Among seaweeds, the brown seaweed Ascophyllum nodosum is the most popular source used in agriculture. There are several seaweed-based liquid fertilizer formulations on the market. Preparing their extracts requires using water, acid or alkaline treatments, low temperatures, or heating, or disruption using physical methods.
Salinity and drought are the most important abiotic stresses. Together, they are responsible for up to a 75% reduction in global crop production. Moreover, their impact will increase due to climate change. 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.
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. Among all phytohormones, ABA has a crucial role in coping with drought stress.
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.
Seaweeds
The specimens included two species from the phylum Rhodophyta (red algae), namely Bonnemaisonia hamifera Hariot and Galaxaura rugosa (J. Ellis & Solander); two species from the phylum Chlorophyta (green algae), namely Dasycladus vermicularis (Scopoli) Krasser and Ulva clathrata (Roth) C.Agardh; and five species from the phylum Ochrophyta (brown algae), namely Cystoseira foeniculacea (Linnaeus) Greville, Cystoseira humilis Schousboe ex Kützing, Lobophora dagamae C.W.Vieira, Colpomenia sinuosa (Mertens ex Roth) Derbès & Solier, and Halopteris scoparia (Linnaeus) Sauvageau.
Chemical analysis of crude extracts
The main metabolites detected in each extract are listed in Table 1, together with their 1H and C-13 chemical shifts.
| Metabolite | δH (J in Hz) | δC | Species |
|---|---|---|---|
| Alanine | 3.82 (m), 1.50 (d, 7.4) | 50.7, 16.1, 175.7 | B. hamifera |
| Valine | 3.63 (m), 2.33 (m), 1.00 (d, 7.8), 1.06 (d, 7.8) | 66.2, 28.9, 16.6, 17.9, 181.5 | B. hamifera |
| Threonine | 3.60 (m), 4.27 (m), 1.33(d) | 60.5, 66.1, 20.3 | B. hamifera |
| Glycine betaine | 3.92 (s), 3.23(s) | 67.4, 53.7 169.3 | B. hamifera |
| Glycine betaine aldehyde | 4.01 (m), 3.23 (s) | 77.0, 53.2, 175.7 | G. rugosa U. clathrata H. scoparia |
| Other betaine | 3.29 (s) | 66.5, 53.3 | B. hamifera L. damagae U. clathrata H. scoparia |
| N,N,N-trimethyl taurine | 3.50 (m), 3.75 (m), 3.23 (s) | 44.8, 61.5, 53.2 | G. rugosa |
| N,N-dimethyltaurine | 3.38 (m), 3.43 (m), 2.86 (s) | 45.4, 53.4, 43.3 | B. hamifera G. rugosa |
| N-monomethyltaurine | 3.30 (m), 3.48 (m), 2.78 (s) | 48.9, 44.3, 33.4 | G. rugosa |
| Taurine | 3.30(t), 3.45(t) | 46.9, 35.7 | G. rugosa |
| Isethionic acid | 3.18 (t), 3.97 (t) | 52.8, 57.0 | B. hamifera G. rugosa H. scoparia C. humilis D. vermicularis |
| Acetic acid | 1.94 (s) | 23.1, 181.4 | B. hamifera |
| Formic acid | 8.47(s) | B. hamifera G. rugosa C. sinuosa | |
| Lactic acid | 4.15 (m), 1.32 (d, 6.8) | 74.2, 22.9, 182.2 | B. hamifera C. foeniculacea |
| Citric acid | 2.54(d), 2.67(d) | 48.5, 78.1, 182.1, 184.7 | C. foeniculacea C. humilis L. dagamae |
| 3-dimethylsulfoniopropionicate | 2.74 (dd, 7.1, 7.1), 3.46 (dd, 7.1, 7.1), 2.92 (s) | 39.2, 29.1, 177.0, 25.8 | U. clathrata |
| 3-hydroxybutyrate | 2.36 (dd, 6.4, 14.6); 2.46 (dd, 7.7, 14.6), 4.21 (m), 1.26 (d, 6.4) | 46.6, 65.8, 21.9, 180.6 | C. sinuosa |
| 2,3-dihydroxypropanesulfonate | 3.05 (dd, 8.0, 14.5), 3.12 (dd, 3.5, 14.5), 4.18 m, 3.60 (dd, 6.5, 11.3), 3.70 (dd, 4.6, 11.3) | 53.7, 68.1, 64.7 | H. scoparia U. clathrata |
| Floridoside | 5.17(d, 3.7), 3.85(m), 3.93 (m), 4.02 (m), 4.13 (m), 3.77 (m), 3.85(m) | 98.1, 68.5, 69.3, 69.2, 71.8, 61.2, 61.4, 78.7, 61.4 | B. hamifera G. rugosa |
| Other saccharides | 4.44 (d, 7.6); 4.90 (brs); 4.60 (d, 7.4); 4.66 (d, 7.6); 5.25 (d, 3.8) | 103.0; 98.5; 96.4; 95.9; 92.1 | B. hamifera |
| α-D-fructofuranose 1,2’:2,3’β-D- dianhydride | 4.06 (d, 13.6), 3.77 m, 4.17 (d, 4.7), 3.90 m, 4.12 (ddd, 2.9, 6.2, 6.2), 3.83 (dd, 3.3, 12.8), 3.66 m, 3.71 m; 3.66 m, 4.34 (d, 7.1), 4.68 (dd, 7.5; 7.5), 3.72 m, 3.90m; 3.77 m | 60.0, 104.6, 82.2, 76.4, 82.9, 62.1, 64.4, 102.4, 80.0, 73.3, 81.2, 61.6 | D. vermiculata |
| Mannitol | 3.85 (dd, 1.9, 11.8), 3.66 (dd, 6.1, 11.8), 3.75 (m); 3.79 (m) | 63.2, 70.8, 69.2 | C. foeniculacea C. humilis L. dagamae |
| Phloroglucinol monosulfate | 6.34 (s), 6.25 (s) | 152.7, 100.7, 158.2, 100.9 | H. scoparia |
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 D2O indicated that this extract contains a complex mixture of metabolites, among which floridoside appeared as the major metabolite present. The HSQC correlations of signals at δC 103.0; 98.5; 96.4; 95.9 and 92.1 ppm, with protons at δH 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, acetic acid, formic and lactic acid and the amino acids alanine, valine, threonine and N,N-dimethyltaurine.
The 1D and 2D NMR spectra analysis of G. rugosa extract in D2O showed that this extract also contained a complex mixture of metabolites, including N,N,N-trimethyl taurine, N,N-dimethyltaurine, N-monomethyltaurine and taurine, 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 α-D-fructofuranose 1,2′:2,3’β-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, but has also been described as a natural product isolated from Lycoris radiata. Although inulin has been found in the green alga Ulva lactuca and inulin-type oligosaccharides have been found in the green alga Acetabularia crenulata, to the best of our knowledge this is the first time that this metabolite has been found in a marine organism.
The 1D and 2D NMR spectra analysis of U. clathrata extract in D2O indicated that 3-dimethylsulfoniopropionate is the major compound present in this extract that dissolves in D2O. Also, 2,3-dihydroxypropanesulfonate can be detected in the crude extract of U. clathrata.
The extracts of C. foeniculacea, C. humilis and L. dagamae in D2O are very similar, mannitol and citric acid 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 δH 4.92 (d, 3.9) ppm and the signal at δC 98.50, indicating the presence of saccharides, and isethionic acid was again detected.
The 1D and 2D NMR spectra analysis of C. sinuosa extract in D2O indicated that 3-hydroxybutyrate is the major compound present in this extract, which dissolves in D2O. Another minor metabolite detected by NMR is formic acid.
By contrast, 1D and 2D NMR spectra analysis of H. scoparia extract in D2O indicated that isethionic acid is the main compound in this extract dissolving in D2O. Other metabolites detected by NMR are: 2,3-dihydroxypropanesulfonate and phloroglucinol monosulfate.
Betaines and sulphonium compounds are common in polar extracts of marine algae. 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 δH 3.26 (s) with both the methyl signal at δC 53.6 ppm and the methylene signal at δC 64.6 ppm indicate the presence of glycine betaine in B. hamifera extract.
The HMBC correlations of the N-Me protons at δH 3.21 (s) with the methyl carbons at δC 53.5 ppm and the methylene signal at δC 78.0 ppm point to another betaine, probably glycine betaine aldehyde in G. rugosa, U. clathrata and H. scoparia extracts. Finally, HMBC correlations of the N-Me protons at δH 3.29 (s) with methyl carbons at δC 54.3 ppm and with the methylene signal at δC 67.0 ppm suggest the presence of another unidentified betaine in B. hamifera, U. clathrata, L. dagamae and H. scoparia extracts.
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 2). Seedling dry weight of WD treatment was reduced by 11.2% after 7 days (Figure 1).
| Treatment | RGR | WUEp |
|---|---|---|
| WW | 0.16 | 2.8 |
| WD | 0.14 | 4.7 |
| Bonnemaisonia hamifera | 0.16 | 5.6 * |
| Cystoseira foeniculacea | 0.15 | 5.3 |
| Dasycladus vermicularis | 0.15 | 5.4 * |
| Cystoseira humilis | 0.16 | 5.5 * |
| Galaxaura rugosa | 0.16 | 5.8 * |
| Lobophora dagamae | 0.15 | 4.9 |
| Colpomenia sinuosa | 0.14 | 4.8 |
| Ulva clathrata | 0.14 | 4.8 |
| Halopteris scoparia | 0.15 | 5.2 |
| Kelpak™ | 0.15 | 4.9 |
| Sealgae™ | 0.16 | 5.8* |
WW represents well-watered conditions (100% field capacity) and WD water deficit conditions (50% field capacity) as indicated in Materials and Methods.
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 2). 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 used as a positive control (Figure 1).
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 2). Furthermore, drought stress indexes, RGR and WUE were also significantly higher for the above treatments (Table 2).
Seaweed 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 2A). 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 2A).
It is noteworthy that extracts collected in different seasons can replicate this protective behavior (Figure 2B). Growth and tolerance index clearly show the tolerance enhancement by root treatment with G. rugosa (Table 3). 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.
| Treatment | RGR | WUEp | SSI | STI |
|---|---|---|---|---|
| WW | 0.18 | 3.1 | ||
| WD | 0.12 | 3.4 | 1.7 | 0.7 |
| A-WW | 0.17 | 2.9 | ||
| A-WD | 0.16 | 5.2 | 0.3 | 1.0 |
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 treatment, respectively.
Mock-treated plants exposed to WD continuously decreased stomatal conductance, transpiration, and net photosynthesis (Figures 3A-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 3A-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 3A-C). We also calculated the iWUE and int WUE parameters, and the efficiency of CO2 assimilation according to the Ci (Figure 4). 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 4A, B). The algal extract also improved the Pn/Ci ratio in A-WD compared to WD plants (Figure 4C).
Seaweed 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.
Recently, an ABA receptor agonist capable of activating ABA signaling in tomato was reported to have biostimulant activity against water deficit stress. 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.
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. We incubated arabidopsis seedlings treated with mock, ABA or seaweed extract for 24 h in a solution containing 100 μM luciferin (Figure 5A).
As a result, we observed induction of the LUC reporter by treatment with a 0.5% G. rugosa extract (Figure 5A). 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. As a result, G. rugosa extract induced ABA-responsive genes in both arabidopsis and tomato (Figure 5B), 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. 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.
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. 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.
For example, in Betula papyrifera (paper birch) seedlings exposed to drought, treatment with an organic biostimulant did not improve tolerance to drought stress. 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.
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. 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. Interestingly, well-watered plants treated with the G. rugosa extract showed reduced gs and E compared to mock-treated plants. 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 CO2 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. Recently, the ABA receptor agonist AMF4 was reported to improve CO2 assimilation and WUE in plants subjected to WD stress.
Interestingly, the seaweed extract behaved analogously to AMF4 in plants subjected to WD stress. 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 CO2 assimilation, relative growth rate and all tolerance and susceptibility indexes compared to WD control plants. These results show that the seaweed extract appears to partially mimic ABA or ABA receptor agonists’ effect to modulate gs to avoid water loss.
G.rugosa extract are consistent with previous work using the seaweed Ascophyllum nodosum in arabidopsis under drought stress conditions. 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 ABA-responsive genes and antioxidant system pathways were responsible for plant tolerance to drought stress.
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. 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. 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.
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. Priming of the ABA response by small molecules such as ß-aminobutyric acid involves ABA accumulation, resulting in stomatal closure and activation of the antioxidant defense enzymes.
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.
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.
The potential of several seaweed extracts in enhancing crop drought tolerance, with mechanisms involving stomatal regulation and activation of the ABA signaling pathway. Building on this foundation, Dora agri has developed a range of seaweed-based biostimulant products, available in both liquid and powder formulations, with some products carrying OMRI organic certification, offering practical solutions for sustainable agriculture. Learn more details from Dora team.