# Transcriptomic profiling of Arabidopsis Thaliana responses to Stutzerimonas stutzeri, chlorella vulgaris, and mixed consortium inoculation under salt stress

Salma Guendouzi, Adrian Gonzalez Ortega-Villaizan, Eoghan King, Mahbouba Benmati, Jésus Vicente-Carbajosa

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Salma Guendouzi, Adrian Gonzalez Ortega-Villaizan, Eoghan King, Mahbouba Benmati, Jésus Vicente-Carbajosa. Transcriptomic profiling of Arabidopsis Thaliana responses to Stutzerimonas stutzeri, chlorella vulgaris, and mixed consortium inoculation under salt stress. Plant Molecular Biology, 2025, 115 (6), pp.125. 10.1007/s11103-025-01656-9. hal-05534563

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Transcriptomic Profiling of *Arabidopsis thaliana* Responses to *Stutzerimonas*

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*stutzeri*, *Chlorella vulgaris*, and Mixed Consortium Inoculation Under Salt Stress

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Authors:

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Salma Guendouzi¹,²*, Adrian Gonzalez Ortega-Villaizan¹, Eoghan King³,

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Mahbouba Benmati², Jésus Vicente-Carbajosa¹

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¹ Centro de Biotecnologia y Genomica de Plantas, Universidad Politecnica de Madrid

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(UPM)-Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentacion

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(INIA/CSIC), Campus de Montegancedo, 28223 Pozuelo de Alarcon (Madrid), Spain.

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² Ecole Nationale Supérieure de Biotechnologie Taoufik KHAZNADAR, nouveau Pôle universitaire Ali Mendjeli, BP. E66, Constantine, 25100, Algeria.

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³ Université Paris-Saclay, INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for

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Plant Sciences (IJPB), 78000, Versailles, France.

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Abstract:

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Plants, constantly exposed to dynamic environmental conditions, encounter various abiotic stresses that significantly affect their growth and development. In response, plants initiate complex physiological and molecular adjustments, including altered gene expression. One of the most influential factors in mitigating stress impacts is the plant-microbe interaction. Among these, plant growth-promoting rhizobacteria (PGPR) are well-studied for their ability to enhance plant resilience. More recently, microalgae have emerged as potential members of the plant microbiome, although their roles remain comparatively underexplored. This study investigates the transcriptomic responses of *Arabidopsis thaliana* to inoculation with the PGPR strain *Stutzerimonas stutzeri*, the green microalgae *Chlorella vulgaris*, and a consortium of both microorganisms under salt stress conditions. Through RNA-seq analysis, we identified a set of core genes commonly regulated across all inoculation treatments, including *SALT OVERLY SENSITIVE 3 (SOS3)*, the potassium channel *AKT2*, and *CBL-INTERACTING PROTEIN KINASE 5 (CIPK5)*, suggesting a shared stress-mitigation mechanism. Additionally, we identified genes uniquely regulated in response to the *S. stutzeri-C. vulgaris* consortium. These included components of the ethylene signaling pathway (*EIN3/EIL1*), detoxification-associated genes such as *β-GLUCOSIDASE (BGLU22)*, and transcription factors linked to stress response, notably *NAC6* and *MYB12*. Together, these findings provide insight into the specific and overlapping transcriptomic changes induced by bacterial, algal, and combined inoculations, contributing to our understanding of plant-microbe interactions under salt stress.

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36 **Keywords**: *Arabidopsis thaliana*, PGPR, microalgae, transcriptomics, salt stress

37 **Key message**: Comparative transcriptomic profiling of *Arabidopsis thaliana* inoculated
38 with plant growth promoting rhizobacteria, *Stutzerimonas stutzeri*, green microalgae
39 *Chlorella vulgaris*, and their consortium under salt stress reveals synergistic
40 transcriptional responses and identifies candidate genes involved in growth
41 promotion and salt stress tolerance.
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**Introduction:**

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Enhancing crop productivity to meet the rising global demand presents a major challenge for modern agriculture. This challenge is exacerbated by various environmental stressors, one of the most critical being soil salinization which is defined as the accumulation of major dissolved ions, primarily Na⁺, Mg²⁺, Ca²⁺, Cl⁻, K⁺, SO₄²⁻, HCO₃⁻, and, in some cases, CO₃²⁻ (Visconti and De Paz, 2016). Elevated salt concentrations in the soil severely impair plant growth and productivity. Climate change has intensified this issue by increasing the demand for irrigation, which often contributes to the further buildup of salts in the soil. Given that advances in agricultural technology are projected to improve crop yields by no more than 25%, a shift toward more sustainable and resilient agricultural practices is becoming increasingly essential (Acosta-Motos et al., 2017).

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Plants form intricate associations with a wide array of microorganisms, organized into diverse taxonomic communities. This plant microbiota encompasses bacteria, fungi, protists, nematodes, and viruses, inhabiting various accessible plant tissues (Trivedi et al., 2020). The microbiome, consisting of microorganisms and their genetic material, occupies the soil, rhizosphere, roots, and other plant tissues, fostering intricate and ever-changing interactions with the host plant, these interactions are significantly shaped by the surrounding environment and have the potential to enhance plant resilience to various environmental stresses (Trivedi et al., 2022).

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Bacteria inhabiting the rhizosphere, known as Plant Growth-Promoting Rhizobacteria (PGPR), are non-pathogenic microorganisms that efficiently interact with plant roots, often residing on the root surface or in the surrounding soil. These bacteria enhance plant growth and yield through a variety of direct and indirect mechanisms, including nutrient solubilization, phytohormone production, and suppression of pathogens. Notably, PGPR have demonstrated significant potential in improving plant tolerance to various environmental stresses, making them a promising tool for sustainable agriculture (Khan et al., 2021).

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In addition to PGPR, certain species of microalgae have been classified as a part of the plant microbiome and were employed to enhance plant growth, improve yields, and increase fruit quality (Kang et al., 2021; Ryu, 2021). This growth-promoting potential is partly attributed to the composition of microalgal extracts, which are rich in macro- and micronutrients considered essential for regulating plant growth and development (Barone et al., 2018). As such, these extracts represent renewable and sustainable biostimulant sources capable of promoting plant health and productivity.
Due to a growing body of research illustrating the capacity of microalgae to enhance plant growth and provide protection against pathogens, numerous microalgae species are now being integrated into the cultivation practices of key vegetables (Kang et al., 2021). Overall, *Chlorella vulgaris*, *Chlorella fusca*, and *Spirulina platensis* have been used in the cultivation of multiple crops, aiming to enhance their yield while maintaining marketable quality (Kang et al., 2021). Since most attention has been given to dry extract, we know very little about how they affect plant growth when considering interactions between microalgae and plants.

The beneficial effects of PGPR strain *Stuterimonas stutzeri* on plant growth and salt stress mitigation have been previously documented in several host species, including *Medicago sativa*, *Medicago polymorpha* (Gil et al., 2023), tomato (Li et al., 2025), as well as barley, lettuce, and sunflower (Szymańska et al., 2022). Similarly, *C. vulgaris* has also been reported to promote plant growth as a symbiont. In a hydroponic culture, tomato plants grown with a consortium of *Chlorella* sp., *Scenedesmus* sp., *Synechocystis* sp., and *Spirulina* sp. exhibited increased plant and algal biomass, highlighting a positive symbiotic interaction (Supraja et al., 2020). *C. vulgaris* co-cultivated with Swiss chard resulted in increased leaf number, total fresh weight, and root volume (Žunić et al., 2022). Under salt stress conditions, *Chlorella ellipsoidea* enhanced the salinity tolerance of wheat plants irrigated with seawater (Abd El-Baky et al., 2010). However, none of these studies have provided an in-depth analysis of the transcriptional responses induced by microalgae inoculation, particularly under stress conditions. In addition, various studies have documented, the mutualistic symbiosis between microalgae and PGPR (Aditya et al., 2022; Amavizca et al., 2017; Dao et al., 2018; Guendouzi et al., 2024; Peng et al., 2021) particularly in environmental and bioprocess applications, such as wastewater treatment (Anand et al., 2023), biomass production (Cho et al., 2015), and the generation of valuable compounds (Fuentes et al., 2016). Nonetheless, the potential of microalgae-bacteria consortia to enhance plant growth under stress conditions, and the underlying molecular mechanisms, remains largely unexplored.

In the current context of transitioning towards agriculture that aims to reduce the reliance on chemical inputs such as fertilizers and pesticides, the interactions between plants and beneficial microorganisms emerge as a promising solution, furthermore, exploring diverse symbiotic inter-kingdom interaction and their effective application for plant growth promotion, especially when confronted with stress conditions is tempting. However, to translate this potential into practical agronomic applications, a thorough understanding of how these symbiotic relationships function
is essential to maximize their benefits. The present study reports the positive stress alleviation effect of the PGPR strain *S. stutzeri* and the green microalgae *C. vulagris*, as well as their combined action in a mixed consortium, on the dicot model plant *Arabidopsis thaliana* through the investigation of transcriptional alterations triggered by microbial inoculations and salt stress. In this study, we report co-expressed genes that are differentially regulated in response to inoculation with *S. stutzeri*, the green microalgae *C. vulgaris*, and their combined consortium, along with a distinct set of genes uniquely modulated by the consortium, indicating a potential synergistic interaction. These gene expression changes are closely associated with the enhanced biomass production observed, highlighting the molecular basis of their plant growth-promoting effects.

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## 125 Materials and methods

### 126 Plant material and growth conditions

In this study *Arabidopsis thaliana* Col-0 was used. Seeds were surface-sterilized through a 10 min agitation in 1 mL of 70% ethanol, followed by agitation in a sterilization solution made of 5% (v/v) sodium hypochlorite, 5% (w/v) SDS for 10 min followed by 2-3 washes in 1 mL of Milli-Q water, and sown in sterile 0.5 × MS media (Murashige and Skoog, 1962) with no sugar, no vitamins or hormones. Plates were stored for 48 h at 4°C in dark for stratification of the seeds, before they were transferred to a growth chamber and incubated vertically for 5 days, in long day condition, 16 h day length at 22°C 100 to 105 µmol photons m⁻² s⁻¹ photosynthetically active radiation. Thereafter, the germinated seedlings were transferred to their corresponding media and growth condition (microbe inoculation and/or salinity stress condition).

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### 138 Microbial material and growth conditions

The green microalgae *Chlorella vulgaris* were kindly provided by the Aquaculture and Bioremediation Laboratory (Aquabior) of the Biotechnology Department at the University of Oran 1 Ahmed Ben Bella, Algeria, cultures were maintained in mBG 11 liquid media (Dao et al., 2018). Cultures were aerated with an aquarium air pump, air was filtered through an 0.22 µm filter. Light intensity was provided with 4 white LED artificial lights with an intensity of 16.5 µmol m⁻² s⁻¹ each. The temperature was kept at an interval of 25-28°C. Stock cultures were renewed monthly and checked for purity with a light microscope to maintain axenic culture.
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In addition, this work involves the bacterial strain *Stutzerimonas stutzeri* J3BG (accession number OP800247). It was isolated from the rhizosphere of wheat fields in eastern Algeria, screened for its plant growth promoting activity, and identified through 16S rRNA gene extraction and sequencing. The strain was cultured on nutrient agar and incubated at 28°C. For long-term preservation, the strain was stored in 20 % glycerol (v/v) stock at -20°C.

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## Plant-microbe co-culture assays

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To establish a robust tripartite culture system involving *A. thaliana*, the PGPR strain *S. stutzeri* J3BG, and the microalgal strain *C. vulgaris*, a cultivation protocol was developed to support the growth of each component. *A. thaliana* plants were grown as described above. After 5 days, the plants were transferred to inoculated solidified 0.5 × MS media, adhering to the protocol outlined by Lian et al. (2021). The culture medium was inoculated with either *S. stutzeri* J3BG, *C. vulgaris*, or both together, with or without 100 mM NaCl, for comparison, non-inoculated control plants were included under both conditions. Once the medium had solidified, seedlings were planted on the surface and cultivated under strictly controlled environmental conditions: a 16 h light and 8 h dark cycle at a constant temperature of 22°C, with a light intensity of 100 to 105 µmol photons m⁻² s⁻¹ of photosynthetically active radiation. After obtaining a differential phenotype 10 days after inoculation and stress induction, the plants were weighed before total RNA was extracted.

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## RNA extraction

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After 10 days of co-cultivation, whole plants were weighted, placed in Eppendorf tubes and frozen in liquid Nitrogen. Whole plants were used for RNA extraction using the protocol reported by Oñate-Sánchez & Vicente-Carbajosa (2008). Briefly, plant tissue was ground using a cell disrupter. After cell disruption, tubes were immediately placed on ice, 600 µL of extraction buffer (0.2 M Tris HCl, 0.4 M LiCl, 25 mM EDTA, 1% (w/v) SDS) were added to each sample. The tubes were then vortexed twice and centrifuged 15,096 × g for 2 min at room temperature. Next, the aqueous phase was transferred to a fresh tube, 600 µL of phenol were added, and tubes were again vortexed twice and centrifuged. Subsequently, the upper phase was transferred into a new tube and 600 µL of Chloroform were added, followed by another vortexing and centrifugation step. Finally, the upper phase was once more transferred to a new tube, before 1/3 of the sample volume of 8 M LiCl was added. After vortexing, the samples were incubated at 4°C for 2 h followed by centrifugation for 30 min at 4°C (14,176 ×g). Next, the supernatant was removed carefully and the pellet was washed with 150 µL
182 of cold 80% (v/v) ethanol and pellets were left to dry for 20 min. Thereafter, each pellet
183 was resuspended 20 µL of autoclaved milliQ water. The concentration and purity of
184 the extracted material was measured photometrically with a Nanodrop ND-258 1000®
185 UV/Vis spectrophotometer (ThermoFisher). RNA integrity was assessed using gel
186 electrophoresis, ensuring the presence of clear 18S and 28S ribosomal bands without
187 smearing. For this, extracted RNA samples were run on an agarose gel for 20 min at
188 220 V and stained with SYBR™ Safe. Additionally, RNA quality was verified using a
189 Bioanalyzer 2100 (Agilent) by Novogene Genomics Service (Cambridge, UK).

190 RNA sequencing

191 In order to evaluate transcriptomic responses, all RNA samples extracted from plants
192 in different biological conditions were subjected to mRNA-sequencing. Library
193 construction and sequencing (150-nt paired-end reads) were carried out on Illumina
194 NovaSeq™ 6000 platforms by the Novogene Genomics Service, RNA libraries are
195 generated through poly(A) selection, followed by reverse transcription into cDNA.
196 Additionally, Novogene provided basic data analysis using their RNA-seq data
197 analysis pipeline. The analysis involved sequence alignment and mapping using
198 HISAT2 (v2.0.5), followed by transcript quantification against the *A. thaliana* reference
199 annotation using featureCounts (v1.5.0-p3).

200 To perform basic comparative analyses of the obtained transcriptomics data, we have
201 used Dashboard for the Inference and Analysis of Networks from Expression data
202 DIANE (Cassan et al., 2021) (https://diane.bpmp.inrae.fr/). Venn Diagrams have been
203 generated using the Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/) online
204 tool, while gene ontology (GO) enrichment analyses were conducted using the
205 g:Profiler (Kolberg et al., 2023) (https://biit.cs.ut.ee/gprofiler/gost) online tool.

206 Statistical analysis

207 All experiments were performed with three independent biological replicates. The
208 statistical analysis of the data was conducted using OriginPro 2021 9.8.0.200 software.
209 Tukey's t-test was used to compare means. Results were considered statistically
210 significant when the p-value was less than 0.05.

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215 **Results**

216 **Growth promoting effects of S. stutzeri, C. vulgaris and mixed the consortium on**
217 **A. thaliana** **under control and salt stress conditions**

218 To assess growth-promoting effects of the PGPR strain S. stutzeri, the green microalgae
219 C. vulgaris and their possible synergistic effects as a consortium, we measured plant
220 biomass under non-stress and salt stress conditions (100 mM NaCl). As illustrated in
221 Fig. 1a, under non-stress condition (0 mM NaCl), the non-inoculated plants showed
222 lower fresh biomass compared to the microbe-inoculated plants, which suggests
223 beneficial interactions between A. thaliana and the tested microbes. The consortium
224 inoculated plants showed significantly higher fresh biomass indicating that the
225 consortium exerts a pronounced positive effect on A. thaliana growth.

226 Although, under salt stress conditions (Fig. 1b), the significant difference in biomass
227 promotion between consortium-inoculated and individually inoculated plants was not
228 evident, all inoculated plants exhibited a similar trend of increased biomass
229 production. Measurements of dry biomass under non-stress conditions, showed slight
230 variations among treatments; however, the differences were not statistically significant
231 (Fig. 1c). In contrast, under salt stress, non-inoculated plants exhibited a marked
232 reduction in dry weight compared to treated plants. Notably, inoculation with S.
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234 improved dry weight, indicating a growth-promoting effect under stress conditions
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236 In addition to plant weight, the total root system length was also assessed for each
237 condition (Fig. 1e,f). Under non-stress condition (0 mM NaCl), plants grown in
238 presence of the consortium exhibited a marked reduction in total root system length
239 compared to non-inoculated control plants (Fig. 1e). A significant reduction was also
240 observed in plants inoculated with S. stutzeri. However, in plants inoculated with
241 C. vulgaris, root length exceeded that of the non-inoculated control, although the
242 differences were not statistically significant. Notably, while the S. stutzeri treatment
243 reduced the total root system length, the lateral roots were considerably well
244 developed (Supplementary Fig. S1). Under salt stress, all three treatments resulted in
245 a reduction in the total root system length (Fig. 1f). Compared to the non-inoculated
246 control, plants inoculated with the consortium exhibited a substantial reduction in
247 total root length, with highly significant differences observed relative to the control.
248 Conversely, inoculation with S. stutzeri led to a slight, albeit statistically insignificant,
249 reduction in root length compared to the control plants, while plants treated with
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C. vulgaris showed a more pronounced decrease in root length relative to the control.

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Furthermore, plants inoculated with the consortium had significantly shorter roots compared to those treated individually with either *S. stutzeri* or *C. vulgaris*.

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In addition to root hair length, a qualitative observation of magnified images presented in (Supplementary Fig. S1) suggested enhanced root hair development under non-stress condition, in the presence of *S. stutzeri*, *C. vulgaris*, and their consortium, however, under saline stress, root hair formation was only maintained in plants treated with *C. vulgaris* and the *S. stutzeri-C. vulgaris* consortium (Supplementary Fig. S1). Collectively, these findings confirm the plant growth-promoting effects of the PGPR *S. stutzeri*, the green microalgae *C. vulgaris*, and their consortium under both control and salt stress conditions.

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Transcriptional responses of *A. thaliana* to differential inoculation with *S. stutzeri*, *C. vulgaris* and their mixed consortium under salt stress

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To evaluate the biological processes altered due to an inoculation with *S. stutzeri*, *C. vulgaris* and their possible synergistic effect used as a consortium under salt stress conditions, a comprehensive transcriptomic approach was taken. RNA was extracted from whole plants and analysed by mRNA-seq. In total, 297 million reads were sequenced with an average of 19 million reads per sample (Table 1). Subsequently, Principal Component Analysis (PCA) was applied to reduce the dimensionality of the multivariate dataset, the analysis discriminates between the differentially inoculated plants as well as between the stressed and the non-stressed controls (Fig. 2a). The analyses revealed a separation between the transcriptomes of plants inoculated with *S. stutzeri* and those inoculated with *C. vulgaris*, indicating clear differences between the two. Additionally, the transcriptomics data from plants inoculated with the mixed consortium clustered between the *S. stutzeri* and *C. vulgaris*-inoculated plant transcriptomes, suggesting similarities between the consortium and each single inoculation.

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In comparison to non-inoculated control plants under salt stress, variations of gene expression in response to the different treatments were observed and expressed as Differentially Expressed Genes (DEGs). In total, 630, 204, and 857 genes were identified as DEGs (FDR<0.05 and a log₂(fold change) ≥1|1)) when inoculated with *S. stutzeri*, *C. vulgaris*, and the mixed consortium, respectively (Fig. 2b), among which 292, 155, 602 were induced genes and 426, 76 and 441 were repressed. To expand on these findings, we have separated the induced and the repressed genes and performed Venn

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diagram analyses through which we were able to identify exclusive and common subsets of DEGs in response to each inoculation condition. As depicted in Fig. 2c, an overlap of 91 DEGs was identified in response to the three different inoculations, of which 52 were upregulated and 39 were downregulated. Moreover, the overlap between *S. stutzeri* inoculation and consortium inoculation encompassed 209 DEGs, nearly twice the number observed between *C. vulgaris* and consortium inoculation, this suggests that *S. stutzeri* exerts a greater influence than *C. vulgaris* on the plant's gene expression profile in the context of the consortium. Nevertheless, 642 DEGs were exclusively regulated in response to consortium infection, suggesting the activation of specific mechanisms, which may imply a tailored response induced by the synergistic interaction between the bacterium and the microalgae.

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## Gene ontology reveal enrichment in salt stress-associated GO terms in response to microbial inoculation

With the aim to pinpoint putatively involved biological processes, we have run GO analyses on the upregulated and downregulated DEGs from each inoculation condition. Fig. 3 provides a visualization of the GO terms enriched in each treatment. Upregulated DEGs induced by the inoculation with *S. stutzeri* were associated with regulatory processes and responses to various stimuli: "response to stimulus", "regulation of primary metabolic process", "regulation of biosynthetic process", and "response to abiotic stimulus". Conversely, in the downregulated DEGs, we identified an enrichment in stress related terms, among which, "response to osmotic stress", "response to salt stress", "response to water deprivation" and "response to oxidative stress", suggested an attenuation of stress-related mechanisms and reduced activation, while promoting regulatory and adaptive processes (Fig. 3a).

Analysis of the repressed DEGs in response to *C. vulgaris* revealed association with GO terms related to "response to abiotic stimulus", "response to stress" and "response to temperature stimulus" while the induced DEGs were predominantly associated with the regulation of oxygen homeostasis, indicated by the enrichment of GO terms such as "cellular response to hypoxia," "cellular response to oxygen levels," and "response to decreased oxygen levels" (Fig. 3b).

Analysis of the upregulated DEGs in response to inoculations with both *S. stutzeri* and *C. vulgaris* in the mixed consortium has revealed an enrichment in the GO terms related to microbial interaction: "response to external biotic stimulus", "response to bacterium", "response to other organisms" and well as "biological process involved
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in interspecies interaction between organisms", this result indicates the triggering of
specific mechanisms to respond to the microbial presence. Conversely, the
downregulated DEGs were categorized in GO terms related to stress such as "response
to salt stress", "response to osmotic stress" and "response to water deprivation" (Fig.
3c).

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Transcriptomic patterns reveal co-expressed genes involved in salt stress adaptation

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Subsequently, gene expression clustering of the RNA-seq data was carried out using
the *coseq* R package (Rau et al., 2015), employing a Poisson mixture model, this
approach enables the identification of candidate genes responsive to each inoculation
by capturing representative expression patterns across the tested conditions. The
clustering procedure resulted in 9 clusters, among these, cluster 3, 5, and 7 showed
expression profiles of particular interest, presented in Fig. 4a. The three clusters
contained 227, 154, and 261 genes, respectively. Among them, we identified genes
associated with biologically relevant GO terms, those genes were used to construct a
functional interaction network represented in Fig. 4b.

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Cluster 3 predominantly contained modestly down regulated genes (Fig. 4c) associated with the GO term "response to salt stress", including CBL-INTERACTING PROTEIN KINASE 5 (CIPK5), CORONATINE INDUCED 3 (CORI3), RESPONSIVE TO DESICCATION 22, and SNF1-RELATED PROTEIN KINASE 2 (SnRK2/SRK2F). In contrast, ARABIDOPSIS K+ TRANSPORTER 2 (AKT2) was associated with the GO term "response to abscisic acid", while Na+/H+ EXCHANGER 2 (NHX2) was linked to "inorganic ion homeostasis", were also repressed. All genes in cluster 5 were associated with the GO term "response to salt stress", and all of the genes were substantially downregulated in response to the three treatments including KINASE-INDUCIBLE GENE 1 (KIN1), OUTER MEMBRANE TRYPTOPHAN-RICH SENSORY PROTEIN (TSPO), GALACTINOL SYNTHASE 2 (GOLS2), ALCOHOL DEHYDROGENASE (ADH1) and the calcium-binding transcription factor NACL-INDUCIBLE GENE 1 (NIG1), whereas, cluster 7, contained genes associated to response to hormone however, their expression was upregulated, including SMALL AUXIN UPREGULATED RNA 1 (SAUR1) and SAUR3, BTB and TAZ DOMAIN PROTEIN 1 and 2 (BT1, BT2), ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN (EBP) and ABA INSENSITIVE 5 (ABI5), the cluster also included SALT OVERLY SENSITIVE 3 (SOS3, other name, CALCINEURIN B-LIKE, CBL4) and the two membrane transporters CHX17 and STELAR K+ OUTWARD RECTIFIER (SKOR).
The analysis of the network topology revealed 17 nodes and 24 edges (Fig. 4b), of which we identified 5 nodes with the highest connectivity: CBL4 (8 edges), RD22 (5 edges), COR15A (5 edges), NHX2 (5 edges) and ABI5 (4 edges). The network exhibited slight centralization around a set of genes, with CBL4 (SOS3) positioned at the centre of this group as a potential key regulator. Notably, CBL4 was connected to three protein kinase (CIPK21, CIPK5, SRK2F), a potassium channel (AKT2), a hormone-related protein (ABI5), and a stress-responsive gene (COR15A) highlighting a cluster of candidate genes potentially involved in salt stress alleviation in response to inoculation with *S. stutzeri*, *C. vulgaris*, and both as a consortium.

## Transcriptome profiling highlights consortium-responsive genes involved in salt stress adaptation

To investigate transcriptional responses associated with improved salt tolerance induced by the *S. stutzeri* + *C. vulgaris* consortium, we performed transcriptome analysis focusing on the unique set of DEGs specific to the *S. stutzeri* + *C. vulgaris* consortium treatment, as identified by the non-overlapping region in the Venn diagram (Fig. 2c). This set represents DEGs that were not shared with single inoculations, indicating transcriptional responses exclusive to the consortium interaction. Gene ontology analysis on these genes revealed enrichment in terms related to salt and osmotic stress (Fig. 5a), consequently, to pinpoint functional contributors to salt tolerance, we focused on DEGs annotated under these stress-related GO terms, Fig. 5b represents the candidate genes with the corresponding log2FC values. Among the identified DEGs, several ones known to be involved in ethylene signaling and hormone response pathways were differentially expressed, such as ETHYLENE INSENSITIVE EIN3/EIL1, a key downstream component of ethylene signaling (Lei et al., 2011), was significantly upregulated. The transcription factor WRKY25, known to be NaCl-inducible (Jiang and Deyholos, 2009) and ABA, Jasmonic acid and Salicylic acid sensitive (Jiang and Deyholos, 2009; Zheng et al., 2007), also showed significant upregulation. MYB20, a transcription factor associated with salt tolerance (Cui et al., 2013), was also upregulated. Conversely, genes with roles in detoxification processes were downregulated, SUPPRESSOR OF MORE AXILLARY GROWTH 4 (SMXL4) involved in strigolactone signaling and phloem development, was downregulated. ANNEXIN 1 (ANN1), a Ca²⁺-dependent membrane-binding protein induced by salt stress (Laohavisit et al., 2012), was downregulated. SALT TOLERANCE-RELATED PROTEIN, STRP associated with oxidative stress protection (Fiorillo et al., 2023), showed reduced expression. β-
GLUCOSIDASE, BGLU22, a involved in defense-related hydrolysis of scopolin (Lin et al., 2024), was also downregulated. Additionally, changes in the expression of transcription factors implicated in stress signaling was observed, NAC6 and NAC042 were both significantly upregulated in response to inoculation. In contrast, MYB12, a regulator of flavonoid biosynthesis (Di Ferdinando et al., 2012), was significantly downregulated. Together, these changes indicate a targeted transcriptional reprogramming involving multiple stress-related pathways, particularly ethylene and oxidative stress signaling, following consortium treatment.

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## 399 Discussion

Plant-microbe interactions are crucial for plant health and development especially under stress. In this study, we report the transcriptional responses of *A. thaliana* to inoculation with *S. stutzeri* strain J3BG, *Chlorella vulgaris*, and their combined consortium under salt stress conditions in attempt to decipher key genes responsible for the observed enhancements in biomass. Previous studies have demonstrated the positive effects *S. stutzeri* and *C. vulgaris* in enhancing plant growth and tolerance in stress separately (Kang et al., 2021; Li et al., 2025; Ryu, 2021; Szymańska et al., 2022). Moreover we have previously proven the symbiotic interaction between *S. stutzeri* and *C. vulgaris* (Guendouzi et al., 2024) opening the possibility of a synergistic effect when co-inoculated. In accordance with this, under salt stress, inoculated plants' fresh and dry weight were increased compared to non-inoculated control (Fig. 1b,d), indeed, microbial inoculation can attenuate the effects of salt stress on plants by maintaining high biomass (Singh et al., 2023). Additionally, total root length was measured and compared. Under non-saline conditions, inoculation with *S. stutzeri* J3BG resulted in primary root inhibition and lateral root stimulation. Similar effects have been previously reported for Arabidopsis inoculated with *Rhizobium* sp. IRBG74 (Zhao et al., 2018), *Serratia marcescens* 90-166 (Shi et al., 2010), and *Micrococcus luteus* LS570 (García-Cárdenas et al., 2022). These changes in root architecture are particularly significant, as the emergence of lateral roots creates natural openings in the main root, which can serve as entry points for beneficial bacteria through “crack entry” (Gyaneshwar et al., 2002; James et al., 2002).

In plants inoculated with *C. vulgaris*, primary root length was maintained, while lateral root development was stimulated. Additionally, an increased presence of visible root hairs was observed (**Supplementary data**). Under salt stress, primary root elongation was promoted by J3BG inoculation, and lateral roots were well developed but lacked
root hairs. In contrast, inoculation with *C. vulgaris* or the microbial consortium maintained root hair formation despite the stress conditions. Intriguingly, a recent study demonstrated that sustained root hair growth under salt stress conditions is one of the key mechanisms triggered by the beneficial fungus *Fusarium* sp. strain K-23 to alleviate salt stress in *A. thaliana*, as the study highlighted that fungus could not exert his growth-promoting effect under salinity stress in the root hairless mutant *rsl2/rsl4* (Onejeme et al., 2024).

To gain deeper insights into the distinct phenotypes induced by individual and combined microbial inoculations, and to elucidate associated gene expression changes, we performed RNA-seq analyses followed by a comprehensive analysis of the resulting transcriptomic data. As illustrated in Fig. 2a, inoculation with the microbial consortium led to a significantly greater number of DEGs compared to single inoculations, with *S. stutzeri* inducing more DEGs than *C. vulgaris*. This suggests that the plant's transcriptional response to *C. vulgaris* was the least pronounced among the treatments, from which it must be concluded that *C. vulgaris* enhanced *A. thaliana* growth through a distinct mechanism or via more subtle transcriptomic modifications. Comparison of DEGs across the three inoculation treatments revealed a subset of commonly regulated genes, representing a core transcriptional response of *A. thaliana* to microbial inoculation under stress. Notably, the number of DEGs in plants treated with the J3BG-C. vulgaris consortium was markedly higher than in those inoculated with either microbe alone, implying the activation of additional, potentially unique processes. This differential expression profile supports the hypothesis of a tailored plant response likely driven by the synergistic interaction between the bacterium and the microalga. This finding further suggests that the plant perceives and responds to the consortium as a unique biological stimulus, rather than merely as an additive effect of individual microbes.

Subsequent GO analyses and clustering of the gene expression data were conducted to elucidate the biological significance of DEGs and to discern patterns of gene expression. In this process, we identified three clusters containing potential candidate genes (Fig. 4b). These clusters were then manually curated to identify genes associated with relevant and enriched GO terms. Cluster 3 consisted of moderately downregulated genes, including the CATION EXCHANGER 3 (CAX3), which encodes a high-affinity vacuolar calcium antiporter. The downregulation of CAX3 suggests reduced Ca²⁺ sequestration into the vacuole, potentially limiting cytosolic calcium availability. Given that Ca²⁺ acts as a crucial secondary messenger in salt stress signalling, its reduced mobilization may attenuate the transduction of stress signals to
downstream pathways, thereby modulating the plant's physiological response (Seifikalhor et al., 2019). Under salt stress, plants experience increased sodium uptake, which disrupts ionic balance by lowering the K+/Na+ ratio and ultimately leads to toxicity (Hasegawa, 2013). In our study, the expression of *AKT2*, a potassium channel involved in K+ loading and unloading in the phloem, was found to be downregulated. This repression may represent a protective mechanism to limit K+ loss under saline conditions. Notably, the downregulation of *AKTs*, including *AKT2*, in rice roots has previously been associated with enhanced salinity tolerance (Musavizadeh et al., 2021), suggesting that downregulation of these K+ channel genes may contribute to maintaining ionic homeostasis under salt stress. Another key component of K+ homeostasis is the family of *NHX* transporters, which play a central role in sequestering K+ into the vacuole through the activity of K+/H+ antiporters, such as *NHX2* (Pinedo et al., 2015). Its downregulation suggests a reduced capacity to sequester K+. The transcription factors *MYB28* and *MYB29*, which are extensively involved in the biosynthesis of aliphatic glucosinolates, were downregulated. This likely leads to a reduction in aliphatic glucosinolate levels, which can alter aquaporin abundance and affect water transport (Martínez-Ballesta et al., 2015) which, in turn, suggests that decreased aquaporin-mediated hydraulic conductivity may slow water uptake down and limit the transport of toxic ions, contributing to enhanced adaptation under salt stress. Taken together, the genes in Cluster 3 were downregulated and predominantly associated with ion homeostasis and ion transport processes. This downregulation suggests an adjustment in the plant's ionic regulation mechanisms, potentially aimed at minimizing ion toxicity and maintaining cellular ion balance under stress conditions. Cluster 5, contains genes with substantially reduced expression. Interestingly, the majority of these genes were stress-related genes, including *RESPONSIVE TO DESICCATION 29B* (*RD29B*) (other name *LTI65*), *KIN1*, *COLD-REGULATED 15A* (*COR15A*), *ADH1* and *GOLS2*. The latter mentioned genes are designated as stress-inducible genes. Thus, their downregulation compared to the non-inoculated control samples points towards a reduction of the stress levels in *A. thaliana*. Similar effects were observed for the salt-tolerance promoting bacterium *Pantoea ananatis* JBR3-16 (Peng et al., 2023). Research on the mechanisms by which stress mitigation is accomplished by JBR3-16 led to the hypothesis that less Na+ is assimilated by inoculated host plants. In consequence, less salt is encountered by the plants, which ultimately leads to a reduced salt stress response. These genes are also responsive to ABA. In a study by (Xiong et al., 2002), a mutation in *LOS6/ABA1*, which encodes a zeaxanthin epoxidase involved in ABA biosynthesis, resulted in the downregulation of these stress-responsive genes under osmotic stress conditions. In
contrast to clusters 3 and 5, cluster 7 contained predominantly upregulated genes. Within this cluster, we identified several key genes associated with ion homeostasis. Among them is CATION/H⁺ EXCHANGER 17 (CHX17), which primarily functions as a K⁺ transporter. Also present is SKOR, a Shaker-like outwardly rectifying K⁺ channel that is rapidly activated in response to membrane depolarization caused by salt stress. Upon activation, SKOR channels mediate K⁺ efflux to help restore the membrane potential to normal levels (Wu et al., 2018). Additionally, we identified a pivotal gene in the SALT OVERLY SENSITIVE pathway (SOS), SOS3 (also known as CBL4), which encodes a Ca²⁺-binding protein that senses elevations in cytosolic Ca²⁺ and initiates downstream signalling (Mahajan et al., 2008). Specifically, increased extracellular ion concentrations lead to Ca²⁺-mediated activation of SOS3, which in turn activates SOS2. SOS2 then stimulates SOS1, a Na⁺/H⁺ antiporter that facilitates Na⁺ extrusion from the cytosol (Amin et al., 2021). In addition, we also identified hormone related genes. The two small auxin upregulated RNA genes SAUR3 and SAUR1 appeared to be upregulated. SAUR genes are early auxin responsive genes (Stortenbeker and Bemer, 2019). In Arabidopsis, SAUR genes are involved in auxin-mediated cell expansion (Xu et al., 2017), although the exact function of SAUR3 and SAUR1 during stress responses is not clarified, their upregulation may indicate the implication of auxin mediated mechanism. Moreover, ABSCISIC ACID-INSENSITIVE 5 (ABI5) was also upregulated, ABI5 encodes a basic leucine zipper (bZIP) transcription factor that plays a central role in abscisic acid (ABA) signalling during seed maturation and germination. In addition, it positively regulates plant responses to drought and high salinity stress (Chang et al., 2019). Moreover, CIPK21, a protein kinase encoding gene inducible by salt stress, was also upregulated. Typically, CIPK21 confers tolerance to salt stress by modulating ion and water homeostasis in vacuole membranes (Pandey et al., 2015). Additionally, (Chen et al., 2014) reported that over-expression of CIPK21 in maize-maintained the cellular Na⁺ and K⁺ ratio.

To elucidate the plant response triggered by microbial inoculation under salinity, we performed transcriptome analysis on the specific region enriched in differentially expressed genes (DEGs) following inoculation with the S. stutzeri + C. vulgaris consortium. Gene Ontology (GO) enrichment revealed an overrepresentation of terms related to salt stress and osmotic regulation (Fig. 5a). Several DEGs were associated with ethylene signaling and response pathways, indicating hormonal regulation as an important mechanism. Notably, the core ethylene-responsive transcription factor EIN3/EIL1 was upregulated. This gene plays a key role in downstream ethylene signaling, and its importance in salinity tolerance was demonstrated by (Lei et al.,
2011), who showed that ein3-1 mutants exhibited increased electrolyte leakage under salt stress, indicating compromised membrane integrity.

In line with this, *WRKY25*, a transcription factor inducible by salt and previously shown to be ethylene-responsive under heat stress (Li et al., 2011), was also upregulated. Overexpression of *WRKY25* in Arabidopsis has been shown to enhance salt tolerance (Jiang and Deyholos, 2009), suggesting that consortium inoculation may prime plants via hormone-mediated transcriptional reprogramming.

Interestingly, SUPPRESSOR OF MORE AXILLARY GROWTH 4 (*SMXL4*) was downregulated. While primarily known as a regulator of phloem development in Arabidopsis (Wang et al., 2025), *SMXL4* has also been implicated in promoting salt and drought tolerance by modulating ROS levels and maintaining $K^+/Na^+$ homeostasis. SMXL4 attenuates strigolactone signaling; therefore, its downregulation may reflect enhanced strigolactone responsiveness, potentially improving stress adaptation under consortium treatment.

Three genes involved in detoxification and cellular homeostasis were also differentially expressed. *ANNEXIN 1* (*ANN1*), a $Ca^{2+}$-dependent membrane-associated protein induced by NaCl (Laohavisit et al., 2012), was downregulated, which may reflect reduced ionic stress perception following inoculation. Similarly, *SALT TOLERANCE-RELATED PROTEIN* (*STRP*), which mitigates oxidative bursts and helps maintain osmotic balance (Fiorillo et al., 2023), was also downregulated, further supporting the hypothesis that consortium-treated plants experience less severe ionic or oxidative stress. *β-GLUCOSIDASE 22* (*BGLU22*), which participates in the hydrolysis of stress-inducible compounds such as scopolin, was upregulated. This suggests activation of secondary metabolic defenses, possibly contributing to enhanced stress resilience (Lin et al., 2024). Transcriptional regulators including *NAC042*, *NAC6*, and *MYB12* were also differentially expressed. *NAC6*, a senescence-associated TF activated under salt stress (Zhang et al., 2017) was induced. In rice, *NAC6* enhances the expression of multiple abiotic stress-responsive genes, including peroxidases (Nakashima et al., 2007), indicating its role in ROS management. *NAC042* (other name *JUNGBRUNNEN 1* (*JUB1*)) known to bind promoters of ROS-scavenging genes and delay leaf senescence (Tikhomirova et al., 2022), was also upregulated, interestingly, this gene was recently linked to sustained root hair development in *A. thaliana* upon interaction with *Fusarium* sp. strain K23. Elevated expression of *JUB1* was shown to repress gibberellin biosynthesis, thereby promoting persistent root hair growth under salt stress conditions (Onejeme et al., 2024), which might be the case for this specific interaction.
Conversely, *MYB12*, a positive regulator of flavonoid biosynthesis, was downregulated. Given that flavonoids act as antioxidants under stress (Di Ferdinando et al., 2012), its reduced expression may indicate lower ROS levels in consortium-treated plants, consistent with reduced oxidative stress. Meanwhile, *MYB20* was upregulated. *MYB20* positively regulates salt tolerance by repressing PP2C-type phosphatases, which are negative regulators of ABA signaling, thereby enhancing ABA-mediated stress responses (Cui et al., 2013).

In conclusion, our work highlights transcriptional alterations in *A. thaliana* following inoculation with the PGPR *S. stutzeri*, the green microalgae *C. vulgaris*, and a consortium comprising both microorganisms. Both individually and in combination, these microbes positively affect the growth and salt stress tolerance of Arabidopsis seedlings. The mixed microbial consortium elicited the most pronounced physiological and transcriptional responses, indicating a synergistic interaction between the bacterium and the microalgae. Notably, to our knowledge, this study represents the first report of transcriptional changes in plants induced by inoculation with *C. vulgaris*, offering novel insights into the molecular basis of microalgae-plant interactions under salt stress conditions.

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834 **Statements & Declarations**

835 **Funding**

836 Work at the CBGP lab was supported TED2021-1299998-100 funded by

837 MCIN/AEI/10.13039/501100011033 and as appropriate, by 'ERDF A way of making

838 Europe', by the 'European Union' or by the 'European Union

839 NextGenerationEU/PRTR' to Jesus Vicente-Carbajosa.

840 **Competing Interests**

841 The authors have no relevant financial or non-financial interests to disclose

842 **Author contributions**

843 SG, JVC and MB designed and conceived the research; SG, AGOV, EK performed the

844 research; JVC was responsible for the acquisition of the required funding to perform

845 the experiments and SG, AGOV wrote and edited the manuscript.

846 **Data Availability**

847 All data supporting the findings of this study are available within the paper and

848 within its Supplementary Data published online.

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868 **Figure captions**

869 **Fig. 1:** Physiological measurements of *A. thaliana* 10 days after microbial inoculation compared to non-inoculated controls at 0 mM and 100 mM. **a)** Box plot of plant fresh weight at 0 mM, and **b)** at 100 mM. **c)** Box plot of plants dry weight at 0 mM and **d)** at 100 mM. **e)** Bars of plant root length at 0 mM, and **f)** at 100 mM. Data are presented as mean n=3 independent biological replicates. Asterisks mark statistically significant differences between differently inoculated plants as determined by Tuckey's test \*p* ≤ 0.05, \*\*p* ≤ 0.01, \*\*\*p* ≤ 0.001

876 **Fig. 2:** Analysis of the transcriptional effects of microbial inoculation on *A. thaliana* under salt stress (100 mM). **a)** Principal component analysis (PCA) of normalized gene expression levels assessing the difference between the differently inoculated plants. **b)** Bar plots of DEG number identified in response to each microbial inoculation. **c)** Venn diagram demonstrating the total number of DEGs in each microbial inoculation, up-regulated genes and down regulated genes.

882 **Fig. 3:** GO enrichment analysis of the upregulated and downregulated DEGs in response to inoculation with **a)** *S. stutzeri*, **b)** *C. vulgaris* and **c)** the mixed consortium. Circles representing GO terms, their size indicates the number of genes enriched in the GO term, the color gradient indicating the FDR (p-value).

886 **Fig. 4:** Analysis of gene clusters associated with differential inoculation. **a)** gene expression profiles in response to each inoculation, expressed as normalized expression divided by the mean of the normalized expression in all tested conditions. **b)** Interaction network generated using STRING app and enhanced using cytoscape, node fill colors indicate genes corresponding GO term. **c)** Heatmaps representing the expression of candidate genes in each cluster, the color is correlated with the value of the differential expression (log2FC).

893 **Fig. 5:** Enrichment analysis of biological processes and expression levels of candidate gene in response to consortium inoculation. **a)** Visualization of enriched Gene Ontology (GO) terms related to Biological Process in response to inoculation with the consortium. ReviGO representation (Supek et al., 2011) of biological process GO terms significantly enriched (FDR<0.05). The GO terms with the lowest p-value are displayed
898 in shades of yellow while the ones with the highest p-value are in shades of purple. The

899 GO terms with higher similarity are closer in the plot. b) Expression levels of salt stress

900 related candidate genes in response to inoculation with the consortium.

901

902 **Table caption :**

903 **Table 1:** RNA-seq data obtained from Arabidopsis thaliana transcriptomes.

904

905
# Transcriptomic Profiling of *Arabidopsis thaliana* Responses to *Stutzerimonas stutzeri*, *Chlorella vulgaris*, and Mixed Consortium Inoculation Under Salt Stress

Journal name: Plant Molecular Biology

Authors:

Salma Guendouzi<sup>1,2*</sup>, Adrian Gonzalez Ortega-Villaizan<sup>1</sup>, Eoghan King<sup>3</sup>, Mahbouba Benmati<sup>2</sup>, Jésus Vicente-Carbajosa<sup>1</sup>

## Corresponding author

Salma Guendouzi

Email: selma.guendouzi@alumnos.upm.es

<footnote n="1"><sup>1</sup>Centro de Biotecnologia y Genomica de Plantas, Universidad Politecnica de Madrid (UPM)-Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentacion (INIA/CSIC), Campus de Montegancedo, 28223 Pozuelo de Alarcon (Madrid), Spain.</footnote>

<footnote n="2"><sup>2</sup> Ecole Nationale Supérieure de Biotechnologie Taoufik KHAZNADAR, nouveau Pôle universitaire Ali Mendjeli, BP. E66, Constantine, 25100, Algeria.</footnote>

<footnote n="3"><sup>3</sup> Université Paris-Saclay, INRAE, AgroParisTech, Institute Jean-Pierre Bourgin for Plant Sciences (IJPB), 78000, Versailles, France.</footnote>
<figure><img src="image_2.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>c)</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>d)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>e)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>f)</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>c)</figcaption></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>
<caption>a)</caption>

Upregulated

<figure><img src="image_13.png" /></figure>

Downregulated

<figure><img src="image_14.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>c)</figcaption></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>
<figure><img src="image_21.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Normalized expression profiles</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>Normalized expression profiles</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>c)</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>Cluster 3</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>Cluster 5</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>Cluster 7</figcaption></figure>
<figure><img src="image_28.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_29.png" /></figure>