Management of Rhizosphere Microbiota and Plant Production under Drought Stress: A Comprehensive Review

Drought generates a complex scenario worldwide in which agriculture should urgently be reframed from an integrative point of view. It includes the search for new water resources and the use of tolerant crops and genotypes, improved irrigation systems, and other less explored alternatives that are very important, such as biotechnological tools that may increase the water use efficiency. Currently, a large body of evidence highlights the role of specific strains in the main microbial rhizosphere groups (arbuscular mycorrhizal fungi, yeasts, and bacteria) on increasing the drought tolerance of their host plants through diverse plant growth-promoting (PGP) characteristics. With this background, it is possible to suggest that the joint use of distinct PGP microbes could produce positive interactions or additive beneficial effects on their host plants if their co-inoculation does not generate antagonistic responses. To date, such effects have only been partially analyzed by using single omics tools, such as genomics, metabolomics, or proteomics. However, there is a gap of information in the use of multi-omics approaches to detect interactions between PGP and host plants. This approach must be the next scale-jump in the study of the interaction of soil–plant–microorganism. In this review, we analyzed the constraints posed by drought in the framework of an increasing global demand for plant production, integrating the important role played by the rhizosphere biota as a PGP agent. Using multi-omics approaches to understand in depth the processes that occur in plants in the presence of microorganisms can allow us to modulate their combined use and drive it to increase crop yields, improving production processes to attend the growing global demand for food.


Introduction
Projections of agroclimatic models indicate a strong impact of global climate change (GCC), represented by both temperature increases of 0.5 to 2 • C by 2100 and a significant decrease in rainfall [1,2]. This change will undoubtedly promote a reconversion of agronomic practices to produce the necessary food for a growing world population, both in volume and requirements of high-quality products [3]. Regardless of the plant species cropped, agricultural production depends on a significant proportion of water being supplied mainly The above scenario generates double uncertainty: (i) whether the inoculant can be beneficial to more than one host plant species; (ii) whether the plant responses can be mechanistically equivalent between different host plants using the same inoculant. Currently, the multiple soil-microorganism-plant interactions represent an unexplored "black box". Much less effort has been put into the validation at the field level of an inoculant's effectiveness beyond the argued socially friendly decrease in the use of chemical inputs, as commonly advertised by marketing agricultural companies.
Against this scenario, the current "omics" tools emerge as an attainable alternative to clarify "what is happening in this black box", allowing us to describe and predict behaviors in plants based on powerful genomic, transcriptomic, proteomic and metabolomic tools. Therefore, with this focused review, we hoped to summarize the current and updated knowledge regarding the role of microbial tools used as bioinoculants to improve the plant production. In addition, we highlight the projections based on the use of single-omics platforms and multi-omics approaches. With this, our aim was to elucidate the mechanisms that explain the improvements in yield and food quality under drought stress, as one the main sensible effects of climate change in agriculture.

The Drought as Main Constraint for Plant Production in a Global Climate Change Scenario
Agriculture meets our food demands (food security), being the main practice that contributes to the economy in many countries worldwide. However, the intensification of agriculture has also led to the degradation and exhaustion of soils; moreover, about 38% of suitable agricultural lands around the world have been degraded by inadequate management practices [25,26]. Supplying the increasing world population with sufficient food is mandatory. Because of this, food security is the most important challenge in the 21st century. However, nowadays, food security is strongly threatened by global warming [27].
Global warming and associated climate change not only affect air temperature but also influence the amount and distribution of rainfall [28]. Predictive models of GCC have shown that the frequency of precipitation events and net volumes have drastically changed during the past one hundred years. It has caused frequent and severe periods of drought in large areas around the world [29].
The agricultural sector is responsible for about 75% of the total global consumption of water [30]. However, in the wake of GCC, drought has emerged as one of the major abiotic stresses and is considered the strongest environmental stress that limits the plant growth, reducing crop productivity [31,32]. Due to the droughts, yield reductions have been reported in the order of 21 to 40%, from 1980 to 2015, for wheat (Triticum aestivum) and maize (Zea mays) productions, two of the most important crops worldwide [33]. Moreover, considering that among 80-95% of the fresh weight of plants consists of water, it is evident that drought stress leads to changes on a multidimensional level, modifying different physiological, biochemical, molecular, and morphological processes in plants [34][35][36].
The responses of plants to water stress depend on the length and severity of the water deficiency [37]. Many plants have developed mechanisms to tolerate water stress, but these mechanisms are varied and depend on the plant species. Included among them are developmental, physiological, morphological, ecological, biochemical and molecular mechanisms [38]. Mainly, the mechanisms involved in plant tolerance to drought are based on maintaining cell water homeostasis under drought conditions, allowing diminished water loss and increasing the water inlet to the plants [39].
To improve plant responses against the water stress, different approaches are currently being studied, such as traditional breeding methods, transgenic technology, and priming methods, among others. However, because of the complexity of drought effects on plants and the specific responses of plants to the water stress, each method has some problems and limitations [40][41][42].
In addition, the role of root-associated microbial communities able to improve plant drought tolerance has only been explored in recent years [43]. In this way, different studies have reported that beneficial soil microorganisms improve plant tolerance to abiotic stresses by producing a root-soil interface that directly or indirectly enhances the absorption of water and nutrients. The most important groups of microorganisms related to increased tolerance to water scarcity include the PGP rhizobacteria and AM fungi [5].

Rhizosphere Microbial Groups and Ecological Roles for Plant Production under Drought Conditions
Currently, due to the importance of the functions that soil provides, it is widely recognized that soil represents one of the main frontiers of science. The functions that soil provides strongly determine the productivity of terrestrial ecosystems, both natural and agricultural-modified ones [44,45]. The sustainable development of agriculture is mediated by biotic and abiotic factors, the rhizosphere being an environment that can be considered the common point shared by both factors. The health of the rhizosphere matrix is closely related to the state of its present PGP microbial communities [46].
In this ecological niche, factors such as the availability of oxygen, nutrients (C:N:P ratio) and water modulate a significant fraction of the total interactions in the rhizosphere [47]. In addition, in this volume of soil, it is possible to find exudates from plant roots (rhizodeposits), which are considered an essential component of the rhizosphere [48,49]. The presence or absence of rhizodeposits can significantly affect rhizosphere interactions since they fulfill signaling functions to give way to the symbiotic association between the plant and PGP microorganisms [50].
Another fundamental characteristic in the ecology of the rhizosphere is the restriction interactions of microorganisms. It has the characteristic of restricting other microorganisms from interacting with the plant (e.g., plant pathogen microorganisms). This converts the rhizosphere into a bridge of chemical communication between the plant and the PGP microorganisms for the formation of protective microbial biofilms, acting as biocontrol agents in both the defense and resistance process of the host plant [51].
The rhizosphere comprises a complex and dynamic microenvironment, with mediating characteristics in the selection of organisms that interact with the host plant. The rhizosphere can be understood as a holobiont assemblage, which is formed by the association of a host and its microbial communities [52,53]. The holobiont impacts the stability, adaptation and evolution of the organisms involved in the assemblage [54].
Under stress conditions, such as drought, salt, and poor nutrient availability, the proper functioning of the holobiont is essential for the development and growth of plants, and thus, for improving their tolerance and nutrient-obtaining capacities [55]. In summary, the ecological relationship between the rhizosphere and the characteristics of PGP microorganisms is essential for the growth, nutrition, and quality of crops, both under favorable and stress conditions. This importance is reflected even more in a context of climate change, where interactions at the rhizosphere level play a fundamental role in the health of crops [56], carbon sequestration, nutrient cycling, and rhizosphere ecosystem functioning [57,58].
Plants are closely associated with soil microorganisms both externally and internally in various ways [59]. Numerous works have demonstrated the important role played by rhizosphere microorganisms in plant growth, highlighting those with PGP capacity (Figure 1). These PGP traits have been evidenced in several studies using different rhizosphere microorganisms (Tables 1 and 2). PGP microorganisms, in addition to benefiting plant growth, are also characterized by having the ability to produce hormones and lytic enzymes, showing a promising use as bioinoculants [60]. Some microorganisms are also involved in the production of secondary metabolites, thus promoting carbon sequestration, nitrogen fixation, and phosphorus solubilization in the rhizosphere [61,62]. PGP microorganisms (fungi, yeasts, and bacteria) are present in the soil and can contribute to maintaining or enhancing soil health, improving plant growth, inducing systemic resistance in plants, and increasing stress tolerance against different unfavorable environmental conditions, both biotic and abiotic [63]. Several works have described these PGP attributes, emphasizing mainly their ability to: (i) ensure greater nutrient availability for plants [64], (ii) stimulate changes in their root structure [13], (iii) promote the establishment and growth of plants under conditions of abiotic stress (e.g., potentially toxic elements, salinity, and drought) [5,12,[65][66][67], or (iv) help with the control of phytopathogens [68].
All the above beneficial traits are normally observed at rhizosphere level, with scarce reports that include the effects on host plants. The use of PGP microbes has been analyzed through effects on plants such as: (i) improvements in photosynthetic variables [7,8,76,77], (ii) biofortification of plants with essential [78,79] and beneficial nutrients [19,20], (iii) modification of antioxidant responses [66,80], and (iv) changes in secondary metabolite profiles [81][82][83], among others. PGP microorganisms (fungi, yeasts, and bacteria) are present in the soil and can contribute to maintaining or enhancing soil health, improving plant growth, inducing systemic resistance in plants, and increasing stress tolerance against different unfavorable environmental conditions, both biotic and abiotic [63]. Several works have described these PGP attributes, emphasizing mainly their ability to: (i) ensure greater nutrient availability for plants [64], (ii) stimulate changes in their root structure [13], (iii) promote the establishment and growth of plants under conditions of abiotic stress (e.g., potentially toxic elements, salinity, and drought) [5,12,[65][66][67], or (iv) help with the control of phytopathogens [68].
All the above beneficial traits are normally observed at rhizosphere level, with scarce reports that include the effects on host plants. The use of PGP microbes has been analyzed through effects on plants such as: (i) improvements in photosynthetic variables [7,8,76,77], (ii) biofortification of plants with essential [78,79] and beneficial nutrients [19,20], (iii) modification of antioxidant responses [66,80], and (iv) changes in secondary metabolite profiles [81][82][83], among others.
However, it should be noticed that the most recent reports highlight the increased tolerance of plants against water stress, such as those based in salinity and drought, because of direct and indirect effects by the above-described traits. In this sense, microorganisms with PGP capabilities have been shown to promote plant growth under water deficit stress conditions, suggesting that they are naturally acclimatized to the stressed environment [84].
In order to enhance plant growth, the most common effect of bacteria on plants exposed to drought conditions is the improvement in the antioxidant plant responses, reducing the cell damage (mainly in membranes) and alleviating their status of stress (Table 1).  AM fungi are another important microbial group studied by its PGP effects. AM fungi are an obligate biotroph that depend on living root tissue for carbohydrate supply, which allows them to complete their life cycle [101]. AM fungal colonization on plant roots occurs when its hyphae penetrate the epidermis and grow extensively between and within living cortical cells, forming a very large and dynamic interface between both symbionts. It enhances plant growth and yield and also decreases the effects of several abiotic stresses [102].
This obligate symbiosis can promote the formation of stable aggregates and improve water storage in the soil through the production of glomalin, which is released in large amounts into the soil [5,75,103]. Moreover, the formation of AM symbiosis can change the efficiency of water uptake by modifying the ionic balances (Na/K) in the host plant, as well as modifying the relative expression of PIP aquaporin and ionic NHX antiporter genes under osmotic stress [5,104]. New reports using AM fungi in association with other PGP microorganisms have also shown a beneficial (synergic or additive) effect on plant growth and development when established in water-scarce environments ( Table 2).  Trifolium repens L. On the other hand, the yeast microbial group has also been studied for their PGP capacity. However, there are fewer studies compared to the groups described above, mainly including some works exploring its PGP attributes under water stress conditions. For instance, Silambarasan et al. [7,18] demonstrated the ability of EPS produced by yeasts to promote the formation of stable aggregates and improve the storage of water in soil.
Furthermore, recent evidence showed that yeast application can upregulate soil enzymes under drought stress conditions, which increased the nutrient content in the soil, also improving the osmotic state of roots and the activity of antioxidant enzymes in the plants treated with the strains [63]. PGP microorganisms are undoubtedly a key element for the adaptation of plants to new unfavorable environmental scenarios. Deepening studies oriented to evaluate the interspecies synergistic potential of PGP microorganisms in the current framework of climate change are required.

The "Omics" Approaches and the Development of Optimized Bioinoculants
As stated above, the use of microorganism for improved drought tolerance has shown promising results in different crops [120,121]. In this sense, rhizosphere engineering (modification in the microbial rhizosphere community by known PGP microorganisms) represents a faster and more advantageous alternative to improve drought tolerance in crops than other tools, such as genetic engineering or genetic improvement [122].
The interaction of the newly added microorganisms with the rhizosphere is the first barrier to overpass to establish a relation with the already naturally present microbiome, and in the better situation, form a mutualistic and cooperative relationship [22,123]. Then, the newly formed microbiome interacts with the plant root and may produce an increase in the physiological traits that allow the plant to confront the drought stress. This promotes, for instance, the formation of lateral roots [124], generates increases in hormone levels (ABA, cytokinin, and gibberellin) associated with increases in the water content and the hydric status of plant organs [125][126][127], improves the expression of osmotic adjustment systems (proline), and promotes the production of antioxidant enzymes [128], among others.
However, a negative effect on the plant growth and development is also a possibility [129]. The way how this microbial community increases the tolerance to abiotic stress is not completely understood yet. This gap in information is due to the complex media where microbial communities are developing, which is the most complex compartment on the Earth's surface [24]. In this sense, understanding the interaction among microbial communities, plants, and the biotic and abiotic factors is an essential step to improve the design of bioinoculants [130,131].
A modern approach to study this interaction is the use of "Omics" technologies, which are also being applied in microbial science (Table 3). This set of new techniques allows to integrate the information of genome, proteome, transcriptome, and metabolome and gives information about the biological changes that underlie the drought tolerance produced by advanced bioinoculants [24,132]. Metabolomics is based on tools that allow the identification of the complete metabolites synthesized by an organism and can be used for the determination of how this metabolic profile changes in different conditions, such as drought stress [139]. The usual workflow to determine a metabolic profile starts with sample acquisition, sample preparation, data acquisition, bioinformatic analysis, and biochemical interpretation [140].
For example, the application of the metabolomics approach in the study of drought condition in the root of the trifoliate orange and the interaction with the AM fungus Rhizophagus intraradices showed a total of 88 and 17 metabolites upregulated and downregulated, respectively, also showing an improvement in the physiological status of the mycorrhized plants [142]. In the same way, the use of metabolomics tools in the application of a consortium of Bacillus subtilis, B. thuringiensis, and B. megaterium in chickpea showed an accumulation of riboflavin, L-asparagine, aspartate, glycerol, nicotinamide, and 3-hydroxy-3-methyglutarate under drought condition, together with the reduction in the deleterious impact on the plant status [143].
On the other hand, proteomics is the use of different technics that allows to determine the complete contents of the different proteins present in an organism under specific circumstances [144]. Proteomics analysis can be used in certain ways, such as for translational proteomics, protein-protein interaction, post-translational modification, and proteomics studies at a comparative level for the comparison of protein profiles, including drought stress [141].
The typical workflow of proteomics analysis starts with the extraction of proteins in a plant tissue of interest, digestion of proteins with specific enzymes into peptides and identification of resulting peptides by mass spectrometry, quantification of protein expression, and determination of post-translational modifications [145]. The widely used techniques used for the study of proteomes include 1D and 2D gel electrophoresis, followed by mass spectrometry with different ionization sources. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS/MS) and electrospray ionization (ESI) are the main mass spectrometry tools used in proteomics approaches [141].
With the use of proteomics approaches, it was possible to find in pepper plants inoculated with B. licheniformis K11 a total of 15 differential expressed proteins that confer tolerance to drought stress [145]. Similar results were observed for the inoculation of Rhizobium leguminosarum and Pseudomonas putida to Vicia faba, which produced changes in the proteomic profile with an improvement in the tolerance to drought stress [145].
Genomics is the study of all genes present in an organism with the respective identification of sequences, intragenic sequences, and genes structures [145]; meanwhile, transcriptomics is focused on the determination of the RNA present in the specific organs, which is highly dependent on the specific environmental condition that makes the transcriptomic highly variable [146]. In this sense, the study of genomics allows to know the potential of microorganisms to produce secondary metabolites or proteins to enhance the growth and development of drought tolerance with plant interaction.
Furthermore, transcriptomics allows to determine how this genomic potential is expressed under specific circumstances. In both cases, the starting point is the extraction of the nucleic acid, but for RNA, it is necessary to synthesize the cDNA and then sequence it in a next-generation sequencing platform [141]. The use of the integrated genomic and transcriptomic approach showed that some colonization genes such as FixL/FixK/FixJ and NodD were upregulated in the presence of beneficial microorganisms [147]. Moreover, there are several types of plant growth-promoting rhizobacteria (PGPR) with the full genome assembly, which previously have shown an improvement in the plant tolerance to drought stress, such as B. amyloliquefaciens [148], Serratia plymuthica [149], Hartmannibacter diazotrophicus [150], and Rhizophagus irregularis [151].
The omics approaches still have important bottlenecks, such as the need of different, focused, and specialized researchers; and the gap in both information and integration of data available in the worldwide databases (e.g., one-stop shop) [152]. Currently, data integration can be performed post-analysis by performing "networking" after individual analyses [153] or carrying out an integrated data analysis in parallel. This last one requires specialized tools to merge data from different platforms prior to the final interpretations of the results [132,154]. For instance, MetaboAnalyst allows for multiple integration of the metabolome, transcriptome, proteome, and genome into a wide spectrum of biological samples, including plants [155], as well as data processing and statistical analyses based on the R platform [132].
Undoubtedly, the research possibilities with the incorporation of multi-omics approaches will be a strong basis for the functional interpretation of the effects that advanced biofertilizers have on their host plants. Based on the strong progress that these approaches have had in other sciences (mainly medicine and human health), it is feasible to state that they may represent the starting point for the design of optimized biofertilizers in a wide range of plant species of agricultural and environmental interest.

Perspectives and Conclusions
As stated above, a large body of evidence highlights the role of specific microbial strains within the main rhizosphere microbial groups in conferring drought tolerance to plants. However, much less known are the physiological, molecular, and biochemical mechanisms and responses displayed by plants as a consequence of the presence and action of these PGP microorganisms.
In the case of yeasts, these microorganisms have very interesting PGP characteristics that have not been widely reported, especially regarding water stress. Nevertheless, recent studies have demonstrated their great potential as coadjutants in plant growth to cope with other abiotic stresses. Such evidence makes us presume that yeasts can be a key element with a great biotechnological value that needs to be explored as a tool to face the food shortage that promises to be accentuated with the advance of GCC. Therefore, the next steps for the designing of biofertilizers supported by the use of multi-omics approaches could represent a significant leap in the research regarding the role of rhizosphere microbial communities in plant production under drought conditions. In this sense, the integration of omics platforms will strongly support the mechanistic understanding that underlies the use of beneficial microorganisms in plant production through rhizosphere engineering.
While it is necessary to realistically recognize that the tangible results could primarily be framed at the local level, considering particular soil conditions, rhizospheres, and crop plants, the research at the pilot scale will establish the basis for the generation and massification of optimized biofertilizers. However, some considerations must be addressed, as the development of local biological collections for the ex situ maintenance of PGP microbes able to enhance drought tolerance represents a valuable resource for testing their applicability in different crops, in line with the global advice for the maintenance of genetic resources oriented to agriculture and food production [156].
Moreover, the projection of the fundamental and mechanistic bases studied in the plant, considering different efficient rhizosphere modifications, can also be focused on different types of environmental stress, such as the low availability of nutrients, salinity, heavy metals, extreme pH values, and many other environmental and soil constraints that currently affect enormous areas of arable land surfaces worldwide.