Irregular climate changes, the increasing population, and intensive agriculture are directly connected to land degradation and food shortages, inducing extreme weather events and environmental impacts in many countries (Munir et al., 2021). Increasing agricultural production, food security and protecting water reserves are critical for sustainable agriculture and environmental safety. Decreased water supply due to declining rainfall affects biological systems, nutrient supply, and crop productivity (Peña-Gallardo et al., 2019; Tyagi and Pandey, 2022). For example, barley (Hordeum vulgare L.) biomass and grain yield have substantially declined in arid regions. Limited food crop productivity is associated with reduced water and nutrient flux, causing significant economic losses and socio-economic issues (Kour and Yadav, 2022). New emerging agricultural techniques are acquired to overcome land degradation and increase crop biomass production (Pittelkow et al., 2015; Siddiqui et al., 2021). One strategy is to explore endophytic bacteria, which establish a symbiotic relationship with the host plant and synthesis of nutrients that offer favorable conditions to resist water stress in plants (Rahman et al., 2018; Kour and Yadav, 2022).

Microbial biotechnology is a promising approach for increasing edible plant biomass under stress conditions (Cardoso et al., 2018; Goudarzi et al., 2023). Microbial supplementation can significantly promote bioremediation, control phytopathogens, and increase plant physiological performance and productivity on degraded arid lands (Oleńska et al., 2020). Soil microbial supplementation is influenced by root exudates that produce different enzymes and metabolites, nutrient accumulation, and hormone production (Singh and Gupta, 2018; Zayed et al., 2022). Plant growth-promoting bacteria (PGPB) improve growth and can protect plants against biotic and abiotic stresses by producing volatile compounds, siderophores, growth hormones, biological nitrogen fixation, and reducing plant ethylene synthesis (Khatoon et al., 2020; Das et al., 2022). Identifying the signaling pathways governing the associations between plants and different PGPB can play an important role in improving agricultural production sustainably (Wiggins et al., 2022).

Changes in root development or leaf elongation can be modulated under drought stress but are closely associated with leaf metabolic status and growth portioning (Knutzen et al., 2015; Abideen et al., 2021). Plant-available nutrients (K⁺, Ca²⁺, Mg²⁺ and N) and carbon metablosim with water accessibility through osmotic balance are important mechanisms of plants for photosynthesis and leaf metabolites production under drought (Abdelaal et al., 2021b). However, the ecophysiological responses of plants to reach a new homeostasis after PGPB inoculation under water deficit are not well understood. Inoculation with PGPB could facilitate water uptake, protecting leaf desiccation and thus improving turgidity and plant growth (Abdelaal et al., 2021). Microbial inoculation improves plant ion flux and the synthesis and use of organic solutes for osmotic adjustments (Santander et al., 2017). In addition, microbial interactions improve stomatal regulation, leaf water use efficiency, and oxidative stress management (Paneque et al., 2016; Tak et al., 2021). Plants protect photosystem II (PSII) activity by regulating light harvesting mechanisms critical for biomass production (Saccon et al., 2022). Low water availability reduces the potential agricultural uses of arable land. However, soil and plants can be supplemented with beneficial bacteria to increase food production. The suitability of selected microbes depends on soil type, bacterial strain and concentration, plant species, and stress conditions. Plant microbiomes are integrated within the host into single units of evolution called holobionts. The seed endosphere is a little-investigated plant microhabitat, more recently receiving attention for its potential as a reservoir and vector of beneficial microbes (Berg and Raijmakers, 2018; Rahman et al., 2018). Seed endophytes have been detected in many crop plants, including cereals and legumes, and can improve plant growth and ecophysiological parameters (Alibrandi et al., 2018; Liu et al., 2020). In barley, seed endophytes increased plant biomass activities when used as inoculants (Rahman et al., 2018).

The two bacterial strains used in this work (Pantoea sp. “ITS group 2” and Pseudomonas sp. “ITS group 11”) were selected as best candidates among a series of new isolates from barley seeds (Rahman et al., 2018). They were demonstrated to be consistently associated to barley seed across a variety of cultivars and years (Rahman et al., 2018). Taxonomical identification was performed by 16S rRNA gene sequencing and the isolates were characterized at strain level by ITS polymorphism analysis. Due to their superior performance in barley growth promotion and biocontrol activity (Rahman et al., 2018), these two strains were chosen for the current study. Moreover, they showed ability to efficiently colonize barley roots upon seed germination (Rahman et al., 2018). This study investigates the potential of selected endophytes to improve biomass and physiology of barley under drought stress. The selection of bacterial strain and appropriate level in dry soil is the key component of this manuscript. Barley was selected due to its importance as a global staple food and the availability of preliminary data using seed endophytes as PGPB (Rahman et al., 2018). We tested the following hypotheses: 1) Bacterial inoculation enhances soil conditions (soil CO₂ flux), barley growth, nutrient acquisition and plant survival under drought stress; 2) Water limitation improves stomatal resistance and photosynthesis by regulating antioxidiant defense in barley with bacterial inoculation.

Drought is a major abiotic factor that inhibits crop yields but association of seed-associated bacterial endophytes of Hordeum vulgare are beneficial for rhizosphere soil health and its proper application, relieved the adverse effects of water deficit on barley grown under water limited areas to ensure productivity of such an important food crops (Chandra et al., 2021; Tyagi and Pandey, 2022; Kour and Yadav, 2022). In this study, bacterial inoculation enhanced both soil carbon flux and soil moisture under drought stress, regulating the ecophysiological performance (growth, net photosynthesis, and mineral acquisition) of barley seedlings. Stimulation of soil carbon flux due to Pseudomonas and Pantoea inoculation in dry arid areas in barley was might be associated with increased soil respiration that triggers higher microbial activity. Higher soil metabolic output with microbial inoculation indicates that barley cultivation with PGPB is a suitable strategy for enhancing carbon sequestration and thus contributing to climate change mitigation (Radicetti et al., 2020). Improvement of soil parameters especially soil water acquisition (especially Pantoea treatment) under stressed and non-stressed conditions was also as reported for maize (Naseem and Bano, 2014; Goudarzi et al., 2023), which has been associated with exopolysaccharides (EPS) production. EPS significantly enhance plant growth (Naseem and Bano, 2014; Zayed et al., 2022) by colonizing plant roots, forming hydrophilic biofilms, and providing plant immune response (Sun et al., 2022). In addition, PGPB= use several mechanisms to improve plant growth such as maintaining sufficient nutrient supply or regulating hormone levels (Forni et al., 2017; Siddiqui et al., 2021). In this study, the introduced bacterial endophytes Pseudomonas and Pantoea emerged as mediators for enhancing total foliage biomass, as reported in Capsicum annuum ( Figure 2 ) (Datta et al., 2011; Lin et al., 2020). Application of microbes can improve fruit quality by upregulating nitrogen metabolism and producing specific hormones that trigger water and mineral balance and promote belowground biomass (Ahemad and Kibret, 2014). In the present study, Pseudomonas (under drought and control both condition) and Pantoea inoculations (well water condition) increased leaf fresh biomass and other growth parameters which was reported earlier in maize under water deficit condition (Jeong et al., 2021). Plants inoculated with Pseudomonas or Pantoea improved plant root elongation under water deficit compared to the other treatments. Higher root production under drought suggests that barley seeds benefit from soil microbe/plant interactions with Pseudomonas and Pantoea endophytes to access optimum water and nutrient which is critical for biomass production (Lin et al., 2020; Verma et al., 2021). Increased root development from microbial amendments can also support seedling emergence and long-term survival of barley in poorly degraded, dry areas that appear futile for agriculture and thus improve sustainable agriculture to avoid food insecurity (Calvo et al., 2017; Abdelfadil et al., 2022). In addition, it was reported that PGPB strains enhance phytohormone production and other signals to modify root system architecture, such as increased lateral root branching and root hair development (Siddiqui et al., 2022). The proper root modification stimulated the leaf development that favors the photosynthesis and the activity of photochemical reaction. Chlorophyll is the main photosynthetic pigment that was stimulated due to PGPB application in barley under drought-stressed and well-watered conditions due to increased nutrient acquisition, as reported elsewhere (Dawwam et al., 2013; Dao et al., 2020). Enhanced synthesis of chlorophyll pigments and their accessory components improve photosynthetic rate as well asPSII efficiency and the protein-pigment complex function. Plants inoculated with PGPR developed drought tolerance, suggesting that plants treated with bacterial strains enhance CO₂ assimilation and reduce water release by leaves (Armada et al., 2015; Mehrasa et al., 2022). In addition, under suboptimum conditions, plants release some photosynthetic assimilates as root exudates, which helps maintain bacterial colonization in the root zone, promoting mutual benefits such as increased plant resistance against abiotic stress (Samaniego-Gámez et al., 2016). It was reported that PGPR elevate photosynthesis in in plants by regulating endogenous sugar/abscisic acid signaling (Sati et al., 2021). In the present study, plants inoculated with Pseudomonas and Pantoea increased gas exchange, respiration, stomatal conductance and leaf transpiration under drought-stressed and well-watered conditions. However, they only increased WUE under well-watered conditions ( Table 3 ), indicating that bacterial-inoculated plants improve plant growth by enhancing photosynthetic performance (Chandra et al., 2021). It was reported that bacterial strains in the root zone synthesize auxins (indole-3-acetic acid/indole acetic acid/IAA) that increase tissue cell division, photosynthetic pigment synthesis, and photosynthesis (Ahemad and Kibret, 2014; Mitra et al., 2022). In tobacco, it was established that CO₂ produced in roots was transported to shoots for photosynthesis via the vascular system instead of stomata (Andrade et al., 2022). It was suggested that endophyte colonization changed the host plant’s photosynthetic apparatus, increasing the activity of light harvesting complexes and enhancing photosynthetic performance (Chaturvedi et al., 2022). Similarly, Liu et al. (2021) indicated that seed endophytes stimulate PSII efficiency in plants.

Bacterial inoculation of pepper plants increased ETR and NPQ which could be a consequence of the positive effect of PGPB (Samaniego-Gámez et al., 2016). Further, NPQ helped minimize the over-synthesis of O₂ in PSII antenna, increasing NPQ in plants inoculated with bacterial strains to reduce excess light energy (Radhakrishnan and Baek, 2017). In our study, bacterial inoculation increased Fv/Fm under well-watered conditions but increased ETR and NPQ under drought stress. Interestingly, drought stress produced higher photoinhibition and ETR/A ratios than well-watered conditions but were substantially lower in plants inoculated with Pseudomonas and Pantoea than the other treatments. ETR increases due to high oxidation of the quinone acceptor (Qa) and its excitation energy, reducing oxidative damage (Garcia-Caparros et al., 2021). Yang et al. (2017) reported that PGPR provoke systemic tolerance of plants during abiotic stress (salt and drought). Abiotic stresses such as drought increase ROS formation, causing oxidative stress (Chiappero et al., 2019). Increased ROS production affects plants due to the oxidation of photosynthetic pigments in membrane lipids, proteins, and nucleic acids (Jajic et al., 2015; Mukarram et al., 2021). The upregulation of antioxidant enzymes, such as SOD and APX, is a significant plant response to drought Mukarram et al., 2021). Increased CAT, GR, and APX activities were reported in drought-stressed Ocimun basilicum inoculated with PGPR (Chiappero et al., 2019). In the present study, drought stress increased SOD, CAT, APX, GPX, and GR activities, and MDA content. Drought stress and bacterial inoculation combined reduced ROS production, as indicated by the decreased SOD activity and MDA content and increased APX, GPX, and GR activities.

Leaf growth and metabolite production are important parameters under water deficiency (Zulfiqar et al., 2020). In the present study, leaf area increased under drought-stressed and well-watered conditions, which may be linked to P uptake triggered by Pseudomonas or Pantoea inoculation, as reported for maize (Chaudhary et al., 2022). Microbial inoculum improves nutritional assimilation (N, P and K contents) in plants relative to uninoculated plants, possibly because the soil microbes compensate for nutrient deficiency, enhancing plant growth in nutrient-deficient environments (Bargaz et al., 2018). In the present study, Pseudomonas inoculation under well-watered conditions increased shoot and root K⁺ concentration but decreased shoot Na⁺ concentration. Sequestration of Na⁺ in roots and higher uptake of K⁺ in leaves increased the shoot K⁺/Na⁺ ratio in well-watered plants inoculated with Pseudomonas, as reported elsewhere for maize (Shahzad et al., 2022). Similarly, under water deficit, Pseudomonas preferentially increased shoot K+, retained root Na⁺, and enhanced the shott K⁺/Na⁺ ratio relative to the other treatments. De Inoculation of Pseudomonas increased seedling growth in low fertile soil by compensating for nutrient deficiency through the synthesis of plant growth-promoting hormones at the root interface, stimulating root development and increasing soil water and nutrient absorption (Amora-Lazcano et al., 2022; Mehrasa et al., 2022).

In addition, Ca²⁺ is critical for plant growth, playing an important role in cell wall and membrane development, photosynthesis and ion homeostasis and acting as a signaling molecule in the cytosol (Shabbir et al., 2022). Recently, Ahmed et al. (2021) showed that Ca²⁺ acts as a signaling agent, enhancing plant stress resistance in unfavorable environmental conditions. In the present study, plant Ca²⁺ concentration increased substantially in bacterial-treated plants compared to uninoculated plants and even under drought stress. In addition to Ca²⁺, Mg²⁺ plays an important role in carbohydrate partitioning, CO₂ fixation during photosynthesis, and reactive oxygen species (ROS) formation (Tewari et al., 2021). Mg²⁺ increases root surface area and overall root growth, enhancing photosynthetic assimilate synthesis and transport and carbohydrate translocation, alleviating drought stress (Alrashidi et al., 2022). In the present study, shoot Mg²⁺ increased in drought-stressed plants inoculated with Pseudomonas while root Mg²⁺ did not change. Well-watered inoculated plants increased shoot Mg²⁺ relative to uninoculated plants, particularly in plants inoculated with Pantoea. Well-watered plants inoculated with Pseudomonas increased root Mg²⁺ relative to uninoculated plants.

Our results suggest that the appropriate selection of endophytes and their respective response is important for inducing drought stress resistance in barley. Pseudomonas and Pantoea inoculations improved growth, metal acquisition, photosynthesis, and oxidative stress tolerance in drought-stressed barley. The improved biomass production and crop yield with endophytic bacterial inoculation could be a solution for growing barley on poorly degraded lands. Further research is needed to confirm our findings under field conditions in saline and waterlogged areas to unlock the full potential of PGPB on crop performance.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ZA, MC and SR: Conceptualization, Investigation. ZA and SR: Formal analysis, Methodology, Writing- original draft. MC, FaZ, H-WK, KH, FeZ and KHMS: Supervision, Conceptualization, Resources, Writing – review and editing, Funding acquisition. WD and FaZ: Formal analysis. All authors contributed to the article and approved the submitted version.

This work was supported by the DAAD (The German Academic Exchange Service) Fund (grant P21067). The authors also acknowledge Taif University Researchers Supporting Project number (TURSP-2020/94), Taif University, Taif, Saudi Arabia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.