Currently, biostimulants in the horticultural sector are a tool that is being used to improve the yield and quality of vegetables under optimal and stressful growth conditions. In the present study, we evaluate the effects of foliar application of a hydroethanolic extract of Sargassum spp., a commercial extract based on Ascophyllum nodosum, and a control with distilled water on growth and biomass, stomatal conductance, photosynthetic pigments, enzymatic and non-enzymatic antioxidants, protein content, and the expression of defense genes in tomato plants (Solanum lycopersicum L.) without stress and with high-temperature stress (45 ℃). The results indicate that extracts of Sargassum spp. and Ascophyllum nodosum are effective in mitigating high-temperature stress, making their use a promising alternative for inducing resistance in plants to the daily adversities of climate change.
Introduction
Among abiotic factors, temperature is one of the most important factors that significantly affects the physiological, morphological, and biochemical processes of plants. Temperature stresses are normally classified into low- and high-temperature stress, which are below and above the optimal temperature range of plants.
High temperatures increase metabolic activity (high speed of vibration and translation of molecules). In addition to this, they cause a very high vapor pressure deficit in plants, which causes high transpiration, which can sometimes exceed the absorption of water by the roots, which results in stomatal closure, low photosynthetic activity, decreased growth, and burning of leaves, stems and fruits, causing low yields and poor quality of fruits. At the molecular level, high temperatures compromise the functionality of biomolecules by reducing their catalytic capacity, or in the worst case, they can inactivate or denature them when the tolerance threshold is exceeded. For terrestrial plants, the threshold where high temperatures begin to negatively affect biomolecules and the interaction processes between them is between 35 ℃ for those with C3 metabolism and 40 ℃ for those with C4 metabolism.
When plants are subjected to stress due to high temperatures, they activate the antioxidant system (enzymatic and non-enzymatic) to eliminate or neutralize the reactive oxygen species (ROS) produced, but sometimes the stress is greater and requires other measures to overcome it. These situations have led to the search for alternatives to mitigate this type of stress, and among the most promising is the use of plant biostimulants. A biostimulant is any substance or microorganism applied to plants with the objective of improving nutritional efficiency, stress tolerance, and quality traits of crops, regardless of their nutrient content. Among the most used biostimulants today are algae extracts, which represent a promising option due to the large amount of biomolecules they contain. The most used extracts are those of brown algae (Ascophyllum nodosum, Ecklonia maxima, Fucus spp., Lessonia nigrescens, Macrocystis pyrifera, Laminaria spp., and Sargassum spp.) due to the high concentration of metabolites they contain such as carbohydrates, proteins, amino acids, phytohormones, carotenoids, vitamins, phenols, and inorganic compounds. These metabolites have the ability to stimulate natural processes that improve nutrient absorption and assimilation, provide stress tolerance, and improve plant growth and yield.
The Sargassum spp. algae is currently causing different problems. The Sargassum spp. seaweed extracts have been used in horticultural plants to improve yield, quality, and give plants tolerance to different types of stress; however, mitigating stress due to high temperatures has not yet been studied. However, there are studies that show that its application can increase the synthesis of total phenolic compounds, flavonoids, carotenoids, and glutathione, compounds that can reduce ROS produced during stress. This makes them a viable option to combat high-temperature stress in horticultural plants. In addition to this, there are some studies with other genera of brown algae that address this problem. In a study on tomato plants (Solanum lycopersicum L.), extracts of Ascophyllum nodosum were shown to mitigate high-temperature stress by improving pollen viability, fruit yield, and the expression of genes encoding heat shock proteins (HSP). Ascophyllum nodosum is among the most widely used algae in agriculture, as it has been shown to induce plants to produce their own hormones, which contributes to improving growth, mitigating stress, and improving the absorption and translocation of nutrients.
The mechanism of action of brown algae, which induces resistance to abiotic stress in plants, can have significant effects, such as reducing agrochemical use and lessening environmental impacts, in addition to addressing the problems of atypical Sargassum upwelling along coasts. In line with the above, the objective of the present investigation was to evaluate the effect of an extract of Sargassum spp. and Ascophyllum nodosum on the induction of tolerance to high-temperature stress in tomato plants. The hypothesis was proposed that seaweed extracts would improve morphological, physiological, biochemical, and transcriptional aspects that would give tomato plants greater tolerance to high-temperature stress. The use of biostimulants derived from natural resources such as brown algae is promising, as they can help address the current challenges facing vegetable production.
Results
Plant Growth and Biomass
Table 1 shows the results of the height, stem diameter, and number of leaves of the tomato plants. For the height of the plants at 11 days after transplant (DAT), there were no differences. At 21 and 31 DAT, there were only differences in the non-stress treatments, where Sargassum spp. seaweed extract (SSE) plants exceeded control (AC) plants by 4.90% and 2.71%, respectively (p ≤ 0.05). For stem diameter and number of leaves, there were no differences for any of the two treatment groups (without and with stress) in any evaluation.
Figure 1 shows the results of the dry biomass of the plants. Regarding the aerial dry biomass in the treatments without stress, the SSE exceeded the AC by 9.56%, and in the treatments with stress, the SSE + 45 ℃ exceeded the AC + 45 ℃ by 6.66% (p ≤ 0.05). For the dry root biomass, there was no difference for any of the two treatments groups. For the total dry biomass, there were only differences for the non-stress treatments, where the SSE exceeded the AC by 8.58% (p ≤ 0.05). Figure 2 shows images of the tomato plants used in the experiment.
Stomatal Conductance
Regarding stomatal conductance, a significant decrease was observed in the stress treatments (p ≤ 0.05) (Figure 3), which is normal, since it is part of the plant’s defense mechanism. In non-stress treatments, a very marked difference was observed between the application of the extracts (Ascophyllum nodosum commercial product (ANCP) and SSE) and the AC, where the latter presented the lowest values in the three evaluations. In this case, stress was applied only once, between 20 and 21 DAT, at which time the stressed treatments were equal, and a difference was found at 31 DAT, where ANCP + 45 ℃ and SSE + 45 ℃ were higher than AC + 45 ℃ by 58.34% and 63.57%, respectively (p ≤ 0.05).
0.05). DAT: days after transplant; SC: stomatal conductance; AC: control; ANCP: Ascophyllum nodosum commercial product; SSE: Sargassum spp. seaweed extract; Values are presented as means ± SD, n = 4.
Photosynthetic Pigments
Figure 4 shows the results of photosynthetic pigments, where significant differences between treatments are observed. For chlorophyll a, in the non-stress treatments, SSE increased the concentration by 33.76% (11 DAT), 26.66% (21 DAT), and 13.22% (31 DAT), compared to the AC (p ≤ 0.05). In the stress treatments there were only differences at 21 and 31 DAT, where the SSE + 45 ℃ group concentration exceeded that of the AC + 45 ℃ group by 22.05% and 12.38%, respectively (p ≤ 0.05). At 21 DAT the ANCP + 45 ℃ group concentration was also higher than that of the AC + 45 ℃ group (p ≤ 0.05).
Regarding chlorophyll b, there were only differences in the non-stress treatments, where the SSE group concentration was greater than the AC at 11 and 21 DAT by 12.40% and 11.96%, respectively (p ≤ 0.05).
For total chlorophyll, in the non-stress treatments, SSE increased the concentration by 23.71% (11 DAT), 19.84% (21 DAT), and 10.58% (31 DAT), compared to AC (p ≤ 0.05). In the stress treatments, SSE + 45 ℃ increased the concentration by 13.33% (21 DAT) and 7.45% (31 DAT), compared to AC + 45 ℃ (p ≤ 0.05).
Enzymatic Activity
The experiment also quantified the activity of ROS-scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) (Figure 5). It is noticeable that the three enzymatic activities increase with stress and with the application of algae extracts. In this sense, the effects of applying SSE without stress was statistically higher than AC, and likewise the effect of SSE + 45 ℃ was higher than AC + 45 ℃ (p ≤ 0.05), for SOD, CAT, and APX in the three samplings (11, 21, and 31 DAT), which tells us about the biostimulant effect of SSE under standard growth conditions and low stress due to high temperatures. The ANCP had a similar effect to the SSE for CAT, where in conditions without stress and with stress, it was higher than AC and AC + 45 ℃, respectively. ANCP application under stressful conditions caused a significant increase (p ≤ 0.05) in SOD at 11 and 21 DAT compared to AC + 45 ℃. However, for APX, ANCP without stress was equal to AC and ANCP + 45 ℃ was equal to AC + 45 ℃.
≤ 0.05). DAT: days after transplant; SOD: superoxide dismutase; CAT: catalase; APX: ascorbate peroxidase; AC: control; ANCP: Ascophyllum nodosum commercial product; SSE: Sargassum spp. seaweed extract; Values are presented as means ± SD, n = 4.
Total Proteins
In the treatments without stress, the effects of SSE in the three samplings was superior to the AC, and the effects of the ANCP only surpassed the AC at 21 and 31 DAT (p ≤ 0.05) (Figure 6). With respect to the treatments with stress, only at 31 DAT were there differences, with ANCP and SSE being superior to AC (p ≤ 0.05) (Figure 6).
Non-Enzymatic Antioxidants and Antioxidant Capacity
Table 2 shows the results for non-enzymatic antioxidants and antioxidant capacity in the leaves of tomato plants. For the content of total phenols, there were only differences at 21 and 31 DAT in the stressed treatments, where the SSE + 45 ℃ exceeded the AC + 45 ℃ by 18.17% and 19.33%, respectively (p ≤ 0.05).
For ascorbic acid, in the three samplings, ANCP and SSE were superior to AC in the treatments without stress, and likewise in the treatments with stress, ANCP + 45 ℃ and SSE + 45 ℃ were superior to AC + 45 ℃ (p ≤ 0.05).
For antioxidant capacity, in the treatments without stress, there were only differences at 11 DAT, with ANCP and SSE being superior to AC (p ≤ 0.05). For the treatments with stress, ANCP and SSE were superior to AC in all three samplings (p ≤ 0.05).
Expression of Defense Genes
Given the greater capacity of seaweed extracts to neutralize ROS by stimulating the enzymatic and non-enzymatic antioxidant systems, the transcript levels of nine stress-related genes (NCED1, HSP70, PIP2, P5CS1, ERD15, Fe-SOD, CAT1, cAPX2 and PAL5-3) were examined (Figure 7).
It can be seen that in the first sampling at 11 DAT, where the plants were not yet subjected to stress, gene expression was somewhat dispersed among all treatments; however, at 21 and 31 DAT, when the plants had already been subjected to stress, gene expression spiked in the treatments with application of extracts and stress by high temperatures.
At 21 DAT, which was after stress and the third application of the extracts, the ANCP and SSE treatments under stressful conditions overexpressed most of the studied genes, with HSP70, ERD15, and Fe-SOD being the most notable. At 31 DAT, the use of the extracts under stressful conditions also increased the expression of the genes evaluated, with NCED1, HSP70, P5CS1, and ERD15 being the most prominent. Overall, SSE under stress conditions induced increased expression of all genes at 21 and 31 DAT.
Furthermore, the application of the algal extracts under stress-free conditions, mainly at 21 and 31 DAT, repressed the expression of the genes PIP2, P5CS1, Fe-SOD, CAT1, cAPX2, and PAL5-3.
These transcriptional findings support the data observed through the physiological and biochemical analyses in the study and argue for a strong involvement of brown seaweed extracts in the activation of the defense system of tomato plants subjected to stress due to high temperatures.
Discussion
Currently, the use of biostimulants in agricultural crops is a recurrent practice due to the multiple benefits they provide to plants and the environment. Brown algae extracts, thanks to the large amounts of biomolecules they contain, are among the most promising biostimulants.
These positive effects on plant growth, biomass, and stomatal conductance induced by brown algae extracts are mainly due to their contents of carbohydrates, proline, and glycine-betaine, which are osmolytes that help plants retain water under stress conditions, improving water regulation, photosynthesis and the development of foliar, floral, and root meristems. In addition, brown algae extracts can stimulate carbon and nitrogen metabolism, which can improve plant growth and biomass.
High temperatures cause a very high vapor pressure deficit, which results in high transpiration that competes with the flow of water from other organs. When transpiration exceeds water absorption, there is burning of leaves, stems, flowers, and fruits, as well as root damage, resulting in lower agronomic development and poor quality productions. With stress from high temperatures, plants can eliminate certain parts of their organs or sometimes eliminate them completely. This may be caused by burns or as a defense response to avoid having to consume energy to maintain these organs. To do this, plants have a network of chain signals in their defense system that informs them about high-temperature stress, and in this way, levels of ethylene and abscisic acid (ABA), compounds responsible for organ senescence and abscission, are immediately elevated.
When high-temperature stress is prolonged and plant transpiration has already exceeded water absorption by the roots, plants close their stomata as a defense measure to avoid water loss as much as possible, which results in a decrease in stomatal conductance and a subsequent increase in leaf temperature. However, brown seaweed extracts can improve this condition due to the considerable concentration of osmolytes they contain.
Regarding pigments, it is observed that they increase with the application of brown algae extracts. In this sense, it is common that the application of low doses of brown algae extracts increases the concentration of pigments in plants, and this is mainly due to glycine-betaine, which acts to protect the extrinsic protein structure of the photosynthetic complex, particularly in photosystem II. In addition, glycine-betaine maintains photosynthetic activity by improving RUBISCO activity and chloroplast stability.
When high-temperature stress is prolonged, chlorophylls tend to degrade. This is due to the activation of enzymes such as chlorophyllases and peroxidases, which break down chlorophyll structures. High temperatures also increase the synthesis of ROS, which affect chloroplast membranes and directly oxidize chlorophylls. In addition, key genes in chlorophyll biosynthesis are repressed by high temperatures.
Among the effects that can be obtained with the application of brown algae extracts and high-temperature stress are the agronomic and photosynthetic parameters already mentioned; however, there is also an influence on the antioxidant system (enzymatic and non-enzymatic), where all these effects are a response to the expression of defense genes. The study showed that brown algae extracts increased the antioxidant system (enzymatic and non-enzymatic) and the expression of defense genes and in some cases there was repression.
It is evident that high-temperature stress and the application of algal extracts influenced the antioxidant system and the expression of the genes evaluated. There are different routes through which the expression of defense genes can be achieved and the best known route is through membrane receptors with the ability to bind to the metabolites of the extracts (amino acids, phenols, carotenoids, carbohydrates, phytohormones, and inorganic compounds) and perceive thermal stress. When this happens, the apoplastic Ca2+ and the one found in the vacuoles is transported to the cytoplasm and binds to proteins called calmodulins, forming a complex that activates protein kinases with the capacity to phosphorylate transcription factors, which travel to the nucleus to bind to specific DNA sequences, thus regulating gene expression. This action allows the synthesis of proteins with antioxidant activity such as SOD, CAT, APX, glutathione peroxidase (GPX), etc., which are the first line of defense against stress by neutralizing ROS. Proteins such as phenylalanine ammonia lyase (PAL), mitochondrial L-GalL dehydrogenase, and glutathione synthase, which synthesize phenolic compounds, ascorbic acid, and reduced glutathione, respectively, compounds that have the highest antioxidant activity in plant cells, can also be synthesized. However, biomolecules contained in algal extracts can enter at the cellular level and directly initiate the signaling process for the subsequent expression of defense genes or act directly in the reduction in ROS.
The genes evaluated have primary functions in the plant defense system. The NCED1 gene encodes for the enzyme involved in the synthesis of ABA, a phytohormone that plays a crucial role in the response to osmotic stress by regulating stomatal opening and closing. The HSP70 gene encodes for a heat shock protein whose function is to prevent misfolding and denaturation of other biomolecules under heat stress conditions. The PIP2 gene encodes for an aquaporin with the ability to regulate water transport in all plant organs. The P5CS1 gene codes for the enzyme that synthesizes the osmolyte proline, a compound of great importance in plant water regulation. The ERD15 gene encodes for an early response dehydration protein responsible for renaturation and protection of biomolecules exposed to thermal and osmotic stress.
The application of brown seaweed extracts to tomato plants not only promoted increased growth and biomass under standard growth conditions and high-temperature stress but was also accompanied by key physiological and biochemical improvements. Increased chlorophyll concentration is directly related to higher photosynthetic activity, which induces the synthesis of photoassimilates essential for plant growth and development. Furthermore, increased activity of antioxidant enzymes (SOD, CAT, and APX), along with increased non-enzymatic antioxidants, provides the plants with a better ability to mitigate oxidative stress, helping to maintain cellular integrity and prolong tissue functionality. Likewise, the overexpression of defense genes reflects a molecular response that underpins the observed physiological changes. Together, these integrated responses explain how stimulation with brown seaweed extracts strengthens both photosynthetic processes and defense mechanisms, resulting in increased growth and biomass accumulation in tomato plants subjected to high-temperature stress.
Conclusions
In this study, it was demonstrated that the application of extracts of Sargassum spp. and Ascophyllum nodosum improved some growth and biomass parameters of tomato plants under optimal growth conditions and high-temperature stress. In addition, stomatal conductance, chlorophylls, antioxidants, and gene expression increased with the extracts under both growth conditions, with the Sargassum extracts being the most effective. It represents a promising alternative for mitigating this type of stress in plants. Therefore, further studies are needed on the use of brown algae extracts in plants subjected to the aforementioned stress and to analyze other compounds such as ROS, phytohormones, osmolytes, minerals, and some other antioxidants and genes that indicate the state of plants.
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