1. Introduction
It is expected that climate change will increase the occurrence of unfavourable environmental conditions for crop cultivation around the world. Furthermore, as these alterations continue in the future, substantial areas of high-quality agricultural land will presumably be destroyed by erosion, desertification, rising seas, and salinisation [
1]. Moreover, agricultural production is also under threat due to the increasing prevalence of diseases and pests [
2] and the unbalanced usage of mineral fertilisers and pesticides [
3]. The level of food production needs to be increased, regardless of the smaller available area for farming and more severe conditions for plant growth [
4]. Therefore, novel approaches are needed to maintain the food, fibre, and fuel requirements of an increasing world population, which is expected to reach 9.7 billion in 2050 [
5].
This goal may prove to be an exceedingly challenging task as, during growth, plants are exposed to biotic and abiotic stresses, which can be of natural or anthropogenic origin [
6]. It has been estimated that adverse environmental effects caused by climate change have reduced crop yields by up to 70% since 1982 [
7]. Stress significantly affects the biochemical, morphological, and physiological mechanisms of plants, along with gene regulation [
8]. In response to unfavourable conditions, a complex range of responses is triggered, which includes various physiological pathways of primary and secondary metabolism [
9]. For instance, under stressful conditions, the level of reactive oxygen species (ROS), which originate from oxidation processes (like photosynthesis and respiration), substantially rises and may result in damage to lipid membranes, nucleic acids, and proteins [
10]. To overcome the stress conditions and ROS assemblage, plants have evolved several mechanisms, which include the accumulation of ascorbic acid, carotenoids, flavonoids, glucosinolates, osmolytes, specific proteins, and sugars and the activation of hormone-mediated responsive networks that involve jasmonates and other signalling molecules [
11].
In addition to a sufficient amount of food, it is also essential to ensure that plant-based products are of high quality [
12] and free of synthetic chemical residues that can induce detrimental health effects [
13]. Nowadays, consumer awareness and requirements for food quality and safety are constantly growing, which simultaneously impose the necessity of substituting synthetic chemicals with products based on natural derivatives, called biostimulants [
14]. A plant biostimulant is defined as: “
any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrients content” [
15]. These preparations are a rich source of bioactive compounds (e.g., acids, antioxidants, hormones, fats, minerals, oils, pigments, polysaccharides, and vitamins) which exhibit diverse activities [
16]. They are usually applied in the form of seed priming or coating, foliar spray, or root dipping and/or direct applications to the soil [
17]. Treatment with biostimulants benefits crop cultivation by improving germination rates, seedling vigour, nutrient uptake, growth, development, and plant metabolism, as well as increasing tolerance to biotic and abiotic stresses [
18]. In recent decades, a tremendous increase in their use has been observed [
19]. This is evidenced by the fact that the global biostimulant market is assessed at USD 3 billion in 2021 and is expected to exceed more than USD 5.1 billion by 2027 [
20]. Therefore, understanding their mechanism of action is crucial and often necessitates a multidisciplinary approach due to the plethora of interactions between a great variety of bioactive compounds within the same extract [
21]. For this reason, within the scope of the conducted research, our aim was to discover the mechanism of action of potential biostimulants that exerted favourable effects on plants growing under controlled laboratory conditions, as well as plants subjected to osmotic stress by sorbitol. Most of the adverse growth conditions enforce osmotic stress on plants by reducing the water potential of the environment [
22]. In our research, one of the most frequently found polyols in plants, namely sorbitol, was used to induce stress. This sugar is a direct product of photosynthesis in leaves and serves vital functions, such as the translocation of carbon skeletons and energy between sources and sink organs. The enhanced transport of polyols in the xylem and phloem occurs as a result of salt or drought stress [
23]. Understanding plant responses to various unfavourable growth conditions and the underlying mechanism of action of biostimulating products is crucial for the development of innovative approaches to minimise the adverse effects of mineral fertilisers and plant protection products, as well as for progress in the individualisation of crop production. This is the first work that presents the effects of the application of botanical extracts on plants growing under stress conditions and attempts to decipher their mechanism of action under controlled laboratory conditions.
3. Discussion
Accessible reports on the effects of biostimulant application to the highest extent indicate alterations in plant growth, development, and quality [
14,
24,
25] and the amplification of crop stress tolerance [
25,
26], however, very often without addressing functional aspects [
14]. Knowledge about the mechanism of action of most biostimulants is negligible or even unrecognised [
14,
25,
27,
28,
29]. In most instances, the total composition of these products remains unidentified [
27], and the analysis of individual bioactive compounds does not provide any direct correlation between their presence and the beneficial effects of biostimulant application that can be found in plants [
21,
28,
29]. Moreover, the separation of the influence of one or more bioactive compounds from the effects of additional ingredients is usually highly complicated [
27,
30] and unfounded as their efficacy is presumably the result of the actions of several molecules [
30]. This issue is also hampered by the presence of naturally occurring or commercially added amino acids, micronutrients, sugars, etc., which may exhibit synergistic, complementary, or no effects or may have been added for marketing or commercial registration purposes only [
27]. For this reason, to obtain more stable bioproducts, it is of the utmost importance to attempt to reduce the heterogeneity of raw materials and to standardise protocols for their preparation and extraction [
30]. Our previously published works focused on exploring the chemical composition of the botanical extracts [
31,
32]. The presence of the following bioactive compounds was confirmed: saponins (Ur L, Sg L, To F, To L, Tp F, Vo R, Hp H, So R, Ps S, and Lc S), oils and fats (Sg L, To F, To L, Tp F, Vo R, Hp H, and So R), alkaloids (Sg L, To L, Vo R, Hp H, So R, Ps S, and Lc S), triterpenes (Ur L and Tp F), terpenoids (Vo R), phytosterols (Sg L, To L, Hp H, and So R), steroids (Tp F and Ps S), phenolic compounds (Ur L, Sg L, To F, To L, Tp F, Vo R, Hp H, and So R), tannins (Ur L, Sg L, To F, To L, Tp F, Vo R, Hp H, So R, Ps S, and Lc S), anthocyanins (Ur L, Sg L, To F, Tp F, Hp H, and So R), coumarins (Ur L, Tp F, Hp H, Ps S, and Lc S), flavones (Ur L, Sg L, To F, Tp F, Hp H, So R, Ps S, and Lc S), flavonoids (Ur L, Sg L, To F, To L, Tp F, Vo R, Hp H, So R, Ps S, and Lc S), quinones (To L and So R), glycosides (Ur L, Sg L, Tp F, So R, Ps S, and Lc S), cardiac glycosides (Ur L, Sg L, To F, To L, Vo R, Hp H, and So R), proteins and amino acids (To F, To L, Tp F, Vo R, So R, Ps S, and Lc S), resin (To F, Vo R, So R, Ps S, and Lc S), sugars (Ur L, Sg L, To F, To L, Tp F, Vo R, Hp H, So R, Ps S, and Lc S), and vitamin C (Ps S and Lc S). In addition, the analyses of plant hormones were also provided.
This article is the beginning of a series of works aimed at clarifying the mechanisms of the action of extracts. In future articles, we will attempt to decipher the mechanism of their action under osmotic stress. This research has shown that the botanical extracts, obtained by means of ultrasound-assisted extraction, stimulated plant growth under controlled laboratory conditions, which was confirmed in Petri dish tests. To obtain the longest aboveground parts of crops, it is recommended to use bioproducts manufactured based on So R and Ur L, and to reduce the growth of unwanted plants, the Hp H extract could be considered (even at higher concentrations). For the cultivation of crops in which the largest root system is desired, we suggest the use of extracts obtained from To L, Tp F, and To F. The application of the Lc S-based extract should not significantly affect the development of the underground parts. The usage of botanical extracts can also provide positive results in increasing the fresh mass of cultivated plants. Extracts based on Lc S, Ps S, So R, and Ur L can stimulate the production of fresh mass by plants, while those based on Hp H and Ur L can contribute to its significant reduction (in the case of Hp H, the length of the shoots also decreased). However, the application of Ur L-based extracts caused the highest increase in root mass, while those based on Vo R caused the lowest increase in mass. Along with the improvement of plant growth, beneficial effects of the extracts on the content of photosynthetic pigments were also observed. For example, the application of extracts obtained from Ps S can increase the content of chlorophyll a, chlorophyll b, and carotenoids, while foliar spraying with Sg L showed one of the lowest stimulating activities of the photosynthetic process. To increase the content of total phenolic compounds, the application of Hp H, To L, and Ur L can be considered. The use of Lc S and Vo R did not exert statistically significant effects on plants. In general, regarding the increase in antioxidant activity, the extracts derived from To L, Ps S, Vo R, Lc S, and So R (DPPH assay); Ps S, Hp H, and To L (ABTS assay); To L, Hp H, Ps S, and Lc S can be successfully applied on plants grown in stress-free conditions. However, the use of Sg L and To F (ABTS assay) and Vo R (FRAP assay) may exhibit the least stimulating properties. The bioactivity and composition of biostimulants are not identical and depend on many factors, such as raw material type, location of raw material acquisition, extraction method, dose, concentration, application method, species and cultivar of crops, and environmental conditions [
33]. For this reason, an attempt to chemically standardise bioproducts is of utmost importance. The presence of bioactive compounds, contributing to the beneficial effects of biostimulants, was evaluated in our previous works [
31,
32]. The performed analysis of botanical extracts indicated the presence of alkaloids, amino acids, carboxylic acids, elements, glycosides, hormones (abscisic acid, benzoic acid, gibberellic acid, indole acetic acid, jasmonic acid, salicylic acid, zeatin, zeatin riboside, and isipentenyl adenine), oils and fats, phenolic compounds (phenols, tannins, anthocyanins, coumarins, flavones, and flavonoids), proteins, quinines, quinones, resins, saponins, steroids, sugars, and vitamin C. Additionally, the antioxidant activity was identified. The observed positive effects of the obtained botanical extracts could be due to the activity of individual components or their synergistic action. Within the framework of this work and to strengthen our knowledge of the mechanism of action of botanical extracts, the transcriptomic analysis was conducted. These analyses allow the assessment of the alterations in the expression of genes; however, the special emphasis on the interplay of various phytohormones involved in signal transduction is necessitated [
30]. The expression levels of known genes associated with the specific metabolic pathways induced by the bioproduct treatments can also be examined by qRT-PCR analysis. Sequencing techniques (NGS, especially RNA-seq) are increasingly being applied to explore mechanisms of action of biostimulants in light of the limitations of the microarray technique [
30,
34]. In addition, to clarify the unknown mechanisms of the activity of bioproducts, the RNA-seq approach could be a successful strategy for the development of innovative formulations [
30].
Crops are frequently subjected to various types of adverse conditions (e.g., salinity and drought), which lead to reduced plant growth and productivity. The majority of these environmental constraints reduce the environmental water potential and cause plant osmotic stress. The consequences of osmotic stress manifest themselves in the inhibition of cell elongation, stomatal closure, reduction of photosynthetic activity, disruptions in water and ion uptake, translocation of assimilates, and changes in various metabolic processes [
22]. Drought stress affects all plant growth parameters and response genes, as well as decreases membrane integrity and cell size, which leads to the formation of reactive oxygen species (including superoxide, hydroxyl, perhydroxy, and alkoxy radicals) and eventually cell death [
35,
36]. In order to combat the negative effects of environmental disturbances, plants develop comprehensive defence mechanisms, using physical adaptation and integrated molecular and cell reactions. The perception of abiotic stress activates the production of reactive oxygen species, an important secondary transmitter and early response mechanism. Under optimal growth conditions, ROS levels are at a moderate and balanced level due to the action of antioxidants and enzymes, making these molecules great signal transmitters. Nevertheless, abiotic stress leads to excessive ROS production that can lead to cell death. Reactive oxygen species include hydrogen peroxide (H
2O
2), hydrogen radical (OH), singlet oxygen (
1O
2), and superoxide anion (O
2), which are formed in a certain pathway. Their accumulation allows plants to survive and adapt to various stressful factors [
37]. Plants can also amplify the initial stress signals with phytohormones, such as ABA (abscisic acid). The phytohormone accumulation is connected to early plant stress signalling incidents (e.g., rapid ROS production). For example, the accumulation of ABA under a water deficit is contingent on ROS production via the NADPH oxidase. This ABA-induced ROS accumulation can enter guard cells and activate Ca
2+ channels, which leads to an increase in the cytoplasmic Ca
2+ concentration and, therefore, induce stomatal closure [
37,
38]. Studies show that biostimulants, due to their anti-stress nature, induce changes in the production of ROS and associated enzyme scavenging activities working to control oxidative damage [
14,
25], along with membrane stability, osmo-protection, and ion homeostasis [
25]. The application of biostimulants evokes a wide array of changes in the abundance of mRNA transcripts, activating various biochemical pathways and physiological responses [
30], which leads to a series of metabolic intracellular changes [
25,
30].
In recent years, new studies are beginning to change our understanding of the mechanisms of action of biostimulants. Despite this, it remains a major challenge to fully delineate their modes and mechanisms of action in plants. It is worth noting that the mode of action is the anatomical or functional alteration arising from the exposure to various factors (e.g., substances), while the mechanism of action specifies these changes at the molecular level [
14]. Due to diverse mechanisms of action, within this discussion, we will focus on the action of one of the main categories of plant biostimulants—seaweed extracts and botanicals. However, attempts to elucidate the mechanism of action of biostimulants are mainly carried out for algal extracts, and such studies are lacking for plant extracts. Therefore, references to our literature will focus on the brown alga
Ascophyllum nodosum (AN), which is the most widely used raw material for biostimulant production. In the literature, a growing number of reports on the mechanisms of action of its extracts (ANEs) appeared in recent years. One of the extensive reviews, provided by De Saeger et al. [
39], showed that the application of ANEs in the cultivation of, among others,
Arabidopsis thaliana (thale cress),
Brassica napus (oilseed rape),
Cucumis sativus (cucumber),
Daucus carota (carrot),
Glycine max (soybean),
Nicotiana tabacum (tobacco),
Solanum lycopersicum (tomato), and
Spinacia oleracea (spinach), subjected to various abiotic (freezing, drought, and salinity) and biotic stresses, modify the expression of genes involved in the transportation of nutrients across the cell membrane, influence the endogenous balance of hormones, alleviate stress-related reactions, and enhance photosynthesis. They also drew attention to the fact that the wide variety of plant responses to bioproducts is stymied by many factors, such as (1) the non-unified experimental setups, (2) the different concentrations of ANE, (3) the incomplete information on composition, (4) the low replicability caused by the seasonal variations in algae compositions, (5) the occurrence of species-dependent effects, and (6) the complex composition of extracts [
39]. In more detail, the use of ANEs in the cultivation of
A. thaliana under drought stress reduced the expression of
NCED3 (
At3g14440), involved in ABA biosynthesis, and
MYB60 (
At1g08810), a transcription factor involved in stomatal regulation and increased the expression of the ABA-responsive genes
RAB18 (
At5g66400) and
RD29A (
At5g52310). The authors also noted a decrease in gene expression of photosynthesis-related
RBCS1A (
At1g67090) and
RCA (
At2g39730) and of
PIP1;2 (
At2g45960) and
βCA1 (
At3g01500), which are involved in the regulation of CO
2 diffusion in the mesophyll. Moreover, an increase in the expression of
PsbS (
At1g44575) and
VDE (
At1g08550) in the experimental groups indicated energy dissipation and improved
DRF (
At5g42800) and
SOD (
At1g8830) expression at the activation of the antioxidant defence system that prevented oxidative damage to PSII. The observed changes in the molecular pathways connected to enhanced drought tolerance after the application of ANE were revealed [
40]. The currently available articles on the activity of
Ascophyllum nodosum-based extracts showed a differential influence on
A. thaliana transcriptome after the use of
Ascophyllum nodosum extracts. The modification of the expression of genes involved in the gluconeogenesis/glyoxylate cycle, oxidative stress, and hormone metabolism was observed. An upregulation of the glutaredoxin family genes (
At1g0320 and
GRXC2) and the cold-regulated gene
COR15A was presented [
41]. Another study on the activity of these brown algae products was conducted by Rasul et al. [
42]. The authors noted a strong decrease in drought-induced damages in
Arabidopsis thaliana induced by the regulation of key genes involved in the coordination of plant growth (
RD26 and
BES1) and cell cycle (
CYCP2;1). Elansary et al. [
35] investigated the foliar application of extracts from
Ascophyllum nodosum on
Paspalum vaginatum (seashore paspalum) during prolonged irrigation intervals and under saline conditions. The authors observed significant increases in antioxidant activities, SOD (superoxide dismutase), CAT (catalase), and APX (ascorbate peroxidase) enzyme activities, leading to ROS (H
2O
2) depletion in plants treated with seaweed extract. The survey conducted by Shukla et al. [
43] revealed that the application of the AN-based extract on
Glycine max led to changes in the expression of stress-responsive
GmCYP707A1a and
GmCYP707A3b genes, involved in ABA catabolism. Moreover, the expression of the ABA-inducible
GmDREB1B and the
BURP domain protein-encoding
GmRD22 increased, while the expression of the ABA-independent stress-responsive gene
GmRD20 remained unchanged. Induced expression of the ABA-responsive fibrillin gene
FIB1a, the aquaporin gene
GmPIP1b, the glutathione S-transferase gene
GmGST, the molecular chaperone
GmBIP, and the antiquitin-like
GmTP55 was also observed in plants treated with ANE. Furthermore, extracts obtained from
Ascophyllum nodosum modify the expression of
RbCS1A and
RCA genes (associated with the
RuBisCO activation) [
44], positively regulate the expression of
P5CS1 and
P5CS2, genes linked to the biosynthesis of proline, negatively regulate the expression of other genes related to proline catabolism in
Arabidopsis [
45], and enhance the activity of antioxidant enzymes (e.g., CAT, SOD, and APX) and the production of antioxidants (e.g., ascorbate), leading to lower ROS accumulation and membrane damage in plants (e.g.,
Phaseolus vulgaris—common bean and
Paspalum vaginatum) [
35,
46,
47]. In the study by Ali et al. [
48], the application of ANE in the cultivation of tomato and sweet pepper increased the upregulation of gene transcripts
Ga2Ox,
IAA, and
IPT implicated in the biosynthesis of gibberellin, auxin, and cytokinin, respectively [
48], while the treatment of oilseed rape caused an increase in the expression of
COPT2, a gene coding for a Cu transporter,
BnSULTR1.1 and
BnSULTR1.2, genes coding for sulfate transporters, and
BnNRT1.1 and
BnNRT2.1, genes coding for nitrate transporters [
49].
5. Conclusions
This is the first study investigating the mechanisms driving the positive effects of novel botanical extracts applied to cabbage seedlings grown under controlled laboratory conditions. It was observed that under drought conditions (induced by the addition of sorbitol), the treatment with biostimulants significantly improved the performance of the model plant, indicating an increase in plant tolerance to adverse growth conditions. Foliar spraying with To L (under 100 mM of sorbitol), Tp F (under 200 mM of sorbitol), and To F (under 300 mM) extracts stimulated the length of the shoots in comparison to the control group treated with water. The least stimulating activity was exhibited by Hp H extracts. In the case of roots, the use of the extract based on Sg L increased their length to the highest extent, while the Ur L-based extract was the only product that significantly reduced the root length (only under 300 mM of sorbitol). In general, the usage of novel botanical extracts can also enhance the fresh mass of shoots and roots of plants. For example, the treatment with Ps S-derived product resulted in favourable effects on the aboveground biomass in low- as well as high-stress conditions, while Hp H extracts exhibited the least stimulating activity. The use of Sg L (under 100 mM of sorbitol), Hp H (200 mM of sorbitol), and Ps S (300 mM of sorbitol) stimulated root mass growth to the greatest extent, while the application of Tp F (100 mM), Tp F, So R (200 mM of sorbitol), and To F (300 mM of sorbitol) showed the lowest activity. Treatment with Ps S (under 100 mM of sorbitol), Lc S (under 200 mM of sorbitol), and Hp H (under 300 mM of sorbitol) increased the photosynthetic process in plants, while the application of So R (under 100 mM of sorbitol), To F (under 200 mM of sorbitol), and Tp F (under 300 mM of sorbitol) exhibited the lowest activity in the enhancement of photosynthesis. Plants treated with botanical extracts responded differently to the stressful conditions. Under the lowest stressful conditions (100 mM of sorbitol), none of the obtained bioproducts increased the concentrations of total phenolic compounds. Under the mid-stressful conditions (200 mM of sorbitol), the application of Hp H stimulated the content of TPC to the highest extent, while the use of Ur L, Ps S, and Sg L exhibited the lowest activity. Under the highest stressful conditions (300 mM of sorbitol), To L and Ur L stimulated an increase in the content of these compounds. As shown by the DPPH assay, the use of all extracts under 100 mM of sorbitol can increase the antioxidant activity, and those with the highest values included Lc S, Ps S, and Sg L. Similar observations can be made for 200 mM of sorbitol in the majority of experimental groups for plants treated with Ps S, To L, Ur L, and Hp H. Only four extracts (To F, Tp F, Sg L, and Lc S) decreased antioxidant activity. The opposite trend can be noted for 300 mM of sorbitol, excluding the extract based on To L. The lowest antioxidant activity was found in the group sprayed with products obtained from Tp F. The highest antioxidant activity, measured with the use of the ABTS assay, under exposure to 100 mM of sorbitol, was observed after the application of the extract obtained from Tp F, So R, and Vo R, while the extract based on To L was the only product that decreased antioxidant activity. Plants exposed to both 200 mM and 300 mM concentrations of sorbitol and treated with botanical extracts (e.g., with Ps S and Lc S under medium stress and with So R and Tp F under the highest stress) showed greater antioxidant activity compared to plants sprayed with only water under the same conditions. However, under the low-stress conditions (100 mM of sorbitol), the antioxidant activity measured with the FRAP assay was higher only in the group treated with So R, while for other bioproducts (e.g., Sg L), it was lower. In mid-stress conditions (200 mM of sorbitol), most of the extracts increased the antioxidant activity (e.g., Vo R, Ur L, To L, and So R), while under the highest stress (300 mM of sorbitol) the observed differences were mostly not statistically significant, with the exception of the application of Hp H and To F. Additionally, our findings were confirmed by transcriptome studies, which showed that the application of a selected extract (So R) modified the expression of the following genes: Bol029651 (glutathione S-transferase), Bol027348 (chlorophyll A-B binding protein), Bol015841 (S-adenosylmethionine-dependent methyltransferases), Bol009860 (chlorophyll A-B binding protein), Bol022819 (GDSL lipase/esterase), Bol036512 (heat shock protein 70 family), Bol005916 (DnaJ Chaperone), Bol028754 (pre-mRNA splicing Prp18-interacting factor), Bol009568 (heat shock protein Hsp90 family), Bol039362 (gibberellin regulated protein), Bol007693 (B-box-type zinc finger), Bol034610 (RmlC-like cupin domain superfamily), Bol019811 (myb_SHAQKYF: myb-like DNA-binding domain, SHAQKYF class), and Bol028965 (DA1-like Protein). The GO functional analysis indicated that the application of extracts leads to a decrease in the expression of many genes related to the response to stress and photosynthetic systems, which may confirm a reduction in the level of oxidative stress in plants treated with biostimulants. Based on the current study, we are convinced that botanical extracts can help crops alleviate the side effects of prolonged drought periods observed in most agricultural areas of the world.