ALKBH10B mediated epitranscriptomic regulation enhances drought tolerance of Arabidopsis thaliana via hormonal cross-talk and sustained photosynthesis

ALKBH10B mediated epitranscriptomic regulation enhances drought tolerance of Arabidopsis thaliana via hormonal cross-talk and sustained photosynthesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ALKBH10B mediated epitranscriptomic regulation enhances drought tolerance of Arabidopsis thaliana via hormonal cross-talk and sustained photosynthesis Yasira Shoaib, Rongpeng Han, Sabarna Bhattacharyya, Alexandra Finkenauer, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8969427/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Epitranscriptomic N⁶-methyladenosine (m⁶A) mRNA modification has emerged as an important regulatory layer of gene expression and protein synthesis in plant stress adaptation. In this study, expression profiling revealed that drought stress induces a global reprogramming of the m 6 A machinery in Arabidopsis thaliana , with the m 6 A demethylase ALKBH10B being the most strongly up-regulated component. ALKBH10B transcription is induced by ABA, most likely via ABRE and MYB cis-elements, whereas jasmonate alleviates this activation. Moreover, ALKBH10B overexpressing plants (OX-1) accumulated wild type (WT) levels of ABA under drought stress but displayed a markedly reduced content in jasmonic acid (JA) and jasmonoyl-isoleucine (JA-Ile). They furthermore exhibit enhanced stomatal closure, improved water-use efficiency, and thus an enhanced drought tolerance. Transcriptome analysis revealed that drought stress induces fewer transcriptional changes in OX-1 plants compared to WT and the drought-sensitive alkbh10b-1 mutant. Differential expression and GO enrichment analyses highlight that photosynthesis-related processes were most affected by ALKBH10B activity, correlating with a preserved chlorophyll content and PSII efficiency in OX-1. Analysis by m⁶A-IP-qPCR showed that ALKBH10B directly demethylates specific photosynthesis-related and drought-responsive genes, thus promoting their sustained expression during stress. Other transcripts are indirectly regulated by ALKBH10B-dependent processes. Moreover, transcripts associated with respiration were enriched in OX‑1, indicating that ALKBH10B also supports mitochondrial energy metabolism during drought. Together, our results suggest that ALKBH10B enhances drought tolerance by coordinating m⁶A-dependent post-transcriptional regulation to maintain photosynthetic capacity and mitochondrial energy metabolism, as well as fine-tuning ABA-jasmonate crosstalk. abiotic stress phytohormones transcriptomics epitranscriptomics (m6A) post-transcriptional modification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key message The m⁶A demethylase ALKBH10B emerges as a crucial post-transcriptional regulator of hormonal crosstalk, photosynthetic maintenance, and mitochondrial energy metabolism to fine-tune plant stress adaptation to drought . Introduction Plants, being sessile organisms, are subjected to various biotic and abiotic stresses. Drought is considered one of the major abiotic stresses, causing a series of morphological, physiological, biochemical, and molecular changes that affect plant development and crop yield (Lozano-Elena et al. 2022 ). Thus, with an increasing human population and a decrease in arable land, there is an intense need for the development of drought-resilient crops to maintain adequate food production. Plants can sense the lack of water early on under progressive drought conditions and trigger complex signalling cascades, which ultimately regulate molecular processes including gene expression to facilitate an appropriate response (Osakabe et al. 2014 ; Nakashima et al. 2014 ). The aim is to prevent water loss, e.g., by stomata closure, adjustment of water-utilizing processes such as photosynthesis, and/or initiation of escape mechanisms such as early flowering (Haghpanah et al. 2024 ). Various physiological and molecular analyses, not only in Arabidopsis but also many other plants, have identified phytohormones as key signalling molecules that regulate drought stress responses (Aimar et al. 2011 ; Gupta et al. 2020 ). Among these, ABA has been shown to play a critical role in drought tolerance both through direct regulation of stomatal closure as well as regulating production of various drought-responsive proteins (Nakashima et al. 2014 ; Chen et al. 2020 ). Promoter regions of drought-responsive genes often contain two cis-acting elements, namely ABRE (Abscisic Acid Responsive Element) and DRE (Dehydration Responsive Element), to facilitate the drought-stress related regulation of gene expression (Nakashima and Yamaguchi-Shinozaki 2010 ). However, other phytohormones have also been shown to be involved in the drought response, including jasmonates, especially JA-Ile, the biologically active derivative of JA (Alam et al. 2014 ; Cheong and Choi 2003 ; Mahmud et al. 2022 ). These hormones do not act independently from each other and cross-talk has been described between jasmonate and ABA signalling with regards to drought responses. It includes co-regulation of transcriptional ABA responses by alleviating the ABA-mediated induction of drought responsive genes such as RD29A in Arabidopsis and StRD29 in Solanum tuberosum (Bleker et al. 2024 ). In recent years, it has been discovered that plants experiencing environmental stresses extensively reprogram post-transcriptional gene regulatory processes. Among around 170 known post-transcriptional mRNA modifications (Boccaletto et al. 2018 ; Xuan et al. 2018 ) N6-methyladenosine (m 6 A) is the most abundant, dynamic, and reversible internal mRNA modification in eukaryotes (Liu and Pan 2016 ; Arribas-Hernandez and Brodersen 2020 ). The role of m 6 A has been explored in various abiotic stress responses such as salinity, drought, and cold stress (Lu et al. 2020 ; Hu et al. 2019 ; Zheng et al. 2021 ; Zhang et al. 2021 ; Shoaib et al. 2021 ; Vicente et al. 2023 ; Pan et al. 2024 ; Cai et al. 2024 ). m 6 A is installed by methyltransferases known as writers, removed by demethylases called erasers, and read by RNA-binding proteins called readers (Hu et al. 2019 ). In plants, a m 6 A writer complex has been identified whose core components includes METHYLTRANSFERASE A and B (MTA, MTB), the adapter protein VIRILIZER (VIR), FKBP12-INTERACTING PROTEIN 37 (FIP37), and a ubiquitin E3 ligase called HAKAI (Zhong et al. 2008 ; Shen et al. 2016 ; Zhang et al. 2019 ; Wang et al. 2022 ; Xu et al. 2022 ). Recently, MTA has been shown to contribute to drought stress resistance in Arabidopsis, apple, and poplar (Lu et al. 2020 ; Hou et al. 2022 ; Ganguly et al. 2025 ). Notable reader proteins that recognize m 6 A modification belong to YTH domain proteins and are named Evolutionary Conserved C-Terminal Region (ECT) (Reichel et al. 2019 ). FLK is another reader protein belonging to the KH domain family (Fabian et al. 2023 ). These reader proteins decode the m 6 A modification and mediate the downstream processes such as splicing, alternative polyadenylation, RNA export, stability and translation (Hu et al. 2019 ). Demethylases involved in m 6 A mRNA modification belong to the family of ALKYLATION B HOMOLOG proteins (ALKBH), whose members are involved in various cellular processes related to DNA repair and gene regulation. Among the 14 ALKBH proteins in Arabidopsis, ALKBH9B, ALKBH9C and ALKBH10B are well-characterized m 6 A eraser (Martinez-Perez et al. 2021 ; Duan et al. 2017 ; Amara et al. 2022 ). ALKBH9B is important in viral defence and also involved in promoting the mobilization of heat-activated long terminal repeat retrotransposons (Martinez-Perez et al. 2021 ; Fan et al. 2023 ). ALKBH9C is crucial for seed germination and seedling growth under salt, osmotic stress and ABA by affecting the stability of various stress-responsive transcripts (Amara et al. 2022 ). ALKBH10B plays an important role in floral transition by demethylation of transcripts of flowering related genes such as FLOWERING LOCUS T and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 and 9 (Duan et al. 2017 ). Moreover, ALKBH10B also affects the expression of various genes involved in salt, osmotic stress, or ABA responses (Shoaib et al. 2021 ; Tang et al. 2021 ; Han et al. 2023 ). While recently shown to positively regulate drought resistance in Arabidopsis (Han et al. 2023 ), the exact contribution of ALKBH10B to drought tolerance is not well defined. Here, we show that constitutive increased expression of ALKBH10B in OX-1 plants leads to improved water retention, reduced jasmonate accumulation, and maintenance of photosynthetic efficiency compared to WT and the alkbh10b-1 null-mutant despite comparable ABA levels across genotypes. RNA-seq analysis showed large alteration in the transcriptional landscape of the different lines, affecting expression of genes related to photosynthesis and drought response. Analysis by m 6 A-IP-qPCR identified direct alteration in m 6 A enrichment in some of these genes in OX-1 and alkbh10b-1 . However, it also manifests that changes in transcript levels of many genes seems rather indirectly affected by ALKBH10B . RNA-seq analysis also revealed that genes associated with mitochondrial respiration and ATP synthesis were up-regulated in OX-1, indicating that increased removal of m 6 A mRNA modification by ALKBH10B can support energy metabolism during drought. Collectively, our findings demonstrate that ALKBH10B-driven mRNA demethylation regulates hormonal crosstalk, photosynthetic maintenance, and mitochondrial energy metabolism and establishes ALKBH10B as a key epitranscriptomic regulator in drought response that fine-tunes plant stress adaptation by coordinating transcriptional regulation and hormone balance. Materials and Methods Plant materials and growth conditions In this study, we used Arabidopsis thaliana Columbia ecotype (Col-0), the T-DNA insertion line alkbh10b (Salk_004215c, Fig. S1A) and two over-expression lines (OX-1 and OX-2) expressing an ALKBH10B-FLAG fusion under the control of CaMV 35S promoter (Han et al. 2023). The aba2-1 and jar1-11 mutant lines were previously described (Cheng et al. 2002; Mahmud et al. 2022). Nicotiana benthamiana was used for transient infiltration. For drought stress, Arabidopsis seeds were sown directly in potting soil whereas for other experiments, they were surface sterilized with 70% (v/v) ethanol for 3 minutes, 6% (v/v) sodium hypochlorite (NaOCl) for 5 minutes and then five times rinsed with distilled water and sown on ½ MS (Duchefa Biochemie, The Netherlands) plates containing 1% (w/v) sucrose and 0.4% (w/v) phytoagar. The plates were maintained in the dark at 4 °C for three days for vernalization before being transferred to the growth chamber. To study the regulation by hormones, Arabidopsis seedlings were grown for three weeks on ½ MS plates and treated either with 50 µM (+)-ABA, 50 µM MeJA (SERVA, Germany), or 50 µM ABA + 50 µM MeJA or with the same volume of H 2 O as a mock treatment. Plants were harvested after 3-, 6-, and 24-hours treatment, frozen in liquid nitrogen, and RNA was extracted as described below. All plants were grown at 22±2 °C in a climate-controlled growth chamber under long-day conditions (16h light /8h dark) with a light intensity of 100 μmol photon*m -2 *s -1 (Philips TLD 18W lamps of alternating 830/840 light temperature). Phenotyping under drought stress, measurement of relative leaf water content, and stomatal aperture and density Seven-day-old seedlings of WT, alkbh10b-1 , and OX-1 grown on soil were transferred to individual pots containing an equal amount of soil. Plants were watered regularly until they were 18 days old and drought stress was applied by withholding water. After 10-12 days plants were rewatered for three days, and the survival rate was measured. The survival percentage was calculated as (number of survived plants / total number of plants)*100. The relative leaf water content (RWC) was quantified and calculated as previously described (Mahmud et al. 2022). In general, leaves no. 5-8 were used from 5 individual plants for each genotype. For the determination of stomatal aperture, plants from WT, alkbh10b-1 mutant, and OX-1 lines grown under normal conditions (control) were incubated in imaging buffer (10 mM MES, 5 mM KCl, 50 μM CaCl 2 , pH 6.15) for two hours. The lower epidermis from the 6 th & 7 th leaves was carefully peeled, separated from mesophyll cells, and depicted using bright field in a Confocal Laser Scanning Flourescence Microscope (Leica SP8 Lightning, Leica, Germany) using the internal LAS X software. Stomatal aperture was measured using ImageJ software (Schindelin et al. 2012). Stomatal density was determined as described in (Bhattacharyya et al. 2025) using data from 6 individual plants for each genotype. Measurement of chlorophyll content, photosystem II activity and stomatal conductance The chlorophyll content and photosystem II activity were measured in control and drought-exposed plants. Chlorophyll was measured through a DUALEX optical leafclip meter (Pessl Instruments, Austria). For the measurement of stomatal transpiration and photosystem II activity, a LI-COR (LI-6000) porometer/fluorometer (LI-COR Environmental GmbH, Germany) was used. All measurements were performed on 2 leaves per plant and 10 independent plants from each line were used. Promoter analysis and molecular cloning in pBIN19-ANX vector The 1-kb promoter region upstream of the ALKBH10B transcription initiation site was analysed for cis-acting elements through PlantCARE (https://bioinformatics.psb.ugent.be/ webtools/plantcare/html/). To measure the promoter activity under different hormone treatments, the 1kb promoter region of ALKBH10B (p ALKBH10B ) was amplified with forward and reverse primers containing Apa1 and Kpn1 restriction sites, respectively, using gDNA as a template and ligated into a pBIN19 vector containing the firefly luciferase (FLUC) reporter gene. The construct cassette is shown in Figure S1D, and the primer sequences are listed in Supplementary Table S1. After confirming the correct sequence, the vector containing p ALKBH10B ::fluc was transformed into Agrobacterium strain GV3101. Transient transformation of Nicotiana benthamiana and luciferase assays Agrobacterium cells carrying the pBIN-p ALKBH10B ::fluc construct were grown overnight at 28°C in YEB medium containing 100 µg/ml rifampicin, 25 µg/ml kanamycin and 1M MgCl 2 . The culture was then centrifuged at 3500 rpm for 5 minutes, resuspended in infiltration medium (20 mM citric acid, 2% (w/v) sucrose) and adjusted to OD 600 = 0.5 for infiltration into the leaves of four-week-old Nicotiana benthamiana plants. Three days after infiltration, the leaves of Nicotiana were cut into discs (ø 6 mm) and immersed in autoclaved water containing 30 µM D-luciferin together with either 50 µM ABA, 50 µM MeJA, 50 µM ABA+ 50 µM MeJA or 50 µM ABA+25 µM MeJA and H 2 O as mock control in a 96 well plate. FLUC luminescence was recorded in a Multi-Mode Microplate reader (Tristar 2 , Berthold GmbH, Germany) after three hours. During the measurement the leaf discs were kept in darkness. RNA extraction, cDNA synthesis and RT-qPCR Total RNA of four-week-old plants grown under control and drought stress conditions was extracted using the Genematrix Universal RNA Purification Kit (Roboklon, Germany) according to the manufacturer’s instructions. RNA integrity was assessed on a 1% agarose gel and the concentration was measured with a Nanopdrop photometer (Nanondrop™ One, Thermo Fisher Scientific). cDNA was synthesized from 1.5 µg of total RNA with oligodT- 18 and random hexamer primers following the manufacturer’s instructions of the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Germany). RT-qPCR was performed in a 96-wells plate using the CFX96 real-time thermal cycler system (Bio Rad, Germany) and SYBR Green PCR master mix (Thermo Fisher Scientific, USA). All transcript levels were normalized against the geometric means of two reference genes, namely ACTIN2 and TUBULIN 2, and the relative expression level was quantified using the 2^- ΔΔCT method. All primers are listed in Supplementary Table S1. Measurement of phytohormones Whole rosette leaves of four-week-old plants grown under control and drought stress conditions, two plants for each sample, were frozen in liquid nitrogen and ground in a pre-cooled mortar with a pestle. Extraction and quantification of hormones were done as previously described (Pan et al. 2010). To 50 mg of the plant powder, 25 µl of internal standards was added and hormones were extracted with 500 µl of extraction solvent consisting of 2-propanol/H 2 O/conc. HCl (2:1:0.002, v/v/v) and shaken at 100 rpm for 30 minutes at 4°C. To each sample, 1 ml of dichloromethane was added and the samples were shaken for 30 minutes at 4°C. The samples were then centrifuged at 13000 x g for 5 minutes at 4°C to achieve phase separation. From the lower phase, 900 µl were transferred to a screw-cap vial and the sample was dried completely using a nitrogen evaporator. Residual tissues were resuspended in 0.1 ml methanol: 0.1% formic acid in water (1:1, v/v). An aliquot of 50 µl of the sample solution was injected onto the reverse-phase C18 Gemini HPLC column and analysed using a QTRAP 6500+ LC-MS/MS system (Sciex, Germany). The concentrations of ABA, JA, JA-Ile and SA (salicylic acid) were quantified relative to the internal standards and expressed as ng/g F.W. RNA-sequencing and data analysis RNA sequencing was performed using three biological replicates, each consisting of pooled RNA extracted from the rosette leaves of two individual plants grown for 4 weeks under well-watered and drought stress conditions using the Direct-zol RNA Purification kit (Zymo Research Corporation, U.S.A.). An RNA quality check was carried out using a Tapestation 4200 (Agilent, U.S.A.). Approximately 100 ng of RNA was used for library preparation and 3’ mRNA sequencing was done by the NGS Core Facility (Medical Faculty at the University of Bonn, Germany) using a NOVASEQ6000 (Illumina, USA) sequencer. An average sequencing depth of ~10 million raw reads per sample (Supplementary Table S2) was generated. The FASTQ raw files were processed to remove low-quality bases and adapter sequences through the Trimmomatic method (Bolger et al. 2014). To confirm the removal of adapter sequence after trimmomatic processing, FastQC was done on a linux system. The reads were aligned through HiSAT2 software against the concatenated reference genome of Arabidopsis (http://plants.ensembl.org/info/data/ftp/index.html). The Gene Counts were generated from the aligned BAM files through the Feature Counts function in RStudio (Liao et al. 2014). The differential gene expression was carried out using EdgeR. The p-values were corrected using the False Discovery Rate (FDR) method (Benjamini and Hochberg 1995) and subsequently the FDR and |Log 2 FC| cut offs were set to 0.05 and 1 respectively. Volcano plots were generated through the SRplot web tool (Tang et al. 2023) whereas the Principal Component Analysis (PCA) plot and clusters were generated with the iDep2 web tool (Ge et al. 2018). Visualization of heatmaps was performed using the Morpheus webtool (https://software.broadinstitute.org/morpheus/). GO analyses of the obtained DEGs were performed using ShinyGO (Ge et al. 2020). m 6 A immunoprecipitation - qPCR The m 6 A- immunoprecipitation (IP)-qPCR was performed as described previously (Hu et al. 2021). Briefly, mRNA was extracted from total RNA using oligo-d(T) 25 magnetic beads (New England Biolabs, U.S.A.) and 5 µg was fragmented into around 300-nucleotide-long mRNA fragments by incubation in RNA fragmentation buffer (100 mM Tris-HCl, pH=7.0, 100 mM ZnCl 2 ). The fragmented mRNA was mixed with an m 6 A-specific antibody (Synaptic Systems, Germany), and the bound RNAs were eluted. The input and eluted mRNA were reverse transcribed using reverse transcriptase with random hexamer primers and the level of each transcript was measured by RT-qPCR conducted on a Rotor-Gene Q thermal cycle (Qiagen, Germany) using gene-specific primers listed in Supplementary Table S1. The level of m 6 A enrichment of target genes was quantified based on the PCR cycle threshold. The value of the immunoprecipitated sample was normalised against that of the input. Values are the means ± SE of three biological replicates. Results Drought stress induces changes in the expression level of m 6 A regulatory components To explore the role of m 6 A mRNA modification in the drought stress response, we quantified the expression of established m 6 A regulatory genes using RT-qPCR. Transcript levels were analysed in rosette leaves of Arabidopsis WT at different time periods of progressive drought treatment and compared to well-watered control conditions (Fig. 1 ). The results show a moderate increase in the expression levels of genes coding for the m 6 A writers MTA , MTB , FIP37 , VIR and HAKAI , the m 6 A readers ECT2 , ECT3 , ECT4 , and FLK , and the m 6 A erasers ALKBH9B and ALKBH9C . By contrast, the m 6 A eraser ALKBH10B was moderately upregulated after 6 days but substantially upregulated after 12 days of withholding water (Fig. 1 A). Under well-watered conditions, only minor changes in the expression of all these genes occurred (Fig. 1 B), suggesting that the drought-induced expression of the m 6 A regulatory genes is not due to developmental effects. Collectively, these results indicate that drought stress induces coordinated reprogramming of the whole m 6 A regulatory machinery, with ALKBH10B emerging as the most strongly induced component. We therefore focused subsequent analyses on ALKBH10B as a key candidate for mediating m 6 A-dependent responses during prolonged drought stress. ALKBH10B confers drought stress tolerance and regulates water loss through modulation of stomatal aperture To further investigate the role of ALKBH10B we used the Arabidopsis T-DNA insertion mutant alkbh10b-1 (SALK_004215C, Fig. S1 A) and 35S::ALKBH10B transgenic plants (OX-1), which were reported in previous studies as knockout and overexpression lines, respectively (Duan et al. 2017 ; Shoaib et al. 2021 ; Han et al. 2023 ). RT-qPCR corroborated a complete loss of ALKBH10B transcript in the alkbh10b-1 mutant and an increased expression in the OX-1 line compared to WT (Fig. S1 B). When progressive drought was applied to 18-day-old WT, alkbh10b-1 , and OX-1 plants by withholding water, alkbh10b-1 plants showed clear symptoms of wilting on day 12, followed by WT at day 15, whereas the OX-1 plants were still green and showed only slight wilting (Fig. 2 A). Consequently, after three days of rewatering, the survival rate of OX-1 plants was approximately 80%, whereas survival rate of WT and alkbh10b-1 plants were only about 33% and 19%, respectively (Fig. 2 B and S2A). The contrasting phenotype of null mutant and overexpression was confirmed using a second OX line (Fig. S2 A, OX-2). These results confirm that ALKBH10B plays a role as a positive regulator of drought tolerance. Reduced water loss through transpiration is a key determinant for drought tolerance. Stomatal density was modestly increased only in alkbh10b-1 but not in WT or the OX lines (Fig. 2 C, Fig. S2 B). Stomatal aperture measurements, however, revealed wider apertures in alkbh10b-1 and reduced apertures in OX compared to WT (Fig. 2 D, Fig. S2 C). Although the reduction in stomatal aperture in the OX lines was moderate in magnitude, it was consistent and statistically significant across biological replicates. These parameters likely contribute to reduced water loss through stomatal transpiration as measured in OX-1 (Fig. 2 E), resulting in delayed wilting and decreased drought sensitivity. Accordingly, the relative water content (RWC) on day 12 of progressive drought was higher in OX-1 and reduced in alkbh10b-1 plants (Fig. 2 F ). We therefore interpret stomatal regulation as one, albeit not the only, contributing component of ALKBH10B-mediated drought tolerance. ALKBH10B expression is regulated by ABA and jasmonates ABA and jasmonate are important regulators of drought responses (Ullah et al. 2018 ) and treatment of WT seedlings grown on ½ MS medium confirmed that ALKBH10B expression is strongly induced by ABA (Fig. 3 A). MeJA application had no influence on ALKBH10B expression but co-application of both hormones strongly reduced ABA-mediated ALKBH10B induction suggesting an antagonistic interaction. In accordance with these results, drought-induction of ALKBH10B was absent in a mutant defective in ABA biosynthesis ( aba2-1) (Cheng et al. 2002 ) and enhanced in jar1-11 , a plant line defective in JA-Ile formation (Mahmud et al. 2022 ) (Fig. 3 B). These data indicate that ABA is a major contributor to drought-induced ALKBH10B expression, while jasmonate modulates the amplitude of this induction. Expression of a luciferase reporter driven by the ALKBH10B promoter (Fig. S1 C). confirmed induction by ABA and attenuation by jasmonate (Fig. 3 C) consistent with a regulation mediated by cis-acting promoter elements. Analysis of the 1-kb promoter region of ALKBH10B using the PlantCARE database revealed multiple ABRE (Abscisic Acid Responsive Element) and MYB binding motifs as well as a MYC motif and AS-1 element (Fig. 3 D). These elements have been associated with ABA and/or jasmonate dependent transcription and often act combinatorically in response to different hormones. Further regulatory elements include DRE (dehydration-responsive element), which is associated with ABA-independent stress response and often occurs alongside ABREs. ALKBH10B affects accumulation of ABA and JA Phytohormones such as ABA and jasmonate regulate extensive transcriptional networks, and a subset of these hormone-responsive genes encode proteins that also modulate the hormone’s own levels, thereby establishing feedback loops of hormone content regulation (Zong et al. 2016 ; Xie et al. 2021 ; Zhao et al. 2024 ). We thus examined whether ALKBH10B also affects hormonal dynamics under drought stress. To this end, the levels of ABA, JA, and JA-Ile were quantified in WT, alkbh10b-1 and OX-1 plants under control and drought stress conditions (Fig. 4 ). Under control conditions, ABA, JA, and JA-Ile levels were low and similar across genotypes (Fig. 4 ). Under drought, ABA level increased in all genotypes, with a slightly higher accumulation in alkbh10b-1 mutant (Fig. 4 A). By contrast, levels of JA and JA-Ile increased in the WT and the alkbh10b-1 mutant under drought but remained unchanged in OX-1 (Fig. 4 B and C). The reduced jasmonate accumulation in OX-1 may reflect either direct regulation of specific transcripts by ALKBH10B or indirect metabolic consequences of altered transcriptional or physiological states. At present, our data do not allow us to distinguish between these possibilities. RNA-seq analysis reveals effect of ALKBH10B on respiration and photosynthesis under drought stress m 6 A mRNA modification can change transcript levels of genes either directly, e.g. by affecting mRNA stability of a specific gene, or indirectly by modulating transcripts of regulatory components/hubs that in turn alter transcription of a larger set of genes. We thus performed RNA-seq analysis on plants of all three genotypes at day 12 of progressive drought compared to well-watered control conditions. The sequencing yielded approximately ~ 10 million raw reads per sample, with around 85% of the reads successfully aligned to the Arabidopsis reference genome TAIR 10 over all samples (Supplementary Table S2 ). Principal Component Analysis (PCA) revealed a clear separation of samples based on treatment (PC1, 71.3% variance). The variance among genotypes was much smaller (PC2, 5.1% variance), but the WT was positioned between alkhb10b-1 and OX-1 under both conditions (Fig. 5 A). This indicates that drought stress is the dominant factor shaping the transcriptomic landscape, while genotypic effects are more subtle. First, we determined the differentially expressed genes (DEGs) between drought and control conditions for each line using a cut-off of FDR < 0.05 and │Log 2 FC│ ≥ 1 (Supplementary Table S3 ). This comparison revealed that alkbh10b-1 exhibited the highest number of DEGs (6205), followed by WT (5343) and OX-1 (4152) (Fig. 5 B). Within each genotype the DEGs were comprised of a similar number of up- and down-regulated genes (Fig. 5 B and C). We also analysed transcript levels under drought of alkhb10b-1 and OX-1 relative to WT. Notably, only four DEGs were detected between WT and alkbh10b-1 , and 34 between WT and OX-1 under drought conditions (Fig. 5 D). Between alkbh10b-1 and WT these included two genes with higher and two with lower expression in the mutant, of which three were functionally uncharacterised and the fourth is DERLIN-1 , encoding a protein that is part of the ERAD / ER protein quality-control machinery. By contrast, between WT and OX-1all genes were higher expressed in OX-1 and mostly functionally annotated (Supplementary Table S4 ). Of these, 14 genes encode mitochondrial proteins, most of them associated with the mitochondrial electron transport chain and oxidative phosphorylation (Fig. 5 E and F). These findings suggest that enhanced mitochondrial processes in OX-1 may provide an energetic advantage that helps to maintain cellular ATP homeostasis under drought, thereby contributing to its higher stress tolerance. For the DEGs (drought vs. control) among the three genotypes, we next calculated their relative expression change (ΔLog₂FC) in the functional variants compared to WT. By directly contrasting ΔLog₂FC for WT vs. alkbh10b-1 and WT vs. OX-1, and omitting those genes that showed an identical behaviour in OX-1 and alkbh10b-1 ( ΔΔlog 2 FC < 1), we detected 3395 genes (ΔΔlog2FC ≥ 1) specifically differentially regulated in only one of the two mutant lines (Supplementary Table S5 ). To understand the functional significance of these genes under drought stress, we performed GO enrichment analyses separately for three classes categorized as biological process, cellular component, and molecular function (Fig. 6 A-C). Remarkably, photosynthesis related processes emerged as the most strongly enriched term in all three classes such as light harvesting and photosystem I within biological processes, photosystem I + II and light harvesting within cellular components, and chlorophyll and tetrapyrrole binding within molecular functions. Other term within these classes relate to water transport and water channel activity but also responses to biotic and abiotic stress. KEGG analysis, which annotates genes at pathway level, revealed photosynthesis antenna proteins as the most significantly enriched pathway but also biosynthesis of glucosinolates and phenylpropanoids which are associated with stomata regulation and drought tolerance (Fig. 6 D). Cluster analysis of the genes that were specifically regulated in only one of the lines (ΔΔLog 2 FC ≥ 1) identified four clusters with similar amounts of genes ranging from about 400 to about 650 (Fig. 6 E and Supplementary Table S5 ). Cluster 1 and 2 were defined by genes that in the alkbh10b-1 mutant were differently expressed from WT and OX-1 (Fig. 6 E, right panel), while cluster 3 and 4 contains genes where OX1 differed from WT and alkbh10b-1 (Fig. 6 E, left panel). Cluster 1 comprised genes that were either up-regulated in alkbh10b-1 and unchanged in the two other lines or unchanged in alkbh10b-1 and down-regulated in the other two lines (Table S5 ). Cluster 2 comprised genes that were either down-regulated in alkbh10b-1 and unchanged in the two other lines or unchanged in alkbh10b-1 and up-regulated in the other two lines (Fig. 6 E). Cluster 3 and 4 represent the same for OX-1. No counter-regulated genes were found in our analysis. Overexpression of ALKBH10B preserves photosynthetic capacity under drought stress and modulates m 6 A levels of photosynthesis-related genes Since photosynthesis emerged as the most prominent GO category among the DEGs, we had a closer look at the RNA-seq data of 11 photosynthesis-related genes with differential expression in alkbh10b-1 and OX-1. They all belong to cluster 3 and their expression levels remained stable in the OX-1 plants under drought stress, whereas they were consistently down-regulated in WT and alkbh10b-1 (Fig. 7 A and Supplementary Table S6 ). To validate the RNA-seq results, we quantified the transcript levels of LHCB4.1 , PSBQ-2 and PSBO1 using RT-qPCR (Fig. 7 B). Their expression patterns matched those observed in the RNA-seq data, supporting the effect of ALKBH10B on a certain subset of photosynthesis-related genes. In line with the transcript analysis, WT and the alkbh10b-1 mutant showed a significant reduction in chlorophyll levels and PSII activity under drought stress compared to control conditions, whereas OX-1 plants were much less affected (Fig. 7 C, D). To elucidate whether ALKBH10B directly affects the m 6 A modification of these photosynthesis-related transcripts, we screened published m 6 A databases and identified potential m 6 A sites in LHCB2.3 , LHCB4.1 , PSAD-2 , PNSL3 , PSBQ-2 and PSBO1 (Fig. 7 A). Quantification of m 6 A enrichment of these genes using m 6 A-IP-qPCR showed significantly decreased m 6 A levels only for PSAD-2 and PSBQ-2 in OX-1 plants compared to WT (Fig. 7 E). Both genes had increased levels of m 6 A in the alkbh10b-1 mutant in line with a stronger down-regulation in the expression (Supplementary Table S5 ), indicating direct regulation by ALKBH10B-mediated demethylation. By contrast, the m 6 A levels of LHCB2.3 , LHCB4.1 , PNSL3 and PSBO1 remained largely unaffected, suggesting that they are not direct targets of ALKBH10B. These data indicate that rather than acting globally ALKBH10B selectively demethylates a subset of photosynthesis-related mRNAs, while other genes are indirectly affected. ALKBH10B modulates the expression and m 6 A enrichment of drought-responsive genes We also examined the effect of ALKBH10B on the regulation of known m 6 A modified drought related transcripts (Fig. 8 A). Several of these genes can be classified as positive and negative drought regulators (Han et al. 2023 ), and our RNA-seq analysis demonstrated that mostly the expression levels of these genes were not significantly different in all three plant lines (Fig. 8 A). However, among the positive regulators the expression in OX-1 was sustained for COR15B , and CIPK20 or up-regulated for VSP2 under drought stress. By contrast, the expression of these genes in WT and alkbh10b-1 was down-regulated for COR15B , and CIPK20 or sustained for VSP2. Moreover, expression levels of the negative regulators CYP707A1 and CML20 were up-regulated in WT and alkbh10b- 1 but sustained in OX-1. With regard to m 6 A modification of their transcript, m 6 A levels of the positive regulators were either decreased in OX-1, increased in alkbh10b-1 or both (Fig. 8 B) with similar patterns observed under control and drought conditions. The m 6 A levels of the two negative regulators CYP707A1 and CML20 remained unchanged in all lines, indicating that they are not directly regulated by ALKBH10B. Discussion In this study, our integrative approach combining transcriptomics, m⁶A profiling, physiological assays, and hormone quantification reveals a multi-layered mechanism in which the m 6 A demethylase ALKBH10B promotes drought stress resilience in Arabidopsis. While drought stress globally alters the expression of the whole m⁶A machinery in Arabidopsis, ALKBH10B stands out as the most strongly induced component (Fig. 1 ). This is in line with an elevated ALKBH10B expression shown previously under drought for sea buckthorn (Zhang et al. 2021 ) and tomato (Shen et al. 2023 ), where the plant prioritizes active m⁶A demethylation as part of its adaptive strategy. Since m⁶A demethylases have been implicated in fine-tuning stress-responsive transcripts (Shoaib et al. 2021 ; Han et al. 2023 ; Tang et al. 2021 ) this dynamic control likely enables the plant to rapidly modulate the stability and translation of stress-related mRNAs during water deficit. Characterization of plant lines with altered ALKBH10B content, revealed its role in modulating stomatal dynamics (Fig. 2 ), which is a critical factor governing water loss and drought resistance. Although differences in stomatal aperture between WT and OX-1 were moderate, the consistently reduced aperture in OX-1 seems sufficient to lower transpirational water loss during progressive drought periods. Despite the difference in stomatal apertures, Interestingly, ABA levels were similar between WT and OX-1 plants under control and drought conditions (Fig. 4 ), indicating that ABA abundance alone does not explain the observed stomatal phenotype. Accordingly, we also did not observe distinctive changes in the expression levels of ABA-dependent genes coding for proteins involved in stomata closure such as TGG1 , SLAC1 or KAT1 (Supplementary Table S3 ). Reduced jasmonate accumulation in OX-1 may alter guard cell signaling dynamics and influence stomatal behavior under drought conditions (Fig. 4 B and C). However, although jasmonates are known to cross-talk extensively with ABA signalling in guard cells, the experimental evidence for their precise role in stomatal regulation is mixed. Depending on the system and conditions, jasmonates have been reported to both potentiate and antagonize ABA-induced stomatal closure (Munemasa et al. 2011 ). At present and based on our data, we cannot establish a direct causal hierarchy between ABA levels, jasmonate accumulation, and stomatal behavior, we therefore interpret these interactions as part of a complex hormonal network. It is also unclear, how the reduced jasmonate content relates to ALKBH10B activity, since there is no clear correlation between jasmonate content and expression of genes related to jasmonate biosynthesis or catabolism (Supplementary Table S7 ). However, altered translational efficiency resulting from changes in m 6 A demethylation could influence protein abundance and cannot be excluded. Distinguishing between these direct and indirect mechanisms will require future integration of m⁶A epitranscriptomic profiling with targeted analyses of jasmonate pathway components including proteomic studies. What has become evident lately is that jasmonates can negatively modulate ABA induced genes expression, as demonstrated for RD29A/StRD29 in Arabidopsis and potato via external applications of ABA and MeJA (Bleker et al. 2024 ). This is likely driven by enhanced JA-Ile content to which MeJA is converted intracellularly (Tamogami et al. 2012 ). Although the underlying molecular mechanism for this modulation of gene expression remains unresolved, we observed the same effect for ALKBH10B expression in this work (Fig. 3 ). Moreover, an elevated drought-induced expression of ALKBH10B and RD29A occurs in the jar1-11 mutant, highlighting the negative regulatory role of endogenous JA-Ile (Mahmud et al. 2022 ). We did not observe any induction of ALKBH10B expression by MeJA itself, which seems to contradict results from Tang et al. ( 2021 ) showing that MeJA up-regulates ALKBH10B expression in seven-day-old seedlings. However, this apparent discrepancy with our findings can likely be attributed to differences in the developmental stage of the plants given that m 6 A dynamics vary across developmental stages (Zhang et al. 2024 ), ALKBH10B may participate in distinct jasmonate-responsive regulatory circuits during early seedling development versus prolonged drought stress in mature plants. Promoter analysis indicates that transcriptional regulation of ALKBH10B by ABA and jasmonate might be directly promoted via ABRE, MYB and MYC cis-elements (Fig. 3 ).The promoter of ALKBH10B also contains an AS-1 cis-regulatory element related to salicylic acid (SA), however, the expression of ALKBH10B was not notably affected by exogenous SA application in line with a lack of SA regulation shown for ALKBH10B in tomato leaves (Shen et al. 2023 ). Also, no significant differences in SA content were observed among all the genotypes under either drought and control growth conditions (Fig. S3 ), indicating that under our experimental conditions, i.e., age and tissue, ALKBH10B function is unlikely to be mediated through SA signalling. Our physiological analyses suggest a likely role of m 6 A removal by ALKBH10B in hormone-dependent regulation of transpiration. However, our transcriptomic analysis reveals that ALKBH10B exerts effects beyond hormonal crosstalk. It is established that mitochondrial respiration plays a vital role in drought tolerance (Atkin and Macherel 2009 ) and our results show that OX-1 plants activate a transcriptional program that enhances expression of multiple genes related to mitochondrial respiration and energy metabolism (Fig. 5 ). This indicates an influence of m 6 A mRNA modification on this process. This is so far not investigated in plants, but m⁶A mRNA modification has recently been shown to regulate transcripts of genes coding for mitochondrial proteins in animal systems (Kahl et al. 2024 ). While this indicates an evolutionary conservation beyond the plant-animal divide, it gains another layer of complexity in plants due to the mitochondrial-chloroplast interplay in plant energy metabolism. Indeed, our transcriptional analysis showed that in the OX-1 plants certain photosynthesis-related genes remain stably expressed and PSII performance is maintained under drought, in contrast to the WT and alkbh10b-1 mutant (Figs. 6 and 7 ). Similar findings have been observed for the ALTERNATIVE OXIDASE (AOX), where over-expression caused drought tolerance by maintaining not only mitochondrial but also chloroplast functions (Dahal and Vanlerberghe 2017 ). In a recent study, it has been revealed that the m 6 A writer VIR regulates m 6 A modification of numerous photosynthesis-related transcripts during high light-induced photodamage, thereby regulating their gene expression at post-transcriptional level (Zhang et al. 2022 ). However, in our study, only the stable expression of PSAD2 and PSBQ-2 can be linked to direct modulation of m 6 A mRNA methylation by ALKBH10B, while other photosynthesis-related transcripts likely reflect indirect regulatory effects (Fig. 8 ). These effects may, for example, arise from demethylation of transcripts encoding transcriptional regulators, which subsequently modulate broader photosynthesis-related gene networks. m 6 A modification has been observed for various transcription factors (Zhang et al. 2022 ) and we observed differential expression between WT and the function mutants for members of different transcription factor families (Supplementary Table S6 ). This distinction between direct m 6 A-mediated regulation and secondary transcriptional reprogramming is critical for interpreting epitranscriptomic datasets. ALKBH10B also regulates expression and m 6 A levels of certain drought-responsive transcripts such as the kinase encoded by CIPK20 (Fig. 8 ), which was shown to mediate stomata closure through regulation of microtubule stability (Li et al. 2024 ). Sustained expression of CIPK20 in OX-1 may contribute to reduced stomatal aperture compared to WT and alkbh10b-1 (Fig. 2 D). The function of these transcripts as well as their differential regulation and m 6 A mRNA modification by ALKBH10B are thus easily linked to the drought phenotype of the OX-1 and alkbh10b-1 mutant lines. Nevertheless, as with the photosynthesis-related genes, not all expression changes correlate directly with m6A enrichment, reinforcing the likelihood indirect downstream effects, such as post-transcriptional regulation, secondary signalling pathways, or downstream consequences of improved photosynthetic performance in OX-1 plants. Interestingly, it was shown very recently that the m 6 A writer, MTA, also imparts drought tolerance through m 6 A-dependent and -independent impacts on mRNA regulation (Ganguly et al. 2025 ). Similar to VIR mentioned above, MTA appears to act more globally than ALKBH10B. Moreover, whereas VIR primarily influences photosynthesis-related genes and MTA affects drought-responsive transcripts, ALKBH10B impacts both processes as well as mitochondrial respiration. Importantly, the limited overlap between m 6 A-modified transcripts and differentially expressed genes underscores the importance of distinguishing direct epitranscriptomic regulation from from secondary transcriptional reprogramming driven by altered m 6 A modification. Declarations Funding This work was supported by Deutscher Akademischer Austauschdienst (DAAD) for providing funding to YS (Grant agreement 57588370). The publication cost was ensured by University of Bonn Projekt DEAL. Authors’ contributions YS contributed to conceptualization, investigation (responsible for most experimental work), formal analysis, validation, visualization, and writing - original draft as well as review & editing. RH and HK contributed to investigation (m 6 A IP-qPCR) and writing – review and editing. SB contributed to RNA-seq analysis (bioinformatics). AF contributed to investigation (molecular cloning). KG and PD contributed to investigation (hormonal analysis), and writing – review and editing. UCV contributed to conceptualization, validation, project administration, supervision, and writing - review and editing. FC contributed to conceptualization, validation, supervision, and writing - original draft as well as review and editing. All authors contributed to the article and approved the submitted version. Acknowledgments We would like to thank Prof. Wan Hsing Cheng, (University of Taiwan) for providing the aba2-1 line and the NGS Core Facility of the Medical Faculty at the University of Bonn for providing support and instrumentation funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation). Competing interests The authors hereby declare no conflict of interests. Data availability Raw RNA seq data used in this study are available under (PRJNA1357100) at SRA repository from NCBI. References Aimar D, Calafat M, Andrade A, Carassay L, Abdala G, Molas M (2011) Drought tolerance and stress hormones: From model organisms to forage crops. 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Supplementary Files SupplementaryFile.docx SupplementaryTable1.xlsx SupplementaryTable4.xlsx SupplementaryTable3.xlsx SupplementaryTable5.xlsx SupplementaryTable6.xlsx SupplementaryTable7.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviews received at journal 16 Mar, 2026 Reviewers agreed at journal 11 Mar, 2026 Reviewers agreed at journal 01 Mar, 2026 Reviewers invited by journal 01 Mar, 2026 Editor assigned by journal 26 Feb, 2026 Submission checks completed at journal 26 Feb, 2026 First submitted to journal 25 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8969427","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599683685,"identity":"b78e6d10-be49-474c-bab7-b375c6955297","order_by":0,"name":"Yasira Shoaib","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Yasira","middleName":"","lastName":"Shoaib","suffix":""},{"id":599683686,"identity":"baf31d77-e839-4e99-83ae-907118274fec","order_by":1,"name":"Rongpeng Han","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Rongpeng","middleName":"","lastName":"Han","suffix":""},{"id":599683689,"identity":"b1b7cf18-f0a9-4f9f-bb99-f6786b98e1fa","order_by":2,"name":"Sabarna Bhattacharyya","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Sabarna","middleName":"","lastName":"Bhattacharyya","suffix":""},{"id":599683691,"identity":"04b4a4c7-cd1a-49f5-ada4-aa4788ac0014","order_by":3,"name":"Alexandra Finkenauer","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Finkenauer","suffix":""},{"id":599683693,"identity":"8511502d-5bdf-4f2a-8952-2c0e76bb3bd2","order_by":4,"name":"Katharina Gutbrod","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Katharina","middleName":"","lastName":"Gutbrod","suffix":""},{"id":599683694,"identity":"bcbe0b3e-9757-46f8-a816-fac055d7e186","order_by":5,"name":"Peter Dörmann","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Dörmann","suffix":""},{"id":599683695,"identity":"a70fc8e9-3a73-4ebd-96e5-a8fce8c5a805","order_by":6,"name":"Hunseung Kang","email":"","orcid":"","institution":"Chonnam National University","correspondingAuthor":false,"prefix":"","firstName":"Hunseung","middleName":"","lastName":"Kang","suffix":""},{"id":599683696,"identity":"404d2dd8-41a8-41eb-b8dd-aa7ff25758a1","order_by":7,"name":"Ute C. Vothknecht","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYHACNsYGIGnAwHwARCaAxXjw6mCGamFjSyBZC48BiEtYi8Hx/mMPZ+6wkTeX7/kmXVDAkMcv3XuA4U0FHi1nDrMbbjyTZrizjXeb9AwDhmLJOecSGOecwa1FckYym+TDtsMJBseAWngM/iduuJFjwMzbRlDLf6AWnmdALQxQLf9wa+GXAGrZ2HYApIUNSUsDHi08h80kZ7YlG244lmZszQPyy4wcg4NzjuHWwsbe+Eyyt81O3uDw4Ye3ef4AQ0wix/DBmxrcWrCDA6RqGAWjYBSMglGACgAVrEsFEwHGuAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Bonn","correspondingAuthor":true,"prefix":"","firstName":"Ute","middleName":"C.","lastName":"Vothknecht","suffix":""},{"id":599683697,"identity":"e93fce86-cb22-4ac0-901b-8c068f0c5ed4","order_by":8,"name":"Fatima Chigri","email":"","orcid":"","institution":"University of Bonn","correspondingAuthor":false,"prefix":"","firstName":"Fatima","middleName":"","lastName":"Chigri","suffix":""}],"badges":[],"createdAt":"2026-02-25 15:39:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8969427/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8969427/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103977916,"identity":"d61185e1-368a-49e5-bfac-3e066ec528ff","added_by":"auto","created_at":"2026-03-05 08:57:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":960554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA regulatory components under drought stress. \u003c/strong\u003eThe relative expression level of \u003cstrong\u003em\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA\u003c/strong\u003e writers, readers and erasers was analysed in 18-day-old WT plants grown on soil and exposed to different time periods of \u003cstrong\u003eA\u003c/strong\u003e progressive drought stress and \u003cstrong\u003eB\u003c/strong\u003e well-watered conditions. The relative expression was analysed through RT-qPCR after 0, 6, 9, and 12 days of treatment. Values are the mean ± SE of at least three independent biological replicates (n=3). Data were analysed through two-tailed student’s t- test (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/d6a1241d443572187c24d85e.png"},{"id":103978044,"identity":"6a8d2e16-0867-4881-844a-f76511cff52e","added_by":"auto","created_at":"2026-03-05 08:58:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1997076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eALKBH10B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is a positive regulator of drought stress. A \u003c/strong\u003ePhenotypes of WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e, and OX-1 plants under well-watered and drought stress conditions applied to 18-day-old plants. \u003cstrong\u003eB\u003c/strong\u003e Survival rate of the plants measured after three days of rewatering. Values are the mean ± SE of three independent biological replicates. \u003cstrong\u003eC\u003c/strong\u003e Representative pictures showing the stomatal aperture, \u003cstrong\u003eD\u003c/strong\u003e density and \u003cstrong\u003eE \u003c/strong\u003etranspiration in leaf (no. 6, 7 and 8) of five individual 28-day-old plants under control conditions. Values are the mean ± SE of n=100 for aperture, and n=29 for density. \u003cstrong\u003eF \u003c/strong\u003eLeaf relative water content (RWC) calculated after 12 days of drought stress using three biological replicates each consisting of two plants (n=3). All data were analysed through one-way ANOVA and Tukey’s Post-Hoc HSD tests (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/03ca6bde6c762d7209c74687.png"},{"id":103978059,"identity":"94bb1a25-79c0-4c15-a8b5-bd3b9747ae67","added_by":"auto","created_at":"2026-03-05 08:58:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":632230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of ABA and MeJA on the expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eALKBH10B. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e The relative expression of \u003cem\u003eALKBH10B \u003c/em\u003ewas\u003cem\u003e \u003c/em\u003equantified\u003cem\u003e \u003c/em\u003ein WT plants grown on ½ MS plates and treated for different time points with water (mock), 50 µM ABA, 50 µM MeJA, or 50 µM of each (ABA+MeJA) \u003cstrong\u003eB \u003c/strong\u003eRelative expression of \u003cem\u003eALKBH10B\u003c/em\u003e in WT\u003cem\u003e, jar1.11, \u003c/em\u003eand \u003cem\u003eaba2-1 \u003c/em\u003eplants grown on soil under control and drought stress conditions \u003cstrong\u003eC\u003c/strong\u003e Relative luciferase activity in tobacco leaves transformed with p\u003cem\u003eALKBH10B::FLUC\u003c/em\u003e and treated for 3 hours with either water (mock), 50 µM ABA, 50 µM MeJA or 50 µM ABA + different concentration of MeJA. Values are the mean ± SE of three independent biological replicates (n=3). Data were analysed through two-way ANOVA followed by Tukey’s Post-Hoc HSD tests (P\u0026lt;0.05). \u003cstrong\u003eD \u003c/strong\u003eAnalysis of the 1kb promoter region of \u003cem\u003eALKBH10B\u003c/em\u003e showing various cis-regulatory elements responsive to hormones including ABA (ABRE and MYB), JA (MYC), and salicylic acid (AS-1) as well as drought (DRE) and light (TCT) responsive element.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/fda801c44cfc61200d0f19e3.png"},{"id":103978038,"identity":"9e35da64-4614-4fde-8754-433887aa0183","added_by":"auto","created_at":"2026-03-05 08:58:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":396499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhytohormone content in WT, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealkbh10b-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and OX-1 lines. \u003c/strong\u003eThe content of \u003cstrong\u003eA\u003c/strong\u003e ABA, \u003cstrong\u003eB \u003c/strong\u003eJA, and \u003cstrong\u003eC \u003c/strong\u003eJA-Ile m\u003csup\u003e6\u003c/sup\u003eA was analysed in plants grown on soil under control or 12 days of drought stress conditions\u003cstrong\u003e. \u003c/strong\u003eData represent mean ± SE of 5 biological replicates (n=5) each containing pooled extracts from 2 plants. Data were analysed through two-way ANOVA followed by Tukey’s Post-Hoc HSD test (P\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/f1abeba6fd69be84e024ad27.png"},{"id":103978152,"identity":"a97cb74b-f2ef-46b1-a4af-325183ca5c8e","added_by":"auto","created_at":"2026-03-05 08:58:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2717575,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes in WT, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ealkbh10b-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and OX-1. A \u003c/strong\u003ePrincipal component analysis of WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e and OX-1 samples under control (C) and drought (D) conditions\u003cstrong\u003e,\u003c/strong\u003e PC1 represents variation based on treatment and PC2 represents variation based on genotype\u003cstrong\u003e B \u003c/strong\u003eNumber of up- and down-regulated genes in WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e and OX-1 (drought \u003cem\u003evs\u003c/em\u003e control) \u003cstrong\u003eC \u003c/strong\u003eVolcano plots showing differentially expressed genes (DEGs) in drought \u003cem\u003evs.\u003c/em\u003e control samples defined by Log\u003csub\u003e2\u003c/sub\u003eFC ≥1 and FDR\u0026lt;0.05 \u003cstrong\u003eD \u003c/strong\u003eNumber of up- and down-regulated genes in WT-drought \u003cem\u003evs.\u003c/em\u003e \u003cem\u003ealkbh10b-1-\u003c/em\u003edrought or WT-drought \u003cem\u003evs.\u003c/em\u003e OX-1-drought \u003cstrong\u003eE\u003c/strong\u003e Heatmap and \u003cstrong\u003eF \u003c/strong\u003eGO biological processes of the up-regulated genes in WT-drought \u003cem\u003evs\u003c/em\u003e OX-1-drought.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/ef721a26e4290ae023fae7d5.png"},{"id":103978017,"identity":"32ed4d7c-0f1a-40ff-ace4-d85a9dc33162","added_by":"auto","created_at":"2026-03-05 08:58:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1617159,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eALKBH10B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eon transcriptomic variations between drought stress and control conditions.\u003c/strong\u003e DEGs identified between WT/\u003cem\u003ealkbh10b-1\u003c/em\u003eand WT/OX-1 were used for GO enrichment analyses classified into \u003cstrong\u003eA\u003c/strong\u003e biological process, \u003cstrong\u003eB\u003c/strong\u003e cellular components and \u003cstrong\u003eC\u003c/strong\u003e molecular functions and\u003cstrong\u003e \u003c/strong\u003efor\u003cstrong\u003eD \u003c/strong\u003eKEGG pathways analysis \u003cstrong\u003eE \u003c/strong\u003eClustering of DEGS into 4 individual clusters.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/443e90c0e8f1a757669fbb8f.png"},{"id":103977917,"identity":"dbc13892-6faa-44fa-affb-9b3a0b15cd1c","added_by":"auto","created_at":"2026-03-05 08:57:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1275975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eALKBH10B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on photosynthesis. A\u003c/strong\u003e Heatmap representing the differential expression of photosynthesis-related genes in WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e and OX-1 between drought (D) and control (C) conditions determined by RNAseq analysis. \u003cstrong\u003eB\u003c/strong\u003e Expression level of selected photosynthesis -related genes quantified by RT-qPCR under normal and drought stress conditions. Values are the mean ± SE of three biological replicates (n=3) \u003cstrong\u003eC\u003c/strong\u003e Measurement of chlorophyll content and \u003cstrong\u003eD\u003c/strong\u003e photosystem II activity. Values are the mean ± SE of n=10. Data were analysed through two-way ANOVA followed by Tukey’s multiple comparison test (P\u0026lt;0.05) \u003cstrong\u003eE\u003c/strong\u003e m\u003csup\u003e6\u003c/sup\u003eA level enrichment quantified using m\u003csup\u003e6\u003c/sup\u003eA-IP-qPCR under control and drought conditions. Values are the mean ± SE of three biological replicates (n=3) and asterisks indicate significant differences (Student’s t-test; *P \u0026lt; 0.05, **P \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/b3c2c9efcd38950bb95086d6.png"},{"id":103977976,"identity":"9e7de8a4-f84c-4699-b8c9-4235aaf245ed","added_by":"auto","created_at":"2026-03-05 08:57:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1023917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eALKBH10B\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on the regulation of known drought-related genes. A \u003c/strong\u003eHeatmap representing the expression of drought-related genes in WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e and OX-1 between drought (D) and control (C) conditions \u003cstrong\u003eB \u003c/strong\u003em\u003csup\u003e6\u003c/sup\u003eA level enrichment quantified using m\u003csup\u003e6\u003c/sup\u003eA IP-qPCR under control and drought conditions. Values are the mean ± SE of three biological replicates (n=3) and asterisks indicate significant differences between WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant or OX-1 lines (Student’s t-test; *P \u0026lt; 0.05, **P \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/be20bb3d4d1e307c19d601a0.png"},{"id":103978196,"identity":"858cf259-8011-4de7-ad3e-44be23e8ad2e","added_by":"auto","created_at":"2026-03-05 08:58:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12455655,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/44eecd45-1da9-4b88-8baa-512ee1740317.pdf"},{"id":103977998,"identity":"16ddf0a0-c5d4-427e-a438-1e72b1bb0236","added_by":"auto","created_at":"2026-03-05 08:58:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":851398,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/ae56ec289d04caf17c15704d.docx"},{"id":103978035,"identity":"5e9b6d1d-c5e7-4ff0-9005-1082cc3a2781","added_by":"auto","created_at":"2026-03-05 08:58:15","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12604,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/e6ed2e3d7b454f0ab1b0a231.xlsx"},{"id":103978026,"identity":"f291b887-b769-4f26-ac75-00384a08908b","added_by":"auto","created_at":"2026-03-05 08:58:12","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21896,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/0a4fbd1cca040d7ab30e3a70.xlsx"},{"id":103978004,"identity":"5311cd1c-ce30-4d6c-9181-7e6dd0d18f84","added_by":"auto","created_at":"2026-03-05 08:58:09","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":690905,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/6b525c1a17a7faa222965a1b.xlsx"},{"id":103977975,"identity":"25f0ee1d-e81a-4604-bcd9-7d9634cb5e01","added_by":"auto","created_at":"2026-03-05 08:57:58","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":352135,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/9f07c91196e708d546b9b69e.xlsx"},{"id":103977913,"identity":"9e7b3319-8d4c-460c-bc3c-d49e12f73503","added_by":"auto","created_at":"2026-03-05 08:57:54","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":22056,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/16edc361bb06f8f3c3a7e8e5.xlsx"},{"id":103978006,"identity":"e29f62cd-3fe3-49d6-96e5-a65c5fca8dfc","added_by":"auto","created_at":"2026-03-05 08:58:09","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":19462,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8969427/v1/4c24f202e63681038cbeb657.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ALKBH10B mediated epitranscriptomic regulation enhances drought tolerance of Arabidopsis thaliana via hormonal cross-talk and sustained photosynthesis ","fulltext":[{"header":"Key message","content":"\u003cp\u003eThe m⁶A demethylase ALKBH10B emerges as a crucial post-transcriptional regulator of hormonal crosstalk, photosynthetic maintenance, and mitochondrial energy metabolism to fine-tune plant stress adaptation to drought\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePlants, being sessile organisms, are subjected to various biotic and abiotic stresses. Drought is considered one of the major abiotic stresses, causing a series of morphological, physiological, biochemical, and molecular changes that affect plant development and crop yield (Lozano-Elena et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Thus, with an increasing human population and a decrease in arable land, there is an intense need for the development of drought-resilient crops to maintain adequate food production.\u003c/p\u003e \u003cp\u003ePlants can sense the lack of water early on under progressive drought conditions and trigger complex signalling cascades, which ultimately regulate molecular processes including gene expression to facilitate an appropriate response (Osakabe et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nakashima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The aim is to prevent water loss, e.g., by stomata closure, adjustment of water-utilizing processes such as photosynthesis, and/or initiation of escape mechanisms such as early flowering (Haghpanah et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Various physiological and molecular analyses, not only in Arabidopsis but also many other plants, have identified phytohormones as key signalling molecules that regulate drought stress responses (Aimar et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Gupta et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among these, ABA has been shown to play a critical role in drought tolerance both through direct regulation of stomatal closure as well as regulating production of various drought-responsive proteins (Nakashima et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Promoter regions of drought-responsive genes often contain two cis-acting elements, namely ABRE (Abscisic Acid Responsive Element) and DRE (Dehydration Responsive Element), to facilitate the drought-stress related regulation of gene expression (Nakashima and Yamaguchi-Shinozaki \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, other phytohormones have also been shown to be involved in the drought response, including jasmonates, especially JA-Ile, the biologically active derivative of JA (Alam et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cheong and Choi \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Mahmud et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These hormones do not act independently from each other and cross-talk has been described between jasmonate and ABA signalling with regards to drought responses. It includes co-regulation of transcriptional ABA responses by alleviating the ABA-mediated induction of drought responsive genes such as \u003cem\u003eRD29A\u003c/em\u003e in Arabidopsis and \u003cem\u003eStRD29\u003c/em\u003e in \u003cem\u003eSolanum tuberosum\u003c/em\u003e (Bleker et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent years, it has been discovered that plants experiencing environmental stresses extensively reprogram post-transcriptional gene regulatory processes. Among around 170 known post-transcriptional mRNA modifications (Boccaletto et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xuan et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) N6-methyladenosine (m\u003csup\u003e6\u003c/sup\u003eA) is the most abundant, dynamic, and reversible internal mRNA modification in eukaryotes (Liu and Pan \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Arribas-Hernandez and Brodersen \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The role of m\u003csup\u003e6\u003c/sup\u003eA has been explored in various abiotic stress responses such as salinity, drought, and cold stress (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Shoaib et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vicente et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Cai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). m\u003csup\u003e6\u003c/sup\u003eA is installed by methyltransferases known as writers, removed by demethylases called erasers, and read by RNA-binding proteins called readers (Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In plants, a m\u003csup\u003e6\u003c/sup\u003eA writer complex has been identified whose core components includes METHYLTRANSFERASE A and B (MTA, MTB), the adapter protein VIRILIZER (VIR), FKBP12-INTERACTING PROTEIN 37 (FIP37), and a ubiquitin E3 ligase called HAKAI (Zhong et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Recently, MTA has been shown to contribute to drought stress resistance in Arabidopsis, apple, and poplar (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ganguly et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Notable reader proteins that recognize m\u003csup\u003e6\u003c/sup\u003eA modification belong to YTH domain proteins and are named Evolutionary Conserved C-Terminal Region (ECT) (Reichel et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). FLK is another reader protein belonging to the KH domain family (Fabian et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These reader proteins decode the m\u003csup\u003e6\u003c/sup\u003eA modification and mediate the downstream processes such as splicing, alternative polyadenylation, RNA export, stability and translation (Hu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDemethylases involved in m\u003csup\u003e6\u003c/sup\u003eA mRNA modification belong to the family of ALKYLATION B HOMOLOG proteins (ALKBH), whose members are involved in various cellular processes related to DNA repair and gene regulation. Among the 14 ALKBH proteins in Arabidopsis, ALKBH9B, ALKBH9C and ALKBH10B are well-characterized m\u003csup\u003e6\u003c/sup\u003eA eraser (Martinez-Perez et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Duan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Amara et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ALKBH9B is important in viral defence and also involved in promoting the mobilization of heat-activated long terminal repeat retrotransposons (Martinez-Perez et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Fan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). ALKBH9C is crucial for seed germination and seedling growth under salt, osmotic stress and ABA by affecting the stability of various stress-responsive transcripts (Amara et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ALKBH10B plays an important role in floral transition by demethylation of transcripts of flowering related genes such as \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e and \u003cem\u003eSQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3\u003c/em\u003e and \u003cem\u003e9\u003c/em\u003e (Duan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, ALKBH10B also affects the expression of various genes involved in salt, osmotic stress, or ABA responses (Shoaib et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile recently shown to positively regulate drought resistance in Arabidopsis (Han et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the exact contribution of ALKBH10B to drought tolerance is not well defined. Here, we show that constitutive increased expression of \u003cem\u003eALKBH10B\u003c/em\u003e in OX-1 plants leads to improved water retention, reduced jasmonate accumulation, and maintenance of photosynthetic efficiency compared to WT and the \u003cem\u003ealkbh10b-1\u003c/em\u003e null-mutant despite comparable ABA levels across genotypes. RNA-seq analysis showed large alteration in the transcriptional landscape of the different lines, affecting expression of genes related to photosynthesis and drought response. Analysis by m\u003csup\u003e6\u003c/sup\u003eA-IP-qPCR identified direct alteration in m\u003csup\u003e6\u003c/sup\u003eA enrichment in some of these genes in OX-1 and \u003cem\u003ealkbh10b-1\u003c/em\u003e. However, it also manifests that changes in transcript levels of many genes seems rather indirectly affected by \u003cem\u003eALKBH10B\u003c/em\u003e. RNA-seq analysis also revealed that genes associated with mitochondrial respiration and ATP synthesis were up-regulated in OX-1, indicating that increased removal of m\u003csup\u003e6\u003c/sup\u003eA mRNA modification by ALKBH10B can support energy metabolism during drought. Collectively, our findings demonstrate that ALKBH10B-driven mRNA demethylation regulates hormonal crosstalk, photosynthetic maintenance, and mitochondrial energy metabolism and establishes ALKBH10B as a key epitranscriptomic regulator in drought response that fine-tunes plant stress adaptation by coordinating transcriptional regulation and hormone balance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we used\u003cem\u003e\u0026nbsp;Arabidopsis thaliana\u0026nbsp;\u003c/em\u003eColumbia ecotype\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Col-0), the T-DNA insertion line \u003cem\u003ealkbh10b\u003c/em\u003e (Salk_004215c, Fig. S1A) and two over-expression lines (OX-1 and OX-2) expressing an \u003cem\u003eALKBH10B-FLAG\u003c/em\u003e fusion under the control of CaMV 35S promoter (Han et al. 2023). The \u003cem\u003eaba2-1\u003c/em\u003e and \u003cem\u003ejar1-11\u003c/em\u003e mutant lines were previously described (Cheng et al. 2002; Mahmud et al. 2022). \u003cem\u003eNicotiana benthamiana\u003c/em\u003e was used for transient infiltration. For drought stress, Arabidopsis seeds were sown directly in potting soil whereas for other experiments, they were surface sterilized with 70% (v/v) ethanol for 3 minutes, 6% (v/v) sodium hypochlorite (NaOCl) for 5 minutes and then five times rinsed with distilled water and sown on \u0026frac12; MS (Duchefa Biochemie, The Netherlands) plates containing 1% (w/v) sucrose and 0.4% (w/v) phytoagar. \u0026nbsp;The plates were maintained in the dark at 4 \u0026deg;C for three days for vernalization before being transferred to the growth chamber. To study the regulation by hormones, Arabidopsis seedlings were grown for three weeks on \u0026frac12; MS plates and treated either with 50 \u0026micro;M (+)-ABA, 50\u0026nbsp;\u0026micro;M MeJA (SERVA, Germany), or 50\u0026nbsp;\u0026micro;M ABA + 50\u0026nbsp;\u0026micro;M MeJA or with the same volume of H\u003csub\u003e2\u003c/sub\u003eO as a mock treatment. Plants were harvested after 3-, 6-, and 24-hours treatment, frozen in liquid nitrogen, and RNA was extracted as described below. All plants were grown at 22\u0026plusmn;2 \u0026deg;C in a climate-controlled growth chamber under long-day conditions (16h light /8h dark) with a light intensity of 100 \u0026mu;mol photon*m\u003csup\u003e-2\u003c/sup\u003e*s\u003csup\u003e-1\u003c/sup\u003e (Philips TLD 18W lamps of alternating 830/840 light temperature).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotyping under drought stress, measurement of relative leaf water content, and stomatal aperture and density\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven-day-old seedlings of WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e, and OX-1 grown on soil were transferred to individual pots containing an equal amount of soil. Plants were watered regularly until they were 18 days old and drought stress was applied by withholding water. After 10-12 days plants were rewatered for three days, and the survival rate was measured. The survival percentage was calculated as (number of survived plants / total number of plants)*100.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe relative leaf water content (RWC) was quantified and calculated as previously described (Mahmud et al. 2022). In general, leaves no. 5-8 were used from 5 individual plants for each genotype. For the determination of stomatal aperture, plants from WT, \u003cem\u003ealkbh10b-1\u0026nbsp;\u003c/em\u003emutant, and OX-1 lines grown under normal conditions (control) were incubated in imaging buffer (10 mM MES, 5 mM KCl, 50 \u0026mu;M CaCl\u003csub\u003e2\u003c/sub\u003e, pH 6.15) for two hours. The lower epidermis from the 6\u003csup\u003eth\u003c/sup\u003e \u0026amp; 7\u003csup\u003eth\u003c/sup\u003e leaves was carefully peeled, separated from mesophyll cells, and depicted using bright field in a Confocal Laser Scanning Flourescence Microscope (Leica SP8 Lightning, Leica, Germany) using the internal LAS X software. Stomatal aperture was measured using ImageJ software (Schindelin et al. 2012). Stomatal density was determined as described in (Bhattacharyya et al. 2025) using data from 6 individual plants for each genotype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of chlorophyll content, photosystem II activity and stomatal conductance\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chlorophyll content and photosystem II activity were measured in control and drought-exposed plants. Chlorophyll was measured through a DUALEX optical leafclip meter (Pessl Instruments, Austria). For the measurement of stomatal transpiration and photosystem II activity, a LI-COR (LI-6000) porometer/fluorometer (LI-COR Environmental GmbH, Germany) was used. All measurements were performed on 2 leaves per plant and 10 independent plants from each line were used.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePromoter analysis and molecular cloning in pBIN19-ANX vector\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 1-kb promoter region upstream of the \u003cem\u003eALKBH10B\u003c/em\u003e transcription initiation site was analysed for cis-acting elements through PlantCARE (https://bioinformatics.psb.ugent.be/ webtools/plantcare/html/). To measure the promoter activity under different hormone treatments, the 1kb promoter region of \u003cem\u003eALKBH10B\u003c/em\u003e (p\u003cem\u003eALKBH10B\u003c/em\u003e) was amplified with forward and reverse primers containing Apa1 and Kpn1 restriction sites, respectively, using gDNA as a template and ligated into a pBIN19 vector containing the firefly luciferase (FLUC) reporter gene. The construct cassette is shown in Figure S1D, and the primer sequences are listed in Supplementary Table S1. After confirming the correct sequence, the vector containing p\u003cem\u003eALKBH10B\u003c/em\u003e::fluc was transformed into Agrobacterium strain GV3101.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient transformation of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e and luciferase assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAgrobacterium cells carrying the pBIN-p\u003cem\u003eALKBH10B\u003c/em\u003e::fluc construct were grown overnight at 28\u0026deg;C in YEB medium containing 100\u0026nbsp;\u0026micro;g/ml rifampicin, 25\u0026nbsp;\u0026micro;g/ml kanamycin and 1M MgCl\u003csub\u003e2\u003c/sub\u003e. The culture was then centrifuged at 3500 rpm for 5 minutes, resuspended in infiltration medium (20 mM citric acid, 2% (w/v) sucrose) and adjusted to OD\u003csub\u003e600\u003c/sub\u003e = 0.5 for infiltration into the leaves of four-week-old \u003cem\u003eNicotiana benthamiana\u0026nbsp;\u003c/em\u003eplants. Three days after infiltration, the leaves of Nicotiana were cut into discs (\u0026oslash; 6 mm) and immersed in autoclaved water containing 30 \u0026micro;M D-luciferin together with either 50 \u0026micro;M ABA, 50 \u0026micro;M MeJA, 50 \u0026micro;M ABA+ 50 \u0026micro;M MeJA or 50 \u0026micro;M ABA+25 \u0026micro;M MeJA and H\u003csub\u003e2\u003c/sub\u003eO as mock control in a 96 well plate. FLUC luminescence was recorded in a Multi-Mode Microplate reader (Tristar\u003csup\u003e2\u003c/sup\u003e, Berthold GmbH, Germany) after three hours. During the measurement the leaf discs were kept in darkness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction, cDNA synthesis and RT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA of four-week-old plants grown under control and drought stress conditions was extracted using the Genematrix Universal RNA Purification Kit (Roboklon, Germany) according to the manufacturer\u0026rsquo;s instructions. RNA integrity was assessed on a 1% agarose gel and the concentration was measured with a Nanopdrop photometer (Nanondrop\u0026trade; One, Thermo Fisher Scientific). cDNA was synthesized from 1.5 \u0026micro;g of total RNA with oligodT-\u003csub\u003e18\u003c/sub\u003e and random hexamer primers following the manufacturer\u0026rsquo;s instructions of the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Germany). RT-qPCR was performed in a 96-wells plate using the CFX96 real-time thermal cycler system (Bio Rad, Germany) and SYBR Green PCR master mix (Thermo Fisher Scientific, USA). All transcript levels were normalized against the geometric means of two reference genes, namely \u003cem\u003eACTIN2\u003c/em\u003e and \u003cem\u003eTUBULIN 2,\u0026nbsp;\u003c/em\u003eand the relative expression level was quantified using the 2^-\u003csup\u003e\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method. All primers are listed in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of phytohormones\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole rosette leaves of four-week-old plants grown under control and drought stress conditions, two plants for each sample, were frozen in liquid nitrogen and ground in a pre-cooled mortar with a pestle. Extraction and quantification of hormones were done as previously described (Pan et al. 2010). To 50 mg of the plant powder, 25 \u0026micro;l of internal standards was added and hormones were extracted with 500 \u0026micro;l of extraction solvent consisting of 2-propanol/H\u003csub\u003e2\u003c/sub\u003eO/conc. HCl (2:1:0.002, v/v/v) and shaken at 100 rpm for 30 minutes at 4\u0026deg;C. To each sample, 1 ml of dichloromethane was added and the samples were shaken for 30 minutes at 4\u0026deg;C. The samples were then centrifuged at 13000 x g for 5 minutes at 4\u0026deg;C to achieve phase separation. From the lower phase, 900 \u0026micro;l were transferred to a screw-cap vial and the sample was dried completely using a nitrogen evaporator. Residual tissues were resuspended in 0.1 ml methanol: 0.1% formic acid in water (1:1, v/v). An aliquot of 50 \u0026micro;l of the sample solution was injected onto the reverse-phase C18 Gemini HPLC column and analysed using a QTRAP 6500+ LC-MS/MS system (Sciex, Germany). The concentrations of ABA, JA, JA-Ile and SA (salicylic acid) were quantified relative to the internal standards and expressed as ng/g F.W.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-sequencing and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA sequencing was performed using three biological replicates, each consisting of pooled RNA extracted from the rosette leaves of two individual plants grown for 4 weeks under well-watered and drought stress conditions using the Direct-zol RNA Purification kit (Zymo Research Corporation, U.S.A.). An RNA quality check was carried out using a Tapestation 4200 (Agilent, U.S.A.). Approximately 100 ng of RNA was used for library preparation and 3\u0026rsquo; mRNA sequencing was done by the NGS Core Facility (Medical Faculty at the University of Bonn, Germany) using a NOVASEQ6000 (Illumina, USA) sequencer. An average sequencing depth of ~10 million raw reads per sample (Supplementary Table S2) was generated. The FASTQ raw files were processed to remove low-quality bases and adapter sequences through the Trimmomatic method (Bolger et al. 2014). To confirm the removal of adapter sequence after trimmomatic processing, FastQC was done on a linux system. The reads were aligned through HiSAT2 software against the concatenated reference genome of Arabidopsis\u003cem\u003e\u0026nbsp;\u003c/em\u003e(http://plants.ensembl.org/info/data/ftp/index.html). The Gene Counts were generated from the aligned BAM files through the Feature Counts function in RStudio (Liao et al. 2014). The differential gene expression was carried out using EdgeR. The p-values were corrected using the False Discovery Rate (FDR) method (Benjamini and Hochberg 1995) and subsequently the FDR and |Log\u003csub\u003e2\u003c/sub\u003eFC| cut offs were set to 0.05 and 1 respectively. Volcano plots were generated through the SRplot web tool\u0026nbsp;(Tang et al. 2023)\u0026nbsp;whereas the Principal Component Analysis (PCA) plot and clusters were generated with the iDep2 web tool\u0026nbsp;(Ge et al. 2018). Visualization of heatmaps was performed using the Morpheus webtool (https://software.broadinstitute.org/morpheus/). GO analyses of the obtained DEGs were performed using ShinyGO\u0026nbsp;(Ge et al. 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003em\u003csup\u003e6\u003c/sup\u003eA immunoprecipitation\u003c/strong\u003e-\u003cstrong\u003eqPCR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe m\u003csup\u003e6\u003c/sup\u003eA-\u0026nbsp;immunoprecipitation (IP)-qPCR was performed as described previously (Hu et al. 2021). Briefly, mRNA was extracted from total RNA using oligo-d(T)\u003csub\u003e25\u003c/sub\u003e magnetic beads (New England Biolabs, U.S.A.) and 5 \u0026micro;g was fragmented into around 300-nucleotide-long mRNA fragments by incubation in RNA fragmentation buffer (100 mM Tris-HCl, pH=7.0, 100 mM ZnCl\u003csub\u003e2\u003c/sub\u003e). The fragmented mRNA was mixed with an m\u003csup\u003e6\u003c/sup\u003eA-specific antibody (Synaptic Systems, Germany), and the bound RNAs were eluted. The input and eluted mRNA were reverse transcribed using reverse transcriptase with random hexamer primers and the level of each transcript was measured by RT-qPCR conducted on a Rotor-Gene Q thermal cycle (Qiagen, Germany) using gene-specific primers listed in Supplementary Table S1. The level of m\u003csup\u003e6\u003c/sup\u003eA enrichment of target genes was quantified based on the PCR cycle threshold. The value of the immunoprecipitated sample was normalised against that of the input. Values are the means \u0026plusmn; SE of three biological replicates.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDrought stress induces changes in the expression level of m\u003csup\u003e6\u003c/sup\u003eA regulatory components\u003c/h2\u003e \u003cp\u003eTo explore the role of m\u003csup\u003e6\u003c/sup\u003eA mRNA modification in the drought stress response, we quantified the expression of established m\u003csup\u003e6\u003c/sup\u003eA regulatory genes using RT-qPCR. Transcript levels were analysed in rosette leaves of Arabidopsis WT at different time periods of progressive drought treatment and compared to well-watered control conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results show a moderate increase in the expression levels of genes coding for the m\u003csup\u003e6\u003c/sup\u003eA writers \u003cem\u003eMTA\u003c/em\u003e, \u003cem\u003eMTB\u003c/em\u003e, \u003cem\u003eFIP37\u003c/em\u003e, \u003cem\u003eVIR\u003c/em\u003e and \u003cem\u003eHAKAI\u003c/em\u003e, the m\u003csup\u003e6\u003c/sup\u003eA readers \u003cem\u003eECT2\u003c/em\u003e, \u003cem\u003eECT3\u003c/em\u003e, \u003cem\u003eECT4\u003c/em\u003e, and \u003cem\u003eFLK\u003c/em\u003e, and the m\u003csup\u003e6\u003c/sup\u003eA erasers \u003cem\u003eALKBH9B\u003c/em\u003e and \u003cem\u003eALKBH9C\u003c/em\u003e. By contrast, the m\u003csup\u003e6\u003c/sup\u003eA eraser \u003cem\u003eALKBH10B\u003c/em\u003e was moderately upregulated after 6 days but substantially upregulated after 12 days of withholding water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Under well-watered conditions, only minor changes in the expression of all these genes occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), suggesting that the drought-induced expression of the m\u003csup\u003e6\u003c/sup\u003eA regulatory genes is not due to developmental effects. Collectively, these results indicate that drought stress induces coordinated reprogramming of the whole m\u003csup\u003e6\u003c/sup\u003eA regulatory machinery, with ALKBH10B emerging as the most strongly induced component. We therefore focused subsequent analyses on ALKBH10B as a key candidate for mediating m\u003csup\u003e6\u003c/sup\u003eA-dependent responses during prolonged drought stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eALKBH10B\u003c/b\u003e \u003cb\u003econfers drought stress tolerance and regulates water loss through modulation of stomatal aperture\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further investigate the role of \u003cem\u003eALKBH10B\u003c/em\u003e we used the Arabidopsis T-DNA insertion mutant \u003cem\u003ealkbh10b-1\u003c/em\u003e (SALK_004215C, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and \u003cem\u003e35S::ALKBH10B\u003c/em\u003e transgenic plants (OX-1), which were reported in previous studies as knockout and overexpression lines, respectively (Duan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shoaib et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). RT-qPCR corroborated a complete loss of \u003cem\u003eALKBH10B\u003c/em\u003e transcript in the \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant and an increased expression in the OX-1 line compared to WT (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). When progressive drought was applied to 18-day-old WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e, and OX-1 plants by withholding water, \u003cem\u003ealkbh10b-1\u003c/em\u003e plants showed clear symptoms of wilting on day 12, followed by WT at day 15, whereas the OX-1 plants were still green and showed only slight wilting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Consequently, after three days of rewatering, the survival rate of OX-1 plants was approximately 80%, whereas survival rate of WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e plants were only about 33% and 19%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and S2A). The contrasting phenotype of null mutant and overexpression was confirmed using a second OX line (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, OX-2). These results confirm that \u003cem\u003eALKBH10B\u003c/em\u003e plays a role as a positive regulator of drought tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eReduced water loss through transpiration is a key determinant for drought tolerance. Stomatal density was modestly increased only in \u003cem\u003ealkbh10b-1\u003c/em\u003e but not in WT or the OX lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Stomatal aperture measurements, however, revealed wider apertures in \u003cem\u003ealkbh10b-1\u003c/em\u003e and reduced apertures in OX compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Although the reduction in stomatal aperture in the OX lines was moderate in magnitude, it was consistent and statistically significant across biological replicates. These parameters likely contribute to reduced water loss through stomatal transpiration as measured in OX-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), resulting in delayed wilting and decreased drought sensitivity. Accordingly, the relative water content (RWC) on day 12 of progressive drought was higher in OX-1 and reduced in \u003cem\u003ealkbh10b-1\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e).\u003c/b\u003e We therefore interpret stomatal regulation as one, albeit not the only, contributing component of ALKBH10B-mediated drought tolerance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eALKBH10B\u003c/b\u003e \u003cb\u003eexpression is regulated by ABA and jasmonates\u003c/b\u003e\u003c/p\u003e \u003cp\u003eABA and jasmonate are important regulators of drought responses (Ullah et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and treatment of WT seedlings grown on \u0026frac12; MS medium confirmed that \u003cem\u003eALKBH10B\u003c/em\u003e expression is strongly induced by ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). MeJA application had no influence on \u003cem\u003eALKBH10B\u003c/em\u003e expression but co-application of both hormones strongly reduced ABA-mediated \u003cem\u003eALKBH10B\u003c/em\u003e induction suggesting an antagonistic interaction. In accordance with these results, drought-induction of \u003cem\u003eALKBH10B\u003c/em\u003e was absent in a mutant defective in ABA biosynthesis (\u003cem\u003eaba2-1)\u003c/em\u003e (Cheng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and enhanced in \u003cem\u003ejar1-11\u003c/em\u003e, a plant line defective in JA-Ile formation (Mahmud et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). These data indicate that ABA is a major contributor to drought-induced \u003cem\u003eALKBH10B\u003c/em\u003e expression, while jasmonate modulates the amplitude of this induction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExpression of a luciferase reporter driven by the \u003cem\u003eALKBH10B\u003c/em\u003e promoter (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). confirmed induction by ABA and attenuation by jasmonate (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) consistent with a regulation mediated by cis-acting promoter elements. Analysis of the 1-kb promoter region of \u003cem\u003eALKBH10B\u003c/em\u003e using the PlantCARE database revealed multiple ABRE (Abscisic Acid Responsive Element) and MYB binding motifs as well as a MYC motif and AS-1 element (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These elements have been associated with ABA and/or jasmonate dependent transcription and often act combinatorically in response to different hormones. Further regulatory elements include DRE (dehydration-responsive element), which is associated with ABA-independent stress response and often occurs alongside ABREs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eALKBH10B\u003c/b\u003e \u003cb\u003eaffects accumulation of ABA and JA\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhytohormones such as ABA and jasmonate regulate extensive transcriptional networks, and a subset of these hormone-responsive genes encode proteins that also modulate the hormone\u0026rsquo;s own levels, thereby establishing feedback loops of hormone content regulation (Zong et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We thus examined whether \u003cem\u003eALKBH10B\u003c/em\u003e also affects hormonal dynamics under drought stress. To this end, the levels of ABA, JA, and JA-Ile were quantified in WT, \u003cem\u003ealkbh10b-1\u003c/em\u003e and OX-1 plants under control and drought stress conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Under control conditions, ABA, JA, and JA-Ile levels were low and similar across genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Under drought, ABA level increased in all genotypes, with a slightly higher accumulation in \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). By contrast, levels of JA and JA-Ile increased in the WT and the \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant under drought but remained unchanged in OX-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). The reduced jasmonate accumulation in OX-1 may reflect either direct regulation of specific transcripts by ALKBH10B or indirect metabolic consequences of altered transcriptional or physiological states. At present, our data do not allow us to distinguish between these possibilities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq analysis reveals effect of\u003c/b\u003e \u003cb\u003eALKBH10B\u003c/b\u003e \u003cb\u003eon respiration and photosynthesis under drought stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003em\u003csup\u003e6\u003c/sup\u003eA mRNA modification can change transcript levels of genes either directly, e.g. by affecting mRNA stability of a specific gene, or indirectly by modulating transcripts of regulatory components/hubs that in turn alter transcription of a larger set of genes. We thus performed RNA-seq analysis on plants of all three genotypes at day 12 of progressive drought compared to well-watered control conditions. The sequencing yielded approximately\u0026thinsp;~\u0026thinsp;10\u0026nbsp;million raw reads per sample, with around 85% of the reads successfully aligned to the Arabidopsis reference genome TAIR 10 over all samples (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Principal Component Analysis (PCA) revealed a clear separation of samples based on treatment (PC1, 71.3% variance). The variance among genotypes was much smaller (PC2, 5.1% variance), but the WT was positioned between \u003cem\u003ealkhb10b-1\u003c/em\u003e and OX-1 under both conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This indicates that drought stress is the dominant factor shaping the transcriptomic landscape, while genotypic effects are more subtle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFirst, we determined the differentially expressed genes (DEGs) between drought and control conditions for each line using a cut-off of FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and │Log\u003csub\u003e2\u003c/sub\u003eFC│\u003cem\u003e\u0026ge;\u003c/em\u003e1 (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). This comparison revealed that \u003cem\u003ealkbh10b-1\u003c/em\u003e exhibited the highest number of DEGs (6205), followed by WT (5343) and OX-1 (4152) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Within each genotype the DEGs were comprised of a similar number of up- and down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and C).\u003c/p\u003e \u003cp\u003eWe also analysed transcript levels under drought of \u003cem\u003ealkhb10b-1\u003c/em\u003e and OX-1 relative to WT. Notably, only four DEGs were detected between WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e, and 34 between WT and OX-1 under drought conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Between \u003cem\u003ealkbh10b-1\u003c/em\u003e and WT these included two genes with higher and two with lower expression in the mutant, of which three were functionally uncharacterised and the fourth is \u003cem\u003eDERLIN-1\u003c/em\u003e, encoding a protein that is part of the ERAD / ER protein quality-control machinery. By contrast, between WT and OX-1all genes were higher expressed in OX-1 and mostly functionally annotated (Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Of these, 14 genes encode mitochondrial proteins, most of them associated with the mitochondrial electron transport chain and oxidative phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). These findings suggest that enhanced mitochondrial processes in OX-1 may provide an energetic advantage that helps to maintain cellular ATP homeostasis under drought, thereby contributing to its higher stress tolerance.\u003c/p\u003e \u003cp\u003eFor the DEGs (drought vs. control) among the three genotypes, we next calculated their relative expression change (ΔLog₂FC) in the functional variants compared to WT. By directly contrasting ΔLog₂FC for WT vs. \u003cem\u003ealkbh10b-1\u003c/em\u003e and WT vs. OX-1, and omitting those genes that showed an identical behaviour in OX-1 and \u003cem\u003ealkbh10b-1 (\u003c/em\u003eΔΔlog\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026lt;\u0026thinsp;1), we detected 3395 genes (ΔΔlog2FC\u0026thinsp;\u0026ge;\u0026thinsp;1) specifically differentially regulated in only one of the two mutant lines (Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). To understand the functional significance of these genes under drought stress, we performed GO enrichment analyses separately for three classes categorized as biological process, cellular component, and molecular function (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Remarkably, photosynthesis related processes emerged as the most strongly enriched term in all three classes such as light harvesting and photosystem I within biological processes, photosystem I\u0026thinsp;+\u0026thinsp;II and light harvesting within cellular components, and chlorophyll and tetrapyrrole binding within molecular functions. Other term within these classes relate to water transport and water channel activity but also responses to biotic and abiotic stress. KEGG analysis, which annotates genes at pathway level, revealed photosynthesis antenna proteins as the most significantly enriched pathway but also biosynthesis of glucosinolates and phenylpropanoids which are associated with stomata regulation and drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCluster analysis of the genes that were specifically regulated in only one of the lines (ΔΔLog\u003csub\u003e2\u003c/sub\u003eFC\u0026thinsp;\u0026ge;\u0026thinsp;1) identified four clusters with similar amounts of genes ranging from about 400 to about 650 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Cluster 1 and 2 were defined by genes that in the \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant were differently expressed from WT and OX-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, right panel), while cluster 3 and 4 contains genes where OX1 differed from WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, left panel). Cluster 1 comprised genes that were either up-regulated in \u003cem\u003ealkbh10b-1\u003c/em\u003e and unchanged in the two other lines or unchanged in \u003cem\u003ealkbh10b-1\u003c/em\u003e and down-regulated in the other two lines (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). Cluster 2 comprised genes that were either down-regulated in \u003cem\u003ealkbh10b-1\u003c/em\u003e and unchanged in the two other lines or unchanged in \u003cem\u003ealkbh10b-1\u003c/em\u003e and up-regulated in the other two lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Cluster 3 and 4 represent the same for OX-1. No counter-regulated genes were found in our analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of ALKBH10B preserves photosynthetic capacity under drought stress and modulates m\u003c/b\u003e \u003csup\u003e \u003cb\u003e6\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eA levels of photosynthesis-related genes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince photosynthesis emerged as the most prominent GO category among the DEGs, we had a closer look at the RNA-seq data of 11 photosynthesis-related genes with differential expression in \u003cem\u003ealkbh10b-1\u003c/em\u003e and OX-1. They all belong to cluster 3 and their expression levels remained stable in the OX-1 plants under drought stress, whereas they were consistently down-regulated in WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Supplementary Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). To validate the RNA-seq results, we quantified the transcript levels of \u003cem\u003eLHCB4.1\u003c/em\u003e, \u003cem\u003ePSBQ-2\u003c/em\u003e and \u003cem\u003ePSBO1\u003c/em\u003e using RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Their expression patterns matched those observed in the RNA-seq data, supporting the effect of ALKBH10B on a certain subset of photosynthesis-related genes. In line with the transcript analysis, WT and the \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant showed a significant reduction in chlorophyll levels and PSII activity under drought stress compared to control conditions, whereas OX-1 plants were much less affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). To elucidate whether ALKBH10B directly affects the m\u003csup\u003e6\u003c/sup\u003eA modification of these photosynthesis-related transcripts, we screened published m\u003csup\u003e6\u003c/sup\u003eA databases and identified potential m\u003csup\u003e6\u003c/sup\u003eA sites in \u003cem\u003eLHCB2.3\u003c/em\u003e, \u003cem\u003eLHCB4.1\u003c/em\u003e, \u003cem\u003ePSAD-2\u003c/em\u003e, \u003cem\u003ePNSL3\u003c/em\u003e, \u003cem\u003ePSBQ-2\u003c/em\u003e and \u003cem\u003ePSBO1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Quantification of m\u003csup\u003e6\u003c/sup\u003eA enrichment of these genes using m\u003csup\u003e6\u003c/sup\u003eA-IP-qPCR showed significantly decreased m\u003csup\u003e6\u003c/sup\u003eA levels only for \u003cem\u003ePSAD-2\u003c/em\u003e and \u003cem\u003ePSBQ-2\u003c/em\u003e in OX-1 plants compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Both genes had increased levels of m\u003csup\u003e6\u003c/sup\u003eA in the \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant in line with a stronger down-regulation in the expression (Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), indicating direct regulation by ALKBH10B-mediated demethylation. By contrast, the m\u003csup\u003e6\u003c/sup\u003eA levels of \u003cem\u003eLHCB2.3\u003c/em\u003e, \u003cem\u003eLHCB4.1\u003c/em\u003e, \u003cem\u003ePNSL3\u003c/em\u003e and \u003cem\u003ePSBO1\u003c/em\u003e remained largely unaffected, suggesting that they are not direct targets of ALKBH10B. These data indicate that rather than acting globally ALKBH10B selectively demethylates a subset of photosynthesis-related mRNAs, while other genes are indirectly affected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eALKBH10B modulates the expression and m\u003csup\u003e6\u003c/sup\u003eA enrichment of drought-responsive genes\u003c/h2\u003e \u003cp\u003eWe also examined the effect of ALKBH10B on the regulation of known m\u003csup\u003e6\u003c/sup\u003eA modified drought related transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Several of these genes can be classified as positive and negative drought regulators (Han et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and our RNA-seq analysis demonstrated that mostly the expression levels of these genes were not significantly different in all three plant lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). However, among the positive regulators the expression in OX-1 was sustained for \u003cem\u003eCOR15B\u003c/em\u003e, and \u003cem\u003eCIPK20\u003c/em\u003e or up-regulated for \u003cem\u003eVSP2\u003c/em\u003e under drought stress. By contrast, the expression of these genes in WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e was down-regulated for \u003cem\u003eCOR15B\u003c/em\u003e, and \u003cem\u003eCIPK20\u003c/em\u003e or sustained for \u003cem\u003eVSP2.\u003c/em\u003e Moreover, expression levels of the negative regulators \u003cem\u003eCYP707A1\u003c/em\u003e and \u003cem\u003eCML20\u003c/em\u003e were up-regulated in WT and \u003cem\u003ealkbh10b-\u003c/em\u003e1 but sustained in OX-1. With regard to m\u003csup\u003e6\u003c/sup\u003eA modification of their transcript, m\u003csup\u003e6\u003c/sup\u003eA levels of the positive regulators were either decreased in OX-1, increased in \u003cem\u003ealkbh10b-1\u003c/em\u003e or both (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) with similar patterns observed under control and drought conditions. The m\u003csup\u003e6\u003c/sup\u003eA levels of the two negative regulators \u003cem\u003eCYP707A1\u003c/em\u003e and \u003cem\u003eCML20\u003c/em\u003e remained unchanged in all lines, indicating that they are not directly regulated by ALKBH10B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, our integrative approach combining transcriptomics, m⁶A profiling, physiological assays, and hormone quantification reveals a multi-layered mechanism in which the m\u003csup\u003e6\u003c/sup\u003eA demethylase ALKBH10B promotes drought stress resilience in Arabidopsis.\u003c/p\u003e \u003cp\u003eWhile drought stress globally alters the expression of the whole m⁶A machinery in Arabidopsis, \u003cem\u003eALKBH10B\u003c/em\u003e stands out as the most strongly induced component (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is in line with an elevated \u003cem\u003eALKBH10B\u003c/em\u003e expression shown previously under drought for sea buckthorn (Zhang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and tomato (Shen et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), where the plant prioritizes active m⁶A demethylation as part of its adaptive strategy. Since m⁶A demethylases have been implicated in fine-tuning stress-responsive transcripts (Shoaib et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) this dynamic control likely enables the plant to rapidly modulate the stability and translation of stress-related mRNAs during water deficit.\u003c/p\u003e \u003cp\u003eCharacterization of plant lines with altered ALKBH10B content, revealed its role in modulating stomatal dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which is a critical factor governing water loss and drought resistance. Although differences in stomatal aperture between WT and OX-1 were moderate, the consistently reduced aperture in OX-1 seems sufficient to lower transpirational water loss during progressive drought periods. Despite the difference in stomatal apertures, Interestingly, ABA levels were similar between WT and OX-1 plants under control and drought conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), indicating that ABA abundance alone does not explain the observed stomatal phenotype. Accordingly, we also did not observe distinctive changes in the expression levels of ABA-dependent genes coding for proteins involved in stomata closure such as \u003cem\u003eTGG1\u003c/em\u003e, \u003cem\u003eSLAC1\u003c/em\u003e or \u003cem\u003eKAT1\u003c/em\u003e (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eReduced jasmonate accumulation in OX-1 may alter guard cell signaling dynamics and influence stomatal behavior under drought conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C). However, although jasmonates are known to cross-talk extensively with ABA signalling in guard cells, the experimental evidence for their precise role in stomatal regulation is mixed. Depending on the system and conditions, jasmonates have been reported to both potentiate and antagonize ABA-induced stomatal closure (Munemasa et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). At present and based on our data, we cannot establish a direct causal hierarchy between ABA levels, jasmonate accumulation, and stomatal behavior, we therefore interpret these interactions as part of a complex hormonal network. It is also unclear, how the reduced jasmonate content relates to ALKBH10B activity, since there is no clear correlation between jasmonate content and expression of genes related to jasmonate biosynthesis or catabolism (Supplementary Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). However, altered translational efficiency resulting from changes in m\u003csup\u003e6\u003c/sup\u003eA demethylation could influence protein abundance and cannot be excluded. Distinguishing between these direct and indirect mechanisms will require future integration of m⁶A epitranscriptomic profiling with targeted analyses of jasmonate pathway components including proteomic studies.\u003c/p\u003e \u003cp\u003eWhat has become evident lately is that jasmonates can negatively modulate ABA induced genes expression, as demonstrated for \u003cem\u003eRD29A/StRD29\u003c/em\u003e in Arabidopsis and potato via external applications of ABA and MeJA (Bleker et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This is likely driven by enhanced JA-Ile content to which MeJA is converted intracellularly (Tamogami et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Although the underlying molecular mechanism for this modulation of gene expression remains unresolved, we observed the same effect for \u003cem\u003eALKBH10B\u003c/em\u003e expression in this work (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, an elevated drought-induced expression of \u003cem\u003eALKBH10B\u003c/em\u003e and \u003cem\u003eRD29A\u003c/em\u003e occurs in the \u003cem\u003ejar1-11\u003c/em\u003e mutant, highlighting the negative regulatory role of endogenous JA-Ile (Mahmud et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We did not observe any induction of \u003cem\u003eALKBH10B\u003c/em\u003e expression by MeJA itself, which seems to contradict results from Tang et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) showing that MeJA up-regulates \u003cem\u003eALKBH10B\u003c/em\u003e expression in seven-day-old seedlings. However, this apparent discrepancy with our findings can likely be attributed to differences in the developmental stage of the plants given that m\u003csup\u003e6\u003c/sup\u003eA dynamics vary across developmental stages (Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), ALKBH10B may participate in distinct jasmonate-responsive regulatory circuits during early seedling development versus prolonged drought stress in mature plants.\u003c/p\u003e \u003cp\u003ePromoter analysis indicates that transcriptional regulation of ALKBH10B by ABA and jasmonate might be directly promoted via ABRE, MYB and MYC cis-elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).The promoter of \u003cem\u003eALKBH10B\u003c/em\u003e also contains an AS-1 cis-regulatory element related to salicylic acid (SA), however, the expression of ALKBH10B was not notably affected by exogenous SA application in line with a lack of SA regulation shown for ALKBH10B in tomato leaves (Shen et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Also, no significant differences in SA content were observed among all the genotypes under either drought and control growth conditions (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), indicating that under our experimental conditions, i.e., age and tissue, ALKBH10B function is unlikely to be mediated through SA signalling.\u003c/p\u003e \u003cp\u003eOur physiological analyses suggest a likely role of m\u003csup\u003e6\u003c/sup\u003eA removal by ALKBH10B in hormone-dependent regulation of transpiration. However, our transcriptomic analysis reveals that ALKBH10B exerts effects beyond hormonal crosstalk. It is established that mitochondrial respiration plays a vital role in drought tolerance (Atkin and Macherel \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and our results show that OX-1 plants activate a transcriptional program that enhances expression of multiple genes related to mitochondrial respiration and energy metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This indicates an influence of m\u003csup\u003e6\u003c/sup\u003eA mRNA modification on this process. This is so far not investigated in plants, but m⁶A mRNA modification has recently been shown to regulate transcripts of genes coding for mitochondrial proteins in animal systems (Kahl et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While this indicates an evolutionary conservation beyond the plant-animal divide, it gains another layer of complexity in plants due to the mitochondrial-chloroplast interplay in plant energy metabolism. Indeed, our transcriptional analysis showed that in the OX-1 plants certain photosynthesis-related genes remain stably expressed and PSII performance is maintained under drought, in contrast to the WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Similar findings have been observed for the ALTERNATIVE OXIDASE (AOX), where over-expression caused drought tolerance by maintaining not only mitochondrial but also chloroplast functions (Dahal and Vanlerberghe \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In a recent study, it has been revealed that the m\u003csup\u003e6\u003c/sup\u003eA writer VIR regulates m\u003csup\u003e6\u003c/sup\u003eA modification of numerous photosynthesis-related transcripts during high light-induced photodamage, thereby regulating their gene expression at post-transcriptional level (Zhang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, in our study, only the stable expression of \u003cem\u003ePSAD2\u003c/em\u003e and \u003cem\u003ePSBQ-2\u003c/em\u003e can be linked to direct modulation of m\u003csup\u003e6\u003c/sup\u003eA mRNA methylation by ALKBH10B, while other photosynthesis-related transcripts likely reflect indirect regulatory effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These effects may, for example, arise from demethylation of transcripts encoding transcriptional regulators, which subsequently modulate broader photosynthesis-related gene networks. m\u003csup\u003e6\u003c/sup\u003eA modification has been observed for various transcription factors (Zhang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and we observed differential expression between WT and the function mutants for members of different transcription factor families (Supplementary Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). This distinction between direct m\u003csup\u003e6\u003c/sup\u003eA-mediated regulation and secondary transcriptional reprogramming is critical for interpreting epitranscriptomic datasets.\u003c/p\u003e \u003cp\u003eALKBH10B also regulates expression and m\u003csup\u003e6\u003c/sup\u003eA levels of certain drought-responsive transcripts such as the kinase encoded by \u003cem\u003eCIPK20\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), which was shown to mediate stomata closure through regulation of microtubule stability (Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Sustained expression of \u003cem\u003eCIPK20\u003c/em\u003e in OX-1 may contribute to reduced stomatal aperture compared to WT and \u003cem\u003ealkbh10b-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The function of these transcripts as well as their differential regulation and m\u003csup\u003e6\u003c/sup\u003eA mRNA modification by ALKBH10B are thus easily linked to the drought phenotype of the OX-1 and \u003cem\u003ealkbh10b-1\u003c/em\u003e mutant lines. Nevertheless, as with the photosynthesis-related genes, not all expression changes correlate directly with m6A enrichment, reinforcing the likelihood indirect downstream effects, such as post-transcriptional regulation, secondary signalling pathways, or downstream consequences of improved photosynthetic performance in OX-1 plants.\u003c/p\u003e \u003cp\u003eInterestingly, it was shown very recently that the m\u003csup\u003e6\u003c/sup\u003eA writer, MTA, also imparts drought tolerance through m\u003csup\u003e6\u003c/sup\u003eA-dependent and -independent impacts on mRNA regulation (Ganguly et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Similar to VIR mentioned above, MTA appears to act more globally than ALKBH10B. Moreover, whereas VIR primarily influences photosynthesis-related genes and MTA affects drought-responsive transcripts, ALKBH10B impacts both processes as well as mitochondrial respiration. Importantly, the limited overlap between m\u003csup\u003e6\u003c/sup\u003eA-modified transcripts and differentially expressed genes underscores the importance of distinguishing direct epitranscriptomic regulation from from secondary transcriptional reprogramming driven by altered m\u003csup\u003e6\u003c/sup\u003eA modification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by Deutscher Akademischer Austauschdienst (DAAD) for providing funding to YS (Grant agreement 57588370). The publication cost was ensured by University of Bonn Projekt DEAL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u0026nbsp;\u003c/strong\u003eYS contributed to conceptualization, investigation (responsible for most experimental work), formal analysis, validation, visualization, and writing - original draft as well as review \u0026amp; editing. RH and HK contributed to investigation (m\u003csup\u003e6\u003c/sup\u003eA IP-qPCR) and writing \u0026ndash; review and editing. SB contributed to RNA-seq analysis (bioinformatics). AF contributed to investigation (molecular cloning). KG and PD contributed to investigation (hormonal analysis), and writing \u0026ndash; review and editing. UCV contributed to conceptualization, validation, project administration, supervision, and writing - review and editing. FC contributed to conceptualization, validation, supervision, and writing - original draft as well as review and editing. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe would like to thank\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eProf. Wan Hsing Cheng, (University of Taiwan) for providing the \u003cem\u003eaba2-1\u003c/em\u003e line and the NGS Core Facility of the Medical Faculty at the University of Bonn for providing support and instrumentation funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors hereby declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eRaw RNA seq data used in this study are available under (PRJNA1357100) at SRA repository from NCBI.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAimar D, Calafat M, Andrade A, Carassay L, Abdala G, Molas M (2011) Drought tolerance and stress hormones: From model organisms to forage crops. 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Plant physiology 171 (4):2810-2825\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"abiotic stress, phytohormones, transcriptomics, epitranscriptomics (m6A), post-transcriptional modification","lastPublishedDoi":"10.21203/rs.3.rs-8969427/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8969427/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpitranscriptomic N⁶-methyladenosine (m⁶A) mRNA modification has emerged as an important regulatory layer of gene expression and protein synthesis in plant stress adaptation. In this study, expression profiling revealed that drought stress induces a global reprogramming of the m\u003csup\u003e6\u003c/sup\u003eA machinery in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, with the m\u003csup\u003e6\u003c/sup\u003eA demethylase \u003cem\u003eALKBH10B\u003c/em\u003e being the most strongly up-regulated component. \u003cem\u003eALKBH10B\u003c/em\u003e transcription is induced by ABA, most likely via ABRE and MYB cis-elements, whereas jasmonate alleviates this activation. Moreover, \u003cem\u003eALKBH10B\u003c/em\u003e overexpressing plants (OX-1) accumulated wild type (WT) levels of ABA under drought stress but displayed a markedly reduced content in jasmonic acid (JA) and jasmonoyl-isoleucine (JA-Ile). They furthermore exhibit enhanced stomatal closure, improved water-use efficiency, and thus an enhanced drought tolerance. Transcriptome analysis revealed that drought stress induces fewer transcriptional changes in OX-1 plants compared to WT and the drought-sensitive \u003cem\u003ealkbh10b-1 \u003c/em\u003emutant. Differential expression and GO enrichment analyses highlight that photosynthesis-related processes were most affected by ALKBH10B activity, correlating with a preserved chlorophyll content and PSII efficiency in OX-1. Analysis by m⁶A-IP-qPCR showed that ALKBH10B directly demethylates specific photosynthesis-related and drought-responsive genes, thus promoting their sustained expression during stress. Other transcripts are indirectly regulated by ALKBH10B-dependent processes. Moreover, transcripts associated with respiration were enriched in OX‑1, indicating that ALKBH10B also supports mitochondrial energy metabolism during drought. Together, our results suggest that ALKBH10B enhances drought tolerance by coordinating m⁶A-dependent post-transcriptional regulation to maintain photosynthetic capacity and mitochondrial energy metabolism, as well as fine-tuning ABA-jasmonate crosstalk.\u003c/p\u003e","manuscriptTitle":"ALKBH10B mediated epitranscriptomic regulation enhances drought tolerance of Arabidopsis thaliana via hormonal cross-talk and sustained photosynthesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 08:55:39","doi":"10.21203/rs.3.rs-8969427/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-25T02:43:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-19T06:32:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T18:16:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74718635954163993657901387736145750245","date":"2026-03-12T02:21:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28417660852731465516986266855739596824","date":"2026-03-02T04:15:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-02T03:15:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-26T14:04:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-26T13:59:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-02-25T15:32:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a9c8c847-ba77-4b8d-9b33-f7731a59e2c5","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T01:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 08:55:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8969427","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8969427","identity":"rs-8969427","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}