infection with Phytophthora infestans interrupts the tomato growing period, mainly when tomato production takes place in the open field in humid environments. The production of tomato is therefore limited to the early spring-summer period but its length can be extended to late summer, since tomato plants have been proven to have great ability to compensate for fluctuating temperatures.14 Extending the growing season provides growers with an opportunity to extend revenue into a normally unproductive period and to benefit from out-ofseason price premiums. Genetic and environmental factors modulate the physiology and metabolism of crop plants, but it is not clear how technological factors (cultivar, grafting) combined with abiotic stress (salinity, environmental factors) affect tomato’s natural metabolic composition. The objective of our research was to evaluate season-dependent differences in the response of grafts to increased salinity. We therefore hypothesized that the problems of salinity are differently expressed when tomato is stressed in mid- or late summer, which is the most popular tomato growing season in southeast European countries.15 Furthermore, we presumed that this response may be rootstock and environment specific. Knowing the effect of grafting on important physiological and fruit quality traits, especially changes in the metabolic profile in terms of the growing period, could also be very helpful for rootstock breeders, who have


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stressed the market value of fruits of grafted plants as a focus of most rootstock breeding efforts. 16 MATERIALS AND METHODS Plant material. The research was conducted on the experimental field (298 m above sea level) of the Biotechnical Faculty in Ljubljana, Slovenia (latitude 46°2’ N, longitude 14°28’ E) in two different growing periods of the season, the first from May until July and the second from June until September. Seeds of the scions were sown on March 28th for the first growing period and on May 10th for the second growing period. Two commercial hybrid tomato cultivars, ‘Belle’ Enza Zaden, The Netherlands) and ‘Gardel’ (Royal Sluis, The Netherlands) were used as scions and self-grafted control. Both cultivars belong to Solanum lycopersicum L., beef type group, and are medium maturing with round and oblate shape, respectively, and with fine beef quality. Two interspecific hybrids, ‘Beaufort’ and ‘Maxifort’ (Solanum lycopersicum L. × Solanum habrochaites S. Knapp and D.M. Spooner) (De Ruiter, The Netherlands), were used as rootstocks. Both rootstocks were selected as the most representative commercial rootstocks used for tomato scions, in the Mediterranean basin and Asia, respectively.17 They are highly resistant to biotic and abiotic stresses, however ‘Maxifort’ supposed to be more tolerant to drought and salinity compared to ‘Beaufort’ (De Ruiter, The Netherlands). In both experiments, the seeds of rootstocks were sown a week later than those of scions. Four weeks later, for both experiments, scions were grafted onto rootstocks or onto themselves (self-grafted), applying the cleft grafting method described by Lee et al.17. Successfully grafted and acclimatized seedlings were transplanted (on May 12th in the first experiment and on June 29th in the second experiment) into 10 L pots filled with a 1:2 (v/v) mixture of vermiculite and rockwool flocks. Pots were placed in a polycarbonate greenhouse, at a density of 3.2 plants m-2. The seedlings were supplied with a complete nutrient solution after they had been transplanted. The complete standard nutrient solution for



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plant irrigation contained the following macronutrients (in mM): 6 KNO3, 1 KH2PO4, 4 Ca(NO3)2, 1 NH4NO3 and 2 MgSO4.7H2O. The micronutrient concentrations (µM) were: 30 H3BO3, 10 MnSO4, 5 ZnSO4, 0.75 CuSO4, 0.5 MoCl and 15 Fe-EDTHA. Standard electroconductivity (EC) was 2 dS m-1. The salinity treatments began in both growing periods 4 weeks after transplanting (DAT). Two salinity levels, 4 and 6 dS m-1, were applied in a single step by adding 2337.6 mg L-1 NaCl (4 dS m-1 or 40 mM NaCl) and 3506.4 mg L-1 NaCl (6 dS m-1 or 60 mM NaCl) to the nutrient solution. Non-stressed plants were grown in the standard solution (2 dS m-1 or 20 mM NaCl) for the whole period of the experiment. Nutrient solutions containing different concentrations of NaCl were supplied every third day or daily and the amount of nutrient solution used for watering plants in the first growing period was 2.5 L plant-1 of standard solution (20 mM NaCl) and 1.9 L plant-1 of nutrient solution containing different concentrations of NaCl (40 or 60 mM NaCl). The amount of nutrient solution used for watering plants in the second growing period was 2.1 L plant-1 of standard solution and 1.3 L plant-1 for salinity solutions (40 mM NaCl or 60 mM NaCl). Salinity stress conditions was maintained for 5 weeks and lasted from 28 to 84 DAT (from June 9th until July 13th in the first growing period and from July 29th until September 2nd in the second growing period). The pH and EC of the nutrient solution were measured at the time of each watering using hand held pH and EC meters (Hana Instruments, Inc., Woonsocket, RI, USA). The pH of the nutrient solution ranged from 5.6 (at 60 mM NaCl) to 7.1 (at 20 mM NaCl). Fruit Sampling. Fruits were harvested at a stages, suitable for fresh market (light red to red). For each cultivar, each salinity level, each graft combination and each of four replications, three tomatoes were randomly selected from among marketable and undamaged fruits. Analysis of sugars, organic acids and phenolics were performed on fresh samples and analysis of carotenoids was performed on dry samples, after the samples had been chopped, frozen in liquid nitrogen and stored at -20 °C. For detection of dry matter, 2 g of the frozen


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sample was freeze-dried for 22 h in a Gamma 2-20 lyophiliser (Christ, Germany) and the water content (%) was calculated from the difference between the masses before and after lyophilisation. Weather conditions: Average daily temperature and total daily solar irradiation (Figure 1) during the experimental period from March to September were taken from the meteorological station on the experimental field of the Biotechnical Faculty in Ljubljana, Slovenia. It can be assumed that the environmental conditions in the greenhouse were very similar to those in the open field but, based on experience from previous years, 3 to 4 °C higher. The weather during the experimental period was comparable to the long-term average in terms of temperatures. The highest temperatures compared to the long-term average (more than 3 °C higher) were measured in April, May, August and September. More solar irradiation compared to the longterm average was measured in all months during the experimental period. Photosynthetic measurements. Physiological measurements (water potential (Ψ), stomatal conductance (gs), transpiration (E) and net-photosynthesis (A)) on plants were subsequently taken with the Li-6400 (Li-Cor Biosciences, Lincoln, USA) portable measuring system. The leaf above the second inflorescence was enclosed in the chamber and measurements were taken under the following conditions: temperature 27 °C; relative humidity18 in the chamber 70%; external CO2 concentration 380 µmol mol-1; and saturating light intensity, photosynthetic photon flux density of 800 µmol m-2 s-1. The midday leaf water potential (Ψ) was measured using a Scholander pressure chamber, on the same leaf after gas-exchange measurements, to check the water status of plants. Chemicals: The following standards were used for the determination of metabolics: Fluka Chemie (Buchs, Switzerland): L-proline, sucrose, fructose, and glucose; citric, malic and fumaric acid, p-coumaric acid, quercetin 3-O-glucoside, kaempferol-3-O-glucoside; SigmaAldrich (Steinheim, Germany): ortho-phosphoric acid, shikimic acid, chlorogenic acid, 3-



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caffeoylquinic acid, quercetin-3-O-glucoside, naringenin; DHI LAB products (Hørsholm, Denmark): α-carotene, β-carotene; Merck (Darmstadt, Germany): lycopene; Serva Feinbiochemica GmbH & Co. (Heidelberg, Germany): ninhidrin reagent. Methanol for the extraction of phenolics was acquired from Sigma-Aldrich Chemicals and acetone for the extraction of carotenoids from Merck (Darmstadt, Germany). The chemicals for the mobile phases were HPLC-MS grade acetonitrile and formic acid from Fluka Chemie GmBH. Water for the mobile phase was double-distilled and purified with a Milli-Q system (Millipore, Bedford, MA, USA). Proline analysis. In first experiment, leaf samples for proline analysis were collected 6 weeks after applying salinity, and in the second experiment 5 weeks after applying salinity, above the fruits of the second cluster from which fruits had been collected for chemical analysis. Proline was determined as described by Bates et al.19. The extraction procedure and colorimetric determination with an acidic ninhydrin reagent (Serva Feinbiochemica GmbH˛Co, Heidelberg, Germany) 2.5 g ninhydrin/100 mL of a solution containing glacial acetic acid (Sigma-Aldrich, Steinheim, Germany), distilled water (Millipore, Bedford, USA) and ortho-phosphoric acid (Sigma-Aldrich, Steinheim, Germany) 85% at a ratio of 6:3:1) were carried out as follows: samples of 1g frozen leaves were ground in a mortar after the addition of a small amount of quartz sand and 10 mL of a 3% (w/v) aqueous sulfosalicylic acid (Sigma-Aldrich, Steinheim, Germany) solution. The homogenate was centrifuged for 12 min at 11000 g at 4°C. Glacial acetic acid and the ninhydrin reagent (1 mL each) were added to 1 mL of the supernatant. Closed tubes with the reaction mixture were kept in a boiling water bath for 1 h, and the reaction was terminated in ice for 10 min. Readings were taken immediately at a wavelength of 546 nm. The proline concentration was calculated from a standard curve plotted with a known concentration of L-proline from Fluka (BioChemika, Switzerland) as standard and expressed as µmol g-1 fresh weight (FW).


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Extraction and analysis of sugars and organic acids. The tomato fruit was finely chopped, and 10 g of the fresh weight was immersed in 25 mL of double distilled water, and homogenized with T-25 Ultra turrax (IKA® - Labortechnik, Staufen, Germany) for 1 min at 9000 rpm. The samples were left for half an hour at room temperature, with frequent stirring. The extracted samples were centrifuged at 10,000 rpm for 6 min, at 4 oC (Eppendorf Centrifuge 5801R, Hamburg, Germany). The supernatant was filtered through a polyamide filter Chromafil A-20/25 (Macherey-Nagel, Düren, Germany) into vials and analysed according to the method described by Mikulic-Petkovsek et al.20 using HPLC (Thermo Scientific, Finnigan Spectra System, Waltham, MA, USA). For each analysis, 20 µL of sample was used. Analysis of sugars was carried out using a Rezex RCM-monosaccharide column (300 mm × 7.8 mm; Phenomenex, Torrance, CA) with a flow rate of 0.6 mL min-1 and column temperature maintained at 65 °C. Double distilled water was used as the mobile phase and an RI (refractive index) detector for identification. Organic acids were analysed with Rezex ROA-Organic H+(8%) (300 mm × 7.8 mm; Phenomenex) and the UV detector was set at 210 nm with a flow rate of 0.6 mL min-1. For the mobile phase, 4 mM H2SO4 was used. The method of external standards was applied to calculate the concentration of organic acids and sugars, which was then presented in g kg-1 fresh weight. Extraction and analysis of ascorbic acid. The tomato fresh fruit was chopped with a ceramic knife into small pieces and 2.5 g of the fresh weight was immediately immersed in 5 mL of 2% meta-phosphoric acid and ground thoroughly in a ceramic mortar. The samples were left for extraction on a shaker for half an hour at room temperature and centrifuged at 10,000 rpm for 5 min at 4oC (Eppendorf Centrifuge 5801R, Hamburg, Germany). The supernatant was filtered through cellulose filters Chromafil A-20/25 (Macherey-Nagel, Düren, Germany), poured into a vial and analysed using high performance liquid chromatography (HPLC; Thermo Scientific, Finnigan Spectra System, Waltham, MA, USA),



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as previously reported by Mikulic-Petkovsek et al. (2012)20. Separation of ascorbic acid was carried out using a Rezex ROA-organic acid H+ (8%) column (300 mm × 7.8 mm) from Phenomenex (Torrance, CA). The column temperature was set at 20 °C, and a 245nm wavelength UV detector was used for identification. The duration of analysis was 30 min. The concentrations of ascorbic acid were calculated with the help of the corresponding external standard and expressed in mg kg-1 fresh weight (FW). Extraction and analysis of carotenoids. Carotenoids were determined using the method described in Sircelj and Batic.21 Pigments were extracted from the dry fruit powder with icecold acetone. All extraction procedures were performed in dim light. Acetone extracts were subjected to HPLC gradient analysis (column Spherisorb S5 ODS-2 250 x 4.6 mm with precolumn S5 ODS-2 50 x 4.6 mm). Pigments were separated using the following solvents: solvent A: acetonitrile/methanol/water (100/10/5, v/v/v); solvent B: acetone/ethylacetate (2/1, v/v), at a flow rate of 1 mL min-1. A linear gradient from 10% solvent B to 70% solvent B in 18 min was applied, run time 30 min, and photometric detection at 440 nm. Extraction and HPLC-MS/MS identification of phenolic compounds. Extraction of fruit samples (peel with pulp together) was performed as described by Mikulic-Petkovsek et al.20 with some modifications. Fresh tomato samples were finely chopped and immersed in 10 mL of extraction solution (methanol containing 1% (w/v) 2.6-di-tert-butyl-4-methylphenol (BTH) and 3% (v/v) formic acid) and extracted for 60 min in an ultrasonic ice bath. Samples were then centrifuged for 7 min at 9,000 rpm and filtered through a 0.20 µm polyamide Cromafil AO-20/25 filter, produced by Macherey-Nagel (Düren, Germany), transferred to a vial and injected into the HPLC system. The individual phenolic compounds were analysed using the Thermo Finnigan Surveyor HPLC system (Thermo Scientific, San Jose, USA) with a diode array detector at 310 nm (hydroxycinnamic acid derivatives) and 350 nm (flavonols and flavanones). The injection volume was 20 µL and the flow rate was maintained at 0.6 mL


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min−1. The elution solvents were aqueous 0.1% formic acid in double distilled water (A) and 0.1% formic acid in acetonitrile (B). A Gemini C18 (Phenomenex) column was used for phenolic determination, operated at 25°C. The samples were eluted according to the linear gradient described by Wang, et al.,22 The identity of the phenolic compounds was confirmed using a mass spectrometer (Thermo Scientific, LCQ Deca XP MAX) with electrospray ionization operating in negative ion mode. Full-scan data dependent MS scanning from m/z 115 to 1100 was used for the analyses. Quantification was achieved based on the concentrations of the corresponding external standard. The concentrations of phenolic compounds were expressed in mg kg-1 of fresh weight (FW). Statistical analysis. For a clearer survey of the results, we analysed the collected data for individual cultivars and treated the experiments as a three-factorial experiment in four randomized blocks. The factor growing season had two levels, growing period May-July and June-September; the factor rootstock had three levels, ‘Beaufort’ and ‘Maxifort’ and selfgrafted plants and the factor salinity had three levels: 20 mM NaCl, 40 mM NaCl, 60 mM NaCl. The blocks were a random factor. The data were analysed by analysis of variance (ANOVA). Where necessary, the data were logarithmically transformed to reach the assumptions of ANOVA. Duncan’s multiple range test at a significance level of p

Photosynthetic traits of plants and the biochemical profile of tomato

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