Recent advances in PGPR and molecular mechanisms involved in drought stress tolerance

Increased severity of droughts, due to anthropogenic activities and global warming has imposed a severe threat on agricultural productivity ever before. This has further advanced the need for some eco-friendly approaches to ensure global food security. In this regard, application of plant growth-promoting rhizobacteria (PGPR) can be beneficial. PGPR through various mechanisms viz. osmotic adjustments, increased antioxidant, phytohormone production, regulating stomatal conductivity, increased nutrient uptake, releasing Volatile organic compounds (VOCs), and Exo-polysaccharide (EPS) production, etc not only ensures the plant’s survival during drought but also augment its growth. This review, extensively discusses the various mechanisms of PGPR in drought stress tolerance. We have also summarized the recent molecular and omics-based approaches for elucidating the role of drought responsive genes. The manuscript presents an in-depth mechanistic approach to combat the drought stress and also deals with designing PGPR based bioinoculants. Lastly, we present a possible sequence of steps for increasing the success rate of bioinoculants. study in Bacillus thuringiensis briefed wheat plants confirmed elongation of root hairs and showed remarkable improvement in root hair density by auxin and ACC deaminase (ACCd) synthesis by rhizobacteria. These results are in synchronization with previously reported work, suggesting about the improved IAA level by a consortium of Bacillus megatherium and Pseudomonas putida in clover plants (55), Phyllobacterium brassicacearum in Arabidopsis plants (56) and B. thuringiensis in Lavandula plants (57). The common IAA-producing bacteria Azospirillum , Bradyrhizobium , and Pseudomonas can encourage root development (58). hydro-absorbers, increased germination potential of on the potential of various microbial inoculants to productivity enhancement and resistance against various biotic and abiotic stresses, a limited to mass-scale agricultural use. common limiting factors for mass-production of bio-inoculants failure of growth enhancement by lab-selected PGPR strains on the field the short expiry period of the formulation, destruction of microbial ecology by fumigants and mineral-based pesticides and fertilizers. These challenges are to be for the widespread use of bio-inoculants sustainable agricultural


Introduction
Various abiotic stresses are increasing in magnitude due to frequent climate changes and anthropogenic activities, among which drought stress is particularly challenging. Drought is a climate-specific term characterized by less than normal or no precipitation within a given time and is broadly categorized into 4 types viz. meteorological, agricultural, hydrological, and socio-economic drought (1). About one-third proportion of the net land area falls into arid and semi-arid regions, though sudden meteorological drought is prevalent in other normal regions. Lack of adequate water supply not only proves fatal to plants but also causes massive societal problems and yield losses (Table 1). Drought is likely to put severe plant growth constraint for about one half of the cultivable lands by 2052 (2). Majorly our food relies on cereals and pulses which need excess water during their cultivation. Drought incidence in such crops can bring serious agricultural damage and can negatively affect global food security. For that reason, the scarceness of water resources has been reflected as a critical worldwide environmental problem.
Droughts have begun to intensify on a global scale, disrupting both ecosystem framework and functioning.
Especially, in arid and semi-arid areas, adverse climatic incidents such as droughts are expected to become more severe, more rapid, and of longer duration (3). As per the report of the Intergovernmental Panel on Climate Change (IPCC) 2018, industrialization has raised the global mean temperature by 1.0 °C. It was also indicated that, by 2052, maintaining anthropogenic activity at the present level could raise the global temperature by 1.5 ° C (4).
Consequently, new strategies are needed more than ever, to uplift production so as to fulfill global food requirements. Extensive studies and policy build-up are being carried out globally to muddle through drought stress.
Development of drought-tolerant varieties, practicing better resource organization, changing the crop calendars, etc are some of the significant strategies used in combating drought. The integration of microbes as a part of cultivation system can be yet another significant as well as eco-friendly strategy against drought (5, 6) economic and sustainable way to uplift production during extreme water deficit. Inside the soil environment, plant-microbe relations are decisive in maintaining primary productivity. Integrating microbes is an effective approach in drought stress mitigation. Up to 41% yield increase was shown in wheat plants after applying a consortium of PGPR and arbuscular mycorrhizal fungi (AMF) (7). Similarly, 40% root enlargement and increased tolerance to water deficit, was observed in pepper plants primed with PGPR (8). Likewise, many other studies highlighting rhizobacterial role in drought stress amelioration were done in peppermint (9), maize (10), foxtail millet (11), guinea grass (12), and pulses (13). However, under severe drought, which is often accompanied by critically low soil water content, PGPRs help plants to survive but fails to maintain crop productivity.
In this review, we are therefore seeking to shed more light on the various mechanisms employed by bacteria to ameliorate the harmful impacts of drought stress on plants and further using them for agricultural sustainability (14).

Drought adversities in plants
Since it is concerned with tissue metabolism, the relative water content (RWC) of leaves is thought to be a good parameter for calculating plant water status. The actual amount of soil water (which remains available for absorption by plants) is known as relative water content, which lies somewhere between field capacity (FC) and permanent wilting potential (PWP). Field capacity is represented by the moisture content left in the soil following gravitational pull, which is stored in the macropores, after being completely saturated with water and reflects the upper limit of available water. The permanent wilting point (PWP) on the other hand, is the matric potential at which plants are unable to pull out water content from the soil. Apart from RWC, drought effects can be studied via various plant parameters like the total anhydrous leaf weight, total anhydrous stem weight, leaf area index, the total number of nodes present, quality of plant fiber, the canopy of plant, and its root development (25,26). However, its generalized effects rely upon various factors like plant species and their genetic constitution, the size, and age of the plant along with the period and stringency of stress (27). Dought can affect various sub-cellular compartments, cell organs, and whole plant levels (28). Water deficit can interfere with seed germination in the early stages of plant development and slows down the plant growth in later stages, by hampering cell elongation (29 (31) . Moreover, many non-stomatal factors also affect the net photosynthesis such as a drop in catalytic activity of RuBisco, amount of CO2 present in the chloroplast, and effectiveness of PSII photosystem (32). Closure of stomata can also trigger reactive oxygen species (ROS) based damage like lipid peroxidation, elevated malondialdehyde (MDA) level, loss of membrane integrity, and degrade protein, lipids, and nucleic acids (33) . Oxidation of photosynthetic machinery frequently causes a decline in chlorophyll level under drought. It also heightens the stress ethylene stature in plants, restraining growth of the plant by different means (34). Drought stress also influences carbohydrates, energy level, respiration, and abscisic acid (ABA) level in the cell (35). Drought can become more detrimental during certain stages of plant growth e.g. if drought abrogates photosynthesis during anther development, it can cause male sterility as pollen maturation ceases due to an insufficient level of carbohydrates inside the cell (36). Thus, drought negatively affects both quantity as well as the quality of growth in plants. Therefore, to ensure global food security, drought stress mitigation is highly desirable.

Mechanistic outlook of PGPR in drought stressed plants
Several mechanisms are known so far, which enable microbes to exert tolerance towards drought in plants, including alteration in osmotic tuning, antioxidant machinery, plant hormonal level, stomatal conductivity, nutrient uptake, release of volatile organic compound (VOC), and exo-polysaccharide production (EPS) ( Figure 1). Besides these, the synergic application of plant growth promoting rhizobacteria (PGPRs) and silicon (Si) also helps in sustaining the physiological fitness of plants for improved carboxylation linked with the increased growth rate in various abiotic stresses like salinity and drought (37).

Osmotic tuning & turgor pressure re-establishment
Osmolytes synthesized by rhizobacteria under drought conditions can enhance the growth of the plant (38). Proline , a crucial osmolyte and can be accumulated during drought condition in plants and owing to its hydration potential, it can easily bind to proteins, making them soluble and thus averting their chances of denaturation. Proline also regulates the redox potential of cells and curtails cell acidity by neutralizing free radicals. When a dehydrated cucumber plant was inoculated with a consortium of Bacillus subtilis, B. cereus, and Serratia sp., a 4-fold increase in proline level was observed (39). Various recent studies suggest a positive correlation between the drought stress tolerance of the plants and their proline content e.g. proline level improved under drought in garden pea (40, 41), chickpea (42), rice (43), and soybean (44). On the molecular level , Δ1-pyrroline-5-carboxylate synthetase (P5CS) and PROLIN DEHYDROGENASE (ProDH) are 2 major genes that adjust proline formation (45). Trehalose is a reducing disaccharide that prevents solidification of the lipid bilayer and also secures other cellular machinery and proteins during drought. It is synthesized by the trehalose-6-phosphate synthase gene (46). Also, betaine a watersoluble alkaloid quaternary ammonium compound (47) aids in strengthening the photosynthetic apparatus, by elevating the synthesis of protective enzymes, and stabilizing membrane integrity under drought. Polyamines like cadaverine, spermine (Spm), spermidine (Spd), and putrescine (Put) are additional osmolytes improving osmotic tolerance during drought stress. Thus, advanced portrayal of crucial genes regulating the synthetic and metabolic pathways of these compatible osmoprotectants are to be characterized on the genetic level, for further improving drought stress endurance in plants. in ROS-quenching enzymatic components involving ascorbate peroxidase and catalase. Similarly, the specific enzyme activity of numerous ROS-quenching enzymes comprising APX, CAT, and SOD was found escalated in PGPR primed plants (48). A similar correlation of drought endurance and CAT profile was shown in cucumber (49) , maize (50), and wheat (51). However, some other studies focused on the assessment of enzymatic antioxidants to investigate the ROS damage that occurred during drought conditions. Lipid peroxidation caused by ROS damage can be evaluated by MDA content. PGPR-treated cucumber exhibited declined MDA level and reduced electrical conductance in leaves for uninoculated plants. These studies clearly illustrate the function of enzymes as ROS quenchers and how PGPR stimulate the oversupply of enzymatic antioxidant gadgetry in plants, to exert drought endurance.

Modulating phytohormonal level
Various chemical regulators are capable of modulating the plant's development and growth. These are better known as phytohormones and comprise a diverse range of chemicals including auxins, cytokinins, abscisic acid, gibberellins, and ethylene, etc. By changing the plant hormone level, PGPR imparts drought resistance in plants.
Water deficit triggered by drought causes photosynthetic drop and badly affects the root metabolism. PGPR  Pseudomonas fluorescens DR7 in foxtail millet showed better adhereance of soil aggregates to root tissue and also increased soil dampness (11).
Salicylic acid (SA) has also been important in plant defense and hence plays a significant role in drought endurance (75). The doubled SA level in barley roots was accountable for improved tolerance under drought (76).
By triggering the activation of various genes like PR ,WRKY and TGA, , SA helps the plant to better cope up with various stressors (77). Similarly, jasmonic acid (JA) successfully advances the plant drought endurance by reinforcing enzymatic antioxidants and increasing the activity of organic osmoprotectants (78).
To conclude, phytohormones have immense potential and are crucial for achieving the target of drought-surviving crops. Further plant's reaction towards drought can be altered by decoding their complex interplay and by increasing or decreasing the regulation of phytohormone-related genes.

Attuned Stomatal conductivity
Drought onset reduces soil water content, leading to the closing of stomata thereby affecting the photosynthesis rate; conversely priming of plants with PGPR augments the photosynthesis rate by enhancing water level, greater CO2 now disperses in the mesophyll area due to extended stomatal conductivity. The stomatal retort to drought occurs by and turgor pressure. The cell membrane of guard cells is placed with numerous protein complexes (e.g. acetylcholine receptors, ABA-binding proteins, light receptors, and GTP-binding proteins), channels, and pumps for smooth trans-membranal K + transport. During drought, stomatal closure helps in water preservation in plants.
Bacterial mediated stomatal movements helped in improved stomatal regulation e.g. the closing of stomata was also observed in Pseudomonas chlororaphis O6 primed plants regardless of the plants ABA stature (79). Similarly, when both normal and ABA-deficient Arabidopsis were inoculated, reduced stomatal opening was observed leading to, reducing transpirational loss and thus delivering drought endurance. Prolonged water stress conditions often generate free radicals and ROS, which irreversibly harm the photosynthetic apparatus after reacting to them.
Rhizobacteria priming can be useful under such conditions since it elevates plant water level and enhances photosynthesis by escalating stomatal conductivity. However, further investigation is needed for a clear picture of the exact pathways which trigger the microbial regulation of the stomata movement in plants under water deficit.

Enhanced nutrient uptake
Water deficit during drought impedes the movement and nutrients diffusion inside the soil due to compromised soil of plants may pave the way to bio-fortification in an environment-friendly manner, along with increasing the green cover and reducing the global carbon footprint.

Release of Volatile Organic Compounds (VOCs)
Bacteria  (84). Plants also produce VOCs in very limited amounts and continually release them from leaves. However, their synthesis is up-regulated during stress which leads to a sharp fall in the photosynthetically fixed carbon. These VOCs work as messengers to elicit stress-related defense responses.

Exopolysaccharide (EPS) fabrication
EPS fabrication by rhizobacteria is a crucial characteristic for growth enhancement during drought as it helps in forming hydrophilic biofilms while inhabiting the roots of the plant, thus shielding them from soil dehydrating conditions (85

Molecular advances to study drought responsive genes
Gene expression studies are a prominent tool to identify and evaluate the all-inclusive reaction of an organism to the environment (113). The transcriptome comprises the complete mRNA, present in a cell at any particular stage or under any environmental conditions. Different techniques for accessing the transcriptome are hybridization-based microarrays, RNA sequencing (114). Whole transcriptome sequencing of a potato colonizing strain i.e. Burkholderia phytofirmans PsJN was also made to examine its effect on drought tolerance (115). The study of rapeseed and its symbiote, Stenotrophomonas rhizophila through transcriptomic analysis helped in the recognition of spermidine, a growth regulator, formed under abiotic stress (116). Proteomics study under drought stress is useful in identifying target proteins and systems can be supported by a relative study of stress, non-stressed, and PGPR-primed plants (117). The increased expression of bab18 (encoding late embryogenesis abundant protein), and erd15 (encoding early response to dehydration 15), in Paenibacillus polymyxa B2 primed Arabidopsis, leading to improved drought tolerance (118). environmental conditions induces metabolism variance which leads towards the alteration exudation pattern (120).
It has been well-documented that under the drought stress, dehydrin-like genes in sunflower (Helianthus annuus) roots and leaves are differentially expressed using RNA sequencing (121). Further, using 2-D polyacrylamide gel electrophoresis (2D-PAGE) some unique proteins responding towards drought has also been recognized in roots of wheat plants (122). In a combinatorial study, utilizing 2D-PAGE and differential display polymerase chain reaction

Seed-coated bioinoculants in drought stress mitigation
The preparation of bio-formulations for plant growth promotion, soil fertility, and the destruction of plant pathogens provide environment-friendly substitute to classical agricultural chemicals (126). Yet, their extensive use is limited by the huge quantity of bio-inoculum required per plant (127). Additionally, lesser research on efficient inoculation procedures in large-acre crops further slow down their commercial level utilization. Bio-inoculants can directly be applied on soil (direct soil application), the seed (seed-applied inoculant), or on the whole plant (e.g., root dipping and foliar spray) (128). A recent study of simultaneous application of biochar and PGPR has reported an increase in RWC and essential nutrient uptake (129). Direct utilization of bio-inoculants on soil is beneficial when there are chances of seed damage by bio-inoculant, but is a cost-intensive method as a higher amount is required per land area  (130). Root dipping and foliar spray are commonly employed for inoculation of plants but the necessity of plant nursery formation and requirement of a higher amount of formulation, are its common drawbacks. Alternatively, seed-applied inoculants is an economical method and effectively places PGPR in its niche i.e. rhizosphere of desired crop (131). Seed coating is an effective technique for the generation of seed-applied inoculants (132). Microbial seed coating is done by combining binders, fillers, and inoculants along with seeds in basic mixing equipment (e.g., cement mixer) or more specialized machinery (e.g., fluidized bed). Binders are common adhesive compounds like Methylcellulose, CMC, Gum Arabic, polysaccharide PelGel, etc. Fillers are bulking agents like Peat, Talc, Lime, etc. Some compounds like alginate work as both filler and binder (133,134). Recently, biochar and chitosan are also used for serving the dual purpose of filler and binders (135)(136)(137). Binders are applied to extend microbial survival.
The right arrangement of the type and concentration of binder and filler is crucial for normal seed germination and further plant growth. A recent report using Pseudomonas putida coated chickpea seeds using starch and SiO2 resulted in increased yield due to elevated biomass under drought (138). Paenibacillus lentimorbus B-30488 coated chickpea along with sodium alginate and calcium chloride (CaCl2) improved the germination rate and colonyforming units (cfu) number, and resulted in better drought stress endurance (139). The report suggested the potent role of sodium alginate and CaCl2 in stimulating the B-30488 biofilm and the coping against drought.
Superabsorbent polymers (SAPs) are water -soluble polymeric compounds that are capable of withstanding over 100 times their weight in water and were used in agriculture since earlier times (140). SAPs are mixed with additive material in seed coating technologies to create encrusted or pelleted seeds (141,142). By enhancing availability of water near the sown seed, SAP and hydro-absorbers, increased germination potential (143). Despite the plethora of published studies on the potential of various microbial inoculants to productivity enhancement and resistance against various biotic and abiotic stresses, only a limited portion has been expanded to mass-scale agricultural use. The common limiting factors for mass-production of bio-inoculants include failure of growth enhancement by labselected PGPR strains on the field (144,145), the short expiry period of the formulation, destruction of microbial ecology by fumigants , and mineral-based pesticides and fertilizers. These challenges are to be overcome for allowing the widespread use of bio-inoculants sustainable agricultural systems.

Discussion
Drought is a significant limiting factor in agricultural productivity. This review provides a detailed study of drought effects on crop plants and encourages the implementation of plant growth-promoting rhizobacteria as an environment-friendly approach for tackling drought and leading to agricultural sustainability. Further, using molecular and omics-based techniques for the study of several drought-responsive genes has broadened our knowledge of diverse functional attributes of rhizobacteria under drought. For more ecologically responsible and sustainable agricultural activities, and cleaner & better food choices, microbial inoculants are the best potential candidates. Recent research indicates that rhizobacteria can impart drought tolerance in plants. Rhizobacteria-primed plants employ multifarious means including, osmotic adjustment, increased antioxidant level, phytohormone production, increased nutrient uptake, EPS production, releasing VOCs, etc confer resistance against desiccation.
All these above mentioned mechanisms almost invariably function by improving RWC of the plant under drought.
While it is well-established that PGPR treatment is associated with increased RWC, the exact mechanism behind this shift is unclear. PGPR-driven ABA surge in plants, followed by quick stomatal closure is widely believed to increase RWC under drought (146).However, another accepted viewpoint relates the spike in RWC with the changes in responsiveness of physiological functions including stomatal closure (147). Such divergent opinions highlight the importance of deciphering the mechanisms underlying bacterial-mediated drought tolerance through increased RWC. Moreover, designing PGPR-based bio-inoculants to assist plants in dealing with drought -stress is a new avenue in microbial use in dry land agriculture. Seed coating could be an efficient approach for rhizobacteria-based bio-inoculants since it effectively translocates the PGPR into the plant's rhizosphere. However, very fewer studies focused on the rhizobacterial inoculants and their effectiveness under drought has been made so far. This review provides a possible sequence of steps for increasing the success rate of PGPR based bioinoculants ( Figure 2). Using the present clues, a more focused study is required about determining the appropriate rhizobacterial strains, their effective delivery mechanisms, and field testing of potential candidates.