Abiotic stress, provoked by extreme temperatures (heat or cold), water shortage or excess caused by drought or flooding, high light and UV radiation, salinity, heavy metals and other pollutants, or their combinations, has been recognized as a major source of economic damage to agriculture [1
]. A common feature of all these events is that they lead to the accumulation of the so-called reactive oxygen species (ROS) in plants, including hydrogen peroxide—H2
; hydroxyl radical—HO∙
, singlet oxygen—1
, etc. [3
]. In low concentrations ROS exert signaling functions and may prime the plants for enhanced stress tolerance[5
]. However, due to their high oxidizing potential, at high doses they can damage numerous cell constituents and even trigger programmed cell death (PCD) [6
]. Treatment of plants with ROS-inducing agents like aminotriazole, menadione and paraquat (PQ), or directly with H2
, is a widespread approach in studies related to abiotic stress since it allows the easy and reliable identification of sensitive or tolerant individuals in different experimental setups [9
In recent years, an elegant solution to optimize plant growth and performance, which attracts the attention of farmers, the industry and the research community, is the treatment with metabolic enhancers called biostimulants [10
]. According to the European Biostimulant Industry Council (EBIC) these products “contain substance(s) and/or microorganisms whose function when applied to plants or the rhizosphere is to stimulate natural processes to enhance/benefit nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality”. Biostimulants are reported to improve yield both in normal conditions and under slight to moderate stress [11
]. Moreover, they are also regarded as an alternative to traditional agro-chemical practices with the potential to reduce the usage of pesticides and fertilizers [13
]. Biostimulants are obtained from natural sources and have undefined chemical composition based on a mixture of multiple chemicals like vitamins, minerals, carbohydrates, amino acids and peptides, phenolic compounds, etc. [11
]. Due to their complexity, it is possible that the observed beneficial properties of these products may be caused by the synergistic action of their different components rather than by the properties of individual constituents. Depending on their source, biostimulants can be divided into different categories, the most prominent of which are prepared from: microorganism isolates, protein hydrolysates of animal or plant origin, seaweed extracts, humic/fulvic acids, etc. [14
Among these different categories, one of the most successful on the market includes the products harvested from seaweeds [15
]. This is a very diverse group of biostimulants whose exact properties depend on the source species, the methods used for extraction, the conditions in the areas used for harvesting, etc. [14
]. Some of the reported beneficial effects of seaweed extracts on plant vigor involve improvement of the root development, synchronization of the fruits, acceleration of fruit onset and flowering, delayed senescence and enhanced stress tolerance [17
]. Another important aspect that contributes to the popularity of these products is their easy biodegradability and lack of toxicity to the environment and human health [20
]. Crops, for which positive influence of seaweed extracts on yield and quality has been described in the literature, include wheat, rice, tomato, cucumber, broccoli, spinach, bean, etc. [14
Biostimulants produced from the brown inter-tidal alga species Ascophyllum nodosum
(L.) of the Fucaceae
family not only have significant commercial success but are also widely studied [12
]. As a result, several modes of action of Ascophyllum nodosum
extracts (ANE) in eliciting plant stress mitigation was recently proposed [12
]. A specific ANE product available on the market, called SuperFifty (SF), has demonstrated high potential as a potent promoter of plant growth and productivity [18
] mainly by reducing stress and enhancing fruit setting processes in plants. SF has been shown to ameliorate plant water holding capacity via modulating the ABA-dependent pathway and partial closure of stomata (unpublished data). However, details on the protective properties of SF on different economically important crops as well as its precise mechanism of action are necessary in order to fully characterize this product. Since the effects of a given biostimulant may differ among different species [11
], in the current work two important crops—tomato (Solanum lycopersicum
cv. MoneyMaker) and sweet pepper (Capsicum annuum
cv. Kurtovska Kapiya-1), as well as the model plant A. thaliana
(ecotype Col-0) were studied. Their physiological and biochemical responses to oxidative stress provoked by PQ and the ability of SF to alleviate the resulting stress symptoms were compared. Details on the underlying molecular mechanisms were obtained by monitoring the primary metabolite profiles and their rearrangements after treatments with SF and/or PQ in all three species.
(SF) is a commercially available biostimulant which contains highly concentrated A. nodosum extract (500 g/L) [33
]. It is a biodegradable product, highly rich in functional bioactives and antioxidant substances such as phlorotannins and fucoidans [12
], as well as proline (involved in drought and salt stress tolerance), mannitol (osmoprotectant and ROS scavenger protecting photosynthesis during abiotic stress) and sorbitol (free radical scavenger) [18
]. The stress reducing potential of SF is possibly due to the presence of uncommon or unique polysaccharides such as fucoidan [19
]. These polysaccharide molecules may prime and trigger plant signaling pathways [19
Due to these properties, SF is proven to enhance abiotic stress tolerance in various plants and helps to improve fruit quality, crop yield, root growth and plant growth [12
]. The polyphenol content and antioxidant capacity were found to be much higher in SF than in the similar product Ecolicitor®
]. ANE extracts also induce nutrient uptake in plants and it was shown that SF application significantly enhanced the accumulation of antioxidants, minerals and essential amino acids in tomato fruits [18
]. The effects of SF and other biostimulants on the metabolome are also increasingly being assessed ([19
] and references therein).
The antioxidant characteristics of SF and several of its components make it a suitable candidate for investigating its relationship with an oxidative stress simulation system. Indeed, a recent study demonstrated that SuperFifty foliar application rescued paraquat-induced oxidative damage in Arabidopsis plants [19
]. To our knowledge, this is the first comparative survey simultaneously carried out with three species, which demonstrates the similarities and differences in their physiological and biochemical responses after individual and combined application of a ROS-inducer and the biostimulant. As the aim of this work was not to study influences on the yield, seed production, germination or any other agricultural properties of the plants, but rather to examine some of their faster reactions, the vegetative phase of their development which was selected for investigation, had to serve several purposes: to allow comparison with previous studies, to be a relatively early stage of development, and to ensure convenient plant material management. Considering these requirements and the interest in the short-term responses, a scheme with pre-treatment (preventive action) with SF was chosen, rather than post-treatment (healing effect). The rationale is to be able to prevent the detrimental effects of the stress rather than repair the already received damages, which is preferable from a practical point of view.
The three investigated plant species manifested specific reactions to the same stressor and displayed dissimilar amounts and type of damages, including shape, pattern, distribution and coloring of the affected areas. Arabidopsis formed the largest lesions, widely distributed among the whole leaf area, while pepper leaves developed only small round lesions, grouped mostly on the basal half of the leaf blades. Tomato had an intermediate position in this aspect (Figure 1
). Based on these observations and considering the lower concentration of PQ in the Arabidopsis treatments, we can suggest that under these conditions, Arabidopsis shows the highest sensitivity to the stress and pepper the lowest.
Pre-treatment with SF produced a well-marked rescue effect, with different strength for the three species. The degree of protection was the strongest for the least sensitive species - pepper, slightly weaker for the most damaged Arabidopsis and the weakest, but still significant, for tomato (Figure 2
). The reasons for the pronounced individual responses to the stressor and the biostimulant may be related to the differences in species anatomy and/or physiology, such as thinner or thicker cuticle, absence or presence of trichomes, number and size of stomata on treated surfaces, etc. Another plausible explanation is the potentially different mechanisms of action, through which SF promotes its protective effect.
The DAB-staining experiment showed regions of H2
accumulation with a very similar pattern, size and shape to the visible lesions. The high degree of their overlap supports that the lesions are caused by locally forming ROS bursts. SF pre-treatment causes a noticeable decrease of the H2
-stained areas, suggesting that its mechanism of action includes prevention of ROS buildup (Figure 3
Additionally, by measuring the electrolyte leakage we could demonstrate higher membrane damage in the PQ-stressed variants, coupled with various, but generally strong levels of rescue effect, caused by the SF pre-treatment (Figure 2
). Considering this parameter, pepper shows the least damages, while tomato appears to be the most sensitive of the three species. The highest level of rescue is observed in Arabidopsis, and the lowest in pepper.
The maximum quantum yield and fluorescence decrease ratio were assessed by PAM fluorometry and showed considerable suppression of photosynthesis in PQ-only treated plants. Unlike the results from the other examination methods though, in this case pre-application of SF, despite producing a significant rescue effect, led to a weaker recovery of the observed parameters (Figure 4
). Possible reasons could include the mode of action of PQ, which leads to superoxide production directly in the chloroplasts, and subsequently sensitizes the photosynthetic apparatus.
The combination of results from all physiological experiments lead to the conclusion that there is a common response to SF: when applied alone it does not seem to provoke any (or small) stimulating effects on the measured parameters in these particular conditions. This is probably due to the experimental setup, which includes a too short time window after the treatment, insufficient to allow monitoring of slower plant growth and development processes. Nevertheless, SF displays a universal protective effect against oxidative stress in all three species, which, however, has some specificities expressed in differences in the level of the rescue. The reason is probably due to the differences of the plants’ reactions to the stressor, combined with species-specific mechanisms via which SF achieves its shielding function.
For each of the studied species, the accumulation patterns of primary metabolites, visualized on unsupervised PCA plots, form four putative groups—one per each treatment (Supplementary Figure S2
). Some of them exhibit partially overlapping areas, from which −SF/+PQ shows the most distinct separation. Using supervised sPLSDA graphs the focus falls on the specific differences between the groups and this creates distinct metabolic profiles for each treatment with much less interceding areas (Figure 5
). A unique pattern is characteristic only for the −SF/+PQ variants, which reflects а massive response occurring after PQ-induction of oxidative stress. Remarkably, these PQ-provoked metabolic fingerprints between three different organisms are comparable. This is further supported by the separate inter-species grouping of the −SF/+PQ variants from all three organisms on the clustered heat maps: they are closer on the dendrogram than the intra-species treatment groups (Figure 7
). Nevertheless, in some cases, several metabolites characteristically change in the −SF/+PQ variant of one species only (see Results). This might exemplify the differences in the modes of action, specific for every particular plant. The closer association of +SF/+PQ profiles to the control groups rather than to the stressed ones proves the high degree of rescue effect acquired by the SF pre-treatment, which prevents this variant from developing the distinct pattern of metabolic rearrangement, characteristic for ROS stressed plants. Assessment of sPLSDA component 1 values indicates that the metabolic changes induced by SF are strongly correlated with the evaluated plant phenotypes, an effect observed for all three plant species.
Considering the specific composition of the changed metabolomes and the metabolite categories within them, the major sub-group in the −SF/+PQ variant for Arabidopsis is formed by amino acids, all of which (11) are increasing. This is presumably due to protein degradation resulting from the oxidative stress-induced cell death [37
]. For the +SF/+PQ variant, 10 of these AA also accumulate, but to a lesser amount, pointing to an incomplete rescue. The same observation is true for pepper, where two out of six selected metabolites are AAs, which are more elevated in −SF/+PQ and less in +SF/+PQ. Similarly, 10 out of the 11 PQ-modulated AAs in in tomato have higher abundance, with a well-pronounced recovery effect in the +SF/+PQ variant, where all AA amounts are tending to the base levels, with five of them slightly above and six—somewhat below the control quantities (Supplementary Figure S8
Another significant metabolite group in Arabidopsis is formed by other organic acids and sugars, but this time most of them (8) decrease in −SF/+PQ and only five increase. For +SF/+PQ the numbers are almost the same: seven and six respectively. The rest of the biochemical substances are from the categories of polyols, amino alcohols, esters, nucleobases, etc., and one group (5) of unknown compounds.
Considering the selected tomato metabolites, they are distributed in the same general biochemical groups (AAs, organic acids, sugars, etc.), with a notable difference that only two compounds are down-accumulated in −SF/+PQ (aspartic acid and cis-3-caffeoyl-quinic acid), while nine decrease in +SF/+PQ: the abovementioned AAs, a keto acid (pyruvic acid), a sugar (rhamnose) and an unknown substance that is probably also a sugar.
The same biochemical groups, supplemented by one ketone (oxo-glutaric acid), are seen in pepper, but with two major differences—no unknown metabolites are present and all but the AAs increase in −SF/+PQ and slightly decrease in +SF/+PQ.
A notable common pattern is that salicylic acid (SA) considerably rises in the −SF/+PQ variant for all three species and goes back to normal in +SF/+PQ. SA is a phytohormone that plays a vital role in enhancement of the plant antioxidant defense system [38
]. It can be regarded as а typical “stress metabolite”, with various functions in the regulation and mitigation of ROS induction, elicited by abiotic stresses such as heavy metals and UV-B [39
], salinity, temperature, drought, etc. [38
]. This explains its elevated levels in all PQ-treated samples.
It is noteworthy that four other differentially accumulated substances in Arabidopsis (beta alanine, gamma aminobutyric acid (GABA), myo
-inositol and nicotinic acid) and three in tomato (beta-alanine, putrescine and trehalose) are considered stress metabolites as well, with various roles in oxidative and other types of stress. Beta-alanine is a well-documented stress response molecule involved in protecting plants from a variety of adverse conditions like extreme temperatures, drought, hypoxia, heavy metals, and even some biotic stresses [43
]. GABA has а rapid stress-specific pattern of accumulation (in some cases with 300% for 15 s [44
]) and plays a role in stress mitigation by triggering preventive metabolic changes, taking place before irreversible damage to tissues may occur [45
]. During salt stress for example, it was demonstrated that the changes in GABA amounts precede the accumulation of other important salt-stress defense metabolites such as soluble sugars and proline [46
-inositol (and the precursor of trehalose - trehalose 6-phosphate) takes part in a broad regulatory network that contributes to oxidative stress homeostasis and plant stress tolerance [47
]. Its broad range of derivatives can assume a dual role, acting both as structural lipids for the cell membrane and as diverse signal molecules, involved in a wide variety of cellular processes, including a crosstalk between sugar and lipid signaling. Trehalose itself is reported to play a role against oxidative stress caused by different abiotic stresses in several species, such as drought and salt in tomato [48
] and heavy metal stress in tobacco [49
]. Nicotinic acid and its metabolites are crucial for many vital functions as energy metabolism, oxidation-reduction reactions and various metabolic regulations. Moreover, nicotinic acid is the building block of many simple pyridine compounds [50
]. Some of its metabolites take part in the signal transduction pathway regulating plants’ response to DNA strand breakage, which is caused mainly by oxidative stress [51
]. Putrescine is known to reduce oxidative damage during strong abiotic stress, such as flooding [52
], aluminium excess [53
] and salt excess [54
Overall, our study shows that SuperFifty’s protective role is not just limited to the model plant Arabidopsis thaliana but it successfully prevents PQ-induced oxidative damage in the important crops tomato and pepper as well. The metabolome reconfigurations, following different treatments, display patterns much more similar among the negative control, SF-treated and double-treated variants, than to the PQ-stressed one, suggesting that SF effectively mitigates the stress responses at the molecular level. These observations are equally valid for all three studied species, although some characteristic variations were observed.
Further steps need to be taken for additional elucidation of the exact mechanism through which SF positively affects the plants and mitigates oxidative stress. RNA-sequencing analyses, verified by qRT-PCR, could uncover more information about the transcriptomic changes, ruling the metabolomics rearrangements reported here. Furthermore, analyses of the activities of particular antioxidant enzymes and other stress markers could help narrow down the alternative pathways leading to the observed effects.