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Amelioration of tomato plants cultivated in organic-matter impoverished soil by supplementation with Undaria pinnatifida
Algal Research 46 (2020) 101785
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Amelioration of tomato plants cultivated in organic-matter impoverished soil by supplementation with Undaria pinnatifida
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María Florencia Salcedoa, Silvana Lorena Colmana, Andrea Yamila Mansillaa, ⁎ María Alejandra Martínezc, Diego Fernando Fiola, , Vera Alejandra Alvarezb, ⁎ Claudia Anahí Casalonguéa, a Instituto de Investigaciones Biológicas, UE CONICET-UNMDP, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Deán Funes 3250, 7600 Mar del Plata, Argentina b Grupo de Materiales Compuestos Termoplásticos (CoMP), Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA), Facultad de Ingeniería, Universidad Nacional de Mar del Plata (UNMdP) y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Colón 10850, 7600 Mar del Plata, Argentina c Universidad Nacional de Mar del Plata, Argentina
A R T I C LE I N FO
A B S T R A C T
Keywords: Growth promoter Redox status Soil amendment Tomato Undaria pinnatifida
Rising demands of food together with modern practices in agriculture require the inclusion of areas with suboptimal soil qualities for cultivation and the replacement of contaminant chemical fertilizers. In this work, we evaluated the use of the seaweed Undaria as a soil amendment of organic-matter impoverished soil. We demonstrated that Undaria supplementation to a substrate containing vermiculite:organic soil mix (95:5) promotes the growth of tomato plants evidenced in increases in aerial and root biomass and the restoration of redox status. By means of chemical and functional analysis we identified in Undaria extract the presence of plant nutrients, minerals, vitamins, antioxidant capacity and phytohormone-like activities. All together these compounds would be able to promote growth and contribute to redox homeostasis in early plant developmental stages which are critical for tomato production yield. We propose Undaria supplementation as an efficient way to provide nutritional and growth promoter compounds to tomato crops growing on impoverished soils.
1. Introduction Tomato is one of the most extensively produced horticultural crops. Tomato originated in South America but nowadays is distributed worldwide and consumed as a human food rich in nutrients and antioxidants compounds [1]. Tomato is produced either in greenhouses or in open field and demands vast quantities of fertilizers to achieve current production rates [2]. When grown in greenhouses, tomato seedlings are propagated by about 4–5 weeks multi-well trays before they are transplanted to the field. Development of a healthy root system is critical during this period, and any improvement made during the first stages of plant growth and development can also enhance its fitness and further crop productivity. Increasing food necessities claim for the expansion of cultivable regions including areas with sub-optimal soil qualities. In addition, modern practices in agriculture require the replacement of synthetic fertilizers that cause detrimental soil effects to sustainable types of growth promoters and biostimulants. However, the development of
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organic agents meeting the requirements of functionality, applicability and effect reproducibility stays behind of its demand of application. Marine algae (seaweed) species were employed as soil conditioners for centuries; however, its use has been recently reinforced because of the need of non-contaminant fertilizers [3]. A number of seaweed extracts have been applied to horticultural crops and resulted in improvements of plant productivity and stress tolerance [4–6]. In this context, commercial products consisting in processed seaweeds presented as liquid extract or soluble powder are already available and keep gaining market, partially driven for a crescent demand of organic food worldwide. Undaria pinnatifida (Undaria) is a brown seaweed species native from China, Japan and Korea. In these countries Undaria, also known as wakame, is widely consumed as a human food and constitutes a good source of proteins, vitamins, dietary fibers and minerals [7]. The highly invasive behavior of Undaria have caused its dissemination worldwide, many times challenging natural ecosystems, but also empowered its cultivation with industrial purposes [8]. The pharmaceutical industry
Corresponding authors. E-mail addresses: diefi[email protected] (D.F. Fiol), [email protected] (C.A. Casalongué).
https://doi.org/10.1016/j.algal.2019.101785 Received 4 July 2019; Received in revised form 11 October 2019; Accepted 28 December 2019 2211-9264/ © 2020 Elsevier B.V. All rights reserved.
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dark, followed by centrifugation at 10,000g for 15 min. The supernatants were collected and diluted with acetone 80% before measuring the absorbance at 645 and 663 nm in a spectrophotometer (GeneQuant 1300, GE Healthcare, USA). Equation for the determination of total chlorophyll (chlorophyll a + chlorophyll b) was used [11]. The total area of the first and second leaf from 30-day-old tomato plants was photographed with a camera (Nikon Coolpix T80, Indonesia) and quantified using the ImageJ image-analysis software (National Institutes of Health, USA).
has also taken advantage of Undaria and its byproduct fucoidan. However, in agriculture its application has been relegated [9]. In this work, we investigated the application of Undaria as a soil amendment to be used as a growth stimulator of tomato plants cultivated in a substrate impoverished in organic matter. Mineral nutrients, vitamins and hormone-like compounds altogether may trigger the activation of a set of biochemical responses improving growth and development in tomato plants. 2. Materials and methods
2.5. H2O2 and anthocyanins measurements 2.1. Plant material To quantify H2O2, 0.1 g of leaf tissue was ground with liquid N2 in a mortar and extracted with H2O for 30 min in the dark, followed by centrifugation at 10,000g for 20 min. The H2O2 content was quantified in each supernatant according to Bellincampi et al.[12] based on the peroxide-mediated oxidation of Fe2+, followed by the reaction of Fe3+ with xylenol orange (3,3′-Bis[N,N-bis (carboxymethyl) aminomethyl]o-cresolsulfonephthalein disodium salt, Sigma-Aldrich, St Louis, USA). For anthocyanins, 0.1 g of leaf tissue was extracted with 1% HCl in methanol, stirring for 1 min and incubated at 4 °C for 2 h in the dark. Monomeric anthocyanin content was determined using a pH- differential method [13]. A microplate reader (ELX 800, Biotek, USA) was used for spectral measurements at 530 and 700 nm. The content of chlorophyll was expressed as mg cyanidin-3-glucoside/g FW, using an extinction coefficient of 34,300 L/cm.mol and molecular weight of 449.2 g/mol.
Seeds of tomato (Solanum lycopersicum L. cv. Platense) were commercially obtained from FECOAGRO Ltda., San Juan , Argentina. Tomato seeds (Solanum lycopersicum L, cv. Micro-Tom) hormone transgenic lines DR5:GUS and ARR5:GUS were kindly donated by Dr. Agustin Zsögön (Universidade Federal de Viçosa, Brazil). DR5:GUS tomato plants express the reporter gene beta-glucuronidase (GUS) under a synthetic auxin-responsive promoter [10]. ARR5:GUS plants express GUS under an Arabidopsis cytokinin-responsive promoter. ARR5:GUS plants were generated by the laboratory of Dr. Lázaro Eustáquio Pereira Peres (Universidade de São Paulo, Brazil). 2.2. Undaria material and preparation Algae biomass of Undaria pinnatifida was obtained from Soriano S.A. (Chubut, Argentina). Seaweed was extracted during February every year from Gaiman Cost, Chubut, Argentina. Seaweed was washed with tap water to remove shell, debris and sand; then air-dried at 26 °C for 2–3 days and finally hand crushed and powdered with high-speed universal grinder. For the assays, Undaria was applied as powder to the soil substrate or alternatively, it was used as water liquid extract suspending Undaria powder in distiller water.
2.6. Protein extraction and quantification of antioxidant enzymes To quantify catalase (CAT) and superoxide dismutase (SOD) enzymatic activities, 0.1 g of leaf tissue were ground with liquid N2 in a mortar and extracted with 3 volumes of buffer (50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl2, 1 mM ethylene diamine tetra acetic acid, and 1.5% polyvinylpolypyrrolidone (PVPP, Fluka/Sigma-Aldrich, St Louis, USA). The homogenate was then centrifuged at 25,000g for 20 min, and the supernatant was used to measure the antioxidant enzymes. CAT activity was quantified as described Beers and Sizer [14] with some modifications. The reaction mixture contained 600 μL of 50 mM phosphate buffer pH 7.4, 0.1 mL of 1% H2O2 and 25 μL of enzyme extract diluted to keep measurements within the linear range of the analysis. A blank was used for each sample, which contained 50 mM phosphate buffer and 25 μL of enzyme extract. H2O2 levels were followed measuring absorbance at 240 nm in a spectrophotometer (GeneQuant 1300, GE Healthcare, USA). SOD activity was measured as described Beauchamp and Fridovich [15]. The reaction mixture contained 50 mM phosphate buffer pH 7.8, 75 μM nitroblue tetrazolium chloride (NBT), 2 μM riboflavin, 13 mM methionine, 0.1 mM EDTA and 2 μL of protein sample. Enzymatic reactions were carried out at 37 °C for 15 min in a water-bath fitted with a 22 W Phillips fluorescent lamp. The absorbance was measured at 550 nm using a microplate reader (ELX 800, Biotek, USA). One unit of SOD activity was defined as the amount of enzyme that produced a 50% decrease, with respect to the control, in the absorbance at 550 nm and it was expressed as Activity of SOD (U/mL) = (Acontrol * Aassay/Acontrol) * (1/50%) * (Vtotal/VSOD). Where Acontrol and Aassay are the absorbance units at 550 nm of control and sample, respectively; Vtotal is the total volume; and VSOD is the enzyme volume.
2.3. Experimental design and treatments Tomato seeds var. Platense were germinated in Petri dishes on filter paper soaked in sterile water and incubated in growth chamber for 4 days at 25 °C with 16:8 h light:dark cycles. Germinated seeds were transferred to plastic pots (0.18 L) containing the different soil substrates: impoverished substrate, referred as a control, prepared with a vermiculite:Growmix (95:5) mix; Undaria supplemented substrate, prepared adding the indicated amounts of Undaria powder to the impoverished substrate; and organic-matter rich substrate, consisting in Growmix. Growmix (Multipro Commercial –Terrafertil, Mar del Plata, Argentina) is commonly used by regional tomato producers (Buenos Aires, Argentina) and is composed of peat moss Sphagnum medium fibers, bark compost, calcite, dolomite lime and wetting agents. Its main physicochemical characteristics include a pH in the range of 5.0–5.8, humidity of 55–65% and water retention capacity of 60%. Tomato seedlings were grown at 25 °C under 250 μmol photons/m2/s with 16:8 h light: dark cycles daily and watered controlled during 30 days. After plant harvesting, measurements of leaf fresh weight (FW) were obtained. Root and leaf dry weights (DW) were determined by drying samples for 6 days at 60 °C in a convection oven. Chlorophyll content, growth and redox parameters were analyzed at the end of experiment when plants were 30-day-old and had three-four true leaves. Second and third leaf was used to measure chlorophyll and H2O2 contents and antioxidant compounds.
2.7. Measurements of germination and vigor index Tomato seeds were incubated on filter paper soaked in Petri dishes containing 3 mL of 0.01%, 0.1% or 1% Undaria liquid extract or H2O as control. Plates were incubated in a growth chamber at 25 °C with 16:8 h light:dark cycles. The percentage of seed germination (% germination) and vigor index of seedlings were calculated at 6 and 11 days,
2.4. Chlorophyll and leaf area measurements The level of total chlorophyll (Total Chl) was quantified from 0.1 g of leaf tissue. Samples were ground with liquid N2 in a mortar and chlorophyll was extracted with acetone 80% for 60 min at 4 °C in the 2
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under same growth conditions. Seedlings were removed washed twice with 50 mM sodium phosphate buffer pH 7.0 and incubated in staining buffer (50 mM Na phosphate pH 7.0, 0.1% Triton X-100, 5 mM EDTA, 0.5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6) and 1 mg/mL X-Gluc (5-bromo-4chloro-3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt, Gold Biotechnology, USA) as substrate at 37 °C for 2 h to overnight. Images were taken using a scanner (Epson Perfection V600 Photo, Indonesia).
respectively after initial treatments. Seed was scored as germinated when its radicle had emerged from within the seed coat. Vigor index was calculated on percentage of germination and mean of root and shoot lengths of seedlings according to the equation: Vigor index = (mean root length + mean shoot length) * % germination [16]. 2.8. Characterization of Undaria liquid extract Compositional analysis of Undaria was carried out at Fares Taie Laboratory services (Mar del Plata, Argentina). Magnesium was determined following standards IRAM 22409 (1964); calcium was determined following standards IRAM 15714–1 (1981), potassium, iron, copper, cadmiun nickel, zinc, mercury and sodium were determined by atomic absorption following standards EPA SW 846 (U.S. Environmental Protection Agency, 1986); phosphorous was determined following standards IRAM 15013 (1985), iodine was determined following standards AOAC 992.24 (1990), vitamin E was determined by gas chromatography, vitamin A was determined by HPLC-DAD, vitamin B2 (riboflavin) was determined HPLC-DAD, proline was determined by spectrophotometry (acid ninhydrin-based method). All values are expressed in (mg/100 g of dry algae).
2.12. Statistical analysis Trials with organic-matter impoverished soil were done in three independent experiments (biological replicates) with at least 15 plants per treatment (n = 45). Values shown in figures are mean values +/− standard deviation (SD). The data were subjected to analysis ANOVA with Tukey post hoc comparisons against control by R Studio Team (2015). Germination percentage was evaluated by generalized linear mixed model with binomial distribution, with at least 30 seeds per treatment in three independent experiments (N = 90). 3. Results and discussion 3.1. Undaria used as a soil amendment promotes growth and development in tomato plants
2.9. DPPH-radical scavenging assay
We evaluated the potential of Undaria as a soil amendment to promote the growth of tomato cultivated in an organic-matter impoverished substrate. Tomato plants were grown for 30 days either in a substrate impoverished in organic matter, containing a 95:5 proportion of vermiculite:organic soil used as control or in the same substrate supplemented with 0.1% or 1% Undaria powder (Fig. 1). Analysis of plant growth parameters as aerial FW and DW, root DW, leaf area and total chlorophyll content showed that Undaria supplementation resulted beneficial for tomato growth and development (Fig. 2). Biomass, evaluated as aerial FW and aerial DW, increased by 38% and 67% respectively in plants grown on 1% Undaria supplemented substrate (Fig. 2A and B). Likewise, the total area of leaves from plants grown with 1% Undaria supplemented substrate increased by 145% respect to the control (Fig. 2E). On the other hand, 0.1% Undaria supplemented substrate did not result in significant changes over control plants in these parameters. Analysis of root FW indicated that both, 0.1% and 1% Undaria supplements resulted in increases of about 50% compared with control (Fig. 2C). In agreement with our results, it was reported that application of seaweed extracts enhanced root development in tomato. Foliar spray of Ulva lactuca and Padina gymnospora extracts increased root length and fresh weight of tomato seedlings [1]. In addition, application of seaweed extract also improved root development and growth in other species including Triticum aestivum [18], grape [19], mung bean [20] and maize [21].These effects on plant growth have been related with the presence of phytohormones in the extract [3,22] as well as to the stimulation of mineral nutrient uptake mediated by seaweed extract in lettuce [23] and in tomato [24]. Chlorophyll concentration in leaves has been considered as a reliable indicator of metabolic and energetic imbalance in tomato plants under stress [25]. In concordance with plant growth parameters, total chlorophyll content also increased between 30 and 50% in plants growing in 0.1% and 1% Undaria supplemented substrate compared with control (Fig. 2D). A typical response of different seaweed extracts has been associated to the promotion of the chlorophyll content in plants and this fact could be related with the presence of cytokinin-like compounds in seaweed extracts [26].
The compound 2,2-diphenyl-1-picrylhydrazyl (DPPH, SigmaAldrich, USA) radical scavenging activity was determined following the method of Molyneux et al. [17]. Briefly, 3 mg of DPPH radical were dissolved in 50 mL of HPLC-grade methanol (Merck, Germany) and adjusted to 1.3 absorbance units at 517 nm. Then, 50 μL of Undaria water extract at concentrations from 1 to 10 mg/mL were mixed with 150 μL of DPPH in single wells of a 96-well plate. The plate was kept for 30 min in the dark. The absorbance was measured at 517 nm using microplate reader (ELX 800, Biotek, USA). The antioxidant capacity was expressed as percentage of the radical scavenging activity and calculated according to the following equation: DPPH scavenged (%) = 1 − [(Asample − ABlank sample)/ AControl] × 100, where Asample, Ablank sample and Acontrol are the absorbance values for the sample, sample without DPPH and control, respectively. Trolox (6-hydroxy-2,5,7,8-tetramethychromane-2-carboxylic acid, Sigma-Aldrich, USA) solution at concentration range of 0–15 μM was used as standard. 2.10. Superoxide radical scavenging assay The superoxide scavenging ability of Undaria extract was measured according to Beauchamp and Fridovich [15]. The reaction mixture contained 50 mM phosphate buffer pH 7.8, 13 mM L-methionine, 75 μM NBT (Sigma-Aldrich, St Louis, USA), 2 μM riboflavin, 0.1 mM EDTA, and 20 μL of 10 mg/mL Undaria. Reactions were carried out at 37 °C for 15 min in a water-bath fitted with a 22 W Phillips fluorescent lamp. The absorbance was measured at 550 nm against a blank. The capability of scavenging the superoxide radical was calculated using the following equation: Scavenging effect (%) = 1 − [(Asample)/AControl] × 100. 2.11. Hormone-like activities measured in ARR5:GUS and DR5:GUS tomato reporter seedlings ARR5:GUS and DR5:GUS tomato seeds were surface-sterilized in 30% hypochlorite solution for 10 min followed by 3 washing steps in sterilized distilled H2O. Sterilized seeds were placed on Petri dishes containing half strength Murashige and Skoog (½ MS) medium at 25 °C. Seven-day-old ARR5:GUS and DR5:GUS seedlings were transferred to liquid ½ MS medium supplemented with 0.1% and 1% Undaria for 24 h
3.2. Undaria supplementation affects redox status in tomato plants Impoverished soil conditions restrict the growth of the plant but also 3
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Fig. 1. Effect of Undaria supplementation on tomato plants cultivated in organic matter- impverished soil. Plants were cultivated in control substrate (A-C), substrate supplemented with 0.1% Undaria (D-F) or substrate supplemented with 1% Undaria (G-I). Representative aerial parts (A, D, G), leaves (B, E, H) and roots (C, F, I) of 30 day old tomato plants are shown. Bar = 0.5 cm.
concentrations of Undaria liquid extract at 0.01%, 0.1% or 1% and then, germination and the vigor index of seedlings were quantified. No significant differences were observed under our assayed conditions suggesting that tomato seeds could not be highly sensitive to the action of Undaria at least during the early stages of development likely facilitating its possible application from the beginning of the plant life cycle (Fig. 4A and B).
generates in the plant oxidative stress that can be reflected in the levels of reactive oxidative species (ROS) as H2O2 and antioxidant molecules as anthocyanins and antioxidant. In order to assess if Undaria soil supplementation is beneficial for the redox status in tomato plants, we analyzed the content of H2O2, anthocyanins as well as CAT and SOD activities. H2O2 and anthocyanins contents in tomato plants decreased about 30% and 50% respectively when impoverished substrates were supplemented with either 0.1% or 1% Undaria (Fig. 3A and C). On the other hand, antioxidant activities CAT and SOD behaved differently. While CAT activity of plants supplement with both, 0.1% or 1% Undaria, showed a 3.5-fold increase compared to control plants (Fig. 3B), SOD activity declined between 15 and 40% in plants cultivated with 0.1 and 1% Undaria, respectively (Fig. 3D). Similar values for the measured parameters were obtained when tomato plants were grown in organicmatter rich soil, under optimal growth conditions, indicating the capability of Undaria supplement of restore the redox balance of the plant (Fig. 3). Since it is well accepted that specific compounds of the antioxidant metabolism such as CAT and SOD activities and anthocyanins modify their levels favoring the scavenger of H2O2 along the plant life cycle [27] we hypothesized that Undaria contributes to improve the redox status by triggering specific redox compounds in tomato plants. In agreement with our results, application of commercial extract of the brown seaweed Ascophyllum nodosum enhances antioxidant activity of spinach [27,28].
3.4. Undaria supplementation does not change soil pH nor moisture Positive effects of algal supplementation may rely on changes that the supplement causes to the conditions of the substrate, on direct effects on the plant metabolism, or on a combination of both. In order to dissect the nature of the observed effects soil parameters such as, pH and moisture were determined. However, neither pH nor moisture drastically changed between samples from non-supplemented and supplemented substrates at the assayed time (Fig. S1). Notably, pH values remained close to 8.0 independently of the addition of Undaria, suggesting that the positive effect of Undaria could be caused through direct effects on plant physiology and metabolism rather than through the modification of soil conditions. 3.5. Chemical characterization of Undaria In order to shed light on the causes that may promote tomato growth when Undaria was added as soil supplement, we analyzed the chemical composition of Undaria extract (Table 1). In agreement to previous reports, Undaria extract presented a vast set of components such as salts, minerals and vitamins [30,31]. Concentrations of detected components were also comparable to other seaweed studied [19,26,32]. Of interest, a wide set of elements considered as plant nutrients such as calcium, potassium, phosphorous and sodium, as well as
3.3. Undaria supplementation does not inhibit tomato seed germination Depending on applied concentrations, the algal supplements may promote or alternatively, cause inhibitory action in seed germination [1,29]. In this work, tomato seeds were soaked with different 4
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Fig. 2. Effect of Undaria supplementation on growth parameters of tomato plants cultivated in organic-matter impoverished soil. Aerial FW (A), aerial and root DW (B, and C), total chlorophyll (D) and leaf area (E) measured in 30 day old tomato plants cultivated in organic-matter impoverished soil supplemented with Undaria. Values represent the mean ± SD of three independent biological replicates (n = 15). Different letters indicate significant differences between treatments based on ANOVA and Tukey-HSD test (p ≤ .05).
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Fig. 3. Effect of Undaria on redox status of tomato plants. H2O2 content (A), CAT activity (B) anthocyanins (C) and SOD activity (D) were measured in 30 day old tomato plants cultivated in organic-matter impoverished soil (Ctrl), organic-matter rich soil (Growmix -G) or organic-matter impoverished soil supplemented with Undaria 0.1% or 1%. Values are the mean ( ± SE) of three independent experiments (n = 3). Different letters indicate significant difference between treatments (p < .05; Tukey-test).
Fig. 4. Seed germination and vigor index of tomato seedlings. Percentage of germination of tomato seeds was calculated at 6 days after initial treatments (A). Vigor index was calculated on percentage of germination and mean of root and shoot lengths in 11 day old tomato seedlings (B). Values represent the mean and SD of three biological replicates (n = 30). Data was analyzed by ANOVA. 6
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antioxidant reference, vitamin E analog Trolox was used and comparable DPPH radical scavenging activity (64.66%) was obtained at 0.01 mg/mL. In agreement with our findings, similar DPPH radical scavenging activity ranging from 26% to 57% at 2.0 mg/mL for enzymatic extracts of Undaria were reported by [20]. Superoxide radical is toxic to cellular components and causes serious damage to the biomolecules such as DNA, proteins and lipids [34]. For this reason, the superoxide radical scavenging activity was measured for Undaria water extract resulting in 54% at 1 mg/mL (Table S1). Inversely to our results [20] Undaria extract acquired with both proteases and carbohydrases showed low superoxide radical scavenging activity ranging from 10% to 20% at 1 mg/mL showing that this scavenging activity is highly sensitive to the biochemical conditions of Undaria extract.
Table 1 Compositional analysis of Undaria extract. Component
g/100 g
Calcium Potassium Phosphorous Iron Copper Cadmiun Nickel Zinc Mercury Sodium Iodine Vitamin A Riboflavin *UI/mg
0.794 1.183 0.266 0.0028 0.0014 < 2.10−6 < 5.10−6 0.0015 < 1.10−6 8.254 0.036 353⁎ 0.0013
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3.7. Undaria contains active auxin, cytokinin- and gibberellin-like compounds
UI/mg
In addition to minerals and vitamins (Table 1) different hormones or hormone-like molecules may also be present in Undaria liquid extract [35]. In order to identify the presence of auxin- and cytokinin-like compounds, we performed a functional assay taking advantage of tomato (cv. Micro Tom) transgenic lines DR5:GUS and ARR5:GUS [10]. These lines express a GUS gene reporter under either auxin (DR5:GUS) or cytokinin (ARR5:GUS) responsive promoters. Undaria liquid extract was able to activate both DR5- and ARR5- promoters indicating the presence of active auxin- and cytokinin-like molecules (Fig. 5). GUS staining, indicating the activation of DR5:GUS, was detected in the root tips of 0.1% Undaria-treated seedlings (Fig. 5A and B). On the other hand cytokinin promoter was highly induced in roots treated with 1% Undaria (Fig. 5C and D). Untreated control seedlings showed minimal
vitamin A and riboflavin were identified in Undaria extract. Different concentrations of Undaria aqueous solutions registered a pH of approximately 7.0.
3.6. Antioxidant activity of Undaria Presence of antioxidant activities was previously reported in Undaria and in other seaweeds [33]. We detected antioxidant activity in Undaria extract using two different methods, a DPPH radical scavenging assay and measuring superoxide radical scavenging activity in riboflavin-light-NBT system (Table S1). Undaria water extract removed 56.40% of DPPH radical at a concentration of 2.5 mg/mL. As an
Fig. 5. Detection of active auxin-like and cytokininlike activities in Undaria extract. Six dpg DR5:GUS (A) and ARR5:GUS (C) tomato seedlings were transferred to liquid ½ MS medium supplemented or not with 0.1 or 1% Undaria. Hormone responsive promoters were measured by GUS activity. GUS activation in roots was quantified at 24 h post-treatment as indicates histograms for auxin (B) and cytokinin (D). Values are the mean ( ± SE) of three independent experiments (n = 3).
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research was funded by grants from Agencia Nacional de Promoción Científica y Técnica Argentina (ANPCyT, PICT Start Up 008), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); and Universidad Nacional de Mar del Plata. MFS is a fellow from ANPCyT; SLC is a fellow from CONICET; YM, DFF, AVA and CAC are researchers from CONICET.
GUS staining values (Fig. 5). In addition, we confirmed the presence of active auxin (or molecules with auxin-like activity) in Undaria extract using a root growth bioassay. In this assay we tested the ability of the Undaria extract to mimic a well-known auxin response consisting in the inhibition of the primary root growth concomitantly with the induction of lateral roots [17]. We observed that Undaria extract treatments were able to mimic auxin responses in a dose-dependent fashion, confirming the presence of an auxin or auxin-like molecules (Fig. Supplemental figure 2A and B). We also performed a functional assay with Undaria extract to evaluate the presence of gibberellins which are essentials in developmental processes [36]. Active gibberellins or gibberellin-like activities were identified based on the ability to promote the growth of lettuce hypocotyls [37]. While Undaria treatments at 0.001% and 0.1% did not differ from control, 0.01% Undaria showed a significant increment in hypocotyl length respect to control and was similar to gibberellic acid (GA3) treatment (Fig. S2C). This result suggests that a gibberellin-like activity, capable to trigger a GA3-dependent response is present in Undaria extracts.
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Giusti, Wrolstad, Characterization and measurement of anthocyanins by UVvisible spectroscopy, in: R.E. Wrolstad, T.E. Acree, H. An, E.A. Decker, M.H. Penner, D.S. Reid, S.J. Schwartz, C.F. Shoemaker, P. Sporns (Eds.), Current Protocols in Food Analytical Chemistry, John Wiley & Sons, New York, USA, 2001, pp. F1.2.1–F1.2.13. [14] R.F. Beers, I.W. Sizer, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J. Biol. Chem. 195 (1952) 133–140. [15] C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels, Anal. Biochem. 44 (1971) 276–287. [16] C.N. Siddaiah, K.V.H. Prasanth, N.R. Satyanarayana, V. Mudili, V.K. Gupta, N.K. Kalagatur, T. Satyavati, X.-F. Dai, J.-Y. Chen, A. Mocan, Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation, Sci. Rep. 8 (2018) 2485. [17] N. Dharmasiri, S. Dharmasiri, D. Weijers, E. 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4. Conclusions Undaria supplementation to impoverished soils promotes the growth of tomato plants during their early developmental stages. Plant growth promotion was evidenced in increases in plant biomass and chlorophyll as well as in the restoration of the plant redox status. This outcome could be caused by the integration of a set of plant responses triggered by components of the algal extract, in agreement with the model proposed by Yakhin et al. [38]. Chemical analysis of Undaria extract revealed the presence of plant nutrients including mineral and vitamins. In addition, by means of functional assays, three phytohormone-like activities (auxin, cytokinin and gibberellin) that have capacity to promote plant growth were identified in Undaria extract. Finally, antioxidant activities were identified in the seaweed extract which also may contribute by reducing the exposition of roots to oxidative compounds. Hence, in spite of the difficulty in deciphering the precise mode of action of Undaria we demonstrated its efficacy to activate the intrinsic hormonal mechanisms of tomato plants resulting in favor to improve their growth and development. Further studies on the possible effects of Undaria extracts on the availability, uptake and assimilation of nutrients will allow a better understanding of their active compounds and the mechanism of action that mediate growth and tolerance to environmental stress. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2019.101785. Author contributions MFS, MAM, VAM, CAA, conception and design; MFS, SLC, YM, CAA, DFF, analysis and interpretation of the data; DFF, CAA, drafting of the article; DFF, CAA, critical revision of the article for important intellectual content; MFS, SLC, YM, MAM, DFF, VAM, CAA, final approval of the article; MAM, provision of algal extracts; MFS, statistical expertise; VAM, CAA, obtaining of funding. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank Dr. Agustin Zsögön (Universidade Federal de Viçosa, Brazil) and Dr. Lázaro Eustáquio Pereira Peres (Universidade de São Paulo) for providing ARR5:GUS and DR5:GUS Micro-Tom seeds. This 8
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Chromatographia 58 (2003) 159–163. [31] P. Rupérez, Mineral content of edible marine seaweeds, Food Chem. 79 (2002) 23–26. [32] S.S. Ramya, N. Vijayanand, S. Rathinavel, Foliar application of liquid biofertilizer of brown alga Stoechospermum marginatum on growth, biochemical and yield of Solanum melongena, International Journal of Recycling of Organic Waste in Agriculture 4 (2015) 167–173. [33] K. Fujimoto, T. Kaneda, Screening test for antioxygenic compounds from marine algae and fractionation from Eisenia bicyclis and Undaria pinnatifida, Nippon Suisan Gakkaishi 46 (1980) 1125–1130. [34] İ. Gülçin, Z. Huyut, M. Elmastaş, H.Y. Aboul-Enein, Radical scavenging and antioxidant activity of tannic acid, Arab. J. Chem. 3 (2010) 43–53. [35] E. Zizkova, M. Kubes, P.I. Dobrev, P. Pribyl, J. Simura, L. Zahajska, L. Zaveska Drabkova, O. Novak, V. Motyka, Control of cytokinin and auxin homeostasis in cyanobacteria and algae, Ann. Bot. 119 (2017) 151–166. [36] J. Patra, G. Das, K.-H. Baek, Chemical composition and antioxidant and antibacterial activities of an essential oil extracted from an edible seaweed, Laminaria japonica L, Molecules 20 (2015) 12093–12113. [37] W.K. Silk, R.L. Jones, Gibberellin response in lettuce hypocotyl sections, Plant Physiol. 56 (1975) 267–272. [38] O.I. Yakhin, A.A. Lubyanov, I.A. Yakhin, P.H. Brown, Biostimulants in plant science: a global perspective, Front. Plant Sci. 7 (2016) 2049.
269–272. [24] S. Zodape, A. Gupta, S. Bhandari, U. Rawat, D. Chaudhary, K. Eswaran, J. Chikara, Foliar application of seaweed sap as biostimulant for enhancement of yieldand quality of tomato (Lycopersicon esculentum Mill.), J. Sci. Ind. Res. 70 (2011) 215–219. [25] O. Goni, P. Quille, S. O’Connell, Ascophyllum nodosum extract biostimulants and their role in enhancing tolerance to drought stress in tomato plants, Plant Physiol. Biochem. 126 (2018) 63–73. [26] C. Kalaivanan, V. Venkatesalu, Utilization of seaweed Sargassum myriocystum extracts as a stimulant of seedlings of Vigna mungo (L.) Hepper, Span. J. Agric. Res. 10 (2012) 466–470. [27] H. Yamasaki, Y. Sakihama, N. Ikehara, Flavonoid-peroxidase reaction as a detoxification mechanism of plant cells against H2O2, Plant Physiol. 115 (1997) 1405–1412. [28] S.S. Ramya, S. Nagaraj, N. Vijayanand, Biofertilizing efficiency of brown and green algae on growth, biochemical and yield parameters of Cyamopsis tetragonolaba (L.) Taub, Recent Res. Sci. Technol. 2 (2010). [29] V. Cecchetti, M.M. Altamura, G. Falasca, P. Costantino, M. Cardarelli, Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation, Plant Cell 20 (2008) 1760–1774. [30] D. Sánchez-Machado, J. López-Cervantes, J. López-Hernández, P. Paseiro-Losada, J. Simal-Lozano, High-performance liquid chromatographic analysis of amino acids in edible seaweeds after derivatization with phenyl isothiocyanate,
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Amelioration of tomato plants cultivated in organic-matter impoverished soil by supplementation with Undaria pinnatifida
T
María Florencia Salcedoa, Silvana Lorena Colmana, Andrea Yamila Mansillaa, ⁎ María Alejandra Martínezc, Diego Fernando Fiola, , Vera Alejandra Alvarezb, ⁎ Claudia Anahí Casalonguéa, a Instituto de Investigaciones Biológicas, UE CONICET-UNMDP, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Deán Funes 3250, 7600 Mar del Plata, Argentina b Grupo de Materiales Compuestos Termoplásticos (CoMP), Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA), Facultad de Ingeniería, Universidad Nacional de Mar del Plata (UNMdP) y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Colón 10850, 7600 Mar del Plata, Argentina c Universidad Nacional de Mar del Plata, Argentina
A R T I C LE I N FO
A B S T R A C T
Keywords: Growth promoter Redox status Soil amendment Tomato Undaria pinnatifida
Rising demands of food together with modern practices in agriculture require the inclusion of areas with suboptimal soil qualities for cultivation and the replacement of contaminant chemical fertilizers. In this work, we evaluated the use of the seaweed Undaria as a soil amendment of organic-matter impoverished soil. We demonstrated that Undaria supplementation to a substrate containing vermiculite:organic soil mix (95:5) promotes the growth of tomato plants evidenced in increases in aerial and root biomass and the restoration of redox status. By means of chemical and functional analysis we identified in Undaria extract the presence of plant nutrients, minerals, vitamins, antioxidant capacity and phytohormone-like activities. All together these compounds would be able to promote growth and contribute to redox homeostasis in early plant developmental stages which are critical for tomato production yield. We propose Undaria supplementation as an efficient way to provide nutritional and growth promoter compounds to tomato crops growing on impoverished soils.
1. Introduction Tomato is one of the most extensively produced horticultural crops. Tomato originated in South America but nowadays is distributed worldwide and consumed as a human food rich in nutrients and antioxidants compounds [1]. Tomato is produced either in greenhouses or in open field and demands vast quantities of fertilizers to achieve current production rates [2]. When grown in greenhouses, tomato seedlings are propagated by about 4–5 weeks multi-well trays before they are transplanted to the field. Development of a healthy root system is critical during this period, and any improvement made during the first stages of plant growth and development can also enhance its fitness and further crop productivity. Increasing food necessities claim for the expansion of cultivable regions including areas with sub-optimal soil qualities. In addition, modern practices in agriculture require the replacement of synthetic fertilizers that cause detrimental soil effects to sustainable types of growth promoters and biostimulants. However, the development of
⁎
organic agents meeting the requirements of functionality, applicability and effect reproducibility stays behind of its demand of application. Marine algae (seaweed) species were employed as soil conditioners for centuries; however, its use has been recently reinforced because of the need of non-contaminant fertilizers [3]. A number of seaweed extracts have been applied to horticultural crops and resulted in improvements of plant productivity and stress tolerance [4–6]. In this context, commercial products consisting in processed seaweeds presented as liquid extract or soluble powder are already available and keep gaining market, partially driven for a crescent demand of organic food worldwide. Undaria pinnatifida (Undaria) is a brown seaweed species native from China, Japan and Korea. In these countries Undaria, also known as wakame, is widely consumed as a human food and constitutes a good source of proteins, vitamins, dietary fibers and minerals [7]. The highly invasive behavior of Undaria have caused its dissemination worldwide, many times challenging natural ecosystems, but also empowered its cultivation with industrial purposes [8]. The pharmaceutical industry
Corresponding authors. E-mail addresses: diefi[email protected] (D.F. Fiol), [email protected] (C.A. Casalongué).
https://doi.org/10.1016/j.algal.2019.101785 Received 4 July 2019; Received in revised form 11 October 2019; Accepted 28 December 2019 2211-9264/ © 2020 Elsevier B.V. All rights reserved.
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dark, followed by centrifugation at 10,000g for 15 min. The supernatants were collected and diluted with acetone 80% before measuring the absorbance at 645 and 663 nm in a spectrophotometer (GeneQuant 1300, GE Healthcare, USA). Equation for the determination of total chlorophyll (chlorophyll a + chlorophyll b) was used [11]. The total area of the first and second leaf from 30-day-old tomato plants was photographed with a camera (Nikon Coolpix T80, Indonesia) and quantified using the ImageJ image-analysis software (National Institutes of Health, USA).
has also taken advantage of Undaria and its byproduct fucoidan. However, in agriculture its application has been relegated [9]. In this work, we investigated the application of Undaria as a soil amendment to be used as a growth stimulator of tomato plants cultivated in a substrate impoverished in organic matter. Mineral nutrients, vitamins and hormone-like compounds altogether may trigger the activation of a set of biochemical responses improving growth and development in tomato plants. 2. Materials and methods
2.5. H2O2 and anthocyanins measurements 2.1. Plant material To quantify H2O2, 0.1 g of leaf tissue was ground with liquid N2 in a mortar and extracted with H2O for 30 min in the dark, followed by centrifugation at 10,000g for 20 min. The H2O2 content was quantified in each supernatant according to Bellincampi et al.[12] based on the peroxide-mediated oxidation of Fe2+, followed by the reaction of Fe3+ with xylenol orange (3,3′-Bis[N,N-bis (carboxymethyl) aminomethyl]o-cresolsulfonephthalein disodium salt, Sigma-Aldrich, St Louis, USA). For anthocyanins, 0.1 g of leaf tissue was extracted with 1% HCl in methanol, stirring for 1 min and incubated at 4 °C for 2 h in the dark. Monomeric anthocyanin content was determined using a pH- differential method [13]. A microplate reader (ELX 800, Biotek, USA) was used for spectral measurements at 530 and 700 nm. The content of chlorophyll was expressed as mg cyanidin-3-glucoside/g FW, using an extinction coefficient of 34,300 L/cm.mol and molecular weight of 449.2 g/mol.
Seeds of tomato (Solanum lycopersicum L. cv. Platense) were commercially obtained from FECOAGRO Ltda., San Juan , Argentina. Tomato seeds (Solanum lycopersicum L, cv. Micro-Tom) hormone transgenic lines DR5:GUS and ARR5:GUS were kindly donated by Dr. Agustin Zsögön (Universidade Federal de Viçosa, Brazil). DR5:GUS tomato plants express the reporter gene beta-glucuronidase (GUS) under a synthetic auxin-responsive promoter [10]. ARR5:GUS plants express GUS under an Arabidopsis cytokinin-responsive promoter. ARR5:GUS plants were generated by the laboratory of Dr. Lázaro Eustáquio Pereira Peres (Universidade de São Paulo, Brazil). 2.2. Undaria material and preparation Algae biomass of Undaria pinnatifida was obtained from Soriano S.A. (Chubut, Argentina). Seaweed was extracted during February every year from Gaiman Cost, Chubut, Argentina. Seaweed was washed with tap water to remove shell, debris and sand; then air-dried at 26 °C for 2–3 days and finally hand crushed and powdered with high-speed universal grinder. For the assays, Undaria was applied as powder to the soil substrate or alternatively, it was used as water liquid extract suspending Undaria powder in distiller water.
2.6. Protein extraction and quantification of antioxidant enzymes To quantify catalase (CAT) and superoxide dismutase (SOD) enzymatic activities, 0.1 g of leaf tissue were ground with liquid N2 in a mortar and extracted with 3 volumes of buffer (50 mM Tris-HCl buffer, pH 7.5, 3 mM MgCl2, 1 mM ethylene diamine tetra acetic acid, and 1.5% polyvinylpolypyrrolidone (PVPP, Fluka/Sigma-Aldrich, St Louis, USA). The homogenate was then centrifuged at 25,000g for 20 min, and the supernatant was used to measure the antioxidant enzymes. CAT activity was quantified as described Beers and Sizer [14] with some modifications. The reaction mixture contained 600 μL of 50 mM phosphate buffer pH 7.4, 0.1 mL of 1% H2O2 and 25 μL of enzyme extract diluted to keep measurements within the linear range of the analysis. A blank was used for each sample, which contained 50 mM phosphate buffer and 25 μL of enzyme extract. H2O2 levels were followed measuring absorbance at 240 nm in a spectrophotometer (GeneQuant 1300, GE Healthcare, USA). SOD activity was measured as described Beauchamp and Fridovich [15]. The reaction mixture contained 50 mM phosphate buffer pH 7.8, 75 μM nitroblue tetrazolium chloride (NBT), 2 μM riboflavin, 13 mM methionine, 0.1 mM EDTA and 2 μL of protein sample. Enzymatic reactions were carried out at 37 °C for 15 min in a water-bath fitted with a 22 W Phillips fluorescent lamp. The absorbance was measured at 550 nm using a microplate reader (ELX 800, Biotek, USA). One unit of SOD activity was defined as the amount of enzyme that produced a 50% decrease, with respect to the control, in the absorbance at 550 nm and it was expressed as Activity of SOD (U/mL) = (Acontrol * Aassay/Acontrol) * (1/50%) * (Vtotal/VSOD). Where Acontrol and Aassay are the absorbance units at 550 nm of control and sample, respectively; Vtotal is the total volume; and VSOD is the enzyme volume.
2.3. Experimental design and treatments Tomato seeds var. Platense were germinated in Petri dishes on filter paper soaked in sterile water and incubated in growth chamber for 4 days at 25 °C with 16:8 h light:dark cycles. Germinated seeds were transferred to plastic pots (0.18 L) containing the different soil substrates: impoverished substrate, referred as a control, prepared with a vermiculite:Growmix (95:5) mix; Undaria supplemented substrate, prepared adding the indicated amounts of Undaria powder to the impoverished substrate; and organic-matter rich substrate, consisting in Growmix. Growmix (Multipro Commercial –Terrafertil, Mar del Plata, Argentina) is commonly used by regional tomato producers (Buenos Aires, Argentina) and is composed of peat moss Sphagnum medium fibers, bark compost, calcite, dolomite lime and wetting agents. Its main physicochemical characteristics include a pH in the range of 5.0–5.8, humidity of 55–65% and water retention capacity of 60%. Tomato seedlings were grown at 25 °C under 250 μmol photons/m2/s with 16:8 h light: dark cycles daily and watered controlled during 30 days. After plant harvesting, measurements of leaf fresh weight (FW) were obtained. Root and leaf dry weights (DW) were determined by drying samples for 6 days at 60 °C in a convection oven. Chlorophyll content, growth and redox parameters were analyzed at the end of experiment when plants were 30-day-old and had three-four true leaves. Second and third leaf was used to measure chlorophyll and H2O2 contents and antioxidant compounds.
2.7. Measurements of germination and vigor index Tomato seeds were incubated on filter paper soaked in Petri dishes containing 3 mL of 0.01%, 0.1% or 1% Undaria liquid extract or H2O as control. Plates were incubated in a growth chamber at 25 °C with 16:8 h light:dark cycles. The percentage of seed germination (% germination) and vigor index of seedlings were calculated at 6 and 11 days,
2.4. Chlorophyll and leaf area measurements The level of total chlorophyll (Total Chl) was quantified from 0.1 g of leaf tissue. Samples were ground with liquid N2 in a mortar and chlorophyll was extracted with acetone 80% for 60 min at 4 °C in the 2
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under same growth conditions. Seedlings were removed washed twice with 50 mM sodium phosphate buffer pH 7.0 and incubated in staining buffer (50 mM Na phosphate pH 7.0, 0.1% Triton X-100, 5 mM EDTA, 0.5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6) and 1 mg/mL X-Gluc (5-bromo-4chloro-3-indolyl-beta-D-glucuronic acid, cyclohexylammonium salt, Gold Biotechnology, USA) as substrate at 37 °C for 2 h to overnight. Images were taken using a scanner (Epson Perfection V600 Photo, Indonesia).
respectively after initial treatments. Seed was scored as germinated when its radicle had emerged from within the seed coat. Vigor index was calculated on percentage of germination and mean of root and shoot lengths of seedlings according to the equation: Vigor index = (mean root length + mean shoot length) * % germination [16]. 2.8. Characterization of Undaria liquid extract Compositional analysis of Undaria was carried out at Fares Taie Laboratory services (Mar del Plata, Argentina). Magnesium was determined following standards IRAM 22409 (1964); calcium was determined following standards IRAM 15714–1 (1981), potassium, iron, copper, cadmiun nickel, zinc, mercury and sodium were determined by atomic absorption following standards EPA SW 846 (U.S. Environmental Protection Agency, 1986); phosphorous was determined following standards IRAM 15013 (1985), iodine was determined following standards AOAC 992.24 (1990), vitamin E was determined by gas chromatography, vitamin A was determined by HPLC-DAD, vitamin B2 (riboflavin) was determined HPLC-DAD, proline was determined by spectrophotometry (acid ninhydrin-based method). All values are expressed in (mg/100 g of dry algae).
2.12. Statistical analysis Trials with organic-matter impoverished soil were done in three independent experiments (biological replicates) with at least 15 plants per treatment (n = 45). Values shown in figures are mean values +/− standard deviation (SD). The data were subjected to analysis ANOVA with Tukey post hoc comparisons against control by R Studio Team (2015). Germination percentage was evaluated by generalized linear mixed model with binomial distribution, with at least 30 seeds per treatment in three independent experiments (N = 90). 3. Results and discussion 3.1. Undaria used as a soil amendment promotes growth and development in tomato plants
2.9. DPPH-radical scavenging assay
We evaluated the potential of Undaria as a soil amendment to promote the growth of tomato cultivated in an organic-matter impoverished substrate. Tomato plants were grown for 30 days either in a substrate impoverished in organic matter, containing a 95:5 proportion of vermiculite:organic soil used as control or in the same substrate supplemented with 0.1% or 1% Undaria powder (Fig. 1). Analysis of plant growth parameters as aerial FW and DW, root DW, leaf area and total chlorophyll content showed that Undaria supplementation resulted beneficial for tomato growth and development (Fig. 2). Biomass, evaluated as aerial FW and aerial DW, increased by 38% and 67% respectively in plants grown on 1% Undaria supplemented substrate (Fig. 2A and B). Likewise, the total area of leaves from plants grown with 1% Undaria supplemented substrate increased by 145% respect to the control (Fig. 2E). On the other hand, 0.1% Undaria supplemented substrate did not result in significant changes over control plants in these parameters. Analysis of root FW indicated that both, 0.1% and 1% Undaria supplements resulted in increases of about 50% compared with control (Fig. 2C). In agreement with our results, it was reported that application of seaweed extracts enhanced root development in tomato. Foliar spray of Ulva lactuca and Padina gymnospora extracts increased root length and fresh weight of tomato seedlings [1]. In addition, application of seaweed extract also improved root development and growth in other species including Triticum aestivum [18], grape [19], mung bean [20] and maize [21].These effects on plant growth have been related with the presence of phytohormones in the extract [3,22] as well as to the stimulation of mineral nutrient uptake mediated by seaweed extract in lettuce [23] and in tomato [24]. Chlorophyll concentration in leaves has been considered as a reliable indicator of metabolic and energetic imbalance in tomato plants under stress [25]. In concordance with plant growth parameters, total chlorophyll content also increased between 30 and 50% in plants growing in 0.1% and 1% Undaria supplemented substrate compared with control (Fig. 2D). A typical response of different seaweed extracts has been associated to the promotion of the chlorophyll content in plants and this fact could be related with the presence of cytokinin-like compounds in seaweed extracts [26].
The compound 2,2-diphenyl-1-picrylhydrazyl (DPPH, SigmaAldrich, USA) radical scavenging activity was determined following the method of Molyneux et al. [17]. Briefly, 3 mg of DPPH radical were dissolved in 50 mL of HPLC-grade methanol (Merck, Germany) and adjusted to 1.3 absorbance units at 517 nm. Then, 50 μL of Undaria water extract at concentrations from 1 to 10 mg/mL were mixed with 150 μL of DPPH in single wells of a 96-well plate. The plate was kept for 30 min in the dark. The absorbance was measured at 517 nm using microplate reader (ELX 800, Biotek, USA). The antioxidant capacity was expressed as percentage of the radical scavenging activity and calculated according to the following equation: DPPH scavenged (%) = 1 − [(Asample − ABlank sample)/ AControl] × 100, where Asample, Ablank sample and Acontrol are the absorbance values for the sample, sample without DPPH and control, respectively. Trolox (6-hydroxy-2,5,7,8-tetramethychromane-2-carboxylic acid, Sigma-Aldrich, USA) solution at concentration range of 0–15 μM was used as standard. 2.10. Superoxide radical scavenging assay The superoxide scavenging ability of Undaria extract was measured according to Beauchamp and Fridovich [15]. The reaction mixture contained 50 mM phosphate buffer pH 7.8, 13 mM L-methionine, 75 μM NBT (Sigma-Aldrich, St Louis, USA), 2 μM riboflavin, 0.1 mM EDTA, and 20 μL of 10 mg/mL Undaria. Reactions were carried out at 37 °C for 15 min in a water-bath fitted with a 22 W Phillips fluorescent lamp. The absorbance was measured at 550 nm against a blank. The capability of scavenging the superoxide radical was calculated using the following equation: Scavenging effect (%) = 1 − [(Asample)/AControl] × 100. 2.11. Hormone-like activities measured in ARR5:GUS and DR5:GUS tomato reporter seedlings ARR5:GUS and DR5:GUS tomato seeds were surface-sterilized in 30% hypochlorite solution for 10 min followed by 3 washing steps in sterilized distilled H2O. Sterilized seeds were placed on Petri dishes containing half strength Murashige and Skoog (½ MS) medium at 25 °C. Seven-day-old ARR5:GUS and DR5:GUS seedlings were transferred to liquid ½ MS medium supplemented with 0.1% and 1% Undaria for 24 h
3.2. Undaria supplementation affects redox status in tomato plants Impoverished soil conditions restrict the growth of the plant but also 3
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Fig. 1. Effect of Undaria supplementation on tomato plants cultivated in organic matter- impverished soil. Plants were cultivated in control substrate (A-C), substrate supplemented with 0.1% Undaria (D-F) or substrate supplemented with 1% Undaria (G-I). Representative aerial parts (A, D, G), leaves (B, E, H) and roots (C, F, I) of 30 day old tomato plants are shown. Bar = 0.5 cm.
concentrations of Undaria liquid extract at 0.01%, 0.1% or 1% and then, germination and the vigor index of seedlings were quantified. No significant differences were observed under our assayed conditions suggesting that tomato seeds could not be highly sensitive to the action of Undaria at least during the early stages of development likely facilitating its possible application from the beginning of the plant life cycle (Fig. 4A and B).
generates in the plant oxidative stress that can be reflected in the levels of reactive oxidative species (ROS) as H2O2 and antioxidant molecules as anthocyanins and antioxidant. In order to assess if Undaria soil supplementation is beneficial for the redox status in tomato plants, we analyzed the content of H2O2, anthocyanins as well as CAT and SOD activities. H2O2 and anthocyanins contents in tomato plants decreased about 30% and 50% respectively when impoverished substrates were supplemented with either 0.1% or 1% Undaria (Fig. 3A and C). On the other hand, antioxidant activities CAT and SOD behaved differently. While CAT activity of plants supplement with both, 0.1% or 1% Undaria, showed a 3.5-fold increase compared to control plants (Fig. 3B), SOD activity declined between 15 and 40% in plants cultivated with 0.1 and 1% Undaria, respectively (Fig. 3D). Similar values for the measured parameters were obtained when tomato plants were grown in organicmatter rich soil, under optimal growth conditions, indicating the capability of Undaria supplement of restore the redox balance of the plant (Fig. 3). Since it is well accepted that specific compounds of the antioxidant metabolism such as CAT and SOD activities and anthocyanins modify their levels favoring the scavenger of H2O2 along the plant life cycle [27] we hypothesized that Undaria contributes to improve the redox status by triggering specific redox compounds in tomato plants. In agreement with our results, application of commercial extract of the brown seaweed Ascophyllum nodosum enhances antioxidant activity of spinach [27,28].
3.4. Undaria supplementation does not change soil pH nor moisture Positive effects of algal supplementation may rely on changes that the supplement causes to the conditions of the substrate, on direct effects on the plant metabolism, or on a combination of both. In order to dissect the nature of the observed effects soil parameters such as, pH and moisture were determined. However, neither pH nor moisture drastically changed between samples from non-supplemented and supplemented substrates at the assayed time (Fig. S1). Notably, pH values remained close to 8.0 independently of the addition of Undaria, suggesting that the positive effect of Undaria could be caused through direct effects on plant physiology and metabolism rather than through the modification of soil conditions. 3.5. Chemical characterization of Undaria In order to shed light on the causes that may promote tomato growth when Undaria was added as soil supplement, we analyzed the chemical composition of Undaria extract (Table 1). In agreement to previous reports, Undaria extract presented a vast set of components such as salts, minerals and vitamins [30,31]. Concentrations of detected components were also comparable to other seaweed studied [19,26,32]. Of interest, a wide set of elements considered as plant nutrients such as calcium, potassium, phosphorous and sodium, as well as
3.3. Undaria supplementation does not inhibit tomato seed germination Depending on applied concentrations, the algal supplements may promote or alternatively, cause inhibitory action in seed germination [1,29]. In this work, tomato seeds were soaked with different 4
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Fig. 2. Effect of Undaria supplementation on growth parameters of tomato plants cultivated in organic-matter impoverished soil. Aerial FW (A), aerial and root DW (B, and C), total chlorophyll (D) and leaf area (E) measured in 30 day old tomato plants cultivated in organic-matter impoverished soil supplemented with Undaria. Values represent the mean ± SD of three independent biological replicates (n = 15). Different letters indicate significant differences between treatments based on ANOVA and Tukey-HSD test (p ≤ .05).
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Fig. 3. Effect of Undaria on redox status of tomato plants. H2O2 content (A), CAT activity (B) anthocyanins (C) and SOD activity (D) were measured in 30 day old tomato plants cultivated in organic-matter impoverished soil (Ctrl), organic-matter rich soil (Growmix -G) or organic-matter impoverished soil supplemented with Undaria 0.1% or 1%. Values are the mean ( ± SE) of three independent experiments (n = 3). Different letters indicate significant difference between treatments (p < .05; Tukey-test).
Fig. 4. Seed germination and vigor index of tomato seedlings. Percentage of germination of tomato seeds was calculated at 6 days after initial treatments (A). Vigor index was calculated on percentage of germination and mean of root and shoot lengths in 11 day old tomato seedlings (B). Values represent the mean and SD of three biological replicates (n = 30). Data was analyzed by ANOVA. 6
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antioxidant reference, vitamin E analog Trolox was used and comparable DPPH radical scavenging activity (64.66%) was obtained at 0.01 mg/mL. In agreement with our findings, similar DPPH radical scavenging activity ranging from 26% to 57% at 2.0 mg/mL for enzymatic extracts of Undaria were reported by [20]. Superoxide radical is toxic to cellular components and causes serious damage to the biomolecules such as DNA, proteins and lipids [34]. For this reason, the superoxide radical scavenging activity was measured for Undaria water extract resulting in 54% at 1 mg/mL (Table S1). Inversely to our results [20] Undaria extract acquired with both proteases and carbohydrases showed low superoxide radical scavenging activity ranging from 10% to 20% at 1 mg/mL showing that this scavenging activity is highly sensitive to the biochemical conditions of Undaria extract.
Table 1 Compositional analysis of Undaria extract. Component
g/100 g
Calcium Potassium Phosphorous Iron Copper Cadmiun Nickel Zinc Mercury Sodium Iodine Vitamin A Riboflavin *UI/mg
0.794 1.183 0.266 0.0028 0.0014 < 2.10−6 < 5.10−6 0.0015 < 1.10−6 8.254 0.036 353⁎ 0.0013
⁎
3.7. Undaria contains active auxin, cytokinin- and gibberellin-like compounds
UI/mg
In addition to minerals and vitamins (Table 1) different hormones or hormone-like molecules may also be present in Undaria liquid extract [35]. In order to identify the presence of auxin- and cytokinin-like compounds, we performed a functional assay taking advantage of tomato (cv. Micro Tom) transgenic lines DR5:GUS and ARR5:GUS [10]. These lines express a GUS gene reporter under either auxin (DR5:GUS) or cytokinin (ARR5:GUS) responsive promoters. Undaria liquid extract was able to activate both DR5- and ARR5- promoters indicating the presence of active auxin- and cytokinin-like molecules (Fig. 5). GUS staining, indicating the activation of DR5:GUS, was detected in the root tips of 0.1% Undaria-treated seedlings (Fig. 5A and B). On the other hand cytokinin promoter was highly induced in roots treated with 1% Undaria (Fig. 5C and D). Untreated control seedlings showed minimal
vitamin A and riboflavin were identified in Undaria extract. Different concentrations of Undaria aqueous solutions registered a pH of approximately 7.0.
3.6. Antioxidant activity of Undaria Presence of antioxidant activities was previously reported in Undaria and in other seaweeds [33]. We detected antioxidant activity in Undaria extract using two different methods, a DPPH radical scavenging assay and measuring superoxide radical scavenging activity in riboflavin-light-NBT system (Table S1). Undaria water extract removed 56.40% of DPPH radical at a concentration of 2.5 mg/mL. As an
Fig. 5. Detection of active auxin-like and cytokininlike activities in Undaria extract. Six dpg DR5:GUS (A) and ARR5:GUS (C) tomato seedlings were transferred to liquid ½ MS medium supplemented or not with 0.1 or 1% Undaria. Hormone responsive promoters were measured by GUS activity. GUS activation in roots was quantified at 24 h post-treatment as indicates histograms for auxin (B) and cytokinin (D). Values are the mean ( ± SE) of three independent experiments (n = 3).
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research was funded by grants from Agencia Nacional de Promoción Científica y Técnica Argentina (ANPCyT, PICT Start Up 008), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); and Universidad Nacional de Mar del Plata. MFS is a fellow from ANPCyT; SLC is a fellow from CONICET; YM, DFF, AVA and CAC are researchers from CONICET.
GUS staining values (Fig. 5). In addition, we confirmed the presence of active auxin (or molecules with auxin-like activity) in Undaria extract using a root growth bioassay. In this assay we tested the ability of the Undaria extract to mimic a well-known auxin response consisting in the inhibition of the primary root growth concomitantly with the induction of lateral roots [17]. We observed that Undaria extract treatments were able to mimic auxin responses in a dose-dependent fashion, confirming the presence of an auxin or auxin-like molecules (Fig. Supplemental figure 2A and B). We also performed a functional assay with Undaria extract to evaluate the presence of gibberellins which are essentials in developmental processes [36]. Active gibberellins or gibberellin-like activities were identified based on the ability to promote the growth of lettuce hypocotyls [37]. While Undaria treatments at 0.001% and 0.1% did not differ from control, 0.01% Undaria showed a significant increment in hypocotyl length respect to control and was similar to gibberellic acid (GA3) treatment (Fig. S2C). This result suggests that a gibberellin-like activity, capable to trigger a GA3-dependent response is present in Undaria extracts.
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4. Conclusions Undaria supplementation to impoverished soils promotes the growth of tomato plants during their early developmental stages. Plant growth promotion was evidenced in increases in plant biomass and chlorophyll as well as in the restoration of the plant redox status. This outcome could be caused by the integration of a set of plant responses triggered by components of the algal extract, in agreement with the model proposed by Yakhin et al. [38]. Chemical analysis of Undaria extract revealed the presence of plant nutrients including mineral and vitamins. In addition, by means of functional assays, three phytohormone-like activities (auxin, cytokinin and gibberellin) that have capacity to promote plant growth were identified in Undaria extract. Finally, antioxidant activities were identified in the seaweed extract which also may contribute by reducing the exposition of roots to oxidative compounds. Hence, in spite of the difficulty in deciphering the precise mode of action of Undaria we demonstrated its efficacy to activate the intrinsic hormonal mechanisms of tomato plants resulting in favor to improve their growth and development. Further studies on the possible effects of Undaria extracts on the availability, uptake and assimilation of nutrients will allow a better understanding of their active compounds and the mechanism of action that mediate growth and tolerance to environmental stress. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2019.101785. Author contributions MFS, MAM, VAM, CAA, conception and design; MFS, SLC, YM, CAA, DFF, analysis and interpretation of the data; DFF, CAA, drafting of the article; DFF, CAA, critical revision of the article for important intellectual content; MFS, SLC, YM, MAM, DFF, VAM, CAA, final approval of the article; MAM, provision of algal extracts; MFS, statistical expertise; VAM, CAA, obtaining of funding. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank Dr. Agustin Zsögön (Universidade Federal de Viçosa, Brazil) and Dr. Lázaro Eustáquio Pereira Peres (Universidade de São Paulo) for providing ARR5:GUS and DR5:GUS Micro-Tom seeds. This 8
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