Application of seaweed organic components increases tolerance to Fe deficiency in tomato plants

The beneficial effects of seaweed extracts have been related to plant growth regulators present in seaweeds. However, algae extracts comprise other organic compounds such as phenols, mannitol, alginates, laminarins and fucoidans that may have a relevant role regarding abiotic stress tolerance due to Fe deficiency. Therefore, we evaluated the individual effect of these organic compounds on the mitigation of Fe deficiency applying a range of concentrations (x1/10, x1, x10) in agar Petri dishes (in tomato seeds) and in the nutrient solution of a hydroponic system (tomato plants). Germination and plant growth promotion, root morphology, chlorophyll content and antioxidant activity were determined. Results showed that the lowest concentration x1/10 and phenolics, laminarin and fucose compounds contributed to increase the tolerance to Fe deficiency in tomato plants.


Introduction
low Fe availability, especially in calcareous soils with alkaline pH, results in a reduction of plant productivity and quality [32]. Nutrient imbalances such as Fe deficiency may be alleviated by the use of SWE products, enhancing the defense mechanism to reduce the oxidative stress and the chlorosis. Moreover, the addition of SWEs may promote the root development and the photosynthesis, improving the nutrient uptake.
Therefore, the objective of this work was to study the effect of individual application of organic components present in the algae extracts on: (1) plant growth development and (2) mitigation of abiotic stress due to Fe deficiency (nutrient imbalance). Also, (3) identify the concentration range at which these compounds may have positive effects. This work will contribute to scientific basis for establishing criteria for the production, use, and regulation of new seaweed extract products, which guarantee farmers the benefits indicated on the label package of these type of products.

Organic compounds
A set of organic compounds present in SWEs that have been related to the beneficial effects of the SWE application in agriculture by several authors (see introduction section) were selected: Fucose (L-(-)-Fucose, Sigma-Aldrich) that is the fundamental sub-unit of the fucoidan; alginic acid polysaccharide (alginic acid sodium salt, Sigma-Aldrich), laminarin polysaccharide (from Laminaria digitate, Sigma-Aldrich); mannitol that is a sugar alcohol (D-Mannitol, Merck); salicylic acid (Panreac) and gallic acid (Sigma-Aldrich) that are phenolic compounds. The organic compounds were applied at three concentrations x1/10, x1, x10 ( Table 1). The concentration x1/10 (1/10-fold with respect to x1) and x10 (10-fold with respect to x1). The concentration x1 was calculated based on the concentration of these organic compounds in commercial SWEs of A. nodosum [4,33] and the average dose of application (by root) of commercial SWEs for tomato plants. Control treatment without any organic compound application was also performed.
Tomato seeds (Solanum lycopersicum L. Moneymaker) were surface sterilized, vernalized at 4 o C for 24 h in darkness and placed on Petri dishes (8 seeds per Petri dish) with the respective organic compounds treatments and concentrations. Control treatment without any organic compound application was also performed. The sowing was carried out in a laminar air flow cabinet to avoid bacterial contamination. Petri dishes were placed in vertical position with a slight inclination for 7 days in a growth chamber with a photosynthetic photon flux density at leaf height of 1000 μmol m −2 s −1 photosynthetically active radiation, 16-h, 25 °C, 40% humidity/ 8-h, 20 °C, 60% humidity day/night regime.
A total of 2 Petri dishes per treatment and concentration was performed. The germination percentage (%) was measured at day 2 and the growth promotion (+%) or growth inhibition (-%) root seedlings at day 3, 4, 5 and 7 after sowing. The growth promotion and growth inhibition were calculated as follow: Growth (%) = [(treated root length -control root length)/control root length]×100

Plant material and growth conditions
Tomato (Solanum lycopersicum cv. Moneymaker) plants were grown in a growth chamber with a photosynthetic photon flux density at leaf height of 1000 μmol m −2 s −1 photosynthetically active radiation, 16- Organic compounds treatments (salicylic acid (SA), gallic acid (GA), sodium alginate (SoA), mannitol (MA), laminarin (LA) and fucose (FU)) were applied three times during the experiment at two different concentrations (x1/10 and x1). The concentration x10 produced inhibition of germination and growth development in seedling during the Petri dish assay, so that concentration was dismissed in hydroponic experiment. First application of the organic compounds was the first day of growth period with completed Hoagland solution, the second application was after five days of growth period with the renewal of nutrient solution, and the third application was at the beginning of Fe-deficient period. A control treatment without organic compound application was also performed. A total of 4 pots per treatment and concentration were used.
The morphology of tomato roots treated with the organic compounds was analysed in Fe sufficiency and after 6 days of Fe deficiency. Fresh roots were washed and blotted with filter paper. Then, root tips and root pieces (3 cm length) cut at five cm from the root tip were mounted on microscope glass slides and analysed by a stereomicroscope (Leica MZ12.5, Wetzlar, Germany) connected to a video camera (Olympus UC30, Tokio, Japan).
Leaf chlorophyll index was assessed at day 0, 3 and 5 of Fe deficiency using a SPAD 502 apparatus (Minolta Co., Osaka, Japan) after applying the organic compounds at two amount of enzyme that causes 50% NBT reduction by superoxide radicals, and the specific activity was expressed as units mg −1 of protein. Catalase activity (CAT, EC 1.11.1.6) was determined according to Aebi [35] with some modifications. CAT activity was assayed in a 3 mL reaction volume at 25 °C by adding 0.1 mL of diluted extract to a solution containing 50 mM phospate buffer pH 7.0 and 10 mM H 2 O 2 The activity was measured by monitoring the decrease in absorbance at 240 nm as a consequence of H 2 O 2 consumption using a spectrophotometer (Spectro start nano, BMG Labtech, Germany). Activity was expressed as units (mmol of H 2 O 2 decomposed per minute) per mg of protein.

Statistical analyses
Statistical analysis was carried out with SPSS for Windows (v. 21.0), using a Levene test for checking homogeneity of variances, and ANOVA or Welch´s tests (p < 0.10) were performed. Post hoc multiple comparisons of means were carried out using Duncan´s or Games-Howell´s test (p < 0.10) as appropriate.

Germination assay in Petri dish
In general, the individual application of the organic compounds at different concentration (x1/10, x1 and x10) in tomato seeds showed a higher germination (>6-37 %) with respect to untreated control, except in the case of SA (x1 and x10) and SoA (x1) that was similar to untreated control ( Table 2). The GA (x1/10) treatment showed the highest germination (37%) compared to the control, followed by GA (x1), SoA (x10), MA (x1 and x10) with a 25% of germination. After the seed sowing and organic compounds application, length of root seedlings was measured at day 3, 4, 5 and 7, and compared to the control. The growth promotion and growth inhibition were calculated. The application of organic compounds promoted the root growth during the 7 days compared to the control, with the exception of SA (x10), GA (x10) and FU (x1/10) that inhibited the root growth (Fig 1). At day 3, GA (x1/10; x1) and LA (x1/10) significantly increased the root length (>40%), but SA (x10) treatment totally inhibited the root growth compared to the control. At day 4 and 5, SA (x10) maintained almost total inhibition of root growth compared to the control, and at day 7, SA (x10) and also GA (x10) inhibited the root growth (80% and 40% respectively) with respect to the control.

Fresh weight
The FW of root, stem, developed leaves and new leaves was determined after six days of

Morphology of tomato roots
The morphology of roots treated with organic compounds was evaluated in Fe sufficiency and after 6 days of Fe deficiency compared to untreated control (Fig 3). Under

Leaf chlorophyll index
During Fe sufficiency, LA (x1/10) and SA (x1) significantly decreased the chlorophyll index compared to the untreated control, but the rest of treatments maintained the chlorophyll index similar to the control (Fig 4). During Fe deficiency, GA (x1/10) and LA (x1) significantly decreased the chlorophyll index compared to the untreated control at day 3, but FU (x1/10; x1) significantly increased the chlorophyll index compared to the untreated control at day 5 of Fe deficiency.

Oxidative stress parameters
The oxidative stress was measured in roots and new developed leaves after six days of Fe deficiency by the determination of SOD and CAT activity. Neither of organic compounds treatments increased the SOD activity in root and new leaves, but SA, GA, SoA and LA (x1) significantly decreased the SOD activity in new leaves compared to the untreated control ( Fig 5). Moreover, GA and FU (x1/10) in root, and GA (x1/10) in new developed leaves increased the CAT activity with respect to the control. However, neither of organic compounds treatments significantly decreased the CAT activity in root and new leaves compared to the untreated control.

Discussion
Seaweed extract application may improve germination and plant growth development [36,37]. This promotion effect may be caused not only by phytohormones, but also by other compounds present in SWEs [5,38]. In fact, all organic compounds applied at different concentrations (x1/10, x1 and x10) in tomato seeds significantly increased the germination with respect to untreated control, except in the case of SA (x1 and x10) and SoA (x1). It has been reported that the positive effect of these compounds depends on the concentration applied. In an experiment with bean seedlings, the application of 0.1 mM SA (14 mg/L) inhibited germination and initial growth, but SA concentrations lower than (only 25%) [40]. SoA (x1/10 and x10) applications increased the germination, and also SoA (x1/10 and x1) promoted the root growth between 10 and 30% during 5 days in tomato seedlings. In concordance with the presented data, it has been reported that alginate derived oligosaccharides enhanced seed germination in maize [41], and increased the root growth in barley [42], lettuce [43] carrot and rice [44]. All MA treatments increased the germination and also MA (x1 and x10) enhanced, but not significantly, the root length until day 5 of growth. Contrary with our results, Johnson and Kane [45] indicated that MA application did not improve the germination in pine-pink seeds, and even very high concentration (7-9% (w/v)) inhibited the seed germination in celery plants by causing osmotic stress [46]. LA treatments enhanced the seed germination and also LA (x1/10 and x10) increased the root length, especially after 3 days of growth. Some authors indicated that LA might be used as a seed germination and plant growth accelerator in many plants [47]. FU treatments showed a very slight increase in seed germination, and except FU (x1/10), FU did not promote the root growth compared to the control. Stevenson and Harrington [48] applied FU in Arabidopsis thaliana seeds and showed a significant decrease in the hypocotyl and root length. medium under salt and heat stress showed an increase of Arabidopsis thaliana Col-0 FW [24]. As far as we know, the evaluation of FU application on plant biomass has been not described in the literature.
Iron deficiency induce root morphological alterations that results in a greater formation of root hairs [55] and secondary roots, a shorter length of the lateral roots [56], a decrease of the distance between secondary roots [57] and a thickening of root tips [56,58]. Under Related to this, some authors showed an increase of secondary roots formation after the application of seaweed extracts in Arabidopsis thaliana [5], grapevine [59] and strawberry [37]. This result suggests that the application of these organic compounds may contribute to the improvement of Fe availability, regulating the morphological adaptive responses of roots to Fe deficient conditions.
Despite several studies reported that the application of SWEs increased the chlorophyll content [5,26,60,61], others did not. For example, Carrasco-Gil et al. [62] did not obtained any change in the chlorophyll content after applying commercial SWEs to tomato plants after 7 days of Fe deficiency, and suggested that the application doses should be increased for attenuating chlorosis symptoms. In the present study, GA x1/10 (4.7 mg/L) and LA x1 Iron deficiency enhanced the production of reactive oxygen species (ROS) in plants [65,66]. However, the role of ROS in Fe response regulation has not been well defined, and it may play multiple roles [67]. Plants have an enzymatic antioxidant system for scavenging the ROS excess and prevent damages to cells [68]. Superoxide dismutase (SOD), and catalase (CAT) are the first enzymes in the detoxification pathway and contain Fe in heme (CAT) and non-heme (Fe-SODs) form. Iron deficiency in plants increased total SOD activity (decreasing Fe-SOD and increasing CuZn-SOD and Mn-SOD), and reduced CAT activity since the synthesis of these enzyme is inhibited [66,[69][70][71]. Several studies reported an increase of total SOD activity in leaves after the application of seaweed extracts in healthy plants [72], in Fe deficient plants [62] and in drought or water stressed plants [74,75]. In the present work, total SOD activity significantly decreased in Fe deficient tomato leaves after the application of SA, GA, SoA and LA (x1; 47, 47, 90 mg/L respectively) compared to untreated leaves. It could be possible that these compounds at concentration x1 balanced the ROS production (superoxide radical; O2 •-), decreasing total SOD activity. Contrary to the results obtained, several authors reported an increase of total SOD activity after the application of GA and SoA in plants. Application of 1-2 mM (170-240 mg/L) GA in soybean grown in normal and cold stressed condition [22] and the application of 1000 mg/L alginate derived oligosaccharides in wheat grown in normal, drought and Cd stressed conditions [52,53], increased the SOD activity. In the case of SA, contradictory results have been found. Some studied showed an increase of total SOD respectively of Fe deficient peanut [76] or 0.5 mM SA in soybean roots under arsenic toxicity [77]. However, other studies reported that 0.5 mM SA applied in maize plants under low temperatures did not affect the SOD activity [78,79], and even high concentrations of SA (2.5 mM; 345 mg/L) applied in wheat seedlings decreased total SOD activity [73]. On the other hand, CAT activity decreased in Fe deficient plants. However, application of SA, GA and FU x1/10 significantly increased the CAT activity in roots compared to Fe deficient control. Similar results were reported in salt stressed rosemary plants where the application of SA (100-300 mg/L) increased CAT activity [50]. The CAT enzyme catalyze the decomposition of hydrogen peroxide (H 2 O 2 ) into oxygen and water [30]. Some authors reported a decrease of H 2 O 2 in plants grown in salt [20] and cold [22] stressed condition after the application of GA suggesting an increase of CAT activity. Therefore, these phenolic compounds at concentration x1/10 may improve the antioxidant system enhancing the plant tolerance to Fe deficient stress. As far as we know, direct effects of fucoidan on plants have not yet been reported.

Conclusions
In summary, the results of this research point out the importance of the concentration applied and the type of organic compounds present in a SWE in relation to its effectiveness to enhance the tolerance to iron deficiency (nutrient imbalance). The lowest concentration x1/10 of organic compounds showed the best results regarding growth promotion seedlings, fresh weight, secondary root elongation, chlorophyll content and CAT antioxidant activity. Moreover, from among all organic compounds evaluated, the phenolic compounds (salicylic acid and gallic acid), laminarin and fucose contributed in a greater extent to increase the tolerance to Fe deficiency in tomato plants. Although, it is necessary to carry out more studies in this regard, since it is possible that the effects of the algae extracts are not only due to the presence of discrete compounds, but the synergy produced by the interaction between them. It would also be of interest to test these compounds on other crops and substrates. In addition, experiments must be carried out to establish the optimal application times for these compounds, both in relation to the vegetative phase and the frequency of application. The achievement of these studies would be of great importance to establish greater control over the existing marine algae extracts, as well as to develop second generation algae extracts, designed with specific compositions for the needs of each crop.

Funding
Authors gratefully acknowledge the financial support by Spanish Ministry of Economy and  Increases or decreases > 40% are indicated by asterisk (*).