Osmotic stress and melatonin in Pfaffia glomerata: biochemical responses and 20-hydroxyecdysone modulation.

We investigated the influence of different levels of osmotic stress on growth and development in selected wild almond species (eight Prunus spp.) grown in vitro. The study, while endorsing the efficacy of in vitro screening of auxiliary buds of wild almond for osmotic stress tolerance, showed species variability in its response to osmotic stress. Osmotic stress reduced growth and development of all the species. How- ever, the putative tolerant Prunus spp. showed better performance than the putative susceptible genotypes. On average there was an 80% de- crease in shoot dry weight at -1.2 MPa. Reduction in shoot weight was more common in osmotic stress-susceptible species in the section labeled 'Euamygdalus'. The tolerant Prunus species produced smaller changes in biochemical responses than the sensitive cultivars for malondialdehyde content, catalase activity, relative permeability of protoplast membranes, and net photosynthetic rate. The tolerant species maintained cell integrity better than drought sensitive species. Wild almond species in the section labeled 'Spartioides' (Prunus arabica (Olivier) Neikle, Prunus glauca

Aim This study examined the gamma-aminobutyric acid (GABA) shunt pathway in response to salt and osmotic stresses in three barley (Hordeum vulgare L.) genotypes (Acsad176, Athroh, and Rum) in terms of seed germination, seedlings growth, oxidative damage through malondialdehyde (MDA) accumulation as an indicator for reactive oxygen species (ROS), GABA metabolite accumulation, chlorophyll level, total proteins, total carbohydrates and the expression of glutamate decarboxylase gene (GAD) analysis. Background GABA is a secondary metabolite that modulates nitrogen metabolism, protects against oxidative damage, and cytosolic pH in response to various abiotic and biotic stress in plants. Methods The effects of salt and osmotic stresses imposed by different concentrations of mannitol, sorbitol, and NaCl on the three barley genotypes were studied. Seed germination, seedling length, fresh weight, and dry mass were recorded. The physiological and biochemical responses as per GABA and MDA accumulation, total chlorophyll, proteins and carbohydrates, and the level of GAD expression were also characterized and determined. Results Mannitol, sorbitol, and NaCl treatments decreased seed germination and seedling growth for the three barely genotypes used in this study. MDA concentration was increased in seedlings of all genotypes with increasing NaCl, mannitol, and sorbitol concentrations. Acsad 176 showed high GABA accumulation under NaCl treatment. Mannitol treatment significantly increased GABA accumulation in the Rum genotype. All salt and osmotic treatments decreased chlorophyll a and b and carbohydrate content and significantly increased GAD transcription in all barley genotypes. Salt and osmotic stresses affected the total protein content in all genotypes. Conclusion Acsad 176 genotype may adapt to NaCl stress by accumulating carbohydrates more than Athroh and Rum. GABA shunt is a crucial signaling and metabolic pathway facilitating barley's adaptation to salt and osmotic stress. In soil with high salt and osmotic contents, the Acsad 176 genotype is the recommended genotype for cultivation.

Osmotic stress is a major factor reducing the growth and yield of many horticultural crops worldwide. To reveal reliable markers of tolerant genotypes, we need a comprehensive understanding of the responsive mechanisms in crops. In vitro stress induction can be an efficient tool to study the mechanisms of responses in plants to help gain a better understanding of the physiological and genetic responses of plant tissues against each stress factor. In the present study, the osmotic stress was induced by addition of mannitol into the culture media to reveal biochemical and genetic responses of tea microplants. The contents of proline, threonine, epigallocatechin, and epigallocatechin gallate were increased in leaves during mannitol treatment. The expression level of several genes, namely DHN2, LOX1, LOX6, BAM, SUS1, TPS11, RS1, RS2, and SnRK1.3, was elevated by 2–10 times under mannitol-induced osmotic stress, while the expression of many other stress-related genes was not changed significantly. Surprisingly, down-regulation of the following genes, viz. bHLH12, bHLH7, bHLH21, bHLH43, CBF1, WRKY2, SWEET1, SWEET2, SWEET3, INV5, and LOX7, was observed. During this study, two major groups of highly correlated genes were observed. The first group included seven genes, namely CBF1, DHN3, HXK2,SnRK1.1, SPS, SWEET3, and SWEET1. The second group comprised eight genes, viz. DHN2, SnRK1.3, HXK3, RS1, RS2,LOX6, SUS4, and BAM5. A high level of correlation indicates the high strength connection of the genes which can be co-expressed or can be linked to the joint regulons. The present study demonstrates that tea plants develop several adaptations to cope under osmotic stress in vitro; however, some important stress-related genes were silent or downregulated in microplants.

Anthropogenic activities have been catalyzing climate changes, owing to increased atmospheric carbon dioxide concentration ([CO 2 ]). In respect to CO 2 , its general effects on the regulation of plant growth, development and metabolism are widely explored, especially in agricultural crops. However, the changes in medicinal species under elevated [CO 2 ] are still poorly understood. Pfaffia glomerata (Spreng.) Pedersen, a plant species native to Brazil and popularly known as Brazilian ginseng, stands out by the production of 20-hydroxyecdysone (20E), a phytoecdysteroid molecule with proven therapeutic and nutraceutical activities in mammals. Previous findings from our research group revealed that in vitro CO 2 -enrichment led to physiological changes in P. glomerata. We hypothesize that CO 2 -enrichment affects the production of primary and secondary metabolites, such as 20E, in P. glomerata. Therefore, the objective here was to evaluate the effect of the CO 2 -enriched atmosphere on morphophysiological, biochemical, structural, molecular aspects, as well as in water deficit tolerance responses. For this, two independent experiments were conducted in open top chambers (OTCs). For both experiments, clonally micropropagation-derived plantlets from the in vitro germplasm bank were used. In experiment I, we assessed the influence of two CO 2 concentrations: ambient (a[CO 2 ], ± 400 µmol mol -1 CO 2 ) and elevated (e[CO 2 ] ± 800 µmol mol -1 CO 2 ). After approximately 21 days of cultivation in OTC, our data showed that e[CO 2 ] increased photosynthesis (A N ), and water use efficiency (WUE), biomass accumulation, photosynthetic pigments, carbohydrate content, expression of genes of the lignin biosynthetic pathway, and induced changes in the proteomic profile. On the other hand, e[CO 2 ] promoted reduction in total protein, amino acids and 20E contents. In experiment II, the physiological and biochemical responses of plants exposed to combined e[CO 2 ] and under water deficit were analyzed. During the first 14 days of cultivation, all plants were regularly irrigated to maintain soil water at field capacity. Then, the plants were subjected for 21 days to the following treatments: (I) a[CO 2 ] and well-watered (a[CO 2 ]WW); (II) e[CO 2 ] and well- watered (e[CO 2 ]WW); (III) a[CO 2 ] and drought stressed (a[CO 2 ]D); and (IV) e[CO 2 ] and drought stressed (e[CO 2 ]D). Our data demonstrate that e[CO 2 ] mitigates drought impacts by reducing stomatal conductance (g S ), improving WUE, and promoting less negative water potentials. Although the reduction in gs decreased water loss by evapotranspiration, it negatively affected A N , and consequently plant growth. Hyperspectral analysis corroborates these findings and is a great non-destructive tool to analyse the effects of drought on plants. We found that P. glomerata exhibits different drought response mechanisms depending on [CO 2 ]. Plants exposed to a[CO 2 ]D increased root/shoot ratio, stem rustification, non- enzymatic antioxidant system by increased anthocyanin content and proteins of ascorbate and glutathione metabolism. Conversely, plants under e[CO 2 ]D invested strongly in osmoregulatory metabolites (e.g. soluble sugars and amino acids). We found that e[CO 2 ] associated to drought promoted changes in the proteomic profile, dramatically affecting the accumulation of stress response proteins. On the other hand, we show that drought positively affected the activity of antioxidant enzymes (CAT and SOD) and 20E content in both [CO 2 ] in P. glomerata plants. However, only in plants exposed to e[CO 2 ]D was there an increase in 20E production, overcoming the biomass limitation caused by this stress. Here, we report for the first time, the up-regulation of cytochrome P450 CYP72A219-like protein in plants grown in the combination e[CO 2 ]D. Therefore, we hypothesize a relationship of this protein with 20E biosynthesis and hypothesize possible ROS signaling (indirectly by increased CAT and SOD) under abiotic stress condition. These data provide relevant information in elucidating the pathway of 20E biosynthesis, as well as enabling biotechnological strategies to increase the production of this metabolite in P. glomerata plants. Keywords: Brazilian ginseng. 20-hydroxyecdysone. Phytoecdysteroids. Water stress. CO 2 enrichment. Secondary metabolism.

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPK alpha in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK.

Stress tolerance is an important trait, that determines the productivity of plants under drought, hypothermia, mineral deficiency, and salinity. Numerous studies of various agricultural crops (J.K. Zhu, 2016; E. Fleta-Soriano, S. Munné-Bosch, 2016), including tea crop (Camellia sinensis L.), were aimed at solving this problem due to the global aridization of the climate. (T.K. Maritim et al., 2015; L.S. Samarina et al., 2019). Along with the sufficiently detailed physiological, biochemical and molecular studies of tea drought tolerance, the exogenous regulation of tolerance by using of chemical and biological substances is still not investigated. In addition, the important role of calcium ions (Ca2+) in the cell recognition of an external stressor by the triggering signal transduction has been shown in many crops (M.C. Kim, 2009; E.G. Rikhvanov et al., 2014). In these studies, tissue culture media supplemented with the osmotically active substances (R.M. Pérez-Clemente et al., 2012; M.K. Rai et al., 2011) and artificial biosystems (microshoots and tissues in vitro), are often used as "drought models" to reveal cellular adaptation mechanisms. However, just a few studies were conducted aimed at deciphering the biochemical and molecular responses of tea plant to stress using tissue culture tool (L.S. Samarina et al., 2018; M.V. Gvasaliya et al., 2019). In this article, for the first time, we investigated the role of calcium in plant adaptation to long-term osmotic stress based on earlier published protocols of tea tissue culture (M.V. Gvasaliya, 2013) and osmotic stress induction protocols. We also demonstrated the prospect of studying the role of exogenous inducers in increasing plant tolerance using "drought models". This work aimed to identify the effect of different concentrations of calcium (Ca2+) in the culture medium on the functional state of tea microshoots grown under mannitol-induced osmotic stress in vitro comparing with control. The changes in morphophysiological state of the leaves, leaves water content, cells membrane permeability, malondialdehyde, proline, and photosynthetic pigments were analyzed. It was found that increased Ca2+content in the nutrient medium (from 440 to 880 mg/l) resulted the slower leaves development and significant decrease of malondialdehyde and cell membranes permeability of tea microshoots (by 50 %, р ≤ 0.05) during the long-term cultivation of tea microshoots in vitro (4 months), indicating inhibition of lipid peroxidation processes. The addition of mannitol (40 g/l) to the culture medium reduced the water content of the shoots (on average by 2 %, р ≤ 0.05), thereby forming light osmotic stress, which led to the accumulation of proline (an increase of 30-40 %, р ≤ 0.05), as well as to the structural and functional rearrangement of the photosynthetic apparatus (a decrease in the amount of photosynthetic pigments by an average of 35-40 %). In addition, a significant decrease of malondialdehyde (by 50-70 %, p ≤ 0.05) and the intensity of electrolyte leakage from leaf tissues (on average by 50 %, p £ 0.05) were observed, indicating a less pronounced oxidative stress in comparison with control (without mannitol). An increase in the Ca2+ concentration in the nutrient medium (from 440 to 880 mg/l) (in the presence of mannitol) did not significantly affect the water content in the leaves and the photosynthetic apparatus (content and ratio of chlorophylls/carotenoids). An insignificant effect of calcium (in the presence of mannitol) manifested itself in a significant decrease in malondialdehyde by 20 μmol/g dry weight. Consequently, the increased concentration of calcium (660-880 mg/l) in the nutrient medium provides an improvement in the functional state of long-term cultivated tea microshoots in vitro (4 months) by reducing the activity of lipid peroxidation in membranes and increasing their stability. The revealed patterns confirm the positive role of calcium ions in the reduction of combined oxidative stress caused by long-term cultivation of plants in vitro in combination with osmotic stress.

Drought tolerance mechanisms are crucial for global crop production under increasing water scarcity. It is important to understand these mechanisms in raspberry (Rubus idaeus L.) cultivars to support their water-limited stress tolerance. This study assessed the physiological, biochemical, and leaf morphological responses of two commercial cultivars, ‘Diamond Jubilee’ and ‘Jade’, across two seasons (2022 and 2024) under controlled irrigation: full irrigation (100%), moderate drought (50%), and PEG-induced osmotic stress in 2022 and two treatments (100% and PEG) in 2024. The responses were significantly influenced by both genotype and treatment. Under PEG stress conditions, ‘Jade’ maintained superior water status with RWC of 48.1% in 2022 and 66.7% in 2024 compared to ‘Diamond Jubilee’ (56.0% in 2022 and 32.4% in 2024), representing 37.4% reduction vs 63.8% reduction relative to their respective controls, indicating greater physiological stability. In contrast, ‘Diamond Jubilee’ showed stronger biochemical responses, with proline increasing from 0.037 to 0.114 μmol/g (1,171% increase) and peroxidase activity rising from 24.4 to 93.9 U/g/min (284.8% increase) in 2022, suggesting enhanced antioxidant defense through multiple enzymatic and non-enzymatic components. Both cultivars accumulated soluble sugars under drought stress, with glucose content increasing from 2.56 to 4.25% (66.0% increase) in 2022 and from 2.59 to 3.09% (19.5% increase) in 2024, indicating osmotic adjustment mechanisms. Total phenolic content increased from 432 to 620 mg GAE/100 g (43.6% increase) in 2024 under PEG treatment. Organic acid analysis in 2024 revealed cultivar-specific responses: citric acid increased from 4.41 to 7.10 mg/g DW (61.0% increase) in ‘Diamond Jubilee’ and from 2.83 to 3.77 mg/g DW (33.2% increase) in ‘Jade’, while ascorbic acid was completely depleted from 0.31 and 0.21 mg/g DW to 0.00 mg/g DW in both cultivars. Oxalic acid showed contrasting responses, increasing from 2.54 to 3.33 mg/g DW in ‘Diamond Jubilee’ but decreasing from 4.12 to 3.60 mg/g DW in ‘Jade’. Principal Component Analysis captured 77.1% of variance in 2022 and 90.2% in 2024, clearly separating cultivars and treatments. Based on superior water retention capacity and maintenance of photosynthetic efficiency across both years, ‘Jade’ demonstrated greater physiological resilience, while ‘Diamond Jubilee’ showed enhanced metabolic plasticity through active osmotic and biochemical stress responses. These findings highlight key traits—relative water content, proline accumulation, phenolic compounds, peroxidase activity, and cultivar-specific organic acid profiles—that can support drought-tolerant raspberry cultivar selection in breeding programs.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-27434-7.

A simple method set for assessing biochemical changes associated with osmotic stress responses was developed using coffee (Coffea arabica L.) leaf disks. Stress was induced by polyethylene glycol (PEG) exposure. Quantitative evaluation of tissue physiological stress parameters was carried out using analytical methods to validate the conversion of classic qualitative histochemical tests for localizing lipid peroxidation, hydrogen peroxide, and total xanthine alkaloids into semi-quantitative assays. Relative electrolyte leakage (EL%) and chlorophyll content (SPAD index) were also recorded. EL% levels of treated disks were higher than those of control ones, whereas SPAD indexes were comparable. Histochemical localization indicated that levels of lipid peroxidation, H2O2, and total xanthines were also higher under osmotic stress than in control conditions. Semi-quantitative data obtained by image processing of histochemical staining consistently matched quantitative evaluations. Chromatographic analyses revealed that theophylline and caffeine concentrations increased in the presence of PEG, whereas theobromine remained constant in relation to the control. The methods herein described can be useful to rapidly acquire initial data regarding biochemical osmotic stress responses in coffee tissues based on simple staining and imaging steps. Moreover, it is likely that the same method may be applicable to other types of stresses and plant species upon minor adjustments.

Osmotic stress is one of the most important abiotic factors which inhibit growth and development in both the vegetative and reproductive stages of many plant species. The aim of this investigation was to compare the biochemical and physiological responses in C3 rice and C4 sorghum to water deficit. Chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (TC) and total carotenoid (Cx+c) contents in both rice and sorghum seedlings under osmotic stress were adversely affected, related to increasing osmotic pressure in the culture media. In addition, the chlorophyll’s fluorescence parameters and net photosynthetic rate (Pn) decreased, leading to growth reduction. Also, a positive correlation was found between physiological and biochemical data, while proline accumulation showed a negative relationship. The Chlb, Pn and fresh weight were maintained better in osmotic-stressed (− 1.205 MPa) C4 sorghum seedlings than those in C3 rice seedlings. The growth and physiological responses of C3 rice and C4 sorghum decreased depending on the plant species, the osmotic pressure in the media and their interactions. Pigment content and Pn ability in C4 sorghum grown under mannitol-induced osmotic stress increased to a greater degree than in C3 rice, resulting in maintenance of growth.

GATA factors are evolutionarily conserved transcription regulators that are implicated in the regulation of physiological changes under abiotic stress. Unfortunately, there are few studies investigating the potential role of GATA genes in potato plants responding to salt and osmotic stresses. The physicochemical properties, chromosomal distribution, gene duplication, evolutionary relationships and classification, conserved motifs, gene structure, interspecific collinearity relationship, and cis-regulatory elements were analyzed. Potato plants were treated with NaCl and PEG to induce salinity and osmotic stress responses. qRT-PCR was carried out to characterize the expression pattern of StGATA family genes in potato plants subjected to salinity and osmotic stress. StGATA12 loss-of-function and gain-of-function plants were established. Morphological phenotypes and growth were indicated. Photosynthetic gas exchange was suggested by the net photosynthetic rate, transpiration rate, and stomatal conductance. Physiological indicators and the corresponding genes were indicated by enzyme activity and mRNA expression of genes encoding CAT, SOD, POD, and P5CS, and contents of H2O2, MDA, and proline. The expression patterns of StGATA family genes were altered in response to salinity and osmotic stress. StGATA12 protein is located in the nucleus. StGATA12 is involved in the regulation of potato plant growth in response to salinity and osmotic stress. Overexpression of StGATA12 promoted photosynthesis, transpiration, and stomatal conductance under salinity and osmotic stress. StGATA12 overexpression induced biochemical responses of potato plants to salinity and osmotic stress by regulating the levels of H2O2, MDA, and proline and the activity of CAT, SOD, and POD. StGATA12 overexpression induced the up-regulation of StCAT, StSOD, StPOD, and StP5CS against salinity and osmotic stress. StGATA12 could reinforce the ability of potato plants to resist salinity and osmosis-induced damages, which may provide an effective strategy to engineer potato plants for better adaptability to adverse salinity and osmotic conditions.

Nitrogen (N) is one of the most important nutrients affecting maize productivity. The effects of osmotic stress on nitrogen assimilation still remain unclear. The aim of this study is to characterize the physiological and biochemical responses to the N level and examine the expression of the genes involved in the N assimilation pathway under osmotic stress. Maize seedlings were supplied with three N levels; low N (LN, 0.5 mM), moderate N (MN, 10 mM) and high N (HN, 20 mM) with or without 10% of PEG6000. Results showed that osmotic stress reduced photosynthesis, and transpiration rate as well as stomatal conductance while increased N assimilation at HN. The activities of NR and GPT enzymes were negatively correlated with the N level. However, a positive correlation was observed between the activities of other N assimilation related enzymes and HN level under osmotic stress. The relative expression of genes involved directly in nitrate transport, for example, ZmNRT1.2, ZmNRT2.2, and ZmNRT2.3 were higher at LN level. At LN application, the genes involved in the N assimilation pathway, like ZmGln4, ZmGln5, ZmGs1.3, ZmGs1.4, and ZmSupp showed higher expression levels under osmotic stress. Overall, the expression level of osmotic-stress related genes was decreased at HN level. Taken together, we concluded that though the mRNA levels and enzymatic activities of N assimilation related enzymes were varied and not typically correlated at various nitrogen levels under osmotic stress. However, the expression level of major N assimilation related genes could be used as biomarkers for the identification of maize genotypes or mutants with high nitrogen assimilation under osmotic stress.

Among abiotic factors, salinity poses a serious danger to agriculture on a worldwide scale, seriously impairing crop productivity. Salinity has a significant impact on food security, making potato (Solanum tuberosum) a promising crop for the future. Due to ion toxicity brought on by osmotic stress during salt stress, potato plant growth is impeded. High salinity levels induce osmotic stress, significantly disrupting the overall physiological health of plants. This disruption manifests in various ways, including the onset of nutritional imbalances, hindrance in detoxifying reactive oxygen species (ROS), membrane impairment, and decreased photosynthetic activity. The broad spectrum of impact adversely influences crucial physiological and biochemical processes in plants. These encompass maintaining water balance, regulating transpiration and respiration, optimizing water usage efficiency, preserving hormonal balance, controlling leaf area, overseeing germination, and hindering the production of antioxidants. The increased permeability of the plasma membrane and subsequent chemical leakage due to ROS during salinity stress result in water imbalance and plasmolysis. However, potato plants effectively manage oxidative stress induced by salinity by upregulating both enzymatic and non-enzymatic antioxidant activities. In response to counteracting the detrimental effects of salinity, plants synthesize osmoprotectants such as proline, polyols (including sorbitol, mannitol, xylitol, lactitol, and maltitol), and quaternary ammonium compounds such as glycine betaine. Many proteins and their interactions regulate the complex and varied pathways that contribute to the salt response and tolerance. This review intends to refocus emphasis on the need to investigate the physiological, biochemical, and molecular responses now in place and subsequently create viable mitigating solutions for salt stress in potatoes. © 2018 The Author(s)

Medicago truncatula is a forage crop of choice for farmers, and it is a model species for molecular research. The growth and development and subsequent yields are limited by water availability mainly in arid and semi-arid regions. Our study aims to evaluate the morpho-physiological, biochemical and molecular responses to water deficit stress in four lines (TN6.18, JA17, TN1.11 and A10) of M. truncatula. The results showed that the treatment factor explained the majority of the variation for the measured traits. It appeared that the line A10 was the most sensitive and therefore adversely affected by water deficit stress, which reduced its growth and yield parameters, whereas the tolerant line TN6.18 exhibited the highest root biomass production, a significantly higher increase in its total protein and soluble sugar contents, and lower levels of lipid peroxidation with greater cell membrane integrity. The expression analysis of the DREB1B gene using RT-qPCR revealed a tissue-differential expression in the four lines under osmotic stress, with a higher induction rate in roots of TN6.18 and JA17 than in A10 roots, suggesting a key role for DREB1B in water deficit tolerance in M. truncatula.

Global climate change poses a significant threat to plant growth and crop yield and is exacerbated by environmental factors, such as drought, salinity, greenhouse gasses, and extreme temperatures. Plant growth-promoting rhizobacteria (PGPR) help plants withstand drought. However, the mechanisms underlying PGPR-plant interactions remain unclear. Thus, this study aimed to isolate PGPR, Bacillus megaterium strains CACC109 and CACC119, from a ginseng field and investigate the mechanisms underlying PGPR-stimulated tolerance to drought stress by evaluating their plant growth-promoting activities and effects on rice growth and stress tolerance through in vitro assays, pot experiments, and physiological and molecular analyses. Compared with B. megaterium type strain ATCC14581, CACC109 and CACC119 exhibited higher survival rates under osmotic stress, indicating their potential to enhance drought tolerance. Additionally, CACC109 and CACC119 strains exhibited various plant growth-promoting activities, including phosphate solubilization, nitrogen fixation, indole-3-acetic acid production, siderophore secretion, 1-aminocyclopropane-1-carboxylate deaminase activity, and exopolysaccharide production. After inoculation, CACC109 and CACC119 significantly improved the seed germination of rice (Oryza sativa L.) under osmotic stress and promoted root growth under stressed and non-stressed conditions. They also facilitated plant growth in pot experiments, as evidenced by increased shoot and root lengths, weights, and leaf widths. Furthermore, CACC109 and CACC119 improved plant physiological characteristics, such as chlorophyll levels, and production of osmolytes, such as proline. In particular, CACC109- and CACC119-treated rice plants showed better drought tolerance, as evidenced by their higher survival rates, greater chlorophyll contents, and lower water loss rates, compared with mock-treated rice plants. Application of CACC109 and CACC119 upregulated the expression of antioxidant-related genes (e.g., OsCAT, OsPOD, OsAPX, and OsSOD) and drought-responsive genes (e.g., OsWRKY47, OsZIP23, OsDREB2, OsNAC066, OsAREB1, and OsAREB2). In conclusion, CACC109 and CACC119 are promising biostimulants for enhancing plant growth and conferring resistance to abiotic stresses in crop production. Future studies should conduct field trials to validate these findings under real agricultural conditions, optimize inoculation methods for practical use, and further investigate the biochemical and physiological responses underlying the observed benefits.

Tissue-specific markers of salinity-induced allostasis in the very euryhaline and widespread mussel Mytilus galloprovincialis.