Improvement of salt tolerance of Arabidopsis thaliana seedlings inoculated with endophytic Bacillus cereus KP120

ABSTRACT In our previous reports, an endophytic bacterium, Bacillus cereus KP120 was isolated from the halophyte species Kosteletzkya virginica. In this study, the effect of KP120 colonization on Arabidopsis thaliana seedlings was investigated. Our results showed that inoculation with KP120 could promote the growth of A. thaliana seedlings plants under salt-stress conditions, compared with uninoculated controls. After salt treatment, chlorophyll, proline, the activity of antioxidant enzymes, Indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate-deaminase in plants inoculated were increased significantly but malondialdehyde content was decreased compared with the plants under salt stress lonely. Similarly, under non-salt stress, physiological indices above except for MDA in plants inoculated with KP120 were increased compared with control. B. cereus also induced the up-regulation of key genes involved in IAA biosynthesis, responses, transport, down-regulated expression of genes related with ethylene synthesis and response. Our work principally demonstrates that Bacillus cereus KP120 significantly enhances plant growth and increases plant tolerance to salt stress.


Introduction
In modern agricultural production, soil salinization is a compelling worldwide environmental problem ) that inhibits plant growth and development (Sherameti et al. 2008), and decreases the yield of crops, including maize, wheat, cotton, soybean, rice, and more (Baek et al. 2020;Fan 2020). Furthermore, salt stress may damage plant physiological processes such as photosynthesis, protein synthesis, and energy metabolism (Song et al. 2016) because of overaccumulation of ROS, osmotic stress, water deficits, and membrane degeneration (Abdel Latef et al. 2019). Therefore, it is necessary to alleviate the adverse effect of salinity stress by different strategies.
Often, genetic modification can be used to improve plant tolerance. However, this approach is time-consuming and costly (Dong et al. 2019). The utilization of plant growthpromoting bacteria (PGPB) is to mitigate severe salt stress in a cost-effective and environmentally friendly approach (Qin et al. 2016). Beneficial microorganisms can colonize and proliferate in the host plant tissues and protect plants from salt stress through various direct and indirect mechanisms (Jaemsaeng et al. 2018;Singh et al. 2019;Yao et al. 2010). Salt stress, manifesting in water uptake reduction via osmotic stress, triggers a range of metabolic and molecular cascades such as inhibition of photosynthetic activity, indole-3-acetic acid (IAA)-mediated responses followed by stimulation of SOD, POD and CAT activities, incensement of membrane permeability, and accumulation of osmolytes like proline. PGPB has been also shown to alleviate salt stress in plants by producing different bioactive secondary metabolites, i.e. volatile organic compounds (VOCs) (Khan et al. 2012), and increasing the activity of antioxidant enzymes, including peroxidase and catalase, that counteract stress-induced reactive oxygen (Bacon and White 2016). PGPB has been found to alleviate the adverse effect of salinity stress in plants such as Piriformospora indica (Mohd et al. 2017), Bacillus flexus (Xiong et al. 2020), Aspergillus terreus (Khushdil et al. 2019), Streptomyces sp. (Jaemsaeng et al. 2018) and Pseudomonas putida (Kumar et al. 2020). PGPB enhances salt stress tolerance by modulating the levels of phytohormones (e.g. IAA, gibberellins. ethylene), or by maintaining K + /Na + balance (Abhinandan et al. 2018;Khan et al. 2012). In addition to plant growth-promoting bacteria, endophytic fungi in ryegrass has been reported to significantly increase phenol content and antioxidant enzyme activity (Qawasmeh et al. 2012), which counteracts plant growth and development repression by reactive oxygen species (ROS) Zheng et al. 2016). Exopolysaccharides (EPS) are mainly secreted by microorganisms during growth and metabolism and released into the surrounding environment. Some studies have shown that the production of EPS in bacteria is one of the strategies used by plants to survive under stress conditions (Kumari and Khanna 2015), by increasing microbial ability to attach to plant rhizospheres, and improving nutrient utilization and water absorption through biofilm formation on the root surface. For example, Sun Liang found that EPS producing bacteria Pantoea alhagi NX-11 could promote rice growth and reduce the toxic effect of salt stress, compared with the EPS-deficient strain NX-11 eps- (Sun et al. 2020). The protective mechanism was believed to relate to the high molecular weight of EPS helping maintain high moisture content in the rhizosphere. As an acidic polysaccharides with negative charges, EPS has strong adsorption capacities for metallic cations such as Na + and Mn 2+ (Sun et al. 2020).
Plant hormones can alleviate plant damage caused by high salinity to some extent. Plant cell growth, development, division, and nutrient absorption are all related to plant hormones. High salinity limits the synthesis of auxin (Cackett et al. 2022). Furthermore, the synthesis, transport, metabolism, and activity regulation of plant auxin were affected under abiotic stress (Mateo-Bonmatí et al. 2021). Auxin (IAA) is widely distributed in higher plants and plays a key role in physiological processes such as cell growth, cell division, and embryonic development. The YUC gene family catalyzes the process of indole-3-pyruvate (IPA) producing auxin and regulates the synthesis of IAA. PIN is an important carrier element to regulate the polar transport of auxin, which is closely related to the growth and development of plants. Auxin regulates plant growth by inducing a series of responsive genes, among which SAUR (Small Auxin-up RNA) is one of the early corresponding genes of auxin (Hagen and Guilfoyle 2002) involved in the regulation of various biological processes (Li et al. 2017).
Some plant rhizosphere-promoting bacteria (PGPB) use 1-aminocyclopropane-1-carboxylate (ACC) as the source to synthesize ACC deaminase, which can reduce the ethylene concentrations of plants and stimulate plant growth. Bacillus cereus can secrete ACC deaminase, which can significantly promote the growth of plants (Jaemsaeng et al. 2018;Singh et al. 2020) such as tomatoes, cucumbers, wheat, beans, and other crops. Ethylene is synthesized from methionine by catalysis mediated by S-adenosyl-L-methionine synthetase, 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) . ACO and ACS are the rate-limiting enzymes that regulate ethylene biosynthesis, and several regulators influence ethylene production by changing the activities and gene expression levels of ACS and ACO. Ethylene response factors (ERFs) participate in abiotic and biotic stress responses and play key roles in various stresses, especially salt stress (Han et al. 2020). Similarly, EILs have a conserved binding sequence of plant-specific transcription factors in the downstream gene promoter involved in ethylene response .
In previous studies, we have isolated and identified an endophytic bacterium, B. cereus KP120, from the salt-tolerant Kosteletzkya pentacarpos (Han et al. 2015). In the present study, we aim to investigate whether the PGPB B. cereus KP120 can alleviate the adverse effects of salt stress on the growth of Arabidopsis thaliana plants. Physiological indices including chlorophyll, proline, EPS content, and plant growth and development under salt stress were investigated. Additionally, IAA content in A. thaliana seedlings was examined, and the expression of SAUR family genes and key genes in auxin synthesis (YUCCA) and transport (PIN) were analyzed. Similarly, the content of ACC deaminas, the expression of some genes involved in the degradation of precursors in ethylene synthesis (ACO, ACS) and in ethylene signal response pathways (ERF and EIL) were analyzed. Our results help elucidate the molecular basis of growth in plants inoculated with B. cereus KP120.

Cultivation of Bacillus cereus KP120
Bacillus cereus KP120, isolated and identified from the salttolerant Kosteletzkya pentacarpos, was used in the present study (Han et al. 2015). Bacterial strains were grown in 50 mL LB medium at 37°C for 12 h.

Plant materials and growth conditions
The Arabidopsis thaliana used in this study was a wild type of Columbia stored in our laboratory. Arabidopsis thaliana seeds were surface-sterilized with 75% ethanol for 1 min, followed by NaClO (5%) for 10 min, and then washed at least 5-10 times with sterilized water (Woo et al. 2020). The seeds were placed on Murashige and Skoog (MS) agar medium (pH 5.8-6.0) for 7 days at 28°C. The seedlings were moved into soil for further assessment (Egamberdieva et al. 2017).

Treatment with B. cereus KP120 and NaCl
Seven-day-old Arabidopsis plants were initially treated with either sterile water or strain KP120, then used for further treatment with salt stresses. For salt stress, from 7 days after inoculation with the bacteria, each plant in a pot was treated with 250 mM NaCl solution or sterile water. After 1 week of growth, the Arabidopsis seedlings were randomly divided into four groups: non-inoculated control group (CK); group treated with 250 mM NaCl solution (Salt); inoculated with B. cereus KP120 (BC); and group treated with 250 mM NaCl solution, then inoculated with B. cereus KP120 (BCS).

Measurement of chlorophyll, proline and malondialdehyde content
Chlorophyll content in leaves of control and Bacillus cereustreated Arabidopsis was quantified according to the method of Moran et al (Moran 1982). The proline contents of Arabidopsis seedlings were determined according to the method described by Jaemsaeng et al (Jaemsaeng et al. 2018). Leaf malondialdehyde (MDA) content was estimated by the method of thiobarbituric (TBA) (Diao et al. 2015).

Measurement of antioxidant enzyme and ACC deaminase activity
To quantify protective enzyme activity, fresh roots and leaves samples (0.5 g) were immediately pulverized with liquid nitrogen. The samples were added to 50 μM potassium phosphate buffer, and then were centrifuged for 20 min at 12,000 g (4°C). Afterwards, the supernatant was collected as a crude enzyme extract.
The method in (Donahue et al. 1997) was carried out to determine the SOD activity by Nitroblue tetrazolium photochemical reduction method at 560 nm. Catalase activity (CAT) was quantified by determining the disintegration of hydrogen peroxide at 240 nm as described in (He et al. 2014). The ascorbate peroxidase (APX) activity was calculated by the decline in optical density at 240 nm according to the method of He et al. (2014). The activity of peroxidase (POD) was analyzed by the guaiacol method (Fujita et al. 1995). The optical density at 240 nm was used to estimate the activity.
The method of Penrose and Glick -2, 4-Dinitrophenylhydrazine Colorimetry was applied to measure the activity of ACC deaminase (Penrose and Glick 2003). That is, the quantification of ACC deaminase production was estimated through the amount of α-ketobutyrate released as a result of the hydrolysis of ACC by ACC deaminase.
Secretion of indole acetic acid (IAA) in strain KP120 and measurement of IAA in leaves and root IAA production was assessed according to Gordon and Weber (Saleem et al. 2021). B. cereus KP120 was inoculated into 50 ml LB medium supplemented with tryptophan and incubated at 37°C at 150 rpm on a rotary shaker for 12 h. 100 μL of bacterium suspension was added into a tube supplemented with 100 μL of Salkowkiwas reagents followed by incubation at room temperature for 15 min to have color reaction. Color change from pale yellow to pinkish red was the indication of IAA production. The amount of IAA produced by the B. cereus KP120 was calculated using a standard curve of commercial IAA at 530 nm in spectrophotometer UV-1800PC.
IAA content in Arabidopsis seedlings was measured via the ELISA kit. IAA was extracted according to a modification of the method of Ertani et al. (2019).

Extraction and measurement of EPS in Bacillus cereus KP120
The isolated strain Bacillus cereus KP120 was inoculated in the fermentation medium at 37°C in an orbital shaker at 180 rpm. After a 40 h incubation period, the culture medium was boiled 4 times for 30 min each time. After ultrasonic extraction for 30 min, the medium was centrifuged at 1800g for 20 min at 4°C. The supernatant was evaporated on a rotary evaporator and then added three volumes of 95% ethanol overnight at 4°C. The precipitated EPS was collected by centrifugation at 1800g for 20 min at 4°C. The precipitate was dissolved in 800 μL distilled water, and the phenol-sulphuric acid method was used for determining the content of the exopolysaccharide (Adesulu-Dahunsi et al. 2018).

RNA isolation and RT-qPCR analysis
Total RNA was isolated from Arabidopsis plants using HiFi-MMLV cDNA kit CW0744s according to the manufacturer's instructions. Reverse transcriptase quantitative RCR was performed using an Ultra SYBR Mixture ((LOW ROX) CW2601M). RT-qPCR analyses were performed using Rotor-Gene (RG-3000). The data were normalized to an internal control gene, AT-actin. The primer sequence information for analysis is shown in Table S1.

Statistical analysis
The obtained data were statistically analyzed by ANOVA method and turkey test with Graphpad Prism 8. All statistical experiments were performed for three biological replicates. The data were presented as the mean values ± S.E.

Effect of Bacillus cereus KP120 on A. thaliana growth under salt stress
During the salt stress condition, the treated plants shriveled and displayed chlorosis in leaves compared with those grown under normal conditions. However, the application of the endophytic KP120 improved the growth of A. thaliana under both normal and salt-stress conditions (Figure 1). In normal conditions (without NaCl), the root lengths, plant height, leaf number, and branch number of seedlings inoculated with strain KP120 increased by 8.86%, 9.75%, 12.50%, and 75.18%, respectively, compared with the control group . Under salt-stress conditions, root lengths, plant height, leaf number, and branch number of seedlings inoculated with KP120 increased significantly by 14.40%, 182.24%, 14.28%, and 53.84% compared with the control seedlings (Figure 2c-f).
Colonization of B. cereus KP120 (+KP120) increased significantly fresh and dry weight of shoot under normal (control) and salt stress (salt) condition. Inoculated plants had obvious enhancements in total fresh and dry mass, which was 1.5-2.2 times and 2.5-2.8 times that of uninoculated plants without NaCl, respectively. Under salt-stress treatment, after application of strain KP120, all growth parameters of A. thaliana were increased (Figure 2a-d).
Bacillus cereus KP120 enhanced proline and chlorophyll, while decreased MDA under salt stress The chlorophyll content in salt stress conditions was lower than that in control conditions. However, the chlorophyll content of A. thaliana inoculated with strain KP120 increased by 23.14% and 30.48% compared with that of non-inoculated under non-salt conditions and salt-stress conditions, respectively ( Figure 3a). These results indicate that KP120 enhanced plant tolerance to salt stress by means of chlorophyll accumulation.
A slight increase in proline content was observed in the plants inoculated with KP120 under non-salt stress or salt stress treatment in leaves and significantly increase in roots under salt treatment. The proline content in A. thaliana leaves and roots were increased by 1.3-fold and 2.0-fold in inoculated plants when compared with non-inoculated plants, respectively ( Figure 3b). Taken together, these results indicate that KP120 improved salt tolerance by increasing proline content.
There was no obvious difference in malondialdehyde (MDA) content under normal conditions with strain KP120 treatment compared with that inoculated with KP120. Compared with control, MDA content was significantly increased by about 3.63-fold (leaves) and 2.27-fold (roots) in salt conditions, respectively. However, the MDA content decreased notably by 76.66% in leaves, and 53.87% in roots in the plants inoculated with strains KP120 under salt-stress conditions (Figure 3c). These results indicate that strains KP120 improved plant tolerance to membrane oxidative damage under salt-stress conditions.

Enhancement of the antioxidant enzyme activities in inoculated plant
Previous studies suggested that antioxidant enzyme activities have a positive effect on B. cereus-induced abiotic stress resistance . Our results clearly showed that the SOD and POD activities in the roots and leaves of colonized plants were higher than the activities of all other treatments (including only salt, no salt, no strain KP120) under salt-stress conditions (Figure 4a and b). A similar result was observed for the CAT and APX activity (Figure 4c and d). Enzyme activity of SOD, POD, CAT, and APX increased by about 26.92%, 46.36%, 11.81%, and 76.85% in the roots of salt-treated plant, respectively, after B. cereus KP120 colonization. Therefore, these results indicate that KP120 promoted plant tolerance to salt stress through the accumulation of antioxidant enzymes.
IAA production of Bacillus cereus and increases in IAA content of Arabidopsis thaliana inoculated with KP120 under salt stress The concentration of IAA was about 0.98 × 10 -13 µg/mL, suggesting that B. cereus KP120 has certain ability for IAA secretion. The concentration of IAA in both leaves and roots was lower in the salinity treatment compared with control treatment (Figure 5a). On the other hand, IAA concentration significantly increased by 8.41% (leaves) and 35.83% (roots) in plants treated with KP120, compared with noninoculated plants under salt stress conditions. Similarly, Figure 1. The growth of Arabidopsis thaliana seedlings after inoculation with B. cereus KP120 under salt stress. CK, BC, Salt, BCS designated respectively as A. thaliana group non-inoculated, inoculated with Bacillus cereus KP120 lonely, under treatment with 250 mM NaCl and under 250 mM NaCl with Bacillus cereus KP120. Figure 2. The fresh weight (a), and the dry weight (b), plant high (c), root length (d), the branch number (e) and leaf number (f) of shoot and root of A. thaliana after inoculation with (+KP120) and without (-KP120) B. cereus KP120 under normal (control) and salt stress(salt) condition. The data represents means ± standard error (SE). Different letters indicate significant differences by Turkey's test (P-value < 0.05).
under normal conditions, the application of strain KP120 induced higher IAA concentration in A. thaliana plants compared with non-inoculated plants.
Bacillus cereus KP120 to reduced ACC content in Arabidopsis thaliana under salt stress As shown in Figure 6(a), there was a significant positive correlation between the level of α-ketobutyrate and the activity of ACC deaminase in Arabidopsis seedlings under control and salt stress conditions. In stress conditions, the concentration of α-ketobutyrate decreased by 6.27% and 19.59% in A. thaliana leaves and roots, respectively, compared with the unstressed conditions (Figure 5b). This shows that the plant produced more ethylene precursor substance ACC, resulting in an increase in ethylene content under salt stress conditions. Compared with controls, the ACC content of plants inoculated with B. cereus KP120 was reduced. In particular, the amount of α-ketobutyrate increased by 17.52% in the roots of plants inoculated with strains KP120 under salt stress conditions (Figure 5b). Overall, these results suggest that strains KP120 reduced ethylene precursor ACC content, thus decreasing ethylene concentrations.

The production of EPS by cultured Bacillus cereus KP120 strain
Because the composition of the medium plays an important role in the yield of EPS in B. cereus KP120, the components of culture medium (C and N sources and inorganic salts) were optimized. With the optimal medium, the accumulation of EPS reached 37.80 mg/L in the fermentation broth, which was 3.9-fold higher than the initial yield of 9.64 mg/L. These results suggest that B. cereus KP120 can produce EPS.

Effect of B. cereus KP120 colonization in seedlings under salt stress on the expression of genes involved in IAA and ethylene signal pathway
Under different conditions, the expression levels of some genes in the SAUR gene family of Arabidopsis thaliana change significantly. To further study the expression changes of SAUR gene family members in Arabidopsis thaliana after inoculation with B. cereus KP120 under saline conditions, the relative expression levels of the measured genes were normalized and analyzed. Fifty members of SAUR gene family in Arabidopsis thaliana can be detected in roots and leaves. As shown in Figure 6a, the expression of SAUR04, SAUR14, SAUR15, SAUR49, SAUR51, SAUR54, SAUR60, SAUR69, SAUR72, and SAUR79 were significantly downregulated in leaves and roots under saline conditions ( Figure  6a), while the genes were upregulated after colonization with B. cereus KP120. In addition, after inoculation with B. cereus KP120 under normal conditions, the expression of SAUR12, SAUR49, SAUR62, SAUR69, SAUR71, SAUR78, and SAUR79 were significantly upregulated in the Arabidopsis seedlings. After inoculation with B. cereus KP120, the expression of SAUR04, SAUR09, SAUR14, SAUR15, SAUR23, SAUR27, SAUR29, SAUR31, SAUR32, SAUR42, SAUR45, SAUR51, SAUR53, SAUR54, SAUR60, SAUR65, SAUR76, and SAUR72 were significantly upregulated in the Arabidopsis seedlings under salt stress conditions. In summary, after inoculation with B. cereus KP120 under saline conditions, the expression profiles of many members of the SAUR gene family in Arabidopsis thaliana change significantly (Figure 6a).
The YUCCA genes encode a flavin monooxygenase-like enzyme that plays a key role in auxin biosynthesis and regulates many aspects of plant growth. Our results demonstrate that the expression of YUCCA2 and YUCCA6 were significantly down-regulated in the Arabidopsis seedlings under the salt stress conditions. However, after inoculation with B. cereus KP120, the expression of YUCCA2 and YUCCA6 was up-regulated ( Figure 6b). The PIN gene family is involved in various developmental processes in plants. Our results demonstrate that the expression of PIN2, PIN4, and PIN5 was lower in seedlings under salt stress compared with the control plants. However, these genes were up-regulated after colonization with B. cereus KP120 (Figure 6b). Therefore, the colonization of B. cereus KP120 in Arabidopsis seedlings regulated the expression of some genes to increase the content of IAA in seedlings.
As shown in Figure 6(c), most of the key genes in ethylene synthesis (ACO12, ACO9, ACS5, ACS1, etc.) and signal transduction (ERF8, ERF5, EIL1) were significantly changed under saline conditions. The expression of ACO3, ACO6, ACO9, ACO12, ACO13, ACS1, ACS2, ACS5, and ACS6 was significantly up-regulated in seedlings under saline conditions, while these genes were down-regulated after colonization with B. cereus KP120 (Figure 6c). This suggests that these two gene families may be involved in the salt response in Arabidopsis. Similarly, our results revealed that the expression of ERF1, ERF, ERF5, EIL1, and EIL3 was upregulated in seedlings under salt stress compared with the control plants. However, these genes were down-regulated after colonization with B. cereus KP120 (Figure 6c). This indicates Figure 3. The chlorophyll content (a), proline (b) and MDA (c)contents of leaves and roots in A. thaliana seedlings inoculated with (+KP120) and without (-KP120) B. cereus KP120 under normal (control) and salt stress (salt). The data represents means ± standard error (SE). Different letters indicate significant differences by Turkey's test (P value < 0.05).
that these genes play important and specific roles in Arabidopsis responses to salt stress.

Discussion
Soil salinization is one of the most important abiotic stresses that seriously affect crop production. Two-thirds of the world's countries and regions have been affected by soil salinization (Ripa et al. 2019). Presently, research shows that PGPB can promote plant tolerance to salt stress (Kumar and Verma 2018;Mesa-Marín et al. 2019). Bacillus contains a substantial number of PGPR strains, which are capable of promoting plant growth by suppressing phytopathogens (Ding et al. 2016;Madriz-Ordeñana et al. 2022;Zhou et al. 2021), causing the death of nematodes (Gao et al. 2016), producing various valuable enzymes and metabolites, degrading heavy metals ) and alleviating abiotic stress to promote plant growth (Vílchez et al. 2018). Soil salinity impacts plant growth through chlorophyll degradation and severely reduced photosynthesis (Zouhaier et al. 2015). In our study, inoculation with B. cereus KP120 increased chlorophyll, suggesting that KP120 can counteract the suppression of photosynthesis under salt stress conditions. Hence, root length, shoot length, and biomass of Arabidopsis thaliana seedlings treated with B. cereus KP120 were increased compared with that non-inoculated plants, implying that the strain KP120 has the potential to cope with the negative effects of salinity stress.
Salt stress disturbs the balance of active oxygen metabolism system in plants, resulting in the generation of reactive oxygen species (ROS), which induces membrane lipid peroxidation in plant tissues, resulting in the destruction of a series of biochemical processes and subsequently reduced plant growth . MDA is one of the main products of membrane lipid peroxidation and can reflect the extent of lipid peroxidation (Wani et al. 2019). The antioxidant enzymes of the ROS scavenging in plants are comprised of SOD, POD, CAT and APX. Most  (salt), with inoculation with (+KP120) and without (-KP120) B. cereus KP120. SOD activity (a), POD (b), CAT (c) and APX (d) activity of the leaves and roots. The data represents means ± standard error (SE). Different letters indicate significant differences by Turkey's test (P value < 0.05). Figure 5. The IAA and a-ketobutyrate contents of Arabidopsis thaliana after inoculation with (+KP120) and without (-KP120) B. cereus KP120 under normal (control) and salt stress (salt). (a) represent IAA contents of the leaves and roots; (b) represent a-ketobutyrate contents: the leaves and roots. The data represents means ± standard error (SE). Different letters indicate significant differences by Turkey's test (P value < 0.05).
plants cannot produce an adequate amount of antioxidants to decrease oxidation damage under stress conditions. Our study clearly showed that the SOD, POD, CAT and APX activities in the roots and leaves of inoculated plants under salt stress conditions are higher than that of non-inoculated plants. KP120 must induce all of these plant antioxidant enzymes. Therefore, reduction of MDA content and mitigation of oxidation damage were observed. Proline, a widely distributed osmotic regulating substance, plays a key role in the scavenging free radicals, protecting plant from osmotic stress under stress conditions (Shin et al. 2020). Proline content is used not only as a marker of plant salt stress, but as signal molecule during stress owing to plant growth regulator by triggering cascade processes . In our study, the presence of B. cereus KP120 led to an increase in proline content, which may help plants prevent osmotic stress under salt stress conditions. IAA is a plant hormone involved in regulating plant life activities in plants and affects the development of the root system of the host plant under abiotic stress (Etesami et al. 2015). Some evidence suggests that IAA production is beneficial to improve plant-microbe interactions . IAA produced by PGPB plays a significant role in the enhancement of root elongation, lateral roots, and root surface area, therefore helping provide soil nutrients and water for plants (Chen et al. 2017;Orozco-Mosqueda et al. 2020). In our results, B. cereus KP120 produced IAA, which may have directly promoted seedling growth. In addition, B. cereus KP120 promoted increase in IAA content of plants. It appeared that IAA accumulation promoted the growth of Arabidopsis thaliana seedlings or mitigated saltinduced damage in the plant. Moreover, our findings show a reduction of ACC in plants inoculated with KP120, which would decrease plant ethylene levels through catabolizing ACC into ammonia and alpha-ketobutyrate, and significantly improve stress resistance and crop yields under various abiotic stresses (Ali and Kim 2018;Zheng et al. 2021). Ethylene levels were likely reduced and saltinduced damage mitigated in plants under treatment with KP120.
EPS are a type of extracellular polymeric substances. It is well known that some endophytes can produce extracellular polysaccharides such as Gluconacetobacter diazotrophicus (Meneses et al. 2017), Azotobacter chrococcum (Rojas-Tapias et al. 2012), Rhodotorula sp. (Silambarasan et al. 2019), and Bacillus licheniformis (Singh and Jha 2016). EPS facilitates microbe-plant interaction, enhances cell adhesion, retains water, forms a protective barrier, and provides a source of nutrients to support plant growth (Bhagat et al. 2021). Bacterial EPS has been shown to bind to sodium ions, thus maintaining Na+/K+ balance, and consequently improving plant salinity tolerance (Jhuma et al. 2021;Singh and Jha 2016). Previous reports also show that bacterial EPS have strong antioxidant activities and scavenging activities on superoxide and hydroxyl radicals and play an important role in sustaining plant growth in salt stress environments (Zheng et al. 2016). We find that KP120 produced EPS, which may help B. cereus KP120 colonize plants to confer salt tolerance by maintaining Na + /K + balance and promoting scavenging activities on superoxide and hydroxyl radicals.
Because of increases in the total amount of IAA and ACCdeaminase in plants inoculated with KP120, we performed real-time RT-PCR to investigate the expression of genes involved in auxin synthesis and transport, IAA signal pathway, ethylene synthesis, and ethylene response in Arabidopsis seedlings inoculated with B. cereus KP120. These analytical results can help us shed light on the molecular mechanism of endophytic bacteria-related plant growth. We assessed the expression of key genes involved in tryptophan-dependent IAA biosynthetic pathways, and auxinresponding factor families such as flavin monooxygenaselike enzyme (YUCCA) and small auxin-upregulated RNAs (SAURs) (Cackett et al. 2022). SAURs may be the largest family of early auxin response genes and play multi-functional roles in plants (Van Mourik et al. 2017). In addition to auxin induction, the Arabidopsis SAURs gene also responds to abscisic acid, gibberellin, ethylene, and other abiotic stresses (anaerobic, low temperature, drought, salt), indicating that SAUR genes participate in the regulation of abiotic stress-tolerance responses (Stamm and Kumar 2013). The expression of most SAUR genes is reduced under salt stress in non-inoculated A. thaliana seedlings. However, SAUR gene expression was increased under salt stress with the inoculation of B. cereus KP120. This demonstrates that KP120 regulates SAURs. Similarly, we observe that the transcript level accumulation of different YUCCA genes was higher in seedlings inoculated with B. cereus KP120 under saline conditions. PIN proteins play a critical role in facilitating auxin efflux from cells. Once IAA is biosynthesized, it is transported with the help of cell-to-cell auxin transport mediated by PINs (Stamm and Kumar 2013). Consistent with the up-regulation of SAURs and YUCCA, the expression of auxin transport genes, particularly PIN2, PIN4, and PIN5 were also up-regulated in inoculated plants under saline conditions. These results indicate that KP120 promotes plant growth under saline conditions through improvements in IAA synthesis and transport.
ACO and ACS are two of the rate-limiting enzymes in the biosynthesis of ethylene, and they play an essential role in the regulation of plant growth and development Sofy et al. 2021). It is well known that expression of ACCdeaminase from B. cereus can improve the growth performance of A. thaliana under normal and salt stress conditions . In this study, salt stress increased the expression of ACS genes and ACO genes in Arabidopsis, while these ACO and ACS genes were down-regulated in Arabidopsis seedlings with inoculation with B. cereus KP120 under saline conditions. Ethylene response factors (ERFs) are widely involved in the response of plants to various stresses (Jiang et al. 2019). EIL proteins are the key components of ethylene signal transduction , which play an important role in plant response to abiotic stress. In our study, we observed down-regulation of ERF and EIL genes in Arabidopsis seedlings inoculated with B. cereus KP120, which suggests that inoculation with B. cereus KP120 improved the tolerance of seedlings to salt stress by decreasing ethylene synthesis.

Conclusion
Salt stress-mediated decreases in the growth of A. thaliana seedlings were significantly alleviated with the supplement of the beneficial B. cereus KP120. The beneficial role of KP120 was reflected by increased proline, chlorophyll, EPS production, and antioxidant enzyme activity, and decreased MDA content in A. thaliana seedlings. These effects all promote plant growth and enhance plant tolerance to salt stress. In addition, the IAA and ACC-deaminase producing strain of KP120 protects A. thaliana seedlings from salt stress through the down-regulation of genes encoding ACS, ACO, and ERF and overall decreases in ACC and ethylene biosynthesis. In addition, there is up-regulation of genes encoding IAA as well as the concentration of IAA in seedling roots to enhance A. thaliana seedling response to salt stress. Our results clarify the function of endophytic bacteria isolated from coastal halophytes K. virginica, and can guide their application as biostimulators in crops to improve salt tolerance in saline soil areas.

Contributions
Yuqi Guo and Zengyuan Tian designed the research, perfoermed data analyses. Yran Zhang performed the most of the experiments. .All authors reviewed and approved the final manuscript.

Notes on contributors
Yaran Zhang obtained her Master's degree in Engineering, Zhengzhou University. She worked on plant-microbe interactions and molecular mechanisms of plant stress resistance.
Zengyuan Tian is Associate Professor at Zhengzhou University. His research interests lie in the area of maize heterosis, epigenetic regulation and molecular mechanisms of plant stress resistance.
Yu Xi is Associate Professor at Zhengzhou University. His research interests are environmental microbiology and ecological toxicology.
Xiaomin Wang obtained her Master's degree in Engineering, Zhengzhou University. Her research interests are plant-microbe interactions and molecular mechanisms of plant stress resistance.
Shuai Chen obtained his Master's degree in Engineering, Zhengzhou University. He worked on plant-microbe interactions and molecular mechanisms of plant stress resistance.
Mengting He obtained her Master's degree in Engineering, Zhengzhou University. Her research interests are plant-microbe interactions and molecular mechanisms of plant stress resistance.
Yange Chen obtained her Master's degree in Agriculture Sciences, Zhengzhou University. Her research interests are plant-microbe interactions and molecular mechanisms of plant stress resistance.
Yuqi Guo is Associate Professor at Zhengzhou University. Her research interests are plant-microbe interactions, molecular mechanisms and signal transduction of plant stress resistance, extraction and application of plant active ingredients.