Application of microbial synthesized phytohormones in the management of environmental impacts on soils

With the world's population growing at an exponential rate, pollution of the ecosystem by heavy metals from anthropogenic activities poses a major threat to agricultural and food security worldwide. Phytohormones are biochemical signal molecules that alter plant responses to different biotic and abiotic stresses. Exogenous use of microbially produced phytohormone in heavy metal remediation and stress tolerance induction, has gained popularity due to its environmental friendliness and sustainability. Microbially produced phytohormones have huge biotechnological potentials and have been exploited in phytoremediation assisted removal of heavy metals, and inducing stress tolerance to plants. This paper exhaustively discusses the remedial roles of microbial phytohormones in heavy metal removal and enhancing plant tolerance to stress. However, the exact mechanism of action and the genetic interplay during the process need to be further studied to better understand the specific key pathways involved in the process.


INTRODUCTION
Climate change, which is usually driven by abiotic stressors, poses global threat to the environment and agricultural systems. Nigeria, like many other developing countries, is unprepared f or the ef f ects of global warming and loss in biodiversity as evidenced by rising sea levels and erosion along the nation's coastline (Olaniyi et al., 2013). Abiotic stress ref ers to environmental variables such as extremes in temperature, water, nutrients and any other environmental elements that af fect plants and limit their development and output below optimal levels. One of such abiotic stresses is soil pollution by heavy metals, which is a major recurrent concern across the world, with negative consequences. These pollutants negatively af fect available arable land, limiting the world food supply chain. The world's population is rapidly growing, and it is predicted to reach 9 to 10 billion people by 2050 (UN, 2019). This projected exponential increase in the global population poses serious challenge to the world f ood supply. Abiotic stressors, in general, have the potential to signif icantly limit agricultural plant development and production, resulting in signif icant yield reductions and presenting a threat to agricultural systems' long-term sustainability and global warming (Mahalingam, 2015). Besides the secondary consequences of climate change, rising Carbon dioxide (CO2) levels and heat wave due to increased temperature have an immediate inf luence on plant growth, propagation, and tolerance.
Plants are rooted in the soil and must respond to abiotic stress using a variety of techniques, if they are to f lourish. Their adaptation strategies against dif f erent stressors include morphological and biochemical changes as well as changes in physiological processes. These changes in morphological and biochemical processes are regulated by certain molecules known as phytohormones that work as chemical messengers and promote plant growth and development (Vob et al., 2014). Phytohormones such as auxins, abscisic acid and gibberellins operate as growth regulators by promoting seed germination and initiating plant vegetative development in response to oxidative stress caused by heavy metals and other abiotic stressors (Li et al., 2010;Adato and Gazit, 2014;Kang et al., 2015). Pirog et al. (2017) noted that these growth regulators are getting prominence in the agro-industrial sectors of advanced economies due to their capacity to improve crop yield and regulate plant metabolism.
Phytohormones, also known as secondary metabolites, have been discovered in algae, as well as bacteria and f ungi associated with plants (Wang et al., 2015;Egamberdieva et al., 2017). Plant growth promoting rhizobacteria (PGPR) predominate the plant rhizosphere, which are capable of boosting plant development directly (via phytohormone synthesis) or indirectly (via the antimicrobial agents that can suppress the growth of pathogens). Greenberg et al. (2008) linked these PGPRs to the reduction of the deleterious ef f ects of salinity stress on plant growth, while Arkhipova et al. (2019) noted that though the relative importance of specif ic bacterial trait was unclear, f ield experiments revealed that inoculation of wheat seeds with auxin-producing or phosphate-solubilizing strains raised crop yield by 10% to 36%.
In addition to promoting and modulating plant growth, microbial synthesized phytohormones also have unique remediation potentials by signaling the presence and stimulating the production of an endogenous def ensive mechanism. This consists of dif f erent enzymatic (superoxide dismutase, catalase, dehydrogenases, glutathione reductase, etc.) and non-enzymatic (ascorbic acid, alkalo ids, f lavonoids, α-tocopherol, etc.) antioxidants (Gill and Tuteja, 2010;Hasanuzzaman et al., 2020). Plant cells maintain steady-state homeostasis through an antioxidant def ense mechanism and the buildup of reactive oxygen species (ROS) (Hasanuzzaman et al., 2012). Plant hormone levels in plant tissue are modulated by microbial regulators, which have been discovered to have ef f ects comparable to exogenous phytohormones (Shahzad et al., 2016;Egamberdieva et al., 2017).
This review describes possible roles of microbial phytohormones in controlling plant growth and enhancing plant tolerance to stress. We highlighted the current status of the application of phytohormone-producing microbes in imparting tolerance against several environmental stressors whilst modulating plant growth.

PHYTOHORMONES
Since the discovery of phytohormones, plant biologists have been captivated by their regulatory ability. Researchers have been intrigued by the possibility that hormone levels or responses may be changed to enhance desired plant traits (Gray, 2004). It has long been known that their capacity to promote plant adaptation to continuously changing climates by inf luencing growth, development, and nutrient allocation is important. Phytohormones are "signal molecules" that inf luence plant growth and stress tolerance ( Figure 1).

Plant Synthesized Phytohormones
Among the f ive "classical phytohormones", auxins were the f irst to be discovered, f ollowed by gibberellins, ethylene, cytokinins and abscisic acid. Other phytohormones discovered recently include strigolactones, salicylic acid, jasmonates and brassinosteroids, polyamines, and nitric oxide. Abscisic acid, salicylic acid, jasmonates, and ethylene have all been shown to play critical roles in controlling plant def ense responses to pathogens and abiotic stresses (Bari and Jones, 2009;Nakashima and Yamaguchi-Shinozaki, 2013). These phytohormones work in harmony with one another, responding to developmental and environmental signals, through synergistic and antagonistic activities known as signaling cross talk. Knowledge of the key communication signals that def ine f avorable or negative plantmicroorganism interactions is critical for improving def ensive responses while maintaining advantageous relationships such as symbiosis (Boivin et al., 2016).
Auxin (indole-3-acetic acid; IAA) was the f irst phytohormone to be discovered and can stimulate plant growth at low concentrations. Since auxin plays a vital role in plant growth, the search to unravel the processes behind its action is both exciting and daunting (Perrot-Rechenmann, 2010). It has been noted that Auxin might be more of a signal transduction molecule, which triggers a pre-set system, than a hormone with a specif ic f unction (Bennett and Leyser, 2014). Auxin can cause extremely quick nontranscriptional reactions, like stimulation of the cellular membranes' proton gradient and signal transduction, as well as microtubule reorientation (Weijers and Wagner, 2016). It can, in combination with cytokinin at physiological concentrations, induce cell division which increases plant cell growth, and plays key roles in nutrient source-sink interactions, root shape, and rhizosphere activity (Paque and Weijers, 2016). Cytokinins and auxins have been noted to induce cell division and undif f erentiated growth in tissue cultures by interacting synergistically. Similarly, cytokinin and gibberellin stimulate seed germination in certain plants. Auxin also interacts antagonistically with cytokinins in the regulation of growth of stem and hypocotyl segments and opposes lateral bud development in plants (Splivallo et al., 2009).
Since most of these plant hormones do not work alone on a specif ic developmental process, maintaining balance amongst them is usually paramount, in order to avoid reversibly inactivating any phytohormone. There is more research on the ef f ect of exogenous applications of plant produced phytohormones and how they inf luence plant development than studies on endogenous concentrations and how they af fect plant growth. There should be more studies f ocused on understanding how endogenously produced phytohormones inf luence the molecular and physiological responses of plants. This would go a long way in enhancing phytoremediation of toxic metals and organic pollutants f rom the environment.

Microbial Synthesized Phytohormone
Microbial phytohormones inf luence the metabolism of plant hormones produced endogenously by the plant's tissues and are involved in root morphological changes caused by drought, salinity, heavy metal toxicity and severe temperatures (Sorty et al., 2016). However, the degree of substantiation f or their participation might vary considerably depending on the individual plant hormone and the examined microbial strain (Spaepen, 2015). The use of microbial mutant strain def ective in phytohormonal production, f urther establishes the microbial phytohormone's participation, as detecting if a particular phytohormone in a microbial culture's supernatant is inadequate.
Historically, there are f ive dif f erent classes of phytohormones produced by rhizobia microbes (symbiotic, f ree-living or endophytic) namely; auxins, gibberellins, cytokinins, abscisic acid and ethylene. Jasmonic and salicylic acid are two more modulators discovered as microbial phytohormones that contribute to long -term plant growth and production. The f ungus Lasiodiplodia theobromae produces jasmonic acid, while Penicillium patulum produces salicylic acid. The microbial synthesis of phytohormones such as auxin (one of the most important phytohormones) occurs through numerous pathways with tryptophan being the most important precursor. With tryptophan as the precursor, only six biosynthetic routes f or the synthesis of IAA in microorganisms have been identif ied, with majority of the pathways predicated on the presence of metabolic intermediates in the culture medium and most of the pathways resembling those f ound in plants, while some intermediates may dif f er (Spaepen et al., 2007). IAA synthesis by rhizobia is regulated by certain genes, and overexpression of this auxin has been observed in a number of bacterial mutants, notably Ensifer (Sinorhizobium) meliloti RD64 as compared to the wild type (Def ez et al., 2019). Free amino acids such as valine, alanine, aspartic acid, and glutamic acid, as well as photosynthetic products, have been signif icantly enhanced by IAA-producing rhizobia (Tsikou et al., 2013;Erice et al., 2014;Def ez et al., 2019).
Plant benef icial bacteria such as Azospirillum, Bacillus, Bradyrhizobium, Enterobacter cloacae, Paenibacillus, Pseudomonas, and Rhizobium make use of a pathway where an aromatic aminotransf erase transaminase, the precursor tryptophan to IPyA in the IPyA pathway as the f irst step. The second step which is the rate limiting stage involves the decarboxylation of indole-3-pyruvate IPyA to indole-3-acetaldehyde (IAAld) by an important enzyme indole-3pyruvate decarboxylase (encoded by the ipdC gene). IAAld is f inally transf ormed to IAA. The regulation and biochemical characterization of the second stage in this pathway has been researched extensively in a variety of bacterial species (Patten et al., 2013).

APPLICATIONS OF MICROBIAL SYNTHESIZED PHYTOHORMONES IN PHYTOREMEDIATION
In the light of global climate change and unsustainable f ood security, exogenous phytohormone supplementation plays a pivotal role in remediation of heavy metal contaminated soils and in modulating plant stress response (Saini et al., 2021). Heavy metal concentrations in uncontaminated soils are quite low (Ma et al., 2016a). Rapid urbanization, uncontrolled use of agricultural chemicals, and anthropogenic activities due to industrialization ultimately lead to increased accumulation of heavy metals in soils (Kumar and Verma, 2018). These have detrimental impact on plants and humans. Thus, f or conservation and protection of the ecosystem f rom the deleterious impacts of these pollutants, it is of utmost importance that sustainable and ef f ective remediation techniques be employed for the removal of the pollutants. Multif aceted traits have proved the ef f ectiveness of microbially synthesized phytohormones in combating these challenges (Chirakkara et al., 2016;Ma et al., 2016b).

Microbial synthesized phytohormones and mechanism of heavy metal removal
Plants have been known to exist in benef icial synergistic relationship with rhizobacteria, and exudates f rom such interactions have been implicated in variety of biogeochemical processes that have a signif icant impact on plant development and survival (Glick, 2010;Dharni et al., 2014). It has been proposed that using phytohormone-producing heavy metal-tolerant PGPR may be a f easible method f or tackling the serious problem of heavy metals contamination in agroecosystems (Sytar et al., 2019;Nazli et al., 2020).
Apart f rom growth promotion, phytohormone heavy metal-tolerant rhizobacteria have the potential to remediate heavy metal polluted soils and improve agricultural system productivity. Phytohormones produced by these rhizobacteria can be utilized exogenously in removing contaminating heavy metals through biosorption and bioaccumulation (Luo et al., 2011). The possibility of using such exogenous synthesized phytohormones in heavy metal removal and stress endurance in plants holds great promise due to its sustainability and cost ef f ectiveness (Nguyen et al., 2021).
Varieties of microalgae produce phytohormones which enable them to withstand and survive biotic and abiotic stress. It has been observed that in multi-metal-challenged environments, phytohormones increase heavy metal uptake by raising the concentrations of primary metabolites in the microalgae Chlorella vulgaris L. (Tassi et al., 2008;Cassina et al., 2011). Similar studies also showed a reduction in mercury toxicity by the exogenous application of salicyclic acid (Zhou et al., 2009). Exogenously administered cytokinins assist in heavy metal uptake by raising plant transpiration rate (Pospisilova, 2003). The majority of heavy metals taken up f rom the soil are retained in root cells, where they are detoxif ied via chelation in the cytoplasm or sequestration into vacuoles, whereas hyperaccumulator plants translocate these elements to the shoot via the xylem quickly and ef f iciently (Monf errán and Wunderlin, 2013). As a result, the f low of water-soluble soil components or pollutants to the plant's upper portions (through xylem sap) rises (Dodd, 2003).
Metal chelation process is quite crucial in heavy metal entrapment and deposition in plant cell vacuoles.
An Auxin derivative, 1-Naphthaleneacetic acid (NAA), has been reported to increases the concentration of hemicellulose 1 in plant root cell wall. This enhances cadmium (Cd) f ixation in roots, decreasing its upward migration towards the shoot and providing resistance to heavy metals (Saini et al., 2021). These phytohormones stimulate the release of root exudates, which are metal chelators (Mahmood et al., 2015). Alterations in phytohormones concentrations, in most cases, signal the activation of heavy metal chelators. Abscisic acid has been suggested to be a stress signaling molecule which induces plant responses under stress conditions (Hayward et al. 2013). Heavy metals are absorbed into the roots where they f orm complexes with various chelators, such as organic acids. These complexes are immobilized in the apoplastic cellular walls or vacuoles, where they can be degraded by intracellular enzymes (Rascio and Navari-Izzo, 2011;Seth, 2012;Ali et al., 2013;van der Ent et al., 2013).
The phytoremediation of lead (Pb) using maize crop (Zea mays) was studied by Fuentes et al. (2000) and they reported an increased uptake by bioaccumulation when treated with either indolebutyric acid (IBA) or naphthylacetic acid (NAA). Treatment with microbially synthesized IBA showed a 41.2% Pb removal, and 87.4% Pb removal using NAA. Some other study showed that application of 28-homobrassinolide, a phytohormone, reduced soil nickel and cadmium heavy metal concentration (Janeczko et al., 2005).

Mechanisms of action of Auxin during phytoremediation
Apart f rom its pivotal role in plant growth regulation, exogenously applied auxin induces plant response to stressors by regulating biosynthetic, transduction, and disintegration activities (Potters et al., 2007;Saini et al., 2017;Bücker-Neto et al., 2017). In mutant plants, Krishnamurthy and Rathinasabapathi (2013) discovered that aux1 seedlings were more resistant to arsenic. Similar study by Elobeid et al.
(2012) f ound enhanced bioaccumulation and absorption of Cadmium (Cd) by cottonwood (Poplar) f ollowing exogenous auxin treatment Cd was shown to increase Gretchen Hagen 3 (GH3) activity, f acilitating auxin conjugation and degradation. The specif ic role of endogenous levels of auxin in plants is unknown due to heavy metal stress; however, amendments with exogenous auxins have been demonstrated to decrease the detrimental ef f ect of these heavy metals in plants ( Saini et al., 2013). Exogenous application of indole-3-acetic acid (IAA) also reduced heavy metal toxicity, stimulating the increased upregulation of the AUX1 and PIN2 genes (Wang et al., 2015).
Aside f rom direct phytoremediative action, phytohormones can also stimulate rhizobacteria to produce gluconic, oxalic and citric acids. These have been implicated in the mobilization and enhancement of the availability of heavy metals to plants (Janoušková et al., 2006;Ullah et al., 2015).
Phytohormones can also induce rhizobacteria to bio-methylate heavy metals like lead (Pb), mercury (Hg), selenium (Sn) and arsenic (As). This invariably increases the bioavailability of the methylated heavy metals for phytoremediation.

Mechanisms of action of exogenously applied cytokinins (CK) during phytoremediation
Even though the precise role of cytokinin application in relieving heavy metal stress is still not well def ined, investigations have demonstrated that its treatment signif icantly improves phytoremediation of Zn, As, and Pb (Vitti et al., 2013;Mohan et al., 2016;Piotrowska-Niczyporuk et al., 2020). Zhou et al. (2019) reported the modulation of heavy metal stress adaptation and biosorption action of exogenously applied cytokinins. Similarly, when supplemented with abscisic acid, cytokinin stimulates tomato plants to bioaccumulate Co through regulating the mechanism of its absorption and translocation. The mechanism of action of exogenously applied cytokinin during Cd toxicity has been suggested to be by increasing the expression concentration of IPT gene and the upregulation of CKX gene in roots and downregulation in shoots. This enhanced the CK content, leading to ef f ective translocation of Cd in Arabidopsis plant (Vitti et al., 2013).

Roles of other phytohormones during phytoremediation
Phytohormones have been known to stimulate the activation of metal chelating agents which contribute signif icantly in heavy metal sequestration and subsequent absorption in plant intracellular vesicles (Nguyen et al., 2021). Exogenously applied Gibberellin has been shown to stimulate the phytoextraction of copper (Cu) f rom polluted soil using pea plants (Javed et al., 2021). According to Bücker-Neto et al. (2017), thiol/phytochelatin production was promoted by exogenous application of abscisic acid, thus enhancing heavy metal resistance and bioaccumulation. Likewise, increasing abscisic acid concentration during Cd contamination stimulated the synthesis of phytochelatin in Deschampsia cespitosa (Hayward et al., 2013). Multiple plant def ense mechanisms were elicited af ter soil treatment with ethylene during Zn contamination, through the uptake regulation of peroxidase, ascorbate peroxidase (APX), glutathione peroxidase ( Brassinosteroids were shown to regulate the accumulation of heavy metals such as Pb, Cu, Cr, Cd, and Zn in plants during heavy metal contamination (Hasan et al., 2011;Hayat et al., 2010;Ramakrishna and Rao, 2015). To maintain BR homeostasis, brassinosteroids biosynthesis genes are expressed at high BR levels (Kim et al., 2006). Exogenous applications of certain phytohormones during phytoremediation have been linked to enhanced absorption and bioaccumulation of heavy metals in dose dependent manner, but the major challenge lies in understanding the intricate signaling mechanisms of action.

Current Status of Exogenous Applications of Phytohormone-Producing Microbes in Growth Modulation or Stress Tolerance
To meet up with the various demands of an exponentially growing global population, together with the challenges of climate change, a "new" bio-revolution would be needed which will be characterized by less inputs with minimal environmental impacts. One of such benef icial approaches is the inoculation of PGPRs in heavy metal stressed environments, both as a f orm of bioremediation and enhancer of plant growth (Kumar and Trivedi, 2016). Rhizobacteriaproduced phytohormones have been demonstrated to enhance stress tolerance and plant development under a variety of abiotic and biotic stress conditions (Liu et al., 2013;Sgroy et al., 2019) Auxins are presently the most researched microbially synthesized phytohormones, with most microorganisms being able to produce them and having signif icant impact on root structure and growth (Gupta et al., 2015). The ef f ects of phytohormones concentrations have been investigated and reported in many studies, with high levels sometimes encouraging f avorable root growth benef its. It has been f ound that auxins regulate plant growth and development through alterations of the plant gene expression patterns (Ljung, 2013). Despite the f act that its production may be decreased under stress situations, it stimulates growth modulation by the induction of abscisic acid (ABA) (Kazan, 2013). Application of auxin has been used to mitigate the harmf ul ef f ect of Pb on sunf lower. This increased the shoot biomass, indicating the metal phytoextractive nature of the phytohormone (Fässler et al., 2010). Singh and Jha, (2016) and Sorty et al. (2016) showed the ability of auxin generated by the root-colonizing halotolerant bacteria B. licheniformis B. subtilis,and Arthrobacter spp, salt-tolerant strain Enterobacter sp. NIASMVII to improve the salinity stress of wheat plants.
IAA-producing bacteria like Pseudomonas species, Serratia sp. and B. megaterium also induced drought and nutrient limited tolerance in clovers plants (Marulanda et al., 2009;Zaheer et al., 2016). Salicylic acid, ethylene, and brassinosteroids have been shown to increase photosynthesis in heavy metal-stressed plants by lowering ROS levels and lipid peroxidation by improving its antioxidative enzyme systems (Bashar et al., 2019). Brassinosteroids were shown to decrease copper buildup in plants under copper stress in a study conducted by Sharma and Bhardwaj, (2018).
Similar study was undertaken by Choudhary et al. (2007) and it showed reduced copper in Raphanus raphanistrum subsp. Nickel heavy metal concentration has been reported to be reduced by the exogenous application of 28homobrassinolide, a phytohormone. An increase in the antioxidative enzyme system, with a proportionate rise in glutathione reductase, superoxide dismutase, peroxidase, catalase, carbonic anhydrase, and nitrate reductase enzymes, resulted in enhanced plant growth, chlorophyll content, and photosynthesis (Yadav et al., 2018). Salicylic acid and Jasmonates, multif unctional hormones generated in plants under abiotic stressors, have long been known to reduce heavy metal toxicity (Dar et al., 2015).
Most bacteria are capable of producing a variety of phytohormones in response to various physiological and biochemical changes in plants, particularly electrolyte and chemical balance (Egamberdieva et al., 2017). All of these investigations revealed the role of phytohormone regulation in plant tissue by plant-associated microorganisms in inducing plant stress tolerance.

CONCLUSION AND FUTURE PROSPECTS
The knowledge of activities around the rhizosphere of plants has helped to explain how soil microbes interact with plants, thereby enhancing plant growth and productivity. This has f acilitated in-depth understanding of hormone signaling and how plants react to unf avorable conditions in the soil. Microbial synthesis of phytohormones is a powerf ul tool used in inf luencing plant physiology and resistance to pathogens. To cope with the dif f erent soil environmental stressors that plants are exposed to, plants utilize microbially synthesized phytohormones. As a result, plant-associated microorganisms can alter hormone balance and physiology in plant cells, notably in metabolic activities that might shield plants against the negative ef f ects of environmental stimuli.
Exogenous treatments of microbially produced phytohormones have been demonstrated to promote abiotic and biotic stress tolerance in plants, providing signif icant practical advantages amid f luctuating or severe abiotic f actors. As stated above, the production and participation of phytohormones in plant interactions have been thoroughly researched and conf irmed by genetic evidence. The use of phytohormone-producing microorganisms under heavy metal stress is one of the sustainable crop production strategies in changing conditions. Phytohormone-mediated remediations have been demonstrated to be potentially viable methods f or inducing stress tolerance in agricultural plants under severe environmental conditions. Notwithstanding, f or a better knowledge of phytohormone metabolism, it is essential that a mutant def ective in each pathway is isolated and that the stability and bioavailability of phytohormones in the soil environment are studied. Further research on dif f erent plant species is needed to see if microbial phytohormones are plant-specif ic f eatures and to learn more about the interactions at work between them. There is a large gap in our understanding of these f eatures that have to be f illed through agronomical research at the grass root level. Furthermore, there is need to select and evaluate possible strains with the ability to produce specif ic phytohormones so that an appropriate strain f or a specif ic purpose can be employed ef f iciently. More research is needed to discover the benef icial soil microbial populations capable of producing phytohormones that can create signif icant metal stress tolerance in certain plant species.
Furthermore, more study is required to identify the benef icial soil microorganisms with the ability to generate phytohormones that can promote heavy metal stress tolerance in certain agricultural crop specie. It is important to conduct specif ic research to examine the processes involved in the synthesis of dif ferent metabolites, as well as their harmf ul and benef icial associations with selected plant species. Plantmicrobe interactions should be enhanced by the use of molecular genetics, bioinf ormatics, and modeling techniques to increase ag ricultural production, soil and environmental conservation.

Conflicts of interest
The authors declare no conf licts of interest.

Acknowledgements
Authors are thankf ul to the Centre for Environmental Management and Control, University of Nigeria, Nsukka f or the mentorship opportunity and signif icant contributions to the success of this work. No specif ic f unding was received f or this research.

AUTHOR CONTRIBUTIONS
All the authors contributed to the study conception and design. The review was conceived and designed by MAN and ECC. The f irst draf t was written by OCJ and ECC. MAN revised the manuscript. The f inal draf t was read and approved by all the authors bef ore submission.   Khadri, M., Tejera, N.A. and Luch C. (2006).

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