Bacillus subtilis strain GOT9 confers enhanced tolerance to drought and salt stresses in Arabidopsis thaliana and Brassica campestris

Journal Pre-proof Bacillus subtilis strain GOT9 confers enhanced tolerance to drought and salt stresses in Arabidopsis thaliana and Brassica campestris Og-Geum Woo, Hani Kim, Jong-Shik Kim, Hye Lim Keum, Kyu-Chan Lee, Woo Jun Sul, Jae-Hoon Lee PII:

S0981-9428(20)30040-1

DOI:

https://doi.org/10.1016/j.plaphy.2020.01.032

Reference:

PLAPHY 6026

To appear in:

Plant Physiology and Biochemistry

Received Date: 8 January 2020 Revised Date:

21 January 2020

Accepted Date: 22 January 2020

Please cite this article as: O.-G. Woo, H. Kim, J.-S. Kim, H.L. Keum, K.-C. Lee, W.J. Sul, J.-H. Lee, Bacillus subtilis strain GOT9 confers enhanced tolerance to drought and salt stresses in Arabidopsis thaliana and Brassica campestris, Plant Physiology et Biochemistry (2020), doi: https://doi.org/10.1016/ j.plaphy.2020.01.032. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Bacillus subtilis strain GOT9 confers enhanced tolerance to drought and salt stresses in Arabidopsis thaliana and Brassica campestris

Og-Geum Wooa,b, Hani Kima,b, Jong-Shik Kimc, Hye Lim Keumd, Kyu-Chan Leed, Woo Jun Suld,*, Jae-Hoon Lee a,*

a

Department of Biology Education, Pusan National University, Busan 46241, Korea

b

Department of Integrated Biological Science, Pusan National University, Busan 46241, Korea

c

Marine Industry Research Institute for East Sea Rim, Uljin 36315, Korea

d

Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Korea

*Corresponding authors; Jae-Hoon Lee and Woo Jun Sul E-mail address: [email protected] and [email protected]

ABSTRACT Soil is a primary source of water and inorganic nutrients vital for plant growth. In particular, the rhizosphere, a microecological region around the plant roots, is enriched with root exudates that enable beneficial microbial communities to form. Plant growth-promoting rhizobacteria (PGPR) are rhizosphere bacteria that contribute to the improvement of plant growth through diverse physiological mechanisms. Identifying PGPR is beneficial for agriculture because their use can effectively increase the productivity of plants without the harmful side effects of chemical fertilizers. To further enrich the pool of PGPR that contribute to abiotic stress resistance in plants, we screened roughly 491 bacteria that had previously been isolated in soil from Gotjawal in Jeju island, South Korea. Among several candidates, the application of Bacillus subtilis strain GOT9, led to the enhancement of drought and salt stress tolerance in Arabidopsis. In agreement with the increased stress tolerance phenotypes, its application resulted in increases in the transcripts of various drought stress- and salt stress-inducible genes in the absence or presence of the stresses. Furthermore, the treatment resulted in improved lateral root growth and development in Arabidopsis. GOT9 also led to enhanced tolerance against drought and salt stresses and to upregulation of drought-inducible genes in Brassica, a closely related crop to Arabidopsis. Taken together, these results show that GOT9 could be utilized as a biotic resource that effectively minimizes damage to plants from environmental stresses.

Key words: Bacillus subtilis GOT9, drought stress, salt stress, Arabidopsis, Brassica

1. Introduction Plant growth-promoting rhizobacteria (PGPR) have specific symbiotic relationships with plants, and therefore positively affect plant life cycles in direct and indirect manners. They directly promote plant growth by enhancing acquisition of soil nutrients, nitrogen fixation and mobilization of key nutrients (phosphorus, potassium and iron), and indirectly by mitigating deleterious effects from pathogenic agents and abiotic stresses such as drought and high salt (Singh et al., 2015; Rashid et al., 2016; Santoyo et al., 2016; Shameer and Prasad, 2018). The possible mechanism of drought and salt tolerance in plants triggered by PGPR includes regulation of level of phytohormones (such as ABA, IAA and ethylene), production of exopolysaccharides and ACC deaminase by PGPR, accumulation of various osmolytes (such as proline, sugars, amino acids, polyamines and betaines), alteration of the antioxidant defense system, and induction of stress responsive genes in plants by PGPR (Vurukonda et al., 2016; Kumar and Verma, 2018). PGPR include strains that inhabit the rhizosphere, rhizobia that develop nodules on legumes, and endophytes that can colonize the interior tissues of plants. As biocontrol agents, PGPR are advantageous because they are cheap and easily available and alleviate adverse effects from various stresses that plants encounter, indicating that they can be an efficient economical tool for increasing the productivity of important agriculture crops (Reed and Glick, 2013; Calvo et al., 2014). Throughout their life cycle, plants are frequently exposed to a variety of abiotic stresses such as high temperature, chilling, freezing, high salinity, drought, and UV-B. To effectively overcome these stresses, plants have developed a complex regulatory system. Among such abiotic stresses, drought has been regarded as one of the most detrimental stresses in terms of adversely affecting crop yield (Boyer, 1982; Zhu, 2002). Drought is expected to give rise to serious problems for plant growth and crop productivity in more than half of the world’s arable lands by the year 2050 (Ashraf, 1994; Vinocur and Altman, 2005; Vurukonda et al., 2016). Therefore, it is imperative that we further our understanding of signal transduction

during drought and develop methods to increase drought stress tolerance in plants. Decreasing water loss via stomatal closure, which is mainly regulated by the phytohormone abscisic acid (ABA), is one of the key methods of enhancing plant drought tolerance (Schroeder et al., 2001). High salinity is also an important detrimental factor influencing crop productivity. Indeed, it is estimated that around 20% of the world’s cultivated land is adversely affected by salt stress (Shrivastava and Kumar, 2015). The effects of high salinity on plants consist of disrupted membrane ion transport, disrupted ion homeostasis, osmotic imbalance, and direct toxicity of high Na+ concentrations on cellular metabolism (Volkov and Beilby, 2017). Since exposing plants to high salinity triggers osmotic imbalance, the effects of salt stress largely overlap with those of drought stress. Moreover, the cellular signaling pathways triggered by drought and salt stresses share many signaling components that are responsible for generating tolerance mechanisms against such stresses (Agarwal et al., 2006). Several studies have shown that a variety of PGPR endow drought and salt stress tolerance in diverse plant species, including Arabidopsis (Vurukonda et al., 2016; Numan et al., 2018). Application of Phyllobacterium brassicacearum strain STM196 onto Arabidopsis led to a significant increase in ABA content and a decrease in transpiration rate in the plants, thereby improving the drought tolerance (Bresson et al., 2013). Inoculation with Paenibacillus yonginensis DCY84T resulted in upregulation of salt-, drought-, and heavy metal-stress response genes and consequently promoted tolerance against salt, drought, and heavy metal stresses in Arabidopsis (Sukweenadhi et al., 2015). Furthermore, treatment with two PGPR that produce ACC deaminase (Aneurinibacillus aneurinilyticus ACC02 and Paenibacillus sp. ACC06) alleviated the adverse effects of salt stress in French beans (Gupta and Pandey, 2019). Diverse members from Bacillus genus, which is one of the most widespread ones (Garbeva et al., 2003), have also been reported to contribute to drought and salt stress tolerance in plants. B. megaterium BOFC15 induces ABA synthesis and enhances plant drought tolerance in Arabidopsis. As well as increasing drought tolerance, BOFC15 is also involved in improvement of growth and photosynthetic capacity in Arabidopsis (Zhou et al., 2016). B. subtilis B26 and B. pumilus ameliorate the growth impairment of Phleum

pratense and Glycyrrhiza uralensis under drought stress conditions, through modifying osmolyte and antioxidants accumulations, respectively (Gagné-Bourque et al., 2016; Xie et al., 2019). Moreover, B. licheniformis K11 producing auxin and ACC deaminase alleviates harmful effects from drought stress in pepper (Lim and Kim, 2013). On the other hand, B. subtilis GB03 improves the growth of white clover under salt stress condition, and B. amyloliquefaciens NBRISN13 confers salt tolerance by modulating gene expression profile in rice (Nautiyal et al., 2013; Han et al., 2014). Collectively, these results show that a variety of PGPR strains interact with their specific plant hosts and contribute to enhanced tolerance against a range of pathogenic bacteria and abiotic stresses. To select a PGPR with the potential to improve abiotic stress tolerance in plants, we used hundreds of bacteria isolated in Gotjawal soil from the volcanic Jeju Island, South Korea. We screened rhizobacteria that led to altered expressions of abiotic stress marker genes in Arabidopsis in response to various abiotic stresses. Among them, Bacillus subtilis strain GOT9 triggered upregulation of various drought stress- and salt stress-inducible genes in the presence or absence of such stresses. Moreover, the application of GOT9 contributed to enhanced tolerance against drought and salt stresses in Arabidopsis. GOT9 treatment also led to amelioration of tolerance to drought and salt stresses in Brassica. Based on these findings, we suggest that GOT9 could act as an effective tool to increase productivity of plants under drought and salt stress. 2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana ecotype Col-0 seeds were surface-sterilized with 75% ethanol and 0.1% Triton X-100 for 5 min. After washing once with 75% ethanol, they were further washed at least ten times with sterilized water. They were then placed on 1× Murashige and Skoog (MS) agar medium supplemented with 1% sucrose and 0.8% bactoagar (pH 5.8). After stratification, seeds were grown for 10 days under long-day conditions (16 h light/8 h dark) in a growth chamber at 23°C. The seedlings were then transferred into a pot (6.5-cm-diameter

for top, 4-cm-diameter for base, 6-cm-height) filled with horticultural soil mix (cocopeat 51.5%, peat moss 10%, vermiculite 13%, perlite 15%, zeolite 10%, humic acid 0.1%, fertilizer 0.4%) for further analysis. The cabbage (Brassica campestris L. ssp. pekinensis, cultivar; ‘Chun Yeon Gold’) seeds were surface sterilized with 70% ethanol for 1 min and subsequently with 50% bleach (3-3.5% sodium hypochlorite) for 30 min. After that, they were washed at least ten times with sterilized water and placed on 1× MS agar medium supplemented with 1% sucrose and 0.8% bactoagar (pH 5.8) for 7 days under the same conditions as Arabidopsis. The seedlings were moved into soil for further assessment. In the case of Arabidopsis, two plants per pot were grown for drought treatment, and one plant per pot for salt treatment. Two plants per pot were used to monitor the expression pattern of UVB and heavy metal stress marker genes. For Brassica, one plant per pot was used. 2.2. Bacterial culture and inoculation into Arabidopsis, and abiotic stress tolerance assays Bacillus sp. strain GOT9 was isolated from soil collected from Gotjawal in Jeju island, South Korea, and was identified by 16S rRNA gene sequencing. Isolated GOT9 were grown on R2A agar media at 37℃ for 16 h. The grown bacteria were then collected, washed, and diluted to an OD600 of 0.02~0.03 with MS liquid solution. Then, 1 mL of bacterial diluent was applied into soil adjacent to the roots of 11-day-old Arabidopsis seedlings. For drought tolerance assay, Arabidopsis plants were treated with GOT9 or MS liquid broth (control) and were then regularly watered for another two weeks. After that, the plants were exposed to stress by withholding watering for two weeks, followed by re-watering for two days. After one week of inoculation with bacteria, salt stress tolerance assays were conducted by applying 50 ml of a 300 mM NaCl solution or water into the pots, three times over six days (with a 3-day interval). Images shown in Fig.4 were retrieved 1 day after the final salt treatment. 2.3. Inoculation of GOT9 into Brassica, and drought/salt stress tolerance assays The collected GOT9 were diluted to an OD600 of 0.02~0.03 with MS liquid solution.

Then, 1 mL of bacterial diluent or MS solution was applied to the soil adjacent to the roots of 8-day-old Brassica seedlings. For drought stress, Brassica plants were regularly watered for another 11 days. After that, the plants were stressed by withholding watering for 13 days, followed by re-watering for three days. In the case of salt stress, starting one week after inoculation with bacteria, the salt stress tolerance was checked by adding 100 ml of a 300 mM NaCl solution or water into the pots, three times over 6 days (with a 3-day interval). Images shown in Fig.6C were retrieved 1 day after the final salt treatment. 2.4. Stomatal aperture analysis Detached leaves from 5-week-old Arabidopsis plants were incubated in the absence or presence of GOT9 (OD600 = 0.3) for 2 h, and the stomatal apertures were monitored using a microscope (DM750, Leica) from epidermal peels of the leaves. 2.5. RNA isolation and quantitative real-time RT-PCR (RT-qPCR) analysis Total RNA was isolated from Arabidopsis and Brassica plants using Trizol reagent (Ambion) according to the manufacturer’s instructions. After eliminating the genomic DNA in the isolated RNA using DNase I (Promega), cDNA was synthesized from 1 µg of RNA using RevertAidTM Reverse Transcriptase (Fermentase). Quantitative real-time RT-PCR analyses were conducted as previously described by Woo et al. (2018). The analyses were performed using the Solg™ 2 × real-time PCR Smart Mix (SolGent) according to the manufacturer’s protocol and the Rotor-Gene Q system (Qiagen). Relative amounts of transcripts were determined using the comparative CT method, which was normalized against those of ACTIN2 (Arabidopsis) or ACT7 (Brassica) transcripts from the same sample. Information on the primer pairs used for RT-qPCR analysis are presented in Supplementary Table S1. 2.6. Quantification of chlorophyll contents Starting one week after inoculation with bacteria, salt stress analyses were conducted by

adding 50 ml of a 300 mM NaCl solution to the pots over four weeks with 3-day intervals. The plants were then harvested, and their chlorophyll contents were measured as previously described by Guleria et al. (2014). Seedlings were homogenized with 80% acetone and the extracts were kept in the dark for 30 min. After centrifugation, the supernatant was retrieved for the quantification assay. The equations used for calculations were as follows: Chlorophyll a = 12.7(A663) - 2.69(A645) Chlorophyll b = 22.9(A645) - 4.48(A663) 2.7. GOT9 genome sequencing and analysis The whole genome sequence of B. subtilis strain GOT9 was obtained using PacBio RS II and WGS-Illumina NovaSeq 6000 sequencing (2×100bp) systems by DNA Link Inc. (Seoul, Korea). To calculate Average Nucleotide Identity (ANI) value of GOT9 with other B. subtilis strains, a total of 130 complete genomes was downloaded from NCBI RefSeq database. ANI values were calculated using fastANI (https://github.com/ParBLiSS/FastANI). Among 130 genomes, 10 B. subtilis strains were selected to generate a phylogenetic tree with GOT9. The phylogenetic tree was constructed based on core gene alignment using Roary (Page et al., 2015) pipeline (v 3.11.2). The complete genome sequence of GOT9 has been uploaded in GenBank under accession number CP047325. 3. Results and discussion 3.1. Isolation of Bacillus sp. strain GOT9 To obtain a novel PGPR strain that positively affects the growth of Arabidopsis under adverse environmental conditions, we isolated about 491 microbes from the soil collected in Gotjawal, Jeju island, where there is a unique soil microbial community (Supplementary Table S2) (Kim et al., 2018; Lee et al., 2019). To select a specific PGPR strain that alters the abiotic stress tolerance of Arabidopsis, the microbes were applied to Arabidopsis and the expression patterns of several abiotic stress marker genes were monitored. Among these

microbes, treatment with one of the PGPR candidates led to the upregulation of several drought- and salt stress-inducible genes in Arabidopsis during the initial screening. 16S rRNA gene sequencing showed that the candidate belonged to the species Bacillus subtilis (Supplementary Fig. S1). From the whole genome sequencing, we found that the candidate is composed of a circular 4,119,195 bp chromosome with a GC content of 43.79% and 4,281 coding sequences (CDS). To address how closely related the candidate is to other B. subtilis strains that have already been characterized, we downloaded a total of 130 complete genomes for B. subtilis strains from NCBI RefSeq database and calculated ANI value of the candidate with other B. subtilis strains. Based on the information from 130 complete genomes of B. subtilis strains, we selected 10 strains including a strain most similar to the candidate (B. subtilis TOA), 8 strains associated with stress tolerance in plant, and a type strain (B. subtilis 168T), and generated a phylogenetic tree with 11 strains using core gene alignment (Fig. 1). B. subtilis TOA showed the highest ANI value of 99.90% and all other strains exhibited high ANI value (> 98.07%), as compared with the candidate, confirming that the candidate is a novel strain of the species Bacillus subtilis (Fig. 1). Therefore, we named it as Bacillus subtilis strain GOT9. 3.2. Expression patterns of various abiotic stress-inducible genes after GOT9 application To elucidate the possible role of GOT9 in abiotic stress tolerance in Arabidopsis, the amounts of transcripts for various abiotic stress-inducible genes in Arabidopsis were checked after exposure to GOT9 in more detail. As shown in Figure 2A, four drought stress-inducible genes (RD29B, RAB18, RD20, and NCED3) were upregulated after application of GOT9 in the absence of drought stress. RD29B and RAB18 encode a dehydrin protein that functions as a protector of biomolecules during abiotic stress and their expressions are highly correlated with drought and salt tolerances (Peng et al., 2010; Zhang et al., 2011; Yu et al., 2018). RD20 protein, a caleosin isoform, is widely involved in resistance against biotic and abiotic stresses. Functional loss of RD20 leads to an enhanced stomatal opening and a reduced drought tolerance, indicating its positive role in drought stress tolerance (Aubert et al., 2010). NCED3,

an ABA biosynthesis gene, is also involved in enhancement of drought tolerance (Iuchi et al., 2001; Tan et al., 2003). Since drought- and salt stress-inducible genes largely overlap and the two stress signaling pathways converge at specific points (Agarwal et al., 2006; Roychoudhury et al., 2013), we additionally checked the expression levels of several salt stress-inducible genes (RD29B, SOS1, RD29A, and WRKY8). Similar to the observed expression pattern in drought marker genes, GOT9 led to increases in the expression of salt stress marker genes under nonstress conditions (Fig. 2B). Like RD29B and RAB18, RD29A as a member of dehydrin family is functionally linked to drought and salt tolerance (Peng et al., 2010; Zhang et al., 2011; Yu et al., 2018). WRKY8, a transcription factor, is known to act as a positive regulator in salt stress tolerance (Hu et al., 2013). Collectively, these results imply that GOT9 may be involved in the regulation of drought and salt stress responses, possibly via modulating the expression of genes related to drought and salt tolerance in Arabidopsis, at least in part. It has been reported that drought and salt stresses induce increased levels of endogenous ABA, and that ABA is largely responsible for the expression of drought/salt stress-responsive genes and for tolerance to both stresses (Shinozaki and Yamaguchi-Shinozaki, 2007; Raghavendra et al., 2010; Hwang et al., 2014; Ma et al., 2019). Since GOT9 triggered the upregulation of NCED3 under nonstress conditions (Tan et al., 2003), we think that the upregulation of several ABA-inducible genes, such as RD29B, RD29A, RAB18, RD20, WRKY8, and NCED3, could be achieved via GOT9-induced increased ABA accumulation (Figs. 2A and B). Application of GOT9 did not lead to the upregulation of UV-B stress marker genes such as CHS, DFR, and HY5 (Fig. 2C). Similarly, the expression patterns of heavy metal (aluminum) stress marker genes, such as ALMT1, AIP, and ALS3, were comparable in GOT9-treated samples and control (Fig. 2D). These results indicate that GOT9 may not be involved in tolerance against UV-B and heavy metal stresses in Arabidopsis. 3.3. GOT9 increases resistance against drought and salt stresses in Arabidopsis Since GOT9 triggered the upregulation of drought-responsive genes, we next

investigated if the increased expression of these genes could affect tolerance against drought stress. As shown in Figure 3A, over 35% of GOT9-treated plants survived after drought stress, while just over 6% of control plants survived. Furthermore, in support of the theory that GOT9 enhances drought tolerance, RD29B, RAB18, RD20, and NCED3 were hyper-induced when plants were treated with GOT9 under conditions of drought stress, relative to their expression under drought stress only (Fig. 2A). Collectively, these results show that GOT9 could play a positive role in drought stress tolerance in Arabidopsis. To check whether such an improved tolerance is functionally related to stomatal movement in Arabidopsis, stomatal apertures in the absence or presence of GOT9 were monitored. The stomatal aperture following GOT9 treatment was significantly decreased compared to the control-treatment (Fig. 3B). Therefore, these results reveal that amelioration of drought tolerance by GOT9 may be achieved via enhancement of stomatal closure. Drought stress signaling is largely divided into two pathways: ABA-dependent and ABA-independent pathways (Agarwal et al., 2006). Based on the fact that ABA induces stomatal closure and that GOT9 treatment triggers the upregulation of ABA biosynthesis genes, enhanced drought tolerance by GOT9 is expected to be mediated by the ABA-dependent pathway (Munemasa et al., 2015) (Fig. 2A). It has been reported that ABA and GAs produced by Azospirillum lipoferum ameliorate adverse effects from drought stress in maize, and that ABA produced by Azosprillium brasilense Sp 245 elevates ABA level in Arabidopsis (Cohen et al., 2008, 2009). Moreover, cytokinin-producing Bacillus subtilis confers drought tolerance stress in Platycladus orientalis through increasing ABA content and the stomatal conductance in plants (Liu et al., 2013). These results imply that compounds derived from GOT9, including hormones, may trigger an increase in endogenous ABA level in plants. Based on previous reports that drought- and salt stress-signaling pathways are largely overlapped with each other and our data that GOT9 contributes to enhanced drought tolerance in Arabidopsis (Agarwal et al., 2006; Roychoudhury et al., 2013) (Fig. 3A), we also investigated whether the bacteria affected the response to salt stress in Arabidopsis. Although only GOT9 application did not lead to any phenotypic differences in the plants, the GOT9

plus salt stress treatment resulted in decreased growth retardation in Arabidopsis compared to the salt stress treatment alone (Fig. 4). Moreover, the chlorophyll levels under the GOT9 plus salt stress treatment were significantly higher than those under the salt stress treatment alone (Fig. 4). These data indicate that GOT9 plays a positive role in enhancement of salt stress tolerance in Arabidopsis. Similar to the drought stress results, RD29B, SOS1, RD29A, and WRKY8 were also hyper-induced when plants were treated with GOT9 under conditions of salt stress, relative to their expression under salt stress alone (Fig. 2B). Therefore, we propose that GOT9 is an effective PGPR that enhances tolerance against drought and salt stresses, which commonly lead to increased osmotic stress and subsequent accumulation of endogenous ABA, indicating the possible involvement of ABA in such stress tolerance processes. As shown in Figure 2B, hyper-induction of SOS1 is observed in the presence of GOT9 and salt stress together. SOS1 is a plasma membrane Na+/H+-antiporter known to play an important role in salt stress tolerance, as it reduces the toxic effects caused by excessive sodium ions by removing Na+ from plant cells (Shi et al., 2000, 2002). Therefore, salttolerant phenotypes of Arabidopsis endowed by GOT9 might result from an increased amount of SOS1, at least in part. 3.4. Comparison of growth patterns between control and GOT9-treated Arabidopsis Based on recent findings that PGPR positively regulate the growth and development of primary and lateral roots (Vacheron et al., 2013; Verbon et al., 2016), the ability of GOT9 to modify the root system architecture of Arabidopsis was also investigated (Figs. 5A-C). Although GOT9 application did not alter the growth pattern of primary roots, its treatment led to increases in both the number and the length of lateral roots (Figs. 5A-C). Several reports have shown that a series of PGPR that endow plant drought tolerance ameliorate root growth, including lateral root development (Cohen et al., 2015; Zhou et al., 2016; Lu et al., 2018), raising the possibility that the enhanced drought tolerance and the lateral root development caused by GOT9 may be functionally related to each other. This may be because improved lateral root growth and development caused by GOT9 may contribute to

effective uptake of water and mineral salts. Several PGPR producing IAA, a hormone that plays an important role in lateral root development, contributed to lateral roots formation and/or improved growth in wheat under drought stress condition, implying the possible involvement of auxin in drought tolerance induced by GOT9 (Arzanesh et al., 2011; Hussain et al., 2014; Du and Scheres, 2018). As shown in Figure 5D, the fresh weights of both shoots and roots were not influenced by GOT9 inoculation, showing that GOT9 does not seem to affect primary growth of Arabidopsis, except in terms of lateral root development. 3.5. Application of GOT9 leads to enhanced tolerance against drought and salt stress in Brassica Since GOT9 contributes to enhanced resistance against abiotic stresses such as drought and high salinity in Arabidopsis, we wanted to know whether GOT9 would have the same effect on crop plants. Therefore, we next monitored the effects of GOT9 on abiotic stress tolerance in Brassica crops, which are known to be closely related to Arabidopsis (Lagercrantz et al., 1996). As shown in Figure 6A, treatment with GOT9 markedly improved the survival of Brassica plants under drought stress conditions. Moreover, its application under conditions of drought stress led to the hyper-induction of various drought-inducible genes such as BrDREB1D (Bra028290), BrWRKY7 (Bra013732), and BraCSD3 (Bra002133), supporting the observed enhanced drought-tolerant phenotypes caused by GOT9 (Fig. 6B). The effect of GOT9 on salt stress tolerance of Brassica was also monitored. Even though a marked change between control and GOT9-treated plants was not detected, the growth of aerial parts was slightly improved by GOT9 relative to plants in the absence of GOT9 under salt stress conditions (Fig. 6C). Collectively, these results reveal that GOT9 enhances drought- and salt-tolerance in Brassica, a useful crop closely related to Arabidopsis, as well as in Arabidopsis. Bacillus subtilis has been known to play an important role in enhancing tolerance to biotic stresses in plants (Hashem et al., 2019). Also, it has been shown that several B. subtilis strains involved in abiotic stress tolerance in diverse plant species (Wang et al., 2012; Han et

al., 2014; Gagné-Bourque et al., 2015, 2016; Bokhari et al., 2019). Nevertheless, the reports on B. subtilis strain that commonly contributes to enhanced resistance against both drought and salt stresses are still elusive. Furthermore, the study on B. subtilis strain that is involved in abiotic stress tolerance in Brassica as the second largest oilseed crop has been limited (Maheshwari et al., 2011). Therefore, this research about B. subtilis strain GOT9 is expected to expand the knowledge of PGPRs enhancing tolerance against both drought and salt stresses in plants and increasing productivity in Brassica under drought/salt stress conditions. 4. Conclusion To overcome the adverse effects of environmental pollution by chemical fertilizers and to maximize cost-cutting efficiency, the development of an effective biological agent to replace chemical fertilizers and pesticides is required. In order to find a feasible solution for these problems, studies on PGPR that ameliorate growth and stress resistance in plants have been a research focus in recent years. In this study, we screened PGPR candidates, obtained from the rhizosphere soil collected in Gotjawal, that endowed Arabidopsis plants with abiotic stress tolerance. One PGPR, Bacillus subtilis strain GOT9, markedly enhanced tolerance against drought and salt stress, and improved lateral root development. Moreover, the beneficial role of GOT9 was also observed in Brassica, a useful crop. These results suggest that GOT9 could be utilized as a biological resource in agricultural fields. Nevertheless, the detailed mechanism behind the beneficial effect of GOT9 on stress tolerance in plants remains unclear. Unearthing the compound(s) used for communication between GOT9 and host plants could be the first step in understanding the related mechanism. Identification of GOT9-producing metabolites and/or root exudates from plants treated with GOT9 may help identify the compound(s). Given the several reports that state that PGPR can affect the levels of phytohormones, such as auxin, cytokinin, ethylene and ABA (Kumar and Verma, 2018), monitoring the hormone levels of host plants and the expression patterns of genes related to hormone biosynthesis is also necessary. Author contributions

JHL conceived the study, designed the experiments, and analyzed the data. WJS designed the experiments and analyzed the data. OGW, HK, HLK and KCL performed the experiments and analyzed the data. JSK provided the microbes from Gotjawal. OGW, HLK, WJS and JHL revised the manuscript. JHL wrote the manuscript with contributions from WJS. All authors approve of the manuscript. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by a 2-Year Research Grant of Pusan National University. Figure Legends Fig. 1. A phylogenetic tree among 11 B. subtilis strains was derived from alignment of 3,439 core genes. ANI values to GOT9 and isolation sources are described on the right side of the tree. The strain that is reported to contribute to abiotic stress resistance in plants is indicated in red. The strains reported to contribute to biotic stress resistance in plants are indicated in green. The related references are presented on the right side. Information on their isolation sources was obtained from the references and NCBI (BioSample). Fig. 2. Expression patterns of various abiotic stress-inducible genes after application of GOT9 and/or in response to drought, salt, and UV-B stresses. (A) Hyper-induction of drought-inducible genes in Arabidopsis treated with GOT9 under drought stress. After 1 week of inoculation with GOT9 or adding MS liquid, 18-day-old Col-0 plants were kept in air until loss of approximately 10% of the total fresh weight or were kept without drought stress. The amounts of transcripts from various genes were normalized against those of ACTIN2 transcripts from the same sample. The average value of each gene’s expression from control samples in the absence of drought stress was considered to be 1.0. Values represent means ± SD (n ≥ 6). A Student’s t test was used to identify statistically significant differences

between control and GOT9-treated samples; *P < 0.05; **P < 0.01; ***P < 0.001. Statistical calculations

were

performed

using

GraphPad

QuickCalcs

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(https://www.graphpad.com/quickcalcs/). (B) Hyper-induction of salt stress-inducible genes in plants treated with GOT9 under salt stress. From 1 week after inoculation with bacteria, 50 ml of a 200 mM NaCl solution or water was added to pots, twice over 3 days (with a 3-day interval). The samples for qRT-PCR analysis were harvested three days after the final salt treatment. The average value of each gene’s expression from control samples in the absence of salt stress was considered to be 1.0. Values represent means ± SD (n = 6). (C) Expression patterns of UV-B stress marker genes after application of GOT9. After 1 week of inoculation with GOT9 or adding MS liquid, Col-0 plants were harvested for qRT-PCR analysis. Values represent means ± SD (n ≥ 6). (D) Expression patterns of heavy metal stress marker genes after application of GOT9. After 1 week of inoculation with GOT9 or adding MS liquid, Col0 plants were harvested for qRT-PCR analysis. Values represent means ± SD (n ≥ 7). Fig. 3. Enhancement of drought tolerance by GOT9 in Arabidopsis. (A) Increased survival rate of GOT9-treated Arabidopsis plants in response to drought stress. Results are from three biological replicates and values represent means ± SD (n = 3). (B) Enhanced stomatal closure in Arabidopsis treated with GOT9. The apertures of at least 52 stomata per sample were measured. The average value of the stomatal apertures from control samples was considered to be 1.0. Values represent means ± SD (n = 3). Fig. 4. Increased tolerance of GOT9-treated Arabidopsis plants to salt stress. To quantify chlorophyll contents, from 1 week after inoculation with bacteria, the plants were exposed to salt stress by adding 50 ml of a 300 mM NaCl solution to pots over 4 weeks with 3-day intervals. Results are from three biological replicates and values represent means ± SD (n = 3). Fig. 5. Regulation of lateral root development after GOT9 application. Comparison of primary root length (A), number of lateral roots (B), and lateral root length (C) between control and GOT9-treated Arabidopsis seedlings. Control or GOT9 diluted to an OD600 of

0.2~0.3 with MS liquid solution was applied to the soil adjacent to the roots of four-day-old Arabidopsis seedlings grown on MS plates. Values represent means ± SD (n = 3 for measurement of primary root length and lateral root number; n = 10 for measurement of total lateral root length). The lengths of primary and lateral roots were measured from the scanned images using ImageJ software. (D) Comparison of fresh weights of shoots and roots of control and GOT9-treated seedlings. Control or GOT9 diluted to an OD600 of 0.2~0.3 with MS liquid solution was applied to the soil adjacent to the roots of four-day-old Arabidopsis seedlings grown on MS plates. Fresh weights were determined seven days after application. The average value of fresh weights of shoots or roots from control samples was considered to be 1.0. Values represent means ± SD (n = 3). Fig. 6. Increased drought and salt tolerance and enhanced expression of drought-inducible genes after treatment with GOT9 in Brassica crops. (A) Increased drought stress tolerance in Brassica after GOT9 application. Values represent means ± SD (n = 4). (B) Hyper-induction of drought-inducible genes in Brassica plants treated with GOT9 under drought stress. After 1 week of inoculation with GOT9 or adding MS liquid, 15-day-old Brassica plants were kept in air until the loss of approximately 20% (for BrDREB1D and BrWRKY7) or 40% (for BraCSD3) of the total fresh weight or were kept without drought stress. The amounts of transcripts from various genes were normalized against those of ACT7 transcripts from the same sample. The average value of each gene’s expression from control samples in the absence of drought stress was considered to be 1.0. Values represent means ± SD (n = 6). (C) Improved salt stress tolerance in Brassica following GOT9 treatment. Total leaf area was determined from the scanned images using ImageJ software. Values represent means ± SD (n = 3).

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Highlights • The application of Bacillus subtilis strain GOT9 led to the enhancement of drought and salt stress tolerance in Arabidopsis. • GOT9 treatment also led to amelioration of tolerance to drought and salt stresses in Brassica. • GOT9 treatment resulted in improved lateral root growth and development in Arabidopsis.

Author contributions JHL conceived the study, designed the experiments, and analyzed the data. WJS designed the experiments and analyzed the data. OGW, HK, HLK and KCL performed the experiments and analyzed the data. JSK provided the microbes from Gotjawal. OGW, HLK, WJS and JHL revised the manuscript. JHL wrote the manuscript with contributions from WJS. All authors approve of the manuscript.

Conflict of interest The authors declare that they have no conflict of interest.