Microorganisms (MOs) were traditionally studied as free-living or planktonic cells
[6][1]. The focus has gradually shifted to the study of sessile communities, since planktonic growth appears to be rare in nature
[7][2]. In other words, characterizing an MO in isolation does not contribute to our understanding of its real behavior in nature, where it shares an ecological niche with diverse microbial populations.
Bacteria of the same or different species are known to associate into biofilms, which are well-structured, multicellular communities embedded in a self-produced matrix attached to a living or inert surface
[8,9][3][4]. This extracellular matrix is mostly composed of water, but also contains proteins (including enzymes), DNA, RNA, and EPSs
[10][5]. The latter are particularly important because they stabilize the 3D structure of the biofilm
[11][6], and they can represent between 40 and 95% of the biofilm biomass
[12][7].
The life cycle of a biofilm begins when it attaches to a compatible surface. Planktonic cells make use of their motility mechanisms (pili, flagella, proteins, EPSs) to gather on such a surface and establish reversible connections with other cells
[10][5]. After these initial interactions, the cells adhere irreversibly to the substrate and start reproducing into microcolonies, thanks to the exchange of molecular signals. During this time, they secrete the components of the extracellular matrix, which becomes crisscrossed with water-filled channels that transport nutrients and eliminate waste
[13][8]. Eventually, planktonic cells may become detached from the biofilm for different reasons, e.g., because they have been newly formed
[14][9], and go on to create new colonies elsewhere. This last stage in a biofilm’s life is known as dispersion.
An important phenomenon for the formation and continued existence of a biofilm is “quorum sensing” (QS). This is a collective communication system that relies on the production and reception of small signal molecules by individual bacterial cells. The denser a bacterial population, the higher the concentration of these molecules. QS-related genes are activated only above a certain signal concentration (i.e., when a certain quorum has been reached), which means the process entails a positive feedback loop: when bacteria sense the molecules building up, they synthesize more of them; the more molecules are released, the higher the likelihood that the expression of QS genes will be induced. For this reason, QS molecules are referred to as “autoinducers”
[15][10].
Three types of autoinducers have been extensively studied in bacteria: N-acyl homoserine lactones (AHLs), autoinducer peptides (AIPs), and AI-2. Different ones are specific to different bacterial groups. In general, QS genes are regulated by AHLs in Gram-negative bacteria, and by AIPs in Gram-positive bacteria
[15][10].
A typical AHL-based system works as follows. A QS gene is activated which encodes LuxI synthase. This enzyme synthesizes an N-3-oxohexanoyl-homoserine lactone (3-oxo-C6) that is passively diffused out of the cell and then binds to a LuxR receptor inside another cell, which is encoded by another, separately regulated gene. The AHL-receptor complex binds to a consensus DNA sequence and triggers the expression of luciferase. Many systems of this kind have been described in Gram-negative bacteria, including more than 70 species of Proteobacteria
[15][10].
2. EPSs Synthesized by Plant Growth-Promoting Rhizobacteria
The rhizosphere (from the Greek “rhiza”: root) is a microbe-inhabited microzone located at the soil–plant root interface. Three subzones have been described within it, based on their proximity to the roots: the endorhizosphere, which includes parts of the root cortex and endodermis; the rhizoplane, made up of the root epidermis and mucilage; and the ectorhizosphere, which consists of the soil that closely adheres to the root
[16][11]. The size and structure of the rhizosphere vary according to the complexity and diversity of the root systems developed by different plant species, as well as to the microbial populations that live within it
[17][12].
Between 15 and 40% of the total rhizospheric surface is occupied by microcolonies or biofilms. Root exudates act as chemoattractants that draw MOs to the rhizosphere
[16][11] and provide them with nutrients (e.g., sugars and amino acids)
[18][13]. In turn, these MOs exert positive effects on the plant in the context of symbiotic or nonsymbiotic relationships.
The development of rhizospheric communities is influenced not only by the availability of nutrients produced by the plant, but also by the properties of the soil, especially temperature, pH, aeration, and physicochemical composition
[21][14]. These communities include fungi, protozoa, algae, and plant growth-promoting rhizobacteria or PGPR
[21][14]. The latter were initially identified in nodular formations in desert plants. The EPSs they produced were found to provide long-term protection against desiccation. Since then, many other PGPR have been described, and they are now categorized into two major classes depending on their interaction with the host plant.
PGPR belong to many different genera, such as Agrobacterium, Arthrobacter, Azotobacter, Azospirillum, Enterobacter, Bacillus, Burkholderia, Caulobacter, Chromobacterium, Erwinia, Flavobacterium, Micrococcus, Pseudomonas, Serratia, and Frankia
[22,23][15][16]. All of them synthesize EPSs which, as described in 1.2., allow them to become attached to the root surface in the form of biofilms. These biofilms protect bacteria against adverse conditions (salinity, extreme temperatures, biotic stress, drought). EPSs themselves are produced in response to stressful stimuli
[21][14]. In addition to conforming to the biofilm structure, these sugar macromolecules can benefit the plant
[24][17], both through direct action and indirectly, by improving soil properties. They can form aggregates that make the soil more stable, fertile, and porous, and improve its enzymatic activity. This means there is a larger availability of nutrients and water to fuel plant growth. In addition, some polysaccharides can retain as much as seven times their mass in water, which further contributes to water availability for the roots
[25,26][18][19]. EPSs have also been linked with longer roots and seedlings, a higher chlorophyll content, and an altogether larger plant biomass
[27,28][20][21].
3. PGPR Activity and the Role of EPSs during Abiotic Stress
3.1. Salt Stress
A high concentration of sodium (Na
+) in the soil alters its physical and chemical structure, in a way that reduces its aggregation stability
[31][22], permeability, and hydraulic conductivity
[32][23]. Saline soils are darker (due to organic matter disaggregating), more alkaline, and in general less capable of sustaining plant growth
[33][24].
In plants, the effects of salt stress can be divided into two phases. Osmotic stress occurs when a high concentration of salt in the root area inhibits water absorption. This is followed by ionic toxicity: when the concentration of Na
+ rises in the leaves, other ions cannot be absorbed and different metabolic pathways are affected, including that for photosynthesis. These processes are accompanied by secondary reactions like the activation of Ca
2+ signaling; the accumulation of mineral ions, metabolites, and abscisic acid (ABA); and the production of reactive oxygen species (ROS)
[34,35][25][26]. In turn, these changes may diminish water and nutrient availability; disturb enzymatic activity, protein metabolism, and hormone regulation; and damage the membrane and its cell constituents. Other aspects that are harmed include root architecture, the composition of plant exudates, the structure of the soil, and the plant-associated microbial community (in terms of biomass, enzymatic activity, soil respiration, and the mineralization of carbon and nitrogen)
[36][27]. As a result, plant growth is stunted and yields are lower.
PGPR conserve their growth-promoting abilities in the face of salt stress. Some of the best-known halotolerant genera are
Halomonas,
Bacillus,
Streptomyces,
Oceanobacillus, and
Pseudomonas [35][26]. There are several things these bacteria can do to counteract the effects of salinity. They can induce systemic tolerance (IST) in the host plant; solubilize phosphates; produce siderophores, ammonia, indole-3-acetic acid (IAA), and 1-aminocyclopropane-1-carboxylate deaminase (ACC); and accumulate osmolytes to maintain the balance between the intracellular and the osmotic pressure. Stress may also be relieved through the secretion of biopolymers, e.g., EPSs, polyesters, and polyamides
[37,38][28][29]. These biopolymers constitute a physical barrier that absorbs excess Na
+ and reduces its concentrations in the rhizosphere
[39,40][30][31]. EPSs are able to do this thanks to their anionic nature, which allows them to capture free Na
+ ions. This not only helps plants but also offsets some of the negative consequences of salt stress on the soil. Improved growth in wheat after inoculation with salinity-tolerant PGPR, such as
Bacillus amyloliquefaciens,
Bacillus insolitus,
Microbacterium spp., and
Pseudomonas syringae, was attributed to the formation of a protective EPS sheath around the roots
[24,37,41][17][28][32].
3.2. Drought Stress
Low water availability in the environment causes an imbalance between transpiration and water absorption in plants, and is thus another source of abiotic stress. Drought inflicts mechanical damage on plant cells by decreasing their turgor and volume and increasing their content of solutes. In plants subjected to this kind of stress, the development of leaves and of the apical and lateral meristems is inhibited, and the end result is a reduction in growth
[42][33].
Some of the mechanisms deployed by PGPR to benefit plants during drought are very similar to those that take place during salt stress. For instance, these bacteria may mediate regulations in plant hormones, lead to modifications in root morphology, enhance ACC activity, help the plant accumulate antioxidants and osmolytes, and produce organic volatile compounds, IAA, gibberellins, cytokinins, and EPSs
[43,44][34][35].
EPSs benefit plants exposed to drought through several mechanisms. They induce the production of ABA, which leads to partial stomatal closure and reduces water evaporation through transpiration, a key factor to enhance plant drought resistance
[41][32]. They promote the accumulation of osmolytes, which can increase osmotic pressure in plant cells and, therefore, their capacity to expand. As a result, these cells are better at uptaking and/or conserving water. For example, two osmolytes (proline and soluble sugar) increased significantly in the leaves of rice seedlings treated with bacterial EPSs
[41][32]. These osmolytes also help eliminate free radicals, which tend to build up during drought stress and damage the cell membrane.
3.3. Temperature Stress
Environmental temperature influences several aspects of the soil, e.g., its temperature, humidity, aggregation stability, pH, and nutrient diffusion
[50,51,52][36][37][38]. Naturally, changes in any of these factors will disturb the MOs living in the soil and plant growth. Besides affecting plants through its effects on the soil, temperature has a direct impact on plants, too
[53][39].
Generally speaking, temperature stress can be responsible for low germination, respiration, and photosynthesis, as well as for the inactivation of proteins and detoxification pathways; the accumulation of ROS; alterations in the synthesis of phytohormones; and changes in the lipid composition of the membrane, which render it less fluid and permeable. All of this stunts plant growth and in some cases may even lead to death
[30,54,55][40][41][42].
Stress caused by very low or freezing temperatures has serious consequences for plant germination, growth, and reproduction
[55][42]. It creates irreversible lesions, cell dehydration, and dysfunctions in the membrane. Inoculated PGPR can mitigate this by fostering plant defenses through a series of strategies, which include the upregulation of ABA; lipid peroxidation; proline accumulation; and an increase in chlorophyll, anthocyanins, starch, and/or iron. As for EPSs, certain plant-associated bacteria have actually been found to produce more of them under low temperatures (0–15 °C). Psychrotolerant
Pseudomonas from the northwestern Himalayas, for example, synthesize more EPSs when exposed to the cold than at ambient temperatures.
Stress created by very high temperatures may similarly decrease germination or even prevent it from happening altogether: it reduces cell size and metabolism, damages plumules and radicles, and produces pollen infertility. It may also generate serious damage during the stage of vegetative growth
[56][43]. Once again, PGPR are able to counteract this through many mechanisms. They can promote nutrient absorption and N
2 fixation; produce siderophores; reduce ROS; increase the content of proteins, chlorophyll, and ABA (which leads to stomatal closure), and stimulate the synthesis of phenolic compounds and secondary metabolites involved in the plant’s defense system.
3.4. Stress by Metal(loid)s
In the last few decades, there has been a worldwide increase in metal toxicity in the soil. This is due to a variety of natural phenomena, as well as rapid industrialization and other anthropogenic activities that involve the massive release of different kinds of waste and chemicals into the environment, such as pesticides and fertilizers. Metals, e.g., cadmium (Cd), mercury (Hg), lead (Pb), copper (Cu), chromium (Cr), and nickel (Ni), and metalloids, e.g., arsenic (As), have high molecular density and can be toxic even at low concentrations. When they accumulate in soil that is destined for agricultural activities, they quickly enter the food chain and also accumulate in living organisms. This is known as bioaccumulation, and has direct and indirect effects on human, animal, and plant health.
In plants in particular, stress caused by rising levels of metal(loid)s in the soil can modify physiological and biochemical processes, and thus affect normal growth and yield. The most considerable damage is usually registered in roots, because they are the initial area of contact with these compounds. However, toxicity does not remain localized: it goes on to have an impact on other tissues and organs, like the stem and the leaves
[60][44].
Beyond plants’ own evolutionary responses to this kind of stress, a human-made solution consists of remediating or decontaminating soils through physical, chemical, and biological strategies. Unfortunately, the first two are often costly in terms of materials and labor, and can produce secondary contaminants. Biological remediation, on the other hand, is much more ecofriendly and economically viable, given that it involves harnessing the detoxifying abilities of living microorganisms, such as PGPR
[59][45].
4. PGPR Activity and the Role of EPSs during Biotic Stress
Biotic stress is caused by viruses, fungi, bacteria, and pests, among others. Like its abiotic counterpart, it provokes metabolic changes in plants, and reduces their ability to uptake nutrients, their vigor, and their germination power. In severe cases, it can lead to plant death. Plant diseases brought about by pathogens, both pre- and postharvest, are a leading cause of yield and economic losses worldwide
[63][46].
Research has shown that plants engage in several strategies to deter pathogenic MOs. The first line of defense is passive and physical. Trichomes, waxes, and cuticles are components of the epidermis that make it difficult for pathogens to establish themselves on the plant surface. Another barrier is made up of secondary metabolites and antimicrobial compounds that the plant secretes in response to pathogenic invasion. At this level, there are two possible immune responses triggered by the plant’s molecular signaling system. One of them is systemic acquired resistance (SAR), which relies on the production of salicylic acid (SA).
Besides inducing plant resistance, many rhizosphere MOs are efficient biocontrol agents in their own right, since they inhibit plant pathogens that indirectly interfere with beneficial microbial consortia
[64][47]. They may do this by outcompeting them, or by synthesizing substances with antibiotic, antifungal, and bactericidal properties; biosurfactants; hydrogen cyanide (HCN); and volatile organic compounds (VOCs)
[29,65][48][49].
5. Conclusions
Stress affects different aspects of plants and of plant-associated microbial communities in the soil, such as photosynthesis, respiration, and ion and nutrient absorption. Bacterial EPSs are important constituents of bacterial biofilms on roots, as they can create a physical barrier that prevents the entry of pathogens and excess Na
+ in cases of salinity. They are capable of storing water and thereby improving its availability for the plant, with their synthesis being necessary for the production of proteins that can offset the effects of heat. The aggregates they form in the soil increase its permeability and stability, which benefits the plant by facilitating nutrient uptake and enabling recalcitrant and toxic compounds such as metal(loid)s to be degraded. Moreover, by promoting the accumulation of osmolytes they protect against desiccation and may act as signaling molecules that intervene by triggering the plant’s innate immune response against pathogenic invasion. Although their specific mechanisms of action still need to be fully elucidated, EPSs produced by PGPR can be safe, ecofriendly, and cost-effective biotechnological tools for sustainable agriculture as they can protect plants from stress and stimulate their growth, thus ensuring adequate yields (i.e., food security) in a way that minimizes the impact of agriculture on the environment.