Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response

Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response | 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 Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response Yueh Cho This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4267287/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 Background Plant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gβ subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling’s survival under high salinity remains unclear. Results We showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the agb1 mutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of agb1 and bzip17 and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in s1p s2p double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings. Conclusions Our results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression. Heterotrimeric G protein AGB1 salinity bZIP17 unfolded protein response (UPR) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Like every one of us, plants are facing an ever-changing environment day by day. Adjusting the armory to cope with divergent stresses is essential to equip seedlings with proper transcriptome for their fitness (1). Integrating external stimuli into plant cells depends on hormones and heterotrimeric G proteins to deliver messages and initiate proper cellular responses for better survival (2). The heterotrimeric guanine nucleotide-binding protein (G protein), including G⍺, Gβ, and Gγ subunits, serves as a signal mediator coupling with the plasma membrane-spanning G-protein-coupled receptors (GPCR) and effectors (3). The human genome contains 23 G⍺ genes, 5 Gβ genes and 11 Gγ genes (4). By contrast, Arabidopsis genome contains one canonical G PROTEIN ALPHA SUBUNIT 1 (GPA1) (5) and three non-canonical G⍺ subunits EXTRA-LARGE G-PROTEIN 1 (XLG1) (6), XLG2 and XLG3 (4), one Gβ subunit GTP BINDING PROTEIN BETA 1 (AGB1) (7) and three Gγ subunits G-PROTEIN GAMMA-SUBUNIT 1 (AGG1) (8), AGG2 (9), AGG3 (10). Previous studies have shown that heterotrimeric G protein subunits play vital roles in responses to developmental cues and environmental stresses, including salt stress (11). Soil salinity is one of the major threats to food security by seriously attenuating plant growth and decreasing crop yield (12). Excessive salt in the soil causes numerous negative effects on different plant developmental stages, including germination, vegetative growth, and flowering (13). These negative effects damage plant cells due to ion toxicity and increasing osmotic stress (14, 15). Accordingly, plants have employed various mechanisms for survival in harsh salinity environments. Three signaling pathways constitute the major transduction during salt stress: calcium-dependent signaling pathway that (1) triggers the activation of stress-responsive genes as dehydration-responsive or late embryogenesis abundant (LEA) proteins (16) and (2) salt overly sensitive (SOS) pathway for regulation of ion homeostasis (17), and (3) osmotic stress signaling pathway involving ABA-dependent induction of downstream salt responsive genes through activation of a group of transcription factors like bZIP, NAC, MYB and ABRE families (18). However, our understanding of the whole network of pathways regulating salinity response is far from complete. Recent research based on studies of Arabidopsis AGB1 has suggested a functional link between plant G protein signaling and regulation of the salt stress response. A knockout mutant of AGB1 exhibited more sensitivity to high salinity than wild-type plants (10, 11, 19-21). Under excessive salt, the agb1 mutant accumulates more Na + , translocate more Na + from root to shoot, and has a high transpiration rate with larger stomatal apertures (22, 23); AGB1 is also coupled with AGG1 or AGG2 to regulate stomatal apertures and transpiration (24). Meanwhile, a receptor-like kinase, FERONIA (FER), has been identified by directed interaction with AGB1. FER is required for cell wall integrity, Ca2 + induction, ROS production, and stomata movement under salinity conditions (25) Most G protein signaling studies have focused on canonical effectors localized to the plasma membrane. Recent studies have shown that the G-protein β subunit functions in the nucleus (23, 26). The Arabidopsis AGB1 is localized in the nucleus, where it interacts with B-BOX DOMAIN PROTEIN 21 (BBX21) for hypocotyl elongation (27), with BRI1-EMS-SUPPRESSOR 1 (BES1) for cell division (28), with PHYTOCHROME B (phyB) - PHYTOCHROME INTERACTING FACTOR 3 (PIF3) (29) or CRYPTOCHROME 1 (CRY1) - ELONGATED HYPOCOTYL 5 (HY5) for photomorphogenesis (30) and with MAP KINASE 6 (MPK6) for drought tolerance (31). Nonetheless, gaining evidence showed that WD domain-containing proteins, including AGB1, directly interacted with bZIP transcription factors like HY5 and VIRE2-INTERACTING PROTEIN 1 (VIP1) to regulate gene expressions (30, 31). The basic region leucine zippers transcription (bZIP) family in Arabidopsis comprised 78 members and assorted into 13 groups (32). bZIPs form as dimers to bind DNA sequences, and heterodimerization results in appreciable regulatory flexibility (33). Among these bZIP transcription factors, three members of group B (bZIP17, bZIP28, bZIP49) perform as important regulators of the evolutionally conserved ER stress response (34), the intrafamily dimerization of group B has been confirmed in yeast cells (35). In particular, under adverse environmental conditions, including high salinity, bZIP17 is reported to relocate from ER to nucleus processing by through regulated intramembrane proteolysis (36). Salt-responsive genes are reported to express in a bZIP17-dependent manner (36, 37). In this study, we observed the nuclei localization and the contributions of AGB1 to induce the expression of bZIP17-mediated salinity-responsive genes. The spliced bZIP17 localization toward nuclei through S1P/S2P-mediated proteolysis was reduced without AGB1. The arrangement of AGB1 pools among different subcellular compartments to involve proper programs to respond to high salinity is crucial for young seedling viability. RESULTS Functional complementation of salinity hypersensitivity in agb1 mutant In this study, the AGB1 transcript was significantly increased under high salt stress after 4 hours, as described previously (Fig. S2A; (19)). The agb1-3 mutant is a null mutant of AGB1 (as agb1 , Fig. S1A, S1B (38)). It shows hypersensitive responses, including poor germination, growth defects, and albino leaves when grown under salt stress (Fig. 1). We produced the genetic complementation lines harboring genomic sequence of AGB1 as previously described ( agb1 pAGB1:AGB1, as AGB1; (38)) with C-terminally fused the triple Venus fluorescent protein in the agb1 mutant ( agb1 pAGB1:AGB1-Venus , as AGB1-V; Fig. S1A, S1B and S1C). To test whether AGB1-V transgene is functional in vivo , we observed shoot, primary root, and lateral roots. The AGB1 and AGB1-V transgenic plants rescued the developmental defects in agb1 mutant (Fig. 1, S1D, S1E, and S1F). We then performed the sequential salt stress tolerance assay on wild-type (WT), agb1 mutant, and AGB1-V plants for 14 days after seeds sowing on ½ MS medium (Fig. 1 and S2). After the salt treatment, AGB1 was induced in 7-d WT seedlings (Fig. S2A). The leaf bleaching phenotype and growth retardation in agb1 mutant were fully rescued in the AGB1-V complementation lines (Fig. 1A, 1C, 1D, S2B, S2C), which were either counted for the albino rate (Fig. 1D) or classified into three groups according to the seedling size and chlorotic phenotypes as Green, Mix (contained at least one white leaf) and White (Fig. S2B, S2C). In the mock treatment (0 mM NaCl), all WT, agb1, and AGB1-V seedlings grew normally and showed green for their aerial part. In contrast, in the salt stress treatment (150 mM NaCl), more agb1 seedlings were categorized significantly into the white group due to the chlorotic phenotype compared to the WT and AGB1-V seedlings (Fig. 1D, S2B, S2C). Since no statistical difference was found between WT and AGB1-V seedlings, we suggested that the AGB1-V was functional and complemented the agb1 mutant in vivo (Fig. 1, S1, and S2) . Subcellular localization of AGB1 at the nuclei in response to salt stress AGB1 is known as a molecular switch through protein-protein interactions to regulate transcriptional programs for plant development and abiotic stress responses, like inhibiting BBX21 activation (27) or binding with PIF3 in the nucleus to promote hypocotyl elongation (29), dephosphorylating BES1 for its nuclei entry and downstream gene expression (28), which prompted us to investigate whether AGB1 localization into nuclei to involve transcriptional regulation under high salinity. The 7-day-old transgenic AGB1-V seedlings were used for confocal laser scanning microscopy to observe any changes in subcellular localization of AGB1-Venus that occurred during short-term NaCl treatment. The time series of salt treatment continuously tracked AGB1-Venus’s localization in the root epidermis (Fig. 2A). Most of AGB1-Venus showed network-like localization under normal conditions, which prompted us to consider the possible ER localization of AGB1 as a previous study described ( ProAGB1:CFP-AGB1 (39); Pro35S: AGB1-GFP (26)). In contrast, many AGB1-Venus expressions showed nuclei-like patterns in the AGB1-V root under 1, 2, and 4 h of salt stress (Fig. 2A) as shown in enlarged images (Fig. 2A’). This nuclei localization of AGB1-V can be seen in other tissues like hypocotyl (middle) and also cotyledon (right) in a spatiotemporal manner (Fig. S3A and S3B); the dot-like are confirmed by DAPI staining to mark nuclei (4-h NaCl treatment, red in Fig. S3A). The nuclei-localized AGB1-V was rarely seen in the tissue with 0-h NaCl treatment, found in the tissue with 1- to 16-hours NaCl treatment as a solid triangle indicated in Fig. S3A. To quantify the nuclei-localized AGB1-V, we grouped the ER-like and PM-like-localized AGB1-V cells (black group) and counted the ratio of the different localized AGB1-V in the tissue that has been examined (Fig. S3B). The nuclei-localized AGB1-V cells (white group) were significantly found in all NaCl-treated tissue with various lengths of time for salt stress induction (Fig. S3B). We also stained 7-day-old AGB1-V seedlings with plasma membrane (PM) marker (FM4-64, Fig. 2B, Left) and ER marker (ER-tracker, Fig. 2B, middle), nuclei marker (DAPI, Fig. 2C, right). The AGB1-Venus signals were partially colocalized with organelle and nuclear markers in the presence of salt stress after 4 hours. The FM4-64 patterns matched cell boundaries, ER tracker marked the perinuclear network, and DAPI filled the central pot inside of the ER (Fig.2). Next, we performed the fractionation assay to isolate nuclei from 7-day-old AGB1-V seedlings with 0- or 4-h NaCl treatment, and the immunoblots of ACTIN8 and Histone H3 served as cytosolic and nuclear marker, respectively. The AGB1-Venus proteins were quantified with CBB staining and p35S: Venus acted as a negative control for Venus in nuclei fraction. The proportion of AGB1-Venus increased around 2-fold in nuclei fraction under salt stress (0.73 to 1.47), but there was no obvious change for AGB1-Venus in total and cytosolic fractions. The above results indicate that AGB1-Venus is widely distributed to PM and ER under normal conditions, which is increasingly localized to nuclei and likely reduced ER localization of AGB1-Venus in response to salt stress (Fig. 2). Repression of transcription factor bZIP17-mediated salt stress response in agb1 mutant We wonder whether AGB1 played a role in transcriptional regulation in response to salt stress since gaining portions of AGB1-Venus to nuclei was observed in young seedlings; if so, what would be the corresponding gene sets to develop protection against excessive salt? We therefore performed microarray analyses using 7-day-old WT and agb1 whole seedlings (Fig. S4A) or root-only samples (Fig. 3 and S4B-E) with 0 or 4 hours of 150 mM NaCl treatment. Compared to whole seedlings (Fig. S4A), the salt stress-induced fold changes are more obvious in agb1 versus wild-type roots (R 2 = 0.1882, Fig. 3A). Interestingly, the induction of a set of salt stress-responsive genes in a bZIP17-dependent manner (36) was repressed in agb1 seedlings (Fig. S4A) and root-only (as mark in purple, Fig. 3A) microarray analysis. The salt stress induction of 13 out of 22 bZIP17-regulated genes ((36)) was significantly reduced in the agb1 mutant than in WT roots (Fig. 3A). These gene expressions failed to induce in s1p-3 and bzip17 mutant with 4-h NaCl treatment and were evaluated by RT-qPCR (36, 37). To examine whether AGB1 exercised control over these genes in roots during salt stress, their expression was assessed in agb1 or bzip17 mutants using quantitative RT-PCR (Fig. 3B-D). The seedlings exposure to 150 mM of NaCl for 4 hours elevated the expression of these genes like NAC DOMAIN CONTAINING PROTEIN 19 ( NAC019 , Fig. 3B), RESPONSIVE TO DESICCATION 20 ( RD20 , Fig. 3C), and OUTER MEMBRANE TRYPTOPHAN-RICH SENSORY PROTEIN-RELATED ( TSPO , Fig. 3D) in WT, but much less in agb1 mutant. These findings are consistent with a model that under abiotic stresses or ABA treatment, S1P and/or S2P initiate the proteolytic activation of bZIP17, which directly upregulates genes like NAC019 (40) . To explore whether the downregulated gene network in agb1 roots resulted from a group of transcription factors (TFs) regulated by AGB1, we performed differential expression gene analysis for agb1 root microarray (Fig. S4B). Indeed, 464 genes were down-regulated in agb1 compared to WT under high salinity, including 18 TF genes. We then generated a Venn diagram to cover identified TFs in this study (Fig. S4C) with NaCl-induced genes in Arabidopsis roots (41) and AGB1 interactome based on the BioGrid database (https://thebiogrid.org). Two TFs MYB15 and NAC069 intersected to serve as potential candidates for AGB1 to regulate expressions of salt stress-responsive gene, which used RT-qPCR further to examine salinity induction in WT and agb1 seedlings (Fig. S4E). Both MYB15 and NAC069 reduced expressions in agb1 after 4-h NaCl treatment. The bZIP17 depletion genetically inhibits the agb1 phenotype under salt stress The bZIP17 and AGB1 are signaling modulators involved in various pathways, including the unfolded protein response and salt stress response. Both agb1-2 and bzip17 mutants exhibit hypersensitivity to salt stress (11, 36). To investigate the genetic interactions between AGB1 and bZIP17, we isolated bzip17-4 (as bzip17, (42)) and generated bzip17 pbZIP17:bZIP17 (Fig. S5) and bzip17 pbZIP17: mRFP-bZIP17 lines (Fig. S6A), which displayed a complementary phenotype, rescuing the bzip17 hypersensitive defect to salt stress (Fig. S5 and S6B-E). Crosses between bzip17 and agb1 yielded an agb1 bzip17 double mutant, confirmed via RT-PCR (Fig. 4A). Under mock conditions, agb1 bzip17 phenocopied the agb1 mutant, displaying lower germination rate, higher biomass, longer primary roots, and increased plant width (Fig. 4B-D). However, under high salinity, both agb1 and agb1 bzip17 mutants exhibited salt hypersensitivity, with smaller seedlings and shorter roots, suggesting that agb1 is epistatic to bzip17 (Fig. 4B-D). Furthermore, agb1 displayed a more severe chlorotic phenotype compared to bzip17 under the same growth conditions (Fig. 4C). Treatment of an agb1 bzip17 double mutant with different NaCl concentrations revealed higher survival rates compared to the agb1 single mutant, indicating significant interaction effects between AGB1 and bZIP17 under salt stress. This suggests an antagonistic interaction between AGB1 and bZIP17. The bZIP17 and AGB1 physical interaction in vivo The nuclear localization of AGB1 and its involvement in initiating bZIP17-dependent salinity stress response, but whether AGB1 physically interacts with bZIP17 is unknown. Under high salinity, the ER-localized bZIP17 was transferred to the Golgi apparatus, generating a spliced bZIP17 to move into the nuclei (Fig. 5C and S6F) and trigger a set of gene expressions, while the AGB1 was located in the plasma membrane, ER, and nuclei (Fig. 2, 5A, (36)). We first test whether the nuclei localization of AGB1 required bZIP17 presence in young seedlings; the AGB1-V showed a nuclei-like pattern under salt stress in agb1 and agb1 bzip17 genetic grounds, suggesting that the subcellular localization of AGB1 did not affect by the presence of bZIP17 (Fig. 5B). We next tested whether AGB1 interacts directly with bZIP17, and BiFC assays were performed in Arabidopsis protoplasts utilizing a pUBN/C BiFC system (43) (Fig. 5D, 5E). The coding sequences of AGB1 and AGG1 were cloned into pUBC vectors to act as positive pairs, and bZIP17 was cloned into pUBN vectors with translational fused with split YFP tag protein, respectively. No signal was observed in the negative control when the empty vector was paired with AGB1, AGG1, or bZIP17 BiFC vector. In contrast, a clear YFP signal (green) at the cytosolic region in samples expressing bZIP17 or AGG1 with AGB1 indicated interaction (Fig. 5D, 5E). This data suggests a physiological interaction between AGB1 and bZIP17 in the protoplast system. The repression of proteolytic processing of bZIP17 in agb1 mutant To test whether AGB1 functions in the S1P-S2P pathway to regulate proteolytic processing of bZIP17 for gene induction under salt stress, we first transiently expressed the biological functional mRFP-bZIP17 (Fig. S6) in 7-day-old WT, agb1 , or s1p-1 s2p-1 ( s1p s2p ) seedlings by AGROBEST (44). In the meristemic zone of the WT root epidermis, the dot-like mRFP-bZIP17 (as indicated by the white triangle) was observed in salt-treated plants but rarely seen in s1p s2p or agb1 mutants. We also quantified the cells expressing nuclei-localized mRFP-bZIP17, the WT roots were significantly higher ( P = 0.013) under salt stress (15.5 ± 7.7 %) than under mock condition (1.0 ± 2.8 %), whereas the nuclei-localized mRFP-bZIP17 in agb1 or s1p s2p roots were not different ( agb1: P = 0.691; s1p s2p : P = 0.486) between stressed ( agb1 : 0.0 ± 7.3 % ; s1p s2p: 3.1 ± 15.2 %) and control ( agb1 : 0.0 ± 3.8 %; s1p s2p: 4.8 ± 3.0 %) conditions (Fig. 6B). DISCUSSION This study took salinity-stressed young seedlings to unveil a long-standing question of the functional aspect of the role of AGB1 in transcriptional regulation for plant survival and fitness. Whether the mechanical inductions of a set of salt stress-responsive genes correlated to AGB1 remains unknown. We uncovered the salinity-responding nuclear localization of AGB1 among multiple tissues of young seedlings (Fig. 2). In the absence of AGB1 , gene inductions of salt-responsive transcription factors, antioxidants, and osmoticants were compensated as bzip17 mutant, root (Fig. 3), which may contribute by the physical interaction between AGB1 and bZIP17 (Fig. 5). At the molecular level, the S1P/S2P-mediated bZIP17 proteolytic processing was abolished in agb1 mutant to decrease the nuclear localization of bZIP17 under high salinity (Fig. 6A and 6B). At the genetic level, the agb1 is epistatic to bzip17 as the agb1 bzip17 double mutant phenocopying agb1 but not bzip17 (Fig. 4) . Therefore, our mechanistic study elucidated that AGB1 coordinated transcriptional salinity response to regulate bZIP17 processing and mediating the signaling pathway (Fig. 6C). AGB1 assumes a pivotal role in responding to salt stress. The G protein interactome study identified a gene ontology term linked to the salt stress response (11). Additionally, microarray analysis employing the agb1 null mutant showcased heightened sensitivity to salinity (19). Phenotypic characterization of chlorotic agb1 plants revealed reductions in chlorophyll content, fresh weight, survival rate, and stomatal aperture size (19, 21). Furthermore, AGB1 depletion was associated with increased shoot ABA content and Na+ accumulation (Yu and Assmann, 2015; 2016). Subsequent research elucidated AGB1's role in maintaining ion homeostasis via interactions with FER and RALF1, regulating K+ and ROS levels (25). AGB1 contributes to ER stress responses, aiding in maintaining ER homeostasis under adverse conditions such as unfolded protein accumulation or environmental stressors, and interacts with ER-localized proteins involved in various cellular processes, including calcium signaling and lipid metabolism, such as RALF (25). The ER lumen, calcium pool, and ER membrane integrity conditions may influence the relocation of bZIP17 from the ER toward the Golgi apparatus. Moreover, AGB1 localizes to the Golgi apparatus in both mammalian cells (45) and leaf epidermal cells (21). The transient expression of mRFP-bZIP17 driven by its promoter in agb1 seedlings mimics the deficiency in bZIP17 relocation to the nucleus observed in s1p s2p mutants, suggesting that the S1P/S2P-mediated proteolytic processing may compensate in agb1. However, whether AGB1 directly affects protease activity or physically interacts with bZIP17 to isolate substrates from enzymes remains unknown. Transcriptome analysis of agb1 mutants subjected to short-term salt stress revealed heightened sensitivity to elevated salinity, suggesting a crucial role for AGB1 in orchestrating transcriptional responses to equip seedlings with the necessary defenses for survival ((19) and the present study). Transcription factors such as bZIP17, NAC069, and MYB15 either interact with AGB1 or have their expression regulated by AGB1, making them promising candidates for further investigation into AGB1's involvement in gene expression regulation. Furthermore, during photomorphogenesis, CRY1 inhibits AGB1 to release the transcription factor HY5 (bZIP56), enabling its DNA-binding activity and promoting gene expression for hypocotyl growth (30). AGB1 interacts with bZIP51 (VIP1) to function under drought-stress conditions (31). Limitations of this study The study presents valuable insights into the role of AGB1 in salt stress responses, yet several limitations constrain it. Firstly, the lack of mechanistic understanding hinders a comprehensive elucidation of molecular pathways, such as the influence of AGB1 on S1P/S2P-mediated proteolytic cleavage of bZIP17, bZIP17 intracellular trafficking and its binding affinity to cis-elements of salt stress-responsive genes. Furthermore, focusing on a limited number of time points within a short duration may only partially capture the dynamic nature of salt stress responses over longer periods, impacting the assessment of long-term fitness. Additionally, the transcriptional analysis is confined to a subset of genes regulated by bZIP17, potentially overlooking other target genes and pathways modulated by AGB1. While BiFC assays suggest a physical interaction between AGB1 and bZIP17, further validation in planta is warranted. Lastly, the study's exclusive focus on Arabidopsis thaliana may restrict the generalizability of the findings to other crop species, underscoring the necessity for comparative studies across diverse plant species to ascertain the broader relevance of AGB1-mediated salt stress responses. Addressing these limitations through future experimental investigations will be crucial for comprehensively understanding AGB1's role in plant salt stress adaptation. MATERIALS AND METHODS Plant materials and growth conditions Arabidopsis thaliana Columbia-0 (Col-0) ecotype was used in this study. T-DNA-tagged mutants of agb1-3 (SALK_061896 as agb1 ), bzip17-4 (GABI_220B01 as bzip17 (42)), s1p-1 (SALK_020530 as s1p ), s2p-1 (GABI_459C12 as s2p ) were obtained from Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu) and Center for Biotechnology (http://www.GABI-Kat.de). Seed surfaces were sterilized with 70% (v/v) ethanol and washed thrice with autoclaved ultrapure water. After one day of stratification at 4 °C, seeds were planted directly onto Petri dishes containing half-strength Murashige and Skoog (½ MS, Duchefa Biochemie, Haarlem, Nederland, M0222) medium with 0.86 % (w/v) sucrose (107687; Merck, Darmstadt, Germany) and 0.6% (w/v) agar (214010; Difco, BD, MJ, USA) at pH 5.6 (46). Plants were grown under 16 h / 8 h light-dark cycle at 22 °C with light intensity of 150 µmol m -1 s -1 . Homozygous plants were isolated by PCR-based genotyping using gene-specific primers and T-DNA specific primers. The primers used were for agb1 (KK70/KK71 and KK8/KK71), and bzip17 (KK537/KK538 and YN1016/KK538). See supplemental Table 1 for the oligonucleotide sequences used in this study. Salt stress treatment, germination, and phenotypic analysis For salt stress induction, 7-day-old seedlings were immersed in liquid MS media containing 150 mM NaCl (1.06404, Merck) for the indicated time. For long-term salt stress tolerance assay, seeds were planted on the ½ MS agar plate containing NaCl as indicated concentration for 14 days. Germination rate, seedling fresh weight (FW), primary root length, and plant width (length of two cotyledons) were measured with 11 to 25 seedlings with at least three biological replicates to quantify morphological phenotypes. The salinity tolerance analysis was performed by classifying plants according to leaf color: green, mixed (at least one white leaf), and white (all albino leaves). Vector construction and plant transformation To clone the split YFP variants for bimolecular fluorescence complementation (BiFC) assay, the coding sequences without stop codon of AGB1, bZIP17, and AGG1 were cloned into pENTR as pYC70, pYC, and pYC69, respectively. For the BiFC assay, above-mentioned entry vectors were recombined into a pUBC-nYFP or pUBC-cYFP to obtain pYC73 (pUBC-AGB1-nYFP), pYC74 (pUBC-AGB1-cCFP), pYC78 (pUBN-bZIP17-nYFP), pYC79 (pUBN-ZIP17-cYFP), pYC71 (pUBC-AGG1-nYFP), pYC72 (pUBC-AGG1-cYFP). agb1 pAGB1:AGB1 (AGB1) Same T3 line #5-2 was reported in (38). agb1 pAGB1:AGB1-Venus (AGB1-V) To generate an AGB1-V complementation line, a DNA fragment encoding triple repeats of the Venus (V) fluorescent reporter was inserted in-frame before the stop codon of the AGB1 genomic region at the Sfo I site into pCC87 ( pENTR-pAGB1:AGB1-Sfo I) to express AGB1-Ven. The obtained pCC91 ( pENTR-pAGB1:AGB1-Venus ) was then recombined into a pBGW destination vector (47) using Gateway TM LR Clonase TM II (Invitrogen, Thermo Fisher Scientific, MA, USA) to obtain plasmid pCC95 ( pBGW-pAGB1:AGB1-Venus ). pCC95 was transduced into agb1 via Agrobacterium tumefaciens strain GV3101 mediated transformation. Forty-eight T1 plants were selected on soil by spraying with 0.1% BASTA solution. The obtained T2 seeds were screened using BASTA and selected by genotyping. To distinguish transgenic pAGB1:AGB1-Venus from endogenous AGB1 , specific primers (KK212/KK104) were designed. Line 6 was used for observation. bzip17 pbZIP17:bZIP17 (bZIP17) PCR amplified the 5.8 kb of genomic sequence for bZIP17 ( pbZIP17:bZIP17 ) with primers LC125 and LC126. The fragment was cloned into the pENTR TM /D_TOPO TM plasmid vector (Invitrogen) to obtain pENTR-ProbZIP17:bZIP17, which was then recombined into the pBGW destination vector (Karimi et al , 2005) with the use of Gateway TM LR Clonase TM II (Invitrogen) to obtain pLC25 (pBGW-ProbZIP17:bZIP17). pLC25 was transduced into bzip17 via A. tumefaciens strain GV3101 mediated transformation. In total, 24 T1 plants were selected on soil by spraying with 0.1% BASTA solution. The obtained T2 seeds were screened using BASTA and selected by genotyping. Lines 3, 5, 7 and 18 were used for observation, and Line 18 was selected as a representative line. bzip17 pbZIP17: mRFP-bZIP17 ( m-bZIP17 ) To generate an m-bZIP17 complementation line, a DNA fragment encoding mRFP fluorescent reporter was inserted in-frame after the start codon of the bZIP17 coding sequences at the Sfo I site into pENTR-pbZIP17: Sfo I -bZIP17 to express mRFP-bZIP17. The obtained pYC87 ( pENTR-pbZIP17: mRFP-bZIP17 ) was then recombined into a pKGW destination vector (Karimi et al ., 2005) using Gateway TM LR Clonase TM II (Invitrogen, Thermo Fisher Scientific, MA, USA) to obtain plasmid pYC89 ( pKGW-pbZIP17: mRFP-bZIP17 ). In total, 24 T1 plants were selected on ½ MS agar plate containing the antibiotic Kanamycin. The obtained T2 seeds were screened using Kanamycin and selected by PCR-based genotyping. To distinguish transgenic pbZIP17: mRFP-bZIP17 from endogenous bZIP17 , specific primers (LC12/KK418) were designed. Lines 16, 17, and 18 were used for observation, and Line 16 was selected as a representative line. Confocal laser-microscopy observation For the transient expression assay, a drop of transformed protoplasts was applied onto a glass slide with a ring sticker. The fluorescent signals were observed under confocal laser-scanning microscopy (LSM 510 Meta; Carl Zeiss, Jena, Germany) equipped with a C-Apochromat ×63 objective with a 1.2 numerical aperture. For live imaging in primary root, hypocotyl, and cotyledon, Venus or mRFP fluorescences in 7-day-old AGB1-V or m-bZIP17 seedlings, respectively, were observed under a microscope equipped with a C-Apochromat ×40 objective with 1.2 numerical aperture. For plasma membrane staining, seedlings were immersed in 10 μg/ml of FM4-64 (Invitrogen TM 13320) for 3 min. For ER staining, samples were immersed in 1 μM of ER-tracker TM Blue-White DPX (Invitrogen TM E12353) for 5 min. For nuclei staining, root samples were immersed in 10 μg/ml of DAPI (Thermo Fisher Scientific 62247). After staining the samples were observed under confocal microscopy, and images were captured using an LSM 510 v3.2 (Carl Zeiss) with filters for DAPI or ER-tracker Blue-White DPX (Diode 405 nm laser, band-pass 420-480 nm); Venus or YFP (Argon 514 nm laser, band-pass 520-555 nm); FM 4-64 or mRFP (HeNe 543 nm laser, band-pass 560-615 nm). Immunoblotting and nuclei isolation Ten of 7-day-old seedlings were homogenized in 100 µl lysis buffer [50 mM Tris HCl (Merck 648317), pH 6.8, 2 % (w/v) SDS (Merck 822050), 10 mM 2-mercaptoethanol (2-ME, Merck 805740), 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich P9599). The homogenate was stood on ice for 20 min and centrifuged at 16,000 g for 10 min at 4 °C. The supernatant (100 µl) was added to 2× sample buffer (50 mM Tris-HCl, pH 6.8, 10% (w/v) SDS, 10%(v/v) 2-ME, 526 mM sucrose, 0.1% (w/v) bromophenol blue (Merck 108122)). Samples were boiled for 3 min at 95 °C and loaded and separated by 10% SDS-PAGE, blotted onto 0.45 µm PVDF blotting membrane (10600023; GE Healthcare, PA, USA) and probed with primary and secondary antibodies, as follows: rabbit polyclonal anti-GFP (for AGB1-VEN, 1:3,000, Invitrogen TM A-11122), mouse monoclonal anti-Actin (1:5,000, Agrisera AS10-702), rabbit polyclonal anti-Histone H3 (1:3,000, Agrisera AS10-710), goat anti-rabbit IgG-HRP (1:10,000, Abcam ab6721) and goat anti-mouse IgG-HRP (1:10,000, Abcam). The target proteins were visualized by use of Image Quant LAS4000 (GE Health). For nuclei isolation, CelLytic TM PN Isolation/Extraction Kit (Sigma CELLYTPN1) was used according to the manufacturer’s instructions with minor modifications. In brief, 1 g of fresh weight (~700 of 7-day-old seedlings) were homogenized in 3 ml of NIBA buffer [25% (v/v) 4× Nuclei Isolation Buffer (Sigma-Aldrich N8304), 10 mM Dithiothreitol Merck #805740), 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich P9599)]. The homogenate was filtrated through the Miracloth (Merck) as a total fraction (46) and then centrifuged at 1,300 g for 10 min at 4 °C. The resulting supernatant was kept as a cytosolic fraction, and the pellet was resuspended by NIBA buffer containing 0.3% Triton X-100. After 30 min incubation at 4 °C, the resuspension was centrifuged at 12,000 g for 5 min at 4 °C with three repeats. The resulting pellet was resuspended by 1× SDS sample buffer and incubated at 95 °C for 3 min. After being centrifuged at 12,000 g for 5 min at 4 °C, the supernatant was kept as a nucleus fraction. Microarray analysis For salt stress treatment, 7-day-old Arabidopsis seedlings were subjected to ½ MS liquid medium containing 0 (control) or 150 mM NaCl (stress) for 4 hours, for root samples were dissected right after salt stress treatment and harvested in liquid nitrogen. Total RNA was extracted using RNeasy Plant mini kit according to the manufacturer’s instructions (Qiagen) with in-membrane digestion of DNase (Qiagen) to remove genomic DNA contamination and quantified by 260/280 nm UV light absorption. For quality control, the integrity of RNA was determined by Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies). Preparation of fluorescence-labelled cDNA and microarray experiments were performed at the DNA Microarray Core Facility, Institute of Plant and Microbial Biology, Academia Sinica, Taiwan. Agilent Arabidopsis (V4) Gene Expression Microarray 4×44k chips were used in this study. Labelling of cDNA probes and hybridization experiments were performed according to the single-colour microarray protocols provided by the manufacturer. The Agilent DNA Microarray Scanner G2565CA and Agilent Feature Extraction 10.7.1.1 software detected the fluorescence signals. Three independent biological replicates were conducted using cDNA from control and stress samples. RNA preparation, cDNA synthesis and reverse-transcription PCR (RT-PCR), quantitative RT-PCR (qPCR) Seedlings were frozen by immersion in liquid nitrogen and stored at -80 °C until use. Total RNA was extracted using a standard TRI reagent solution (Invitrogen AM9738). In brief, ten of 7-day-old seedlings were homogenized in 600 µl of TRI reagent, followed by a phase separation step with 120 µl chloroform (Merck 107024). RNA was precipitated with 300 µl isopropanol (Merck 107022) and then 0.3 M sodium acetate (Merck 106268), washed with ethanol and resuspended in 30 µl of diethylpyrocarbonate (DEPC, Merck 298711) -treated water. Genomic contamination was removed using RNase-free DNase set (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Five hundred ng RNA was used for complementary DNA (cDNA) synthesis by the SuperScript III First-Strand Synthesis SuperMix (Invitrogen 1172050). Fifty ng cDNA was used as template for quantitative RT-PCR with SYBRTM Green PCR Master Mix (4309155, Applied Biosystems TM Thermo Fisher Scientific) detection and performed in triplicate using the Applied Biosystems 7500 fast real-time PCR system. Data were analyzed by the comparative threshold cycle method (ΔΔCT methods). The transcript level was normalized to the ACTIN2 gene ( ACT2 , KK129/KK130) for each sample. For RESPONSIVE TO DESICCATION 20 ( RD20 , YC116/YC117), NAC DOMAIN CONTAINING PROTEIN 19 ( NAC019 , YC32/YC33), and TSPO (YC118/YC119), the relative transcript level is expressed as the fold change (mean ± SD) in each genotype under mock (0 mM NaCl) or salt (150 mM NaCl) treatment relative to the mock control in the wild type (set to value as 1) from three biological replicates with three technical replicates. The primer sets for quantitative RT-PCR are listed in supplemental table 1. Protoplast isolation and BiFC assay Protoplasts were isolated from 20- to 22-day-old Arabidopsis WT leaves using fungal cellulase (1% (v/v) ‘Onozuka’ R10, Yakult, Tokyo, Japan) and macerozyme (‘Onozuka’ R10, Yakult) to remove cell walls accordingly with minor modification (Wu, et al ., 2009). DNA transfection was performed using the PEG-calcium solution, followed by 16-hr incubation at 24°C. Transformed protoplasts were observed under a laser-scanning confocal microscope. AGROBEST transient expression For a transient expression to observe the bZIP17 processing in seedlings, the AGROBEST method was used with minor modifications (44). In Brief, seeds were germinated in the MS liquid media 3-day-old WT, agb1 and s1p s2p mutant seedlings were infected with Agrobacteria tumefaciens strain C58C1 (pTiB6S3ΔT) H carrying the pYC89 (pKGW-ProbZIP17: mRFP-bZIP17) in ABM-MS [½ AB-MES, ¼ MS, 0.25% (w/v) sucrose, pH5.5] liquid medium for 2 days. The co-cultivation medium was then replaced with 1 ml fresh ½ MS medium and then incubated for 2 days. For salt stress assay, seedlings were incubated in ½ MS medium containing 0 or 150 mM NaCl for 4 hours and then observed the mRFP-bZIP17 signals under confocal microscopy. ACCESSION NUMBERS AGB1 (At4g34460), bZIP17 (At2g40950), AGG1 (AT3G63420). CONCLUSIONS I n conclusion, this study elucidates the vital role of AGB1 in plant salt stress responses through its interaction with the transcription factor bZIP17 and its regulation of crucial stress-responsive pathways. The findings demonstrate that AGB1's subcellular localization changes under salt stress are essential for initiating specific gene expressions necessary for plant survival. Genetic analyses further highlight the sensitivity of AGB1 mutants to salt stress, emphasizing AGB1's importance in cellular homeostasis and overall plant fitness. This research contributes to our understanding of plant stress mechanisms and opens possibilities for developing crops with improved tolerance to salinity, addressing agricultural challenges posed by environmental stresses. However, more detailed studies are required to fully explore the implications of these mechanisms across various plant species and conditions. Abbreviations ABA Abscisic acid ABRE ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN AGB1 Gβ subunit GTP BINDING PROTEIN BETA 1 AGG G-PROTEIN GAMMA-SUBUNIT BBX21 B-BOX DOMAIN PROTEIN 21 BES1 BRI1-EMS-SUPPRESSOR 1 BiFC Bimolecular Fluorescence Complementation bZIP Basic region leucine zipper CRY1 CRYPTOCHROME 1 FER FERONIA G protein Heterotrimeric guanine nucleotide-binding protein GPCR G-Protein-Coupled Receptor GPA1 G PROTEIN ALPHA SUBUNIT 1 HY5 ELONGATED HYPOCOTYL 5 LEA LATE EMBRYOGENESIS ABUNDANT MYB Myeloblastosis MPK6 MAP KINASE mRFP Monomeric Red Fluorescent Protein NAC NAM, ATAF1/2, and CUC2 PHYB PHYTOCHROME B PIF3 PHYTOCHROME INTERACTING FACTOR 3 RALF1 Rapid Alkalinization Factor 1 RD20 RESPONSIVE TO DESICCATION 20 ROS Reactive Oxygen Species S1P SITE-1 PROTEASE S2P SITE-2 PROTEASE SOS SALT OVERLY SENSITIVE TSPO OUTER MEMBRANE TRYPTOPHAN-RICH SENSORY PROTEIN-RELATED UB Ubiquitin VIP1 VIRE2-INTERACTING PROTEIN 1 XLG EXTRA-LARGE G-PROTEIN YFP Yellow Fluorescent Protein Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions Y.C. designed research, performed experiments, analyzed data, and wrote the manuscript. FUNDING INFORMATION This research received funding from the Institute of Plant and Microbial Biology, Academia Sinica, Taiwan’s core budgets for Dr. Kazue Kanehara and a postdoctoral scholarship for Y.C. from Academia Sinica. ACKNOWLEDGEMENTS The authors thank the Live Cell Imaging Core Lab and Genomic Technology Core Lab at the Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, for their facilities and support. Thanks to Chia-En Chen and Ling Chuang for molecular cloning, the Arabidopsis Biological Resource Center for providing seeds, and Dr. Erh-Min Lai for the Agrobacterium tumefaciens strain C58C1. 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Supplementary Files 240419AGB1bZIP17BMCPlantBiolSup.pdf 240418bZIP17BMCPlantBiolTableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 15 May, 2024 Reviews received at journal 14 May, 2024 Reviewers agreed at journal 06 May, 2024 Reviews received at journal 06 May, 2024 Reviewers agreed at journal 28 Apr, 2024 Reviewers invited by journal 22 Apr, 2024 Editor assigned by journal 22 Apr, 2024 Submission checks completed at journal 22 Apr, 2024 First submitted to journal 15 Apr, 2024 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. We do this by developing innovative software and high quality services for the global research community. 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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-4267287","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295156795,"identity":"b740b42f-5128-4dae-9743-d994372685b6","order_by":0,"name":"Yueh Cho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEklEQVRIiWNgGAWjYDACCTB5QA7OZQPTbIS1GDMwMKNp4SGgJbEBogUG8Gjhn918TJqn4k76fPfzBx8w/LHI42M//IDhQ9lhBnuJBOyW3DmWJs1z5lnuxjPJzAaMbRLFbDxpBowzzh1m4MGhxUAix0yat+1w7saGZDYJxgaJxDYJBgNmoAgDjzQ+Lf8Opxv2P2b/wfAHpIX9A/NfgloaDifISyQDvcwG0sJjwMyIR4vEjbRkyznHDhtukHhsDFKf2MaTU3Cw51w6D8/9B9hDbEbywRtvag7Ly/cnPvzw4U9d4vz24xsf/CizlmPvOYBVCxCwgKPGACQPcwmIjTMmgYD5A4iUb8CjZBSMglEwCkY2AAD1o1kmgvQCTgAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Plant and Microbial Biology, Academia Sinica","correspondingAuthor":true,"prefix":"","firstName":"Yueh","middleName":"","lastName":"Cho","suffix":""}],"badges":[],"createdAt":"2024-04-15 05:15:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4267287/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4267287/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55295295,"identity":"c5f71627-82d5-441f-82c3-fa89cacaa117","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":287134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional complementation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eagb1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ehypersensitivity to salt stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Salinity tolerance assay,\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) germination rates,\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e) fresh weight, and (\u003cstrong\u003eD\u003c/strong\u003e) percentage of the \u003cem\u003ealbino leaf\u003c/em\u003e of \u003cem\u003eagb1-3\u003c/em\u003e (\u003cem\u003eagb1\u003c/em\u003e) mutant and \u003cem\u003eagb1 pAGB1:AGB1-VEN\u003c/em\u003e (AGB1-V) transgenic plant seeds compared with the corresponding Col-0 (WT) seeds. Representative images (\u003cstrong\u003eA\u003c/strong\u003e) of 14-day-old WT, \u003cem\u003eagb1\u003c/em\u003e mutant, and AGB1-V transgenic plant were grown on ½ MS containing NaCl with indicated concentration to induce salt stress. (\u003cstrong\u003eB\u003c/strong\u003e) Each value represents the means ± SD of the germination percentage (with 25 seeds) for six independent experiments. (\u003cstrong\u003eC\u003c/strong\u003e) Representative plots for fresh weight of 14-day-old seedlings grown on 0 (up) or 150 mM NaCl (down) were measured individually with six biological experiments (n=11) and shown as means ± SD. Data with different letters represent significant differences [one-way ANOVA at P \u0026lt; 0.05]. (\u003cstrong\u003eD\u003c/strong\u003e) The albino leaves were counted and displayed as means ± SD. ***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/da649670a16506fc141632f6.png"},{"id":55295298,"identity":"c58202c0-e625-42a0-b683-33455e7f3ca0","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":787010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNuclei localization of AGB1 in response to high salinity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eRepresentative images of \u003cem\u003eProAGB1:AGB1-VENUS \u003c/em\u003e(AGB1-VEN) in a stable transgenic complementation plant in the absence (½ MS medium) or presence of 150 mM NaCl as indicated time for salt stress induction. White arrows indicated the nuclei localization of AGB1. The regions of the root epidermis were marked by dash open boxes and zoomed to show in the right\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA’\u003c/strong\u003e)\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eSubcellular localization of AGB1 in root of 7-day-old AGB1-VEN plant. Fluorescence of AGB1-VEN (Green) and staining of the plasma membrane by FM4-64 (left), endoplasmic reticulum (ER) by ER-tracker (middle), and nuclei by DAPI (right) in treatment of 150 mM NaCl for 0 or 4 hours. Co-localization signals of AGB1-VEN and staining dye were shown in the merge images as white colors in the bottom panel. Scale bars equal to 10 µm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Immunoblot analysis of AGB1-V protein among different sub-cellular fractions. Nuclei isolation of 7-day-old AGB1-VEN seedlings with 0 or 4 hours of 150 mM NaCl treatment, cytosol (C), or nuclei (N) fractions were separated after centrifugation. T: total input. The percentages of each fraction for sample loading were indicated in the bottom per lane. Immunoblots of AGB1-VEN (121 kDa) were detected by anti-GFP antibody, anti-ACTIN8 antibody was used as cytosol marker, and anti-histone H3 antibody (H3: 17 kDa) was used as nuclei marker. CBB staining as the loading control. The relative ratio of AGB1-VEN was normalized to CBB intensity.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/bd51923333f02866c1838f34.png"},{"id":55295296,"identity":"4c3aa448-9c5a-4b86-b8e4-9a8814acc29c","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSalt stress induction of bZIP17-mediated salt stress response in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eagb1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Thirteen of bZIP17-mediated salt stress-responsive gene expressions under salt stress in 7-day-old WT or \u003cem\u003eagb1\u003c/em\u003e mutant. Roots were collected after 0 or 4 hours of 150 mM NaCl treatment for microarray analysis, and genes with significant changes among 0 and 4 hours of salt stress were selected (P \u0026lt; 0.05, n=4,830). The Log2 values indicated the means ± SD of the ratio of the representative transcripts under salt stress (4-h) comparing the mock condition in WT (x-axis) or agb1 mutant (y-axis) from three independent replicates. Quantitative RT-PCR transcript analysis was performed using 7-day-old roots of WT, \u003cem\u003eagb1\u003c/em\u003e, \u003cem\u003ebzip17-4\u003c/em\u003e (\u003cem\u003ebzip17\u003c/em\u003e) treated with mock or 150 mM NaCl containing MS medium for 4 hours. Roots were collected for RNA extraction and cDNA synthesis. Transcription levels of (\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eNAC DOMAIN CONTAINING PROTEIN 19\u003c/em\u003e (\u003cem\u003eNAC019\u003c/em\u003e), (\u003cstrong\u003eC\u003c/strong\u003e) \u003cem\u003eRESPONSIVE TO DESICCATION 20\u003c/em\u003e (\u003cem\u003eRD20\u003c/em\u003e), and (\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eTSPO\u003c/em\u003ewere quantified. The expression of the WT sample at 0 hour set to 1. Three technical replicates averaged data in the same run, and three biological replicates in separate runs were shown in mean ± SD. Data with different letters represent significant differences [one-way ANOVA at P \u0026lt; 0.05].\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/f1dae47b9304e261115967f6.png"},{"id":55295294,"identity":"2464c49f-6502-4db1-9feb-36e9aef0a3c9","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":485359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenetic interaction of AGB1 and bZIP17.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) RT-PCR analysis for the wild type (WT), \u003cem\u003eagb1-3\u003c/em\u003e (\u003cem\u003eagb1\u003c/em\u003e), \u003cem\u003ebzip17-4\u003c/em\u003e (\u003cem\u003ebzip17\u003c/em\u003e) and \u003cem\u003eagb1 bzip17\u003c/em\u003e. \u003cem\u003eACTIN2\u003c/em\u003e (\u003cem\u003eACT2\u003c/em\u003e) was used as the positive control. (\u003cstrong\u003eB\u003c/strong\u003e) Germination rates of \u003cem\u003eagb1\u003c/em\u003e (green), \u003cem\u003ebzip17\u003c/em\u003e (magenta), and \u003cem\u003eagb1 bzip17\u003c/em\u003e (mustard) mutant seeds compared with the corresponding WT seeds grown on MS containing 0 or 125 mM NaCl for 14 days. Each value represents the means ± SD of the germination percentage (with 25 seeds) for four independent experiments. Representative images (\u003cstrong\u003eC\u003c/strong\u003e) and phenotypic quantification (\u003cstrong\u003eD\u003c/strong\u003e) of 14-day-old WT, \u003cem\u003eagb1\u003c/em\u003e, \u003cem\u003ebzip17, \u003c/em\u003eand\u003cem\u003e agb1 bzip17 \u003c/em\u003emutants were grown on ½ MS containing NaCl with indicated concentration to induce salt stress. (\u003cstrong\u003eD\u003c/strong\u003e) seedling fresh weight (left), primary root length (middle), and leave length (right) of 14-day-old seedlings grown on 0 or 125 mM NaCl shown in (D) were measured individually with three biological experiments (n=25), and shown as means ± SD of 3 independent mean value. Data with different letters represent significant differences [one-way ANOVA at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05].\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/5fd26c6add318f53ca3c3df5.png"},{"id":55295301,"identity":"6d79944e-9334-4bce-88c8-1882eacba2d7","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":727464,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAGB1 and bZIP17 protein-protein interaction \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of the UPR pathway is initiated when misfolded proteins are over-accumulated in the ER lumen by activating bZIP17 under salt stress. AGB1 (\u003cem\u003epAGB1:AGB1-VENUS\u003c/em\u003e, AGB1-VEN) was marked as green pattern and bZIP17 (\u003cem\u003epbZIP17:mRFP-bZIP17\u003c/em\u003e, mRFP-bZIP17). (\u003cstrong\u003eB\u003c/strong\u003e) Subcellular localization of AGB1 in the root of 7-day-old AGB1-VEN plant in \u003cem\u003eagb1\u003c/em\u003e (up) or \u003cem\u003eagb1 bzip17\u003c/em\u003e (bottom) background. ER-like and nuclei localization of AGB1-VEN were marked by open or solid arrowheads, respectively. (\u003cstrong\u003eC\u003c/strong\u003e) AGB1 and bZIP17 were transiently expressed in protoplasts to observe subcellular localization after 0 or 4-h of 150 mM NaCl treatment. (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e) Bimolecular Fluorescence Complementation (BiFC, green) assay for physical interaction of AGB1 and bZIP17 after 4 hours of 150 mM NaCl treatment as arrowhead indicated. AGG1 is a positive control for AGB1 interaction with N-YFP and C-YFP combinations. A dashed circle marked protoplast boundaries. bZIP17 were paired with empty BiFC vectors (C-YFP or N-YFP) for negative control. Differential interference contrast (DIC) images to show cellular structures. Scale bars equal to 10 µm.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/a5edf7e02672e62bc7e4d39c.png"},{"id":55295300,"identity":"d096dc8e-9c81-4915-9574-a19c3133c78a","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":241471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ebzip17\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e proteolytic processing in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eagb1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) ARGOBEST transient expression of mRFP-bZIP17 in the 7-day-old WT, \u003cem\u003es1p-1\u003c/em\u003e \u003cem\u003es2p-1\u003c/em\u003e (\u003cem\u003es1p s2p\u003c/em\u003e) and \u003cem\u003eagb1\u003c/em\u003e mutant seedlings followed by 4-h of 0 or 150 mM NaCl treatment. An empty triangle marked the ER-like localization of mRFP-bZIP17, and a solid triangle marked the nuclei-like localization of mRFP-bZIP17. Scale bars equal to 10 µm. The ratio of different subcellular localization of mRFP-bZIP17 was quantified as shown in (\u003cstrong\u003eB\u003c/strong\u003e). Each value represents the means ± SD of the percentage of mRFP-bZIP17 localization (ten seedlings) for three independent experiments per time point. The total numbers of examined epidermal cells for corresponding tissue were shown at the top of each bar. (\u003cstrong\u003eC\u003c/strong\u003e) Schematic representation of AGB1-mediated salinity stress response through bZIP17 signaling. The nuclei localization of AGB1 (green) was observed after short-term salt stress treatment (left), and the bZIP17 (magenta)-regulated expression of salinity-responsive genes like \u003cem\u003eLEA7\u003c/em\u003e, \u003cem\u003eNAC019\u003c/em\u003e, and \u003cem\u003eTSPO\u003c/em\u003e (blue) was reduced in the absence of AGB1. The nuclei localization of bZIP17 through S1P/S2P-mediated proteolytic processing was also compromised in the \u003cem\u003eagb1\u003c/em\u003e mutant (right).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/02da06ecda63db1ac0baf5c9.png"},{"id":55296207,"identity":"af687125-1c8a-41ef-a5c1-3cffb695d430","added_by":"auto","created_at":"2024-04-25 10:40:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3060799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/6c1948e8-0268-4e67-9ef3-e97f4002dd71.pdf"},{"id":55295868,"identity":"aa41fcc5-5e4f-4c79-abc5-0d6fab78239b","added_by":"auto","created_at":"2024-04-25 10:32:35","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12024190,"visible":true,"origin":"","legend":"","description":"","filename":"240419AGB1bZIP17BMCPlantBiolSup.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/96180dcea68f688cc849c9a7.pdf"},{"id":55295293,"identity":"5e98ad33-fadd-4d4b-b1ff-966b6b188448","added_by":"auto","created_at":"2024-04-25 10:24:35","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10883,"visible":true,"origin":"","legend":"","description":"","filename":"240418bZIP17BMCPlantBiolTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4267287/v1/f39fb7e59e920a69321ab7e1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response","fulltext":[{"header":"Background","content":"\u003cp\u003eLike every one of us, plants are facing an ever-changing environment day by day. Adjusting the armory to cope with divergent stresses is essential to equip seedlings with proper transcriptome for their fitness\u0026nbsp;(1). Integrating external stimuli into plant cells depends on hormones and heterotrimeric G proteins to deliver messages and initiate proper cellular responses for better survival\u0026nbsp;(2).\u0026nbsp;The heterotrimeric guanine nucleotide-binding protein (G protein), including G⍺, G\u0026beta;, and G\u0026gamma; subunits, serves as a signal mediator coupling with the plasma membrane-spanning G-protein-coupled receptors (GPCR) and effectors\u0026nbsp;(3). The human genome contains 23 G⍺\u0026nbsp;genes, 5 G\u0026beta; genes and 11 G\u0026gamma; genes\u0026nbsp;(4). By contrast, Arabidopsis genome contains one canonical G PROTEIN ALPHA SUBUNIT 1 (GPA1)\u0026nbsp;(5)\u0026nbsp;and three non-canonical G⍺\u0026nbsp;subunits EXTRA-LARGE G-PROTEIN 1 (XLG1)\u0026nbsp;(6), XLG2 and XLG3\u0026nbsp;(4), one G\u0026beta; subunit GTP BINDING PROTEIN BETA 1 (AGB1)\u0026nbsp;(7)\u0026nbsp;and three G\u0026gamma; subunits G-PROTEIN GAMMA-SUBUNIT 1 (AGG1)\u0026nbsp;(8), AGG2\u0026nbsp;(9), AGG3\u0026nbsp;(10). Previous studies have shown that heterotrimeric G protein subunits play vital roles in responses to developmental cues and environmental stresses, including salt stress\u0026nbsp;(11).\u003c/p\u003e\n\u003cp\u003eSoil salinity is one of the major threats to food security by seriously attenuating plant growth and decreasing crop yield\u0026nbsp;(12). Excessive salt in the soil causes numerous negative effects on different plant developmental stages, including germination, vegetative growth, and flowering\u0026nbsp;(13). These negative effects damage plant cells due to ion toxicity and increasing osmotic stress\u0026nbsp;(14, 15). Accordingly, plants have employed various mechanisms for survival in harsh salinity environments. Three signaling pathways constitute the major transduction during salt stress: calcium-dependent signaling pathway that (1) triggers the activation of stress-responsive genes as dehydration-responsive or late embryogenesis abundant (LEA) proteins\u0026nbsp;(16)\u0026nbsp;and (2) salt overly sensitive (SOS) pathway for regulation of ion homeostasis\u0026nbsp;(17), and (3) osmotic stress signaling pathway involving ABA-dependent induction of downstream salt responsive genes through activation of a group of transcription factors like bZIP, NAC, MYB and ABRE families\u0026nbsp;(18). However, our understanding of the whole network of pathways regulating salinity response is far from complete.\u003c/p\u003e\n\u003cp\u003eRecent research based on studies of Arabidopsis AGB1 has suggested a functional link between plant G protein signaling and regulation of the salt stress response. A knockout mutant of AGB1 exhibited more sensitivity to high salinity than wild-type plants\u0026nbsp;(10, 11, 19-21). Under excessive salt, the \u003cem\u003eagb1\u003c/em\u003e mutant accumulates more Na\u003csup\u003e+\u003c/sup\u003e, translocate more Na\u003csup\u003e+\u003c/sup\u003e from root to shoot, and has a high transpiration rate with larger stomatal apertures\u0026nbsp;(22, 23); AGB1\u0026nbsp;is also coupled with AGG1 or AGG2 to regulate stomatal apertures and transpiration\u0026nbsp;(24).\u0026nbsp;Meanwhile, a receptor-like kinase, FERONIA (FER), has been identified by directed interaction with AGB1. FER is required for cell wall integrity, Ca2\u003csup\u003e+\u003c/sup\u003e induction, ROS production, and stomata movement under salinity conditions\u0026nbsp;(25)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMost G protein signaling studies have focused on canonical effectors localized to the plasma membrane. Recent studies have shown that the G-protein \u0026beta; subunit functions in the nucleus\u0026nbsp;(23, 26). The Arabidopsis AGB1 is localized in the nucleus, where it interacts with B-BOX DOMAIN PROTEIN 21 (BBX21) for hypocotyl elongation\u0026nbsp;(27), with BRI1-EMS-SUPPRESSOR 1 (BES1) for cell division\u0026nbsp;(28), with PHYTOCHROME B (phyB) - PHYTOCHROME INTERACTING FACTOR 3 (PIF3)\u0026nbsp;(29)\u0026nbsp;or CRYPTOCHROME 1 (CRY1) - ELONGATED HYPOCOTYL 5 (HY5) for photomorphogenesis\u0026nbsp;(30)\u0026nbsp;and with MAP KINASE 6 (MPK6) for drought tolerance\u0026nbsp;(31). Nonetheless, gaining evidence showed that WD domain-containing proteins, including AGB1, directly interacted with bZIP transcription factors like HY5 and VIRE2-INTERACTING PROTEIN 1 (VIP1) to regulate gene expressions\u0026nbsp;(30, 31).\u003c/p\u003e\n\u003cp\u003eThe basic region leucine zippers transcription (bZIP) family in Arabidopsis comprised 78 members and assorted into 13 groups (32). bZIPs form as dimers to bind DNA sequences, and heterodimerization results in appreciable regulatory flexibility (33). Among these bZIP transcription factors, three members of group B (bZIP17, bZIP28, bZIP49) perform as important regulators of the evolutionally conserved ER stress response (34), the intrafamily dimerization of group B has been confirmed in yeast cells (35). In particular, under adverse environmental conditions, including high salinity, bZIP17 is reported to relocate from ER to nucleus processing by \u0026nbsp; through regulated intramembrane proteolysis (36). Salt-responsive genes are reported to express in a bZIP17-dependent manner (36, 37). In this study, we observed the nuclei localization and the contributions of AGB1 to induce the expression of bZIP17-mediated salinity-responsive genes. The spliced bZIP17 localization toward nuclei through S1P/S2P-mediated proteolysis was reduced without AGB1. The arrangement of AGB1 pools among different subcellular compartments to involve proper programs to respond to high salinity is crucial for young seedling viability.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eFunctional complementation of salinity hypersensitivity in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, the AGB1 transcript was significantly increased under high salt stress after 4 hours, as described previously (Fig. S2A; (19)). The \u003cem\u003eagb1-3\u0026nbsp;\u003c/em\u003emutant is a null mutant of \u003cem\u003eAGB1\u0026nbsp;\u003c/em\u003e(as \u003cem\u003eagb1\u003c/em\u003e, Fig. S1A, S1B (38)). It shows hypersensitive responses, including poor germination, growth defects, and albino leaves when grown under salt stress (Fig. 1). We produced the genetic complementation lines harboring genomic sequence of \u003cem\u003eAGB1\u0026nbsp;\u003c/em\u003eas previously described (\u003cem\u003eagb1 pAGB1:AGB1,\u0026nbsp;\u003c/em\u003eas AGB1; (38)) with C-terminally fused the triple Venus fluorescent protein in the \u003cem\u003eagb1\u003c/em\u003e mutant (\u003cem\u003eagb1\u003c/em\u003e \u003cem\u003epAGB1:AGB1-Venus\u003c/em\u003e, as AGB1-V; Fig. S1A, S1B and S1C). To test whether AGB1-V transgene is functional \u003cem\u003ein vivo\u003c/em\u003e, we observed shoot, primary root, and lateral roots. The AGB1 and AGB1-V transgenic plants rescued the developmental defects in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant (Fig. 1, S1D, S1E, and S1F). We then performed the sequential salt stress tolerance assay on wild-type (WT), \u003cem\u003eagb1\u003c/em\u003e mutant, and AGB1-V plants for 14 days after seeds sowing on \u0026frac12; MS medium (Fig. 1 and S2). After the salt treatment, \u003cem\u003eAGB1\u0026nbsp;\u003c/em\u003ewas induced in 7-d WT seedlings (Fig. S2A). The leaf bleaching phenotype and growth retardation in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant were fully rescued in the AGB1-V complementation lines (Fig. 1A, 1C, 1D, S2B, S2C), which were either counted for the albino rate (Fig. 1D) or classified into three groups according to the seedling size and chlorotic phenotypes as Green, Mix (contained at least one white leaf) and White (Fig. S2B, S2C). In the mock treatment (0 mM NaCl), all WT, \u003cem\u003eagb1,\u0026nbsp;\u003c/em\u003eand AGB1-V seedlings grew normally and showed green for their aerial part. In contrast, in the salt stress treatment (150 mM NaCl), more \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eseedlings were categorized significantly into the white group due to the chlorotic phenotype compared to the WT and AGB1-V seedlings (Fig. 1D, S2B, S2C). Since no statistical difference was found between WT and AGB1-V seedlings, we suggested that the AGB1-V was functional and complemented the \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003e(Fig. 1, S1, and S2)\u003cem\u003e.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular localization of AGB1 at the nuclei in response to salt stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAGB1 is known as a molecular switch through protein-protein interactions to regulate transcriptional programs for plant development and abiotic stress responses, like inhibiting BBX21 activation\u0026nbsp;(27)\u0026nbsp;or binding with PIF3 in the nucleus to promote hypocotyl elongation\u0026nbsp;(29), dephosphorylating BES1 for its nuclei entry and downstream gene expression\u0026nbsp;(28), which prompted us to investigate whether AGB1 localization into nuclei to involve transcriptional regulation under high salinity. The 7-day-old transgenic AGB1-V seedlings were used for confocal laser scanning microscopy to observe any changes in subcellular localization of AGB1-Venus that occurred during short-term NaCl treatment. The time series of salt treatment continuously tracked AGB1-Venus\u0026rsquo;s localization in the root epidermis (Fig. 2A). Most of AGB1-Venus showed network-like localization under normal conditions, which prompted us to consider the possible ER localization of AGB1 as a previous study described (\u003cem\u003eProAGB1:CFP-AGB1\u003c/em\u003e (39); \u003cem\u003ePro35S: AGB1-GFP\u0026nbsp;\u003c/em\u003e(26)). In contrast, many AGB1-Venus expressions showed nuclei-like patterns in the AGB1-V root under 1, 2, and 4 h of salt stress (Fig. 2A) as shown in enlarged images (Fig. 2A\u0026rsquo;). This nuclei localization of AGB1-V can be seen in other tissues like hypocotyl (middle) and also cotyledon (right) in a spatiotemporal manner (Fig. S3A and S3B); the dot-like are confirmed by DAPI staining to mark nuclei (4-h NaCl treatment, red in Fig. S3A). The nuclei-localized AGB1-V was rarely seen in the tissue with 0-h NaCl treatment, found in the tissue with 1- to 16-hours NaCl treatment as a solid triangle indicated in Fig. S3A. To quantify the nuclei-localized AGB1-V, we grouped the ER-like and PM-like-localized AGB1-V cells (black group) and counted the ratio of the different localized AGB1-V in the tissue that has been examined (Fig. S3B). The nuclei-localized AGB1-V cells (white group) were significantly found in all NaCl-treated tissue with various lengths of time for salt stress induction (Fig. S3B).\u003c/p\u003e\n\u003cp\u003eWe also stained 7-day-old AGB1-V seedlings with plasma membrane (PM) marker (FM4-64, Fig. 2B, Left) and ER marker (ER-tracker, Fig. 2B, middle), nuclei marker (DAPI, Fig. 2C, right). The AGB1-Venus signals were partially colocalized with organelle and nuclear markers in the presence of salt stress after 4 hours. The FM4-64 patterns matched cell boundaries, ER tracker marked the perinuclear network, and DAPI filled the central pot inside of the ER (Fig.2). Next, we performed the fractionation assay to isolate nuclei from 7-day-old AGB1-V seedlings with 0- or 4-h NaCl treatment, and the immunoblots of ACTIN8 and Histone H3 served as cytosolic and nuclear marker, respectively. The AGB1-Venus proteins were quantified with CBB staining and p35S: Venus acted as a negative control for Venus in nuclei fraction. The proportion of AGB1-Venus increased around 2-fold in nuclei fraction under salt stress (0.73 to 1.47), but there was no obvious change for AGB1-Venus in total and cytosolic fractions. The above results indicate that AGB1-Venus is widely distributed to PM and ER under normal conditions, which is increasingly localized to nuclei and likely reduced ER localization of AGB1-Venus in response to salt stress (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepression of transcription factor bZIP17-mediated salt stress response in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe wonder whether AGB1 played a role in transcriptional regulation in response to salt stress since gaining portions of AGB1-Venus to nuclei was observed in young seedlings; if so, what would be the corresponding gene sets to develop protection against excessive salt? We therefore performed microarray analyses using 7-day-old WT and \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003ewhole seedlings (Fig. S4A) or root-only samples (Fig. 3 and S4B-E) with 0 or 4 hours of 150 mM NaCl treatment. Compared to whole seedlings (Fig. S4A), the salt stress-induced fold changes are more obvious in \u003cem\u003eagb1\u003c/em\u003e versus wild-type roots (R\u003csup\u003e2\u003c/sup\u003e = 0.1882, Fig. 3A). Interestingly, the induction of a set of salt stress-responsive genes in a bZIP17-dependent manner (36) was repressed in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eseedlings (Fig. S4A) and root-only (as mark in purple, Fig. 3A) microarray analysis. The salt stress induction of 13 out of 22 bZIP17-regulated genes ((36)) was significantly reduced in the \u003cem\u003eagb1\u003c/em\u003e mutant than in WT roots (Fig. 3A). These gene expressions failed to induce in \u003cem\u003es1p-3\u0026nbsp;\u003c/em\u003eand \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003emutant with 4-h NaCl treatment and were evaluated by RT-qPCR (36, 37). To examine whether AGB1 exercised control over these genes in roots during salt stress, their expression was assessed in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eor \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003emutants using quantitative RT-PCR (Fig. 3B-D). The seedlings exposure to 150 mM of NaCl for 4 hours elevated the expression of these genes like \u003cem\u003eNAC DOMAIN CONTAINING PROTEIN 19\u003c/em\u003e (\u003cem\u003eNAC019\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eFig. 3B), \u003cem\u003eRESPONSIVE TO DESICCATION 20\u0026nbsp;\u003c/em\u003e(\u003cem\u003eRD20\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eFig. 3C), and \u003cem\u003eOUTER MEMBRANE TRYPTOPHAN-RICH SENSORY PROTEIN-RELATED\u003c/em\u003e (\u003cem\u003eTSPO\u003c/em\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003eFig. 3D) in WT, but much less in \u003cem\u003eagb1\u003c/em\u003e mutant. These findings are consistent with a model that under abiotic stresses or ABA treatment, S1P and/or S2P initiate the proteolytic activation of bZIP17, which directly upregulates genes like \u003cem\u003eNAC019\u0026nbsp;\u003c/em\u003e(40)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eTo explore whether the downregulated gene network in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eroots resulted from a group of transcription factors (TFs) regulated by AGB1, we performed differential expression gene analysis for \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eroot microarray (Fig. S4B). Indeed, 464 genes were down-regulated in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003ecompared to WT under high salinity, including 18 TF genes. We then generated a Venn diagram to cover identified TFs in this study (Fig. S4C) with NaCl-induced genes in Arabidopsis roots (41) and AGB1 interactome based on the BioGrid database (https://thebiogrid.org). Two TFs \u003cem\u003eMYB15\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNAC069\u003c/em\u003e intersected to serve as potential candidates for AGB1 to regulate expressions of salt stress-responsive gene, which used RT-qPCR further to examine salinity induction in WT and \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eseedlings (Fig. S4E). Both \u003cem\u003eMYB15\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNAC069\u003c/em\u003e reduced expressions in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eafter 4-h NaCl treatment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe \u003cem\u003ebZIP17\u003c/em\u003e depletion genetically inhibits the \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003ephenotype under salt stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bZIP17 and AGB1 are signaling modulators involved in various pathways, including the unfolded protein response and salt stress response. Both \u003cem\u003eagb1-2\u003c/em\u003e and \u003cem\u003ebzip17\u003c/em\u003e mutants exhibit hypersensitivity to salt stress (11, 36). To investigate the genetic interactions between AGB1 and bZIP17, we isolated \u003cem\u003ebzip17-4\u0026nbsp;\u003c/em\u003e(as \u003cem\u003ebzip17,\u0026nbsp;\u003c/em\u003e(42)) and generated \u003cem\u003ebzip17\u003c/em\u003e \u003cem\u003epbZIP17:bZIP17\u003c/em\u003e (Fig. S5) and \u003cem\u003ebzip17 pbZIP17:\u0026nbsp;\u003c/em\u003emRFP-bZIP17 lines (Fig. S6A), which displayed a complementary phenotype, rescuing the \u003cem\u003ebzip17\u003c/em\u003e hypersensitive defect to salt stress (Fig. S5 and S6B-E). Crosses between \u003cem\u003ebzip17\u003c/em\u003e and \u003cem\u003eagb1\u003c/em\u003e yielded an \u003cem\u003eagb1 bzip17\u003c/em\u003e double mutant, confirmed via RT-PCR (Fig. 4A). Under mock conditions, \u003cem\u003eagb1 bzip17\u003c/em\u003e phenocopied the \u003cem\u003eagb1\u003c/em\u003e mutant, displaying lower germination rate, higher biomass, longer primary roots, and increased plant width (Fig. 4B-D). However, under high salinity, both \u003cem\u003eagb1\u003c/em\u003e and \u003cem\u003eagb1 bzip17\u003c/em\u003e mutants exhibited salt hypersensitivity, with smaller seedlings and shorter roots, suggesting that \u003cem\u003eagb1\u003c/em\u003e is epistatic to \u003cem\u003ebzip17\u003c/em\u003e (Fig. 4B-D). Furthermore, \u003cem\u003eagb1\u003c/em\u003e displayed a more severe chlorotic phenotype compared to \u003cem\u003ebzip17\u003c/em\u003e under the same growth conditions (Fig. 4C). Treatment of an \u003cem\u003eagb1 bzip17\u003c/em\u003e double mutant with different NaCl concentrations revealed higher survival rates compared to the \u003cem\u003eagb1\u003c/em\u003e single mutant, indicating significant interaction effects between AGB1 and bZIP17 under salt stress. This suggests an antagonistic interaction between AGB1 and bZIP17.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe bZIP17 and AGB1 physical interaction \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe nuclear localization of AGB1 and its involvement in initiating bZIP17-dependent salinity stress response, but whether AGB1 physically interacts with bZIP17 is unknown. Under high salinity, the ER-localized bZIP17 was transferred to the Golgi apparatus, generating a spliced bZIP17 to move into the nuclei (Fig. 5C and S6F) and trigger a set of gene expressions, while the AGB1 was located in the plasma membrane, ER, and nuclei (Fig. 2, 5A, (36)). We first test whether the nuclei localization of AGB1 required bZIP17 presence in young seedlings; the AGB1-V showed a nuclei-like pattern under salt stress in \u003cem\u003eagb1\u003c/em\u003e and \u003cem\u003eagb1 bzip17\u003c/em\u003e genetic grounds, suggesting that the subcellular localization of AGB1 did not affect by the presence of bZIP17 (Fig. 5B). We next tested whether AGB1 interacts directly with bZIP17, and BiFC assays were performed in \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003eprotoplasts utilizing a pUBN/C BiFC system (43) (Fig. 5D, 5E). The coding sequences of AGB1 and AGG1 were cloned into pUBC vectors to act as positive pairs, and bZIP17 was cloned into pUBN vectors with translational fused with split YFP tag protein, respectively. No signal was observed in the negative control when the empty vector was paired with AGB1, AGG1, or bZIP17 BiFC vector. In contrast, a clear YFP signal (green) at the cytosolic region in samples expressing bZIP17 or AGG1 with AGB1 indicated interaction (Fig. 5D, 5E). This data suggests a physiological interaction between AGB1 and bZIP17 in the protoplast system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe repression of proteolytic processing of bZIP17 in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether AGB1 functions in the S1P-S2P pathway to regulate proteolytic processing of bZIP17 for gene induction under salt stress, we first transiently expressed the biological functional mRFP-bZIP17 (Fig. S6) in 7-day-old WT, \u003cem\u003eagb1\u003c/em\u003e, or \u003cem\u003es1p-1 s2p-1\u003c/em\u003e (\u003cem\u003es1p s2p\u003c/em\u003e) seedlings by AGROBEST\u0026nbsp;(44). In the meristemic zone of the WT root epidermis, the dot-like mRFP-bZIP17 (as indicated by the white triangle) was observed in salt-treated plants but rarely seen in \u003cem\u003es1p s2p\u0026nbsp;\u003c/em\u003eor \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutants. We also quantified the cells expressing nuclei-localized mRFP-bZIP17, the WT roots were significantly higher (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.013) under salt stress (15.5 \u0026plusmn; 7.7 %) than under mock condition (1.0 \u0026plusmn; 2.8 %), whereas the nuclei-localized mRFP-bZIP17 in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eor \u003cem\u003es1p s2p\u0026nbsp;\u003c/em\u003eroots were not different (\u003cem\u003eagb1: P\u0026nbsp;\u003c/em\u003e= 0.691; \u003cem\u003es1p s2p\u003c/em\u003e: \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.486) between stressed (\u003cem\u003eagb1\u003c/em\u003e: 0.0 \u0026plusmn; 7.3 % ; \u003cem\u003es1p s2p:\u0026nbsp;\u003c/em\u003e3.1 \u0026plusmn; 15.2 %) and control (\u003cem\u003eagb1\u003c/em\u003e: 0.0 \u0026plusmn; 3.8 %; \u003cem\u003es1p s2p:\u0026nbsp;\u003c/em\u003e4.8 \u0026plusmn; 3.0 %) conditions (Fig. 6B).\u003c/p\u003e"},{"header":"DISCUSSION ","content":"\u003cp\u003eThis study took salinity-stressed young seedlings to unveil a long-standing question of the functional aspect of the role of AGB1 in transcriptional regulation for plant survival and fitness.\u003c/p\u003e\n\u003cp\u003eWhether the mechanical inductions of a set of salt stress-responsive genes correlated to AGB1 remains unknown. We uncovered the salinity-responding nuclear localization of AGB1 among multiple tissues of young seedlings (Fig. 2). In the absence of \u003cem\u003eAGB1\u003c/em\u003e, gene inductions of salt-responsive transcription factors, antioxidants, and osmoticants were compensated as \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003emutant, root (Fig. 3), which may contribute by the physical interaction between AGB1 and bZIP17 (Fig. 5). At the molecular level, the S1P/S2P-mediated bZIP17 proteolytic processing was abolished in \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003emutant to decrease the nuclear localization of bZIP17 under high salinity (Fig. 6A and 6B). At the genetic level, the \u003cem\u003eagb1 is\u0026nbsp;\u003c/em\u003eepistatic to \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003eas the \u003cem\u003eagb1 bzip17\u0026nbsp;\u003c/em\u003edouble mutant phenocopying \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003ebut not \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003e(Fig. 4)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eTherefore, our mechanistic study elucidated that AGB1 coordinated transcriptional salinity response to regulate bZIP17 processing and mediating the signaling pathway (Fig. 6C).\u003c/p\u003e\n\u003cp\u003eAGB1 assumes a pivotal role in responding to salt stress. The G protein interactome study identified a gene ontology term linked to the salt stress response (11). Additionally, microarray analysis employing the \u003cem\u003eagb1\u003c/em\u003e null mutant showcased heightened sensitivity to salinity (19). Phenotypic characterization of chlorotic agb1 plants revealed reductions in chlorophyll content, fresh weight, survival rate, and stomatal aperture size (19, 21). Furthermore, AGB1 depletion was associated with increased shoot ABA content and Na+ accumulation (Yu and Assmann, 2015; 2016). Subsequent research elucidated AGB1\u0026apos;s role in maintaining ion homeostasis via interactions with FER and RALF1, regulating K+ and ROS levels (25).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAGB1 contributes to ER stress responses, aiding in maintaining ER homeostasis under adverse conditions such as unfolded protein accumulation or environmental stressors, and interacts with ER-localized proteins involved in various cellular processes, including calcium signaling and lipid metabolism, such as RALF (25). The ER lumen, calcium pool, and ER membrane integrity conditions may influence the relocation of bZIP17 from the ER toward the Golgi apparatus. Moreover, AGB1 localizes to the Golgi apparatus in both mammalian cells (45) and leaf epidermal cells (21). The transient expression of mRFP-bZIP17 driven by its promoter in agb1 seedlings mimics the deficiency in bZIP17 relocation to the nucleus observed in \u003cem\u003es1p s2p\u003c/em\u003e mutants, suggesting that the S1P/S2P-mediated proteolytic processing may compensate in agb1. However, whether AGB1 directly affects protease activity or physically interacts with bZIP17 to isolate substrates from enzymes remains unknown.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranscriptome analysis of \u003cem\u003eagb1\u003c/em\u003e mutants subjected to short-term salt stress revealed heightened sensitivity to elevated salinity, suggesting a crucial role for AGB1 in orchestrating transcriptional responses to equip seedlings with the necessary defenses for survival ((19) and the present study). Transcription factors such as bZIP17, NAC069, and MYB15 either interact with AGB1 or have their expression regulated by AGB1, making them promising candidates for further investigation into AGB1\u0026apos;s involvement in gene expression regulation. Furthermore, during photomorphogenesis, CRY1 inhibits AGB1 to release the transcription factor HY5 (bZIP56), enabling its DNA-binding activity and promoting gene expression for hypocotyl growth (30). AGB1 interacts with bZIP51 (VIP1) to function under drought-stress conditions (31).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations of this study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study presents valuable insights into the role of AGB1 in salt stress responses, yet several limitations constrain it. Firstly, the lack of mechanistic understanding hinders a comprehensive elucidation of molecular pathways, such as the influence of AGB1 on S1P/S2P-mediated proteolytic cleavage of bZIP17, bZIP17 intracellular trafficking and its binding affinity to cis-elements of salt stress-responsive genes. Furthermore, focusing on a limited number of time points within a short duration may only partially capture the dynamic nature of salt stress responses over longer periods, impacting the assessment of long-term fitness. Additionally, the transcriptional analysis is confined to a subset of genes regulated by bZIP17, potentially overlooking other target genes and pathways modulated by AGB1. While BiFC assays suggest a physical interaction between AGB1 and bZIP17, further validation in planta is warranted. Lastly, the study\u0026apos;s exclusive focus on \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003emay restrict the generalizability of the findings to other crop species, underscoring the necessity for comparative studies across diverse plant species to ascertain the broader relevance of AGB1-mediated salt stress responses. Addressing these limitations through future experimental investigations will be crucial for comprehensively understanding AGB1\u0026apos;s role in plant salt stress adaptation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growth conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e Columbia-0 (Col-0) ecotype was used in this study. T-DNA-tagged mutants of \u003cem\u003eagb1-3\u0026nbsp;\u003c/em\u003e(SALK_061896 as \u003cem\u003eagb1\u003c/em\u003e), \u003cem\u003ebzip17-4\u0026nbsp;\u003c/em\u003e(GABI_220B01 as \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003e(42)), \u003cem\u003es1p-1\u0026nbsp;\u003c/em\u003e(SALK_020530 as \u003cem\u003es1p\u003c/em\u003e), \u003cem\u003es2p-1\u0026nbsp;\u003c/em\u003e(GABI_459C12 as \u003cem\u003es2p\u003c/em\u003e) were obtained from Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu) and Center for Biotechnology (http://www.GABI-Kat.de). Seed surfaces were sterilized with 70% (v/v) ethanol and washed thrice with autoclaved ultrapure water. After one day of stratification at 4 \u0026deg;C, seeds were planted directly onto Petri dishes containing half-strength Murashige and Skoog (\u0026frac12; MS, Duchefa Biochemie, Haarlem, Nederland, M0222) medium with 0.86 % (w/v) sucrose (107687; Merck, Darmstadt, Germany) and 0.6% (w/v) agar (214010; Difco, BD, MJ, USA) at pH 5.6 (46). Plants were grown under 16 h / 8 h light-dark cycle at 22 \u0026deg;C with light intensity of 150 \u0026micro;mol m\u003csup\u003e-1\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e. Homozygous plants were isolated by PCR-based genotyping using gene-specific primers and T-DNA specific primers. The primers used were for \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003e(KK70/KK71 and KK8/KK71), and \u003cem\u003ebzip17\u0026nbsp;\u003c/em\u003e(KK537/KK538 and YN1016/KK538). See supplemental Table 1 for the oligonucleotide sequences used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSalt stress treatment, germination, and phenotypic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor salt stress induction, 7-day-old seedlings were immersed in liquid MS media containing 150 mM NaCl (1.06404, Merck) for the indicated time. For long-term salt stress tolerance assay, seeds were planted on the \u0026frac12; MS agar plate containing NaCl as indicated concentration for 14 days. Germination rate, seedling fresh weight (FW), primary root length, and plant width (length of two cotyledons) were measured with 11 to 25 seedlings with at least three biological replicates to quantify morphological phenotypes. The salinity tolerance analysis was performed by classifying plants according to leaf color: green, mixed (at least one white leaf), and white (all albino leaves).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVector construction and plant transformation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo clone the split YFP variants for bimolecular fluorescence complementation (BiFC) assay, the coding sequences without stop codon of AGB1, bZIP17, and AGG1 were cloned into pENTR as pYC70, pYC, and pYC69, respectively. For the BiFC assay, above-mentioned entry vectors were recombined into a pUBC-nYFP or pUBC-cYFP to obtain pYC73 (pUBC-AGB1-nYFP), pYC74 (pUBC-AGB1-cCFP), pYC78 (pUBN-bZIP17-nYFP), pYC79 (pUBN-ZIP17-cYFP), pYC71 (pUBC-AGG1-nYFP), pYC72 (pUBC-AGG1-cYFP).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eagb1 pAGB1:AGB1\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(AGB1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSame T3 line #5-2 was reported in (38).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eagb1 pAGB1:AGB1-Venus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(AGB1-V)\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate an AGB1-V complementation line, a DNA fragment encoding triple repeats of the Venus (V) fluorescent reporter was inserted in-frame before the stop codon of the AGB1 genomic region at the \u003cem\u003eSfo\u003c/em\u003eI site into pCC87 (\u003cem\u003epENTR-pAGB1:AGB1-Sfo\u003c/em\u003eI) to express AGB1-Ven. The obtained pCC91 (\u003cem\u003epENTR-pAGB1:AGB1-Venus\u003c/em\u003e) was then recombined into a pBGW destination vector (47) using Gateway\u003csup\u003eTM\u003c/sup\u003e LR Clonase\u003csup\u003eTM\u003c/sup\u003e II (Invitrogen, Thermo Fisher Scientific, MA, USA) to obtain plasmid pCC95 (\u003cem\u003epBGW-pAGB1:AGB1-Venus\u003c/em\u003e). pCC95 was transduced into \u003cem\u003eagb1\u003c/em\u003e via \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 mediated transformation. Forty-eight T1 plants were selected on soil by spraying with 0.1% BASTA solution. The obtained T2 seeds were screened using BASTA and selected by genotyping. To distinguish transgenic \u003cem\u003epAGB1:AGB1-Venus\u003c/em\u003e from endogenous \u003cem\u003eAGB1\u003c/em\u003e, specific primers (KK212/KK104) were designed. Line 6 was used for observation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ebzip17 pbZIP17:bZIP17\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e(bZIP17)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCR amplified the 5.8 kb of genomic sequence for \u003cem\u003ebZIP17\u003c/em\u003e (\u003cem\u003epbZIP17:bZIP17\u003c/em\u003e) with primers LC125 and LC126. The fragment was cloned into the pENTR\u003csup\u003eTM\u003c/sup\u003e/D_TOPO\u003csup\u003eTM\u003c/sup\u003e plasmid vector (Invitrogen) to obtain pENTR-ProbZIP17:bZIP17, which was then recombined into the pBGW destination vector (Karimi \u003cem\u003eet al\u003c/em\u003e, 2005) with the use of Gateway\u003csup\u003eTM\u003c/sup\u003e LR Clonase\u003csup\u003eTM\u003c/sup\u003e II (Invitrogen) to obtain pLC25 (pBGW-ProbZIP17:bZIP17). pLC25 was transduced into \u003cem\u003ebzip17\u003c/em\u003e via \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101 mediated transformation. In total, 24 T1 plants were selected on soil by spraying with 0.1% BASTA solution. The obtained T2 seeds were screened using BASTA and selected by genotyping. Lines 3, 5, 7 and 18 were used for observation, and Line 18 was selected as a representative line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ebzip17 pbZIP17: mRFP-bZIP17\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(\u003cem\u003em-bZIP17\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo generate an m-bZIP17 complementation line, a DNA fragment encoding mRFP fluorescent reporter was inserted in-frame after the start codon of the bZIP17 coding sequences at the \u003cem\u003eSfo\u003c/em\u003eI site into \u003cem\u003epENTR-pbZIP17: Sfo\u003c/em\u003eI\u003cem\u003e-bZIP17\u003c/em\u003e to express mRFP-bZIP17. The obtained pYC87 (\u003cem\u003epENTR-pbZIP17: mRFP-bZIP17\u003c/em\u003e) was then recombined into a pKGW destination vector (Karimi \u003cem\u003eet al\u003c/em\u003e., 2005) using Gateway\u003csup\u003eTM\u003c/sup\u003e LR Clonase\u003csup\u003eTM\u003c/sup\u003e II (Invitrogen, Thermo Fisher Scientific, MA, USA) to obtain plasmid pYC89 (\u003cem\u003epKGW-pbZIP17: mRFP-bZIP17\u003c/em\u003e). In total, 24 T1 plants were selected on \u0026frac12; MS agar plate containing the antibiotic Kanamycin. The obtained T2 seeds were screened using Kanamycin and selected by PCR-based genotyping. To distinguish transgenic \u003cem\u003epbZIP17: mRFP-bZIP17\u003c/em\u003e from endogenous \u003cem\u003ebZIP17\u003c/em\u003e, specific primers (LC12/KK418) were designed. Lines 16, 17, and 18 were used for observation, and Line 16 was selected as a representative line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal laser-microscopy observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the transient expression assay, a drop of transformed protoplasts was applied onto a glass slide with a ring sticker. The fluorescent signals were observed under confocal laser-scanning microscopy (LSM 510 Meta; Carl Zeiss, Jena, Germany) equipped with a C-Apochromat \u0026times;63 objective with a 1.2 numerical aperture. For live imaging in primary root, hypocotyl, and cotyledon, Venus or mRFP fluorescences in 7-day-old AGB1-V or m-bZIP17 seedlings, respectively, were observed under a microscope equipped with a C-Apochromat \u0026times;40 objective with 1.2 numerical aperture. For plasma membrane staining, seedlings were immersed in 10 \u0026mu;g/ml of FM4-64 (Invitrogen\u003csup\u003eTM\u003c/sup\u003e 13320) for 3 min. For ER staining, samples were immersed in 1 \u0026mu;M of ER-tracker\u003csup\u003eTM\u003c/sup\u003e Blue-White DPX (Invitrogen\u003csup\u003eTM\u003c/sup\u003e E12353) for 5 min. For nuclei staining, root samples were immersed in 10 \u0026mu;g/ml of DAPI (Thermo Fisher Scientific 62247). After staining the samples were observed under confocal microscopy, and images were captured using an LSM 510 v3.2 (Carl Zeiss) with filters for DAPI or ER-tracker Blue-White DPX (Diode 405 nm laser, band-pass 420-480 nm); Venus or YFP (Argon 514 nm laser, band-pass 520-555 nm); FM 4-64 or mRFP (HeNe 543 nm laser, band-pass 560-615 nm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting and nuclei isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTen of 7-day-old seedlings were homogenized in 100 \u0026micro;l lysis buffer [50 mM Tris HCl (Merck 648317), pH 6.8, 2 % (w/v) SDS (Merck 822050), 10 mM 2-mercaptoethanol (2-ME, Merck 805740), 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich P9599). The homogenate was stood on ice for 20 min and centrifuged at 16,000 g for 10 min at 4 \u0026deg;C. The supernatant (100 \u0026micro;l) was added to 2\u0026times; sample buffer (50 mM Tris-HCl, pH 6.8, 10% (w/v) SDS, 10%(v/v) 2-ME, 526 mM sucrose, 0.1% (w/v) bromophenol blue (Merck 108122)). Samples were boiled for 3 min at 95 \u0026deg;C and loaded and separated by 10% SDS-PAGE, blotted onto 0.45 \u0026micro;m PVDF blotting membrane (10600023; GE Healthcare, PA, USA) and probed with primary and secondary antibodies, as follows: rabbit polyclonal anti-GFP (for AGB1-VEN, 1:3,000, Invitrogen\u003csup\u003eTM\u003c/sup\u003e A-11122), mouse monoclonal anti-Actin (1:5,000, Agrisera AS10-702), rabbit polyclonal anti-Histone H3 (1:3,000, Agrisera AS10-710), goat anti-rabbit IgG-HRP (1:10,000, Abcam ab6721) and goat anti-mouse IgG-HRP (1:10,000, Abcam). The target proteins were visualized by use of Image Quant LAS4000 (GE Health). For nuclei isolation, CelLytic\u003csup\u003eTM\u003c/sup\u003e PN Isolation/Extraction Kit (Sigma CELLYTPN1) was used according to the manufacturer\u0026rsquo;s instructions with minor modifications. In brief, 1 g of fresh weight (~700 of 7-day-old seedlings) were homogenized in 3 ml of NIBA buffer [25% (v/v) 4\u0026times; Nuclei Isolation Buffer (Sigma-Aldrich N8304), 10 mM Dithiothreitol Merck #805740), 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich P9599)]. The homogenate was filtrated through the Miracloth (Merck) as a total fraction (46) and then centrifuged at 1,300 g for 10 min at 4 \u0026deg;C. The resulting supernatant was kept as a cytosolic fraction, and the pellet was resuspended by NIBA buffer containing 0.3% Triton X-100. After 30 min incubation at 4 \u0026deg;C, the resuspension was centrifuged at 12,000 g for 5 min at 4 \u0026deg;C with three repeats. The resulting pellet was resuspended by 1\u0026times; SDS sample buffer and incubated at 95 \u0026deg;C for 3 min. After being centrifuged at 12,000 g for 5 min at 4 \u0026deg;C, the supernatant was kept as a nucleus fraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroarray analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor salt stress treatment, 7-day-old Arabidopsis seedlings were subjected to \u0026frac12; MS liquid medium containing 0 (control) or 150 mM NaCl (stress) for 4 hours, for root samples were dissected right after salt stress treatment and harvested in liquid nitrogen. Total RNA was extracted using RNeasy Plant mini kit according to the manufacturer\u0026rsquo;s instructions (Qiagen) with in-membrane digestion of DNase (Qiagen) to remove genomic DNA contamination and quantified by 260/280 nm UV light absorption. For quality control, the integrity of RNA was determined by Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies). Preparation of fluorescence-labelled cDNA and microarray experiments were performed at the DNA Microarray Core Facility, Institute of Plant and Microbial Biology, Academia Sinica, Taiwan. Agilent Arabidopsis (V4) Gene Expression Microarray 4\u0026times;44k chips were used in this study. Labelling of cDNA probes and hybridization experiments were performed according to the single-colour microarray protocols provided by the manufacturer. The Agilent DNA Microarray Scanner G2565CA and Agilent Feature Extraction 10.7.1.1 software detected the fluorescence signals. Three independent biological replicates were conducted using cDNA from control and stress samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA preparation, cDNA synthesis and reverse-transcription PCR (RT-PCR), quantitative RT-PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeedlings were frozen by immersion in liquid nitrogen and stored at -80 \u0026deg;C until use. Total RNA was extracted using a standard TRI reagent solution (Invitrogen AM9738). In brief, ten of 7-day-old seedlings were homogenized in 600 \u0026micro;l of TRI reagent, followed by a phase separation step with 120 \u0026micro;l chloroform (Merck 107024). RNA was precipitated with 300 \u0026micro;l isopropanol (Merck 107022) and then 0.3 M sodium acetate (Merck 106268), washed with ethanol and resuspended in 30 \u0026micro;l of diethylpyrocarbonate (DEPC, Merck 298711) -treated water. Genomic contamination was removed using RNase-free DNase set (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. Five hundred ng RNA was used for complementary DNA (cDNA) synthesis by the SuperScript III First-Strand Synthesis SuperMix (Invitrogen 1172050). Fifty ng cDNA was used as template for quantitative RT-PCR with SYBRTM Green PCR Master Mix (4309155, Applied Biosystems\u003csup\u003eTM\u003c/sup\u003e Thermo Fisher Scientific) detection and performed in triplicate using the Applied Biosystems 7500 fast real-time PCR system. Data were analyzed by the comparative threshold cycle method (\u0026Delta;\u0026Delta;CT methods). The transcript level was normalized to the \u003cem\u003eACTIN2\u003c/em\u003e gene (\u003cem\u003eACT2\u003c/em\u003e, KK129/KK130) for each sample. For \u003cem\u003eRESPONSIVE TO DESICCATION 20\u003c/em\u003e (\u003cem\u003eRD20\u003c/em\u003e, YC116/YC117), \u003cem\u003eNAC DOMAIN CONTAINING PROTEIN 19\u003c/em\u003e (\u003cem\u003eNAC019\u003c/em\u003e, YC32/YC33), and \u003cem\u003eTSPO\u003c/em\u003e (YC118/YC119), the relative transcript level is expressed as the fold change (mean \u0026plusmn; SD) in each genotype under mock (0 mM NaCl) or salt (150 mM NaCl) treatment relative to the mock control in the wild type (set to value as 1) from three biological replicates with three technical replicates. The primer sets for quantitative RT-PCR are listed in supplemental table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtoplast isolation and BiFC assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtoplasts were isolated from 20- to 22-day-old Arabidopsis WT leaves using fungal cellulase (1% (v/v) \u0026lsquo;Onozuka\u0026rsquo; R10, Yakult, Tokyo, Japan) and macerozyme (\u0026lsquo;Onozuka\u0026rsquo; R10, Yakult) to remove cell walls accordingly with minor modification (Wu, \u003cem\u003eet al\u003c/em\u003e., 2009). DNA transfection was performed using the PEG-calcium solution, followed by 16-hr incubation at 24\u0026deg;C. Transformed protoplasts were observed under a laser-scanning confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAGROBEST transient expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor a transient expression to observe the bZIP17 processing in seedlings, the AGROBEST method was used with minor modifications (44). In Brief, seeds were germinated in the MS liquid media 3-day-old WT, \u003cem\u003eagb1\u0026nbsp;\u003c/em\u003eand \u003cem\u003es1p s2p\u003c/em\u003e mutant\u003cem\u003e\u0026nbsp;\u003c/em\u003eseedlings were infected with \u003cem\u003eAgrobacteria tumefaciens\u0026nbsp;\u003c/em\u003estrain C58C1 (pTiB6S3\u0026Delta;T)\u003csup\u003eH\u0026nbsp;\u003c/sup\u003ecarrying the pYC89 (pKGW-ProbZIP17: mRFP-bZIP17) in ABM-MS [\u0026frac12; AB-MES, \u0026frac14; MS, 0.25% (w/v) sucrose, pH5.5] liquid medium for 2 days. The co-cultivation medium was then replaced with 1 ml fresh \u0026frac12; MS medium and then incubated for 2 days. For salt stress assay, seedlings were incubated in \u0026frac12; MS medium containing 0 or 150 mM NaCl for 4 hours and then observed the mRFP-bZIP17 signals under confocal microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACCESSION NUMBERS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAGB1 (At4g34460), bZIP17 (At2g40950), AGG1 (AT3G63420).\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003en conclusion, this study elucidates the vital role of AGB1 in plant salt stress responses through its interaction with the transcription factor bZIP17 and its regulation of crucial stress-responsive pathways. The findings demonstrate that AGB1\u0026apos;s subcellular localization changes under salt stress are essential for initiating specific gene expressions necessary for plant survival. Genetic analyses further highlight the sensitivity of AGB1 mutants to salt stress, emphasizing AGB1\u0026apos;s importance in cellular homeostasis and overall plant fitness. This research contributes to our understanding of plant stress mechanisms and opens possibilities for developing crops with improved tolerance to salinity, addressing agricultural challenges posed by environmental stresses. However, more detailed studies are required to fully explore the implications of these mechanisms across various plant species and conditions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eABA \u003c/strong\u003eAbscisic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eABRE \u003c/strong\u003eABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAGB1 \u003c/strong\u003eG\u0026beta; subunit GTP BINDING PROTEIN BETA 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAGG \u003c/strong\u003eG-PROTEIN GAMMA-SUBUNIT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBBX21 \u003c/strong\u003eB-BOX DOMAIN PROTEIN 21\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBES1 \u003c/strong\u003eBRI1-EMS-SUPPRESSOR 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiFC \u003c/strong\u003eBimolecular Fluorescence Complementation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ebZIP \u003c/strong\u003eBasic region leucine zipper\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRY1 \u003c/strong\u003eCRYPTOCHROME 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFER \u003c/strong\u003eFERONIA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG protein \u003c/strong\u003eHeterotrimeric guanine nucleotide-binding protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGPCR \u003c/strong\u003eG-Protein-Coupled Receptor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGPA1 \u003c/strong\u003eG PROTEIN ALPHA SUBUNIT 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHY5 \u003c/strong\u003eELONGATED HYPOCOTYL 5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLEA \u003c/strong\u003eLATE EMBRYOGENESIS ABUNDANT\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMYB \u003c/strong\u003eMyeloblastosis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMPK6 \u003c/strong\u003eMAP KINASE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRFP \u003c/strong\u003eMonomeric Red Fluorescent Protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNAC \u003c/strong\u003eNAM, ATAF1/2, and CUC2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePHYB \u003c/strong\u003ePHYTOCHROME B\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePIF3 \u003c/strong\u003ePHYTOCHROME INTERACTING FACTOR 3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRALF1 \u003c/strong\u003eRapid Alkalinization Factor 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRD20 \u003c/strong\u003eRESPONSIVE TO DESICCATION 20\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS \u003c/strong\u003eReactive Oxygen Species\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS1P \u003c/strong\u003eSITE-1 PROTEASE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS2P \u003c/strong\u003eSITE-2 PROTEASE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOS \u003c/strong\u003eSALT OVERLY SENSITIVE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSPO \u003c/strong\u003eOUTER MEMBRANE TRYPTOPHAN-RICH SENSORY PROTEIN-RELATED\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUB \u003c/strong\u003eUbiquitin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVIP1 \u003c/strong\u003eVIRE2-INTERACTING PROTEIN 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXLG \u003c/strong\u003eEXTRA-LARGE G-PROTEIN\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYFP \u003c/strong\u003eYellow Fluorescent Protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C. designed research, performed experiments, analyzed data, and wrote the manuscript. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received funding from the Institute of Plant and Microbial Biology, Academia Sinica, Taiwan\u0026rsquo;s core budgets for Dr. Kazue Kanehara and a postdoctoral scholarship for Y.C. from Academia Sinica.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Live Cell Imaging Core Lab and Genomic Technology Core Lab at the Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, for their facilities and support. Thanks to Chia-En Chen and Ling Chuang for molecular cloning, the Arabidopsis Biological Resource Center for providing seeds, and Dr. Erh-Min Lai for the \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain C58C1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing or financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. Lamers, T. van der Meer, C. Testerink, How Plants Sense and Respond to Stressful Environments. \u003cem\u003ePlant Physiol\u003c/em\u003e \u003cstrong\u003e182\u003c/strong\u003e, 1624-1635 (2020).\u003c/li\u003e\n\u003cli\u003eD. Urano, A. M. Jones, Heterotrimeric G protein-coupled signaling in plants. \u003cem\u003eAnnu Rev Plant Biol\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 365-384 (2014).\u003c/li\u003e\n\u003cli\u003eM. J. Marinissen, J. S. 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Skoog, A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. \u003cem\u003ePhysiologia Plantarum\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 473-497 (2006).\u003c/li\u003e\n\u003cli\u003eM. Karimi, B. De Meyer, P. Hilson, Modular cloning in plant cells. \u003cem\u003eTrends Plant Sci\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 103-105 (2005).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Heterotrimeric G protein, AGB1, salinity, bZIP17, unfolded protein response (UPR)","lastPublishedDoi":"10.21203/rs.3.rs-4267287/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4267287/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gβ subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling’s survival under high salinity remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the \u003cem\u003eagb1\u003c/em\u003emutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of \u003cem\u003eagb1\u003c/em\u003e and \u003cem\u003ebzip17\u003c/em\u003e and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in \u003cem\u003es1p s2p\u003c/em\u003e double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression.\u003c/p\u003e","manuscriptTitle":"Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 10:24:30","doi":"10.21203/rs.3.rs-4267287/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-15T10:07:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-14T13:30:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21492672154497951303616330307533147345","date":"2024-05-06T12:49:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-06T08:31:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80f95357-8dd5-4516-8d3c-649c36a66543","date":"2024-04-28T06:53:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-22T15:30:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-22T11:58:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-22T11:57:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-04-15T05:14:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3b52c1a8-9073-4a90-b8b3-33b931fecb4e","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T14:36:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 10:24:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4267287","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4267287","identity":"rs-4267287","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}