Biostimulant activity of Galaxaura rugosa seaweed extracts against water deficit stress in tomato seedlings involves activation of ABA signaling

Water scarcity is a serious constraint for agriculture, and global warming and climate change can exacerbate it in many areas. Therefore, sustainable approaches must be implemented to deal with current and future water scarcity scenarios. Genetic and chemical approaches are being applied to manage this limitation and maintain crop yields. In particular, biostimulants obtained from natural sources such as marine algae are promising aids for coping with water deficit stress in agriculture. Here we present a bioprospection study of extracts of the macroalgae Bonnemaisonia hamifera, Galaxaura rugosa, Dasycladus vermicularis, Ulva clathrata, Cystoseira foeniculacea, Cystoseira humilis, Lobophora dagamae, Colpomenia sinuosa and Halopteris scoparia from the north coast of Tenerife, in the Canary Islands. The aqueous extracts of Bonnemaisonia hamifera, Galaxaura rugosa, Dasycladus vermicularis and Cystoseira humilis show biostimulant activity against water deficit stress in tomato seedlings under controlled conditions, providing higher tolerance than the mock-treated control. The Galaxaura rugosa extract showed the highest biostimulant activity against water deficit stress. We demonstrate that this positive effect involves the activation of the abscisic acid (ABA) pathway in Arabidopsis thaliana (arabidopsis) and Solanum lycopersicum (tomato). Application of G. rugosa extract to the root system by drenching tomato seedlings subjected to water deficit leads to improved CO2 assimilation and water use efficiency (WUEp), compared to mock-treated plants. These results highlight a new potential seaweed source of substances with osmoprotectant properties, useful for biostimulant development. Future studies may provide further insight into which components of the seaweed extract induce activation of the ABA pathway.


Introduction
In recent decades, the exponential development of innovative new technologies has led to the provision of innovative methods for the isolation and identification of natural products.One of the areas of interest is the development of therapeutic bioactive compounds from the marine ecosystem (Molinski et al., 2009).The world's oceans and seas are home to a wide variety of organisms that have evolved complex metabolic capabilities to adapt to their habitat and produce a range of secondary metabolites with useful biological activities.Among them, marine biotechnology is an emerging field based on the exploration and exploitation of marine resources (de Vera et al., 2018).
One application of marine natural products is the use of seaweeds and microalgae as biostimulants for agriculture (Bulgari et al., 2019;Mazepa et al., 2021).In recent years, due to population growth levels that could seriously threaten food security, the agricultural industry has expanded the search for novel compounds with an environmentally friendly profile.This is in line with the "Green Deal" and "Farm to Fork" strategies adopted by EU policies, being driven by global awareness of the need to develop sustainable production systems (Mazepa et al., 2021).For example, this worldwide trend in agricultural markets has increased the extent of certified organic farming, to reach 71.1 million ha, almost doubling the 35.9 million ha in 2013 (Bulgari et al., 2019).
According to the EU Regulation, biostimulants are products that "stimulate plant nutritional processes, irrespective of the nutrient content of the product, with the sole aim of improving one or more of the following characteristics of the plant or its rhizosphere: (a) nutrient use efficiency; (b) tolerance to abiotic stress; (c) quality traits; or (d) availability of limited nutrients in the soil or rhizosphere" According to Du Jardin (du Jardin, 2015), there are seven categories of biostimulants: humic/fulvic acids, seaweed/ botanical extracts, protein hydrolysates, biopolymers, beneficial minerals, beneficial bacteria, and beneficial fungi.Another classification by Bulgari et al., 2019 added a new category including extracts from industrial or food waste, and placed nanomaterials and nanoparticles in the biopolymers category (Jimeńez-Arias et al., 2021).
Among seaweeds, the brown seaweed Ascophyllum nodosum is the most popular source used in agriculture (Xu and Leskovar, 2015;Yang et al., 2019).There are several seaweed-based liquid fertilizer formulations on the market, such as Kelpak ™ (Aremu et al., 2016) and Sealgae ™ .Preparing their extracts requires using water, acid or alkaline treatments, low temperatures, or heating, or disruption using physical methods (Battacharyya et al., 2015).However, for most documented seaweed extracts, the main chemical components responsible for their biostimulant activity are unknown (Ertani et al., 2018).
Salinity and drought are the most important abiotic stresses (Summary for Policymakers, 2014).Together, they are responsible for up to a 75% reduction in global crop production.Moreover, their impact will increase due to climate change (Summary for Policymakers, 2014).These two stresses exert an osmotic shock on plants by lowering the soil water potential due to limited water availability, leading to water deficit, oxidative stress, and nutrient imbalance.The aim of this study was to evaluate the effects of aqueous extracts obtained from nine seaweeds from the north coast of Tenerife in the Canary Islands, and to determine their chemical composition.Among them, the Galaxaura rugosa extract showed the highest activity.We then investigated the possible mechanism underlying this activity and found it to be partly mediated by the activation of the ABA signaling pathway.The defense mechanisms against drought include biophysical, biochemical, cellular and molecular processes integrated in plant stress physiology.The improvement of the root system architecture, leaf structure, osmotic balance, relative water content and stomatal aperture modulation are considered to be the most prominent physiological features for drought resistance in crop plants.Moreover, reactive oxygen scavenging and signaling via calcium and phytohormones such as abscisic acid, salicylic acid, jasmonic acid, auxin, gibberellin, ethylene, brassinosteroids and peptide molecules are crucial mechanisms for coping with drought stress (Iqbal et al., 2022).Among all phytohormones, ABA has a crucial role in coping with drought stress (Mega et al., 2019;Yang et al., 2019;Mao et al., 2022).ABA is a key signaling molecule that mediates plant acclimation to water deficit by reducing transpiration, protecting photosynthesis and triggering other metabolic adjustments, including the induction of stress proteins and osmolytes.Consequently, fine-tuning and modulating ABA responses holds the promise of pre-adapting plants to drought through changes in both short and long-term plant physiology (Yang et al., 2019).
These results highlight a new seaweed source potentially capable of protecting plants against water deficit stress, showing notable osmoprotectant properties.Moreover, we unveil the capability of G. rugosa seaweed as a novel biostimulant source.Both these uses make it or its constituents a promising resource for commercialization.

Algae collection and extraction processes
The nine macroalgae tested in this study were collected along different seasons between 2021 and 2022 at Punta del Hidalgo (28°3 3' 37.32'' N, 16°20' 7.843'' W) on the north coast of the island of Tenerife (Canary Islands) at low tide in the intertidal zone, in accordance with the Nagoya Protocol, permit reference: ESNC102.These algae were selected for their extensive study in the archipelago in terms of morphology and genetics.Specimen identification was confirmed using a Leica DM 500 Microsystems optical microscope (Wetzlar, Germany), following the classification proposed by Guiry and Guiry in 2023.A voucher specimen of each species was deposited at the University of La Laguna (Tenerife, Spain), see Table 1.
Each species was gently washed with deionized water, dried at room temperature, and ground into a powder.Seaweed extracts were prepared by adding the dried macroalgal biomass to 150 mL of distilled water (600 mL flask) and sonicating (J.P. Selecta Ultrasounds 3000513, Abrera, Spain) for 30 min at room temperature, after which the mixture was centrifuged to separate the extract from the biomass.This process was repeated three times, then the three extractions were pooled and after removal of solvent by a rotary evaporator Flawil,Switzerland), the extracts of each species were obtained.Table 1 shows the species, the quantity of dry algae and the yield of each extract.

Extract characterization by NMR (Nuclear Magnetic Resonance)
Each extract was analyzed by NMR using D 2 O as solvent.We measured 1 H NMR and 13 C NMR, HSQC (Heteronuclear Single Quantum Coherence), HMBC (Heteronuclear Multiple Bond Correlation) and COSY (Correlated Spectroscopy) spectra using a Bruker Avance II-500 instrument (Bruker, Karlsruhe, Germany) operating at 500 MHz for 1 H NMR and at 125 MHz for 13 C NMR.The use of standard Bruker software (TOpSpin 2.1, Bruker, Karlsruhe, Germany) aided the provision of two-dimensional NMR spectra.

Plant material and experimental conditions
Tomato (Solanum lycopersicum) var.Robin seedlings were obtained from a local nursery.We sowed tomato seeds in standard 150-cell tomato seedling trays using universal substrate and an automatic sowing machine to ensure germination and growth uniformity.When the seedlings reached the two true leaf stage (two weeks), we started the experiment.The seedling trays were then transferred to a growth chamber with controlled conditions: temperature 24 ± 2°C, photoperiod 16-8 h, humidity 65 ± 2%, and irradiance 300 µmols/m 2 s.All plants received a halfstrength Hoagland solution as water supply (Hoagland and Arnon, 1950).Kelpak ™ (BASF, Germany) and Sealgae ™ (Biovert, Spain) were purchased from a local supplier.

Treatments and water-deficit assays
All water deficit growth tests were carried out over 7 days according to the method described by Jimeńez-Arias et al. (Jimeńez-Arias et al., 2022).Water stress was induced by watering at 50% field capacity with a half-strength Hoagland solution, compared to control plants well-irrigated at 100% field capacity.All treatments consisted of twenty plants (N=20).Results are the mean of three independent experiments (see schema of the experimental set up in Figure 1).
Plant treatment was conducted by dissolving the various algal extracts at 1% w/v in 100 ml distilled water and adding 5 ml directly to the root system, except for the control treatment which received 5 ml of a half-strength Hoagland solution.

Biomass measures and stress index calculations
Seedling biomass was calculated at the beginning and end of the water deficit period (7 days).We removed the seedlings from the cell trays, carefully washed their roots under water to remove peat, then oven-dried them at 70°C for three days.Relative growth rate (RGR) was estimated according to the formula: RGR = (lnW2 -lnW1)/(t2 -t1), where W1 and W2 are the dry weights of the seedling at times t1 and t2 (the beginning and end of the water deficit, respectively) (Jimeńez-Arias et al., 2015).Various indexes were calculated using the weight of the plants at 7 days, such as the stress susceptibility index (SSI) (Gananca et al., 2015), stress tolerance index (TSI) (Farshadfar et al., 2013), relative growth rate (RGR), and plant water use efficiency (WUEp) (Jimeńez-Arias et al., 2022).

Gas exchange measurements
The fully developed leaves (N = 30) were subjected to gas exchange analyses.Photosynthesis (Pn), intracellular CO 2 (Ci), stomatal conductance (gs) and transpiration rate (E) were measured on the attached leaves using a portable infrared gas analyzer (LCPro, BioScientific Ltd., Hoddesdon, UK).Meas urements were at ambient C O 2 conc e ntr at i on , photosynthetic photon flux density (PPFD) of 1000 µmol m -2 s -1 (optimized with a light curve) and cuvette airflow of 500 ml min -1 .The values for instantaneous water use efficiency (iWUE) and intrinsic water use efficiency (intWUE) are the ratios between Pn/ E and Pn/gs, respectively (Seibt et al., 2008).The ratio between Pn and Ci was also calculated.

Induction of the luciferase reporter in Arabidopsis transgenic seedlings
We grew pMAPKKK18-LUC Arabidopsis thaliana seedlings (Vaidya et al., 2017) in 24-well plates (25-35 seeds per well) filled with 1 ml of MS medium supplemented with 1% agar for 7 days (d).Subsequently, Arabidopsis seedlings were treated with mock, 25 mM ABA or 0.5% (w/v) G. rugosa extract for 24 h in a solution containing 100 mM luciferin (potassium salt, GoldBio).Seedlings were incubated for 24 h and luminescence was recorded with a LAS-3000 imager (Fujifilm) equipped with a CCD camera using 2 min exposures.We converted eight-bit images to rainbow false color and quantified using Fiji.The experiment was repeated at least twice.

qRT-PCR analysis of ABA-responsive genes in tomato
Ten-day-old tomato seedlings (cv.Moneymaker) were treated with mock or 0.5% G. rugosa extract for 6.Total RNA was extracted using a NucleoSpin RNA plant kit.Synthesis of cDNA and quantitative real-time PCR (qRT-PCR) analyses were performed as described by González-Guzmán et al. (2014).cDNAs corresponding to the ABA-responsive genes Sl02g084850 (SlRAB18) and Sl06g067980 (SlLEA) genes were amplified using the same primers they used (Gonzaĺez-Guzmań et al., 2014).Treatments and experimental set-up.WW and WD mean well-watered and water deficit treatments, respectively.A-WW and A-WD mean G. rugosa treatment in well-watered and G. rugosa treatment in water deficit conditions, respectively.
Expression was normalized using values obtained with Sl06g009970 (SlEF1a).

Quantification of ABA
The G. rugosa extract was dissolved in 80% methanol-1% acetic acid containing internal standards and mixed by shaking for one hour at 4°C.The extract was stored overnight at -20°C, centrifuged and the supernatant dried in a vacuum evaporator.The dried residue was dissolved in 1% acetic acid and passed through an Oasis HLB (reverse phase) column as described in (Seo et al., 2011).To quantify the hormone ABA, the dried eluate was dissolved in 5% acetonitrile-1% acetic acid and the hormone separated using an autosampler and reverse phase UHPLC chromatography (2.6 µm Accucore RP-MS column, 100 mm length x 2.1 mm i.d.; ThermoFisher Scientific) with a 5 to 50% acetonitrile gradient containing 0.05% acetic acid, at 400 µL/min for 21 min.The hormone was analyzed by selected ion monitoring (SIM) with a Q-Exactive mass spectrometer (Orbitrap detector; ThermoFisher Scientific), and its concentration in the extract determined using embedded calibration curves and the Xcalibur 4.0 and TraceFinder 4.1 SP1 programs.The internal standard for quantification was the deuterium-labeled hormone.

Statistical analyses
After the data normality and homoscedasticity were checked a one-way ANOVA TEST (Duncan´s post hoc, IBM-SPSS24 statistical software package) was used to analyze the differences at p values<0.05 between treatments in all the measures studied.

Chemical analysis of crude extracts
The comprehensive metabolic profile analysis of each crude extract was carried out by 1 HNMR and 13 C NMR, COSY, HSQC and HMBC experiments (Supplementary Material).The main metabolites detected in each extract are listed in Table 2, together with their 1 H and C-13 chemical shifts.The presence of each metabolite was confirmed by comparing its spectroscopic data with those previously described in the literature.NMR spectra analysis of the extract of B. hamifera in D 2 O indicated that this extract contains a complex mixture of metabolites, among which floridoside (Ye et al., 2013) appeared as the major metabolite present.The HSQC correlations of signals at d C 103.0; 98.5; 96.4; 95.9 and 92.1 ppm, with protons at d H 4.44 (d, 7.6); 4.90 (brs); 4.60 (d, 7.4); 4.66 (d, 7.6) and 5.25 (d, 3.8) ppm respectively, indicate the presence of other saccharides in minor amounts.Other metabolites detected were: isethionic acid (Barrow et al., 1993), acetic acid (Sciubba et al., 2020), formic and lactic acid (Ponder and Richards, 1993) and the amino acids alanine (Sciubba et al., 2020), valine (Ryu  (Salvador et al., 2022) and N,Ndimethyltaurine (Blunden et al., 1986).The 1D and 2D NMR spectra analysis of G. rugosa extract in D 2 O showed that this extract also contained a complex mixture of metabolites, including N,N,N-trimethyl taurine (King et al., 1982), N,N-dimethyltaurine, N-monomethyltaurine (McCusker and Klinman, 2009) and taurine (Ye et al., 2013), as well as isethionic acid and floridoside.The NMR data of N-monomethyltaurine and taurine are consistent with those of standard commercial products.Formic acid was also detected.
NMR data for D. vermicularis extract show the presence of a-Dfructofuranose 1,2':2,3'b-D-dianhydride (DFAIII) as the major compound.DFA III has been isolated as a by-product of inulin, obtained from acid hydrolysis, pyrolysis or an enzymatic reaction of inulin (Uchiyama, 1982;Blize et al., 1994), but has also been described as a natural product isolated from Lycoris radiata (Li et al., 1997).Although inulin has been found in the green alga Ulva lactuca (Aguilera Morales et al., 2018) and inulin-type oligosaccharides have been found in the green alga Acetabularia crenulata (Bourne et al., 1972), to the best of our knowledge this is the first time that this metabolite has been found in a marine organism.The 1 H NMR spectrum of D. vermicularis extract also shows low intensity signals at d H 7.96 (s) and 7.40 (s) ppm in the aromatic region, indicating the presence of sulfated coumarins such as dasycladins A and B and signals at d H 7.92 (d, 10.1) and 7.36 (d, 10.1) ppm and d H 7.32 (d, 8.9) and 7.27 (d, 8.9) ppm indicating the presence of 4-(sulfooxy)benzoic acid and 4-(sulfooxy)phenylacetic acid respectively (Hartmann et al., 2018).Another metabolite detected at low levels was isethionic acid.
The 1D and 2D NMR spectra analysis of U. clathrata extract in D 2 O indicated that 3-dimethylsulfoniopropionate (Blunden et al., 1992) is the major compound present in this extract that dissolves in D 2 O. Also, 2,3-dihydroxypropanesulfonate (Edmonds and Francesconi, 1983) can be detected in the crude extract of U. clathrata.
The extracts of C. foeniculacea, C. humilis and L. dagamae in D 2 O are very similar, mannitol (Yesil and Akgül, 2022) and citric acid (Pinto et al., 2021) were found, with mannitol being the major constituent of the two in all three extracts.Lactic acid was also detected in the C. foeniculacea extract.While the HSQC spectrum of C. humilis shows a correlation between the signal at d The 1D and 2D NMR spectra analysis of C. sinuosa extract in D 2 O indicated that 3-hydroxybutyrate (Zweifel et al., 2009)   NMR are: 2,3-dihydroxypropanesulfonate and phloroglucinol monosulfate (Glombitza and Knöss, 1992).
Betaines and sulphonium compounds are common in polar extracts of marine algae (Blunden et al., 1986;Blunden et al., 1992).In addition to the substances mentioned above, three betaines were detected in five of the nine algae according to the HMBC correlations of the N-methyl groups.The HMBC correlations of the N-Me protons at d H 3.26 (s) with both the methyl signal at d C 53.6 ppm and the methylene signal at d C 64.6 ppm indicate the presence of glycine betaine (Blunden et al., 1992) in B. hamifera extract.The HMBC correlations of the N-Me protons at d H 3.21 (s) with the methyl carbons at d C 53.5 ppm and the methylene signal at d C 78.0 ppm point to another betaine, probably glycine betaine aldehyde (Blunden et al., 1992)

Several seaweed extracts improve tomato tolerance to water deficit
In order to test the biostimulant activity of the nine algal extracts, tomato seedlings were subjected to water deficit conditions (WD, 50% field capacity) after treating their root systems with the different extracts (see Table 3).Seedling dry weight of WD treatment was reduced by 11.2% after 7 days (Figure 2).
Seedlings exposed to WD also showed significant differences in water use efficiency (WUEp), compared to the well-watered (WW) plants, which increased by 66.3% (Table 3).However, the seedling dry weight of B. hamifera, D. vermicularis, C. humilis, and G. rugosa extract treatments was significantly higher compared to WD, comparable to the commercial extract Sealgae ™ used as a positive control (Figure 2).Interestingly, treatment with B. hamifera, D. vermicularis, C. humilis, and G. rugosa extracts caused a significant increase in WUE compared to mock-treated WD plants (Table 3).Furthermore, drought stress indexes, RGR and WUE were also significantly higher for the above treatments (Table 3).

Galaxaura rugosa extract improves tomato plant tolerance of water deficit
After the bioprospection results, G. rugosa extract performs best under WD conditions.For this reason, deeper analyses were carried out under WW or WD conditions (Figure 3A).Seedlings treated with the extract show a small but non-significant reduction in plant weight under well-watered conditions, while the protective effect is clearly visible under deficit conditions (Figure 3A).It is noteworthy that extracts collected in different seasons can replicate this protective behavior (Figure 3B).Growth and tolerance index clearly show the tolerance enhancement by root treatment with G. rugosa (Table 4).Under a low watering regime, treated plants increased in RGR, WUEp, and STI by 31, 53 and 40%, respectively, compared to untreated seedlings, showing an 83% decrease in sensitivity as SSI indicated.
Mock-treated plants exposed to WD continuously decreased stomatal conductance, transpiration, and net photosynthesis (Figures 4A-C).Application of the G. rugosa extract led to the recovery of photosynthesis at 5 days after the onset of WD, which also correlated with higher gs and E compared to mock-treated plants subjected to WD (Figures 4A-C).Applying the algal extract to well-watered plants also reduced gs, E and Pn, reaching its minimum three days after stress exposure, indicating that the extract's components likely induce stomatal closure (Figures 4A-C).We also calculated the iWUE and int WUE parameters, and the efficiency of CO 2 assimilation according to the Ci (Figure 5).At 3 and 5 days, we observed that plants subjected to WD and treated with the G. rugosa extract showed higher instantaneous and intrinsic WUE than mock-treated plants (Figures 5A, B).The algal extract also improved the Pn/Ci ratio in A-WD compared to WD plants (Figure 5C).

Galaxaura rugosa extract induces expression of ABA-responsive genes in Arabidopsis and tomato
Seaweed extracts are complex mixtures, so to understand their action mechanisms is challenging.ABA is an important phytohormone for coping with water deficit stress, therefore activation of ABA signaling leads to increased WUE in crops (Mega et al., 2019;Yang 2023).Thus, to further understand the action mechanism of the seaweed extracts reported here, we focused on a sample that showed strong biostimulant potential, i.e., G. rugosa, to establish whether it could activate ABA signaling in either Arabidopsis thaliana (arabidopsis) or tomato.We first measured the ABA concentration in the G. rugosa extract as described in the Materials and Methods section, finding it to be 0.45 ± 0.12 nM.This ABA concentration was used in subsequent mock-treated samples to maintain the background effect due to residual ABA in that extract.
To test the effect on ABA signaling, we used an arabidopsis transgenic line in which the ABA-responsive MAPKKK18 promoter was fused to the LUC reporter (Vaidya et al., 2017).We incubated arabidopsis seedlings treated with mock, ABA or seaweed extract for 24 h in a solution containing 100 mM luciferin (Figure 6A).As a result, we observed induction of the LUC reporter by treatment with a 0.5% G. rugosa extract (Figure 6A).To test the effect of the extract in wild-type tomato seedlings, we treated tomato seedlings with a 0.5% G. rugosa extract for 6 h and assessed the expression of two ABA-responsive genes, namely Sl02g084850 (SlRAB18) and Sl06g067980 (SlLEA).As a result, we found that G. rugosa extract induced ABA-responsive genes in both arabidopsis and tomato  Dry weights of tomato plants treated with different algal extracts under water-deficit stress conditions.Blue and red dashed lines represent WW and WD dry weight average respectively.+ and * means significant differences at p< 0.05 with respect to WW and WD, respectively.
(Figure 6B), which may contribute to the protective effect seen in the water deficit experiment.

Discussion
In recent years, the need to look for environmentally friendly alternatives to protect our crops has become apparent.In the EU at least, measures have been promoted to achieve this goal, recognizing that in order to feed a growing world population and ensure food security we need to optimize yields.One of the most obvious and widespread consequences of climate change and global warming is drought, whose impact on crop yields threatens our future (Summary for Policymakers, 2014).It is therefore important to look for sustainable alternatives to save water in agriculture.One possible solution is the use of biostimulants to increase plant tolerance to water deficit conditions.
From our bioprospection in WD conditions, plants-treated with aqueous extracts of four seaweeds, B. hamifera, D. vermicularis, C. humilis and G. rugosa at 0.1 g/L showed significantly higher dry weights compared to mock-treated or plants-treated with the rest of the extracts (Figure 2).Given these results, it seems obvious that not all extracts are able to increase plant tolerance under the water deficit conditions to which they were exposed.Therefore, the chemical composition of these extracts is crucial in determining how well they confer tolerance in the conditions tested (Ertani et al., 2018).In this context, there are seaweed-based or organic biostimulant treatments in the literature that show different levels of effectiveness under water deficit or drought conditions, even different extracts from the same seaweed led to different activity (Goñi et al., 2016).For example, in Betula papyrifera (paper birch) seedlings exposed to drought, treatment with an organic biostimulant did not improve tolerance to drought stress (Richardson et al., 2004).However, improved tolerance was observed in grapes treated with Ascophyllum extracts and exposed to drought.This latter case was attributable to the beneficial effect of these extracts on plant osmotic status and the effect of the betaines and oligosaccharides in their composition (Norrie et al., 2002).
Chemical study of our extracts showed that some substances are common to several species, such as mannitol and isethionic acid.Mannitol is the main constituent in the extracts from L. dagamae, C. foeniculacea and C. humilis.On correlating their mannitol content with extract activity, we conclude it is not be responsible for the activity of C. humilis extract, since the other two are not active.Similarly, isethionic acid is the main compound in the inactive H. scoparia extract that is also found in B. hamifera, G. rugosa and D. vermicularis extracts, which did improve drought tolerance activity.
We consider that C. humilis is active because of a minor metabolite or the synergistic effect of more than one substance.For additional research progress, it would be necessary to carry out a bioguided fractionation of D. vermicularis, to see if DFAIII or one of the sulfated coumarins could be responsible for its activity.
The two red algae studied, B. hamifera and G. rugosa, share some common products such as N,N-dimethyl taurine and floridoside, which latter is a characteristic component of several red algae species (Ekman et al., 1991).In addition to such compounds, analysis of the betaines detected suggests that glycine betaine could be responsible for the activity of B. hamifera, since this substance was not detected in any other extract.However, glycine betaine aldehyde and the unidentified betaine were detected not only in the active extract of G. rugosa, but also in non-active extracts of U. clathrata and H. scoparia, so it does not appear that these substances alone are responsible for the activity of the G. rugosa extract.We plan to purify the crude extracts following a bioguided fractionation process to examine individual metabolites and determine which are responsible for the observed biological activity, either individually or in combination.
Under our conditions, the G. rugosa extract showed the highest osmoprotectant activity under WD stress (Figure 2).Interestingly, we also observed that well-watered plants treated with the G. rugosa extract showed reduced gs and E compared to mock-treated plants (Figure 2).This suggests that the extract induces stomatal closure, which might indicate priming of ABA signaling after the application of the seaweed extract.Although closure of stomata limits the CO 2 uptake necessary for photosynthesis, one of the first stress responses of plants under drought conditions is indeed to close the stomata to reduce water loss and maintain cell turgor (Mega et al., 2019;Yang et al., 2019;Mao et al., 2022).Recently, the ABA receptor agonist AMF4 was reported to improve CO 2 assimilation and WUE in plants subjected to WD stress (Jimeńez-Arias et al., 2023).Interestingly, the seaweed extract behaved analogously to AMF4 in plants subjected to WD stress (Figure 3, 4).Thus, comparing the gas-exchange values of G. rugosa-treated and mock-treated tomato seedlings under WW or WD stress conditions reveals a clear effect of the seaweed extract on the gs and E values, and photosynthesis protection after 5 days of WD.Whereas the gs and E of mock-treated plants were significantly reduced under WD conditions, leading to a dramatic drop in photosynthesis, the G. rugosa treatment significantly improved CO 2 assimilation, relative growth rate and all tolerance and susceptibility indexes compared to WD control plants (Figures 4,  5; Table 4).These results show that the seaweed extract appears to partially mimic ABA or ABA receptor agonists' effect to modulate gs to avoid water loss (Figure 4A).
Our results with the G. rugosa extract are consistent with previous work using the seaweed Ascophyllum nodosum in arabidopsis under drought stress conditions (Santaniello et al., 2017;Rasul et al., 2021).Santaniello et al. (2017) found that plants treated for 5 days with A. nodosum seaweed extract showed a partial stomatal closure and reduced gs.The authors suggested that this effect together with the preactivation of ABAresponsive genes and antioxidant system pathways were responsible for plant tolerance to drought stress (Santaniello et al., 2017).
Likewise, priming with the biostimulant super fifty (also produced from an A. nodosum seaweed extract) improved drought tolerance in arabidopsis through the maintenance of higher relative water content and expression of ABA-dependent genes (Rasul et al., 2021).Therefore, we also investigated the possible induction of ABA-responsive genes in an arabidopsis LUC reporter line or tomato seedlings.The residual ABA concentration of the extract (below 0.5 nM) was used as mock-treatment as explained.Still, it was not enough to significantly activate the pMAPKKK18-LUC reporter, in contrast to the seaweed treatment (Figure 6A).Two tomato ABA-responsive genes were also induced in response to treatment with the G. rugosa extract.Thus, activating the ABA pathway probably contributes to the protective effect of the G. rugosa extract in plants subjected to water deficit conditions (Figure 6B).These results do not exclude protection effects contributed by other mechanisms, for example by compatible solutes (betaines, amino acids, etc.) that favor osmoadjustment processes.However, future studies should address the mechanism whereby certain seaweed extracts induce activation of the ABA pathway (Santaniello et al., 2017;Rasul et al., 2021;this work).Priming of the ABA response by small molecules such as ßaminobutyric acid involves ABA accumulation, resulting in stomatal closure and activation of the antioxidant defense enzymes (Du et al., 2012).We speculate that some components of the extract might affect ABA biosynthesis or signaling, acting as priming agents.It is also possible that the sensitivity of the stomata to low changes in ABA concentration is increased after seaweed treatment (Santaniello et al., 2017).
The results of this study demonstrate the potential of some of the algae studied as a source of extracts and/or products that biostimulate drought tolerance in tomato plants.This advance prompts us to continue with a more detailed study of the active algae, to determine which substances exert this biostimulatory effect.Synergistic effects aside, the individual activity of the substances identified should be much greater than that of the crude extract and they are likely to have industrial applications.

FIGURE 1
FIGURE 1 H 4.92 (d, 3.9) ppm and the signal at d C 98.50, indicating the presence of saccharides, and isethionic acid was again detected.The L. dagamae extract also shows signals at d C 102.7: 102.4 and 98.1 ppm, which correlate in the HSQC spectrum with protons at d H 4.92: 4.80 and 4.53 ppm respectively, indicating the presence of other saccharides in minor amounts.

FIGURE 3
FIGURE 3 Dry weights of tomato plants treated with G. rugosa extract under well-watered and water-deficit stress conditions.(A) Dry weights of tomato plants treated with different G. rugosa extracts under water-deficit conditions.(B) Blue and red dashed lines represent WW and WD dry weight average respectively.+ and * means significant differences at p< 0.05 with respect to WW and WD respectively.1-3 means G. rugosa extracts from different seasons.

FIGURE 2
FIGURE 2 FIGURE 4 Plant gas exchange measurements and CO 2 assimilation during the experiment.(A) Transpiration (E).(B) Stomatal conductance (gs).(C) Net photosynthesis (Pn).Bars labeled with letters indicate significant differences at p<0.05.Bars labeled by the same letter did not show significant differences at p<0.05.
FIGURE 5 Enhanced WUE and CO 2 assimilation ratio by G. rugosa treatment under WD conditions.(A) Instantaneous water use efficiency, Pn/E.(B) Intrinsic water use efficiency, Pn/gs.(C) Pn/Ci index.Bars labeled with the same letter did not show significant differences at p<0.05.Ratios and standard deviations were obtained from values reported in Figure 3.

TABLE 1
Details of species, biomass, extracts and voucher specimen codes.

TABLE 2
Low-Molecular-weight metabolites detected in the algal extracts and NMR Data in D 2 O [ 1 H 500 MHz, ppm, 13 C 125 MHz D 2 O].
is the major compound present in this extract, which dissolves in D 2 O. Another minor metabolite detected by NMR is formic acid.By contrast, 1D and 2D NMR spectra analysis of H. scoparia extract in D 2 O indicated that isethionic acid is the main compound in this extract dissolving in D 2 O.Other metabolites detected by

TABLE 2 Continued
in G. rugosa, U. clathrata and H. scoparia extracts.Finally, HMBC correlations of the N-Me protons at d H 3.29 (s) with methyl carbons at d C 54.3 ppm and with the methylene signal at d C 67.0 ppm suggest the presence of another unidentified betaine in B. hamifera, U. clathrata, L. dagamae and H. scoparia extracts.

TABLE 3
Relative growth rate and water use efficiency index studied in the different treatments under water deficit conditions.

TABLE 4
Relative growth rate and stress indexes attained with the Galaxaura rugosa treatment under water deficit conditions.