The Multifaceted Role of Methylorubrum in Plant Growth Promotion and Abiotic Stress

Crops face many environmental challenges, both biotic and abiotic stress, including extreme temperatures, salinity, drought, pest and disease incidence, and nutrient deficiencies that significantly impede the growth and development of crops. Global warming and an increase in population pose a global threat to food security. Due to climate variability, global food production has already decreased (Tito et al., 2018). As a result of global warming, irregular rainfall pattern, high and low temperature stress, drought stress, UV stress, and elevated CO₂ levels significantly reduces crop growth and alter their physiology (Madhaiyan et al., 2007a; Jinal et al., 2019; Gopi et al., 2020). Due to the intricate nature of stress tolerance, employing conventional breeding techniques to develop crops with strong stress resistance is impossible. Plants developed sophisticated mechanisms to reduce the impacts to adapt to unfavourable stress conditions. The mechanisms include physiological, biochemical, and molecular changes that help plants maintain cellular homeostasis and ensure survival.  In recent years, the usage of plant growth-promoting microorganisms has emerged as a sustainable approach to improve plant productivity and resilience under both normal and stress conditions (Vandenkoornhuyse et al., 2015).

Plants are not a single entity, but a host of diverse groups of microorganisms that provide both beneficial and detrimental effects through a diverse relationship, including commensal, symbiotic, parasitic, and mutualistic relationships that shape the productivity of crops (Krishnamoorthy et al., 2021; Rani et al., 2021). The microorganisms that can promote plant growth is known as Plant Growth Promoting bacteria (PGPBs). Among these PGPBs, Methylorubrum, a genus of pink-pigmented, facultative methylotrophic bacteria, able to colonise the phyllosphere, are commonly known as pink pigmented facultative methylotrophs (PPFMs) (Fig.1). Methylorubrum has gained major attention due to its significant potential in mitigating abiotic stress and improving plant growth.  These plant-associated microbes help plants to withstand abiotic stress through several mechanisms, including ACC-deaminase production, phytohormones (auxin, cytokinin) synthesis, and by producing free radical scavenging enzymes (Prittesh et al., 2020). In the face of climate change and increasing abiotic stress, the role of Methylorubrum is crucial in improving plant growth and stress mitigation for sustainable agriculture practices. Due to their versatile capabilities, inoculating Methylorubrum alone or in combination, is a valuable tool to improve plant growth and yield. Maintenance of proper plant–microbiome interactions minimizes the use of hazardous agrochemicals and fosters sustainable agriculture practices (Delmotte et al., 2009).

Figure 1. Phyllospheric Colonisation by Methylorubrum

Taxonomy

In 2018, Green and Ardley divided Methylobacterium into two genera, Methylobacterium and Methylorubrum. Based on phenotypic traits, 16s rRNA, multilocus sequence analysis (MLSA), 11 species were included in the genus Methylorubrum, including Methylorubrum extorquens, M. aminovorans, M. podarium, M. populi, M. pseudosasae, M. rhodesianum, M. rhodinum, M. salsuginis, M. suomiense, M. thiocyanatum, and M. zatmanii. (Green and Ardley, 2018).

Ecology

Methylorubrum is a genus of facultative methylotrophs with diverse ecological adaptability. They are ubiquitous, thriving in diverse niches, including water, soil, plants, animals and contaminated environments (Madhaiyan et al., 2007b). Their ability to utilize one-carbon compounds as a carbon source helps them to survive in unfavourable conditions. The ability of Methylorubrum to utilize single-carbon compounds like methanol, methylamine, its ecological adaptability, and metabolic versatility make it a valuable component in sustainable agriculture, bioprocessing and pollution abatement (Schauer et al., 2011; Green et al., 1988). It plays a crucial role in the global carbon cycle by recycling single-carbon compounds. Diversity of Methylorubrum in various environments was presented in Table 1 (Danko et al., 2021).

Table 1 Diversity of Methylorubrum in diverse environments

Metabolism of single-carbon compounds

            Methylotrophs are a group of microorganisms that can utilize single–carbon compounds such as methanol, methylamine, or formate as a primary carbon source. Methanol dehydrogenase (MDHs) is the key enzyme that catalyses methanol oxidation to generate formaldehyde with hydrogen ions and electrons (Zhang et al., 2017). This produced formaldehyde enters the cell and it is assimilated through any one of the following pathways – (i) Serine Cycle, (ii) Ribulose monophosphate cycle, (iii) Xylulose monophosphate cycle (Zhang et al., 2017). In most cases, Methylorubrum employs the serine cycle for the assimilation of formaldehyde through a series of complex biochemical reactions that convert formaldehyde into building blocks of the cell and energy production. This pathway also helps Methylorubrum to produce vital nucleic acids, lipids, and amino acids from the single–carbon compound methanol. Through these metabolic capabilities, Methylorubrum thrives in diverse environments and establishes beneficial interactions with plants, which fosters plant growth and enhances stress tolerance. This capability helps Methylorubrum to stand out among the other microorganisms. In addition to single carbon assimilation, they play a crucial role in mineral solubilisation and plant nutrient uptake. Another essential micronutrient of plants that plays a crucial role in the physiological processes of plants is iron. Plant Growth Promoting microorganisms produce siderophores, a specific type of molecules that enhance iron bioavailability for uptake (Verma et al., 2017).

Plant Growth-Promoting Mechanisms

Methylorubrum employs diverse mechanisms both directly and indirectly to promote plant growth and improve crop productivity (Madhaiyan et al., 2006). Some direct mechanisms include auxin and cytokine production and mineral solubilisation. Phytohormones regulate plant growth and development. Producing indole acetic acid (IAA), a type of auxin essential for cell elongation and root development, promotes plant growth. On the other hand, cytokinins promote cell division, rejuvenate cells, and increase shoot proliferation and biomass (Verma et al., 2017).

The production of organic acids, such as gluconic acid, through metabolism, helps the bacteria to solubilize the insoluble phosphorus from an unavailable form to an available form (Kwak et al., 2014). Iron is an essential micronutrient for plants, promoting plant photosynthesis and chlorophyll synthesis. Methylorubrum enhances iron absorption through the production of siderophores (Shi et al., 2012). Siderophores are iron-chelating organic compounds that bind with the unavailable form of iron and convert it into an available form (Ahmed and Holmström, 2014).

            Methylorubrum also suppresses the phytopathogens by producing antimicrobial compounds and volatile compounds that inhibit the multiplication and activity of various bacterial and fungal pathogens. They also induce systemic resistance in plants by activating plant defence genes and producing defence-related enzymes, such as peroxidase and chitinase, which help plants respond more effectively in the event of pathogen attacks. Methylorubrum species promote plant growth through both direct and indirect pathways (Kazan, 2015).

Phytohormone Production and Regulation

• Auxins: Methylorubrum can synthesize auxins, such as indole-3-acetic acid, which plays a vital role in cell elongation, root development and apical dominance (Mano and Nemoto, 2012). Increased auxin levels stimulate cell division and differentiation, leading to enhanced root and shoot growth (Ivanova et al., 2001).
• Cytokinins: Some Methylorubrum strains produce cytokinin, which promote cell division, rejuvenate cells, and enhance plant nutrient mobilization (Holland and Polacco, 1992). Cytokinin interacts with auxins to regulate various developmental processes.
• Ethylene Regulation: Methylorubrum can modulate ethylene levels in plants by producing ACC deaminase. ACC deaminase breaks the 1-Aminocyclopropane-1-carboxylic acid (ACC), the precursor to ethylene, into ammonia and alpha-ketoglutaric acid, thereby reducing ethylene levels and mitigating its inhibitory effects under stress conditions (Madhaiyan et al., 2006).

Nutrient Acquisition

• Nitrogen Fixation:

Nitrogen-fixing microorganism can convert atmospheric nitrogen into ammonia (Singh et al., 1981). Using these nitrogen-fixing bacteria reduces the necessity of synthetic nitrogen fertilizers. Methylorubrum rhodesianum has the potential to convert atmospheric nitrogen into ammonia; thereby increasing the availability of nitrogen, a crucial nutrient for plant growth (Sy et al., 2001).

• Phosphate Solubilization

Plants take up phosphorus as orthophosphate ions; however, in soil, based on the pH, phosphorus exists in insoluble forms, such as calcium phosphate or aluminium/iron phosphate (Tang et al., 2023). Through producing organic acid, acid and alkyl phosphatase, phytase enzyme Methylorubrum converts insoluble phosphorus into a soluble organic form. Several phosphorous solubilising Methylorubrum strains were isolated and listed in table 3, highlighting their role in sustainable agriculture.

• Siderophore Production:

Iron is a vital micronutrient mainly available in two oxidation forms, Fe³⁺ (soluble) and Fe²⁺ (insoluble) in soil (Shi et al., 2012). The ferric form of iron is insoluble, making iron acquisition a challenge for plants. Under iron-limiting conditions, microorganisms secrete siderophores, an iron-chelating compound that enhances iron uptake. Several strains that can produce siderophores are presented in the table 3.

Table 2. Stress adaptive Methylorubrum with multifarious PGP attributes for alleviation of diverse abiotic stresses in plants (P- Phosphate solubilization; IAA- Indole acetic Acid production; Fe- Siderophores production; ACC- ACC deaminase production

Abiotic stresses

            The Methylorubrum genus has a remarkable ability to mitigate several abiotic stresses, including high and low temperature stress, drought, salinity, and heavy metal toxicity. Abiotic stresses significantly reduce the growth and development of plants, which can cause major yield loss and productivity. Plants associated with a beneficial microbiome are the basic line of defence against any abiotic stresses, especially Methylorubrum association, which mitigates the unfavourable effects of all abiotic stress through several mechanisms. These mechanisms include increased accumulation of osmolytes and antioxidant enzymes, promote nutrient uptake, protection against phytopathogens, and alter the ethylene level through ACC-D production. Figure 2 depicts the potential of Methylorubrum in abiotic stress mitigation.

Figure 2. Role of Methylorubrum in abiotic stress mitigation

Drought stress

Drought stress triggers many physiological and biochemical changes in plants, including reduced water uptake, decreased photosynthesis, and increased oxidative stress. During abiotic stress, plants accumulate the excess amount of ethylene and ROX; alter DNA, RNA, proteins, phytohormones accumulation, osmolytes content, stomatal closure, and reduce transpiration (Silva et al., 2020).. There are several reports of Methylorubrum species, such as M. populi, M. aminovorans and M. extorquens, that have been reported to help plants survive under abiotic stress conditions.

Salinity stress

Regular irrigation of the field with salt water and sea water intrusion develops saline conditions. There are three different salt-affected conditions- saline, sodic, and alkaline. Saline condition refers to increased accumulation of soluble salts. Alkalinity describes soil conditions characterised by increased accumulation of carbonate and bicarbonate ions. Sodic soils have increased exchangeable sodium accumulation, affecting water and air movement. All the critical growth stages of a plant are highly susceptible to salt stress (Kumar et al., 2015; Sorty et al., 2016; Mishra et al., 2016). Salinity stress significantly threatens agriculture, particularly in arid and semi-arid regions. High salt concentrations in the soil can disrupt plant water uptake, nutrient balance, and enzyme activity. Accumulation of salts reduces the water potential in the soil and causes water to move out of the plant cell to the soil (Egamberdieva et al., 2015).

UV stress

            Ozone layer depletion due to anthropogenic greenhouse gas emissions increases UV exposure, which has a significant impact on plants. UV radiation causes DNA damage, reduces photosynthesis, and increases oxidative stress (Sage et al., 2012). UV-B causes nucleoprotein damage. Generates DNA photoproducts, oxidises the plant tissues, and reduces their survival rate (Kosobryukhov et al., 2020).  Methylorubrum protects plants through various protective mechanisms, including astaxanthin pigments and ergothioneine accumulation, which can absorb UV radiation and thereby reduce damage (Bazela et al., 2014).

Mechanism of abiotic stress mitigation by Methylorubrum

• ACC Deaminase Production

Ethylene at lower concentrations promotes root extension, whereas high concentration inhibits root elongation. Under abiotic stress conditions, the endogenous level of ethylene increases significantly. Ethylene, as a stress hormone, can intensify the adverse effects of drought (Glick, 1995). Methylorubrum helps plants reduce ethylene levels and maintains better growth and water use efficiency under drought conditions. Through the production of ACC deaminase, Methylorubrum reduces ethylene levels in plants by converting the precursor of ethylene, ACC, into ammonia and alpha-ketoglutarate (Hardoim et al., 2008).  

• Osmolytes accumulation

Osmolytes are solutes that accumulate in plants to reduce cell damage caused by oxidative stress (Sharma et al., 2019). Glycine betaine, proline, polyamines, and sugars are some osmolytes that can reduce the osmotic pressure induced by abiotic stress conditions. Plants treated with Methylorubrum spp. have increased accumulation of osmolytes like glycine betaine, proline and sugars, which help plants maintain osmotic balance under abiotic stress. Osmolytes protect cellular structures and enzymes from damage caused by dehydration (Chandrasekaran et al., 2017).

·         Stomatal Regulation

Stomata are minute pores on the leaf surface that regulate gas exchange and water loss. Guard cells control the opening and closure of stomata. In abiotic stress conditions, plants accumulate abscisic acid (ABA), which in turn closes the stomatal pore (Daszkowska-Golec and Szarejko, 2013).  Methylorubrum plays a significant role in stomatal regulation through direct and indirect strategies to maintain water balance and gas exchange. Production of ACC deaminase reduces ethylene levels and enhances photosynthesis and water retention (Sivakumar et al., 2017). Accumulation of proline and glycine betaine maintains turgor pressure and osmotic stability. In the direct mechanism, Methylorubrum releases volatile organic compounds that activate defense phytohormones like salicylic acid and jasmonic acid, in turn triggering the opening of stomata through the OST1 signal cascade. It also modulates ion channels, transporters, and signalling proteins through gene expression (Krishnamoorthy, 2020). Opening of stomata reduces the leaf temperature, thereby reducing the adverse effects of the drought (Rajagopalan, 1956).

·         Increased antioxidant enzyme activity

Abiotic stress conditions enhance the production of reactive oxygen species (ROS), such as singlet oxygen, superoxide radical, hydrogen peroxide, and hydroxyl radical (Sivakumar et al., 2017). Low quantities of these ROS can be easily balanced by antioxidant enzymes. However, in abiotic stress conditions, these molecules levels increase and result in oxidative damage. Accumulation of ROS leads to oxidative damage, causes protein degradation, lipid peroxidation, and membrane disruption. Antioxidant enzymes such as peroxidase, superoxide dismutase, catalase and ascorbate peroxidase neutralize the ROS. Application of Methylorubrum enhances plant defence by upregulating the antioxidant-related genes and increasing the antioxidant enzyme activity, thereby facilitating ROS detoxification (Chandrasekaran et al., 2017).

Synergistic Effects and Multi-Stress Tolerance

In natural environments, plants often face multiple stresses. The application of Methylorubrum spp. with plant growth-promoting capabilities has several beneficial effects on plants.

• Reduced reliance on synthetic chemical fertilizers: Enhance nutrient availability and promote plant growth, Methylorubrum reduces the application of synthetic fertilizers, minimizes environmental pollution, and promotes soil health.
• Improved crop yields: Methylorubrum can enhance crop yields under normal and stressful conditions, contributing to food security and economic benefits for farmers.
• Sustainable stress management: Methylorubrum reduces the deleterious effects of abiotic stresses, contributes to sustainable agriculture in the face of global warming and increasing environmental pressures (Chandrasekaran et al., 2017).

Methylorubrum spp. is a group of plant growth-promoting bacteria that play a crucial role in sustainable agriculture.  Accumulation of osmolytes like proline and glycine betaine maintains the cell turgor pressure. It maintains the osmotic balance, increased activity of antioxidants reduces the deleterious effects of reactive oxygen species, and production of phytohormones like auxin and cytokinin increases shoot and root growth. These abilities of Methylorubrum to produce phytohormones, enhance nutrient availability, and improve stress tolerance make them a multifaceted candidate for improving plant growth and stress resilience.  Methylorubrum supports plant resilience against adverse conditions, contributing to sustainable yield. This symbiotic relationship with plants minimizes reliance on chemical fertilizers and promote eco-friendly crop management strategies